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/Title (Effects of Dantrolene Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice � PLOS Currents Muscular Dystrophy)
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BT 15.000 718.042 Td /F2 21.0 Tf  [(Effects of Dantrolene Therapy on Disease Phenotype in )] TJ ET
BT 15.000 693.094 Td /F2 21.0 Tf  [(Dystrophin Deficient mdx Mice)] TJ ET
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BT 15.000 675.088 Td /F3 9.8 Tf  [(November 8, 2013)] TJ ET
BT 91.635 675.088 Td /F3 9.8 Tf  [(�)] TJ ET
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BT 96.510 675.088 Td /F3 9.8 Tf  [(Experimental Therapeutics)] TJ ET
BT 26.250 663.247 Td /F1 9.8 Tf  [(James L Quinn)] TJ ET
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BT 91.819 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 97.240 663.247 Td /F1 9.8 Tf  [(Tony Huynh)] TJ ET
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BT 149.802 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 155.223 663.247 Td /F1 9.8 Tf  [(Kitipong Uaesoontrachoon)] TJ ET
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BT 270.117 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 275.538 663.247 Td /F1 9.8 Tf  [(Kathleen Tatem)] TJ ET
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BT 349.784 663.247 Td /F1 9.8 Tf  [(Aditi Phadke)] TJ ET
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BT 404.521 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 409.942 663.247 Td /F1 9.8 Tf  [(Jack H Van der Meulen)] TJ ET
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BT 511.273 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 516.694 663.247 Td /F1 9.8 Tf  [(Qing Yu)] TJ ET
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BT 551.921 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 26.250 651.342 Td /F1 9.8 Tf  [(Kannaboyina Nagaraju)] TJ ET
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BT 26.250 639.438 Td /F1 9.8 Tf  [(Quinn JL, Huynh T, Uaesoontrachoon K, Tatem K, Phadke A, Van der Meulen JH, Yu Q, Nagaraju K. Effects of Dantrolene )] TJ ET
BT 26.250 627.533 Td /F1 9.8 Tf  [(Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice. PLOS Currents Muscular Dystrophy. 2013 Nov 8 . Edition 1. )] TJ ET
BT 26.250 615.628 Td /F1 9.8 Tf  [(doi: 10.1371/currents.md.e246cf493a7edb1669f42fb735936b46.)] TJ ET
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BT 26.250 586.526 Td /F4 12.0 Tf  [(Abstract)] TJ ET
BT 26.250 566.571 Td /F1 9.8 Tf  [(Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in )] TJ ET
BT 26.250 554.667 Td /F1 9.8 Tf  [(intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor )] TJ ET
BT 26.250 542.762 Td /F1 9.8 Tf  [(\(RyR1\) on the sarcoplasmic reticular \(SR\) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle )] TJ ET
BT 26.250 530.857 Td /F1 9.8 Tf  [(relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release )] TJ ET
BT 26.250 518.952 Td /F1 9.8 Tf  [(by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease )] TJ ET
BT 26.250 507.048 Td /F1 9.8 Tf  [(phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice \(4 weeks old\) with daily intraperitoneal )] TJ ET
BT 26.250 495.143 Td /F1 9.8 Tf  [(injections of 40mg/kg of Dantrolene for 6 weeks and measured functional \(grip strength, in vitro force contractions\), behavioral )] TJ ET
BT 26.250 483.238 Td /F1 9.8 Tf  [(\(open field digiscan\), imagining \(optical imaging for inflammation\), histological \(H&E\), and molecular \(protein and RNA\) )] TJ ET
BT 26.250 471.333 Td /F1 9.8 Tf  [(endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field )] TJ ET
BT 26.250 459.429 Td /F1 9.8 Tf  [(behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of )] TJ ET
BT 26.250 447.524 Td /F1 9.8 Tf  [(cathepsin activity or histological \(H&E\) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific )] TJ ET
BT 26.250 435.619 Td /F1 9.8 Tf  [(force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK )] TJ ET
BT 26.250 423.714 Td /F1 9.8 Tf  [(levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results )] TJ ET
BT 26.250 411.810 Td /F1 9.8 Tf  [(suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice.)] TJ ET
BT 26.250 375.207 Td /F4 12.0 Tf  [(Funding Statement)] TJ ET
BT 26.250 355.253 Td /F1 9.8 Tf  [(This project was supported with funding from the Department of Defense \(W81XWH-11-1-0330\).)] TJ ET
BT 26.250 326.150 Td /F4 12.0 Tf  [(Introduction)] TJ ET
BT 26.250 306.196 Td /F1 9.8 Tf  [(Duchenne muscular dystrophy \(DMD\) is an X-linked genetic disorder that affects 1 in 3500 boys in all populations )] TJ ET
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BT 517.202 307.703 Td /F4 8.7 Tf  [(1)] TJ ET
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BT 522.020 306.196 Td /F1 9.8 Tf  [(. It is caused )] TJ ET
BT 26.250 294.291 Td /F1 9.8 Tf  [(by a mutation in the dystrophin gene, leading to the absence of the cytoskeletal protein dystrophin )] TJ ET
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BT 450.609 295.799 Td /F4 8.7 Tf  [(2)] TJ ET
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BT 455.428 294.291 Td /F1 9.8 Tf  [(. This protein connects the )] TJ ET
BT 26.250 282.387 Td /F1 9.8 Tf  [(cytoskeleton of muscle fibers to the extracellular matrix. Without this anchoring protein, the muscle�s structure is compromised )] TJ ET
BT 26.250 270.482 Td /F1 9.8 Tf  [(which in turn results in contraction-induced injury. This repeated injury leads to cycles of inflammation and muscle degeneration, )] TJ ET
BT 26.250 258.577 Td /F1 9.8 Tf  [(which in turn leads to muscle weakening and loss of function. The repeated damage to the muscle fiber due to contraction also )] TJ ET
BT 26.250 246.672 Td /F1 9.8 Tf  [(results in transient tears in the sarcolemma of dystrophic muscles, thereby allowing the influx of extracellular calcium )] TJ ET
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BT 530.715 248.180 Td /F4 8.7 Tf  [(3)] TJ ET
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BT 535.534 246.672 Td /F1 9.8 Tf  [(. This )] TJ ET
BT 26.250 234.768 Td /F1 9.8 Tf  [(elevated influx of calcium results in a sustained increase in intracellular calcium levels.)] TJ ET
BT 26.250 215.363 Td /F1 9.8 Tf  [(One of the major consequences of this calcium increase is the increased activation of calcium-dependent proteases that lead to )] TJ ET
BT 26.250 203.458 Td /F1 9.8 Tf  [(protein degradation and eventually cell death )] TJ ET
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BT 222.986 204.965 Td /F4 8.7 Tf  [(4)] TJ ET
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BT 227.804 203.458 Td /F1 9.8 Tf  [(. This increased degeneration from calcium-induced necrosis worsens the overall )] TJ ET
BT 26.250 191.553 Td /F1 9.8 Tf  [(phenotype of DMD and hastens the loss of ambulation and eventual death due to respiratory and cardiac complications. )] TJ ET
BT 26.250 179.649 Td /F1 9.8 Tf  [(Therefore, targeting mechanisms that control calcium should have therapeutic benefits. In fact, restoring calcium homeostasis )] TJ ET
BT 26.250 167.744 Td /F1 9.8 Tf  [(by increasing calcium channels or the use of drugs that increase cellular proteins that stabilize calcium channels has been )] TJ ET
BT 26.250 155.839 Td /F1 9.8 Tf  [(shown to improve muscle function and strength )] TJ ET
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BT 232.716 157.346 Td /F4 8.7 Tf  [(5)] TJ ET
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BT 237.535 159.727 Td /F1 8.7 Tf  [(,)] TJ ET
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BT 244.763 159.727 Td /F1 8.7 Tf  [(,)] TJ ET
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BT 247.172 157.346 Td /F4 8.7 Tf  [(7)] TJ ET
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BT 251.991 155.839 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 136.434 Td /F1 9.8 Tf  [(In skeletal muscle, contraction is induced through a process of calcium-induced calcium release. This occurs when the )] TJ ET
BT 26.250 124.530 Td /F1 9.8 Tf  [(Ryanodine receptor \(RyR1\) on the sarcoplasmic reticular \(SR\) membrane interacts with voltage-dependent Ca)] TJ ET
BT 501.991 128.418 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 511.872 124.530 Td /F1 9.8 Tf  [( channels on )] TJ ET
BT 26.250 112.625 Td /F1 9.8 Tf  [(the T-tubules such as dyhydropyridine receptors \(DHPRs\) and allows calcium release from the SR into the sarcoplasm when )] TJ ET
BT 26.250 100.720 Td /F1 9.8 Tf  [(activated )] TJ ET
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BT 67.980 102.227 Td /F4 8.7 Tf  [(8)] TJ ET
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BT 72.799 100.720 Td /F1 9.8 Tf  [(. This complex is activated by signals proliferated along the T-tubules and leads to muscle contraction.)] TJ ET
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BT 15.000 718.042 Td /F2 21.0 Tf  [(Effects of Dantrolene Therapy on Disease Phenotype in )] TJ ET
BT 15.000 693.094 Td /F2 21.0 Tf  [(Dystrophin Deficient mdx Mice)] TJ ET
Q
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BT 15.000 675.088 Td /F3 9.8 Tf  [(November 8, 2013)] TJ ET
BT 91.635 675.088 Td /F3 9.8 Tf  [(�)] TJ ET
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BT 96.510 675.088 Td /F3 9.8 Tf  [(Experimental Therapeutics)] TJ ET
BT 26.250 663.247 Td /F1 9.8 Tf  [(James L Quinn)] TJ ET
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BT 91.819 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 97.240 663.247 Td /F1 9.8 Tf  [(Tony Huynh)] TJ ET
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BT 149.802 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 155.223 663.247 Td /F1 9.8 Tf  [(Kitipong Uaesoontrachoon)] TJ ET
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BT 270.117 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 275.538 663.247 Td /F1 9.8 Tf  [(Kathleen Tatem)] TJ ET
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BT 344.363 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 349.784 663.247 Td /F1 9.8 Tf  [(Aditi Phadke)] TJ ET
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BT 404.521 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 409.942 663.247 Td /F1 9.8 Tf  [(Jack H Van der Meulen)] TJ ET
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BT 511.273 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 516.694 663.247 Td /F1 9.8 Tf  [(Qing Yu)] TJ ET
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BT 551.921 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 26.250 651.342 Td /F1 9.8 Tf  [(Kannaboyina Nagaraju)] TJ ET
0.271 0.267 0.267 rg
BT 26.250 639.438 Td /F1 9.8 Tf  [(Quinn JL, Huynh T, Uaesoontrachoon K, Tatem K, Phadke A, Van der Meulen JH, Yu Q, Nagaraju K. Effects of Dantrolene )] TJ ET
BT 26.250 627.533 Td /F1 9.8 Tf  [(Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice. PLOS Currents Muscular Dystrophy. 2013 Nov 8 . Edition 1. )] TJ ET
BT 26.250 615.628 Td /F1 9.8 Tf  [(doi: 10.1371/currents.md.e246cf493a7edb1669f42fb735936b46.)] TJ ET
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15.000 35.720 577.500 577.527 re W n
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BT 26.250 586.526 Td /F4 12.0 Tf  [(Abstract)] TJ ET
BT 26.250 566.571 Td /F1 9.8 Tf  [(Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in )] TJ ET
BT 26.250 554.667 Td /F1 9.8 Tf  [(intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor )] TJ ET
BT 26.250 542.762 Td /F1 9.8 Tf  [(\(RyR1\) on the sarcoplasmic reticular \(SR\) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle )] TJ ET
BT 26.250 530.857 Td /F1 9.8 Tf  [(relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release )] TJ ET
BT 26.250 518.952 Td /F1 9.8 Tf  [(by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease )] TJ ET
BT 26.250 507.048 Td /F1 9.8 Tf  [(phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice \(4 weeks old\) with daily intraperitoneal )] TJ ET
BT 26.250 495.143 Td /F1 9.8 Tf  [(injections of 40mg/kg of Dantrolene for 6 weeks and measured functional \(grip strength, in vitro force contractions\), behavioral )] TJ ET
BT 26.250 483.238 Td /F1 9.8 Tf  [(\(open field digiscan\), imagining \(optical imaging for inflammation\), histological \(H&E\), and molecular \(protein and RNA\) )] TJ ET
BT 26.250 471.333 Td /F1 9.8 Tf  [(endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field )] TJ ET
BT 26.250 459.429 Td /F1 9.8 Tf  [(behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of )] TJ ET
BT 26.250 447.524 Td /F1 9.8 Tf  [(cathepsin activity or histological \(H&E\) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific )] TJ ET
BT 26.250 435.619 Td /F1 9.8 Tf  [(force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK )] TJ ET
BT 26.250 423.714 Td /F1 9.8 Tf  [(levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results )] TJ ET
BT 26.250 411.810 Td /F1 9.8 Tf  [(suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice.)] TJ ET
BT 26.250 375.207 Td /F4 12.0 Tf  [(Funding Statement)] TJ ET
BT 26.250 355.253 Td /F1 9.8 Tf  [(This project was supported with funding from the Department of Defense \(W81XWH-11-1-0330\).)] TJ ET
BT 26.250 326.150 Td /F4 12.0 Tf  [(Introduction)] TJ ET
BT 26.250 306.196 Td /F1 9.8 Tf  [(Duchenne muscular dystrophy \(DMD\) is an X-linked genetic disorder that affects 1 in 3500 boys in all populations )] TJ ET
0.267 0.267 0.267 rg
BT 517.202 307.703 Td /F4 8.7 Tf  [(1)] TJ ET
0.271 0.267 0.267 rg
BT 522.020 306.196 Td /F1 9.8 Tf  [(. It is caused )] TJ ET
BT 26.250 294.291 Td /F1 9.8 Tf  [(by a mutation in the dystrophin gene, leading to the absence of the cytoskeletal protein dystrophin )] TJ ET
0.267 0.267 0.267 rg
BT 450.609 295.799 Td /F4 8.7 Tf  [(2)] TJ ET
0.271 0.267 0.267 rg
BT 455.428 294.291 Td /F1 9.8 Tf  [(. This protein connects the )] TJ ET
BT 26.250 282.387 Td /F1 9.8 Tf  [(cytoskeleton of muscle fibers to the extracellular matrix. Without this anchoring protein, the muscle�s structure is compromised )] TJ ET
BT 26.250 270.482 Td /F1 9.8 Tf  [(which in turn results in contraction-induced injury. This repeated injury leads to cycles of inflammation and muscle degeneration, )] TJ ET
BT 26.250 258.577 Td /F1 9.8 Tf  [(which in turn leads to muscle weakening and loss of function. The repeated damage to the muscle fiber due to contraction also )] TJ ET
BT 26.250 246.672 Td /F1 9.8 Tf  [(results in transient tears in the sarcolemma of dystrophic muscles, thereby allowing the influx of extracellular calcium )] TJ ET
0.267 0.267 0.267 rg
BT 530.715 248.180 Td /F4 8.7 Tf  [(3)] TJ ET
0.271 0.267 0.267 rg
BT 535.534 246.672 Td /F1 9.8 Tf  [(. This )] TJ ET
BT 26.250 234.768 Td /F1 9.8 Tf  [(elevated influx of calcium results in a sustained increase in intracellular calcium levels.)] TJ ET
BT 26.250 215.363 Td /F1 9.8 Tf  [(One of the major consequences of this calcium increase is the increased activation of calcium-dependent proteases that lead to )] TJ ET
BT 26.250 203.458 Td /F1 9.8 Tf  [(protein degradation and eventually cell death )] TJ ET
0.267 0.267 0.267 rg
BT 222.986 204.965 Td /F4 8.7 Tf  [(4)] TJ ET
0.271 0.267 0.267 rg
BT 227.804 203.458 Td /F1 9.8 Tf  [(. This increased degeneration from calcium-induced necrosis worsens the overall )] TJ ET
BT 26.250 191.553 Td /F1 9.8 Tf  [(phenotype of DMD and hastens the loss of ambulation and eventual death due to respiratory and cardiac complications. )] TJ ET
BT 26.250 179.649 Td /F1 9.8 Tf  [(Therefore, targeting mechanisms that control calcium should have therapeutic benefits. In fact, restoring calcium homeostasis )] TJ ET
BT 26.250 167.744 Td /F1 9.8 Tf  [(by increasing calcium channels or the use of drugs that increase cellular proteins that stabilize calcium channels has been )] TJ ET
BT 26.250 155.839 Td /F1 9.8 Tf  [(shown to improve muscle function and strength )] TJ ET
0.267 0.267 0.267 rg
BT 232.716 157.346 Td /F4 8.7 Tf  [(5)] TJ ET
0.271 0.267 0.267 rg
BT 237.535 159.727 Td /F1 8.7 Tf  [(,)] TJ ET
0.267 0.267 0.267 rg
BT 239.944 157.346 Td /F4 8.7 Tf  [(6)] TJ ET
0.271 0.267 0.267 rg
BT 244.763 159.727 Td /F1 8.7 Tf  [(,)] TJ ET
0.267 0.267 0.267 rg
BT 247.172 157.346 Td /F4 8.7 Tf  [(7)] TJ ET
0.271 0.267 0.267 rg
BT 251.991 155.839 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 136.434 Td /F1 9.8 Tf  [(In skeletal muscle, contraction is induced through a process of calcium-induced calcium release. This occurs when the )] TJ ET
BT 26.250 124.530 Td /F1 9.8 Tf  [(Ryanodine receptor \(RyR1\) on the sarcoplasmic reticular \(SR\) membrane interacts with voltage-dependent Ca)] TJ ET
BT 501.991 128.418 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 511.872 124.530 Td /F1 9.8 Tf  [( channels on )] TJ ET
BT 26.250 112.625 Td /F1 9.8 Tf  [(the T-tubules such as dyhydropyridine receptors \(DHPRs\) and allows calcium release from the SR into the sarcoplasm when )] TJ ET
BT 26.250 100.720 Td /F1 9.8 Tf  [(activated )] TJ ET
0.267 0.267 0.267 rg
BT 67.980 102.227 Td /F4 8.7 Tf  [(8)] TJ ET
0.271 0.267 0.267 rg
BT 72.799 100.720 Td /F1 9.8 Tf  [(. This complex is activated by signals proliferated along the T-tubules and leads to muscle contraction.)] TJ ET
Q
q
15.000 684.354 577.500 53.646 re W n
0.267 0.267 0.267 rg
BT 15.000 718.042 Td /F2 21.0 Tf  [(Effects of Dantrolene Therapy on Disease Phenotype in )] TJ ET
BT 15.000 693.094 Td /F2 21.0 Tf  [(Dystrophin Deficient mdx Mice)] TJ ET
Q
0.271 0.267 0.267 rg
BT 15.000 675.088 Td /F3 9.8 Tf  [(November 8, 2013)] TJ ET
BT 91.635 675.088 Td /F3 9.8 Tf  [(�)] TJ ET
0.267 0.267 0.267 rg
BT 96.510 675.088 Td /F3 9.8 Tf  [(Experimental Therapeutics)] TJ ET
BT 26.250 663.247 Td /F1 9.8 Tf  [(James L Quinn)] TJ ET
0.271 0.267 0.267 rg
BT 91.819 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
0.267 0.267 0.267 rg
BT 97.240 663.247 Td /F1 9.8 Tf  [(Tony Huynh)] TJ ET
0.271 0.267 0.267 rg
BT 149.802 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
0.267 0.267 0.267 rg
BT 155.223 663.247 Td /F1 9.8 Tf  [(Kitipong Uaesoontrachoon)] TJ ET
0.271 0.267 0.267 rg
BT 270.117 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
0.267 0.267 0.267 rg
BT 275.538 663.247 Td /F1 9.8 Tf  [(Kathleen Tatem)] TJ ET
0.271 0.267 0.267 rg
BT 344.363 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
0.267 0.267 0.267 rg
BT 349.784 663.247 Td /F1 9.8 Tf  [(Aditi Phadke)] TJ ET
0.271 0.267 0.267 rg
BT 404.521 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
0.267 0.267 0.267 rg
BT 409.942 663.247 Td /F1 9.8 Tf  [(Jack H Van der Meulen)] TJ ET
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BT 516.694 663.247 Td /F1 9.8 Tf  [(Qing Yu)] TJ ET
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BT 551.921 663.247 Td /F1 9.8 Tf  [(, )] TJ ET
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BT 26.250 651.342 Td /F1 9.8 Tf  [(Kannaboyina Nagaraju)] TJ ET
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BT 26.250 639.438 Td /F1 9.8 Tf  [(Quinn JL, Huynh T, Uaesoontrachoon K, Tatem K, Phadke A, Van der Meulen JH, Yu Q, Nagaraju K. Effects of Dantrolene )] TJ ET
BT 26.250 627.533 Td /F1 9.8 Tf  [(Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice. PLOS Currents Muscular Dystrophy. 2013 Nov 8 . Edition 1. )] TJ ET
BT 26.250 615.628 Td /F1 9.8 Tf  [(doi: 10.1371/currents.md.e246cf493a7edb1669f42fb735936b46.)] TJ ET
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BT 26.250 586.526 Td /F4 12.0 Tf  [(Abstract)] TJ ET
BT 26.250 566.571 Td /F1 9.8 Tf  [(Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in )] TJ ET
BT 26.250 554.667 Td /F1 9.8 Tf  [(intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor )] TJ ET
BT 26.250 542.762 Td /F1 9.8 Tf  [(\(RyR1\) on the sarcoplasmic reticular \(SR\) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle )] TJ ET
BT 26.250 530.857 Td /F1 9.8 Tf  [(relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release )] TJ ET
BT 26.250 518.952 Td /F1 9.8 Tf  [(by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease )] TJ ET
BT 26.250 507.048 Td /F1 9.8 Tf  [(phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice \(4 weeks old\) with daily intraperitoneal )] TJ ET
BT 26.250 495.143 Td /F1 9.8 Tf  [(injections of 40mg/kg of Dantrolene for 6 weeks and measured functional \(grip strength, in vitro force contractions\), behavioral )] TJ ET
BT 26.250 483.238 Td /F1 9.8 Tf  [(\(open field digiscan\), imagining \(optical imaging for inflammation\), histological \(H&E\), and molecular \(protein and RNA\) )] TJ ET
BT 26.250 471.333 Td /F1 9.8 Tf  [(endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field )] TJ ET
BT 26.250 459.429 Td /F1 9.8 Tf  [(behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of )] TJ ET
BT 26.250 447.524 Td /F1 9.8 Tf  [(cathepsin activity or histological \(H&E\) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific )] TJ ET
BT 26.250 435.619 Td /F1 9.8 Tf  [(force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK )] TJ ET
BT 26.250 423.714 Td /F1 9.8 Tf  [(levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results )] TJ ET
BT 26.250 411.810 Td /F1 9.8 Tf  [(suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice.)] TJ ET
BT 26.250 375.207 Td /F4 12.0 Tf  [(Funding Statement)] TJ ET
BT 26.250 355.253 Td /F1 9.8 Tf  [(This project was supported with funding from the Department of Defense \(W81XWH-11-1-0330\).)] TJ ET
BT 26.250 326.150 Td /F4 12.0 Tf  [(Introduction)] TJ ET
BT 26.250 306.196 Td /F1 9.8 Tf  [(Duchenne muscular dystrophy \(DMD\) is an X-linked genetic disorder that affects 1 in 3500 boys in all populations )] TJ ET
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BT 522.020 306.196 Td /F1 9.8 Tf  [(. It is caused )] TJ ET
BT 26.250 294.291 Td /F1 9.8 Tf  [(by a mutation in the dystrophin gene, leading to the absence of the cytoskeletal protein dystrophin )] TJ ET
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BT 450.609 295.799 Td /F4 8.7 Tf  [(2)] TJ ET
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BT 455.428 294.291 Td /F1 9.8 Tf  [(. This protein connects the )] TJ ET
BT 26.250 282.387 Td /F1 9.8 Tf  [(cytoskeleton of muscle fibers to the extracellular matrix. Without this anchoring protein, the muscle�s structure is compromised )] TJ ET
BT 26.250 270.482 Td /F1 9.8 Tf  [(which in turn results in contraction-induced injury. This repeated injury leads to cycles of inflammation and muscle degeneration, )] TJ ET
BT 26.250 258.577 Td /F1 9.8 Tf  [(which in turn leads to muscle weakening and loss of function. The repeated damage to the muscle fiber due to contraction also )] TJ ET
BT 26.250 246.672 Td /F1 9.8 Tf  [(results in transient tears in the sarcolemma of dystrophic muscles, thereby allowing the influx of extracellular calcium )] TJ ET
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BT 530.715 248.180 Td /F4 8.7 Tf  [(3)] TJ ET
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BT 535.534 246.672 Td /F1 9.8 Tf  [(. This )] TJ ET
BT 26.250 234.768 Td /F1 9.8 Tf  [(elevated influx of calcium results in a sustained increase in intracellular calcium levels.)] TJ ET
BT 26.250 215.363 Td /F1 9.8 Tf  [(One of the major consequences of this calcium increase is the increased activation of calcium-dependent proteases that lead to )] TJ ET
BT 26.250 203.458 Td /F1 9.8 Tf  [(protein degradation and eventually cell death )] TJ ET
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BT 222.986 204.965 Td /F4 8.7 Tf  [(4)] TJ ET
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BT 227.804 203.458 Td /F1 9.8 Tf  [(. This increased degeneration from calcium-induced necrosis worsens the overall )] TJ ET
BT 26.250 191.553 Td /F1 9.8 Tf  [(phenotype of DMD and hastens the loss of ambulation and eventual death due to respiratory and cardiac complications. )] TJ ET
BT 26.250 179.649 Td /F1 9.8 Tf  [(Therefore, targeting mechanisms that control calcium should have therapeutic benefits. In fact, restoring calcium homeostasis )] TJ ET
BT 26.250 167.744 Td /F1 9.8 Tf  [(by increasing calcium channels or the use of drugs that increase cellular proteins that stabilize calcium channels has been )] TJ ET
BT 26.250 155.839 Td /F1 9.8 Tf  [(shown to improve muscle function and strength )] TJ ET
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BT 232.716 157.346 Td /F4 8.7 Tf  [(5)] TJ ET
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BT 237.535 159.727 Td /F1 8.7 Tf  [(,)] TJ ET
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BT 239.944 157.346 Td /F4 8.7 Tf  [(6)] TJ ET
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BT 244.763 159.727 Td /F1 8.7 Tf  [(,)] TJ ET
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BT 247.172 157.346 Td /F4 8.7 Tf  [(7)] TJ ET
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BT 251.991 155.839 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 136.434 Td /F1 9.8 Tf  [(In skeletal muscle, contraction is induced through a process of calcium-induced calcium release. This occurs when the )] TJ ET
BT 26.250 124.530 Td /F1 9.8 Tf  [(Ryanodine receptor \(RyR1\) on the sarcoplasmic reticular \(SR\) membrane interacts with voltage-dependent Ca)] TJ ET
BT 501.991 128.418 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 511.872 124.530 Td /F1 9.8 Tf  [( channels on )] TJ ET
BT 26.250 112.625 Td /F1 9.8 Tf  [(the T-tubules such as dyhydropyridine receptors \(DHPRs\) and allows calcium release from the SR into the sarcoplasm when )] TJ ET
BT 26.250 100.720 Td /F1 9.8 Tf  [(activated )] TJ ET
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BT 67.980 102.227 Td /F4 8.7 Tf  [(8)] TJ ET
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BT 26.250 767.476 Td /F1 9.8 Tf  [(Another channel of interest for calcium handling within muscle fibers is the sarco/endoplasmic reticulum Ca)] TJ ET
BT 487.376 771.364 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 497.256 767.476 Td /F1 9.8 Tf  [(-ATPase )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(\(SERCA1\), a calcium ATPase found on the SR membrane in muscle fibers. This protein is responsible for pumping calcium out )] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(of the sarcoplasm and back into the SR following excitation )] TJ ET
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BT 283.660 745.174 Td /F4 8.7 Tf  [(9)] TJ ET
0.271 0.267 0.267 rg
BT 288.478 743.667 Td /F1 9.8 Tf  [(. It has been demonstrated that this protein is dysfunctional in mdx )] TJ ET
BT 26.250 731.762 Td /F1 9.8 Tf  [(mice and further contributes to increased levels of sarcoplasmic calcium )] TJ ET
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BT 339.440 733.269 Td /F4 8.7 Tf  [(10)] TJ ET
0.271 0.267 0.267 rg
BT 349.077 731.762 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 712.357 Td /F1 9.8 Tf  [(Dantrolene Sodium is an FDA approved skeletal muscle relaxant that has been safely administered to DMD patients without )] TJ ET
BT 26.250 700.452 Td /F1 9.8 Tf  [(harmful side effects )] TJ ET
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BT 113.493 701.960 Td /F4 8.7 Tf  [(11)] TJ ET
0.271 0.267 0.267 rg
BT 123.130 700.452 Td /F1 9.8 Tf  [(. Dantrolene binds to the RyR1 receptor and inhibits the release of calcium from the SR during excitation-)] TJ ET
BT 26.250 688.548 Td /F1 9.8 Tf  [(contraction by blocking its activation )] TJ ET
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BT 184.483 690.055 Td /F4 8.7 Tf  [(12)] TJ ET
0.271 0.267 0.267 rg
BT 194.120 688.548 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(We will use Dantrolene treatment to decrease intracellular calcium levels and thus decrease the amount of calcium-induced )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(necrosis and improve the overall phenotype of the treated mice. To test the efficacy of this treatment regime, we have )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(performed a pre-clinical trial in mdx mice and will evaluate improvement using several criteria: functional, behavioral, )] TJ ET
BT 26.250 633.429 Td /F1 9.8 Tf  [(histological, and molecular assays.)] TJ ET
BT 26.250 596.826 Td /F4 12.0 Tf  [(Methods)] TJ ET
BT 26.250 576.872 Td /F4 9.8 Tf  [(Animal and Drug Dosing Regimen:)] TJ ET
BT 26.250 557.467 Td /F1 9.8 Tf  [(Wild type \(C57BL/10ScSn/J\) and mdx \(C57BL/10ScSn-mdx/J\) mice were obtained from Jackson Laboratory \(Bar Harbor, ME\) )] TJ ET
BT 26.250 545.562 Td /F1 9.8 Tf  [(and bred in house in our Animal Facility. All mice were acclimated for four weeks before testing. All handling and )] TJ ET
BT 26.250 533.658 Td /F1 9.8 Tf  [(experimentation with mice was conducted in accordance with our IACUC guidelines under approved protocols. Mdx mice were )] TJ ET
BT 26.250 521.753 Td /F1 9.8 Tf  [(assigned to treatment groups randomly based on bodyweight \(BW\). Beginning at 4 weeks of age, all animals received daily \(7 )] TJ ET
BT 26.250 509.848 Td /F1 9.8 Tf  [(day/week\) single intraperitoneal injections of water or Dantrolene at a concentration of 40mg/kg for 6 weeks. Functional testing )] TJ ET
BT 26.250 497.943 Td /F1 9.8 Tf  [(was conducted at 8 weeks of age followed by optical imaging the next week. At 10 weeks of age the mice were sacrificed and )] TJ ET
BT 26.250 486.039 Td /F1 9.8 Tf  [(tissue samples were immediately collected, weighed, snap frozen in liquid nitrogen-cooled isopentane, and stored at -80?C for )] TJ ET
BT 26.250 474.134 Td /F1 9.8 Tf  [(later processing.)] TJ ET
BT 26.250 454.729 Td /F4 9.8 Tf  [(Grip Strength Measurement:)] TJ ET
BT 26.250 435.324 Td /F1 9.8 Tf  [(After four weeks of treatment, forelimb and hindlimb GSMs were performed as previously described )] TJ ET
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BT 457.570 436.832 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 467.208 435.324 Td /F1 9.8 Tf  [( but with an increase in )] TJ ET
BT 26.250 423.420 Td /F1 9.8 Tf  [(the acclimation period from three to five consecutive days. The GSMs were collected in the morning hours over a 5-day period, )] TJ ET
BT 26.250 411.515 Td /F1 9.8 Tf  [(with maximum values for each day over this period averaged to obtain absolute GSM values \(Kgf\) or normalized to BW )] TJ ET
BT 26.250 399.610 Td /F1 9.8 Tf  [(\(recorded on the first day of testing\) for normalized GSM values \(Kgf/kg\).)] TJ ET
BT 26.250 380.205 Td /F4 9.8 Tf  [(Open-field Behavioral Activity Measurements \(Digiscan\):)] TJ ET
BT 26.250 360.801 Td /F1 9.8 Tf  [(After four weeks of treatment, voluntary locomotor activity was measured using an open-field digiscan apparatus \(Omnitech )] TJ ET
BT 26.250 348.896 Td /F1 9.8 Tf  [(Electronics, Columbus, Ohio\) as previously described )] TJ ET
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BT 259.782 350.403 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 269.419 348.896 Td /F1 9.8 Tf  [( except that the mice were acclimated for four days before data was )] TJ ET
BT 26.250 336.991 Td /F1 9.8 Tf  [(collected. Following acclimation, data was recorded over a 1-hour period each day for four consecutive days. Acclimation and )] TJ ET
BT 26.250 325.086 Td /F1 9.8 Tf  [(collection of data was performed in the morning hours for all mice.)] TJ ET
BT 26.250 305.682 Td /F5 9.8 Tf  [(In vitro)] TJ ET
BT 58.757 305.682 Td /F4 9.8 Tf  [(Force Contraction:)] TJ ET
BT 26.250 286.277 Td /F1 9.8 Tf  [(Prior to sacrifice, the extensor digitorium longus \(EDL\) muscles of the right hindlimbs were removed for comparison of )] TJ ET
BT 536.126 286.277 Td /F6 9.8 Tf  [(in vitro)] TJ ET
BT 26.250 274.372 Td /F1 9.8 Tf  [(force contractions between groups as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 282.568 275.879 Td /F4 8.7 Tf  [(14)] TJ ET
0.271 0.267 0.267 rg
BT 292.205 274.372 Td /F1 9.8 Tf  [(. Briefly, the distal tendon of the muscle was tied securely to the )] TJ ET
BT 26.250 262.467 Td /F1 9.8 Tf  [(lever arm of a servomotor/force transducer \(model 305B\) \(Aurora Scientific, Aurora, Ontario, Canada\) and the proximal tendon )] TJ ET
BT 26.250 250.563 Td /F1 9.8 Tf  [(was fixed to a stationary post in the bath. The muscles were stimulated between two stainless steel plate electrodes. At optimal )] TJ ET
BT 26.250 238.658 Td /F1 9.8 Tf  [(muscle length, the force developed was measured during trains of stimulation \(300 milliseconds\) with increasing frequencies )] TJ ET
BT 26.250 226.753 Td /F1 9.8 Tf  [(until the highest plateau was achieved. The force generated to obtain the highest plateau was used to determine specific force )] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(and is expressed as mN/mm)] TJ ET
BT 149.782 218.737 Td /F1 8.7 Tf  [(2)] TJ ET
BT 154.601 214.848 Td /F1 9.8 Tf  [(. Additionally, the muscle was subjected to a protocol of ten lengthening contractions separated by )] TJ ET
BT 26.250 202.944 Td /F1 9.8 Tf  [(one minute rest intervals. The muscle was stimulated at 250Hz for 300ms allowing the muscle to generate force immediately )] TJ ET
BT 26.250 191.039 Td /F1 9.8 Tf  [(followed by a lengthening over 10% of muscle length at a velocity of two fiber lengths/second. Finally, the muscle was subjected )] TJ ET
BT 26.250 179.134 Td /F1 9.8 Tf  [(to a fatigue protocol consisting of 60 isometric contractions for 300 milliseconds each, once every 5 seconds. The frequency at )] TJ ET
BT 26.250 167.229 Td /F1 9.8 Tf  [(which the EDL muscles were stimulated is 250Hz. The force was recorded every minute during the repetitive contractions and )] TJ ET
BT 26.250 155.325 Td /F1 9.8 Tf  [(again five minutes after to measure recovery.)] TJ ET
BT 26.250 135.920 Td /F4 9.8 Tf  [(Live Animal Optical Imaging:)] TJ ET
BT 26.250 116.515 Td /F1 9.8 Tf  [(After five weeks of treatment, quantification of inflammation in forelimb and hindlimb muscles of mice was achieved using live-)] TJ ET
BT 26.250 104.610 Td /F1 9.8 Tf  [(animal optical imaging of Cathepsin-B \(CTSB\) enzyme activity using a caged near-infrared substrate \(ProSense 680\) as )] TJ ET
BT 26.250 92.706 Td /F1 9.8 Tf  [(previously described )] TJ ET
0.267 0.267 0.267 rg
BT 117.822 94.213 Td /F4 8.7 Tf  [(15)] TJ ET
0.271 0.267 0.267 rg
BT 127.459 92.706 Td /F1 9.8 Tf  [(. Briefly, mice were injected intraperitoneally with 1.5 nmol of ProSense 680 \(VisEn Medical\). Areas of )] TJ ET
BT 26.250 80.801 Td /F1 9.8 Tf  [(scanning were defined at 1.0-mm resolution. Cathepsin activity was measured from forelimb and hindlimb muscles and defined )] TJ ET
BT 26.250 68.896 Td /F1 9.8 Tf  [(as intensity in terms of photon counts/mm)] TJ ET
BT 206.157 72.784 Td /F1 8.7 Tf  [(2)] TJ ET
BT 210.976 68.896 Td /F1 9.8 Tf  [( using the Optiview 2.0 software Optix.)] TJ ET
BT 26.250 49.491 Td /F4 9.8 Tf  [(Serum creatine kinase \(CK\) measurements:)] TJ ET
Q
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BT 26.250 767.476 Td /F1 9.8 Tf  [(Another channel of interest for calcium handling within muscle fibers is the sarco/endoplasmic reticulum Ca)] TJ ET
BT 487.376 771.364 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 497.256 767.476 Td /F1 9.8 Tf  [(-ATPase )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(\(SERCA1\), a calcium ATPase found on the SR membrane in muscle fibers. This protein is responsible for pumping calcium out )] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(of the sarcoplasm and back into the SR following excitation )] TJ ET
0.267 0.267 0.267 rg
BT 283.660 745.174 Td /F4 8.7 Tf  [(9)] TJ ET
0.271 0.267 0.267 rg
BT 288.478 743.667 Td /F1 9.8 Tf  [(. It has been demonstrated that this protein is dysfunctional in mdx )] TJ ET
BT 26.250 731.762 Td /F1 9.8 Tf  [(mice and further contributes to increased levels of sarcoplasmic calcium )] TJ ET
0.267 0.267 0.267 rg
BT 339.440 733.269 Td /F4 8.7 Tf  [(10)] TJ ET
0.271 0.267 0.267 rg
BT 349.077 731.762 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 712.357 Td /F1 9.8 Tf  [(Dantrolene Sodium is an FDA approved skeletal muscle relaxant that has been safely administered to DMD patients without )] TJ ET
BT 26.250 700.452 Td /F1 9.8 Tf  [(harmful side effects )] TJ ET
0.267 0.267 0.267 rg
BT 113.493 701.960 Td /F4 8.7 Tf  [(11)] TJ ET
0.271 0.267 0.267 rg
BT 123.130 700.452 Td /F1 9.8 Tf  [(. Dantrolene binds to the RyR1 receptor and inhibits the release of calcium from the SR during excitation-)] TJ ET
BT 26.250 688.548 Td /F1 9.8 Tf  [(contraction by blocking its activation )] TJ ET
0.267 0.267 0.267 rg
BT 184.483 690.055 Td /F4 8.7 Tf  [(12)] TJ ET
0.271 0.267 0.267 rg
BT 194.120 688.548 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(We will use Dantrolene treatment to decrease intracellular calcium levels and thus decrease the amount of calcium-induced )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(necrosis and improve the overall phenotype of the treated mice. To test the efficacy of this treatment regime, we have )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(performed a pre-clinical trial in mdx mice and will evaluate improvement using several criteria: functional, behavioral, )] TJ ET
BT 26.250 633.429 Td /F1 9.8 Tf  [(histological, and molecular assays.)] TJ ET
BT 26.250 596.826 Td /F4 12.0 Tf  [(Methods)] TJ ET
BT 26.250 576.872 Td /F4 9.8 Tf  [(Animal and Drug Dosing Regimen:)] TJ ET
BT 26.250 557.467 Td /F1 9.8 Tf  [(Wild type \(C57BL/10ScSn/J\) and mdx \(C57BL/10ScSn-mdx/J\) mice were obtained from Jackson Laboratory \(Bar Harbor, ME\) )] TJ ET
BT 26.250 545.562 Td /F1 9.8 Tf  [(and bred in house in our Animal Facility. All mice were acclimated for four weeks before testing. All handling and )] TJ ET
BT 26.250 533.658 Td /F1 9.8 Tf  [(experimentation with mice was conducted in accordance with our IACUC guidelines under approved protocols. Mdx mice were )] TJ ET
BT 26.250 521.753 Td /F1 9.8 Tf  [(assigned to treatment groups randomly based on bodyweight \(BW\). Beginning at 4 weeks of age, all animals received daily \(7 )] TJ ET
BT 26.250 509.848 Td /F1 9.8 Tf  [(day/week\) single intraperitoneal injections of water or Dantrolene at a concentration of 40mg/kg for 6 weeks. Functional testing )] TJ ET
BT 26.250 497.943 Td /F1 9.8 Tf  [(was conducted at 8 weeks of age followed by optical imaging the next week. At 10 weeks of age the mice were sacrificed and )] TJ ET
BT 26.250 486.039 Td /F1 9.8 Tf  [(tissue samples were immediately collected, weighed, snap frozen in liquid nitrogen-cooled isopentane, and stored at -80?C for )] TJ ET
BT 26.250 474.134 Td /F1 9.8 Tf  [(later processing.)] TJ ET
BT 26.250 454.729 Td /F4 9.8 Tf  [(Grip Strength Measurement:)] TJ ET
BT 26.250 435.324 Td /F1 9.8 Tf  [(After four weeks of treatment, forelimb and hindlimb GSMs were performed as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 457.570 436.832 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 467.208 435.324 Td /F1 9.8 Tf  [( but with an increase in )] TJ ET
BT 26.250 423.420 Td /F1 9.8 Tf  [(the acclimation period from three to five consecutive days. The GSMs were collected in the morning hours over a 5-day period, )] TJ ET
BT 26.250 411.515 Td /F1 9.8 Tf  [(with maximum values for each day over this period averaged to obtain absolute GSM values \(Kgf\) or normalized to BW )] TJ ET
BT 26.250 399.610 Td /F1 9.8 Tf  [(\(recorded on the first day of testing\) for normalized GSM values \(Kgf/kg\).)] TJ ET
BT 26.250 380.205 Td /F4 9.8 Tf  [(Open-field Behavioral Activity Measurements \(Digiscan\):)] TJ ET
BT 26.250 360.801 Td /F1 9.8 Tf  [(After four weeks of treatment, voluntary locomotor activity was measured using an open-field digiscan apparatus \(Omnitech )] TJ ET
BT 26.250 348.896 Td /F1 9.8 Tf  [(Electronics, Columbus, Ohio\) as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 259.782 350.403 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 269.419 348.896 Td /F1 9.8 Tf  [( except that the mice were acclimated for four days before data was )] TJ ET
BT 26.250 336.991 Td /F1 9.8 Tf  [(collected. Following acclimation, data was recorded over a 1-hour period each day for four consecutive days. Acclimation and )] TJ ET
BT 26.250 325.086 Td /F1 9.8 Tf  [(collection of data was performed in the morning hours for all mice.)] TJ ET
BT 26.250 305.682 Td /F5 9.8 Tf  [(In vitro)] TJ ET
BT 58.757 305.682 Td /F4 9.8 Tf  [(Force Contraction:)] TJ ET
BT 26.250 286.277 Td /F1 9.8 Tf  [(Prior to sacrifice, the extensor digitorium longus \(EDL\) muscles of the right hindlimbs were removed for comparison of )] TJ ET
BT 536.126 286.277 Td /F6 9.8 Tf  [(in vitro)] TJ ET
BT 26.250 274.372 Td /F1 9.8 Tf  [(force contractions between groups as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 282.568 275.879 Td /F4 8.7 Tf  [(14)] TJ ET
0.271 0.267 0.267 rg
BT 292.205 274.372 Td /F1 9.8 Tf  [(. Briefly, the distal tendon of the muscle was tied securely to the )] TJ ET
BT 26.250 262.467 Td /F1 9.8 Tf  [(lever arm of a servomotor/force transducer \(model 305B\) \(Aurora Scientific, Aurora, Ontario, Canada\) and the proximal tendon )] TJ ET
BT 26.250 250.563 Td /F1 9.8 Tf  [(was fixed to a stationary post in the bath. The muscles were stimulated between two stainless steel plate electrodes. At optimal )] TJ ET
BT 26.250 238.658 Td /F1 9.8 Tf  [(muscle length, the force developed was measured during trains of stimulation \(300 milliseconds\) with increasing frequencies )] TJ ET
BT 26.250 226.753 Td /F1 9.8 Tf  [(until the highest plateau was achieved. The force generated to obtain the highest plateau was used to determine specific force )] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(and is expressed as mN/mm)] TJ ET
BT 149.782 218.737 Td /F1 8.7 Tf  [(2)] TJ ET
BT 154.601 214.848 Td /F1 9.8 Tf  [(. Additionally, the muscle was subjected to a protocol of ten lengthening contractions separated by )] TJ ET
BT 26.250 202.944 Td /F1 9.8 Tf  [(one minute rest intervals. The muscle was stimulated at 250Hz for 300ms allowing the muscle to generate force immediately )] TJ ET
BT 26.250 191.039 Td /F1 9.8 Tf  [(followed by a lengthening over 10% of muscle length at a velocity of two fiber lengths/second. Finally, the muscle was subjected )] TJ ET
BT 26.250 179.134 Td /F1 9.8 Tf  [(to a fatigue protocol consisting of 60 isometric contractions for 300 milliseconds each, once every 5 seconds. The frequency at )] TJ ET
BT 26.250 167.229 Td /F1 9.8 Tf  [(which the EDL muscles were stimulated is 250Hz. The force was recorded every minute during the repetitive contractions and )] TJ ET
BT 26.250 155.325 Td /F1 9.8 Tf  [(again five minutes after to measure recovery.)] TJ ET
BT 26.250 135.920 Td /F4 9.8 Tf  [(Live Animal Optical Imaging:)] TJ ET
BT 26.250 116.515 Td /F1 9.8 Tf  [(After five weeks of treatment, quantification of inflammation in forelimb and hindlimb muscles of mice was achieved using live-)] TJ ET
BT 26.250 104.610 Td /F1 9.8 Tf  [(animal optical imaging of Cathepsin-B \(CTSB\) enzyme activity using a caged near-infrared substrate \(ProSense 680\) as )] TJ ET
BT 26.250 92.706 Td /F1 9.8 Tf  [(previously described )] TJ ET
0.267 0.267 0.267 rg
BT 117.822 94.213 Td /F4 8.7 Tf  [(15)] TJ ET
0.271 0.267 0.267 rg
BT 127.459 92.706 Td /F1 9.8 Tf  [(. Briefly, mice were injected intraperitoneally with 1.5 nmol of ProSense 680 \(VisEn Medical\). Areas of )] TJ ET
BT 26.250 80.801 Td /F1 9.8 Tf  [(scanning were defined at 1.0-mm resolution. Cathepsin activity was measured from forelimb and hindlimb muscles and defined )] TJ ET
BT 26.250 68.896 Td /F1 9.8 Tf  [(as intensity in terms of photon counts/mm)] TJ ET
BT 206.157 72.784 Td /F1 8.7 Tf  [(2)] TJ ET
BT 210.976 68.896 Td /F1 9.8 Tf  [( using the Optiview 2.0 software Optix.)] TJ ET
BT 26.250 49.491 Td /F4 9.8 Tf  [(Serum creatine kinase \(CK\) measurements:)] TJ ET
Q
q
15.000 39.610 577.500 737.390 re W n
0.271 0.267 0.267 rg
BT 26.250 767.476 Td /F1 9.8 Tf  [(Another channel of interest for calcium handling within muscle fibers is the sarco/endoplasmic reticulum Ca)] TJ ET
BT 487.376 771.364 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 497.256 767.476 Td /F1 9.8 Tf  [(-ATPase )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(\(SERCA1\), a calcium ATPase found on the SR membrane in muscle fibers. This protein is responsible for pumping calcium out )] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(of the sarcoplasm and back into the SR following excitation )] TJ ET
0.267 0.267 0.267 rg
BT 283.660 745.174 Td /F4 8.7 Tf  [(9)] TJ ET
0.271 0.267 0.267 rg
BT 288.478 743.667 Td /F1 9.8 Tf  [(. It has been demonstrated that this protein is dysfunctional in mdx )] TJ ET
BT 26.250 731.762 Td /F1 9.8 Tf  [(mice and further contributes to increased levels of sarcoplasmic calcium )] TJ ET
0.267 0.267 0.267 rg
BT 339.440 733.269 Td /F4 8.7 Tf  [(10)] TJ ET
0.271 0.267 0.267 rg
BT 349.077 731.762 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 712.357 Td /F1 9.8 Tf  [(Dantrolene Sodium is an FDA approved skeletal muscle relaxant that has been safely administered to DMD patients without )] TJ ET
BT 26.250 700.452 Td /F1 9.8 Tf  [(harmful side effects )] TJ ET
0.267 0.267 0.267 rg
BT 113.493 701.960 Td /F4 8.7 Tf  [(11)] TJ ET
0.271 0.267 0.267 rg
BT 123.130 700.452 Td /F1 9.8 Tf  [(. Dantrolene binds to the RyR1 receptor and inhibits the release of calcium from the SR during excitation-)] TJ ET
BT 26.250 688.548 Td /F1 9.8 Tf  [(contraction by blocking its activation )] TJ ET
0.267 0.267 0.267 rg
BT 184.483 690.055 Td /F4 8.7 Tf  [(12)] TJ ET
0.271 0.267 0.267 rg
BT 194.120 688.548 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(We will use Dantrolene treatment to decrease intracellular calcium levels and thus decrease the amount of calcium-induced )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(necrosis and improve the overall phenotype of the treated mice. To test the efficacy of this treatment regime, we have )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(performed a pre-clinical trial in mdx mice and will evaluate improvement using several criteria: functional, behavioral, )] TJ ET
BT 26.250 633.429 Td /F1 9.8 Tf  [(histological, and molecular assays.)] TJ ET
BT 26.250 596.826 Td /F4 12.0 Tf  [(Methods)] TJ ET
BT 26.250 576.872 Td /F4 9.8 Tf  [(Animal and Drug Dosing Regimen:)] TJ ET
BT 26.250 557.467 Td /F1 9.8 Tf  [(Wild type \(C57BL/10ScSn/J\) and mdx \(C57BL/10ScSn-mdx/J\) mice were obtained from Jackson Laboratory \(Bar Harbor, ME\) )] TJ ET
BT 26.250 545.562 Td /F1 9.8 Tf  [(and bred in house in our Animal Facility. All mice were acclimated for four weeks before testing. All handling and )] TJ ET
BT 26.250 533.658 Td /F1 9.8 Tf  [(experimentation with mice was conducted in accordance with our IACUC guidelines under approved protocols. Mdx mice were )] TJ ET
BT 26.250 521.753 Td /F1 9.8 Tf  [(assigned to treatment groups randomly based on bodyweight \(BW\). Beginning at 4 weeks of age, all animals received daily \(7 )] TJ ET
BT 26.250 509.848 Td /F1 9.8 Tf  [(day/week\) single intraperitoneal injections of water or Dantrolene at a concentration of 40mg/kg for 6 weeks. Functional testing )] TJ ET
BT 26.250 497.943 Td /F1 9.8 Tf  [(was conducted at 8 weeks of age followed by optical imaging the next week. At 10 weeks of age the mice were sacrificed and )] TJ ET
BT 26.250 486.039 Td /F1 9.8 Tf  [(tissue samples were immediately collected, weighed, snap frozen in liquid nitrogen-cooled isopentane, and stored at -80?C for )] TJ ET
BT 26.250 474.134 Td /F1 9.8 Tf  [(later processing.)] TJ ET
BT 26.250 454.729 Td /F4 9.8 Tf  [(Grip Strength Measurement:)] TJ ET
BT 26.250 435.324 Td /F1 9.8 Tf  [(After four weeks of treatment, forelimb and hindlimb GSMs were performed as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 457.570 436.832 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 467.208 435.324 Td /F1 9.8 Tf  [( but with an increase in )] TJ ET
BT 26.250 423.420 Td /F1 9.8 Tf  [(the acclimation period from three to five consecutive days. The GSMs were collected in the morning hours over a 5-day period, )] TJ ET
BT 26.250 411.515 Td /F1 9.8 Tf  [(with maximum values for each day over this period averaged to obtain absolute GSM values \(Kgf\) or normalized to BW )] TJ ET
BT 26.250 399.610 Td /F1 9.8 Tf  [(\(recorded on the first day of testing\) for normalized GSM values \(Kgf/kg\).)] TJ ET
BT 26.250 380.205 Td /F4 9.8 Tf  [(Open-field Behavioral Activity Measurements \(Digiscan\):)] TJ ET
BT 26.250 360.801 Td /F1 9.8 Tf  [(After four weeks of treatment, voluntary locomotor activity was measured using an open-field digiscan apparatus \(Omnitech )] TJ ET
BT 26.250 348.896 Td /F1 9.8 Tf  [(Electronics, Columbus, Ohio\) as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 259.782 350.403 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 269.419 348.896 Td /F1 9.8 Tf  [( except that the mice were acclimated for four days before data was )] TJ ET
BT 26.250 336.991 Td /F1 9.8 Tf  [(collected. Following acclimation, data was recorded over a 1-hour period each day for four consecutive days. Acclimation and )] TJ ET
BT 26.250 325.086 Td /F1 9.8 Tf  [(collection of data was performed in the morning hours for all mice.)] TJ ET
BT 26.250 305.682 Td /F5 9.8 Tf  [(In vitro)] TJ ET
BT 58.757 305.682 Td /F4 9.8 Tf  [(Force Contraction:)] TJ ET
BT 26.250 286.277 Td /F1 9.8 Tf  [(Prior to sacrifice, the extensor digitorium longus \(EDL\) muscles of the right hindlimbs were removed for comparison of )] TJ ET
BT 536.126 286.277 Td /F6 9.8 Tf  [(in vitro)] TJ ET
BT 26.250 274.372 Td /F1 9.8 Tf  [(force contractions between groups as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 282.568 275.879 Td /F4 8.7 Tf  [(14)] TJ ET
0.271 0.267 0.267 rg
BT 292.205 274.372 Td /F1 9.8 Tf  [(. Briefly, the distal tendon of the muscle was tied securely to the )] TJ ET
BT 26.250 262.467 Td /F1 9.8 Tf  [(lever arm of a servomotor/force transducer \(model 305B\) \(Aurora Scientific, Aurora, Ontario, Canada\) and the proximal tendon )] TJ ET
BT 26.250 250.563 Td /F1 9.8 Tf  [(was fixed to a stationary post in the bath. The muscles were stimulated between two stainless steel plate electrodes. At optimal )] TJ ET
BT 26.250 238.658 Td /F1 9.8 Tf  [(muscle length, the force developed was measured during trains of stimulation \(300 milliseconds\) with increasing frequencies )] TJ ET
BT 26.250 226.753 Td /F1 9.8 Tf  [(until the highest plateau was achieved. The force generated to obtain the highest plateau was used to determine specific force )] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(and is expressed as mN/mm)] TJ ET
BT 149.782 218.737 Td /F1 8.7 Tf  [(2)] TJ ET
BT 154.601 214.848 Td /F1 9.8 Tf  [(. Additionally, the muscle was subjected to a protocol of ten lengthening contractions separated by )] TJ ET
BT 26.250 202.944 Td /F1 9.8 Tf  [(one minute rest intervals. The muscle was stimulated at 250Hz for 300ms allowing the muscle to generate force immediately )] TJ ET
BT 26.250 191.039 Td /F1 9.8 Tf  [(followed by a lengthening over 10% of muscle length at a velocity of two fiber lengths/second. Finally, the muscle was subjected )] TJ ET
BT 26.250 179.134 Td /F1 9.8 Tf  [(to a fatigue protocol consisting of 60 isometric contractions for 300 milliseconds each, once every 5 seconds. The frequency at )] TJ ET
BT 26.250 167.229 Td /F1 9.8 Tf  [(which the EDL muscles were stimulated is 250Hz. The force was recorded every minute during the repetitive contractions and )] TJ ET
BT 26.250 155.325 Td /F1 9.8 Tf  [(again five minutes after to measure recovery.)] TJ ET
BT 26.250 135.920 Td /F4 9.8 Tf  [(Live Animal Optical Imaging:)] TJ ET
BT 26.250 116.515 Td /F1 9.8 Tf  [(After five weeks of treatment, quantification of inflammation in forelimb and hindlimb muscles of mice was achieved using live-)] TJ ET
BT 26.250 104.610 Td /F1 9.8 Tf  [(animal optical imaging of Cathepsin-B \(CTSB\) enzyme activity using a caged near-infrared substrate \(ProSense 680\) as )] TJ ET
BT 26.250 92.706 Td /F1 9.8 Tf  [(previously described )] TJ ET
0.267 0.267 0.267 rg
BT 117.822 94.213 Td /F4 8.7 Tf  [(15)] TJ ET
0.271 0.267 0.267 rg
BT 127.459 92.706 Td /F1 9.8 Tf  [(. Briefly, mice were injected intraperitoneally with 1.5 nmol of ProSense 680 \(VisEn Medical\). Areas of )] TJ ET
BT 26.250 80.801 Td /F1 9.8 Tf  [(scanning were defined at 1.0-mm resolution. Cathepsin activity was measured from forelimb and hindlimb muscles and defined )] TJ ET
BT 26.250 68.896 Td /F1 9.8 Tf  [(as intensity in terms of photon counts/mm)] TJ ET
BT 206.157 72.784 Td /F1 8.7 Tf  [(2)] TJ ET
BT 210.976 68.896 Td /F1 9.8 Tf  [( using the Optiview 2.0 software Optix.)] TJ ET
BT 26.250 49.491 Td /F4 9.8 Tf  [(Serum creatine kinase \(CK\) measurements:)] TJ ET
Q
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BT 291.710 19.825 Td /F1 11.0 Tf  [(2)] TJ ET
BT 25.000 19.825 Td /F1 11.0 Tf  [(PLOS Currents Muscular Dystrophy)] TJ ET

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BT 26.250 767.476 Td /F1 9.8 Tf  [(The blood was collected by cardiac puncture immediately after euthanasia by carbon dioxide inhalation. Blood was spun down )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(and serum collected. Creatine kinase levels \(U/L\) were analyzed using a standard spectrophotometric method with enzyme-)] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(coupled assay reagent, CK10 \(Thermo Fisher Scientific, Waltham, MA\), according to the manufacturer�s instructions )] TJ ET
0.267 0.267 0.267 rg
BT 529.096 745.174 Td /F4 8.7 Tf  [(16)] TJ ET
0.271 0.267 0.267 rg
BT 538.734 743.667 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 724.262 Td /F4 9.8 Tf  [(Histopathology Scoring:)] TJ ET
BT 26.250 704.857 Td /F1 9.8 Tf  [(Directly following euthanasia, the tibialis anterior \(TA\) muscles were removed and preserved in formalin. For histological )] TJ ET
BT 26.250 692.952 Td /F1 9.8 Tf  [(analysis, muscles were embedded into paraffin blocks and subsequently sectioned and stained with haematoxylin and eosin )] TJ ET
BT 26.250 681.048 Td /F1 9.8 Tf  [(\(H&E\) by Histoserv \(Germantown, MD\). The slides were blinded and randomized and five digital images at 20X magnification )] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(were taken of each using an Eclipse E800 \(Nikon, Japan\) microscope, and each was assessed for the nine following criteria; )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(fibers/field, central nuclei/fiber, peripheral nuclei/fiber, total nuclei/fiber, fibers with central nuclei/field, ratio of central to )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(peripheral nuclei/field, regenerating fibers/field, percentage of central nucleated fibers and inflammatory foci using Image J )] TJ ET
BT 26.250 633.429 Td /F1 9.8 Tf  [(\(NIH\) as previously reported )] TJ ET
0.267 0.267 0.267 rg
BT 150.865 634.936 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 160.502 633.429 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 614.024 Td /F4 9.8 Tf  [(Western blotting:)] TJ ET
BT 26.250 594.619 Td /F1 9.8 Tf  [(SERCA1 ATPase protein levels were measured in Gastrocnemius muscles from the mdx treatment and control groups. Frozen )] TJ ET
BT 26.250 582.714 Td /F1 9.8 Tf  [(muscle sections were homogenized as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 287.969 584.222 Td /F4 8.7 Tf  [(17)] TJ ET
0.271 0.267 0.267 rg
BT 297.607 582.714 Td /F1 9.8 Tf  [( and equal amounts \(50ug\) were loaded and run in a reducing )] TJ ET
BT 26.250 570.810 Td /F1 9.8 Tf  [(SDS-PAGE gel \(Invitrogen, Grand Island, NY\) at 150V. The gel was then blotted transferred onto PVDF membranes and )] TJ ET
BT 26.250 558.905 Td /F1 9.8 Tf  [(incubated overnight with anti-SERCA1 ATPase monoclonal antibody \(1:2500, Thermo Scientific, Waltham, MA\) at 4�C. After )] TJ ET
BT 26.250 547.000 Td /F1 9.8 Tf  [(washing, membranes were probed with HRP-conjugated anti-mouse IgG1 \(1:2000, Invitrogen\) and incubated with ECL Western )] TJ ET
BT 26.250 535.095 Td /F1 9.8 Tf  [(Blotting Substrate \(GE Healthcare, Piscataway, NJ\) and processed on X-ray film \(Denville Scientific, South Plainfield, NJ\). )] TJ ET
BT 26.250 523.191 Td /F1 9.8 Tf  [(Densitometry analysis was performed and normalized to ?-actin \(1:1000, Santa Cruz\).)] TJ ET
BT 26.250 503.786 Td /F4 9.8 Tf  [(Quantitative PCR \(qPCR\):)] TJ ET
BT 26.250 484.381 Td /F1 9.8 Tf  [(RNA was isolated from frozen Gastrocnemius muscle using an integrated Trizol and RNeasy minikit \(Qiagen, Valencia, CA\) )] TJ ET
BT 26.250 472.476 Td /F1 9.8 Tf  [(protocol with DNase I \(Qiagen\) treatment. cDNA was obtained using Reverse Transcription System \(Promega, Madison, WI\). )] TJ ET
BT 26.250 460.572 Td /F1 9.8 Tf  [(The Taqman Gene Expression Assays Hprt \(Mm01545399_m1\) and Ryr1 \(Mm01175211_m1\) were used for analysis. All PCR )] TJ ET
BT 26.250 448.667 Td /F1 9.8 Tf  [(reactions were carried out using Taqman Gene Expression Master Mix on the 7900HT Fast Real-Time PCR System under the )] TJ ET
BT 26.250 436.762 Td /F1 9.8 Tf  [(following thermal cycle conditions: initial steps \(UNG activation � 50�C for two minutes; DNA Polymerase activation � 95�C for )] TJ ET
BT 26.250 424.857 Td /F1 9.8 Tf  [(10 minutes\) and 40 cycles with a melting temperature of 95�C for 15 seconds and an annealing/extend temperature of 60�C for )] TJ ET
BT 26.250 412.953 Td /F1 9.8 Tf  [(60 seconds.)] TJ ET
BT 26.250 393.548 Td /F4 9.8 Tf  [(Statistical Analysis:)] TJ ET
BT 26.250 374.143 Td /F1 9.8 Tf  [(For all proposed experiments and procedures, one-way ANOVA with Dunnett�s post-test comparison against a control was used )] TJ ET
BT 26.250 362.238 Td /F1 9.8 Tf  [(for experiments involving more than two groups. A two-tailed Student�s t test was used for experiments involving only two )] TJ ET
BT 26.250 350.334 Td /F1 9.8 Tf  [(groups. Significance was defined as a p-value less than 0.05. All data is expressed as mean � standard error of the mean )] TJ ET
BT 26.250 338.429 Td /F1 9.8 Tf  [(\(S.E.M.\). Prism statistical software \(GraphPad, La Jolla, CA\) was used for statistical analyzes. qPCR results were analyzed for )] TJ ET
BT 26.250 326.524 Td /F1 9.8 Tf  [(significant differences by a 2000-sample pairwise fixed reallocation randomization test using REST software )] TJ ET
0.267 0.267 0.267 rg
BT 493.889 328.031 Td /F4 8.7 Tf  [(18)] TJ ET
0.271 0.267 0.267 rg
BT 503.527 326.524 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 289.922 Td /F4 12.0 Tf  [(Results)] TJ ET
BT 26.250 269.967 Td /F4 9.8 Tf  [(Effect on body weight)] TJ ET
BT 26.250 250.563 Td /F1 9.8 Tf  [(Bodyweight of each mouse was measured at four weeks of age and recorded every week of treatment up until sacrifice at ten )] TJ ET
BT 26.250 238.658 Td /F1 9.8 Tf  [(weeks of age. All three groups weighed approximately the same at the beginning of treatment with BL10 mice averaging 6.47% )] TJ ET
BT 26.250 226.753 Td /F1 9.8 Tf  [(weight increase each week of treatment while untreated and treated mdx mice averaged 10.33% and 10.53% respectively. This )] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(resulted in a significant difference \(P-value = 0.0115\) between the bodyweights of the BL10 group and the mdx groups starting )] TJ ET
BT 26.250 202.944 Td /F1 9.8 Tf  [(at two weeks of treatment and continuing until the end of the trial. There was no significant difference in bodyweight over time )] TJ ET
BT 26.250 191.039 Td /F1 9.8 Tf  [(between the two mdx groups \(Fig. 1\).)] TJ ET
0.965 0.965 0.965 rg
26.250 44.658 555.000 136.500 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 181.158 m 581.250 181.158 l 581.250 180.408 l 26.250 180.408 l  f
q
225.000 0 0 120.750 35.250 50.658 cm /I3 Do
Q 
q
35.250 44.658 537.000 0.000 re W n
Q
Q
q
15.000 44.658 577.500 732.342 re W n
0.271 0.267 0.267 rg
BT 26.250 767.476 Td /F1 9.8 Tf  [(The blood was collected by cardiac puncture immediately after euthanasia by carbon dioxide inhalation. Blood was spun down )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(and serum collected. Creatine kinase levels \(U/L\) were analyzed using a standard spectrophotometric method with enzyme-)] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(coupled assay reagent, CK10 \(Thermo Fisher Scientific, Waltham, MA\), according to the manufacturer�s instructions )] TJ ET
0.267 0.267 0.267 rg
BT 529.096 745.174 Td /F4 8.7 Tf  [(16)] TJ ET
0.271 0.267 0.267 rg
BT 538.734 743.667 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 724.262 Td /F4 9.8 Tf  [(Histopathology Scoring:)] TJ ET
BT 26.250 704.857 Td /F1 9.8 Tf  [(Directly following euthanasia, the tibialis anterior \(TA\) muscles were removed and preserved in formalin. For histological )] TJ ET
BT 26.250 692.952 Td /F1 9.8 Tf  [(analysis, muscles were embedded into paraffin blocks and subsequently sectioned and stained with haematoxylin and eosin )] TJ ET
BT 26.250 681.048 Td /F1 9.8 Tf  [(\(H&E\) by Histoserv \(Germantown, MD\). The slides were blinded and randomized and five digital images at 20X magnification )] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(were taken of each using an Eclipse E800 \(Nikon, Japan\) microscope, and each was assessed for the nine following criteria; )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(fibers/field, central nuclei/fiber, peripheral nuclei/fiber, total nuclei/fiber, fibers with central nuclei/field, ratio of central to )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(peripheral nuclei/field, regenerating fibers/field, percentage of central nucleated fibers and inflammatory foci using Image J )] TJ ET
BT 26.250 633.429 Td /F1 9.8 Tf  [(\(NIH\) as previously reported )] TJ ET
0.267 0.267 0.267 rg
BT 150.865 634.936 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 160.502 633.429 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 614.024 Td /F4 9.8 Tf  [(Western blotting:)] TJ ET
BT 26.250 594.619 Td /F1 9.8 Tf  [(SERCA1 ATPase protein levels were measured in Gastrocnemius muscles from the mdx treatment and control groups. Frozen )] TJ ET
BT 26.250 582.714 Td /F1 9.8 Tf  [(muscle sections were homogenized as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 287.969 584.222 Td /F4 8.7 Tf  [(17)] TJ ET
0.271 0.267 0.267 rg
BT 297.607 582.714 Td /F1 9.8 Tf  [( and equal amounts \(50ug\) were loaded and run in a reducing )] TJ ET
BT 26.250 570.810 Td /F1 9.8 Tf  [(SDS-PAGE gel \(Invitrogen, Grand Island, NY\) at 150V. The gel was then blotted transferred onto PVDF membranes and )] TJ ET
BT 26.250 558.905 Td /F1 9.8 Tf  [(incubated overnight with anti-SERCA1 ATPase monoclonal antibody \(1:2500, Thermo Scientific, Waltham, MA\) at 4�C. After )] TJ ET
BT 26.250 547.000 Td /F1 9.8 Tf  [(washing, membranes were probed with HRP-conjugated anti-mouse IgG1 \(1:2000, Invitrogen\) and incubated with ECL Western )] TJ ET
BT 26.250 535.095 Td /F1 9.8 Tf  [(Blotting Substrate \(GE Healthcare, Piscataway, NJ\) and processed on X-ray film \(Denville Scientific, South Plainfield, NJ\). )] TJ ET
BT 26.250 523.191 Td /F1 9.8 Tf  [(Densitometry analysis was performed and normalized to ?-actin \(1:1000, Santa Cruz\).)] TJ ET
BT 26.250 503.786 Td /F4 9.8 Tf  [(Quantitative PCR \(qPCR\):)] TJ ET
BT 26.250 484.381 Td /F1 9.8 Tf  [(RNA was isolated from frozen Gastrocnemius muscle using an integrated Trizol and RNeasy minikit \(Qiagen, Valencia, CA\) )] TJ ET
BT 26.250 472.476 Td /F1 9.8 Tf  [(protocol with DNase I \(Qiagen\) treatment. cDNA was obtained using Reverse Transcription System \(Promega, Madison, WI\). )] TJ ET
BT 26.250 460.572 Td /F1 9.8 Tf  [(The Taqman Gene Expression Assays Hprt \(Mm01545399_m1\) and Ryr1 \(Mm01175211_m1\) were used for analysis. All PCR )] TJ ET
BT 26.250 448.667 Td /F1 9.8 Tf  [(reactions were carried out using Taqman Gene Expression Master Mix on the 7900HT Fast Real-Time PCR System under the )] TJ ET
BT 26.250 436.762 Td /F1 9.8 Tf  [(following thermal cycle conditions: initial steps \(UNG activation � 50�C for two minutes; DNA Polymerase activation � 95�C for )] TJ ET
BT 26.250 424.857 Td /F1 9.8 Tf  [(10 minutes\) and 40 cycles with a melting temperature of 95�C for 15 seconds and an annealing/extend temperature of 60�C for )] TJ ET
BT 26.250 412.953 Td /F1 9.8 Tf  [(60 seconds.)] TJ ET
BT 26.250 393.548 Td /F4 9.8 Tf  [(Statistical Analysis:)] TJ ET
BT 26.250 374.143 Td /F1 9.8 Tf  [(For all proposed experiments and procedures, one-way ANOVA with Dunnett�s post-test comparison against a control was used )] TJ ET
BT 26.250 362.238 Td /F1 9.8 Tf  [(for experiments involving more than two groups. A two-tailed Student�s t test was used for experiments involving only two )] TJ ET
BT 26.250 350.334 Td /F1 9.8 Tf  [(groups. Significance was defined as a p-value less than 0.05. All data is expressed as mean � standard error of the mean )] TJ ET
BT 26.250 338.429 Td /F1 9.8 Tf  [(\(S.E.M.\). Prism statistical software \(GraphPad, La Jolla, CA\) was used for statistical analyzes. qPCR results were analyzed for )] TJ ET
BT 26.250 326.524 Td /F1 9.8 Tf  [(significant differences by a 2000-sample pairwise fixed reallocation randomization test using REST software )] TJ ET
0.267 0.267 0.267 rg
BT 493.889 328.031 Td /F4 8.7 Tf  [(18)] TJ ET
0.271 0.267 0.267 rg
BT 503.527 326.524 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 289.922 Td /F4 12.0 Tf  [(Results)] TJ ET
BT 26.250 269.967 Td /F4 9.8 Tf  [(Effect on body weight)] TJ ET
BT 26.250 250.563 Td /F1 9.8 Tf  [(Bodyweight of each mouse was measured at four weeks of age and recorded every week of treatment up until sacrifice at ten )] TJ ET
BT 26.250 238.658 Td /F1 9.8 Tf  [(weeks of age. All three groups weighed approximately the same at the beginning of treatment with BL10 mice averaging 6.47% )] TJ ET
BT 26.250 226.753 Td /F1 9.8 Tf  [(weight increase each week of treatment while untreated and treated mdx mice averaged 10.33% and 10.53% respectively. This )] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(resulted in a significant difference \(P-value = 0.0115\) between the bodyweights of the BL10 group and the mdx groups starting )] TJ ET
BT 26.250 202.944 Td /F1 9.8 Tf  [(at two weeks of treatment and continuing until the end of the trial. There was no significant difference in bodyweight over time )] TJ ET
BT 26.250 191.039 Td /F1 9.8 Tf  [(between the two mdx groups \(Fig. 1\).)] TJ ET
0.965 0.965 0.965 rg
26.250 44.658 555.000 136.500 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 181.158 m 581.250 181.158 l 581.250 180.408 l 26.250 180.408 l  f
q
225.000 0 0 120.750 35.250 50.658 cm /I3 Do
Q 
q
35.250 44.658 537.000 0.000 re W n
Q
Q
q
15.000 44.658 577.500 732.342 re W n
0.271 0.267 0.267 rg
BT 26.250 767.476 Td /F1 9.8 Tf  [(The blood was collected by cardiac puncture immediately after euthanasia by carbon dioxide inhalation. Blood was spun down )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(and serum collected. Creatine kinase levels \(U/L\) were analyzed using a standard spectrophotometric method with enzyme-)] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(coupled assay reagent, CK10 \(Thermo Fisher Scientific, Waltham, MA\), according to the manufacturer�s instructions )] TJ ET
0.267 0.267 0.267 rg
BT 529.096 745.174 Td /F4 8.7 Tf  [(16)] TJ ET
0.271 0.267 0.267 rg
BT 538.734 743.667 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 724.262 Td /F4 9.8 Tf  [(Histopathology Scoring:)] TJ ET
BT 26.250 704.857 Td /F1 9.8 Tf  [(Directly following euthanasia, the tibialis anterior \(TA\) muscles were removed and preserved in formalin. For histological )] TJ ET
BT 26.250 692.952 Td /F1 9.8 Tf  [(analysis, muscles were embedded into paraffin blocks and subsequently sectioned and stained with haematoxylin and eosin )] TJ ET
BT 26.250 681.048 Td /F1 9.8 Tf  [(\(H&E\) by Histoserv \(Germantown, MD\). The slides were blinded and randomized and five digital images at 20X magnification )] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(were taken of each using an Eclipse E800 \(Nikon, Japan\) microscope, and each was assessed for the nine following criteria; )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(fibers/field, central nuclei/fiber, peripheral nuclei/fiber, total nuclei/fiber, fibers with central nuclei/field, ratio of central to )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(peripheral nuclei/field, regenerating fibers/field, percentage of central nucleated fibers and inflammatory foci using Image J )] TJ ET
BT 26.250 633.429 Td /F1 9.8 Tf  [(\(NIH\) as previously reported )] TJ ET
0.267 0.267 0.267 rg
BT 150.865 634.936 Td /F4 8.7 Tf  [(13)] TJ ET
0.271 0.267 0.267 rg
BT 160.502 633.429 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 614.024 Td /F4 9.8 Tf  [(Western blotting:)] TJ ET
BT 26.250 594.619 Td /F1 9.8 Tf  [(SERCA1 ATPase protein levels were measured in Gastrocnemius muscles from the mdx treatment and control groups. Frozen )] TJ ET
BT 26.250 582.714 Td /F1 9.8 Tf  [(muscle sections were homogenized as previously described )] TJ ET
0.267 0.267 0.267 rg
BT 287.969 584.222 Td /F4 8.7 Tf  [(17)] TJ ET
0.271 0.267 0.267 rg
BT 297.607 582.714 Td /F1 9.8 Tf  [( and equal amounts \(50ug\) were loaded and run in a reducing )] TJ ET
BT 26.250 570.810 Td /F1 9.8 Tf  [(SDS-PAGE gel \(Invitrogen, Grand Island, NY\) at 150V. The gel was then blotted transferred onto PVDF membranes and )] TJ ET
BT 26.250 558.905 Td /F1 9.8 Tf  [(incubated overnight with anti-SERCA1 ATPase monoclonal antibody \(1:2500, Thermo Scientific, Waltham, MA\) at 4�C. After )] TJ ET
BT 26.250 547.000 Td /F1 9.8 Tf  [(washing, membranes were probed with HRP-conjugated anti-mouse IgG1 \(1:2000, Invitrogen\) and incubated with ECL Western )] TJ ET
BT 26.250 535.095 Td /F1 9.8 Tf  [(Blotting Substrate \(GE Healthcare, Piscataway, NJ\) and processed on X-ray film \(Denville Scientific, South Plainfield, NJ\). )] TJ ET
BT 26.250 523.191 Td /F1 9.8 Tf  [(Densitometry analysis was performed and normalized to ?-actin \(1:1000, Santa Cruz\).)] TJ ET
BT 26.250 503.786 Td /F4 9.8 Tf  [(Quantitative PCR \(qPCR\):)] TJ ET
BT 26.250 484.381 Td /F1 9.8 Tf  [(RNA was isolated from frozen Gastrocnemius muscle using an integrated Trizol and RNeasy minikit \(Qiagen, Valencia, CA\) )] TJ ET
BT 26.250 472.476 Td /F1 9.8 Tf  [(protocol with DNase I \(Qiagen\) treatment. cDNA was obtained using Reverse Transcription System \(Promega, Madison, WI\). )] TJ ET
BT 26.250 460.572 Td /F1 9.8 Tf  [(The Taqman Gene Expression Assays Hprt \(Mm01545399_m1\) and Ryr1 \(Mm01175211_m1\) were used for analysis. All PCR )] TJ ET
BT 26.250 448.667 Td /F1 9.8 Tf  [(reactions were carried out using Taqman Gene Expression Master Mix on the 7900HT Fast Real-Time PCR System under the )] TJ ET
BT 26.250 436.762 Td /F1 9.8 Tf  [(following thermal cycle conditions: initial steps \(UNG activation � 50�C for two minutes; DNA Polymerase activation � 95�C for )] TJ ET
BT 26.250 424.857 Td /F1 9.8 Tf  [(10 minutes\) and 40 cycles with a melting temperature of 95�C for 15 seconds and an annealing/extend temperature of 60�C for )] TJ ET
BT 26.250 412.953 Td /F1 9.8 Tf  [(60 seconds.)] TJ ET
BT 26.250 393.548 Td /F4 9.8 Tf  [(Statistical Analysis:)] TJ ET
BT 26.250 374.143 Td /F1 9.8 Tf  [(For all proposed experiments and procedures, one-way ANOVA with Dunnett�s post-test comparison against a control was used )] TJ ET
BT 26.250 362.238 Td /F1 9.8 Tf  [(for experiments involving more than two groups. A two-tailed Student�s t test was used for experiments involving only two )] TJ ET
BT 26.250 350.334 Td /F1 9.8 Tf  [(groups. Significance was defined as a p-value less than 0.05. All data is expressed as mean � standard error of the mean )] TJ ET
BT 26.250 338.429 Td /F1 9.8 Tf  [(\(S.E.M.\). Prism statistical software \(GraphPad, La Jolla, CA\) was used for statistical analyzes. qPCR results were analyzed for )] TJ ET
BT 26.250 326.524 Td /F1 9.8 Tf  [(significant differences by a 2000-sample pairwise fixed reallocation randomization test using REST software )] TJ ET
0.267 0.267 0.267 rg
BT 493.889 328.031 Td /F4 8.7 Tf  [(18)] TJ ET
0.271 0.267 0.267 rg
BT 503.527 326.524 Td /F1 9.8 Tf  [(.)] TJ ET
BT 26.250 289.922 Td /F4 12.0 Tf  [(Results)] TJ ET
BT 26.250 269.967 Td /F4 9.8 Tf  [(Effect on body weight)] TJ ET
BT 26.250 250.563 Td /F1 9.8 Tf  [(Bodyweight of each mouse was measured at four weeks of age and recorded every week of treatment up until sacrifice at ten )] TJ ET
BT 26.250 238.658 Td /F1 9.8 Tf  [(weeks of age. All three groups weighed approximately the same at the beginning of treatment with BL10 mice averaging 6.47% )] TJ ET
BT 26.250 226.753 Td /F1 9.8 Tf  [(weight increase each week of treatment while untreated and treated mdx mice averaged 10.33% and 10.53% respectively. This )] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(resulted in a significant difference \(P-value = 0.0115\) between the bodyweights of the BL10 group and the mdx groups starting )] TJ ET
BT 26.250 202.944 Td /F1 9.8 Tf  [(at two weeks of treatment and continuing until the end of the trial. There was no significant difference in bodyweight over time )] TJ ET
BT 26.250 191.039 Td /F1 9.8 Tf  [(between the two mdx groups \(Fig. 1\).)] TJ ET
0.965 0.965 0.965 rg
26.250 44.658 555.000 136.500 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 181.158 m 581.250 181.158 l 581.250 180.408 l 26.250 180.408 l  f
q
225.000 0 0 120.750 35.250 50.658 cm /I3 Do
Q 
q
35.250 44.658 537.000 0.000 re W n
Q
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BT 35.250 767.476 Td /F4 9.8 Tf  [(Fig. 1: Body weight measurements during treatment)] TJ ET
BT 35.250 748.106 Td /F1 9.8 Tf  [(Body weight was recorded each week over the entire duration of treatment. The two mdx treatment groups showed no )] TJ ET
BT 35.250 734.370 Td /F1 9.8 Tf  [(significant change in body weight of the course of treatment after the mdx mice were broken up into groups based on )] TJ ET
BT 35.250 720.634 Td /F1 9.8 Tf  [(normalizing bodyweight. N=8)] TJ ET
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BT 26.250 682.113 Td /F4 9.8 Tf  [(Effect on functional and behavioral assays)] TJ ET
BT 26.250 662.708 Td /F1 9.8 Tf  [(Grip strength and open field testing for mdx and BL10 mice was performed after 4 weeks of treatment. Vehicle-treated mdx )] TJ ET
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BT 26.250 626.994 Td /F1 9.8 Tf  [(differences in bodyweight between BL10 and mdx mice, we also normalized strength to body weight. Untreated mdx mice )] TJ ET
BT 26.250 615.089 Td /F1 9.8 Tf  [(showed a significant decrease \(24.23%\) in normalized forelimb strength relative to the BL10 mice. Mdx mice treated with )] TJ ET
BT 26.250 603.184 Td /F1 9.8 Tf  [(Dantrolene showed a 13.58% decrease compared to the untreated mdx group \(Fig. 2B\). Hindlimb measurements were unable )] TJ ET
BT 26.250 591.279 Td /F1 9.8 Tf  [(to ascertain a difference between mdx and BL10 mice even after normalization \(data not shown\).)] TJ ET
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0.267 0.267 0.267 RG
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0.271 0.267 0.267 rg
BT 35.250 440.625 Td /F4 9.8 Tf  [(Fig. 2: GSM results of wild type and mdx mice)] TJ ET
BT 35.250 421.255 Td /F1 9.8 Tf  [(Grip strength measurements were performed at 4 weeks of treatment. A\) Absolute Forelimb B\)Forelimb normalized to body )] TJ ET
BT 35.250 407.518 Td /F1 9.8 Tf  [(weight. * P ? 0.05, *** P ? 0.001. N=8)] TJ ET
Q
BT 26.250 368.997 Td /F1 9.8 Tf  [(Activity was measured using open field activity chambers. There was no significant change in any measured parameters, )] TJ ET
BT 26.250 357.093 Td /F1 9.8 Tf  [(including horizontal and vertical activity as well as movement and rest time, between any of the groups \(Table 1\).)] TJ ET
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0.271 0.267 0.267 rg
BT 26.250 336.223 Td /F1 9.8 Tf  [(Table 1: Open field activity measured parameters)] TJ ET
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0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
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0.271 0.267 0.267 rg
BT 33.000 311.899 Td /F1 9.0 Tf  [(Comparisons between BL10 and mdx Vehicle-treated mice as well as both mdx mice groups yielded no significant differences. N=8)] TJ ET
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0.267 0.267 0.267 rg
26.625 301.443 215.682 0.750 re f
26.625 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 291.588 Td /F1 9.8 Tf  [(Measurement)] TJ ET
0.267 0.267 0.267 rg
241.557 301.443 107.688 0.750 re f
241.557 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 291.588 Td /F1 9.8 Tf  [(BL10 Vehicle)] TJ ET
0.267 0.267 0.267 rg
348.494 301.443 107.688 0.750 re f
348.494 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 291.588 Td /F1 9.8 Tf  [(mdx Vehicle)] TJ ET
0.267 0.267 0.267 rg
455.432 301.443 125.443 0.750 re f
455.432 285.562 0.750 16.631 re f
580.125 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 291.588 Td /F1 9.8 Tf  [(mdx Treatment)] TJ ET
0.267 0.267 0.267 rg
26.625 285.562 215.682 0.750 re f
26.625 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 275.707 Td /F1 9.8 Tf  [(Horizontal Activity \(arbitrary units\))] TJ ET
0.267 0.267 0.267 rg
241.557 285.562 107.688 0.750 re f
241.557 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 275.707 Td /F1 9.8 Tf  [(1785.7 � 81.2)] TJ ET
0.267 0.267 0.267 rg
348.494 285.562 107.688 0.750 re f
348.494 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 275.707 Td /F1 9.8 Tf  [(1463.0 � 99.9)] TJ ET
0.267 0.267 0.267 rg
455.432 285.562 125.443 0.750 re f
455.432 269.680 0.750 16.631 re f
580.125 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 275.707 Td /F1 9.8 Tf  [(1259.2 � 118.7)] TJ ET
0.267 0.267 0.267 rg
26.625 269.680 215.682 0.750 re f
26.625 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 259.825 Td /F1 9.8 Tf  [(Vertical Activity \(arbitrary units\))] TJ ET
0.267 0.267 0.267 rg
241.557 269.680 107.688 0.750 re f
241.557 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 259.825 Td /F1 9.8 Tf  [(26.4 � 5.8)] TJ ET
0.267 0.267 0.267 rg
348.494 269.680 107.688 0.750 re f
348.494 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 259.825 Td /F1 9.8 Tf  [(87.8 � 25.7)] TJ ET
0.267 0.267 0.267 rg
455.432 269.680 125.443 0.750 re f
455.432 253.799 0.750 16.631 re f
580.125 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 259.825 Td /F1 9.8 Tf  [(41.4 � 12.2)] TJ ET
0.267 0.267 0.267 rg
26.625 253.799 215.682 0.750 re f
26.625 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 243.944 Td /F1 9.8 Tf  [(Total Movement Time \(seconds\))] TJ ET
0.267 0.267 0.267 rg
241.557 253.799 107.688 0.750 re f
241.557 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 243.944 Td /F1 9.8 Tf  [(71.9 � 8.5)] TJ ET
0.267 0.267 0.267 rg
348.494 253.799 107.688 0.750 re f
348.494 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 243.944 Td /F1 9.8 Tf  [(64.0 � 8.1)] TJ ET
0.267 0.267 0.267 rg
455.432 253.799 125.443 0.750 re f
455.432 237.918 0.750 16.631 re f
580.125 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 243.944 Td /F1 9.8 Tf  [(53.0 � 8.4)] TJ ET
0.267 0.267 0.267 rg
26.625 237.918 215.682 0.750 re f
26.625 222.037 215.682 0.750 re f
26.625 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 228.063 Td /F1 9.8 Tf  [(Total Rest Time \(seconds\))] TJ ET
0.267 0.267 0.267 rg
241.557 237.918 107.688 0.750 re f
241.557 222.037 107.688 0.750 re f
241.557 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 228.063 Td /F1 9.8 Tf  [(528.0 � 8.5)] TJ ET
0.267 0.267 0.267 rg
348.494 237.918 107.688 0.750 re f
348.494 222.037 107.688 0.750 re f
348.494 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 228.063 Td /F1 9.8 Tf  [(536.0 � 8.0)] TJ ET
0.267 0.267 0.267 rg
455.432 237.918 125.443 0.750 re f
455.432 222.037 125.443 0.750 re f
455.432 222.037 0.750 16.631 re f
580.125 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 228.063 Td /F1 9.8 Tf  [(547.0 � 8.4)] TJ ET
BT 26.250 167.138 Td /F4 9.8 Tf  [(Effect on muscle inflammation)] TJ ET
BT 26.250 147.733 Td /F1 9.8 Tf  [(We have previously shown that cathepsin activity in fore and hindlimbs is significantly increased in mdx mice )] TJ ET
0.267 0.267 0.267 rg
BT 496.044 149.240 Td /F4 8.7 Tf  [(20)] TJ ET
0.271 0.267 0.267 rg
BT 505.681 147.733 Td /F1 9.8 Tf  [(. We assessed )] TJ ET
BT 26.250 135.828 Td /F1 9.8 Tf  [(cathepsin activity using optical imaging after 5 weeks of treatment. Untreated mdx mice showed a significant \(84.87%\) increase )] TJ ET
BT 26.250 123.924 Td /F1 9.8 Tf  [(in cathepsin activity in the forelimbs using optical imaging compared to BL10 mice. They also showed a 42.41% increase in )] TJ ET
BT 26.250 112.019 Td /F1 9.8 Tf  [(hindlimb cathepsin activity. Mice treated with Dantrolene showed no significant decrease \(18.17%\) in inflammation in the )] TJ ET
BT 26.250 100.114 Td /F1 9.8 Tf  [(forelimbs compared to untreated mdx mice. Alternatively, no change was found in the hindlimbs \(Fig. 3\).)] TJ ET
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0.965 0.965 0.965 rg
26.250 699.136 555.000 77.864 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 699.136 m 581.250 699.136 l 581.250 699.886 l 26.250 699.886 l  f
q
35.250 710.386 537.000 66.614 re W n
0.271 0.267 0.267 rg
BT 35.250 767.476 Td /F4 9.8 Tf  [(Fig. 1: Body weight measurements during treatment)] TJ ET
BT 35.250 748.106 Td /F1 9.8 Tf  [(Body weight was recorded each week over the entire duration of treatment. The two mdx treatment groups showed no )] TJ ET
BT 35.250 734.370 Td /F1 9.8 Tf  [(significant change in body weight of the course of treatment after the mdx mice were broken up into groups based on )] TJ ET
BT 35.250 720.634 Td /F1 9.8 Tf  [(normalizing bodyweight. N=8)] TJ ET
Q
BT 26.250 682.113 Td /F4 9.8 Tf  [(Effect on functional and behavioral assays)] TJ ET
BT 26.250 662.708 Td /F1 9.8 Tf  [(Grip strength and open field testing for mdx and BL10 mice was performed after 4 weeks of treatment. Vehicle-treated mdx )] TJ ET
BT 26.250 650.803 Td /F1 9.8 Tf  [(mice showed a reduction in absolute forelimb strength \(7.88%\) compared to BL10 mice. Treatment of mdx mice with Dantrolene )] TJ ET
BT 26.250 638.898 Td /F1 9.8 Tf  [(resulted in a 16.69% reduction in strength compared to the untreated mdx mice \(Fig. 2A\). Because we found significant )] TJ ET
BT 26.250 626.994 Td /F1 9.8 Tf  [(differences in bodyweight between BL10 and mdx mice, we also normalized strength to body weight. Untreated mdx mice )] TJ ET
BT 26.250 615.089 Td /F1 9.8 Tf  [(showed a significant decrease \(24.23%\) in normalized forelimb strength relative to the BL10 mice. Mdx mice treated with )] TJ ET
BT 26.250 603.184 Td /F1 9.8 Tf  [(Dantrolene showed a 13.58% decrease compared to the untreated mdx group \(Fig. 2B\). Hindlimb measurements were unable )] TJ ET
BT 26.250 591.279 Td /F1 9.8 Tf  [(to ascertain a difference between mdx and BL10 mice even after normalization \(data not shown\).)] TJ ET
0.965 0.965 0.965 rg
26.250 386.021 555.000 195.377 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 581.398 m 581.250 581.398 l 581.250 580.648 l 26.250 580.648 l  f
26.250 386.021 m 581.250 386.021 l 581.250 386.771 l 26.250 386.771 l  f
q
225.000 0 0 115.500 35.250 456.148 cm /I4 Do
Q 
q
35.250 397.271 537.000 52.877 re W n
0.271 0.267 0.267 rg
BT 35.250 440.625 Td /F4 9.8 Tf  [(Fig. 2: GSM results of wild type and mdx mice)] TJ ET
BT 35.250 421.255 Td /F1 9.8 Tf  [(Grip strength measurements were performed at 4 weeks of treatment. A\) Absolute Forelimb B\)Forelimb normalized to body )] TJ ET
BT 35.250 407.518 Td /F1 9.8 Tf  [(weight. * P ? 0.05, *** P ? 0.001. N=8)] TJ ET
Q
BT 26.250 368.997 Td /F1 9.8 Tf  [(Activity was measured using open field activity chambers. There was no significant change in any measured parameters, )] TJ ET
BT 26.250 357.093 Td /F1 9.8 Tf  [(including horizontal and vertical activity as well as movement and rest time, between any of the groups \(Table 1\).)] TJ ET
q
26.250 333.475 555.000 13.736 re W n
0.271 0.267 0.267 rg
BT 26.250 336.223 Td /F1 9.8 Tf  [(Table 1: Open field activity measured parameters)] TJ ET
Q
0.965 0.965 0.965 rg
26.250 302.568 555.000 23.408 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 325.975 m 581.250 325.975 l 580.500 325.225 l 27.000 325.225 l  f
581.250 325.975 m 581.250 302.568 l 580.500 302.568 l 580.500 325.225 l  f
26.250 325.975 m 26.250 302.568 l 27.000 302.568 l 27.000 325.225 l  f
0.271 0.267 0.267 rg
BT 33.000 311.899 Td /F1 9.0 Tf  [(Comparisons between BL10 and mdx Vehicle-treated mice as well as both mdx mice groups yielded no significant differences. N=8)] TJ ET
1.000 1.000 1.000 rg
26.250 221.662 555.000 80.906 re f
0.267 0.267 0.267 rg
26.625 301.443 215.682 0.750 re f
26.625 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 291.588 Td /F1 9.8 Tf  [(Measurement)] TJ ET
0.267 0.267 0.267 rg
241.557 301.443 107.688 0.750 re f
241.557 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 291.588 Td /F1 9.8 Tf  [(BL10 Vehicle)] TJ ET
0.267 0.267 0.267 rg
348.494 301.443 107.688 0.750 re f
348.494 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 291.588 Td /F1 9.8 Tf  [(mdx Vehicle)] TJ ET
0.267 0.267 0.267 rg
455.432 301.443 125.443 0.750 re f
455.432 285.562 0.750 16.631 re f
580.125 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 291.588 Td /F1 9.8 Tf  [(mdx Treatment)] TJ ET
0.267 0.267 0.267 rg
26.625 285.562 215.682 0.750 re f
26.625 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 275.707 Td /F1 9.8 Tf  [(Horizontal Activity \(arbitrary units\))] TJ ET
0.267 0.267 0.267 rg
241.557 285.562 107.688 0.750 re f
241.557 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 275.707 Td /F1 9.8 Tf  [(1785.7 � 81.2)] TJ ET
0.267 0.267 0.267 rg
348.494 285.562 107.688 0.750 re f
348.494 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 275.707 Td /F1 9.8 Tf  [(1463.0 � 99.9)] TJ ET
0.267 0.267 0.267 rg
455.432 285.562 125.443 0.750 re f
455.432 269.680 0.750 16.631 re f
580.125 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 275.707 Td /F1 9.8 Tf  [(1259.2 � 118.7)] TJ ET
0.267 0.267 0.267 rg
26.625 269.680 215.682 0.750 re f
26.625 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 259.825 Td /F1 9.8 Tf  [(Vertical Activity \(arbitrary units\))] TJ ET
0.267 0.267 0.267 rg
241.557 269.680 107.688 0.750 re f
241.557 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 259.825 Td /F1 9.8 Tf  [(26.4 � 5.8)] TJ ET
0.267 0.267 0.267 rg
348.494 269.680 107.688 0.750 re f
348.494 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 259.825 Td /F1 9.8 Tf  [(87.8 � 25.7)] TJ ET
0.267 0.267 0.267 rg
455.432 269.680 125.443 0.750 re f
455.432 253.799 0.750 16.631 re f
580.125 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 259.825 Td /F1 9.8 Tf  [(41.4 � 12.2)] TJ ET
0.267 0.267 0.267 rg
26.625 253.799 215.682 0.750 re f
26.625 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 243.944 Td /F1 9.8 Tf  [(Total Movement Time \(seconds\))] TJ ET
0.267 0.267 0.267 rg
241.557 253.799 107.688 0.750 re f
241.557 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 243.944 Td /F1 9.8 Tf  [(71.9 � 8.5)] TJ ET
0.267 0.267 0.267 rg
348.494 253.799 107.688 0.750 re f
348.494 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 243.944 Td /F1 9.8 Tf  [(64.0 � 8.1)] TJ ET
0.267 0.267 0.267 rg
455.432 253.799 125.443 0.750 re f
455.432 237.918 0.750 16.631 re f
580.125 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 243.944 Td /F1 9.8 Tf  [(53.0 � 8.4)] TJ ET
0.267 0.267 0.267 rg
26.625 237.918 215.682 0.750 re f
26.625 222.037 215.682 0.750 re f
26.625 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 228.063 Td /F1 9.8 Tf  [(Total Rest Time \(seconds\))] TJ ET
0.267 0.267 0.267 rg
241.557 237.918 107.688 0.750 re f
241.557 222.037 107.688 0.750 re f
241.557 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 228.063 Td /F1 9.8 Tf  [(528.0 � 8.5)] TJ ET
0.267 0.267 0.267 rg
348.494 237.918 107.688 0.750 re f
348.494 222.037 107.688 0.750 re f
348.494 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 228.063 Td /F1 9.8 Tf  [(536.0 � 8.0)] TJ ET
0.267 0.267 0.267 rg
455.432 237.918 125.443 0.750 re f
455.432 222.037 125.443 0.750 re f
455.432 222.037 0.750 16.631 re f
580.125 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 228.063 Td /F1 9.8 Tf  [(547.0 � 8.4)] TJ ET
BT 26.250 167.138 Td /F4 9.8 Tf  [(Effect on muscle inflammation)] TJ ET
BT 26.250 147.733 Td /F1 9.8 Tf  [(We have previously shown that cathepsin activity in fore and hindlimbs is significantly increased in mdx mice )] TJ ET
0.267 0.267 0.267 rg
BT 496.044 149.240 Td /F4 8.7 Tf  [(20)] TJ ET
0.271 0.267 0.267 rg
BT 505.681 147.733 Td /F1 9.8 Tf  [(. We assessed )] TJ ET
BT 26.250 135.828 Td /F1 9.8 Tf  [(cathepsin activity using optical imaging after 5 weeks of treatment. Untreated mdx mice showed a significant \(84.87%\) increase )] TJ ET
BT 26.250 123.924 Td /F1 9.8 Tf  [(in cathepsin activity in the forelimbs using optical imaging compared to BL10 mice. They also showed a 42.41% increase in )] TJ ET
BT 26.250 112.019 Td /F1 9.8 Tf  [(hindlimb cathepsin activity. Mice treated with Dantrolene showed no significant decrease \(18.17%\) in inflammation in the )] TJ ET
BT 26.250 100.114 Td /F1 9.8 Tf  [(forelimbs compared to untreated mdx mice. Alternatively, no change was found in the hindlimbs \(Fig. 3\).)] TJ ET
0.965 0.965 0.965 rg
26.250 -26.767 555.000 117.000 re f
0.267 0.267 0.267 rg
26.250 90.233 m 581.250 90.233 l 581.250 89.483 l 26.250 89.483 l  f
q
225.000 0 0 101.250 35.250 -20.767 cm /I5 Do
Q 
q
35.250 -26.767 537.000 0.000 re W n
Q
Q
q
15.000 -26.767 577.500 803.767 re W n
0.965 0.965 0.965 rg
26.250 699.136 555.000 77.864 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 699.136 m 581.250 699.136 l 581.250 699.886 l 26.250 699.886 l  f
q
35.250 710.386 537.000 66.614 re W n
0.271 0.267 0.267 rg
BT 35.250 767.476 Td /F4 9.8 Tf  [(Fig. 1: Body weight measurements during treatment)] TJ ET
BT 35.250 748.106 Td /F1 9.8 Tf  [(Body weight was recorded each week over the entire duration of treatment. The two mdx treatment groups showed no )] TJ ET
BT 35.250 734.370 Td /F1 9.8 Tf  [(significant change in body weight of the course of treatment after the mdx mice were broken up into groups based on )] TJ ET
BT 35.250 720.634 Td /F1 9.8 Tf  [(normalizing bodyweight. N=8)] TJ ET
Q
BT 26.250 682.113 Td /F4 9.8 Tf  [(Effect on functional and behavioral assays)] TJ ET
BT 26.250 662.708 Td /F1 9.8 Tf  [(Grip strength and open field testing for mdx and BL10 mice was performed after 4 weeks of treatment. Vehicle-treated mdx )] TJ ET
BT 26.250 650.803 Td /F1 9.8 Tf  [(mice showed a reduction in absolute forelimb strength \(7.88%\) compared to BL10 mice. Treatment of mdx mice with Dantrolene )] TJ ET
BT 26.250 638.898 Td /F1 9.8 Tf  [(resulted in a 16.69% reduction in strength compared to the untreated mdx mice \(Fig. 2A\). Because we found significant )] TJ ET
BT 26.250 626.994 Td /F1 9.8 Tf  [(differences in bodyweight between BL10 and mdx mice, we also normalized strength to body weight. Untreated mdx mice )] TJ ET
BT 26.250 615.089 Td /F1 9.8 Tf  [(showed a significant decrease \(24.23%\) in normalized forelimb strength relative to the BL10 mice. Mdx mice treated with )] TJ ET
BT 26.250 603.184 Td /F1 9.8 Tf  [(Dantrolene showed a 13.58% decrease compared to the untreated mdx group \(Fig. 2B\). Hindlimb measurements were unable )] TJ ET
BT 26.250 591.279 Td /F1 9.8 Tf  [(to ascertain a difference between mdx and BL10 mice even after normalization \(data not shown\).)] TJ ET
0.965 0.965 0.965 rg
26.250 386.021 555.000 195.377 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 581.398 m 581.250 581.398 l 581.250 580.648 l 26.250 580.648 l  f
26.250 386.021 m 581.250 386.021 l 581.250 386.771 l 26.250 386.771 l  f
q
225.000 0 0 115.500 35.250 456.148 cm /I4 Do
Q 
q
35.250 397.271 537.000 52.877 re W n
0.271 0.267 0.267 rg
BT 35.250 440.625 Td /F4 9.8 Tf  [(Fig. 2: GSM results of wild type and mdx mice)] TJ ET
BT 35.250 421.255 Td /F1 9.8 Tf  [(Grip strength measurements were performed at 4 weeks of treatment. A\) Absolute Forelimb B\)Forelimb normalized to body )] TJ ET
BT 35.250 407.518 Td /F1 9.8 Tf  [(weight. * P ? 0.05, *** P ? 0.001. N=8)] TJ ET
Q
BT 26.250 368.997 Td /F1 9.8 Tf  [(Activity was measured using open field activity chambers. There was no significant change in any measured parameters, )] TJ ET
BT 26.250 357.093 Td /F1 9.8 Tf  [(including horizontal and vertical activity as well as movement and rest time, between any of the groups \(Table 1\).)] TJ ET
q
26.250 333.475 555.000 13.736 re W n
0.271 0.267 0.267 rg
BT 26.250 336.223 Td /F1 9.8 Tf  [(Table 1: Open field activity measured parameters)] TJ ET
Q
0.965 0.965 0.965 rg
26.250 302.568 555.000 23.408 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 325.975 m 581.250 325.975 l 580.500 325.225 l 27.000 325.225 l  f
581.250 325.975 m 581.250 302.568 l 580.500 302.568 l 580.500 325.225 l  f
26.250 325.975 m 26.250 302.568 l 27.000 302.568 l 27.000 325.225 l  f
0.271 0.267 0.267 rg
BT 33.000 311.899 Td /F1 9.0 Tf  [(Comparisons between BL10 and mdx Vehicle-treated mice as well as both mdx mice groups yielded no significant differences. N=8)] TJ ET
1.000 1.000 1.000 rg
26.250 221.662 555.000 80.906 re f
0.267 0.267 0.267 rg
26.625 301.443 215.682 0.750 re f
26.625 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 291.588 Td /F1 9.8 Tf  [(Measurement)] TJ ET
0.267 0.267 0.267 rg
241.557 301.443 107.688 0.750 re f
241.557 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 291.588 Td /F1 9.8 Tf  [(BL10 Vehicle)] TJ ET
0.267 0.267 0.267 rg
348.494 301.443 107.688 0.750 re f
348.494 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 291.588 Td /F1 9.8 Tf  [(mdx Vehicle)] TJ ET
0.267 0.267 0.267 rg
455.432 301.443 125.443 0.750 re f
455.432 285.562 0.750 16.631 re f
580.125 285.562 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 291.588 Td /F1 9.8 Tf  [(mdx Treatment)] TJ ET
0.267 0.267 0.267 rg
26.625 285.562 215.682 0.750 re f
26.625 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 275.707 Td /F1 9.8 Tf  [(Horizontal Activity \(arbitrary units\))] TJ ET
0.267 0.267 0.267 rg
241.557 285.562 107.688 0.750 re f
241.557 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 275.707 Td /F1 9.8 Tf  [(1785.7 � 81.2)] TJ ET
0.267 0.267 0.267 rg
348.494 285.562 107.688 0.750 re f
348.494 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 275.707 Td /F1 9.8 Tf  [(1463.0 � 99.9)] TJ ET
0.267 0.267 0.267 rg
455.432 285.562 125.443 0.750 re f
455.432 269.680 0.750 16.631 re f
580.125 269.680 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 275.707 Td /F1 9.8 Tf  [(1259.2 � 118.7)] TJ ET
0.267 0.267 0.267 rg
26.625 269.680 215.682 0.750 re f
26.625 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 259.825 Td /F1 9.8 Tf  [(Vertical Activity \(arbitrary units\))] TJ ET
0.267 0.267 0.267 rg
241.557 269.680 107.688 0.750 re f
241.557 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 259.825 Td /F1 9.8 Tf  [(26.4 � 5.8)] TJ ET
0.267 0.267 0.267 rg
348.494 269.680 107.688 0.750 re f
348.494 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 259.825 Td /F1 9.8 Tf  [(87.8 � 25.7)] TJ ET
0.267 0.267 0.267 rg
455.432 269.680 125.443 0.750 re f
455.432 253.799 0.750 16.631 re f
580.125 253.799 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 259.825 Td /F1 9.8 Tf  [(41.4 � 12.2)] TJ ET
0.267 0.267 0.267 rg
26.625 253.799 215.682 0.750 re f
26.625 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 243.944 Td /F1 9.8 Tf  [(Total Movement Time \(seconds\))] TJ ET
0.267 0.267 0.267 rg
241.557 253.799 107.688 0.750 re f
241.557 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 243.944 Td /F1 9.8 Tf  [(71.9 � 8.5)] TJ ET
0.267 0.267 0.267 rg
348.494 253.799 107.688 0.750 re f
348.494 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 243.944 Td /F1 9.8 Tf  [(64.0 � 8.1)] TJ ET
0.267 0.267 0.267 rg
455.432 253.799 125.443 0.750 re f
455.432 237.918 0.750 16.631 re f
580.125 237.918 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 243.944 Td /F1 9.8 Tf  [(53.0 � 8.4)] TJ ET
0.267 0.267 0.267 rg
26.625 237.918 215.682 0.750 re f
26.625 222.037 215.682 0.750 re f
26.625 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 228.063 Td /F1 9.8 Tf  [(Total Rest Time \(seconds\))] TJ ET
0.267 0.267 0.267 rg
241.557 237.918 107.688 0.750 re f
241.557 222.037 107.688 0.750 re f
241.557 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 246.807 228.063 Td /F1 9.8 Tf  [(528.0 � 8.5)] TJ ET
0.267 0.267 0.267 rg
348.494 237.918 107.688 0.750 re f
348.494 222.037 107.688 0.750 re f
348.494 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 353.744 228.063 Td /F1 9.8 Tf  [(536.0 � 8.0)] TJ ET
0.267 0.267 0.267 rg
455.432 237.918 125.443 0.750 re f
455.432 222.037 125.443 0.750 re f
455.432 222.037 0.750 16.631 re f
580.125 222.037 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 460.682 228.063 Td /F1 9.8 Tf  [(547.0 � 8.4)] TJ ET
BT 26.250 167.138 Td /F4 9.8 Tf  [(Effect on muscle inflammation)] TJ ET
BT 26.250 147.733 Td /F1 9.8 Tf  [(We have previously shown that cathepsin activity in fore and hindlimbs is significantly increased in mdx mice )] TJ ET
0.267 0.267 0.267 rg
BT 496.044 149.240 Td /F4 8.7 Tf  [(20)] TJ ET
0.271 0.267 0.267 rg
BT 505.681 147.733 Td /F1 9.8 Tf  [(. We assessed )] TJ ET
BT 26.250 135.828 Td /F1 9.8 Tf  [(cathepsin activity using optical imaging after 5 weeks of treatment. Untreated mdx mice showed a significant \(84.87%\) increase )] TJ ET
BT 26.250 123.924 Td /F1 9.8 Tf  [(in cathepsin activity in the forelimbs using optical imaging compared to BL10 mice. They also showed a 42.41% increase in )] TJ ET
BT 26.250 112.019 Td /F1 9.8 Tf  [(hindlimb cathepsin activity. Mice treated with Dantrolene showed no significant decrease \(18.17%\) in inflammation in the )] TJ ET
BT 26.250 100.114 Td /F1 9.8 Tf  [(forelimbs compared to untreated mdx mice. Alternatively, no change was found in the hindlimbs \(Fig. 3\).)] TJ ET
0.965 0.965 0.965 rg
26.250 -26.767 555.000 117.000 re f
0.267 0.267 0.267 rg
26.250 90.233 m 581.250 90.233 l 581.250 89.483 l 26.250 89.483 l  f
q
225.000 0 0 101.250 35.250 -20.767 cm /I5 Do
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35.250 -26.767 537.000 0.000 re W n
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225.000 0 0 115.500 35.250 456.148 cm /I4 Do
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225.000 0 0 101.250 35.250 -20.767 cm /I5 Do
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0.000 0.000 0.000 rg
BT 291.710 19.825 Td /F1 11.0 Tf  [(4)] TJ ET
BT 25.000 19.825 Td /F1 11.0 Tf  [(PLOS Currents Muscular Dystrophy)] TJ ET

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0.271 0.267 0.267 rg
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0.271 0.267 0.267 rg
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0.267 0.267 0.267 rg
376.254 480.635 128.437 0.750 re f
376.254 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 470.780 Td /F1 9.8 Tf  [(0.00527 � 0.0003)] TJ ET
0.267 0.267 0.267 rg
503.940 480.635 76.935 0.750 re f
503.940 464.754 0.750 16.631 re f
580.125 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 470.780 Td /F1 9.8 Tf  [(0.4533)] TJ ET
0.267 0.267 0.267 rg
26.625 464.754 231.886 0.750 re f
26.625 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 454.899 Td /F1 9.8 Tf  [(Regenerating Fibers/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 464.754 119.242 0.750 re f
257.761 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 454.899 Td /F1 9.8 Tf  [(2.625 � 0.78)] TJ ET
0.267 0.267 0.267 rg
376.254 464.754 128.437 0.750 re f
376.254 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 454.899 Td /F1 9.8 Tf  [(3.844 � 0.98)] TJ ET
0.267 0.267 0.267 rg
503.940 464.754 76.935 0.750 re f
503.940 448.873 0.750 16.631 re f
580.125 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 454.899 Td /F1 9.8 Tf  [(0.3544)] TJ ET
0.267 0.267 0.267 rg
26.625 448.873 231.886 0.750 re f
26.625 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 439.018 Td /F1 9.8 Tf  [(% of Central Nucleated Fibers)] TJ ET
0.267 0.267 0.267 rg
257.761 448.873 119.242 0.750 re f
257.761 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 439.018 Td /F1 9.8 Tf  [(71.28% � 3.33)] TJ ET
0.267 0.267 0.267 rg
376.254 448.873 128.437 0.750 re f
376.254 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 439.018 Td /F1 9.8 Tf  [(69.33% � 2.63)] TJ ET
0.267 0.267 0.267 rg
503.940 448.873 76.935 0.750 re f
503.940 432.991 0.750 16.631 re f
580.125 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 439.018 Td /F1 9.8 Tf  [(0.6489)] TJ ET
0.267 0.267 0.267 rg
26.625 432.991 231.886 0.750 re f
26.625 417.110 231.886 0.750 re f
26.625 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 423.136 Td /F1 9.8 Tf  [(Inflammation Foci/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 432.991 119.242 0.750 re f
257.761 417.110 119.242 0.750 re f
257.761 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 423.136 Td /F1 9.8 Tf  [(0.675 � 0.12)] TJ ET
0.267 0.267 0.267 rg
376.254 432.991 128.437 0.750 re f
376.254 417.110 128.437 0.750 re f
376.254 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 423.136 Td /F1 9.8 Tf  [(1.133 � 0.51)] TJ ET
0.267 0.267 0.267 rg
503.940 432.991 76.935 0.750 re f
503.940 417.110 76.935 0.750 re f
503.940 417.110 0.750 16.631 re f
580.125 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 423.136 Td /F1 9.8 Tf  [(0.4073)] TJ ET
BT 26.250 362.211 Td /F4 9.8 Tf  [(Effect on)] TJ ET
BT 67.960 362.211 Td /F5 9.8 Tf  [(in vitro)] TJ ET
BT 100.467 362.211 Td /F4 9.8 Tf  [( muscle force, fatigue resistance, and lengthening contractions)] TJ ET
BT 26.250 342.807 Td /F6 9.8 Tf  [(In vitro)] TJ ET
BT 55.510 342.807 Td /F1 9.8 Tf  [( force analysis was performed on the EDL muscle of each mouse prior to sacrifice. The EDL muscles of mdx mice had a )] TJ ET
BT 26.250 330.902 Td /F1 9.8 Tf  [(significant decrease \(31.98%\) in specific force relative to BL10 mice. Dantrolene, however, did not significantly improve it \(Fig. )] TJ ET
BT 26.250 318.997 Td /F1 9.8 Tf  [(4A\). Additionally, mdx mice were significantly less resistant to damage occurring from lengthening compared to BL10 mice \(Fig. )] TJ ET
BT 26.250 307.092 Td /F1 9.8 Tf  [(4B\). Treatment with Dantrolene showed no improvement in muscle force generated after lengthening contractions \(Fig. 4B\). )] TJ ET
BT 26.250 295.188 Td /F1 9.8 Tf  [(Finally, no significant differences were observed between any of the groups in muscle fatigue or recovery \(Fig. 4C\).)] TJ ET
0.965 0.965 0.965 rg
26.250 91.152 555.000 194.155 re f
0.267 0.267 0.267 rg
26.250 285.307 m 581.250 285.307 l 581.250 284.557 l 26.250 284.557 l  f
q
225.000 0 0 160.500 35.250 115.057 cm /I6 Do
Q 
q
35.250 91.152 537.000 17.905 re W n
Q
Q
q
15.000 91.152 577.500 685.848 re W n
0.965 0.965 0.965 rg
26.250 712.873 555.000 64.127 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 712.873 m 581.250 712.873 l 581.250 713.623 l 26.250 713.623 l  f
q
35.250 724.123 537.000 52.877 re W n
0.271 0.267 0.267 rg
BT 35.250 767.476 Td /F4 9.8 Tf  [(Fig. 3: Analysis of muscle inflammation using optical imaging of cathepsin)] TJ ET
BT 35.250 748.106 Td /F1 9.8 Tf  [(Cathepsin activity was measured from A\) forelimb and B\) hindlimb muscles as a quantification of inflammation. Activity is )] TJ ET
BT 35.250 734.370 Td /F1 9.8 Tf  [(defined in terms of photon counts/mm2. ** P ? 0.01)] TJ ET
Q
BT 26.250 695.849 Td /F4 9.8 Tf  [(Effect on measured histological parameters)] TJ ET
BT 26.250 676.444 Td /F1 9.8 Tf  [(Hematoxylin and eosin \(H&E\) stained sections from the TA of each animal were analyzed in blinded fashion. Dantrolene )] TJ ET
BT 26.250 664.539 Td /F1 9.8 Tf  [(treatment did not significantly alter any of these parameters \(fibers/field, central nuclei/fiber, peripheral nuclei/fiber, total )] TJ ET
BT 26.250 652.635 Td /F1 9.8 Tf  [(nuclei/fiber, fibers with central nuclei/field, ratio of central to peripheral nuclei/field, regenerating fibers/field, percentage of )] TJ ET
BT 26.250 640.730 Td /F1 9.8 Tf  [(central nucleated fibers and inflammatory foci\) compared to the vehicle-treated mdx mice \(Table 2\).)] TJ ET
q
26.250 617.113 555.000 13.736 re W n
0.271 0.267 0.267 rg
BT 26.250 619.860 Td /F1 9.8 Tf  [(Table 2: Comparison of histological parameters in TA muscle)] TJ ET
Q
0.965 0.965 0.965 rg
26.250 577.048 555.000 32.565 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 609.613 m 581.250 609.613 l 580.500 608.863 l 27.000 608.863 l  f
581.250 609.613 m 581.250 577.048 l 580.500 577.048 l 580.500 608.863 l  f
26.250 609.613 m 26.250 577.048 l 27.000 577.048 l 27.000 608.863 l  f
0.271 0.267 0.267 rg
BT 33.000 595.537 Td /F1 9.0 Tf  [(Analysis of muscle histology performed on TA muscle of each animal in a blinded fashion. Comparisons of ten standard parameters )] TJ ET
BT 33.000 586.379 Td /F1 9.0 Tf  [(yielded no significant change between the two mdx groups. N=8)] TJ ET
1.000 1.000 1.000 rg
26.250 416.735 555.000 160.313 re f
0.267 0.267 0.267 rg
26.625 575.923 231.886 0.750 re f
26.625 560.041 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 566.068 Td /F1 9.8 Tf  [(Measurement)] TJ ET
0.267 0.267 0.267 rg
257.761 575.923 119.242 0.750 re f
257.761 560.041 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 566.068 Td /F1 9.8 Tf  [(mdx Vehicle)] TJ ET
0.267 0.267 0.267 rg
376.254 575.923 128.437 0.750 re f
376.254 560.041 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 566.068 Td /F1 9.8 Tf  [(mdx Treatment)] TJ ET
0.267 0.267 0.267 rg
503.940 575.923 76.935 0.750 re f
503.940 560.041 0.750 16.631 re f
580.125 560.041 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 566.068 Td /F1 9.8 Tf  [(P-value)] TJ ET
0.267 0.267 0.267 rg
26.625 560.041 231.886 0.750 re f
26.625 544.160 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 550.186 Td /F1 9.8 Tf  [(Fibers/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 560.041 119.242 0.750 re f
257.761 544.160 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 550.186 Td /F1 9.8 Tf  [(179.45 � 4.57)] TJ ET
0.267 0.267 0.267 rg
376.254 560.041 128.437 0.750 re f
376.254 544.160 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 550.186 Td /F1 9.8 Tf  [(191.47 � 8.93)] TJ ET
0.267 0.267 0.267 rg
503.940 560.041 76.935 0.750 re f
503.940 544.160 0.750 16.631 re f
580.125 544.160 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 550.186 Td /F1 9.8 Tf  [(0.2675)] TJ ET
0.267 0.267 0.267 rg
26.625 544.160 231.886 0.750 re f
26.625 528.279 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 534.305 Td /F1 9.8 Tf  [(Central Nuclei/Fiber)] TJ ET
0.267 0.267 0.267 rg
257.761 544.160 119.242 0.750 re f
257.761 528.279 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 534.305 Td /F1 9.8 Tf  [(0.713 � 0.03)] TJ ET
0.267 0.267 0.267 rg
376.254 544.160 128.437 0.750 re f
376.254 528.279 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 534.305 Td /F1 9.8 Tf  [(0.693 � 0.03)] TJ ET
0.267 0.267 0.267 rg
503.940 544.160 76.935 0.750 re f
503.940 528.279 0.750 16.631 re f
580.125 528.279 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 534.305 Td /F1 9.8 Tf  [(0.6490)] TJ ET
0.267 0.267 0.267 rg
26.625 528.279 231.886 0.750 re f
26.625 512.398 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 518.424 Td /F1 9.8 Tf  [(Peripheral Nuclei/Fiber)] TJ ET
0.267 0.267 0.267 rg
257.761 528.279 119.242 0.750 re f
257.761 512.398 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 518.424 Td /F1 9.8 Tf  [(0.717 � 0.03)] TJ ET
0.267 0.267 0.267 rg
376.254 528.279 128.437 0.750 re f
376.254 512.398 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 518.424 Td /F1 9.8 Tf  [(0.702 � 0.03)] TJ ET
0.267 0.267 0.267 rg
503.940 528.279 76.935 0.750 re f
503.940 512.398 0.750 16.631 re f
580.125 512.398 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 518.424 Td /F1 9.8 Tf  [(0.7311)] TJ ET
0.267 0.267 0.267 rg
26.625 512.398 231.886 0.750 re f
26.625 496.516 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 502.543 Td /F1 9.8 Tf  [(Total Nuclei/Fiber)] TJ ET
0.267 0.267 0.267 rg
257.761 512.398 119.242 0.750 re f
257.761 496.516 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 502.543 Td /F1 9.8 Tf  [(1.430 � 0.06)] TJ ET
0.267 0.267 0.267 rg
376.254 512.398 128.437 0.750 re f
376.254 496.516 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 502.543 Td /F1 9.8 Tf  [(1.396 � 0.05)] TJ ET
0.267 0.267 0.267 rg
503.940 512.398 76.935 0.750 re f
503.940 496.516 0.750 16.631 re f
580.125 496.516 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 502.543 Td /F1 9.8 Tf  [(0.6541)] TJ ET
0.267 0.267 0.267 rg
26.625 496.516 231.886 0.750 re f
26.625 480.635 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 486.661 Td /F1 9.8 Tf  [(Fibers with Central Nuclei/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 496.516 119.242 0.750 re f
257.761 480.635 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 486.661 Td /F1 9.8 Tf  [(98.125 � 3.28)] TJ ET
0.267 0.267 0.267 rg
376.254 496.516 128.437 0.750 re f
376.254 480.635 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 486.661 Td /F1 9.8 Tf  [(104.133 � 4.93)] TJ ET
0.267 0.267 0.267 rg
503.940 496.516 76.935 0.750 re f
503.940 480.635 0.750 16.631 re f
580.125 480.635 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 486.661 Td /F1 9.8 Tf  [(0.3392)] TJ ET
0.267 0.267 0.267 rg
26.625 480.635 231.886 0.750 re f
26.625 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 470.780 Td /F1 9.8 Tf  [(Ratio of Central to Peripheral Nuclei/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 480.635 119.242 0.750 re f
257.761 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 470.780 Td /F1 9.8 Tf  [(0.00559 � 0.0003)] TJ ET
0.267 0.267 0.267 rg
376.254 480.635 128.437 0.750 re f
376.254 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 470.780 Td /F1 9.8 Tf  [(0.00527 � 0.0003)] TJ ET
0.267 0.267 0.267 rg
503.940 480.635 76.935 0.750 re f
503.940 464.754 0.750 16.631 re f
580.125 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 470.780 Td /F1 9.8 Tf  [(0.4533)] TJ ET
0.267 0.267 0.267 rg
26.625 464.754 231.886 0.750 re f
26.625 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 454.899 Td /F1 9.8 Tf  [(Regenerating Fibers/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 464.754 119.242 0.750 re f
257.761 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 454.899 Td /F1 9.8 Tf  [(2.625 � 0.78)] TJ ET
0.267 0.267 0.267 rg
376.254 464.754 128.437 0.750 re f
376.254 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 454.899 Td /F1 9.8 Tf  [(3.844 � 0.98)] TJ ET
0.267 0.267 0.267 rg
503.940 464.754 76.935 0.750 re f
503.940 448.873 0.750 16.631 re f
580.125 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 454.899 Td /F1 9.8 Tf  [(0.3544)] TJ ET
0.267 0.267 0.267 rg
26.625 448.873 231.886 0.750 re f
26.625 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 439.018 Td /F1 9.8 Tf  [(% of Central Nucleated Fibers)] TJ ET
0.267 0.267 0.267 rg
257.761 448.873 119.242 0.750 re f
257.761 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 439.018 Td /F1 9.8 Tf  [(71.28% � 3.33)] TJ ET
0.267 0.267 0.267 rg
376.254 448.873 128.437 0.750 re f
376.254 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 439.018 Td /F1 9.8 Tf  [(69.33% � 2.63)] TJ ET
0.267 0.267 0.267 rg
503.940 448.873 76.935 0.750 re f
503.940 432.991 0.750 16.631 re f
580.125 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 439.018 Td /F1 9.8 Tf  [(0.6489)] TJ ET
0.267 0.267 0.267 rg
26.625 432.991 231.886 0.750 re f
26.625 417.110 231.886 0.750 re f
26.625 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 423.136 Td /F1 9.8 Tf  [(Inflammation Foci/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 432.991 119.242 0.750 re f
257.761 417.110 119.242 0.750 re f
257.761 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 423.136 Td /F1 9.8 Tf  [(0.675 � 0.12)] TJ ET
0.267 0.267 0.267 rg
376.254 432.991 128.437 0.750 re f
376.254 417.110 128.437 0.750 re f
376.254 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 423.136 Td /F1 9.8 Tf  [(1.133 � 0.51)] TJ ET
0.267 0.267 0.267 rg
503.940 432.991 76.935 0.750 re f
503.940 417.110 76.935 0.750 re f
503.940 417.110 0.750 16.631 re f
580.125 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 423.136 Td /F1 9.8 Tf  [(0.4073)] TJ ET
BT 26.250 362.211 Td /F4 9.8 Tf  [(Effect on)] TJ ET
BT 67.960 362.211 Td /F5 9.8 Tf  [(in vitro)] TJ ET
BT 100.467 362.211 Td /F4 9.8 Tf  [( muscle force, fatigue resistance, and lengthening contractions)] TJ ET
BT 26.250 342.807 Td /F6 9.8 Tf  [(In vitro)] TJ ET
BT 55.510 342.807 Td /F1 9.8 Tf  [( force analysis was performed on the EDL muscle of each mouse prior to sacrifice. The EDL muscles of mdx mice had a )] TJ ET
BT 26.250 330.902 Td /F1 9.8 Tf  [(significant decrease \(31.98%\) in specific force relative to BL10 mice. Dantrolene, however, did not significantly improve it \(Fig. )] TJ ET
BT 26.250 318.997 Td /F1 9.8 Tf  [(4A\). Additionally, mdx mice were significantly less resistant to damage occurring from lengthening compared to BL10 mice \(Fig. )] TJ ET
BT 26.250 307.092 Td /F1 9.8 Tf  [(4B\). Treatment with Dantrolene showed no improvement in muscle force generated after lengthening contractions \(Fig. 4B\). )] TJ ET
BT 26.250 295.188 Td /F1 9.8 Tf  [(Finally, no significant differences were observed between any of the groups in muscle fatigue or recovery \(Fig. 4C\).)] TJ ET
0.965 0.965 0.965 rg
26.250 91.152 555.000 194.155 re f
0.267 0.267 0.267 rg
26.250 285.307 m 581.250 285.307 l 581.250 284.557 l 26.250 284.557 l  f
q
225.000 0 0 160.500 35.250 115.057 cm /I6 Do
Q 
q
35.250 91.152 537.000 17.905 re W n
Q
Q
q
15.000 91.152 577.500 685.848 re W n
0.965 0.965 0.965 rg
26.250 712.873 555.000 64.127 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 712.873 m 581.250 712.873 l 581.250 713.623 l 26.250 713.623 l  f
q
35.250 724.123 537.000 52.877 re W n
0.271 0.267 0.267 rg
BT 35.250 767.476 Td /F4 9.8 Tf  [(Fig. 3: Analysis of muscle inflammation using optical imaging of cathepsin)] TJ ET
BT 35.250 748.106 Td /F1 9.8 Tf  [(Cathepsin activity was measured from A\) forelimb and B\) hindlimb muscles as a quantification of inflammation. Activity is )] TJ ET
BT 35.250 734.370 Td /F1 9.8 Tf  [(defined in terms of photon counts/mm2. ** P ? 0.01)] TJ ET
Q
BT 26.250 695.849 Td /F4 9.8 Tf  [(Effect on measured histological parameters)] TJ ET
BT 26.250 676.444 Td /F1 9.8 Tf  [(Hematoxylin and eosin \(H&E\) stained sections from the TA of each animal were analyzed in blinded fashion. Dantrolene )] TJ ET
BT 26.250 664.539 Td /F1 9.8 Tf  [(treatment did not significantly alter any of these parameters \(fibers/field, central nuclei/fiber, peripheral nuclei/fiber, total )] TJ ET
BT 26.250 652.635 Td /F1 9.8 Tf  [(nuclei/fiber, fibers with central nuclei/field, ratio of central to peripheral nuclei/field, regenerating fibers/field, percentage of )] TJ ET
BT 26.250 640.730 Td /F1 9.8 Tf  [(central nucleated fibers and inflammatory foci\) compared to the vehicle-treated mdx mice \(Table 2\).)] TJ ET
q
26.250 617.113 555.000 13.736 re W n
0.271 0.267 0.267 rg
BT 26.250 619.860 Td /F1 9.8 Tf  [(Table 2: Comparison of histological parameters in TA muscle)] TJ ET
Q
0.965 0.965 0.965 rg
26.250 577.048 555.000 32.565 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 609.613 m 581.250 609.613 l 580.500 608.863 l 27.000 608.863 l  f
581.250 609.613 m 581.250 577.048 l 580.500 577.048 l 580.500 608.863 l  f
26.250 609.613 m 26.250 577.048 l 27.000 577.048 l 27.000 608.863 l  f
0.271 0.267 0.267 rg
BT 33.000 595.537 Td /F1 9.0 Tf  [(Analysis of muscle histology performed on TA muscle of each animal in a blinded fashion. Comparisons of ten standard parameters )] TJ ET
BT 33.000 586.379 Td /F1 9.0 Tf  [(yielded no significant change between the two mdx groups. N=8)] TJ ET
1.000 1.000 1.000 rg
26.250 416.735 555.000 160.313 re f
0.267 0.267 0.267 rg
26.625 575.923 231.886 0.750 re f
26.625 560.041 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 566.068 Td /F1 9.8 Tf  [(Measurement)] TJ ET
0.267 0.267 0.267 rg
257.761 575.923 119.242 0.750 re f
257.761 560.041 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 566.068 Td /F1 9.8 Tf  [(mdx Vehicle)] TJ ET
0.267 0.267 0.267 rg
376.254 575.923 128.437 0.750 re f
376.254 560.041 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 566.068 Td /F1 9.8 Tf  [(mdx Treatment)] TJ ET
0.267 0.267 0.267 rg
503.940 575.923 76.935 0.750 re f
503.940 560.041 0.750 16.631 re f
580.125 560.041 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 566.068 Td /F1 9.8 Tf  [(P-value)] TJ ET
0.267 0.267 0.267 rg
26.625 560.041 231.886 0.750 re f
26.625 544.160 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 550.186 Td /F1 9.8 Tf  [(Fibers/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 560.041 119.242 0.750 re f
257.761 544.160 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 550.186 Td /F1 9.8 Tf  [(179.45 � 4.57)] TJ ET
0.267 0.267 0.267 rg
376.254 560.041 128.437 0.750 re f
376.254 544.160 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 550.186 Td /F1 9.8 Tf  [(191.47 � 8.93)] TJ ET
0.267 0.267 0.267 rg
503.940 560.041 76.935 0.750 re f
503.940 544.160 0.750 16.631 re f
580.125 544.160 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 550.186 Td /F1 9.8 Tf  [(0.2675)] TJ ET
0.267 0.267 0.267 rg
26.625 544.160 231.886 0.750 re f
26.625 528.279 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 534.305 Td /F1 9.8 Tf  [(Central Nuclei/Fiber)] TJ ET
0.267 0.267 0.267 rg
257.761 544.160 119.242 0.750 re f
257.761 528.279 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 534.305 Td /F1 9.8 Tf  [(0.713 � 0.03)] TJ ET
0.267 0.267 0.267 rg
376.254 544.160 128.437 0.750 re f
376.254 528.279 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 534.305 Td /F1 9.8 Tf  [(0.693 � 0.03)] TJ ET
0.267 0.267 0.267 rg
503.940 544.160 76.935 0.750 re f
503.940 528.279 0.750 16.631 re f
580.125 528.279 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 534.305 Td /F1 9.8 Tf  [(0.6490)] TJ ET
0.267 0.267 0.267 rg
26.625 528.279 231.886 0.750 re f
26.625 512.398 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 518.424 Td /F1 9.8 Tf  [(Peripheral Nuclei/Fiber)] TJ ET
0.267 0.267 0.267 rg
257.761 528.279 119.242 0.750 re f
257.761 512.398 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 518.424 Td /F1 9.8 Tf  [(0.717 � 0.03)] TJ ET
0.267 0.267 0.267 rg
376.254 528.279 128.437 0.750 re f
376.254 512.398 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 518.424 Td /F1 9.8 Tf  [(0.702 � 0.03)] TJ ET
0.267 0.267 0.267 rg
503.940 528.279 76.935 0.750 re f
503.940 512.398 0.750 16.631 re f
580.125 512.398 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 518.424 Td /F1 9.8 Tf  [(0.7311)] TJ ET
0.267 0.267 0.267 rg
26.625 512.398 231.886 0.750 re f
26.625 496.516 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 502.543 Td /F1 9.8 Tf  [(Total Nuclei/Fiber)] TJ ET
0.267 0.267 0.267 rg
257.761 512.398 119.242 0.750 re f
257.761 496.516 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 502.543 Td /F1 9.8 Tf  [(1.430 � 0.06)] TJ ET
0.267 0.267 0.267 rg
376.254 512.398 128.437 0.750 re f
376.254 496.516 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 502.543 Td /F1 9.8 Tf  [(1.396 � 0.05)] TJ ET
0.267 0.267 0.267 rg
503.940 512.398 76.935 0.750 re f
503.940 496.516 0.750 16.631 re f
580.125 496.516 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 502.543 Td /F1 9.8 Tf  [(0.6541)] TJ ET
0.267 0.267 0.267 rg
26.625 496.516 231.886 0.750 re f
26.625 480.635 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 486.661 Td /F1 9.8 Tf  [(Fibers with Central Nuclei/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 496.516 119.242 0.750 re f
257.761 480.635 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 486.661 Td /F1 9.8 Tf  [(98.125 � 3.28)] TJ ET
0.267 0.267 0.267 rg
376.254 496.516 128.437 0.750 re f
376.254 480.635 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 486.661 Td /F1 9.8 Tf  [(104.133 � 4.93)] TJ ET
0.267 0.267 0.267 rg
503.940 496.516 76.935 0.750 re f
503.940 480.635 0.750 16.631 re f
580.125 480.635 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 486.661 Td /F1 9.8 Tf  [(0.3392)] TJ ET
0.267 0.267 0.267 rg
26.625 480.635 231.886 0.750 re f
26.625 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 470.780 Td /F1 9.8 Tf  [(Ratio of Central to Peripheral Nuclei/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 480.635 119.242 0.750 re f
257.761 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 470.780 Td /F1 9.8 Tf  [(0.00559 � 0.0003)] TJ ET
0.267 0.267 0.267 rg
376.254 480.635 128.437 0.750 re f
376.254 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 470.780 Td /F1 9.8 Tf  [(0.00527 � 0.0003)] TJ ET
0.267 0.267 0.267 rg
503.940 480.635 76.935 0.750 re f
503.940 464.754 0.750 16.631 re f
580.125 464.754 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 470.780 Td /F1 9.8 Tf  [(0.4533)] TJ ET
0.267 0.267 0.267 rg
26.625 464.754 231.886 0.750 re f
26.625 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 454.899 Td /F1 9.8 Tf  [(Regenerating Fibers/Field)] TJ ET
0.267 0.267 0.267 rg
257.761 464.754 119.242 0.750 re f
257.761 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 454.899 Td /F1 9.8 Tf  [(2.625 � 0.78)] TJ ET
0.267 0.267 0.267 rg
376.254 464.754 128.437 0.750 re f
376.254 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 454.899 Td /F1 9.8 Tf  [(3.844 � 0.98)] TJ ET
0.267 0.267 0.267 rg
503.940 464.754 76.935 0.750 re f
503.940 448.873 0.750 16.631 re f
580.125 448.873 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 454.899 Td /F1 9.8 Tf  [(0.3544)] TJ ET
0.267 0.267 0.267 rg
26.625 448.873 231.886 0.750 re f
26.625 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 31.875 439.018 Td /F1 9.8 Tf  [(% of Central Nucleated Fibers)] TJ ET
0.267 0.267 0.267 rg
257.761 448.873 119.242 0.750 re f
257.761 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 439.018 Td /F1 9.8 Tf  [(71.28% � 3.33)] TJ ET
0.267 0.267 0.267 rg
376.254 448.873 128.437 0.750 re f
376.254 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 439.018 Td /F1 9.8 Tf  [(69.33% � 2.63)] TJ ET
0.267 0.267 0.267 rg
503.940 448.873 76.935 0.750 re f
503.940 432.991 0.750 16.631 re f
580.125 432.991 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 439.018 Td /F1 9.8 Tf  [(0.6489)] TJ ET
0.267 0.267 0.267 rg
26.625 432.991 231.886 0.750 re f
26.625 417.110 231.886 0.750 re f
26.625 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
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0.267 0.267 0.267 rg
257.761 432.991 119.242 0.750 re f
257.761 417.110 119.242 0.750 re f
257.761 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 263.011 423.136 Td /F1 9.8 Tf  [(0.675 � 0.12)] TJ ET
0.267 0.267 0.267 rg
376.254 432.991 128.437 0.750 re f
376.254 417.110 128.437 0.750 re f
376.254 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 381.504 423.136 Td /F1 9.8 Tf  [(1.133 � 0.51)] TJ ET
0.267 0.267 0.267 rg
503.940 432.991 76.935 0.750 re f
503.940 417.110 76.935 0.750 re f
503.940 417.110 0.750 16.631 re f
580.125 417.110 0.750 16.631 re f
0.271 0.267 0.267 rg
BT 509.190 423.136 Td /F1 9.8 Tf  [(0.4073)] TJ ET
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BT 67.960 362.211 Td /F5 9.8 Tf  [(in vitro)] TJ ET
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BT 35.250 734.370 Td /F1 9.8 Tf  [(contractions to measure how the muscle responds to damage from over lengthening, or C\) measuring the muscle�s ability to )] TJ ET
BT 35.250 720.634 Td /F1 9.8 Tf  [(resist and recover from fatigue. D\) Serum was collected at sacrifice via cardiac puncture and CK levels were measured. *** )] TJ ET
BT 35.250 706.897 Td /F1 9.8 Tf  [(P ? 0.001, **** P ? 0.0001. N=8)] TJ ET
Q
BT 26.250 635.472 Td /F1 9.8 Tf  [(Effect on serum creatine kinase levels)] TJ ET
BT 26.250 616.067 Td /F1 9.8 Tf  [(Serum collected at sacrifice was analyzed to assess creatine kinase levels. In untreated mdx mice, there was an 11.76 fold )] TJ ET
BT 26.250 604.162 Td /F1 9.8 Tf  [(increase in creatine kinase in serum collected from mice prior to sacrifice relative to wild type controls. Treatment with )] TJ ET
BT 26.250 592.257 Td /F1 9.8 Tf  [(Dantrolene resulted in a significant decrease \(76.42%\) in serum CK levels compared to untreated mdx mice \(Fig. 4D\).)] TJ ET
BT 26.250 572.853 Td /F4 9.8 Tf  [(Effect on SERCA1 protein levels in muscle)] TJ ET
BT 26.250 553.448 Td /F1 9.8 Tf  [(To evaluate whether Dantrolene treatment altered SERCA1 expression, we did western blotting and densitometry normalized to )] TJ ET
BT 26.250 541.543 Td /F1 9.8 Tf  [(?-actin. This was performed on protein extracted from frozen gastrocnemius muscle. Dantrolene-treated mice showed a )] TJ ET
BT 26.250 529.638 Td /F1 9.8 Tf  [(significant decrease \(33%\) in SERCA1 levels compared to vehicle-treated mdx mice \(Fig. 5\).)] TJ ET
0.965 0.965 0.965 rg
26.250 283.880 555.000 235.877 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 519.757 m 581.250 519.757 l 581.250 519.007 l 26.250 519.007 l  f
26.250 283.880 m 581.250 283.880 l 581.250 284.630 l 26.250 284.630 l  f
q
225.000 0 0 156.000 35.250 354.007 cm /I7 Do
Q 
q
35.250 295.130 537.000 52.877 re W n
0.271 0.267 0.267 rg
BT 35.250 338.484 Td /F4 9.8 Tf  [(Fig. 5: Western blot and densitometry analysis of SERCA1)] TJ ET
BT 35.250 319.114 Td /F1 9.8 Tf  [(Western blot and densitometry analysis performed on protein extracted from gastrocnemius muscle to measure SERCA1 )] TJ ET
BT 35.250 305.377 Td /F1 9.8 Tf  [(protein levels in skeletal muscle. * P ? 0.05. N=4)] TJ ET
Q
BT 26.250 266.856 Td /F4 9.8 Tf  [(Effect on RyR1 gene expression levels in muscle)] TJ ET
BT 26.250 247.452 Td /F1 9.8 Tf  [(In order to determine the effect of Dantrolene treatment on Ryr1 in skeletal muscle, qPCR to measure RyR1 levels was )] TJ ET
BT 26.250 235.547 Td /F1 9.8 Tf  [(performed. Ryr1 gene expression was normalized to HPRT. We did not find differences in RyR1 expression between the two )] TJ ET
BT 26.250 223.642 Td /F1 9.8 Tf  [(mdx mouse groups \(data not shown\).)] TJ ET
BT 26.250 187.040 Td /F4 12.0 Tf  [(Discussion)] TJ ET
BT 26.250 167.085 Td /F1 9.8 Tf  [(In this study we demonstrated that Dantrolene treatment has no significant beneficial effects at the tested dose in young mdx )] TJ ET
BT 26.250 155.181 Td /F1 9.8 Tf  [(mice. Dantrolene failed to improve the muscle phenotype as measured by functional, behavioral, histological, and imaging )] TJ ET
BT 26.250 143.276 Td /F1 9.8 Tf  [(assays. The exception being CK levels, which were significantly decreased by Dantrolene treatment.)] TJ ET
BT 26.250 123.871 Td /F1 9.8 Tf  [(Dantrolene is a known muscle relaxant and used for the treatment of malignant hyperthermia )] TJ ET
0.267 0.267 0.267 rg
BT 428.886 125.378 Td /F4 8.7 Tf  [(11)] TJ ET
0.271 0.267 0.267 rg
BT 438.523 123.871 Td /F1 9.8 Tf  [(. It decreases the calcium )] TJ ET
BT 26.250 111.966 Td /F1 9.8 Tf  [(released in skeletal muscle upon stimulation, resulting in decreased muscle contraction )] TJ ET
0.267 0.267 0.267 rg
BT 404.482 113.474 Td /F4 8.7 Tf  [(19)] TJ ET
0.271 0.267 0.267 rg
BT 414.119 111.966 Td /F1 9.8 Tf  [(. In fact, functional assessments such )] TJ ET
BT 26.250 100.062 Td /F1 9.8 Tf  [(as grip strength and open field activity were decreased in Dantrolene-treated mice compared to their vehicle-treated )] TJ ET
BT 26.250 88.157 Td /F1 9.8 Tf  [(counterparts. This is likely a result of the muscle relaxant properties of the drug. This further indicates that the treatment does )] TJ ET
BT 26.250 76.252 Td /F1 9.8 Tf  [(not improve muscle function or strength. To minimize variation in the functional and behavioral assays, only female mice were )] TJ ET
BT 26.250 64.347 Td /F1 9.8 Tf  [(used because there are clear gender differences in mdx muscle pathology between sexes )] TJ ET
0.267 0.267 0.267 rg
BT 416.435 65.855 Td /F4 8.7 Tf  [(21)] TJ ET
0.271 0.267 0.267 rg
BT 426.073 64.347 Td /F1 9.8 Tf  [( and to reduce variability others )] TJ ET
BT 26.250 52.443 Td /F1 9.8 Tf  [(and we have suggested that pre-clinical tests should be performed on groups of mdx mice of the same sex )] TJ ET
0.267 0.267 0.267 rg
BT 488.507 53.950 Td /F4 8.7 Tf  [(22)] TJ ET
0.271 0.267 0.267 rg
BT 498.145 52.443 Td /F1 9.8 Tf  [(.)] TJ ET
Q
q
15.000 42.562 577.500 734.438 re W n
0.965 0.965 0.965 rg
26.250 652.495 555.000 124.505 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 652.495 m 581.250 652.495 l 581.250 653.245 l 26.250 653.245 l  f
q
35.250 663.745 537.000 113.255 re W n
0.271 0.267 0.267 rg
BT 35.250 767.476 Td /F4 9.8 Tf  [(Fig. 4: In vitro force assessment performed on the EDL muscle)] TJ ET
BT 35.250 748.106 Td /F1 9.8 Tf  [(No significant difference was observed in any of these measurements including A\) specific muscle force, B\) lengthening )] TJ ET
BT 35.250 734.370 Td /F1 9.8 Tf  [(contractions to measure how the muscle responds to damage from over lengthening, or C\) measuring the muscle�s ability to )] TJ ET
BT 35.250 720.634 Td /F1 9.8 Tf  [(resist and recover from fatigue. D\) Serum was collected at sacrifice via cardiac puncture and CK levels were measured. *** )] TJ ET
BT 35.250 706.897 Td /F1 9.8 Tf  [(P ? 0.001, **** P ? 0.0001. N=8)] TJ ET
Q
BT 26.250 635.472 Td /F1 9.8 Tf  [(Effect on serum creatine kinase levels)] TJ ET
BT 26.250 616.067 Td /F1 9.8 Tf  [(Serum collected at sacrifice was analyzed to assess creatine kinase levels. In untreated mdx mice, there was an 11.76 fold )] TJ ET
BT 26.250 604.162 Td /F1 9.8 Tf  [(increase in creatine kinase in serum collected from mice prior to sacrifice relative to wild type controls. Treatment with )] TJ ET
BT 26.250 592.257 Td /F1 9.8 Tf  [(Dantrolene resulted in a significant decrease \(76.42%\) in serum CK levels compared to untreated mdx mice \(Fig. 4D\).)] TJ ET
BT 26.250 572.853 Td /F4 9.8 Tf  [(Effect on SERCA1 protein levels in muscle)] TJ ET
BT 26.250 553.448 Td /F1 9.8 Tf  [(To evaluate whether Dantrolene treatment altered SERCA1 expression, we did western blotting and densitometry normalized to )] TJ ET
BT 26.250 541.543 Td /F1 9.8 Tf  [(?-actin. This was performed on protein extracted from frozen gastrocnemius muscle. Dantrolene-treated mice showed a )] TJ ET
BT 26.250 529.638 Td /F1 9.8 Tf  [(significant decrease \(33%\) in SERCA1 levels compared to vehicle-treated mdx mice \(Fig. 5\).)] TJ ET
0.965 0.965 0.965 rg
26.250 283.880 555.000 235.877 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 519.757 m 581.250 519.757 l 581.250 519.007 l 26.250 519.007 l  f
26.250 283.880 m 581.250 283.880 l 581.250 284.630 l 26.250 284.630 l  f
q
225.000 0 0 156.000 35.250 354.007 cm /I7 Do
Q 
q
35.250 295.130 537.000 52.877 re W n
0.271 0.267 0.267 rg
BT 35.250 338.484 Td /F4 9.8 Tf  [(Fig. 5: Western blot and densitometry analysis of SERCA1)] TJ ET
BT 35.250 319.114 Td /F1 9.8 Tf  [(Western blot and densitometry analysis performed on protein extracted from gastrocnemius muscle to measure SERCA1 )] TJ ET
BT 35.250 305.377 Td /F1 9.8 Tf  [(protein levels in skeletal muscle. * P ? 0.05. N=4)] TJ ET
Q
BT 26.250 266.856 Td /F4 9.8 Tf  [(Effect on RyR1 gene expression levels in muscle)] TJ ET
BT 26.250 247.452 Td /F1 9.8 Tf  [(In order to determine the effect of Dantrolene treatment on Ryr1 in skeletal muscle, qPCR to measure RyR1 levels was )] TJ ET
BT 26.250 235.547 Td /F1 9.8 Tf  [(performed. Ryr1 gene expression was normalized to HPRT. We did not find differences in RyR1 expression between the two )] TJ ET
BT 26.250 223.642 Td /F1 9.8 Tf  [(mdx mouse groups \(data not shown\).)] TJ ET
BT 26.250 187.040 Td /F4 12.0 Tf  [(Discussion)] TJ ET
BT 26.250 167.085 Td /F1 9.8 Tf  [(In this study we demonstrated that Dantrolene treatment has no significant beneficial effects at the tested dose in young mdx )] TJ ET
BT 26.250 155.181 Td /F1 9.8 Tf  [(mice. Dantrolene failed to improve the muscle phenotype as measured by functional, behavioral, histological, and imaging )] TJ ET
BT 26.250 143.276 Td /F1 9.8 Tf  [(assays. The exception being CK levels, which were significantly decreased by Dantrolene treatment.)] TJ ET
BT 26.250 123.871 Td /F1 9.8 Tf  [(Dantrolene is a known muscle relaxant and used for the treatment of malignant hyperthermia )] TJ ET
0.267 0.267 0.267 rg
BT 428.886 125.378 Td /F4 8.7 Tf  [(11)] TJ ET
0.271 0.267 0.267 rg
BT 438.523 123.871 Td /F1 9.8 Tf  [(. It decreases the calcium )] TJ ET
BT 26.250 111.966 Td /F1 9.8 Tf  [(released in skeletal muscle upon stimulation, resulting in decreased muscle contraction )] TJ ET
0.267 0.267 0.267 rg
BT 404.482 113.474 Td /F4 8.7 Tf  [(19)] TJ ET
0.271 0.267 0.267 rg
BT 414.119 111.966 Td /F1 9.8 Tf  [(. In fact, functional assessments such )] TJ ET
BT 26.250 100.062 Td /F1 9.8 Tf  [(as grip strength and open field activity were decreased in Dantrolene-treated mice compared to their vehicle-treated )] TJ ET
BT 26.250 88.157 Td /F1 9.8 Tf  [(counterparts. This is likely a result of the muscle relaxant properties of the drug. This further indicates that the treatment does )] TJ ET
BT 26.250 76.252 Td /F1 9.8 Tf  [(not improve muscle function or strength. To minimize variation in the functional and behavioral assays, only female mice were )] TJ ET
BT 26.250 64.347 Td /F1 9.8 Tf  [(used because there are clear gender differences in mdx muscle pathology between sexes )] TJ ET
0.267 0.267 0.267 rg
BT 416.435 65.855 Td /F4 8.7 Tf  [(21)] TJ ET
0.271 0.267 0.267 rg
BT 426.073 64.347 Td /F1 9.8 Tf  [( and to reduce variability others )] TJ ET
BT 26.250 52.443 Td /F1 9.8 Tf  [(and we have suggested that pre-clinical tests should be performed on groups of mdx mice of the same sex )] TJ ET
0.267 0.267 0.267 rg
BT 488.507 53.950 Td /F4 8.7 Tf  [(22)] TJ ET
0.271 0.267 0.267 rg
BT 498.145 52.443 Td /F1 9.8 Tf  [(.)] TJ ET
Q
q
15.000 42.562 577.500 734.438 re W n
0.965 0.965 0.965 rg
26.250 652.495 555.000 124.505 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 652.495 m 581.250 652.495 l 581.250 653.245 l 26.250 653.245 l  f
q
35.250 663.745 537.000 113.255 re W n
0.271 0.267 0.267 rg
BT 35.250 767.476 Td /F4 9.8 Tf  [(Fig. 4: In vitro force assessment performed on the EDL muscle)] TJ ET
BT 35.250 748.106 Td /F1 9.8 Tf  [(No significant difference was observed in any of these measurements including A\) specific muscle force, B\) lengthening )] TJ ET
BT 35.250 734.370 Td /F1 9.8 Tf  [(contractions to measure how the muscle responds to damage from over lengthening, or C\) measuring the muscle�s ability to )] TJ ET
BT 35.250 720.634 Td /F1 9.8 Tf  [(resist and recover from fatigue. D\) Serum was collected at sacrifice via cardiac puncture and CK levels were measured. *** )] TJ ET
BT 35.250 706.897 Td /F1 9.8 Tf  [(P ? 0.001, **** P ? 0.0001. N=8)] TJ ET
Q
BT 26.250 635.472 Td /F1 9.8 Tf  [(Effect on serum creatine kinase levels)] TJ ET
BT 26.250 616.067 Td /F1 9.8 Tf  [(Serum collected at sacrifice was analyzed to assess creatine kinase levels. In untreated mdx mice, there was an 11.76 fold )] TJ ET
BT 26.250 604.162 Td /F1 9.8 Tf  [(increase in creatine kinase in serum collected from mice prior to sacrifice relative to wild type controls. Treatment with )] TJ ET
BT 26.250 592.257 Td /F1 9.8 Tf  [(Dantrolene resulted in a significant decrease \(76.42%\) in serum CK levels compared to untreated mdx mice \(Fig. 4D\).)] TJ ET
BT 26.250 572.853 Td /F4 9.8 Tf  [(Effect on SERCA1 protein levels in muscle)] TJ ET
BT 26.250 553.448 Td /F1 9.8 Tf  [(To evaluate whether Dantrolene treatment altered SERCA1 expression, we did western blotting and densitometry normalized to )] TJ ET
BT 26.250 541.543 Td /F1 9.8 Tf  [(?-actin. This was performed on protein extracted from frozen gastrocnemius muscle. Dantrolene-treated mice showed a )] TJ ET
BT 26.250 529.638 Td /F1 9.8 Tf  [(significant decrease \(33%\) in SERCA1 levels compared to vehicle-treated mdx mice \(Fig. 5\).)] TJ ET
0.965 0.965 0.965 rg
26.250 283.880 555.000 235.877 re f
0.267 0.267 0.267 rg
0.267 0.267 0.267 RG
26.250 519.757 m 581.250 519.757 l 581.250 519.007 l 26.250 519.007 l  f
26.250 283.880 m 581.250 283.880 l 581.250 284.630 l 26.250 284.630 l  f
q
225.000 0 0 156.000 35.250 354.007 cm /I7 Do
Q 
q
35.250 295.130 537.000 52.877 re W n
0.271 0.267 0.267 rg
BT 35.250 338.484 Td /F4 9.8 Tf  [(Fig. 5: Western blot and densitometry analysis of SERCA1)] TJ ET
BT 35.250 319.114 Td /F1 9.8 Tf  [(Western blot and densitometry analysis performed on protein extracted from gastrocnemius muscle to measure SERCA1 )] TJ ET
BT 35.250 305.377 Td /F1 9.8 Tf  [(protein levels in skeletal muscle. * P ? 0.05. N=4)] TJ ET
Q
BT 26.250 266.856 Td /F4 9.8 Tf  [(Effect on RyR1 gene expression levels in muscle)] TJ ET
BT 26.250 247.452 Td /F1 9.8 Tf  [(In order to determine the effect of Dantrolene treatment on Ryr1 in skeletal muscle, qPCR to measure RyR1 levels was )] TJ ET
BT 26.250 235.547 Td /F1 9.8 Tf  [(performed. Ryr1 gene expression was normalized to HPRT. We did not find differences in RyR1 expression between the two )] TJ ET
BT 26.250 223.642 Td /F1 9.8 Tf  [(mdx mouse groups \(data not shown\).)] TJ ET
BT 26.250 187.040 Td /F4 12.0 Tf  [(Discussion)] TJ ET
BT 26.250 167.085 Td /F1 9.8 Tf  [(In this study we demonstrated that Dantrolene treatment has no significant beneficial effects at the tested dose in young mdx )] TJ ET
BT 26.250 155.181 Td /F1 9.8 Tf  [(mice. Dantrolene failed to improve the muscle phenotype as measured by functional, behavioral, histological, and imaging )] TJ ET
BT 26.250 143.276 Td /F1 9.8 Tf  [(assays. The exception being CK levels, which were significantly decreased by Dantrolene treatment.)] TJ ET
BT 26.250 123.871 Td /F1 9.8 Tf  [(Dantrolene is a known muscle relaxant and used for the treatment of malignant hyperthermia )] TJ ET
0.267 0.267 0.267 rg
BT 428.886 125.378 Td /F4 8.7 Tf  [(11)] TJ ET
0.271 0.267 0.267 rg
BT 438.523 123.871 Td /F1 9.8 Tf  [(. It decreases the calcium )] TJ ET
BT 26.250 111.966 Td /F1 9.8 Tf  [(released in skeletal muscle upon stimulation, resulting in decreased muscle contraction )] TJ ET
0.267 0.267 0.267 rg
BT 404.482 113.474 Td /F4 8.7 Tf  [(19)] TJ ET
0.271 0.267 0.267 rg
BT 414.119 111.966 Td /F1 9.8 Tf  [(. In fact, functional assessments such )] TJ ET
BT 26.250 100.062 Td /F1 9.8 Tf  [(as grip strength and open field activity were decreased in Dantrolene-treated mice compared to their vehicle-treated )] TJ ET
BT 26.250 88.157 Td /F1 9.8 Tf  [(counterparts. This is likely a result of the muscle relaxant properties of the drug. This further indicates that the treatment does )] TJ ET
BT 26.250 76.252 Td /F1 9.8 Tf  [(not improve muscle function or strength. To minimize variation in the functional and behavioral assays, only female mice were )] TJ ET
BT 26.250 64.347 Td /F1 9.8 Tf  [(used because there are clear gender differences in mdx muscle pathology between sexes )] TJ ET
0.267 0.267 0.267 rg
BT 416.435 65.855 Td /F4 8.7 Tf  [(21)] TJ ET
0.271 0.267 0.267 rg
BT 426.073 64.347 Td /F1 9.8 Tf  [( and to reduce variability others )] TJ ET
BT 26.250 52.443 Td /F1 9.8 Tf  [(and we have suggested that pre-clinical tests should be performed on groups of mdx mice of the same sex )] TJ ET
0.267 0.267 0.267 rg
BT 488.507 53.950 Td /F4 8.7 Tf  [(22)] TJ ET
0.271 0.267 0.267 rg
BT 498.145 52.443 Td /F1 9.8 Tf  [(.)] TJ ET
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BT 291.710 19.825 Td /F1 11.0 Tf  [(6)] TJ ET
BT 25.000 19.825 Td /F1 11.0 Tf  [(PLOS Currents Muscular Dystrophy)] TJ ET

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BT 26.250 767.476 Td /F1 9.8 Tf  [(It is clear from the results that Dantrolene did not reduce inflammation in mdx mice. Cathepsin B activity \(macrophage/muscle\) )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(as measured by optical imaging is unaltered in the two mdx mouse groups. These findings were confirmed by analysis of H&E )] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(sections that also indicate no change in the histological profile, including inflammation, between the two groups.)] TJ ET
BT 26.250 724.262 Td /F1 9.8 Tf  [(Even though it has been shown previously that RyR1 is leaky in dystrophic muscle and contributes to the disease phenotype, )] TJ ET
BT 26.250 712.357 Td /F1 9.8 Tf  [(and drugs that stabilize RyR1 are known to be effective in muscular dystrophy )] TJ ET
0.267 0.267 0.267 rg
BT 364.936 713.864 Td /F4 8.7 Tf  [(23)] TJ ET
0.271 0.267 0.267 rg
BT 374.573 712.357 Td /F1 9.8 Tf  [(, we could not see a similar effect with )] TJ ET
BT 26.250 700.452 Td /F1 9.8 Tf  [(Dantrolene in this experiment. The reasons for this discrepancy are not clear, but could be due to the muscle relaxant properties )] TJ ET
BT 26.250 688.548 Td /F1 9.8 Tf  [(of the drug.)] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(Dantrolene has previously been shown to enhance exon skipping in mdx mice but not improve muscle strength on its own )] TJ ET
0.267 0.267 0.267 rg
BT 552.984 670.650 Td /F4 8.7 Tf  [(24)] TJ ET
0.271 0.267 0.267 rg
BT 562.621 669.143 Td /F1 9.8 Tf  [(. )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(Our study confirms their findings despite using a higher dose of the drug to treat older mdx mice for a longer period of time \(8 )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(weeks\). In addition, we conducted additional functional assays to determine the effect of the drug.)] TJ ET
BT 26.250 625.929 Td /F1 9.8 Tf  [(Lack of efficacy in our study could be a result of beginning treatment too late in the disease progression, at a point where )] TJ ET
BT 26.250 614.024 Td /F1 9.8 Tf  [(significant damage had already occurred in the muscle fibers. Previous studies have demonstrated that calpains, calcium-)] TJ ET
BT 26.250 602.119 Td /F1 9.8 Tf  [(dependent cytosine proteases, are present at elevated levels in mdx muscle by 4 weeks of age )] TJ ET
0.267 0.267 0.267 rg
BT 438.119 603.626 Td /F4 8.7 Tf  [(25)] TJ ET
0.271 0.267 0.267 rg
BT 447.757 602.119 Td /F1 9.8 Tf  [(. This means that calcium-)] TJ ET
BT 26.250 590.214 Td /F1 9.8 Tf  [(induced necrosis had already initiated by the time we started treatment. It is unlikely that improved calcium handling in )] TJ ET
BT 26.250 578.310 Td /F1 9.8 Tf  [(previously damaged fibers would reverse the damage, but only slow further deterioration. Additionally, it is quite possible that a )] TJ ET
BT 26.250 566.405 Td /F1 9.8 Tf  [(longer duration of treatment is necessary to observe any benefit from the drug in mdx mice.)] TJ ET
BT 26.250 547.000 Td /F1 9.8 Tf  [(Our analysis of the serum CK levels did show a significant decrease in the amount of CK in the blood. This trend is consistent )] TJ ET
BT 26.250 535.095 Td /F1 9.8 Tf  [(with previously shown data )] TJ ET
0.267 0.267 0.267 rg
BT 145.463 536.603 Td /F4 8.7 Tf  [(26)] TJ ET
0.271 0.267 0.267 rg
BT 155.101 535.095 Td /F1 9.8 Tf  [(. Based on the histology and functional assays, it is unlikely that this change is based on improved )] TJ ET
BT 26.250 523.191 Td /F1 9.8 Tf  [(muscle health. Instead, it is highly likely that this decrease in serum CK is a result of decreased activity. As a muscle relaxant, )] TJ ET
BT 26.250 511.286 Td /F1 9.8 Tf  [(Dantrolene slowed down the breathing rate and reduced activity in the mice )] TJ ET
0.267 0.267 0.267 rg
BT 354.113 512.793 Td /F4 8.7 Tf  [(19)] TJ ET
0.271 0.267 0.267 rg
BT 363.751 511.286 Td /F1 9.8 Tf  [(, reducing CK levels in the blood.)] TJ ET
BT 26.250 491.881 Td /F1 9.8 Tf  [(To determine whether Dantrolene target, RyR1, was engaged, we evaluated a sarco/endoplasmic reticulum Ca)] TJ ET
BT 504.741 495.769 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 514.621 491.881 Td /F1 9.8 Tf  [(-ATPase )] TJ ET
BT 26.250 479.976 Td /F1 9.8 Tf  [(\(SERCA1\). This calcium ATPase is found on the SR membrane in muscle fibers, and is responsible for pumping calcium out of )] TJ ET
BT 26.250 468.072 Td /F1 9.8 Tf  [(the sarcoplasm and back into the SR. We expected that Dantrolene treatment and the resulting intracellular calcium decrease )] TJ ET
BT 26.250 456.167 Td /F1 9.8 Tf  [(would result in an increase in SERCA1 levels in muscle fibers based on previous research with rat hearts )] TJ ET
0.267 0.267 0.267 rg
BT 481.419 457.674 Td /F4 8.7 Tf  [(27)] TJ ET
0.271 0.267 0.267 rg
BT 491.056 456.167 Td /F1 9.8 Tf  [(. We performed )] TJ ET
BT 26.250 444.262 Td /F1 9.8 Tf  [(Western blotting on gastrocnemius muscles and found a 33% decrease in SERCA1 protein levels compared to mdx vehicle-)] TJ ET
BT 26.250 432.357 Td /F1 9.8 Tf  [(treated mice. We suspect this difference in effect is likely due to the different muscle type, dosage, duration, and model. This )] TJ ET
BT 26.250 420.453 Td /F1 9.8 Tf  [(assay however, establishes that Dantrolene did affect calcium handling within mdx muscle fibers but it did not result in an )] TJ ET
BT 26.250 408.548 Td /F1 9.8 Tf  [(improved disease phenotype.)] TJ ET
BT 26.250 389.143 Td /F1 9.8 Tf  [(Additional assays that we performed on isolated EDL muscles indicate that not only strength, but also muscle recovery during )] TJ ET
BT 26.250 377.238 Td /F6 9.8 Tf  [(in vitro )] TJ ET
BT 57.674 377.238 Td /F1 9.8 Tf  [(fatigue and lengthening contraction protocols demonstrated no effect. Additionally, the histological scoring showed no )] TJ ET
BT 26.250 365.334 Td /F1 9.8 Tf  [(significant change in any of the eight measured parameters, indicating existing damage of the fibers was not altered. Our data )] TJ ET
BT 26.250 353.429 Td /F1 9.8 Tf  [(suggests that during early necrosis when there is cycles of damage, regeneration, and re-injury; administration of Dantrolene is )] TJ ET
BT 26.250 341.524 Td /F1 9.8 Tf  [(ineffective. Therefore, we conclude that Dantrolene alone at this dose does not improve the overall phenotype of the mdx )] TJ ET
BT 26.250 329.619 Td /F1 9.8 Tf  [(mouse.)] TJ ET
BT 26.250 293.017 Td /F4 12.0 Tf  [(Correspondence)] TJ ET
BT 26.250 273.063 Td /F1 9.8 Tf  [(Corresponding author:)] TJ ET
BT 26.250 253.658 Td /F1 9.8 Tf  [(Kanneboyina Nagaraju, DVM, Ph.D.)] TJ ET
BT 26.250 234.253 Td /F1 9.8 Tf  [(Research Center for Genetic Medicine)] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(Children�s National Medical Center)] TJ ET
BT 26.250 195.444 Td /F1 9.8 Tf  [(111 Michigan Ave. NW)] TJ ET
BT 26.250 176.039 Td /F1 9.8 Tf  [(Washington DC 20010)] TJ ET
BT 26.250 156.634 Td /F1 9.8 Tf  [(Email: knagaraju@cnmcresearch.org)] TJ ET
BT 26.250 137.229 Td /F1 9.8 Tf  [(Phone \(202\)476-6220)] TJ ET
BT 26.250 100.627 Td /F4 12.0 Tf  [(Competing Interest Statement)] TJ ET
BT 26.250 80.673 Td /F1 9.8 Tf  [(The authors have declared that no competing interests exist.)] TJ ET
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0.271 0.267 0.267 rg
BT 26.250 767.476 Td /F1 9.8 Tf  [(It is clear from the results that Dantrolene did not reduce inflammation in mdx mice. Cathepsin B activity \(macrophage/muscle\) )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(as measured by optical imaging is unaltered in the two mdx mouse groups. These findings were confirmed by analysis of H&E )] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(sections that also indicate no change in the histological profile, including inflammation, between the two groups.)] TJ ET
BT 26.250 724.262 Td /F1 9.8 Tf  [(Even though it has been shown previously that RyR1 is leaky in dystrophic muscle and contributes to the disease phenotype, )] TJ ET
BT 26.250 712.357 Td /F1 9.8 Tf  [(and drugs that stabilize RyR1 are known to be effective in muscular dystrophy )] TJ ET
0.267 0.267 0.267 rg
BT 364.936 713.864 Td /F4 8.7 Tf  [(23)] TJ ET
0.271 0.267 0.267 rg
BT 374.573 712.357 Td /F1 9.8 Tf  [(, we could not see a similar effect with )] TJ ET
BT 26.250 700.452 Td /F1 9.8 Tf  [(Dantrolene in this experiment. The reasons for this discrepancy are not clear, but could be due to the muscle relaxant properties )] TJ ET
BT 26.250 688.548 Td /F1 9.8 Tf  [(of the drug.)] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(Dantrolene has previously been shown to enhance exon skipping in mdx mice but not improve muscle strength on its own )] TJ ET
0.267 0.267 0.267 rg
BT 552.984 670.650 Td /F4 8.7 Tf  [(24)] TJ ET
0.271 0.267 0.267 rg
BT 562.621 669.143 Td /F1 9.8 Tf  [(. )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(Our study confirms their findings despite using a higher dose of the drug to treat older mdx mice for a longer period of time \(8 )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(weeks\). In addition, we conducted additional functional assays to determine the effect of the drug.)] TJ ET
BT 26.250 625.929 Td /F1 9.8 Tf  [(Lack of efficacy in our study could be a result of beginning treatment too late in the disease progression, at a point where )] TJ ET
BT 26.250 614.024 Td /F1 9.8 Tf  [(significant damage had already occurred in the muscle fibers. Previous studies have demonstrated that calpains, calcium-)] TJ ET
BT 26.250 602.119 Td /F1 9.8 Tf  [(dependent cytosine proteases, are present at elevated levels in mdx muscle by 4 weeks of age )] TJ ET
0.267 0.267 0.267 rg
BT 438.119 603.626 Td /F4 8.7 Tf  [(25)] TJ ET
0.271 0.267 0.267 rg
BT 447.757 602.119 Td /F1 9.8 Tf  [(. This means that calcium-)] TJ ET
BT 26.250 590.214 Td /F1 9.8 Tf  [(induced necrosis had already initiated by the time we started treatment. It is unlikely that improved calcium handling in )] TJ ET
BT 26.250 578.310 Td /F1 9.8 Tf  [(previously damaged fibers would reverse the damage, but only slow further deterioration. Additionally, it is quite possible that a )] TJ ET
BT 26.250 566.405 Td /F1 9.8 Tf  [(longer duration of treatment is necessary to observe any benefit from the drug in mdx mice.)] TJ ET
BT 26.250 547.000 Td /F1 9.8 Tf  [(Our analysis of the serum CK levels did show a significant decrease in the amount of CK in the blood. This trend is consistent )] TJ ET
BT 26.250 535.095 Td /F1 9.8 Tf  [(with previously shown data )] TJ ET
0.267 0.267 0.267 rg
BT 145.463 536.603 Td /F4 8.7 Tf  [(26)] TJ ET
0.271 0.267 0.267 rg
BT 155.101 535.095 Td /F1 9.8 Tf  [(. Based on the histology and functional assays, it is unlikely that this change is based on improved )] TJ ET
BT 26.250 523.191 Td /F1 9.8 Tf  [(muscle health. Instead, it is highly likely that this decrease in serum CK is a result of decreased activity. As a muscle relaxant, )] TJ ET
BT 26.250 511.286 Td /F1 9.8 Tf  [(Dantrolene slowed down the breathing rate and reduced activity in the mice )] TJ ET
0.267 0.267 0.267 rg
BT 354.113 512.793 Td /F4 8.7 Tf  [(19)] TJ ET
0.271 0.267 0.267 rg
BT 363.751 511.286 Td /F1 9.8 Tf  [(, reducing CK levels in the blood.)] TJ ET
BT 26.250 491.881 Td /F1 9.8 Tf  [(To determine whether Dantrolene target, RyR1, was engaged, we evaluated a sarco/endoplasmic reticulum Ca)] TJ ET
BT 504.741 495.769 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 514.621 491.881 Td /F1 9.8 Tf  [(-ATPase )] TJ ET
BT 26.250 479.976 Td /F1 9.8 Tf  [(\(SERCA1\). This calcium ATPase is found on the SR membrane in muscle fibers, and is responsible for pumping calcium out of )] TJ ET
BT 26.250 468.072 Td /F1 9.8 Tf  [(the sarcoplasm and back into the SR. We expected that Dantrolene treatment and the resulting intracellular calcium decrease )] TJ ET
BT 26.250 456.167 Td /F1 9.8 Tf  [(would result in an increase in SERCA1 levels in muscle fibers based on previous research with rat hearts )] TJ ET
0.267 0.267 0.267 rg
BT 481.419 457.674 Td /F4 8.7 Tf  [(27)] TJ ET
0.271 0.267 0.267 rg
BT 491.056 456.167 Td /F1 9.8 Tf  [(. We performed )] TJ ET
BT 26.250 444.262 Td /F1 9.8 Tf  [(Western blotting on gastrocnemius muscles and found a 33% decrease in SERCA1 protein levels compared to mdx vehicle-)] TJ ET
BT 26.250 432.357 Td /F1 9.8 Tf  [(treated mice. We suspect this difference in effect is likely due to the different muscle type, dosage, duration, and model. This )] TJ ET
BT 26.250 420.453 Td /F1 9.8 Tf  [(assay however, establishes that Dantrolene did affect calcium handling within mdx muscle fibers but it did not result in an )] TJ ET
BT 26.250 408.548 Td /F1 9.8 Tf  [(improved disease phenotype.)] TJ ET
BT 26.250 389.143 Td /F1 9.8 Tf  [(Additional assays that we performed on isolated EDL muscles indicate that not only strength, but also muscle recovery during )] TJ ET
BT 26.250 377.238 Td /F6 9.8 Tf  [(in vitro )] TJ ET
BT 57.674 377.238 Td /F1 9.8 Tf  [(fatigue and lengthening contraction protocols demonstrated no effect. Additionally, the histological scoring showed no )] TJ ET
BT 26.250 365.334 Td /F1 9.8 Tf  [(significant change in any of the eight measured parameters, indicating existing damage of the fibers was not altered. Our data )] TJ ET
BT 26.250 353.429 Td /F1 9.8 Tf  [(suggests that during early necrosis when there is cycles of damage, regeneration, and re-injury; administration of Dantrolene is )] TJ ET
BT 26.250 341.524 Td /F1 9.8 Tf  [(ineffective. Therefore, we conclude that Dantrolene alone at this dose does not improve the overall phenotype of the mdx )] TJ ET
BT 26.250 329.619 Td /F1 9.8 Tf  [(mouse.)] TJ ET
BT 26.250 293.017 Td /F4 12.0 Tf  [(Correspondence)] TJ ET
BT 26.250 273.063 Td /F1 9.8 Tf  [(Corresponding author:)] TJ ET
BT 26.250 253.658 Td /F1 9.8 Tf  [(Kanneboyina Nagaraju, DVM, Ph.D.)] TJ ET
BT 26.250 234.253 Td /F1 9.8 Tf  [(Research Center for Genetic Medicine)] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(Children�s National Medical Center)] TJ ET
BT 26.250 195.444 Td /F1 9.8 Tf  [(111 Michigan Ave. NW)] TJ ET
BT 26.250 176.039 Td /F1 9.8 Tf  [(Washington DC 20010)] TJ ET
BT 26.250 156.634 Td /F1 9.8 Tf  [(Email: knagaraju@cnmcresearch.org)] TJ ET
BT 26.250 137.229 Td /F1 9.8 Tf  [(Phone \(202\)476-6220)] TJ ET
BT 26.250 100.627 Td /F4 12.0 Tf  [(Competing Interest Statement)] TJ ET
BT 26.250 80.673 Td /F1 9.8 Tf  [(The authors have declared that no competing interests exist.)] TJ ET
Q
q
15.000 33.640 577.500 743.360 re W n
0.271 0.267 0.267 rg
BT 26.250 767.476 Td /F1 9.8 Tf  [(It is clear from the results that Dantrolene did not reduce inflammation in mdx mice. Cathepsin B activity \(macrophage/muscle\) )] TJ ET
BT 26.250 755.571 Td /F1 9.8 Tf  [(as measured by optical imaging is unaltered in the two mdx mouse groups. These findings were confirmed by analysis of H&E )] TJ ET
BT 26.250 743.667 Td /F1 9.8 Tf  [(sections that also indicate no change in the histological profile, including inflammation, between the two groups.)] TJ ET
BT 26.250 724.262 Td /F1 9.8 Tf  [(Even though it has been shown previously that RyR1 is leaky in dystrophic muscle and contributes to the disease phenotype, )] TJ ET
BT 26.250 712.357 Td /F1 9.8 Tf  [(and drugs that stabilize RyR1 are known to be effective in muscular dystrophy )] TJ ET
0.267 0.267 0.267 rg
BT 364.936 713.864 Td /F4 8.7 Tf  [(23)] TJ ET
0.271 0.267 0.267 rg
BT 374.573 712.357 Td /F1 9.8 Tf  [(, we could not see a similar effect with )] TJ ET
BT 26.250 700.452 Td /F1 9.8 Tf  [(Dantrolene in this experiment. The reasons for this discrepancy are not clear, but could be due to the muscle relaxant properties )] TJ ET
BT 26.250 688.548 Td /F1 9.8 Tf  [(of the drug.)] TJ ET
BT 26.250 669.143 Td /F1 9.8 Tf  [(Dantrolene has previously been shown to enhance exon skipping in mdx mice but not improve muscle strength on its own )] TJ ET
0.267 0.267 0.267 rg
BT 552.984 670.650 Td /F4 8.7 Tf  [(24)] TJ ET
0.271 0.267 0.267 rg
BT 562.621 669.143 Td /F1 9.8 Tf  [(. )] TJ ET
BT 26.250 657.238 Td /F1 9.8 Tf  [(Our study confirms their findings despite using a higher dose of the drug to treat older mdx mice for a longer period of time \(8 )] TJ ET
BT 26.250 645.333 Td /F1 9.8 Tf  [(weeks\). In addition, we conducted additional functional assays to determine the effect of the drug.)] TJ ET
BT 26.250 625.929 Td /F1 9.8 Tf  [(Lack of efficacy in our study could be a result of beginning treatment too late in the disease progression, at a point where )] TJ ET
BT 26.250 614.024 Td /F1 9.8 Tf  [(significant damage had already occurred in the muscle fibers. Previous studies have demonstrated that calpains, calcium-)] TJ ET
BT 26.250 602.119 Td /F1 9.8 Tf  [(dependent cytosine proteases, are present at elevated levels in mdx muscle by 4 weeks of age )] TJ ET
0.267 0.267 0.267 rg
BT 438.119 603.626 Td /F4 8.7 Tf  [(25)] TJ ET
0.271 0.267 0.267 rg
BT 447.757 602.119 Td /F1 9.8 Tf  [(. This means that calcium-)] TJ ET
BT 26.250 590.214 Td /F1 9.8 Tf  [(induced necrosis had already initiated by the time we started treatment. It is unlikely that improved calcium handling in )] TJ ET
BT 26.250 578.310 Td /F1 9.8 Tf  [(previously damaged fibers would reverse the damage, but only slow further deterioration. Additionally, it is quite possible that a )] TJ ET
BT 26.250 566.405 Td /F1 9.8 Tf  [(longer duration of treatment is necessary to observe any benefit from the drug in mdx mice.)] TJ ET
BT 26.250 547.000 Td /F1 9.8 Tf  [(Our analysis of the serum CK levels did show a significant decrease in the amount of CK in the blood. This trend is consistent )] TJ ET
BT 26.250 535.095 Td /F1 9.8 Tf  [(with previously shown data )] TJ ET
0.267 0.267 0.267 rg
BT 145.463 536.603 Td /F4 8.7 Tf  [(26)] TJ ET
0.271 0.267 0.267 rg
BT 155.101 535.095 Td /F1 9.8 Tf  [(. Based on the histology and functional assays, it is unlikely that this change is based on improved )] TJ ET
BT 26.250 523.191 Td /F1 9.8 Tf  [(muscle health. Instead, it is highly likely that this decrease in serum CK is a result of decreased activity. As a muscle relaxant, )] TJ ET
BT 26.250 511.286 Td /F1 9.8 Tf  [(Dantrolene slowed down the breathing rate and reduced activity in the mice )] TJ ET
0.267 0.267 0.267 rg
BT 354.113 512.793 Td /F4 8.7 Tf  [(19)] TJ ET
0.271 0.267 0.267 rg
BT 363.751 511.286 Td /F1 9.8 Tf  [(, reducing CK levels in the blood.)] TJ ET
BT 26.250 491.881 Td /F1 9.8 Tf  [(To determine whether Dantrolene target, RyR1, was engaged, we evaluated a sarco/endoplasmic reticulum Ca)] TJ ET
BT 504.741 495.769 Td /F1 8.7 Tf  [(2+)] TJ ET
BT 514.621 491.881 Td /F1 9.8 Tf  [(-ATPase )] TJ ET
BT 26.250 479.976 Td /F1 9.8 Tf  [(\(SERCA1\). This calcium ATPase is found on the SR membrane in muscle fibers, and is responsible for pumping calcium out of )] TJ ET
BT 26.250 468.072 Td /F1 9.8 Tf  [(the sarcoplasm and back into the SR. We expected that Dantrolene treatment and the resulting intracellular calcium decrease )] TJ ET
BT 26.250 456.167 Td /F1 9.8 Tf  [(would result in an increase in SERCA1 levels in muscle fibers based on previous research with rat hearts )] TJ ET
0.267 0.267 0.267 rg
BT 481.419 457.674 Td /F4 8.7 Tf  [(27)] TJ ET
0.271 0.267 0.267 rg
BT 491.056 456.167 Td /F1 9.8 Tf  [(. We performed )] TJ ET
BT 26.250 444.262 Td /F1 9.8 Tf  [(Western blotting on gastrocnemius muscles and found a 33% decrease in SERCA1 protein levels compared to mdx vehicle-)] TJ ET
BT 26.250 432.357 Td /F1 9.8 Tf  [(treated mice. We suspect this difference in effect is likely due to the different muscle type, dosage, duration, and model. This )] TJ ET
BT 26.250 420.453 Td /F1 9.8 Tf  [(assay however, establishes that Dantrolene did affect calcium handling within mdx muscle fibers but it did not result in an )] TJ ET
BT 26.250 408.548 Td /F1 9.8 Tf  [(improved disease phenotype.)] TJ ET
BT 26.250 389.143 Td /F1 9.8 Tf  [(Additional assays that we performed on isolated EDL muscles indicate that not only strength, but also muscle recovery during )] TJ ET
BT 26.250 377.238 Td /F6 9.8 Tf  [(in vitro )] TJ ET
BT 57.674 377.238 Td /F1 9.8 Tf  [(fatigue and lengthening contraction protocols demonstrated no effect. Additionally, the histological scoring showed no )] TJ ET
BT 26.250 365.334 Td /F1 9.8 Tf  [(significant change in any of the eight measured parameters, indicating existing damage of the fibers was not altered. Our data )] TJ ET
BT 26.250 353.429 Td /F1 9.8 Tf  [(suggests that during early necrosis when there is cycles of damage, regeneration, and re-injury; administration of Dantrolene is )] TJ ET
BT 26.250 341.524 Td /F1 9.8 Tf  [(ineffective. Therefore, we conclude that Dantrolene alone at this dose does not improve the overall phenotype of the mdx )] TJ ET
BT 26.250 329.619 Td /F1 9.8 Tf  [(mouse.)] TJ ET
BT 26.250 293.017 Td /F4 12.0 Tf  [(Correspondence)] TJ ET
BT 26.250 273.063 Td /F1 9.8 Tf  [(Corresponding author:)] TJ ET
BT 26.250 253.658 Td /F1 9.8 Tf  [(Kanneboyina Nagaraju, DVM, Ph.D.)] TJ ET
BT 26.250 234.253 Td /F1 9.8 Tf  [(Research Center for Genetic Medicine)] TJ ET
BT 26.250 214.848 Td /F1 9.8 Tf  [(Children�s National Medical Center)] TJ ET
BT 26.250 195.444 Td /F1 9.8 Tf  [(111 Michigan Ave. NW)] TJ ET
BT 26.250 176.039 Td /F1 9.8 Tf  [(Washington DC 20010)] TJ ET
BT 26.250 156.634 Td /F1 9.8 Tf  [(Email: knagaraju@cnmcresearch.org)] TJ ET
BT 26.250 137.229 Td /F1 9.8 Tf  [(Phone \(202\)476-6220)] TJ ET
BT 26.250 100.627 Td /F4 12.0 Tf  [(Competing Interest Statement)] TJ ET
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BT 26.250 330.801 Td /F1 9.8 Tf  [(a dantrolene-binding sequence on the skeletal muscle ryanodine receptor. J Biol Chem. 2002 Sep 20;277\(38\):34918-23. )] TJ ET
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BT 26.250 299.491 Td /F1 9.8 Tf  [(13.)] TJ ET
BT 43.553 299.491 Td /F1 9.8 Tf  [(Spurney CF, Gordish-Dressman H, Guerron AD, Sali A, Pandey GS, Rawat R, Van Der Meulen JH, Cha HJ, Pistilli EE, )] TJ ET
BT 26.250 287.586 Td /F1 9.8 Tf  [(Partridge TA, Hoffman EP, Nagaraju K. Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome )] TJ ET
BT 26.250 275.682 Td /F1 9.8 Tf  [(measures. Muscle Nerve. 2009 May;39\(5\):591-602. PubMed PMID:19260102.)] TJ ET
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BT 43.553 256.277 Td /F1 9.8 Tf  [(Rayavarapu S, Van der Meulen JH, Gordish-Dressman H, Hoffman EP, Nagaraju K, Knoblach SM. Characterization of )] TJ ET
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BT 43.553 213.063 Td /F1 9.8 Tf  [(Baudy AR, Sali A, Jordan S, Kesari A, Johnston HK, Hoffman EP, Nagaraju K. Non-invasive optical imaging of muscle )] TJ ET
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BT 43.553 169.848 Td /F1 9.8 Tf  [(Spurney CF, Cha HJ, Sali A, Pandey GS, Pistilli E, Guerron AD, Gordish-Dressman H, Hoffman EP, Nagaraju K. Evaluation )] TJ ET
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BT 26.250 750.278 Td /F4 12.0 Tf  [(References)] TJ ET
BT 26.250 722.824 Td /F1 9.8 Tf  [(1.)] TJ ET
BT 38.132 722.824 Td /F1 9.8 Tf  [(Emery AE. Population frequencies of inherited neuromuscular diseases--a world survey. Neuromuscul Disord. 1991;1\(1\):19-)] TJ ET
BT 26.250 710.919 Td /F1 9.8 Tf  [(29. PubMed PMID:1822774.)] TJ ET
BT 26.250 691.515 Td /F1 9.8 Tf  [(2.)] TJ ET
BT 38.132 691.515 Td /F1 9.8 Tf  [(Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987 )] TJ ET
BT 26.250 679.610 Td /F1 9.8 Tf  [(Dec 24;51\(6\):919-28. PubMed PMID:3319190.)] TJ ET
BT 26.250 660.205 Td /F1 9.8 Tf  [(3.)] TJ ET
BT 38.132 660.205 Td /F1 9.8 Tf  [(Allen DG, Gervasio OL, Yeung EW, Whitehead NP. Calcium and the damage pathways in muscular dystrophy. Can J Physiol )] TJ ET
BT 26.250 648.300 Td /F1 9.8 Tf  [(Pharmacol. 2010 Feb;88\(2\):83-91. PubMed PMID:20237582.)] TJ ET
BT 26.250 628.896 Td /F1 9.8 Tf  [(4.)] TJ ET
BT 38.132 628.896 Td /F1 9.8 Tf  [(Turner PR, Westwood T, Regen CM, Steinhardt RA. Increased protein degradation results from elevated free calcium levels )] TJ ET
BT 26.250 616.991 Td /F1 9.8 Tf  [(found in muscle from mdx mice. Nature. 1988 Oct 20;335\(6192\):735-8. PubMed PMID:3173492.)] TJ ET
BT 26.250 597.586 Td /F1 9.8 Tf  [(5.)] TJ ET
BT 38.132 597.586 Td /F1 9.8 Tf  [(Morine KJ, Sleeper MM, Barton ER, Sweeney HL. Overexpression of SERCA1a in the mdx diaphragm reduces susceptibility )] TJ ET
BT 26.250 585.681 Td /F1 9.8 Tf  [(to contraction-induced damage. Hum Gene Ther. 2010 Dec;21\(12\):1735-9. PubMed PMID:20540606.)] TJ ET
BT 26.250 566.277 Td /F1 9.8 Tf  [(6.)] TJ ET
BT 38.132 566.277 Td /F1 9.8 Tf  [(Andersson DC, Meli AC, Reiken S, Betzenhauser MJ, Umanskaya A, Shiomi T, D'Armiento J, Marks AR. Leaky ryanodine )] TJ ET
BT 26.250 554.372 Td /F1 9.8 Tf  [(receptors in ?-sarcoglycan deficient mice: a potential common defect in muscular dystrophy. Skelet Muscle. 2012 May )] TJ ET
BT 26.250 542.467 Td /F1 9.8 Tf  [(28;2\(1\):9. PubMed PMID:22640601.)] TJ ET
BT 26.250 523.062 Td /F1 9.8 Tf  [(7.)] TJ ET
BT 38.132 523.062 Td /F1 9.8 Tf  [(Gehrig SM, van der Poel C, Sayer TA, Schertzer JD, Henstridge DC, Church JE, Lamon S, Russell AP, Davies KE, Febbraio )] TJ ET
BT 26.250 511.158 Td /F1 9.8 Tf  [(MA, Lynch GS. Hsp72 preserves muscle function and slows progression of severe muscular dystrophy. Nature. 2012 Apr )] TJ ET
BT 26.250 499.253 Td /F1 9.8 Tf  [(4;484\(7394\):394-8. PubMed PMID:22495301.)] TJ ET
BT 26.250 479.848 Td /F1 9.8 Tf  [(8.)] TJ ET
BT 38.132 479.848 Td /F1 9.8 Tf  [(Fill M, Copello JA. Ryanodine receptor calcium release channels. Physiol Rev. 2002 Oct;82\(4\):893-922. PubMed )] TJ ET
BT 26.250 467.943 Td /F1 9.8 Tf  [(PMID:12270947.)] TJ ET
BT 26.250 448.539 Td /F1 9.8 Tf  [(9.)] TJ ET
BT 38.132 448.539 Td /F1 9.8 Tf  [(Rossi AE, Dirksen RT. Sarcoplasmic reticulum: the dynamic calcium governor of muscle. Muscle Nerve. 2006 Jun;33\(6\):715-)] TJ ET
BT 26.250 436.634 Td /F1 9.8 Tf  [(31. PubMed PMID:16477617.)] TJ ET
BT 26.250 417.229 Td /F1 9.8 Tf  [(10.)] TJ ET
BT 43.553 417.229 Td /F1 9.8 Tf  [(Gehrig SM, van der Poel C, Sayer TA, Schertzer JD, Henstridge DC, Church JE, Lamon S, Russell AP, Davies KE, )] TJ ET
BT 26.250 405.324 Td /F1 9.8 Tf  [(Febbraio MA, Lynch GS. Hsp72 preserves muscle function and slows progression of severe muscular dystrophy. Nature. 2012 )] TJ ET
BT 26.250 393.420 Td /F1 9.8 Tf  [(Apr 4;484\(7394\):394-8. PubMed PMID:22495301.)] TJ ET
BT 26.250 374.015 Td /F1 9.8 Tf  [(11.)] TJ ET
BT 43.553 374.015 Td /F1 9.8 Tf  [(Bertorini TE, Palmieri GM, Griffin J, Igarashi M, Hinton A, Karas JG. Effect of dantrolene in Duchenne muscular dystrophy. )] TJ ET
BT 26.250 362.110 Td /F1 9.8 Tf  [(Muscle Nerve. 1991 Jun;14\(6\):503-7. PubMed PMID:1852156.)] TJ ET
BT 26.250 342.705 Td /F1 9.8 Tf  [(12.)] TJ ET
BT 43.553 342.705 Td /F1 9.8 Tf  [(Paul-Pletzer K, Yamamoto T, Bhat MB, Ma J, Ikemoto N, Jimenez LS, Morimoto H, Williams PG, Parness J. Identification of )] TJ ET
BT 26.250 330.801 Td /F1 9.8 Tf  [(a dantrolene-binding sequence on the skeletal muscle ryanodine receptor. J Biol Chem. 2002 Sep 20;277\(38\):34918-23. )] TJ ET
BT 26.250 318.896 Td /F1 9.8 Tf  [(PubMed PMID:12167662.)] TJ ET
BT 26.250 299.491 Td /F1 9.8 Tf  [(13.)] TJ ET
BT 43.553 299.491 Td /F1 9.8 Tf  [(Spurney CF, Gordish-Dressman H, Guerron AD, Sali A, Pandey GS, Rawat R, Van Der Meulen JH, Cha HJ, Pistilli EE, )] TJ ET
BT 26.250 287.586 Td /F1 9.8 Tf  [(Partridge TA, Hoffman EP, Nagaraju K. Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome )] TJ ET
BT 26.250 275.682 Td /F1 9.8 Tf  [(measures. Muscle Nerve. 2009 May;39\(5\):591-602. PubMed PMID:19260102.)] TJ ET
BT 26.250 256.277 Td /F1 9.8 Tf  [(14.)] TJ ET
BT 43.553 256.277 Td /F1 9.8 Tf  [(Rayavarapu S, Van der Meulen JH, Gordish-Dressman H, Hoffman EP, Nagaraju K, Knoblach SM. Characterization of )] TJ ET
BT 26.250 244.372 Td /F1 9.8 Tf  [(dysferlin deficient SJL/J mice to assess preclinical drug efficacy: fasudil exacerbates muscle disease phenotype. PLoS One. )] TJ ET
BT 26.250 232.467 Td /F1 9.8 Tf  [(2010 Sep 24;5\(9\):e12981. PubMed PMID:20886045.)] TJ ET
BT 26.250 213.063 Td /F1 9.8 Tf  [(15.)] TJ ET
BT 43.553 213.063 Td /F1 9.8 Tf  [(Baudy AR, Sali A, Jordan S, Kesari A, Johnston HK, Hoffman EP, Nagaraju K. Non-invasive optical imaging of muscle )] TJ ET
BT 26.250 201.158 Td /F1 9.8 Tf  [(pathology in mdx mice using cathepsin caged near-infrared imaging. Mol Imaging Biol. 2011 Jun;13\(3\):462-70. PubMed )] TJ ET
BT 26.250 189.253 Td /F1 9.8 Tf  [(PMID:20661652.)] TJ ET
BT 26.250 169.848 Td /F1 9.8 Tf  [(16.)] TJ ET
BT 43.553 169.848 Td /F1 9.8 Tf  [(Spurney CF, Cha HJ, Sali A, Pandey GS, Pistilli E, Guerron AD, Gordish-Dressman H, Hoffman EP, Nagaraju K. Evaluation )] TJ ET
BT 26.250 157.944 Td /F1 9.8 Tf  [(of skeletal and cardiac muscle function after chronic administration of thymosin beta-4 in the dystrophin deficient mouse. PLoS )] TJ ET
BT 26.250 146.039 Td /F1 9.8 Tf  [(One. 2010 Jan 29;5\(1\):e8976. PubMed PMID:20126456.)] TJ ET
BT 26.250 126.634 Td /F1 9.8 Tf  [(17.)] TJ ET
BT 43.553 126.634 Td /F1 9.8 Tf  [(Rayavarapu S, Coley W, Cakir E, Jahnke V, Takeda S, Aoki Y, Grodish-Dressman H, Jaiswal JK, Hoffman EP, Brown KJ, )] TJ ET
BT 26.250 114.729 Td /F1 9.8 Tf  [(Hathout Y, Nagaraju K. Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx )] TJ ET
BT 26.250 102.825 Td /F1 9.8 Tf  [(mouse. Mol Cell Proteomics. 2013 May;12\(5\):1061-73. PubMed PMID:23297347.)] TJ ET
BT 26.250 83.420 Td /F1 9.8 Tf  [(18.)] TJ ET
BT 43.553 83.420 Td /F1 9.8 Tf  [(Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001 May )] TJ ET
BT 26.250 71.515 Td /F1 9.8 Tf  [(1;29\(9\):e45. PubMed PMID:11328886.)] TJ ET
BT 26.250 52.110 Td /F1 9.8 Tf  [(19.)] TJ ET
BT 43.553 52.110 Td /F1 9.8 Tf  [(Ellis KO, Bryant SH. Excitation-contraction uncoupling in skeletal muscle by dantrolene sodium. Naunyn Schmiedebergs )] TJ ET
Q
q
0.000 0.000 0.000 rg
BT 291.710 19.825 Td /F1 11.0 Tf  [(8)] TJ ET
BT 25.000 19.825 Td /F1 11.0 Tf  [(PLOS Currents Muscular Dystrophy)] TJ ET

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15.000 428.191 577.500 348.809 re W n
0.271 0.267 0.267 rg
BT 26.250 767.476 Td /F1 9.8 Tf  [(Arch Pharmacol. 1972;274\(1\):107-9. PubMed PMID:4262721.)] TJ ET
BT 26.250 748.071 Td /F1 9.8 Tf  [(20.)] TJ ET
BT 43.553 748.071 Td /F1 9.8 Tf  [(Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Lacampagne A, Marks AR. Hypernitrosylated )] TJ ET
BT 26.250 736.167 Td /F1 9.8 Tf  [(ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med. 2009 Mar;15\(3\):325-30. PubMed )] TJ ET
BT 26.250 724.262 Td /F1 9.8 Tf  [(PMID:19198614.)] TJ ET
BT 26.250 704.857 Td /F1 9.8 Tf  [(21.)] TJ ET
BT 43.553 704.857 Td /F1 9.8 Tf  [(Salimena MC, Lagrota-Candido J, Qu�rico-Santos T. Gender dimorphism influences extracellular matrix expression and )] TJ ET
BT 26.250 692.952 Td /F1 9.8 Tf  [(regeneration of muscular tissue in mdx dystrophic mice. Histochem Cell Biol. 2004 Nov;122\(5\):435-44. PubMed )] TJ ET
BT 26.250 681.048 Td /F1 9.8 Tf  [(PMID:15452719.)] TJ ET
BT 26.250 661.643 Td /F1 9.8 Tf  [(22.)] TJ ET
BT 43.553 661.643 Td /F1 9.8 Tf  [(Grounds MD, Radley HG, Lynch GS, Nagaraju K, De Luca A. Towards developing standard operating procedures for pre-)] TJ ET
BT 26.250 649.738 Td /F1 9.8 Tf  [(clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol Dis. 2008 Jul;31\(1\):1-19. PubMed )] TJ ET
BT 26.250 637.833 Td /F1 9.8 Tf  [(PMID:18499465.)] TJ ET
BT 26.250 618.429 Td /F1 9.8 Tf  [(23.)] TJ ET
BT 43.553 618.429 Td /F1 9.8 Tf  [(Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Lacampagne A, Marks AR. Hypernitrosylated )] TJ ET
BT 26.250 606.524 Td /F1 9.8 Tf  [(ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med. 2009 Mar;15\(3\):325-30. PubMed )] TJ ET
BT 26.250 594.619 Td /F1 9.8 Tf  [(PMID:19198614.)] TJ ET
BT 26.250 575.214 Td /F1 9.8 Tf  [(24.)] TJ ET
BT 43.553 575.214 Td /F1 9.8 Tf  [(Kendall GC, Mokhonova EI, Moran M, Sejbuk NE, Wang DW, Silva O, Wang RT, Martinez L, Lu QL, Damoiseaux R, )] TJ ET
BT 26.250 563.310 Td /F1 9.8 Tf  [(Spencer MJ, Nelson SF, Miceli MC. Dantrolene enhances antisense-mediated exon skipping in human and mouse models of )] TJ ET
BT 26.250 551.405 Td /F1 9.8 Tf  [(Duchenne muscular dystrophy. Sci Transl Med. 2012 Dec 12;4\(164\):164ra160. PubMed PMID:23241744.)] TJ ET
BT 26.250 532.000 Td /F1 9.8 Tf  [(25.)] TJ ET
BT 43.553 532.000 Td /F1 9.8 Tf  [(Spencer MJ, Croall DE, Tidball JG. Calpains are activated in necrotic fibers from mdx dystrophic mice. J Biol Chem. 1995 )] TJ ET
BT 26.250 520.095 Td /F1 9.8 Tf  [(May 5;270\(18\):10909-14. PubMed PMID:7738032.)] TJ ET
BT 26.250 500.691 Td /F1 9.8 Tf  [(26.)] TJ ET
BT 43.553 500.691 Td /F1 9.8 Tf  [(Quinlan JG, Johnson SR, Samaha FJ. Dantrolene normalizes serum creatine kinase in MDX mice. Muscle Nerve. 1990 )] TJ ET
BT 26.250 488.786 Td /F1 9.8 Tf  [(Mar;13\(3\):268-9. PubMed PMID:2320049.)] TJ ET
BT 26.250 469.381 Td /F1 9.8 Tf  [(27.)] TJ ET
BT 43.553 469.381 Td /F1 9.8 Tf  [(Min JY, Meissner A, Feng X, Wang J, Malek S, Wang JF, Simon R, Morgan JP. Dantrolene: effects on abnormal )] TJ ET
BT 26.250 457.476 Td /F1 9.8 Tf  [(intracellular Ca\(2+\) handling and inotropy in postinfarcted rat myocardium. Eur J Pharmacol. 2003 Jun 13;471\(1\):41-7. PubMed )] TJ ET
BT 26.250 445.572 Td /F1 9.8 Tf  [(PMID:12809951.)] TJ ET
Q
q
15.000 428.191 577.500 348.809 re W n
0.271 0.267 0.267 rg
BT 26.250 767.476 Td /F1 9.8 Tf  [(Arch Pharmacol. 1972;274\(1\):107-9. PubMed PMID:4262721.)] TJ ET
BT 26.250 748.071 Td /F1 9.8 Tf  [(20.)] TJ ET
BT 43.553 748.071 Td /F1 9.8 Tf  [(Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Lacampagne A, Marks AR. Hypernitrosylated )] TJ ET
BT 26.250 736.167 Td /F1 9.8 Tf  [(ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med. 2009 Mar;15\(3\):325-30. PubMed )] TJ ET
BT 26.250 724.262 Td /F1 9.8 Tf  [(PMID:19198614.)] TJ ET
BT 26.250 704.857 Td /F1 9.8 Tf  [(21.)] TJ ET
BT 43.553 704.857 Td /F1 9.8 Tf  [(Salimena MC, Lagrota-Candido J, Qu�rico-Santos T. Gender dimorphism influences extracellular matrix expression and )] TJ ET
BT 26.250 692.952 Td /F1 9.8 Tf  [(regeneration of muscular tissue in mdx dystrophic mice. Histochem Cell Biol. 2004 Nov;122\(5\):435-44. PubMed )] TJ ET
BT 26.250 681.048 Td /F1 9.8 Tf  [(PMID:15452719.)] TJ ET
BT 26.250 661.643 Td /F1 9.8 Tf  [(22.)] TJ ET
BT 43.553 661.643 Td /F1 9.8 Tf  [(Grounds MD, Radley HG, Lynch GS, Nagaraju K, De Luca A. Towards developing standard operating procedures for pre-)] TJ ET
BT 26.250 649.738 Td /F1 9.8 Tf  [(clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol Dis. 2008 Jul;31\(1\):1-19. PubMed )] TJ ET
BT 26.250 637.833 Td /F1 9.8 Tf  [(PMID:18499465.)] TJ ET
BT 26.250 618.429 Td /F1 9.8 Tf  [(23.)] TJ ET
BT 43.553 618.429 Td /F1 9.8 Tf  [(Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Lacampagne A, Marks AR. Hypernitrosylated )] TJ ET
BT 26.250 606.524 Td /F1 9.8 Tf  [(ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med. 2009 Mar;15\(3\):325-30. PubMed )] TJ ET
BT 26.250 594.619 Td /F1 9.8 Tf  [(PMID:19198614.)] TJ ET
BT 26.250 575.214 Td /F1 9.8 Tf  [(24.)] TJ ET
BT 43.553 575.214 Td /F1 9.8 Tf  [(Kendall GC, Mokhonova EI, Moran M, Sejbuk NE, Wang DW, Silva O, Wang RT, Martinez L, Lu QL, Damoiseaux R, )] TJ ET
BT 26.250 563.310 Td /F1 9.8 Tf  [(Spencer MJ, Nelson SF, Miceli MC. Dantrolene enhances antisense-mediated exon skipping in human and mouse models of )] TJ ET
BT 26.250 551.405 Td /F1 9.8 Tf  [(Duchenne muscular dystrophy. Sci Transl Med. 2012 Dec 12;4\(164\):164ra160. PubMed PMID:23241744.)] TJ ET
BT 26.250 532.000 Td /F1 9.8 Tf  [(25.)] TJ ET
BT 43.553 532.000 Td /F1 9.8 Tf  [(Spencer MJ, Croall DE, Tidball JG. Calpains are activated in necrotic fibers from mdx dystrophic mice. J Biol Chem. 1995 )] TJ ET
BT 26.250 520.095 Td /F1 9.8 Tf  [(May 5;270\(18\):10909-14. PubMed PMID:7738032.)] TJ ET
BT 26.250 500.691 Td /F1 9.8 Tf  [(26.)] TJ ET
BT 43.553 500.691 Td /F1 9.8 Tf  [(Quinlan JG, Johnson SR, Samaha FJ. Dantrolene normalizes serum creatine kinase in MDX mice. Muscle Nerve. 1990 )] TJ ET
BT 26.250 488.786 Td /F1 9.8 Tf  [(Mar;13\(3\):268-9. PubMed PMID:2320049.)] TJ ET
BT 26.250 469.381 Td /F1 9.8 Tf  [(27.)] TJ ET
BT 43.553 469.381 Td /F1 9.8 Tf  [(Min JY, Meissner A, Feng X, Wang J, Malek S, Wang JF, Simon R, Morgan JP. Dantrolene: effects on abnormal )] TJ ET
BT 26.250 457.476 Td /F1 9.8 Tf  [(intracellular Ca\(2+\) handling and inotropy in postinfarcted rat myocardium. Eur J Pharmacol. 2003 Jun 13;471\(1\):41-7. PubMed )] TJ ET
BT 26.250 445.572 Td /F1 9.8 Tf  [(PMID:12809951.)] TJ ET
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BT 26.250 767.476 Td /F1 9.8 Tf  [(Arch Pharmacol. 1972;274\(1\):107-9. PubMed PMID:4262721.)] TJ ET
BT 26.250 748.071 Td /F1 9.8 Tf  [(20.)] TJ ET
BT 43.553 748.071 Td /F1 9.8 Tf  [(Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Lacampagne A, Marks AR. Hypernitrosylated )] TJ ET
BT 26.250 736.167 Td /F1 9.8 Tf  [(ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med. 2009 Mar;15\(3\):325-30. PubMed )] TJ ET
BT 26.250 724.262 Td /F1 9.8 Tf  [(PMID:19198614.)] TJ ET
BT 26.250 704.857 Td /F1 9.8 Tf  [(21.)] TJ ET
BT 43.553 704.857 Td /F1 9.8 Tf  [(Salimena MC, Lagrota-Candido J, Qu�rico-Santos T. Gender dimorphism influences extracellular matrix expression and )] TJ ET
BT 26.250 692.952 Td /F1 9.8 Tf  [(regeneration of muscular tissue in mdx dystrophic mice. Histochem Cell Biol. 2004 Nov;122\(5\):435-44. PubMed )] TJ ET
BT 26.250 681.048 Td /F1 9.8 Tf  [(PMID:15452719.)] TJ ET
BT 26.250 661.643 Td /F1 9.8 Tf  [(22.)] TJ ET
BT 43.553 661.643 Td /F1 9.8 Tf  [(Grounds MD, Radley HG, Lynch GS, Nagaraju K, De Luca A. Towards developing standard operating procedures for pre-)] TJ ET
BT 26.250 649.738 Td /F1 9.8 Tf  [(clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol Dis. 2008 Jul;31\(1\):1-19. PubMed )] TJ ET
BT 26.250 637.833 Td /F1 9.8 Tf  [(PMID:18499465.)] TJ ET
BT 26.250 618.429 Td /F1 9.8 Tf  [(23.)] TJ ET
BT 43.553 618.429 Td /F1 9.8 Tf  [(Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Lacampagne A, Marks AR. Hypernitrosylated )] TJ ET
BT 26.250 606.524 Td /F1 9.8 Tf  [(ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med. 2009 Mar;15\(3\):325-30. PubMed )] TJ ET
BT 26.250 594.619 Td /F1 9.8 Tf  [(PMID:19198614.)] TJ ET
BT 26.250 575.214 Td /F1 9.8 Tf  [(24.)] TJ ET
BT 43.553 575.214 Td /F1 9.8 Tf  [(Kendall GC, Mokhonova EI, Moran M, Sejbuk NE, Wang DW, Silva O, Wang RT, Martinez L, Lu QL, Damoiseaux R, )] TJ ET
BT 26.250 563.310 Td /F1 9.8 Tf  [(Spencer MJ, Nelson SF, Miceli MC. Dantrolene enhances antisense-mediated exon skipping in human and mouse models of )] TJ ET
BT 26.250 551.405 Td /F1 9.8 Tf  [(Duchenne muscular dystrophy. Sci Transl Med. 2012 Dec 12;4\(164\):164ra160. PubMed PMID:23241744.)] TJ ET
BT 26.250 532.000 Td /F1 9.8 Tf  [(25.)] TJ ET
BT 43.553 532.000 Td /F1 9.8 Tf  [(Spencer MJ, Croall DE, Tidball JG. Calpains are activated in necrotic fibers from mdx dystrophic mice. J Biol Chem. 1995 )] TJ ET
BT 26.250 520.095 Td /F1 9.8 Tf  [(May 5;270\(18\):10909-14. PubMed PMID:7738032.)] TJ ET
BT 26.250 500.691 Td /F1 9.8 Tf  [(26.)] TJ ET
BT 43.553 500.691 Td /F1 9.8 Tf  [(Quinlan JG, Johnson SR, Samaha FJ. Dantrolene normalizes serum creatine kinase in MDX mice. Muscle Nerve. 1990 )] TJ ET
BT 26.250 488.786 Td /F1 9.8 Tf  [(Mar;13\(3\):268-9. PubMed PMID:2320049.)] TJ ET
BT 26.250 469.381 Td /F1 9.8 Tf  [(27.)] TJ ET
BT 43.553 469.381 Td /F1 9.8 Tf  [(Min JY, Meissner A, Feng X, Wang J, Malek S, Wang JF, Simon R, Morgan JP. Dantrolene: effects on abnormal )] TJ ET
BT 26.250 457.476 Td /F1 9.8 Tf  [(intracellular Ca\(2+\) handling and inotropy in postinfarcted rat myocardium. Eur J Pharmacol. 2003 Jun 13;471\(1\):41-7. PubMed )] TJ ET
BT 26.250 445.572 Td /F1 9.8 Tf  [(PMID:12809951.)] TJ ET
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BT 25.000 19.825 Td /F1 11.0 Tf  [(PLOS Currents Muscular Dystrophy)] TJ ET

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