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Fibroadipogenic progenitor (FAP) cells are implicated as a major source of fatty infiltration (FI) in murine rotator cuff (RC) injury, but FAP cell response after RC tear in a rabbit model is unknown. This study determined whether changes in FAP cell count after an RC tear predate muscle degeneration in a clinically relevant rabbit model. We hypothesized increases in FAP cell count correlate temporally with RC degeneration. New Zealand white rabbits (n = 26) were evaluated at 1, 2, 4, and 6 weeks after unilateral full-thickness tenotomy of supraspinatus and infraspinatus tendons. FI area and adipocyte size were histologically analyzed, muscle density was measured by computerized tomography, and quantification of FAP cells was measured by flow cytometry and immunohistochemistry. The percentage of intrafascicular adipocyte area increased over time in supraspinatus muscle samples (p = 0.03), significantly between 1- and 6-week samples (p = 0.04). There were no differences in perifascicular adipocyte area percentages between time points. Peak increase in FAP cell count occurred at 1-week (p = 0.03), with a decrease in the following weeks. There was a negative correlation between supraspinatus adipocyte area and FAP cell count (p < 0.05). On computed tomography (CT) scan, maximal decrease in muscle density was observed in the 4th to 6th weeks. In summary, FAP cell response occurred early after tenotomy and did not correlate temporally with increases in FI. This suggests that FAP cell response may predate degenerative changes, and early targeting of FAPs before adipocyte maturation could blunt FI after RC tear., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Human pluripotent stem cell (hPSC)-derived myogenic progenitor cell (MPC) transplantation is a promising therapeutic approach for a variety of degenerative muscle disorders. Here, using an MPC-specific fluorescent reporter system (PAX7::GFP), we demonstrate that hPSC-derived MPCs can contribute to the regeneration of myofibers in mice following local injury and in mice deficient of dystrophin (mdx). We also demonstrate that a subset of PAX7::GFP MPCs engraft within the basal lamina of regenerated myofibers, adopt a quiescent state, and contribute to regeneration upon reinjury and in mdx mouse models. This subset of PAX7::GFP MPCs undergo a maturation process and remodel their molecular characteristics to resemble those of late-stage fetal MPCs/adult satellite cells following in vivo engraftment. These in-vivo-matured PAX7::GFP MPCs retain a cell-autonomous ability to regenerate and can repopulate in the niche of secondary recipient mice, providing a proof of principle for future hPSC-based cell therapy for muscle disorders., Competing Interests: Declaration of interests C.S., P.A., K.R.W., and G.L. are scientific founders and shareholders and P.A. and G.L. serve as scientific advisors of Vita Therapeutics. K.R.W. is a current employee of F. Hoffmann La-Roche. C.S. is a current employee of Vita Therapeutics. C.S., I.Y.C., H.L., P.A., K.R.W., and G.L. are inventors of a patent related to this study. The Lee lab receives a funding from Vita Therapeutics. R.M.L. is a current employee at National Institutes of Health, but this work was prepared while R.M.L. was employed at University of Maryland School of Medicine. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Intermediate filaments (IFs) are a primary structural component of the cytoskeleton extending throughout the muscle cell (myofiber). Mechanotransduction, the process by which mechanical force is translated into a biochemical signal to activate downstream cellular responses, is crucial to myofiber function. Mechanical forces also act on the nuclear cytoskeleton, which is integrated with the myofiber cytoskeleton by the linker of the nucleoskeleton and cytoskeleton (LINC) complexes. Thus, the nucleus serves as the endpoint for the transmission of force through the cell. The nuclear lamina, a dense meshwork of lamin IFs between the nuclear envelope and underlying chromatin, plays a crucial role in responding to mechanical input; myofibers constantly respond to mechanical perturbation via signaling pathways by activation of specific genes. The nucleus is the largest organelle in cells and a master regulator of cell homeostasis, thus an understanding of how it responds to its mechanical environment is of great interest. The importance of the cell nucleus is magnified in skeletal muscle cells due to their syncytial nature and the extreme mechanical environment that muscle contraction creates. In this review, we summarize the bidirectional link between the organization of the nucleoskeleton and the contractile features of skeletal muscle as they relate to muscle function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Iyer, Folker and Lovering.)

The neuromuscular junction (NMJ) is a specialized synapse that bridges the motor neuron and the skeletal muscle fiber and is crucial for conversion of electrical impulses originating in the motor neuron to action potentials in the muscle fiber. The consideration of contributing factors to skeletal muscle injury, muscular dystrophy and sarcopenia cannot be restricted only to processes intrinsic to the muscle, as data show that these conditions incur denervation-like findings, such as fragmented NMJ morphology and corresponding functional changes in neuromuscular transmission. Primary defects in the NMJ also influence functional loss in motor neuron disease, congenital myasthenic syndromes and myasthenia gravis, resulting in skeletal muscle weakness and heightened fatigue. Such findings underscore the role that the NMJ plays in neuromuscular performance. Regardless of cause or effect, functional denervation is now an accepted consequence of sarcopenia and muscle disease. In this short review, we provide an overview of the pathologic etiology, symptoms, and therapeutic strategies related to the NMJ. In particular, we examine the role of the NMJ as a disease modifier and a potential therapeutic target in neuromuscular injury and disease.

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca 2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.

Introduction: Our aim was to assess key muscle imaging and contractility parameters in the Duchenne muscular dystrophy (DMD) rat model (Dmd-KO rat), which have not yet been characterized sufficiently., Methods: We performed in-vivo magnetic resonance imaging (MRI) for thigh and leg muscles, and performed hematoxylin and eosin (H&E) staining and in-vivo muscle contractility testing in specific hindlimb muscles., Results: MRI prior to testing muscle contractility revealed multiple, unevenly distributed focal hyperintensities in the Dmd-KO rat quadriceps and tibialis anterior muscles. H&E staining showed corresponding areas of inflammation and ongoing regeneration. In-vivo contractile testing showed maximal force generated by Dmd-KO muscles was significantly lower, and susceptibility to injury was ~ two-fold greater in the Dmd-KO rats compared to wild-type (WT) rats., Discussion: Together, the MRI findings, histological findings, and the low strength and high susceptibility to injury in muscles support use of the Dmd-KO rat as an animal model of DMD., (© 2020 Wiley Periodicals LLC.)

Isolated myofibers are commonly used to understand the function of skeletal muscle in vivo . This can involve single isolated myofibers obtained from dissection or from enzymatic dissociation. Isolation via dissection allows control of sarcomere length and preserves tendon attachment but is labor-intensive, time-consuming and yields few viable myofibers. In contrast, enzymatic dissociation is fast and facile, produces hundreds of myofibers, and more importantly reduces the number of muscles/animals needed for studies. Biomechanical properties of the sarcolemma have been studied using myofibers from the extensor digitorum longus, but this has been limited to dissected myofibers, making data collection slow and difficult. We have modified this tool to perform biomechanical measurements of the sarcolemma in dissociated myofibers from the flexor digitorum brevis.

The focus of this review is on Duchenne muscular dystrophy (DMD), which is caused by the absence of the protein dystrophin and is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but data show that both human patients and animal models for DMD present with fragmented neuromuscular junction (NMJ) morphology. In addition to pre- and post-synaptic abnormalities, studies indicate increased susceptibility of the NMJ to contraction-induced injury, with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyographic activity. Such findings suggest that alterations in the NMJ of dystrophic muscle may play a role in muscle weakness via impairment of neuromuscular transmission. Further work is needed to fully understand the role of the NMJ in the weakness, susceptibility to injury, and progressive wasting associated with DMD., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In order to repair chronic nerve injuries (injuries repaired after a long delay), the damaged nerve segments are resected and stumps are bridged by grafts. Autografts remain the gold-standard, but outcomes are typically poor, even after long periods of recovery. In a recent study, we described the use of a nerve lengthening device to gradually elongate the proximal stump of a transected nerve towards the distal stump, enabling a tension-free end-to-end repair. This approach showed significantly improved outcomes in comparison to autografts in repairing acutely injured nerves. In this study, we compared the use of nerve lengthening/end-to-end repair (LETER) to isograft repair of chronically transected nerves in a rat model. Structural and functional regenerative outcomes following LETER were comparable to isograft-based repair, with no significant differences found in outcomes involving functional recovery or axon growth. These data demonstrate the feasibility of nerve lengthening as a viable graft-free strategy for repairing chronically injured nerves. Not unexpectedly, outcomes for chronic nerve injuries were less favorable in both groups compared to repair of acutely injured nerves. Nonetheless, the findings provide insight into barriers to restoring function after chronic nerve injury through novel comprehensive characterization of a diverse set of neuromuscular outcomes. This analysis revealed key parameters predicting functional recovery., (Published by Elsevier Inc.)

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by the lack of dystrophin with progressive degeneration of skeletal muscles. Most studies regarding DMD understandably focus on muscle, but dystrophin is also expressed in the central nervous system, potentially resulting in cognitive and behavioral changes. Animal models are being used for developing more comprehensive neuromonitoring protocols and clinical image acquisition procedures. The recently developed DMD rat is an animal model that parallels the progressive muscle wasting seen in DMD. Here, we studied the brain and temporalis muscle structure and neurochemistry of wild type (WT) and dystrophic (DMD) rats using magnetic resonance imaging and spectroscopy. Both structural and neurochemistry alterations were observed in the DMD rat brain and the temporalis muscle. There was a decrease in absolute brain volume ( WT = 1579 mm 3 ; DMD = 1501 mm 3 ; p = 0.039, Cohen's d = 1.867), but not normalized ( WT = 4.27; DMD = 4.02; p = 0.306) brain volume. Diffusion tensor imaging (DTI) revealed structural alterations in the DMD temporalis muscle, with increased mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD). In the DMD rat thalamus, DTI revealed an increase in fractional anisotropy (FA) and a decrease in RD. Smaller normalized brain volume correlated to severity of muscle dystrophy ( r = -0.975). Neurochemical changes in the DMD rat brain included increased GABA and NAA in the prefrontal cortex, and GABA in the hippocampus. Such findings could indicate disturbed motor and sensory signaling, resulting in a dysfunctional GABAergic neurotransmission, and an unstable osmoregulation in the dystrophin-null brain., (Copyright © 2020 Xu, Tang, Li, Iyer and Lovering.)

Background: Clinical use of platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) has gained momentum as treatment for muscle injuries. Exosomes, or small cell-derived vesicles, could be helpful if they could deliver the same or better physiological effect without cell transplantation into the muscle., Hypothesis: Local delivery of exosomes derived from PRP (PRP-exos) or MSCs (MSC-exos) to injured muscles hastens recovery of contractile function., Study Design: Controlled laboratory study., Methods: In a rat model, platelets were isolated from blood, and MSCs were isolated from bone marrow and expanded in culture; exosomes from both were isolated through ultracentrifugation. The tibialis anterior muscles were injured in vivo using maximal lengthening contractions. Muscles were injected with PRP-exos or MSC-exos (immediately after injury and 5 and 10 days after injury); controls received an equal volume of saline. Histological and biochemical analysis was performed on tissues for all groups., Results: Injury resulted in a significant loss of maximal isometric torque (66% ± 3%) that gradually recovered over 2 weeks. Both PRP-exos and MSC-exos accelerated recovery, with similar faster recovery of contractile function over the saline-treated group at 5, 10, and 15 days after injury ( P < .001). A significant increase in centrally nucleated fibers was seen with both types of exosome groups by day 15 ( P < .01). Genes involved in skeletal muscle regeneration were modulated by different exosomes. Muscles treated with PRP-exos had increased expression of Myogenin gene ( P < .05), whereas muscles treated with MSC-exos had reduced expression of TGF-β ( P < .05) at 10 days after muscle injury., Conclusion: Exosomes derived from PRP or MSCs can facilitate recovery after a muscle strain injury in a small-animal model likely because of factors that can modulate inflammation, fibrosis, and myogenesis., Clinical Relevance: Given their small size, low immunogenicity, and ease with which they can be obtained, exosomes could represent a novel therapy for many orthopaedic ailments.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. In the present study, when human induced pluripotent stem cells (hiPSCs) were differentiated into myoblasts, the myoblasts derived from DMD patient hiPSCs (DMD hiPSC-derived myoblasts) exhibited an identifiable DMD-relevant phenotype: myogenic fusion deficiency. Based on this model, we developed a DMD hiPSC-derived myoblast screening platform employing a high-content imaging (BD Pathway 855) approach to generate parameters describing morphological as well as myogenic marker protein expression. Following treatment of the cells with 1524 compounds from the Johns Hopkins Clinical Compound Library, compounds that enhanced myogenic fusion of DMD hiPSC-derived myoblasts were identified. The final hits were ginsenoside Rd and fenofibrate. Transcriptional profiling revealed that ginsenoside Rd is functionally related to FLT3 signaling, while fenofibrate is linked to TGF-β signaling. Preclinical tests in mdx mice showed that treatment with these 2 hit compounds can significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency, supporting their therapeutic potential. Further study revealed that fenofibrate could inhibit mitochondrion-induced apoptosis in DMD hiPSC-derived cardiomyocytes. We have developed a platform based on DMD hiPSC-derived myoblasts for drug screening and identified 2 promising small molecules with in vivo efficacy.

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18 -knockout ( Krt18 -KO) or dominant-negative mutant mice ( Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18 -null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

Myostatin is a negative regulator of muscle growth and metabolism and its inhibition in mice improves insulin sensitivity, increases glucose uptake into skeletal muscle, and decreases total body fat. A recently described mammalian protein called MSS51 is significantly downregulated with myostatin inhibition. In vitro disruption of Mss51 results in increased levels of ATP, β-oxidation, glycolysis, and oxidative phosphorylation. To determine the in vivo biological function of Mss51 in mice, we disrupted the Mss51 gene by CRISPR/Cas9 and found that Mss51-KO mice have normal muscle weights and fiber-type distribution but reduced fat pads. Myofibers isolated from Mss51-KO mice showed an increased oxygen consumption rate compared with WT controls, indicating an accelerated rate of skeletal muscle metabolism. The expression of genes related to oxidative phosphorylation and fatty acid β-oxidation were enhanced in skeletal muscle of Mss51-KO mice compared with that of WT mice. We found that mice lacking Mss51 and challenged with a high-fat diet were resistant to diet-induced weight gain, had increased whole-body glucose turnover and glycolysis rate, and increased systemic insulin sensitivity and fatty acid β-oxidation. These findings demonstrate that MSS51 modulates skeletal muscle mitochondrial respiration and regulates whole-body glucose and fatty acid metabolism, making it a potential target for obesity and diabetes.

Mechanical forces regulate muscle development, hypertrophy, and homeostasis. Force-transmitting structures allow mechanotransduction at the sarcolemma, cytoskeleton, and nuclear envelope. There is growing evidence that Yes-associated protein (YAP) serves as a nuclear relay of mechanical signals and can induce a range of downstream signaling cascades. Dystrophin is a sarcolemma-associated protein, and its absence underlies the pathology in Duchenne muscular dystrophy. We tested the hypothesis that the absence of dystrophin in muscle would result in reduced YAP signaling in response to loading. Following in vivo contractile loading in muscles of healthy (wild-type; WT) mice and mice lacking dystrophin ( mdx ), we performed Western blots of whole and fractionated muscle homogenates to examine the ratio of phospho (cytoplasmic) YAP to total YAP and nuclear YAP, respectively. We show that in vivo contractile loading induced a robust increase in YAP expression and its nuclear localization in WT muscles. Surprisingly, in mdx muscles, active YAP expression was constitutively elevated and unresponsive to load. Results from qRT-PCR analysis support the hyperactivation of YAP in vivo in mdx muscles, as evidenced by increased gene expression of YAP downstream targets. In vitro assays of isolated myofibers plated on substrates with high stiffness showed YAP nuclear labeling for both genotypes, indicating functional YAP signaling in mdx muscles. We conclude that while YAP signaling can occur in the absence of dystrophin, dystrophic muscles have altered mechanotransduction, whereby constitutively active YAP results in a failure to respond to load, which could be attributed to the increased state of "pre-stress" with increased cytoskeletal and extracellular matrix stiffness.

Tissue engineering strategies to treat patients with volumetric muscle loss (VML) aim to recover the structure and contractile function of lost muscle tissue. Here, we assessed the capacity of novel electrospun fibrin hydrogel scaffolds seeded with murine myoblasts to regenerate the structure and function of damaged muscle within VML defects to the mouse tibialis anterior muscle. The electrospun fibrin scaffolds provide pro-myogenic alignment and stiffness cues, myomimetic hierarchical structure, suturability, and scale-up capabilities. Myoblast-seeded scaffolds enabled remarkable muscle regeneration with high myofiber and vascular densities after 2 and 4 weeks, mimicking that of native skeletal muscle, while acellular scaffolds lacked muscle regeneration. Both myoblast-seeded and acellular scaffolds fully recovered muscle contractile function to uninjured values after 2 and 4 weeks. Electrospun scaffolds pre-vascularized with co-cultured human endothelial cells and human adipose-derived stem cells implanted into VML defects for 2 weeks anastomosed with host vasculature and were perfused with host red blood cells. These data demonstrate the significant potential of electrospun fibrin scaffolds seeded with myoblasts to fully regenerate the structure and function of volumetric muscle defects and these scaffolds offer a promising treatment option for patients with VML., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Duchenne muscular dystrophy (DMD), caused by the absence of the protein dystrophin, is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but there is little information to determine if damage from contraction-induced injury also occurs at or near the nerve terminal axon. Interestingly, both human patients and the mouse model for DMD (the mdx mouse) present fragmented neuromuscular junction (NMJ) morphology. Studies of mdx mice have revealed presynaptic and postsynaptic abnormalities, nerve terminal discontinuity, as well as increased susceptibility of the NMJ to contraction-induced injury with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyography. Focusing on the NMJ as a contributor to functional deficits in the muscle represents a paradigm shift from the more prevalent myocentric perspectives. Further studies are needed to determine the extent to which the nerve-muscle interaction is disrupted in DMD and the role of the NMJ in the dystrophic progression. This chapter lists the tools needed for nerve terminal and NMJ structural analysis using fluorescence imaging, and provides a step-by-step outline for how to stain, image, and analyze the NMJ in skeletal muscle, with specific attention to mdx muscle.

Introduction: Dystrophic muscle is particularly susceptible to eccentric contraction-induced injury. We tested the hypothesis that electrical impedance myography (EIM) can detect injury induced by maximal-force lengthening contractions., Methods: We induced injury in the quadriceps of wild-type (WT) and dystrophic (mdx) mice with eccentric contractions using an established model., Results: mdx quadriceps had significantly greater losses in peak twitch and tetany compared with losses in WT quadriceps. Injured muscle showed a significant increase in EIM characteristic frequency in both WT (177 ± 7.7%) and mdx (167 ± 7.8%) quadriceps. EIM also revealed decreased extracellular resistance for both WT and mdx quadriceps after injury., Discussion: Our results show overall agreement between muscle function and EIM measurements of injured muscle, indicating that EIM is a viable tool to assess injury in dystrophic muscle. Muscle Nerve 56: E85-E94, 2017., (© 2017 Wiley Periodicals, Inc.)

While excessive tensile strain can be detrimental to nerve function, strain can be a positive regulator of neuronal outgrowth. We used an in vivo rat model of sciatic nerve strain to investigate signaling mechanisms underlying peripheral nerve response to deformation. Nerves were deformed by 11% and did not demonstrate deficits in compound action potential latency or amplitude during or after 6 h of strain. As revealed by Western blotting, application of strain resulted in significant upregulation of mammalian target of rapamycin (mTOR) and S6 signaling in nerves, increased myelin basic protein (MBP) and β-actin levels, and increased phosphorylation of neurofilament subunit H (NF-H) compared with unstrained (sham) contralateral nerves ( P < 0.05 for all comparisons, paired two-tailed t -test). Strain did not alter neuron-specific β3-tubulin or overall nerve tubulin levels compared with unstrained controls. Systemic rapamycin treatment, thought to selectively target mTOR complex 1 (mTORC1), suppressed mTOR/S6 signaling, reduced levels of MBP and overall tubulin, and decreased NF-H phosphorylation in nerves strained for 6 h, revealing a role for mTOR in increasing MBP expression and NF-H phosphorylation, and maintaining tubulin levels. Consistent with stretch-induced increases in MBP, immunolabeling revealed increased S6 signaling in Schwann cells of stretched nerves compared with unstretched nerves. In addition, application of strain to cultured adult dorsal root ganglion neurons showed an increase in axonal protein synthesis based on a puromycin incorporation assay, suggesting that neuronal translational pathways also respond to strain. This work has important implications for understanding mechanisms underlying nerve response to strain during development and regeneration. NEW & NOTEWORTHY Peripheral nerves experience tensile strain (stretch) during development and movement. Excessive strain impairs neuronal function, but moderate strains are accommodated by nerves and can promote neuronal growth; mechanisms underlying these phenomena are not well understood. We demonstrated that levels of several structural proteins increase following physiological levels of nerve strain and that expression of a subset of these proteins is regulated by mTOR. Our work has important implications for understanding nerve development and strain-based regenerative strategies., (Copyright © 2017 the American Physiological Society.)

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD + ) synthesis, consistent with a potential role for the essential cofactor NAD + in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD + and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD + levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD + biosynthesis. Replenishing NAD + stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr -/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD + repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD + may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures., (Copyright © 2016, American Association for the Advancement of Science.)

Background: Most cultured enzymatically dissociated adult myofibers exhibit spatially uniform (UNI) contractile responses and Ca(2+) transients over the entire myofiber in response to electric field stimuli of either polarity applied via bipolar electrodes. However, some myofibers only exhibit contraction and Ca(2+) transients at alternating (ALT) ends in response to alternating polarity field stimulation. Here, we present for the first time the methodology for identification of ALT myofibers in primary cultures and isolated muscles, as well as a study of their electrophysiological properties., Results: We used high-speed confocal microscopic Ca(2+) imaging, electric field stimulation, microelectrode recordings, immunostaining, and confocal microscopy to characterize the properties of action potential-induced Ca(2+) transients, contractility, resting membrane potential, and staining of T-tubule voltage-gated Na(+) channel distribution applied to cultured adult myofibers. Here, we show for the first time, with high temporal and spatial resolution, that normal control myofibers with UNI responses can be converted to ALT response myofibers by TTX addition or by removal of Na(+) from the bathing medium, with reappearance of the UNI response on return of Na(+). Our results suggest disrupted excitability as the cause of ALT behavior and indicate that the ALT response is due to local depolarization-induced Ca(2+) release, whereas the UNI response is triggered by action potential propagation over the entire myofiber. Consistent with this interpretation, local depolarizing monopolar stimuli give uniform (propagated) responses in UNI myofibers, but only local responses at the electrode in ALT myofibers. The ALT responses in electrically inexcitable myofibers are consistent with expectations of current spread between bipolar stimulating electrodes, entering (hyperpolarizing) one end of a myofiber and leaving (depolarizing) the other end of the myofiber. ALT responses were also detected in some myofibers within intact isolated whole muscles from wild-type and MDX mice, demonstrating that ALT responses can be present before enzymatic dissociation., Conclusions: We suggest that checking for ALT myofiber responsiveness by looking at the end of a myofiber during alternating polarity stimuli provides a test for compromised excitability of myofibers, and could be used to identify inexcitable, damaged or diseased myofibers by ALT behavior in healthy and diseased muscle.

In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Animal models provide unmitigated access to histological samples, which provide a "direct measure" of damage. However, even with unconstrained access to tissue morphology and biochemistry assays, the findings typically do not account for loss of muscle function. Thus, the most comprehensive measure of the overall health of the muscle is assessment of its primary function, which is to produce contractile force. The majority of animal models testing contractile force have been limited to the muscle groups moving the ankle, with advantages and disadvantages depending on the equipment. Here, we describe in vivo methods to measure torque, to produce a reliable muscle injury, and to follow muscle function within the same animal over time. We also describe in vivo methods to measure tension in the leg and thigh muscles.

Duchenne muscular dystrophy (DMD), an X-linked disorder caused by the lack of dystrophin, is characterized by the progressive wasting of skeletal muscles. To date, what is known about dystrophin function is derived from studies of dystrophin-deficient animals, with the most common model being the mdx mouse. Most studies on patients with DMD and in mdx mice have focused on skeletal muscle and the development of therapies to reverse, or at least slow, the severe muscle wasting and progressive degeneration. However, dystrophin is also expressed in the CNS. Both mdx mice and patients with DMD can have cognitive and behavioral changes, but studies in the dystrophic brain are limited. We examined the brain structure and metabolites of mature wild type (WT) and mdx mice using magnetic resonance imaging and spectroscopy (MRI/MRS). Both structural and metabolic alterations were observed in the mdx brain. Enlarged lateral ventricles were detected in mdx mice when compared to WT. Diffusion tensor imaging (DTI) revealed elevations in diffusion diffusivities in the prefrontal cortex and a reduction of fractional anisotropy in the hippocampus. Metabolic changes included elevations in phosphocholine and glutathione, and a reduction in γ-aminobutyric acid in the hippocampus. In addition, an elevation in taurine was observed in the prefrontal cortex. Such findings indicate a regional structural change, altered cellular antioxidant defenses, a dysfunction of GABAergic neurotransmission, and a perturbed osmoregulation in the brain lacking dystrophin., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated in mdx mice (murine model for DMD) significant morphologic alterations at the motor endplate of the neuromuscular junction (NMJ) and corresponding NMJ transmission failure after injury. Here we extend these initial observations at the motor endplate to gain insight into the pre- vs. postsynaptic morphology, as well as the subsynaptic nuclei in healthy (WT) vs. mdx mice. We quantified the discontinuity and branching of the terminal nerve in adult mice. We report mdx- and age-dependent changes for discontinuity and an increase in branching when compared to WT. To examine mdx- and age-dependent changes in the relative localization of pre- and postsynaptic structures, we calculated NMJ occupancy, defined as the ratio of the footprint occupied by presynaptic vesicles vs. that of the underlying motor endplate. The normally congruent coupling between presynaptic and postsynaptic morphology was altered in mdx mice, independent of age. Finally we found an almost two-fold increase in the number of nuclei and an increase in density (nuclei/area) underlying the NMJ. These outcomes suggest substantial remodeling of the NMJ during dystrophic progression. This remodeling reflects plasticity in both pre- and postsynaptic contributors to NMJ structure, and thus perhaps also NM transmission and muscle function.

Mutations in the dysferlin gene (DYSF) lead to human muscular dystrophies known as dysferlinopathies. The dysferlin-deficient A/J mouse develops a mild myopathy after 6 months of age, and when younger models the subclinical phase of the human disease. We subjected the tibialis anterior muscle of 3- to 4-month-old A/J mice to in vivo large-strain injury (LSI) from lengthening contractions and studied the progression of torque loss, myofiber damage, and inflammation afterward. We report that myofiber damage in A/J mice occurs before inflammatory cell infiltration. Peak edema and inflammation, monitored by magnetic resonance imaging and by immunofluorescence labeling of neutrophils and macrophages, respectively, develop 24 to 72 hours after LSI, well after the appearance of damaged myofibers. Cytokine profiles 72 hours after injury are consistent with extensive macrophage infiltration. Dysferlin-sufficient A/WySnJ mice show much less myofiber damage and inflammation and lesser cytokine levels after LSI than do A/J mice. Partial suppression of macrophage infiltration by systemic administration of clodronate-incorporated liposomes fails to suppress LSI-induced damage or to accelerate torque recovery in A/J mice. The findings from our studies suggest that, although macrophage infiltration is prominent in dysferlin-deficient A/J muscle after LSI, it is the consequence and not the cause of progressive myofiber damage., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting secondary to repeated muscle damage and inadequate repair. Elevations in intracellular free Ca²⁺ have been implicated in disease progression, and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1 (SERCA1) overexpression has been shown to ameliorate the dystrophic phenotype in mdx mice. The purpose of this study was to assess the effects of SERCA1 overexpression in the more severe mdx/Utr(-/-) mouse model of DMD. Mice overexpressing SERCA1 were crossed with mdx/Utr ± mice to generate mdx/Utr(-/-)/+SERCA1 mice and compared with wild-type (WT), WT/+SERCA1, mdx/+SERCA1, and genotype controls. Mice were assessed at ∼12 wk of age for changes in Ca²⁺ handling, muscle mass, quadriceps torque, markers of muscle damage, and response to repeated eccentric contractions. SERCA1-overexpressing mice had a two- to threefold increase in maximal sarcoplasmic reticulum Ca²⁺-ATPase activity compared with WT which was associated with normalization in body mass for both mdx/+SERCA1 and mdx/Utr(-/-)/+SERCA1. Torque deficit in the quadriceps after eccentric injury was 2.7-fold greater in mdx/Utr(-/-) vs. WT mice, but only 1.5-fold greater in mdx/Utr(-/-)/+SERCA1 vs. WT mice, an attenuation of 44%. Markers of muscle damage (% centrally nucleated fibers, necrotic area, and serum creatine kinase levels) were higher in both mdx and mdx/Utr(-/-) vs. WT, and all were attenuated by overexpression of SERCA1. These data indicate that SERCA1 overexpression ameliorates functional impairments and cellular markers of damage in a more severe mouse model of DMD. These findings support targeting intracellular Ca²⁺ control as a therapeutic approach for DMD., (Copyright © 2015 the American Physiological Society.)

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle., (Copyright © 2015 the American Physiological Society.)

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated significant alterations in the neuromuscular junction (NMJ) morphology and resulting neuromuscular transmission failure (NTF) 24 h after injury in mdx mice (murine model for DMD). Here we determine the contribution of NMJ morphology and NTF to the recovery of muscle contractile function post-injury. NMJ morphology and NTF rates were assessed day 0 (immediately after injury) and days 1, 7, 14 and 21 after quadriceps injury. Eccentric injury of the quadriceps resulted in a significant loss of maximal torque in both WT (39 ± 6 %) and mdx (76 ± 8 %) with a full recovery in WT by day 7 and in mdx by day 21. Post-injury alterations in NMJ morphology and NTF were found only in mdx, were limited to days 0 and 1, and were independent of changes in MuSK or AChR expression. Such early changes at the NMJ after injury are consistent with mechanical disruption rather than newly forming NMJs. Furthermore, we show that the dense microtubule network that underlies the NMJ is significantly reduced and disorganized in mdx compared to WT. These structural changes at the NMJ may play a role in the increased NMJ disruption and the exaggerated loss of nerve-evoked muscle force seen after injury to dystrophic muscles.

Introduction: We examined the possibility that tetanus toxin can prevent muscle atrophy associated with limb immobility in rats., Methods: While the knee and ankle joints were immobilized unilaterally, the tibialis anterior (TA) muscle on the immobilized side was injected with 1 μl saline or with 1 ng tetanus toxin. After 2 weeks, TA wet weights, contractile forces, and myofiber sizes from the immobilized sides were compared with those from body weight-matched normal animals., Results: Saline group wet weights decreased and produced less absolute twitch and tetanic force and normalized tetanic force compared with the toxin or normal groups. Cross-sectional areas of saline group type I, IIa, and IId myofibers, and the masses of saline group IIa, IId, IIb, and toxin group IIb myofibers, were smaller compared with the normal group., Conclusions: Tetanus toxin prevented common signs of muscle atrophy and may become a useful adjunct to current rehabilitation strategies., (© 2014 Wiley Periodicals, Inc.)

The patient was a 35-year-old man who worked as a pipe fitter. He was referred to a physical therapist by an orthopaedic surgeon for a chief complaint of progressively worsening pain in the medial aspect of the right distal Achilles tendon and heel that began insidiously 12 months earlier, which was consistent with a diagnosis of insertional Achilles tendonitis. Prior radiographs revealed mild calcification at the insertion of the Achilles tendon. Despite physical therapist intervention for 8 weeks, the patient did not improve. Subsequent magnetic resonance imaging revealed a large multiloculated ganglion cyst in the tarsal tunnel.

Introduction: The ability to view individual myofibers is possible with many histological techniques, but not yet with standard in vivo imaging. Optical coherence tomography (OCT) is an emerging technology that can generate high resolution 1-10 μm cross-sectional imaging of tissue in vivo and in real time., Methods: We used OCT to determine architectural differences of tibialis anterior muscles in situ from healthy mice (wild-type [WT], n = 4) and dystrophic mice (mdx, n = 4). After diffusion tensor imaging (DTI) and OCT, muscles were harvested, snap-frozen, and sectioned for staining with wheat germ agglutinin., Results: DTI suggested differences in pennation and OCT was used to confirm this supposition. OCT indicated a shorter intramuscular tendon (WT/mdx ratio of 1.2) and an 18% higher degree of pennation in mdx. Staining confirmed these architectural changes., Conclusions: Architectural changes in mdx muscles, which could contribute to reduction of force, are detectable with OCT., (Copyright © 2012 Wiley Periodicals, Inc.)

Disruptions of ovarian function in women are associated with increased risk of metabolic disease due to dysregulation of peripheral glucose homeostasis in skeletal muscle. Our previous evidence suggests that alterations in skeletal muscle lipid metabolism coupled with altered mitochondrial function may also develop. The objective of this study was to use an integrative metabolic approach to identify potential areas of dysfunction that develop in skeletal muscle from ovariectomized (OVX) female mice compared with age-matched ovary-intact adult female mice (sham). The OVX mice exhibited significant increases in body weight, visceral, and inguinal fat mass compared with sham mice. OVX mice also had significant increases in skeletal muscle intramyocellular lipids (IMCL) compared with the sham animals, which corresponded to significant increases in the protein content of the fatty acid transporters CD36/FAT and FABPpm. A targeted metabolic profiling approach identified significantly lower levels of specific acyl carnitine species and various amino acids in skeletal muscle from OVX mice compared with the sham animals, suggesting a potential dysfunction in lipid and amino acid metabolism, respectively. Basal and maximal mitochondrial oxygen consumption rates were significantly impaired in skeletal muscle fibers from OVX mice compared with sham animals. Collectively, these data indicate that loss of ovarian function results in increased IMCL storage that is coupled with alterations in mitochondrial function and changes in the skeletal muscle metabolic profile.

The most common and severe form of muscular dystrophy is Duchenne muscular dystrophy (DMD), a disorder caused by the absence of dystrophin, a structural protein found on the cytoplasmic surface of the sarcolemma of striated muscle fibres. Considerable attention has been dedicated to studying myofibre damage and muscle plasticity, but there is little information to determine if damage from contraction-induced injury occurs at or near the nerve terminal axon. We used α-bungarotoxin to compare neuromuscular junction (NMJ) morphology in healthy (wild-type, WT) and dystrophic (mdx) mouse quadriceps muscles and evaluated transcript levels of the post-synaptic muscle-specific kinase signalling complex. Our focus was to study changes in NMJs after injury induced with an established in vivo animal injury model. Neuromuscular transmission, electromyography (EMG), and NMJ morphology were assessed 24 h after injury. In non-injured muscle, muscle-specific kinase expression was significantly decreased in mdx compared to WT. Injury resulted in a significant loss of maximal torque in WT (39 ± 6%) and mdx (76 ± 8%) quadriceps, but significant changes in NMJ morphology, neuromuscular transmission and EMG data were found only in mdx following injury. Compared with WT mice, motor end-plates of mdx mice demonstrated less continuous morphology, more disperse acetylcholine receptor aggregates and increased number of individual acetylcholine receptor clusters, an effect that was exacerbated following injury. Neuromuscular transmission failure increased and the EMG measures decreased after injury in mdx mice only. The data show that eccentric contraction-induced injury causes morphological and functional changes to the NMJs in mdx skeletal muscle, which may play a role in excitation-contraction coupling failure and progression of the dystrophic process.

Intermediate filaments (IFs), composed of desmin and keratins, link myofibrils to each other and to the sarcolemma in skeletal muscle. Fast-twitch muscle of mice lacking the IF proteins, desmin and keratin 19 (K19), showed reduced specific force and increased susceptibility to injury in earlier studies. Here we tested the hypothesis that the number of malformed myofibers in mice lacking desmin (Des(-/-)), keratin 19 (K19(-/-)), or both IF proteins (double knockout, DKO) is increased and is coincident with altered excitation-contraction (EC) coupling Ca(2+) kinetics, as reported for mdx mice. We quantified the number of branched myofibers, characterized their organization with confocal and electron microscopy (EM), and compared the Ca(2+) kinetics of EC coupling in flexor digitorum brevis myofibers from adult Des(-/-), K19(-/-), or DKO mice and compared them to age-matched wild type (WT) and mdx myofibers. Consistent with our previous findings, 9.9% of mdx myofibers had visible malformations. Des(-/-) myofibers had more malformations (4.7%) than K19(-/-) (0.9%) or DKO (1.3%) myofibers. Confocal and EM imaging revealed no obvious changes in sarcomere misalignment at the branch points, and the neuromuscular junctions in the mutant mice, while more variably located, were limited to one per myofiber. Global, electrically evoked Ca(2+) signals showed a decrease in the rate of Ca(2+) uptake (decay rate) into the sarcoplasmic reticulum after Ca(2+) release, with the most profound effect in branched DKO myofibers (44% increase in uptake relative to WT). Although branched DKO myofibers showed significantly faster rates of Ca(2+) clearance, the milder branching phenotype observed in DKO muscle suggests that the absence of K19 corrects the defect created by the absence of desmin alone. Thus, there are complex roles for desmin-based and K19-based IFs in skeletal muscle, with the null and DKO mutations having different effects on Ca(2+) reuptake and myofiber branching.

Most animal studies of muscle contractile function utilise the anterior or posterior crural muscle (dorsiflexors and plantarflexors, respectively). An advantage to using these muscles is that the common fibular and tibial nerves are readily accessible, while the small size of the crural muscles is a disadvantage. Working with small muscles not only makes some in vivo imaging and the muscle testing techniques more challenging, but also provides limited amounts of tissue to study. The purpose of this study was to describe a new animal muscle injury model in the quadriceps that results in a significant and reproducible loss of force. The thigh of Sprague Dawley rats (N=5) and C57BL/10 mice (N=5) was immobilised and the ankle was attached to a custom-made lever arm. The femoral nerve was stimulated using subcutaneous electrodes and injury was induced using 50 lengthening ("eccentric") contractions through a 70° arc of knee motion. This protocol produces a significant and reproducible injury, with comparable susceptibility to injury in the rats and mice. This novel model shows that the quadriceps muscle provides a means to study whole muscle contractility, injury, and recovery in vivo. In addition to the usual benefits of an in vivo model, the larger size of the quadriceps facilitates in vivo imaging and provides a significant increase in the amount of tissue available for histology and biochemistry studies. A controlled muscle injury in the quadriceps also allows one to study a muscle, with mixed fibre types, which is extremely relevant to gait in humans and quadruped models., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Neurotrophin-dependent activation of the tyrosine kinase receptor trkB.FL modulates neuromuscular synapse maintenance and function; however, it is unclear what role the alternative splice variant, truncated trkB (trkB.T1), may have in the peripheral neuromuscular axis. We examined this question in trkB.T1 null mice and demonstrate that in vivo neuromuscular performance and nerve-evoked muscle tension are significantly increased. In vitro assays indicated that the gain-in-function in trkB.T1(-/-) animals resulted specifically from an increased muscle contractility, and increased electrically evoked calcium release. In the trkB.T1 null muscle, we identified an increase in Akt activation in resting muscle as well as a significant increase in trkB.FL and Akt activation in response to contractile activity. On the basis of these findings, we conclude that the trkB signaling pathway might represent a novel target for intervention across diseases characterized by deficits in neuromuscular function.

Background: Muscle strains are one of the most common complaints treated by physicians. High-force lengthening contractions can produce very high forces resulting in pain and tissue damage; such strains are the most common cause of muscle injuries. The hamstring muscles are particularly susceptible as they cross two joints and regularly perform lengthening contractions during running. We describe a patient with return to full function after a large hamstring tear., Case Description: We report the case of a 26-year-old man who presented 1 year after a noncontact, left-sided proximal hamstring tear incurred while sprinting. He received no medical treatment or formal rehabilitation. He was able to return to all sports and activities 1 to 2 months after injury, but noted a persistent deformity of the proximal thigh, which led him to seek evaluation. Physical examination, MRI functional tests, and specific muscle tests 1 year after his injury documented a major hamstring tear at the musculotendinous junction with muscle retraction, but no avulsion of the proximal tendon attachment., Literature Review: Surgery often is recommended for major proximal hamstring tendon tears, especially when more than one tendon of origin is ruptured from the ischial tuberosity. Myotendinous tears are treated nonoperatively, but may be associated with decreased strength, prolonged recovery, and recurrence., Purpose and Clinical Relevance: We describe the case of a young man who sustained a hamstring tear, with retraction, at the proximal myotendinous junction, where the biceps femoris and semitendinosus arise from the conjoint tendon. He achieved full functional recovery without medical attention, but had a persistent cosmetic deformity and slight hamstring tightness. This case suggests a benign natural history for this injury and the appropriateness of noninvasive treatment.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins [double knockout (DKO)]. K19-/- mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (-40%) than the absence of K19 (-19%) and played the predominant role in contractile function (-40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (-48%) compared with wild type (-39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle.

The role of S100A1 in skeletal muscle is just beginning to be elucidated. We have previously shown that skeletal muscle fibers from S100A1 knockout (KO) mice exhibit decreased action potential (AP)-evoked Ca(2+) transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulin-binding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that Ca(2+) release was suppressed at all test membrane potentials in S100A1(-/-) fibers. Here we examine the role of S100A1 during physiological AP-induced muscle activity, using an integrative approach spanning AP propagation to muscle force production. With the voltage-sensitive indicator di-8-aminonaphthylethenylpyridinium, we first demonstrate that the AP waveform is not altered in flexor digitorum brevis muscle fibers isolated from S100A1 KO mice. We then use a model for myoplasmic Ca(2+) binding and transport processes to calculate sarcoplasmic reticulum Ca(2+) release flux initiated by APs and demonstrate decreased release flux and greater inactivation of flux in KO fibers. Using in vivo stimulation of tibialis anterior muscles in anesthetized mice, we show that the maximal isometric force response to twitch and tetanic stimulation is decreased in S100A1(-/-) muscles. KO muscles also fatigue more rapidly upon repetitive stimulation than those of wild-type counterparts. We additionally show that fiber diameter, type, and expression of key excitation-contraction coupling proteins are unchanged in S100A1 KO muscle. We conclude that the absence of S100A1 suppresses physiological AP-induced Ca(2+) release flux, resulting in impaired contractile activation and force production in skeletal muscle.

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

Skeletal muscle function is dependent on its highly regular structure. In studies of dystrophic (dy/dy) mice, the proportion of malformed myofibers decreases after prolonged whole muscle stimulation, suggesting that the malformed myofibers are more prone to injury. The aim of this study was to assess morphology and to measure excitation-contraction (EC) coupling (Ca(2+) transients) and susceptibility to osmotic stress (Ca(2+) sparks) of enzymatically isolated muscle fibers of the extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles from young (2-3 mo) and old (8-9 mo) mdx and age-matched control mice (C57BL10). In young mdx EDL, 6% of the myofibers had visible malformations (i.e., interfiber splitting, branched ends, midfiber appendages). In contrast, 65% of myofibers in old mdx EDL contained visible malformations. In the mdx FDB, malformation occurred in only 5% of young myofibers and 11% of old myofibers. Age-matched control mice did not display the altered morphology of mdx muscles. The membrane-associated and cytoplasmic cytoskeletal structures appeared normal in the malformed mdx myofibers. In mdx FDBs with significantly branched ends, an assessment of global, electrically evoked Ca(2+) signals (indo-1PE-AM) revealed an EC coupling deficit in myofibers with significant branching. Interestingly, peak amplitude of electrically evoked Ca(2+) release in the branch of the bifurcated mdx myofiber was significantly decreased compared with the trunk of the same myofiber. No alteration in the basal myoplasmic Ca(2+) concentration (i.e., indo ratio) was seen in malformed vs. normal mdx myofibers. Finally, osmotic stress induced the occurrence of Ca(2+) sparks to a greater extent in the malformed portions of myofibers, which is consistent with deficits in EC coupling control. In summary, our data show that aging mdx myofibers develop morphological malformations. These malformations are not associated with gross disruptions in cytoskeletal or t-tubule structure; however, alterations in myofiber Ca(2+) signaling are evident.

Background: Standard nonoperative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus on how to accelerate recovery., Hypothesis: Local delivery of platelet-rich plasma to injured muscles hastens recovery of function., Study Design: Controlled laboratory study., Methods: In vivo, the tibialis anterior muscles of anesthetized Sprague-Dawley rats were injured by a single (large strain) lengthening contraction or multiple (small strain) lengthening contractions, both of which resulted in a significant injury. The tibialis anterior either was injected with platelet-rich plasma, was injected with platelet-poor plasma as a sham treatment, or received no treatment., Results: Both injury protocols yielded a similar loss of force. The platelet-rich plasma only had a beneficial effect at 1 time point after the single contraction injury protocol. However, platelet-rich plasma had a beneficial effect at 2 time points after the multiple contraction injury protocol and resulted in a faster recovery time to full contractile function. The sham injections had no effect compared with no treatment., Conclusion: Local delivery of platelet-rich plasma can shorten recovery time after a muscle strain injury in a small-animal model. Recovery of muscle from the high-repetition protocol has already been shown to require myogenesis, whereas recovery from a single strain does not. This difference in mechanism of recovery may explain why platelet-rich plasma was more effective in the high-repetition protocol, because platelet-rich plasma is rich in growth factors that can stimulate myogenesis., Clinical Relevance: Because autologous blood products are safe, platelet-rich plasma may be a useful product in clinical treatment of muscle injuries.

The paired pyramidalis muscles are small triangular-shaped muscles that lie between the anterior surface of the rectus abdominus and the posterior surface of the rectus sheath. The precise function of pyramidalis muscles is unclear, but together the muscles are thought to tense the linea alba. The muscles are not always present, or are often unilateral, and vary greatly in size. Their wider inferior margins attach to the pubic symphyses and pubic crests, whereas their narrow superior margins attach to the linea alba. The gross anatomy and innervation of the pyramidalis muscles has been described by others, but their architecture and fiber type have not been determined in previous publications. The purpose of the present paper was therefore to investigate these parameters and place the findings into context for the literature available on this muscle. An example of bilateral pyramidalis muscles was recently encountered in a male cadaver that provided ample tissue for an analysis of its architecture and fiber type. The muscle mass, muscle length, fiber length, and pennation angle of muscle fibers were measured to ascertain physiological cross-sectional area and thereby estimate force production. Fiber type composition was also examined using immunofluorescent labeling. The results show that this is a muscle of mixed fiber type composition, similar to the rectus abdominus, and that the estimated forces generated by this muscle are relatively small.

Objective: To determine if the proliferation of myogenic cells is equally important to recovery of contractile function after 2 different types of contraction-induced muscle injuries., Design: Randomized trial., Setting: Muscle biology laboratory., Animals: Adult male Sprague-Dawley rats., Interventions: Tibialis anterior muscles were injured by a single lengthening contraction with large strain (1R) or multiple lengthening contractions with small strain (MR). The hindlimbs of some animals in each group were irradiated before injury to prevent proliferation of myogenic cells during recovery., Main Outcome Measures: Contractile tension was measured immediately after injury and 3, 7, 14, and 21 days after injury. Permeation to Evans blue dye was used to assay membrane damage. Centrally nucleated fibers and reverse transcriptase-polymerase chain reaction of myoD and myogenin were used as measures of myogenesis., Results: Inhibiting myogenesis prevented the recovery of contractile function after MR, but not after 1R. Both protocols caused Evans blue dye uptake immediately after injury, but Evans blue dye was only retained in fibers for several days after 1R. This suggests that membranes reseal after 1R, but not after MR., Conclusions: The mechanisms that underlie recovery after injuries caused by repeated lengthening contractions and injuries caused by a single lengthening contraction are different. The differences may be important when planning targeted rehabilitation strategies for each type of injury.

Injuries to the anterior cruciate ligament (ACL) result in immediate and long-term morbidity and expense. Young women are more likely to sustain ACL injuries than men who participate in similar athletic and military activities. Although significant attention has focused on the role that female sex hormones may play in this disparity, it is still unclear whether the female ACL also responds to androgens. The purpose of this study was to determine whether the female ACL was an androgen-responsive tissue. To identify and localize androgen receptors in the female ACL, we used Western blotting and immunofluorescent labeling, respectively, of ACL tissue harvested during surgery from young women (n = 3). We then measured ACL stiffness and assessed total testosterone (T) and free [free androgen index (FAI)] testosterone concentrations, as well as relative estradiol to testosterone ratios (E(2)/T and E(2)/FAI) at three consecutive menstrual stages (n = 20). There were significant rank-order correlations between T (0.48, P = 0.031), FAI (0.44, P = 0.053), E(2)/T (-0.71, P < 0.001), E(2)/FAI (-0.63, P = 0.003), and ACL stiffness near ovulation. With the influences of the other variables controlled, there were significant negative partial rank-order correlations between ACL stiffness and E(2)/T (-0.72, P < 0.001) and E(2)/FAI (-0.59, P = 0.012). The partial order residuals for T and FAI were not significant. These findings suggest that the female ACL is an androgen-responsive tissue but that T and FAI are not independent predictors of ACL stiffness near ovulation. Instead, the relationship between T, FAI, and ACL stiffness was likely influenced by another hormone or sex hormone binding globulin.

The purpose of this study was to evaluate the integrity of the muscle membrane and its associated cytoskeleton after a contraction-induced injury. A single eccentric contraction was performed in vivo on the tibialis anterior (TA) of male Sprague-Dawley rats at 900 degrees /s throughout a 90 degrees -arc of motion. Maximal tetanic tension (Po) of the TAs was assessed immediately and at 3, 7, and 21 days after the injury. To evaluate sarcolemmal integrity, we used an Evans blue dye (EBD) assay, and to assess structural changes, we used immunofluorescent labeling with antibodies against contractile (myosin, actin), cytoskeletal (alpha-actinin, desmin, dystrophin, beta-spectrin), integral membrane (alpha- and beta-dystroglycan, sarcoglycan), and extracellular (laminin, fibronectin) proteins. Immediately after injury, P0 was significantly reduced to 4.23 +/- 0.22 N, compared with 8.24 +/- 1.34 N in noninjured controls, and EBD was detected intracellularly in 54 +/- 22% of fibers from the injured TA, compared with 0% in noninjured controls. We found a significant association between EBD-positive fibers and the loss of complete dystrophin labeling. The loss of dystrophin was notable because organization of other components of the subsarcolemmal cytoskeleton was affected minimally (beta-spectrin) or not at all (alpha- and beta-dystroglycan). Labeling with specific antibodies indicated that dystrophin's COOH terminus was selectively more affected than its rod domain. Twenty-one days after injury, contractile properties were normal, fibers did not contain EBD, and dystrophin organization and protein level returned to normal. These data indicate the selective vulnerability of dystrophin after a single eccentric contraction-induced injury and suggest a critical role of dystrophin in force transduction.