Neuromuscular Dysfunction Precedes Cognitive Impairment in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) develops along a continuum that spans years prior to diagnosis. Decreased muscle function and mitochondrial respiration occur years earlier in those that develop AD; however, it is unknown what causes these peripheral phenotypes in a disease of the brain. Exercise promotes muscle, mitochondria, and cognitive health and is proposed to be a potential therapeutic for AD, but no study has investigated how skeletal muscle adapts to exercise training in an AD-like context. Utilizing 5xFAD mice, an AD model that develops ad-like pathology and cognitive impairments around 6 mo of age, we examined in vivo neuromuscular function and exercise adapations (mitochondrial respiration and RNA sequencing) before the manifestation of overt cognitive impairment. We found 5xFAD mice develop neuromuscular dysfunction beginning as early as 4 mo of age, characterized by impaired nerve-stimulated muscle torque production and compound nerve action potential of the sciatic nerve. Furthermore, skeletal muscle in 5xFAD mice had altered, sex-dependent, adaptive responses (mitochondrial respiration and gene expression) to exercise training in the absence of overt cognitive impairment. Changes in peripheral systems, specifically neural communication to skeletal muscle, may be harbingers for AD and have implications for lifestyle interventions, like exercise, in AD.

Dietary Conjugated Linoleic Acid Reduces Body Weight and Fat in Snord116m+/p- and Snord116m-/p- Mouse Models of Prader-Willi Syndrome.

Prader-Willi Syndrome (PWS) is a human genetic condition that affects up to 1 in 10,000 live births. Affected infants present with hypotonia and developmental delay. Hyperphagia and increasing body weight follow unless drastic calorie restriction is initiated. Recently, our laboratory showed that one of the genes in the deleted locus causative for PWS, Snord116, maintains increased expression of hypothalamic Nhlh2, a basic helix-loop-helix transcription factor. We have previously also shown that obese mice with a deletion of Nhlh2 respond to a conjugated linoleic acid (CLA) diet with weight and fat loss. In this study, we investigated whether mice with a paternal deletion of Snord116 (Snord116m+/p-) would respond similarly. We found that while Snord116m+/p- mice and mice with a deletion of both Snord116 alleles were not significantly obese on a high-fat diet, they did lose body weight and fat on a high-fat/CLA diet, suggesting that the genotype did not interfere with CLA actions. There were no changes in food intake or metabolic rate, and only moderate differences in exercise performance. RNA-seq and microbiome analyses identified hypothalamic mRNAs, and differentially populated gut bacteria, that support future mechanistic analyses. CLA may be useful as a food additive to reduce obesity in humans with PWS.

Erratum: Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

[This corrects the article DOI: 10.1016/j.omtm.2021.02.024.].

Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

We tested the hypothesis that voluntary wheel running would complement microdystrophin gene therapy to improve muscle function in young mdx mice, a model of Duchenne muscular dystrophy. mdx mice injected with a single dose of AAV9-CK8-microdystrophin or vehicle at age 7 weeks were assigned to three groups: mdxRGT (run, gene therapy), mdxGT (no run, gene therapy), or mdx (no run, no gene therapy). Wild-type (WT) mice were assigned to WTR (run) and WT (no run) groups. WTR and mdxRGT performed voluntary wheel running for 21 weeks; remaining groups were cage active. Robust expression of microdystrophin occurred in heart and limb muscles of treated mice. mdxRGT versus mdxGT mice showed increased microdystrophin in quadriceps but decreased levels in diaphragm. mdx final treadmill fatigue time was depressed compared to all groups, improved in mdxGT, and highest in mdxRGT. Both weekly running distance (km) and final treadmill fatigue time for mdxRGT and WTR were similar. Remarkably, mdxRGT diaphragm power was only rescued to 60% of WT, suggesting a negative impact of running. However, potential changes in fiber type distribution in mdxRGT diaphragms could indicate an adaptation to trade power for endurance. Post-treatment in vivo maximal plantar flexor torque relative to baseline values was greater for mdxGT and mdxRGT versus all other groups. Mitochondrial respiration rates from red quadriceps fibers were significantly improved in mdxGT animals, but the greatest bioenergetic benefit was observed in the mdxRGT group. Additional assessments revealed partial to full functional restoration in mdxGT and mdxRGT muscles relative to WT. These data demonstrate that voluntary wheel running combined with microdystrophin gene therapy in young mdx mice improved whole-body performance, affected muscle function differentially, mitigated energetic deficits, but also revealed some detrimental effects of exercise. With microdystrophin gene therapy currently in clinical trials, these data may help us understand the potential impact of exercise in treated patients.

A novel murine muscle loading model to investigate Achilles musculotendinous adaptation.

Achilles tendinopathy is a debilitating condition affecting the entire spectrum of society and a condition that increases the risk of tendon rupture. Effective therapies remain elusive, as anti-inflammatory drugs and surgical interventions show poor long-term outcomes. Eccentric loading of the Achilles muscle-tendon unit is an effective physical therapy for treatment of symptomatic human tendinopathy. Here, we introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This model includes the application of tissue (muscle and tendon)-loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human clinical protocols. Under computer control, the foot was rotated through the entire ankle joint range of motion while the plantar flexors simultaneously contracted to simulate body mass loading, consistent with human therapeutic exercises. This approach achieved two key components of the heel drop and raise movement: ankle range of motion coupled with body mass loading. Model development entailed the tuning of parameters such as footplate speed, number of repetitions, number of sets of repetitions, treatment frequency, treatment duration, and treatment timing. Initial model development was carried out on uninjured mice to define a protocol that was well tolerated and nondeleterious to tendon biomechanical function. When applied to a murine Achilles tendinopathy model, muscle loading led to a significant improvement in biomechanical outcome measures, with a decrease in cross-sectional area and an increase in material properties, compared with untreated animals. Our model facilitates the future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.NEW & NOTEWORTHY We introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This innovative model allows for application of muscle loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human loading clinical treatment. Our model facilitates future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.

Potential cross-talk between muscle and tendon in Duchenne muscular dystrophy.

PURPOSE: To describe potential signaling (cross-talk) between dystrophic skeletal muscle and tendon in Duchenne muscular dystrophy. MATERIALS AND METHODS: Review of Duchenne muscular dystrophy and associated literature relevant to muscle-tendon cross-talk. RESULTS AND CONCLUSIONS: Duchenne muscular dystrophy results from the absence of the protein dystrophin and the associated dystrophin - glycoprotein complex, which are thought to provide both structural support and signaling functions for the muscle fiber. In addition, there are other potential signal pathways that could represent cross-talk between muscle and tendon, particularly at the myotendinous junction. Duchenne muscular dystrophy is characterized by multiple pathophysiologic mechanisms. Herein, we explore three of these: (1) the extracellular matrix, fibrosis, and fat deposition; (2) satellite cells; and (3) tensegrity. A key signaling protein that emerged in each was transforming growth factor - beta one (TGF-ß1).].

A novel ex vivo protocol to mimic human walking gait: implications for Duchenne muscular dystrophy.

We developed a novel ex vivo mouse protocol to mimic in vivo human soleus muscle function predicted by musculoskeletal simulations to better understand eccentric contractions during gait and ultimately to better understand their effects in Duchenne muscular dystrophy (DMD) muscles. DMD muscles are susceptible to eccentric injury because the protein dystrophin is absent. The mdx mouse, a DMD model that also lacks dystrophin, is often subjected to ex vivo acute but nonphysiological eccentric injury protocols. It is possible these acute protocols either over- or underestimate eccentric stresses and strains compared with those from humans during gait. To explore this possibility, healthy human soleus excitation, force, and length change profiles during a single walking stride (gait cycle) were simulated using OpenSim and then scaled to an ex vivo mouse soleus preparation based on muscle architectural measurements. Aurora Scientific, Inc., software and a 701C electrical stimulator were modified to discretely modulate muscle stimulation voltage at constant frequency and finely control muscle length changes to produce a force pattern that correctly mimicked the gait cycle from simulations. In a proof-of-principle study, wild-type and mdx mice soleus muscles were subjected to 25 gait cycles. Modest fatigue was evident in the muscles at the 25th versus first gait cycle for both genotypes, but both rapidly recovered isometric force within 1 min of the last cycle. These data indicate that the ex vivo gait protocol was well tolerated. More important, this protocol provides a novel assessment tool to determine the effects of physiological eccentric contractions on dystrophic muscle.NEW & NOTEWORTHY A novel ex vivo mouse soleus protocol that mimics scaled length change and excitation profiles predicted by a mathematical model of human soleus during gait is presented. A custom stimulator was developed that enabled an innovative muscle stimulation technique to modulate voltage to closely match the excitation pattern of human soleus during gait. This ex vivo protocol provides assessment of simulated human movement in mouse muscle, including components of eccentric contractions.

Laminin-111 protein therapy after disease onset slows muscle disease in a mouse model of laminin-α2 related congenital muscular dystrophy.

Laminin-α2 related congenital muscular dystrophy (LAMA2-CMD) is a fatal muscle disease caused by mutations in the LAMA2 gene. Laminin-α2 is critical for the formation of laminin-211 and -221 heterotrimers in the muscle basal lamina. LAMA2-CMD patients exhibit hypotonia from birth and progressive muscle loss that results in developmental delay, confinement to a wheelchair, respiratory insufficiency and premature death. There is currently no cure or effective treatment for LAMA2-CMD. Several studies have shown laminin-111 can serve as an effective protein-replacement therapy for LAMA2-CMD. Studies have demonstrated early treatment with laminin-111 protein results in an increase in life expectancy and improvements in muscle pathology and function. Since LAMA2-CMD patients are often diagnosed after advanced disease, it is unclear if laminin-111 protein therapy at an advanced stage of the disease can have beneficial outcomes. In this study, we tested the efficacy of laminin-111 protein therapy after disease onset in a mouse model of LAMA2-CMD. Our results showed laminin-111 treatment after muscle disease onset increased life expectancy, promoted muscle growth and increased muscle stiffness. Together these studies indicate laminin-111 protein therapy either early or late in the disease process could serve as an effective protein replacement therapy for LAMA2-CMD.

Pro-opiomelanocortin Neurons and the Transcriptional Regulation of Motivated Exercise.

Hypothalamic pro-opiomelanocortin (POMC) neurons are key sensory neurons for energy balance. The basic helix-loop-helix transcription factor NHLH2 is expressed in POMC neurons, and Nhlh2 knockout mice show adult-onset obesity with low exercise behavior. Evidence is presented to explore the hypothesis that NHLH2 transcriptional activity within POMC neurons is crucial for maintaining motivated spontaneous activity and enforced exercise.

AAV-Mediated Gene Transfer Restores a Normal Muscle Transcriptome in a Canine Model of X-Linked Myotubular Myopathy.

Multiple clinical trials employing recombinant adeno-associated viral (rAAV) vectors have been initiated for neuromuscular disorders, including Duchenne and limb-girdle muscular dystrophies, spinal muscular atrophy, and recently X-linked myotubular myopathy (XLMTM). Our previous work on a canine model of XLMTM showed that a single rAAV8-cMTM1 systemic infusion corrected structural abnormalities within the muscle and restored contractile function, with affected dogs surviving more than 4 years post injection. This remarkable therapeutic efficacy presents a unique opportunity to identify the downstream molecular drivers of XLMTM pathology and to what extent the whole muscle transcriptome is restored to normal after gene transfer. Herein, RNA-sequencing was used to examine the transcriptomes of the Biceps femoris and Vastus lateralis in a previously described canine cohort that showed dose-dependent clinical improvements after rAAV8-cMTM1 gene transfer. Our analysis confirmed several dysregulated genes previously observed in XLMTM mice but also identified transcripts linked to XLMTM pathology. We demonstrated XLMTM transcriptome remodeling and dose-dependent normalization of gene expression after gene transfer and created metrics to pinpoint potential biomarkers of disease progression and correction.

Cardioprotective effects of idebenone do not involve ROS scavenging: Evidence for mitochondrial complex I bypass in ischemia/reperfusion injury.

Novel therapeutic strategies to treat mitochondrial deficiencies in acute coronary syndromes are needed. Complex I of the mitochondrial electron transport system is damaged following ischemia/reperfusion (I/R) injury. This disruption contributes to aberrant electron transport, diminished bioenergetics, an altered redox environment, and mitochondrial damage involved in tissue injury. In this study, we determined the cardiac and mitochondrial effects of idebenone, a benzoquinone currently in several clinical trials with purported 'antioxidant' effects. We employed complimentary models of ischemia/reperfusion injury in perfused hearts, permeabilized cardiac fibers, isolated mitochondria, and in cells to elucidate idebenone's cardioprotective mechanism(s). In ex vivo whole hearts, infarct size was markedly reduced with post-ischemic idebenone treatment (25 ±â€¯5% area at risk, AAR) compared to controls (56 ±â€¯6% AAR, P < .05). Several parameters of hemodynamic function were also significantly improved after idebenone treatment. Parallel studies of anoxia/reoxygenation were conducted using isolated mitochondria and permeabilized ventricular fibers. In isolated mitochondria, we simultaneously monitored respiration and ROS emission. Idebenone treatment modestly elevated succinate-derived H2O2 production when compared to vehicle control (1.34 ±â€¯0.05 vs 1.21 ±â€¯0.05%, H2O2/O2 respectively, P < .05). Isolated mitochondria subjected to anoxia/reoxygenation demonstrated higher rates of respiration with idebenone treatment (2360 ±â€¯69 pmol/s*mg) versus vehicle control (1995 ±â€¯101 pmol/s*mg). Both mitochondria and permeabilized cardiac fibers produced high rates of H2O2 after anoxia/reoxygenation, with idebenone showing no discernable attenuation on H2O2 production. These insights were further investigated with studies in mitochondria isolated from reperfused ventricle. The profound decrease in complex-I dependent respiration after ischemia/reperfusion (701 ±â€¯59 pmolO2/s*mg compared to 1816 ±â€¯105 pmol O2/s*mg in normoxic mitochondria) was attenuated with idebenone treatment (994 ±â€¯76 vs pmol O2/s*mg, P < .05). Finally, the effects of idebenone were determined using permeabilized cell models with chemical inhibition of complex I. ADP-dependent oxidative phosphorylation capacity was significantly higher in complex-I inhibited cells treated acutely with idebenone (89.0 ±â€¯4.2 pmol/s*million cells versus 70.1 ±â€¯8.2 pmol/s*million cells in untreated cells). Taken together, these data indicate that the cardioprotective effects of idebenone treatment do not involve ROS-scavenging but appear to involve augmentation of the quinone pool, thus providing reducing equivalents downstream of complex I. As this compound is already in clinical trials for other indications, it may provide a safe and useful approach to mitigate ischemia/reperfusion injury in patients.

Long-term effects of systemic gene therapy in a canine model of myotubular myopathy.

INTRODUCTION: X-linked myotubular myopathy (XLMTM), a devastating pediatric disease caused by the absence of the protein myotubularin, results from mutations in the MTM1 gene. While there is no cure for XLMTM, we previously reported effects of MTM1 gene therapy using adeno-associated virus (AAV) vector on muscle weakness and pathology in MTM1-mutant dogs. Here, we followed 2 AAV-infused dogs over 4 years. METHODS: We evaluated gait, strength, respiration, neurological function, muscle pathology, AAV vector copy number (VCN), and transgene expression. RESULTS: Four years following AAV-mediated gene therapy, gait, respiratory performance, neurological function and pathology in AAV-infused XLMTM dogs remained comparable to their healthy littermate controls despite a decline in VCN and muscle strength. CONCLUSIONS: AAV-mediated gene transfer of MTM1 in young XLMTM dogs results in long-term expression of myotubularin transgene with normal muscular performance and neurological function in the absence of muscle pathology. These findings support a clinical trial in patients. Muscle Nerve 56: 943-953, 2017.

Classical and adaptive control of ex vivo skeletal muscle contractions using Functional Electrical Stimulation (FES).

Functional Electrical Stimulation is a promising approach to treat patients by stimulating the peripheral nerves and their corresponding motor neurons using electrical current. This technique helps maintain muscle mass and promote blood flow in the absence of a functioning nervous system. The goal of this work is to control muscle contractions from FES via three different algorithms and assess the most appropriate controller providing effective stimulation of the muscle. An open-loop system and a closed-loop system with three types of model-free feedback controllers were assessed for tracking control of skeletal muscle contractions: a Proportional-Integral (PI) controller, a Model Reference Adaptive Control algorithm, and an Adaptive Augmented PI system. Furthermore, a mathematical model of a muscle-mass-spring system was implemented in simulation to test the open-loop case and closed-loop controllers. These simulations were carried out and then validated through experiments ex vivo. The experiments included muscle contractions following four distinct trajectories: a step, sine, ramp, and square wave. Overall, the closed-loop controllers followed the stimulation trajectories set for all the simulated and tested muscles. When comparing the experimental outcomes of each controller, we concluded that the Adaptive Augmented PI algorithm provided the best closed-loop performance for speed of convergence and disturbance rejection.

Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

Contractile efficiency of dystrophic mdx mouse muscle: in vivo and ex vivo assessment of adaptation to exercise of functional end points.

Progressive weakness is a typical feature of Duchenne muscular dystrophy (DMD) patients and is exacerbated in the benign mdx mouse model by in vivo treadmill exercise. We hypothesized a different threshold for functional adaptation of mdx muscles in response to the duration of the exercise protocol. In vivo weakness was confirmed by grip strength after 4, 8, and 12 wk of exercise in mdx mice. Torque measurements revealed that exercise-related weakness in mdx mice correlated with the duration of the protocol, while wild-type (WT) mice were stronger. Twitch and tetanic forces of isolated diaphragm and extensor digitorum longus (EDL) muscles were lower in mdx compared with WT mice. In mdx, both muscle types exhibited greater weakness after a single exercise bout, but only in EDL after a long exercise protocol. As opposite to WT muscles, mdx EDL ones did not show any exercise-induced adaptations against eccentric contraction force drop. qRT-PCR analysis confirmed the maladaptation of genes involved in metabolic and structural remodeling, while damage-related genes remained significantly upregulated and angiogenesis impaired. Phosphorylated AMP kinase level increased only in exercised WT muscle. The severe histopathology and the high levels of muscular TGF-ß1 and of plasma matrix metalloproteinase-9 confirmed the persistence of muscle damage in mdx mice. Therefore, dystrophic muscles showed a partial degree of functional adaptation to chronic exercise, although not sufficient to overcome weakness nor signs of damage. The improved understanding of the complex mechanisms underlying maladaptation of dystrophic muscle paves the way to a better managment of DMD patients.NEW & NOTEWORTHY We focused on the adaptation/maladaptation of dystrophic mdx mouse muscles to a standard protocol of exercise to provide guidance in the development of more effective drug and physical therapies in Duchenne muscular dystrophy. The mdx muscles showed a modest functional adaptation to chronic exercise, but it was not sufficient to overcome the progressive in vivo weakness, nor to counter signs of muscle damage. Therefore, a complex involvement of multiple systems underlies the maladaptive response of dystrophic muscle.

In Vitro Assays to Determine Skeletal Muscle Physiologic Function.

In vitro muscle contractile function assays are important to characterize the differences between different muscle types (e.g., slow vs. fast), between a diseased and non-diseased muscle, or importantly, to demonstrate the efficacy of a muscle treatment such as a drug, an overexpressed transgene, or knockout of a specific gene. Fundamental contractile properties can be assessed by twitch, tetanic, force-frequency, force-velocity, and fatigue assays. Many of these assays are conducted with the muscle at a constant length, e.g., an isometric contraction. However, to better represent the dynamic purpose of muscles in vivo (e.g., to move limbs), dynamic assays such as the force-velocity (concentric contractions) or stretch-injury (eccentric contractions) should also be obtained. Characterizing skeletal muscle function in vitro is a powerful approach to demonstrate efficacy of a treatment to rescue diseased muscle and to assess functional regeneration.

Defective excitation-contraction coupling is partially responsible for impaired contractility in hindlimb muscles of Stac3 knockout mice.

The Stac3 gene is exclusively expressed in skeletal muscle, and Stac3 knockout is perinatal lethal in mice. Previous data from Stac3-deleted diaphragms indicated that Stac3-deleted skeletal muscle could not contract because of defective excitation-contraction (EC) coupling. In this study, we determined the contractility of Stac3-deleted hindlimb muscle. In response to frequent electrostimulation, Stac3-deleted hindlimb muscle contracted but the maximal tension generated was only 20% of that in control (wild type or heterozygous) muscle (P < 0.05). In response to high [K(+)], caffeine, and 4-chloro-m-cresol (4-CMC), the maximal tensions generated in Stac3-deleted muscle were 29% (P < 0.05), 58% (P = 0.08), and 55% (P < 0.05) of those in control muscle, respectively. In response to 4-CMC or caffeine, over 90% of myotubes formed from control myoblasts contracted, but only 60% of myotubes formed from Stac3-deleted myoblasts contracted (P = 0.05). However, in response to 4-CMC or caffeine, similar increases in intracellular calcium concentration were observed in Stac3-deleted and control myotubes. Gene expression and histological analyses revealed that Stac3-deleted hindlimb muscle contained more slow type-like fibers than control muscle. These data together confirm a critical role of STAC3 in EC coupling but also suggest that STAC3 may have additional functions in skeletal muscle, at least in the hindlimb muscle.

Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures.

BACKGROUND: Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. METHODS: To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn (+/-)) whippets. A total of four GRippets (dystrophic and Mstn (+/-)), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. RESULTS: Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no differential expression between GRMD and GRippet dogs. Satellite cell exhaustion was not observed in GRippets up to 3 years of age. CONCLUSIONS: Partial myostatin loss may exaggerate selective muscle hypertrophy or atrophy/hypoplasia in GRMD dogs and worsen contractures. While muscle imbalance is not a feature of myostatin inhibition in mdx mice, findings in a larger animal model could translate to human experience with myostatin inhibitors.

The SH3 and cysteine-rich domain 3 (Stac3) gene is important to growth, fiber composition, and calcium release from the sarcoplasmic reticulum in postnatal skeletal muscle.

BACKGROUND: The SH3 and cysteine-rich domain 3 (Stac3) gene is specifically expressed in the skeletal muscle. Stac3 knockout mice die perinatally. In this study, we determined the potential role of Stac3 in postnatal skeletal muscle growth, fiber composition, and contraction by generating conditional Stac3 knockout mice. METHODS: We disrupted the Stac3 gene in 4-week-old male mice using the Flp-FRT and tamoxifen-inducible Cre-loxP systems. RESULTS: RT-qPCR and western blotting analyses of the limb muscles of target mice indicated that nearly all Stac3 mRNA and more than 70 % of STAC3 protein were deleted 4 weeks after tamoxifen injection. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei, with no effect on the total myofiber number. Grip strength and grip time tests indicated that postnatal Stac3 deletion decreased limb muscle strength in mice. Muscle contractile tests revealed that postnatal Stac3 deletion reduced electrostimulation-induced but not the ryanodine receptor agonist caffeine-induced maximal force output in the limb muscles. Calcium imaging analysis of single flexor digitorum brevis myofibers indicated that postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. CONCLUSIONS: This study demonstrates that STAC3 is important to myofiber hypertrophy, myofiber-type composition, contraction, and excitation-induced calcium release from the sarcoplasmic reticulum in the postnatal skeletal muscle.

Muscle pathology, limb strength, walking gait, respiratory function and neurological impairment establish disease progression in the p.N155K canine model of X-linked myotubular myopathy.

BACKGROUND: Loss-of-function mutations in the myotubularin (MTM1) gene cause X-linked myotubular myopathy (XLMTM), a fatal, inherited pediatric disease that affects the entire skeletal musculature. Labrador retriever dogs carrying an MTM1 missense mutation exhibit strongly reduced synthesis of myotubularin, the founder member of a lipid phosphatase required for normal skeletal muscle function. The resulting canine phenotype resembles that of human patients with comparably severe mutations, and survival does not normally exceed 4 months. METHODS: We studied MTM1 mutant dogs (n=7) and their age-matched control littermates (n=6) between the ages of 10 and 25 weeks. Investigators blinded to the animal identities sequentially measured limb muscle pathology, fore- and hind limb strength, walking gait, respiratory function and neurological impairment. RESULTS: MTM1-mutant puppies display centrally-nucleated myofibers of reduced size and disrupted sarcotubular architecture progressing until the end of life, an average of 17 weeks. In-life measures of fore- and hind limb strength establish the rate at which XLMTM muscles weaken, and their corresponding decrease in gait velocity and stride length. Pulmonary function tests in affected dogs reveal a right-shifted relationship between peak inspiratory flow (PIF) and inspiratory time (TI); neurological assessments indicate that affected puppies as young as 10 weeks show early signs of neurological impairment (neurological severity score, NSS =8.6±0.9) with progressive decline (NSS =5.6±1.7 at 17 weeks-of-age). CONCLUSIONS: Our findings document the rate of disease progression in a large animal model of XLMTM and lay a foundation for preclinical studies.