Duchenne muscular dystrophy is a fatal disease characterized by persistent skeletal muscle degeneration, inflammation, and fibrosis. Gene therapy using an adeno-associated virus-derived vector and a microdystrophin transgene is currently under investigation in patients, but the impact of physical activity on long-term therapeutic outcome remains poorly understood. Recently, we reported that 21 weeks of voluntary wheel running complemented the positive endurance and muscle function outcomes of gene therapy in mdx mice. In the present study, we performed a transcriptomic analysis of the gene expression changes associated with functional recovery in the diaphragm with a focus on genes and signaling pathways related to the inflammatory response. RNA sequencing and bioinformatic analysis revealed 2,881 dysregulated genes in untreated and unexercised mdx mice including inflammatory and fibrotic signaling pathways frequently affected in Duchenne muscular dystrophy patients. Among the dysregulated genes, 774 were rescued toward WT level after adeno-associated virus microdystrophin injection. Importantly, 93% of the rescued genes were maintained by voluntary running, which indicates that physical exercise has no significant impact on the outcome of gene therapy-rescued genes in the mdx diaphragm. Our study provides vital information that could help guide DMD patient follow-up protocols after treatment with gene therapy.

Achilles tendinopathy limits mobility and decreases quality of life. Physical therapy (eccentric muscle loading) improves tendon function; however, the underlying mechanisms are unknown. This study investigated the effect of load magnitude and treatment duration in a mouse Achilles tendinopathy model. We hypothesized that loading would upregulate signaling and metabolic transcriptional networks associated with improved tendon healing. Mice were randomly assigned to muscle loading groups (50 or 100% body weight (BW)) or age-matched injured/untreated (IU) and naïve control groups. Following induction of Achilles tendinopathy via paired TGFB-β1 injections, loading was performed for 1, 2, or 4 weeks, mice euthanized, and Achilles tendons harvested for transcriptomics. The exercised groups exhibited relatively converging transcriptional patterns at 4 weeks, while the IU group was tightly associated with the naïve group over time, and diverging from both exercised groups at 2 and 4 weeks. Two weeks of exercise at either 50 or 100% BW load resulted in uniquely expressed gene networks not present in unexercised controls. Comparative assessment of the expression profile and functional annotation of networks across groups revealed that exercise differentially affected the innate immune response, sensory innervation and collagen biosynthesis during tendon repair. Ingenuity Pathway Analysis further suggests that 50% BW loading is associated with a shorter pro-inflammatory response and early matrix deposition in healing tendons compared to 100% BW loading. The transcriptional alterations seen in response to 50% BW eccentric muscle loading support the benefits of controlled loading exercises when treating Achilles tendinopathy.

Alzheimer's disease (AD) progresses along a continuum for years to possibly decades prior to cognitive decline. Although AD is primarily an age-related brain pathology, increasing evidence indicates dysfunction in peripheral nerves and skeletal muscle may manifest early in the disease progression. However, the underlying cause(s) for peripheral nerve dysfunction leading to impaired skeletal muscle torque production are not understood. Sciatic nerves from 5xFAD and wild-type (WT) mice were analyzed by tandem mass tag (TMT)-labeled proteomics at 3, 4, and 7 months, identifying proteome remodeling coincides with functional declines at 4 months particularly in pathways linked to mitochondrial turnover, calcium handling, and inflammation. We hypothesized either voluntary wheel running or donepezil treatment, begun prior to neuromuscular decline, would delay manifestation of neuromuscular impairment in 5xFAD mice. Separate cohorts, using 3-month-old 5xFAD mice and WT littermates, were given voluntary wheel access for 4 weeks or treated with the acetylcholinesterase inhibitor donepezil. We assessed tibial nerve stimulated plantar flexion torque and sciatic nerve compound (motor) neuron action potential (CNAP) in vivo at 4 months. Both exercise and donepezil attenuated in vivo nerve-stimulated muscle torque and CNAP dysfunction. Further, both exercise and donepezil attenuated the proteomic remodeling of the sciatic nerve through both shared and independent mechanisms that converged on mitochondria-centric pathways. Our findings in the 5xFAD model of AD support the notion that early phenotypes of AD are evident in the periphery that may have implications for timing of interventions.

Although eccentric contraction protocols are widely used to study the pathophysiology and potential treatments for Duchenne muscular dystrophy (DMD), they do not reflect the stresses, strains, strain rates, and excitation profiles that DMD muscles experience during human daily functional tasks, like walking. This limitation of eccentric contractions may impede our understanding of disease progression in DMD and proper assessment of treatment efficacy. The goals of this study were to examine the extent of force loss induced by a gait cycling protocol we developed, and compare to that from a typical eccentric contraction protocol in soleus and extensor digitorum longus (EDL) muscles of mdx mice. To achieve this goal, mdx soleus and EDL muscles were subjected to eccentric contractions at three levels of strain (10%, 20% and 30% optimal length Lo) and up to 200 cycles of our gait cycling protocol that mimicked the length changes and excitation patterns of the corresponding muscles during human walking gait. Our results showed that EDL but not soleus muscles had significant losses in isometric tetanic forces after the cycling protocols. Compared to the eccentric contraction protocol, the decrements in contractile performance from the cycling protocol were similar to those from the eccentric contractions at 10% in soleus and 20% Lo in EDL. Together, these results indicated the gait cycling protocol is a valuable experimental approach to better understand disease progression and to screen and evaluate efficacy of novel therapeutics for DMD.

Background: Alzheimer's disease (AD) progresses along a continuum for years to possibly decades prior to cognitive decline and clinical diagnosis. Preclinical AD is associated with neuromuscular dysfunction. We previously characterized early neuromuscular impairment prior to cognitive decline at 4 months of age in the 5xFAD mouse model of AD. However, the underlying cause(s) for peripheral nerve dysfunction leading to impaired skeletal muscle torque production are not understood, therefore limiting interventional capacity. We hypothesized that either voluntary wheel running or donepezil treatment, begun prior to neuromuscular decline, would delay manifestation of neuromuscular impairment in 5xFAD mice. Methods: Sciatic nerves from 5xFAD and wild-type (WT) mice were analyzed by tandem mass tag (TMT)-labeled proteomics at 3, 4, and 7 months to investigate proteome remodeling. Separate cohorts, using 3-month-old 5xFAD mice and WT littermates given voluntary wheel access for 4 weeks or treated with the acetylcholinesterase inhibitor donepezil to test if neuromuscular dysfunction could be attenuated. Afterwards, we assessed tibial nerve stimulated plantar flexion torque and sciatic nerve compound (motor) neuron action potential (CNAP) in-vivo at 4 months. Additionally, we performed TMT-labeled proteomics to ascertain the effect of exercise and donepezil treatments on sciatic proteome. Results: Sciatic nerves in 5xFAD mice exhibited proteomic remodeling from 3 to 4 months, particularly in pathways linked to mitochondrial turnover, calcium handling, lipid metabolism, and inflammation, coinciding with onset of neuromuscular dysfunction. Both exercise and donepezil attenuated in nerve-stimulated muscle torque and CNAP dysfunction. Both exercise and donepezil attenuated proteomic remodeling of the sciatic nerve involving mitochondrial-centric processes through both shared and independent mechanisms. Conclusions: Declines in neuromuscular function may be pre-clinical identifiers for AD that share pathway similarities with noted central effects of the pathology on the brain. Our findings highlight the importance of a systemic approach to AD pathology and importance of disease state in interventional efficacy. Graphical abstract:Created in Biorender.

Body movement relies on skeletal muscles generating power to move limbs effectively. Power is defined as force multiplied by velocity: a muscle produces force at a specific velocity (the speed of muscle shortening) and this results in power. In diseases like Duchenne Muscular Dystrophy (DMD), the absence of dystrophin weakens muscles and impairs their shortening velocity, leading to decreased power and consequently, impaired movement. Additionally, the diaphragm and heart muscles are also affected in DMD, causing difficulty breathing and impaired cardiac function. Compromised cardiorespiratory function can ultimately lead to death. Given the complex etiology of DMD and the essential role of power in all affected muscles, it is crucial to assess potential treatments for their effectiveness in improving muscle function. This review focuses on fundamental physiological assays used to evaluate muscle function in skeletal and diaphragm muscles. Common assays include force-frequency, force-velocity, power, and eccentric protocols, which are conducted ex vivo , in situ , and in vivo in small rodents (such as mice and rats) and larger intermediate animal models such as the Golden Retriever Muscular Dystrophy dog. Existing data support the use of skeletal muscle contractile assays as objective tools for assessing the efficacy of treatments.

Abstract 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.

We tested the effects of prolonged voluntary wheel running on the muscle function of mdx mice treated with one of two different microdystrophin constructs. At 7 weeks of age mdx mice were injected with a single dose of AAV9-CK8-microdystrophin with (gene therapy 1, GT1) or without (gene therapy 2, GT2) the nNOS-binding domain and were assigned to one of four gene therapy treated groups: mdxRGT1 (run, GT1), mdxGT1 (no run, GT1), or mdxRGT2 (run,GT2), mdxGT2 (no run, GT2). There were two mdx untreated groups injected with excipient: mdxR (run, no gene therapy) and mdx (no run, no gene therapy). A third no treatment group, Wildtype (WT) received no injection and did not run. mdxRGT1, mdxRGT2 and mdxR performed voluntary wheel running for 52 weeks; WT and remaining mdx groups were cage active. Robust expression of microdystrophin occurred in diaphragm, quadriceps, and heart muscles of all treated mice. Dystrophic muscle pathology was high in diaphragms of non-treated mdx and mdxR mice and improved in all treated groups. Endurance capacity was rescued by both voluntary wheel running and gene therapy alone, but their combination was most beneficial. All treated groups increased in vivo plantarflexor torque over both mdx and mdxR mice. mdx and mdxR mice displayed ∼3-fold lower diaphragm force and power compared to WT values. Treated groups demonstrated partial improvements in diaphragm force and power, with mdxRGT2 mice experiencing the greatest improvement at ∼60% of WT values. Evaluation of oxidative red quadriceps fibers revealed the greatest improvements in mitochondrial respiration in mdxRGT1 mice, reaching WT levels. Interestingly, mdxGT2 mice displayed diaphragm mitochondrial respiration values similar to WT but mdxRGT2 animals showed relative decreases compared to the no run group. Collectively, these data demonstrate that either microdystrophin construct combined with voluntary wheel running increased in vivo maximal muscle strength, power, and endurance. However, these data also highlighted important differences between the two microdystrophin constructs. GT1, with the nNOS-binding site, improved more markers of exercise-driven adaptations in metabolic enzyme activity of limb muscles, while GT2, without the nNOS-binding site, demonstrated greater protection of diaphragm strength after chronic voluntary endurance exercise but decreased mitochondrial respiration in the context of running.

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.

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-beta 1).].

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.

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.

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.

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). Previous work from our laboratory on a canine model of XLMTM showed that a single rAAV8-cMTM1 systemic infusion corrects structural abnormalities within the muscle and restores contractile function, with affected dogs surviving more than four years post injection. This exceptional 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 showing dose-dependent clinical improvements after rAAV8-cMTM1 gene transfer. Our analysis confirmed several dysregulated genes previously observed in XLMTM mice, but also identified new transcripts linked to XLMTM pathology. We demonstrated XLMTM transcriptome remodeling and dose-dependent normalization of gene expression after gene transfer and created new metrics to pinpoint potential biomarkers of disease progression and correction.

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.

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.

Background: Heart failure is associated with inflammation, but the precise immune mechanisms that contribute to cardiomyopathy remain elusive. Myocarditis is a rare toxicity of checkpoint inhibitors, which are oncologic therapies that block signaling through the immunoinhibitory molecule PD-1. We investigated whether PD-1 attenuates cardiac immune responses. Methods: Expression of PD-1 and its ligand PD-L1 on cardiac cell populations were assessed by flow cytometry. The functional implications of PD-1 signaling were determined using PD-1 blocking antibodies (Abs) in two surgical models of cardiac injury: ischemia/reperfusion and transverse aortic constriction (TAC). Ischemic injury was measured by troponin release and histology. Systolic function was assayed by echocardiography, diastolic function by invasive pressure-volume loops, and fibrosis by histology. Results: PD-L1 is expressed on most cardiac endothelial cells and a small fraction of cardiomyocytes. While intracardiac T cells do not express PD-1 under baseline conditions, 15% of both CD4 and CD8 T cells express PD-1 after TAC. Blocking PD-1 signaling after ischemic injury did not affect troponin levels (5.8 ng/ml in mice receiving isotype control (IC) Ab vs 7.8 ng/ml in mice receiving anti-PD-1 Ab, p=0.26), size of infarct (1.6% of ventricular cross-sectional area for IC Ab vs 2.0% for anti-PD-1 Ab, p=0.27), or systolic function (fractional shortening 52.6% for IC Ab vs 53.2% for anti-PD-1 Ab, p=0.92). Similarly, abrogation of PD-1 signaling after TAC did not worsen systolic dysfunction (fractional shortening 36.8% for IC Ab vs 32.4% for anti-PD-1 Ab, p=0.38), diastolic function (dP/dT minimum -9445 mmHg/s vs -10950 mmHg/s, p=0.44; Tau 6.8 ms for IC Ab vs 6.0 ms for anti-PD-1 Ab, p=0.45), or the extent of cardiac fibrosis (1.4% of ventricular cross-sectional area for IC vs 1.7% for anti-PD-1 Ab, p=0.41). Conclusion: Collectively, these data do not implicate PD-1 as an important regulator of intracardiac immune responses in the injury models tested. This suggests that additional pathways may contribute to myocarditis occurring in cancer patients treated with anti-PD-1 Ab, the identification of which could provide insight into immune mechanisms active in heart failure.