Our study was to characterize sarcopenia in C57BL/6J mice using a clinically relevant definition to investigate the underlying molecular mechanisms. Aged male (23-32 months old) and female (27-28 months old) C57BL/6J mice were classified as non-, probable-, or sarcopenic based on assessments of grip strength, muscle mass, and treadmill running time, using 2 SDs below the mean of their young counterparts as cutoff points. A 9%-22% prevalence of sarcopenia was identified in 23-26 month-old male mice, with more severe age-related declines in muscle function than mass. Females aged 27-28 months showed fewer sarcopenic but more probable cases compared with the males. As sarcopenia progressed, a decrease in muscle contractility and a trend toward lower type IIB fiber size were observed in males. Mitochondrial biogenesis, oxidative capacity, and AMPK-autophagy signaling decreased as sarcopenia progressed in males, with pathways linked to mitochondrial metabolism positively correlated with muscle mass. No age- or sarcopenia-related changes were observed in mitochondrial biogenesis, OXPHOS complexes, AMPK signaling, mitophagy, or atrogenes in females. Our results highlight the different trajectories of age-related declines in muscle mass and function, providing insights into sex-dependent molecular changes associated with sarcopenia progression, which may inform the future development of novel therapeutic interventions.

Pulmonary disorders affect 40%-80% of individuals with obesity. Respiratory muscle dysfunction is linked to these conditions; however, its pathophysiology remains largely undefined. Mice subjected to diet-induced obesity (DIO) develop diaphragm muscle weakness. Increased intradiaphragmatic adiposity and extracellular matrix (ECM) content correlate with reductions in contractile force. Thrombospondin-1 (THBS1) is an obesity-associated matricellular protein linked with muscular damage in genetic myopathies. THBS1 induces proliferation of fibro-adipogenic progenitors (FAPs) - mesenchymal cells that differentiate into adipocytes and fibroblasts. We hypothesized that THBS1 drives FAP-mediated diaphragm remodeling and contractile dysfunction in DIO. We tested this by comparing the effects of dietary challenge on diaphragms of wild-type (WT) and Thbs1-knockout (Thbs1-/-) mice. Bulk and single-cell transcriptomics demonstrated DIO-induced stromal expansion in WT diaphragms. Diaphragm FAPs displayed upregulation of ECM and TGF-β-related expression signatures and augmentation of a Thy1-expressing subpopulation previously linked to type 2 diabetes. Despite similar weight gain, Thbs1-/- mice were protected from these transcriptomic changes and from obesity-induced increases in diaphragm adiposity and ECM deposition. Unlike WT controls, Thbs1-/- diaphragms maintained normal contractile force and motion after DIO challenge. THBS1 is therefore a necessary mediator of diaphragm stromal remodeling and contractile dysfunction in overnutrition and a potential therapeutic target in obesity-associated respiratory dysfunction.

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.

The complex compositional and functional nature of skeletal muscle makes this organ an essential topic of study for biomedical researchers and clinicians. An additional layer of complexity is added with the consideration of sex as a biological variable. Recent research advances have revealed sexual dimorphisms in developmental biology, muscle homeostasis, adaptive responses, and disorders relating to skeletal muscle. Many of the observed sex differences have hormonal and molecular mechanistic underpinnings, whereas others have yet to be elucidated. Future research is needed to investigate the mechanisms dictating sex-based differences in the various aspects of skeletal muscle. As such, it is necessary that skeletal muscle biologists ensure that both female and male subjects are represented in biomedical and clinical studies to facilitate the successful testing and development of therapeutics for all patients.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected Dmdmdx mouse induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Notably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Sarcopenia burdens the older population through loss of muscle energy and mass, yet treatments to functionally rescue both parameters are lacking. The glucocorticoid prednisone remodels muscle metabolism on the basis of frequency of intake, but its mechanisms in sarcopenia are unknown. We found that once-weekly intermittent prednisone administration rescued muscle quality in aged 24-month-old mice to a level comparable to that seen in young 4-month-old mice. We discovered an age- and sex-independent glucocorticoid receptor transactivation program in muscle encompassing peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α) and its cofactor Lipin1. Treatment coordinately improved mitochondrial abundance through isoform 1 and muscle mass through isoform 4 of the myocyte-specific PGC1α, which was required for the treatment-driven increase in carbon shuttling from glucose oxidation to amino acid biogenesis. We also probed myocyte-specific Lipin1 as a nonredundant factor coaxing PGC1α upregulation to the stimulation of both oxidative and anabolic effects. Our study unveils an aging-resistant druggable program in myocytes for the coordinated rescue of energy and mass in sarcopenia.

Proline substitutions within the coiled-coil rod region of the β-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Chronic kidney disease (CKD) causes accumulation of uremic metabolites that negatively affect skeletal muscle. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR), which has been shown to be activated in CKD. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared with controls with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD, and the magnitude of AHR activation was inversely correlated with mitochondrial respiration. In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was markedly impaired and strongly correlated with the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR substantially improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD + probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite/AHR/mitochondrial axis in skeletal muscle was verified using muscle-specific AHR knockdown in C57BL/6J mice harboring a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were present only when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significantly increased pyruvate dehydrogenase kinase 4 (Pdk4) expression and phosphorylation of pyruvate dehydrogenase enzyme. These findings establish a uremic metabolite/AHR/Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.

Skeletal muscle wasting results from numerous pathological conditions affecting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role of E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss-of-function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 resulted in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fiber integrity. We further implicated a disruption in atrogin-1-mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We revealed that BiP was not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorated pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicate atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy and highlight atrogin-1\'s essential role in maintaining muscle homeostasis.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.