Skeletal muscle fibers are commonly classified according to myosin heavy chain (MyHC) isoform, but fiber-type identity also involves coordinated expression of calcium-handling proteins such as sarco/endoplasmic reticulum calcium ATPases (SERCAs). Because fiber-type-associated proteins differ in turnover rate, incomplete remodeling may leave molecular traces of previous fiber identities. MyHC–SERCA mismatch may therefore provide insight into fiber-type remodeling, but its prevalence and morphology in aging human muscle are poorly defined. Vastus lateralis biopsies from young sedentary adults (Y-SED), older sedentary adults (O-SED) and older lifelong recreational exercisers (O-LLEX) were analyzed using immunofluorescence staining for MyHC-I, MyHC-IIa, SERCA1 and SERCA2 on serial sections. Fibers were classified as matched type I, type IIa or type IIx, or as MyHC–SERCA mismatched. Mismatched fibers were grouped into four categories, and fiber cross-sectional area and shape factor index were quantified. MyHC–SERCA mismatched fibers were present in all groups but were more prevalent in older adults than Y-SED. Mismatch did not follow a single staining pattern but comprised several distinct profiles. The most prevalent profile expressed type I MyHC with fast-type or dual SERCA expression. A smaller subset expressed type II MyHC with slow-type or dual SERCA expression and was less prevalent in O-LLEX than O-SED. In older adults, mismatched fibers showed heterogeneous morphology. Aging human skeletal muscle contains more MyHC–SERCA mismatched fibers than young muscle. These fibers show heterogeneous staining profiles and morphology. Lifelong recreational exercise was associated with fewer type II MyHC fibers expressing slow-type or dual SERCA. MyHC–SERCA mismatch may reflect different stages of incomplete fiber-type remodeling during aging.
The primary source of fibrosis in skeletal muscle is fibro-adipogenic progenitors (FAPs) which deposit extensive extracellular matrix (ECM). FAPs are key in supporting regeneration and myogenesis in healthy muscle but become pathogenic in fibrosis. The goal of this study was to determine the effect of fibrotic FAPs on muscle stem cells (MuSC) based myogenesis while distinguishing between signaling through fibrotic FAP ECM deposition or FAP soluble factors. Primary MuSCs and FAPs were isolated from wildtype and D2.mdx mice for in vitro assays. FAPs pro-fibrotic activation was modulated in vitro with transforming growth factor beta (TGF-β) and verteporfin for activation and inhibition of activation respectively. FAP activation state was also modulated by substrate stiffness. Transwell co-culture systems of FAPs and MuSCs were used to interrogate soluble factor signaling. Cell-derived matrices were synthesized by FAPs and used as a substrate to query MuSC myogenesis. Imaging collagen deposition was via second harmonic generation to measure ECM architecture of FAP-derived matrices. Profibrotic activation of FAPs by TGF-β impaired myogenesis in transwell co-cultures. FAPs induced with TGF-β produced ECM with increased collagen deposition, whereas FAPs derived from D2.mdx mice produced ECM with increased collagen fiber diameter. Verteporfin, a suppressor of the YAP-TEAD complex, decreased collagen deposition and impaired collagen fibrillogenesis in FAPs. Across conditions, there was a negative relationship between FAP activation state and myogenic differentiation of MuSCs on the cell-derived matrices (CDMs) synthesized. Further, ECM synthesized by FAPs on underlying substrates matching patho-fibrotic stiffness significantly impaired myogenic differentiation. Overall, profibrotic FAPs produce soluble factors and altered ECM that impairs myogenic differentiation of MuSCs.
Nemaline myopathy (NM) is a clinically and genetically heterogeneous disease caused by mutations in sarcomere-associated genes, with congenital forms representing the most severe end of the spectrum. Recessive mutations in KLHL41 underlie a severe congenital NM characterized by sarcomeric disorganization and impaired muscle growth. The in vivo mechanisms by which KLHL41 governs sarcomere assembly and growth remain poorly understood. We generated zebrafish models of KLHL41 deficiency by the CRISPR-Cas9 approach to examine the temporal and molecular basis of sarcomere assembly defects in NM. The phenotypic and physiological characterization of skeletal muscle defects was investigated by birefringence, spontaneous coiling, spontaneous swimming behavior, and force generation assays. Structural analyses by electron microscopy and immunofluorescence assessed myofibril organization and sarcomere structures across developmental time points. Quantitative proteomics and biochemical assays were performed to identify perturbations in sarcomeric proteins and associated molecular pathways. We show that Klhl41b is essential for the formation and organization of sarcomeres, supporting both radial and longitudinal muscle growth. Loss of Klhl41b resulted in reduced sarcomere size, defective sarcomere assembly followed by progressive destabilization of sarcomeres, nemaline body formation, and reduced muscle function. Proteomic analysis in Klhl41b-deficient zebrafish revealed reduced abundance of the sarcomeric troponin complex proteins, altered abundance of components of the translational machinery, and increased abundance of components of atrophy-associated pathways. These data show that Klhl41b is required for sarcomere assembly and growth, and its deficiency drives a cascade of structural and molecular changes underlying skeletal muscle hypotrophy and NM pathology. These findings advance our understanding of disease mechanisms and may inform targeted therapeutic strategies to restore sarcomere integrity and muscle function.
Nitric oxide (NO), a potent vasodilating agent, plays an essential role in regulation of mitochondrial respiration, and is crucial for proper skeletal muscle function. In skeletal muscle, neuronal NO synthase (nNOS) produces NO and regulates the nitrate/nitrite reductive pathway. nNOS is anchored to the sarcolemma in skeletal muscle by the dystrophin complex. When nNOS dissociates from cell membrane such as in Duchenne muscular dystrophy, nNOS ceases to fulfil its protective role on muscle function and blood flow regulation. We tested the hypothesis that high nitrate diet could enhance NO in skeletal muscle of nNOS knockout (KO) mice. We used gene expression analysis to identify other potential pathways that may be involved to enhance NO production after 7-day high nitrate diet in mice lacking nNOS compared to the wild type (WT) mice. We provided WT mice and nNOS KO mice with normal chow and either standard or high nitrate water (1 g/L nitrate) for seven days. Following the treatment, we collected quadriceps tissues, and isolated the RNA, before performing bulk RNA-sequencing (Illumina). We performed a differential gene expression analysis using DEseq2, then identified pathways related to significantly differentially expressed genes (DEGs) using ShinyGO enrichment analysis. Finally, we validated candidate genes from potential enriched gene pathways using qPCR. Enrichment analysis of DEGs identified 3 potential enriched gene pathways, along with candidate genes associated with NO production and skeletal muscle development and function: (I) muscle system process, (II) cellular response to endogenous stimulus, and (III) tissue development. qPCR validation confirmed that nitrate supplementation alters gene expression of key markers of skeletal muscle development and function, potentially reversing deficits in nNOS KO mice. While nNOS KO control mice showed a downregulation of Ryr1 and Dmd genes compared to WT mice, nitrate supplementation in nNOS KO mice indicated increased expression of these and other candidate genes, including Pax7, Myc, Notch1, and Vangl2. Our findings suggest that dietary nitrate modulates key pathways involved in skeletal muscle maintenance of development in nNOS deficient mice, compared to control mice. Nitrate supplementation may support muscle homeostasis by upregulating gene programs associated with muscle function and provide a potential molecular basis for therapeutic interventions in diseases characterized by nNOS dysfunction.
Exercise intolerance is a hallmark of heart failure with preserved ejection fraction (HFpEF), particularly in its cardiometabolic form, where obesity, metabolic dysfunction, and impaired peripheral oxygen utilization contribute to functional limitation. Skeletal muscle mitochondrial dysfunction is a recognized contributor to this phenotype, yet whether it is shared across muscles with contrasting oxidative profiles, or accompanied by muscle-specific stress responses, has been little explored. We therefore examined gastrocnemius and soleus from male cardiometabolic HFpEF mice using a descriptive and integrative approach. Male C57BL/6J mice received a 60
