Isolated individual myofibers are valuable experimental models that can be used in various conditions to understand skeletal muscle physiology and pathophysiology at the tissue and cellular level. This report details a time- and cost-effective method for isolation of single myofibers from the flexor digitorum brevis (FDB) muscle in both young and aged mice. The FDB muscle was chosen for its documented history in single myofiber experiments. By modifying published methods for FDB myofiber isolation, we have optimized the protocol by first separating FDB muscle into individual bundles before the digestion, followed by optimizing the subsequent digestion medium conditions to ensure reproducibility. Morphological and functional assessments demonstrate a high yield of isolated FDB myofibers with sarcolemma integrity achieved in a shorter time frame than previous published procedures. This method could be also adapted to other types of skeletal muscle. Additionally, this highly reproducible method can greatly reduce the number of animals needed to yield adequate numbers of myofibers for experiments. Thus, this advanced method for myofiber isolation has the potential to accelerate research in skeletal muscle physiology and screening potential therapeutics “ ex vivo ” for muscle diseases and regeneration.

Sarcopenia is characterized by progressive loss of skeletal muscle mass and function and is a major contributor to frailty, disability, and mortality in older adults. Store-operated calcium entry (SOCE) is a crucial regulator of skeletal muscle calcium homeostasis, and impaired SOCE has been linked to age-related muscle weakness. Here, we investigated the role of the synaptophysin family member synaptophysin-like protein 2, also known as mitsugumin 29 (MG29/SYPL2) in regulating SOCE, muscle structure, and lipid signaling during aging. Using knockout mice (mg29−/−) as a model of accelerated sarcopenia, in combination with RNA interference against MG29/SYPL2 in adult muscle and primary myotubes, we quantified changes in muscle morphology, contractile function, SOCE activity, and targeted lipidomic profiles. We found that reduced MG29/SYPL2 expression leads to decreased muscle fiber cross-sectional area, reduced specific force, blunted SOCE, and marked alterations to membrane cholesterol content as well as fatty acid–derived lipid mediators. Cholesterol depletion by methyl-β-cyclodextrin in wild-type myotubes produced similar SOCE defects as those observed in aged wild-type and young mg29−/− muscles, indicating that MG29/SYPL2-dependent maintenance of membrane cholesterol is required for normal SOCE. Acute MG29/SYPL2 knockdown also modified myogenic differentiation, expression of calcium-handling and stress-response genes, and the release and consumption of specific polyunsaturated fatty acid–derived lipid mediators. Together, these findings identify MG29/SYPL2 as a critical regulator of SOCE and lipid signaling in skeletal muscle and suggest that its age-related decline contributes to sarcopenia by disrupting triad membrane organization and excitation-contraction coupling.

Sarcopenia is characterized by progressive loss of skeletal muscle mass and function and is a major contributor to frailty, disability, and mortality in older adults. Store-operated calcium entry (SOCE) is a crucial regulator of skeletal muscle calcium homeostasis, and impaired SOCE has been linked to age-related muscle weakness. Here, we identify the synaptophysin family member synaptophysin-like protein 2, also known as mitsugumin 29 (MG29; encoded by the human gene SYPL2 and the mouse ortholog Mg29), as a key organizer of triad membrane cholesterol and lipid signaling required for normal SOCE during aging. Using Mg29^−/− mice as a model of accelerated sarcopenia, together with RNA interference against Mg29 in adult muscle and primary myotubes, we quantified changes in muscle morphology, contractile function, SOCE activity, and targeted lipidomic profiles. Reduced MG29 expression led to decreased muscle fiber cross-sectional area, reduced specific force, blunted SOCE, and marked alterations in membrane cholesterol content and fatty acid-derived lipid mediators. Cholesterol depletion by methyl-β-cyclodextrin in wild-type myotubes produced SOCE defects similar to those observed in aged wild-type and young Mg29^−/− muscles, indicating that MG29-dependent maintenance of membrane cholesterol is required for normal SOCE. Acute Mg29 knockdown also altered myogenic differentiation, the expression of calcium-handling and stress-response genes, and the release and consumption of specific polyunsaturated fatty acid-derived lipid mediators. Together, these findings identify MG29 as a critical regulator of SOCE and lipid signaling in skeletal muscle and suggest that its age-related decline contributes to sarcopenia by disrupting triad membrane organization and excitation–contraction coupling.