Objectives

Skeletal muscle is a central regulator of metabolic health, serving as the primary site of postprandial glucose uptake and playing a critical role in whole-body insulin sensitivity. Despite its importance, the molecular mechanisms governing muscle differentiation (myogenesis) and their modulation by metabolic interventions remain poorly defined. This study identifies the clathrin adaptor protein Picalm (phosphatidylinositol-binding clathrin assembly protein) as a novel regulator of myogenesis and investigates its regulation in response to exercise training and intermittent fasting.

Methods

Functional characterization of Picalm was conducted in C2C12 myoblasts and primary myocytes using siRNA-mediated knockdown. Clathrin-mediated endocytosis was performed using dynamin inhibition (Dyngo-4a) and via an EGF internalization assay. Surface proteome alterations were analyzed by plasma membrane proteomics, and autophagy dynamics were assessed via immunoblotting and fluorescence imaging. Jasplakinolide was used to rescue differentiation defects by enhancing actin polymerization.

Results

Picalm-depleted C2C12 myoblasts exhibited impaired differentiation, presumably due to diminished intracellular trafficking dynamics of cell surface proteins. Inhibition of dynamin-dependent endocytosis phenocopied the differentiation defect and further aggravated myogenesis in Picalm-depleted cells, indicating that Picalm-dependent endocytic function is required for efficient differentiation. Consistent with this, Picalm knockdown significantly decreased clathrin-dependent uptake of EGF. Proteome analysis of a plasma membrane-enriched fraction revealed increased abundance of over 100 proteins after Picalm knockdown, particularly candidates involved in vesicular trafficking (Vamp3, Vamp5), actin remodeling (Actn1, Actn4, Rhog, Rock1, Rock2) and cell adhesion (integrin receptors). In line with this, Picalm knockdown resulted in impaired maturation and lysosomal degradation of autophagic vesicles. Remarkably, pharmacological stabilization of actin filaments with Jasplakinolide restored myogenic differentiation in Picalm-deficient cells, highlighting a functional link between actin remodeling and myogenesis.

Conclusions

Picalm regulates skeletal muscle differentiation by supporting clathrin-mediated endocytosis and plasma membrane remodeling, thereby maintaining trafficking-dependent control of actin organization. Its expression is responsive to metabolic cues such as exercise and intermittent fasting. These findings reveal a novel molecular link between nutrient signaling and myogenesis, with implications for metabolic disease and muscle regeneration.