The development of skeletal muscle (myogenesis) is a well-orchestrated process where myoblasts withdraw from the cell cycle and differentiate into myotubes. Signaling by fluxes in intracellular calcium (Ca2+) is known to contribute to myogenesis, and increased mitochondrial biogenesis is required to meet the metabolic demand of mature myotubes. However, gaps remain in the understanding of how intracellular Ca2+ signals can govern myogenesis. Polycystin-2 (PC2 or TRPP1) is a nonselective cation channel permeable to Ca2+. It can interact with intracellular calcium channels to control Ca2+ release and concurrently modulates mitochondrial function and remodeling. Due to these features, we hypothesized that PC2 is a central protein in mediating both the intracellular Ca2+ responses and mitochondrial changes seen in myogenesis. To test this hypothesis, we created CRISPR/Cas9 knockout (KO) C2C12 murine myoblast cell lines. PC2 KO cells were unable to differentiate into myotubes, had impaired spontaneous Ca2+ oscillations, and did not develop depolarization-evoked Ca2+ transients. The autophagic-associated pathway beclin-1 was downregulated in PC2 KO cells, and direct activation of the autophagic pathway resulted in decreased mitochondrial remodeling. Re-expression of full-length PC2, but not a calcium channel dead pathologic mutant, restored the differentiation phenotype and increased the expression of mitochondrial proteins. Our results establish that PC2 is a novel regulator of in vitro myogenesis by integrating PC2-dependent Ca2+ signals and metabolic pathways.

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sarcoplasmic reticulum (SR). A separate group of cardiomyocytes were fixed and key proteins were labelled with antibodies for subsequent confocal imaging. MCT myocytes had the largest cross-sectional area (4338 5 292mm 2) versus CON (3170 5 262mm 2 , P=0.01), although no difference in cell shortening was observed in response to 1 Hz stimulation. MCT myocytes had larger Ca 2þ transients at all stimulation frequencies (P= 0.004) and increased spontaneous activity during ß-adrenergic stimulation. No difference between groups was found in Ca 2þ store content, although the time constant of caffeine transient decay was prolonged in MCT (17.3 5 1.5s) versus CON (13.7 5 0.6s, P=0.05). Although there was no difference in shortening or SR Ca 2þ content, there was evidence of changes in Ca 2þ handling in MCT myocytes during inotropic stimulation. Furthermore, hypertrophic MCT myocytes exhibited slower transsarcolemmal Ca 2þ removal during 20mM caffeine, potentially as a result of disorganized T-tubular arrangement and/or reduced Na þ /Ca 2þ exchanger abundance.