Distinct transcriptomic changes in E14.5 mouse skeletal muscle lacking RYR1 or Ca(v)1.1 converge at E18.5

In skeletal muscle the coordinated actions of two mechanically coupled Ca(2+) channels—the 1,4-dihydropyridine receptor (Ca(v)1.1) and the type 1 ryanodine receptor (RYR1)–underlie the molecular mechanism of rapid cytosolic [Ca(2+)] increase leading to contraction. While both [Ca(2+)](i) and contractile activity have been implicated in the regulation of myogenesis, less is known about potential specific roles of Ca(v)1.1 and RYR1 in skeletal muscle development. In this study, we analyzed the histology and the transcriptomic changes occurring at E14.5 –the end of primary myogenesis and around the onset of intrauterine limb movement, and at E18.5 –the end of secondary myogenesis, in WT, RYR1(-/-), and Ca(v)1.1(-/-) murine limb skeletal muscle. At E14.5 the muscle histology of both mutants exhibited initial alterations, which became much more severe at E18.5. Immunohistological analysis also revealed higher levels of activated caspase-3 in the Ca(v)1.1(-/-) muscles at E14.5, indicating an increase in apoptosis. With WT littermates as controls, microarray analyses identified 61 and 97 differentially regulated genes (DEGs) at E14.5, and 493 and 1047 DEGs at E18.5, in RYR1(-/-) and Ca(v)1.1(-/-) samples, respectively. Gene enrichment analysis detected no overlap in the affected biological processes and pathways in the two mutants at E14.5, whereas at E18.5 there was a significant overlap of DEGs in both mutants, affecting predominantly processes linked to muscle contraction. Moreover, the E18.5 vs. E14.5 comparison revealed multiple genotype-specific DEGs involved in contraction, cell cycle and miRNA-mediated signaling in WT, neuronal and bone development in RYR1(-/-), and lipid metabolism in Ca(v)1.1(-/-) samples. Taken together, our study reveals discrete changes in the global transcriptome occurring in limb skeletal muscle from E14.5 to E18.5 in WT, RYR1(-/-) and Ca(v)1.1(-/-) mice. Our results suggest distinct functional roles for RYR1 and Ca(v)1.1 in skeletal primary and secondary myogenesis.