Abstract
In skeletal muscle the coordinated actions of two mechanically coupled Ca2+ channels—the 1,4-dihydropyridine receptor (Cav1.1) and the type 1 ryanodine receptor (RYR1)–underlie the molecular mechanism of rapid cytosolic [Ca2+] increase leading to contraction. While both [Ca2+]i and contractile activity have been implicated in the regulation of myogenesis, less is known about potential specific roles of Cav1.1 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 Cav1.1-/- 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 Cav1.1-/- 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 Cav1.1-/- 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 Cav1.1-/- 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 Cav1.1-/- mice. Our results suggest distinct functional roles for RYR1 and Cav1.1 in skeletal primary and secondary myogenesis.
Highlights
Skeletal muscle is the largest organ in the vertebrate body
While we find almost no overlap in the transcriptomic changes of ryanodine receptor type 1 (RYR1)-/- and Cav1.1-/- samples at E14.5, at E18.5 we reveal a significant convergence of differential gene expression in both mutants, with common differentially regulated genes (DEGs) primarily associated with muscle contraction
For this assessment 3 littermates from each genotype, RYR1+/+ (WT), RYR1+/- and RYR1-/, as well as Cav1.1+/+ (WT), Cav1.1+/- and Cav1.1-/, at both E14.5 and E18.5, were used for whole embryos preparations (Fig 1)
Summary
Skeletal muscle is the largest organ in the vertebrate body. the most prominent association coming into mind when one thinks of skeletal muscle is probably that related to contraction and movement, the functional repertoire of this organ is by far more versatile. The proper formation of the skeletal muscle organ is a complex, only partially understood multistep process, subjected to a strict spatiotemporal regulation throughout development [4]. The primary myogenesis lays the foundation of the developing skeletal muscle and during the secondary myogenesis the muscle grows and differentiates. In line with this notion, a genome wide expression analysis has shown that embryonic and fetal myoblasts have distinct transcriptomic profiles [9]. The proper transition between the different stages in myogenesis is regulated by the strict spatiotemporal expression of canonical myogenic regulatory factors (MRFs) [10] and involves a crosstalk with the surrounding connective and neuronal tissue [4]. Around the time of late embryonic and early fetal development the first skeletal muscle contractions effective in limb movement start appearing in mouse [11]
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