Distinct transcriptomic changes in E14.5 mouse skeletal muscle lacking RYR1 or Ca_v1.1 converge at E18.5

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.在骨骼肌中，两种机械耦联的钙离子通道——1,4-二氢吡啶受体（1,4-dihydropyridine receptor，Cav1.1）与1型兰尼碱受体（type 1 ryanodine receptor，RYR1）——的协同作用，是引发胞质钙离子浓度快速升高进而导致肌肉收缩的分子机制基础。尽管胞内钙离子浓度（[Ca²⁺]i）与收缩活动均被证实参与肌发生（myogenesis）的调控，但目前对于Cav1.1与RYR1在骨骼肌发育中潜在的特异性功能仍知之甚少。本研究针对野生型（wild type，WT）、RYR1基因敲除（RYR1-/-）及Cav1.1基因敲除（Cav1.1-/-）小鼠的肢体骨骼肌，分析了两个发育节点的组织学与转录组变化：E14.5（原代肌发生末期、宫内肢体运动启动前后）与E18.5（继代肌发生末期）。在E14.5时，两种突变体的肌肉组织学均出现初始异常，而该异常在E18.5时显著加重。免疫组织化学分析还显示，E14.5时Cav1.1-/-小鼠肌肉中活化的半胱天冬酶-3（activated caspase-3）水平更高，表明细胞凋亡水平升高。以同窝野生型小鼠作为对照，微阵列（microarray）分析显示，RYR1-/-与Cav1.1-/-样本在E14.5时分别鉴定出61个与97个差异表达基因（differentially regulated genes，DEGs），在E18.5时则分别为493个与1047个差异表达基因。基因富集分析发现，E14.5时两种突变体受影响的生物学过程与通路并无重叠；而在E18.5时，两种突变体的差异表达基因出现显著重叠，且主要涉及与肌肉收缩相关的生物学过程。此外，对E18.5与E14.5样本的比较分析显示，不同基因型存在大量特异性差异表达基因：野生型样本中涉及收缩、细胞周期与miRNA介导的信号通路；RYR1-/-样本中涉及神经元与骨骼发育；Cav1.1-/-样本中则涉及脂质代谢。综上，本研究揭示了野生型、RYR1-/-及Cav1.1-/-小鼠肢体骨骼肌从E14.5至E18.5发育过程中，全局转录组发生的特异性变化。研究结果表明，RYR1与Cav1.1在骨骼肌原代与继代肌发生中发挥着截然不同的功能。