Transcriptomic profiling of skeletal muscle in nNOS-deficient mice implies nitrate-induced activation of muscle development pathways

Abstract

Nitric oxide (NO), a potent vasodilating agent, plays an essential role in regulation of mitochondrial respiration, and is crucial for proper skeletal muscle function. In skeletal muscle, neuronal NO synthase (nNOS) produces NO and regulates the nitrate/nitrite reductive pathway. nNOS is anchored to the sarcolemma in skeletal muscle by the dystrophin complex. When nNOS dissociates from cell membrane such as in Duchenne muscular dystrophy, nNOS ceases to fulfil its protective role on muscle function and blood flow regulation. We tested the hypothesis that high nitrate diet could enhance NO in skeletal muscle of nNOS knockout (KO) mice. We used gene expression analysis to identify other potential pathways that may be involved to enhance NO production after 7-day high nitrate diet in mice lacking nNOS compared to the wild type (WT) mice. We provided WT mice and nNOS KO mice with normal chow and either standard or high nitrate water (1 g/L nitrate) for seven days. Following the treatment, we collected quadriceps tissues, and isolated the RNA, before performing bulk RNA-sequencing (Illumina). We performed a differential gene expression analysis using DEseq2, then identified pathways related to significantly differentially expressed genes (DEGs) using ShinyGO enrichment analysis. Finally, we validated candidate genes from potential enriched gene pathways using qPCR. Enrichment analysis of DEGs identified 3 potential enriched gene pathways, along with candidate genes associated with NO production and skeletal muscle development and function: (I) muscle system process , (II) cellular response to endogenous stimulus, and (III) tissue development. qPCR validation confirmed that nitrate supplementation alters gene expression of key markers of skeletal muscle development and function, potentially reversing deficits in nNOS KO mice. While nNOS KO control mice showed a downregulation of Ryr1 and Dmd genes compared to WT mice, nitrate supplementation in nNOS KO mice indicated increased expression of these and other candidate genes, including Pax7 , Myc , Notch1 , and Vangl2 . Our findings suggest that dietary nitrate modulates key pathways involved in skeletal muscle maintenance of development in nNOS deficient mice, compared to control mice. Nitrate supplementation may support muscle homeostasis by upregulating gene programs associated with muscle function and provide a potential molecular basis for therapeutic interventions in diseases characterized by nNOS dysfunction.