Transcriptomic analysis of human pectoral muscle reveals a positive association of NOS3 and PIEZO1 gene expression
N Almulhim, C W Cheng, F Bartoli, N Makava, O I Brown, S Straw, S Kamalathasan, M Paton, R Cubbon, K Witte, M T Kearney, D J Beech, J GierulaAbstract
Background
Recent research focused on elucidating the mechanisms involved in endothelial nitric oxide synthase (NOS3) and its activation by the mechanosensitive cation channel PIEZO1. This activation promotes blood flow through endothelial relaxation, blood pressure homeostasis and exercise performance. However, the transcriptional relationship between NOS3 and PIEZO1 in skeletal muscle and its relevance to human muscle remains largely unexplored.
Purpose
To explore the transcriptional relationships between NOS (NOS1-3) and PIEZO (PIEZO1-2) isoforms in human skeletal muscle.
Methods
Pectoral skeletal muscle biopsies were collected from 30 participants who underwent surgical implantation of cardiac electronic devices. Transcriptome quantification was performed using bulk RNA-Seq and co-expression analysis using Spearman correlation. DESeq2 Package was used to perform data processing and variance-stabilization transformation (VST). For validation, an independent cohort (n =19) was used for RT-qPCR analysis.
Results
Transcriptome analysis resulting from RNA-Seq highlighted expression of NOS1, NOS2, NOS3 and PIEZO1 transcripts across all participants, but not PIEZO2. Mean VST-normalized expression was 66.89 ± 25.97 for NOS3 and 241.13 ± 82.37 for PIEZO1. Co-expression analysis suggested a positive association of NOS3 and PIEZO1 (Spearman's r = 0.62, p < 0.01, FDR q 0.008). RT-qPCR results confirmed a positive association (Spearman's r = 0.83, p < 0.0001). In contrast, no correlation was observed between PIEZO1 and NOS1 (Spearman’s r = -0.01, p = 0.94) or PIEZO1 and NOS2 (spearman’s r = 0.3, p = 0.13).
Gene Set Enrichment Analysis (GSEA) based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated NOS3 enrichment in pathways of cardiovascular development, inflammation, apoptosis and P13K-AKT signalling (False Discovery Rate, FDR < 0.05), whereas PIEZO1 showed no significant enrichment.
Conclusion
The results suggest a transcriptional association between mechanosensing by PIEZO1 and nitric oxide regulation by NOS3 in human skeletal muscle. Further studies are warranted to determine the significance and relationship to cardiovascular disease.