Transcriptomic Responses to Neuromuscular Electrical Stimulation (NMES) in Muscle, Brain and Plasma Exosomes in WT and Klotho Deficient Mice [ncRNA]

Previous studies indicate extracellular vesicles (EVs) play a role in cellular communication which may be impacted by neuromuscular electrical stimulation (NMES), a noninvasive approach to improve daily living and functional motor activities. Therefore, we utilized NMES to stimulate the tibialis anterior of WT and KlHET mice and evaluated transcriptomic profiles of muscle tissue, brain tissue, and plasma-derived EVs. Muscle mRNA indicate that genes related to metabolic processes are upregulated in response to NMES in but have little overlap in WT and KlHET NMES versus control. Brain mRNA in WT shows upregulation of several genes related to blood flow and cell adhesion processes while KlHET shows downregulation in cell adhesion processes and immune function. In plasma-derived EVs, little overlap in ncRNA, with the exception of let-7d-5p which regulates insulin signaling targets and immune responses, was present. Findings indicate local and distal transcriptomic metabolic effects of NMES are mediated by KLOTHO deficiency. Overall design: NMES was administered to using Empi 300 PV, Boston, USA at 9mA under 2.5% isoflurane anesthesia for 2 sets of 10 consecutive stimulations per side. A total of 5 sessions were performed. Following sample collection, isolation of brain, muscle, and plasma RNA took place. Extracted muscle and brain RNA was sent to Novogene Co. for total RNA library generation on NovaSeq X Plus Series (PE150). Brain and muscle mRNA data were aligned with Rsubread (v 2.16.1), annotated with the org.Mm.eg.db package (v 3.19.1) and analyzed with edgeR (v 4.0.16). Gene ontology (GO) terms were generated in DAVID with a p-value cut-off of <0.05 followed by a fold-change cut-off of >0.5. Plasma EV ncRNA libraries were sequenced by the UPMC Genome Center (Pittsburgh, PA) on NextSeq 2000. ncRNA alignment results were then checked by STAR (v 2.5.3a), analyzed with COMPSRA (v 1.0.3) and DESeq2 (v 1.42.1). RNA analysis from DAVID and REVIGO Gene Ontology were visualized in GraphPad Prism, and RStudio. ncRNA library select gene targets were analyzed in TargetScanMouse 8.0 and were visualized using STRING.