Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome

Abstract

Electrical pulse stimulation (EPS) has proven to be a useful tool to interrogate cell-specific responses to muscle contraction. In the present study, we aimed to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes from young male subjects were exposed to EPS protocol 1 (i.e. 2 ms, 10 V, and 0.1 Hz for 24 h), whereas myotubes from middle-aged women and men were exposed to protocol 2 (i.e. 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling as well as the transcriptome, cellular proteome, and secreted proteins of EPS-treated myotubes from young male subjects were analyzed using a combination of high-throughput RNA sequencing, high-resolution liquid chromatography-tandem mass spectrometry, oxidation assay, and immunoblotting. The data showed that oxidative metabolism was enhanced in EPS-exposed myotubes from young male subjects. Moreover, a total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in these cells after EPS protocol 1. We also found 61 overlapping genes while comparing the DEGs to mRNA expression in myotubes from the middle-aged group exposed to protocol 2, assessed by microarray. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Additionally, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes from young male subjects exposed to EPS protocol 1. Selected putative myokines were measured using ELISA or multiplex assay to validate the results. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.

Mimicking exercise in vitro: effects of myotube contractions and mechanical stretch on omics

“…Based on RNA-seq, we (18) and others (16,26) observed that processes related to cytokine and inflammatory pathways were upregulated after EL-EPS in C2C12 myotubes. Local inflammation plays a role in SkM adaptation to exercise (27).…”

“…Genes related to pro-inflammatory pathways were upregulated in myotubes originating from females with polycystic ovary syndrome relative to healthy controls (25). Although EL-EPS responses may be affected by donor characteristics (1,3), a recent study suggests that age, sex, and EL-EPS parameters may not have substantial transcriptomic and proteomic effects (26).…”

“…Two recent studies examined the transcriptome after EL-EPS in primary human myotubes (25,26). Genes related to pro-inflammatory pathways were upregulated in myotubes originating from females with polycystic ovary syndrome relative to healthy controls (25).…”

“…After acute aerobic exercise, these effects might be partly mediated by monocyte attractants, such as cytokines from the cytokine families CXC and CC (23). Indeed, upregulation of Cxcl1, Cxcl5 (CXC family) and Ccl2 (CC family) in response to EL-EPS has been reported using transcriptomic (16,18,26) and targeted methods (20)(21)(22). These specific cytokines have been associated with exercise-induced macrophage infiltration into SkM to promote tissue regeneration and/or hypertrophy in vivo (5,21,28).…”

Translating In Vitro Models of Exercise in Human Muscle Cells: A Mitocentric View

“…Studies that used EPS as an in vitro model of exercise mimicking acute versus chronic or aerobic versus resistance training have mostly been described in the immortalized rodent cell lines C2C12 and L6 (24,25). From our review of the literature, 10 studies defined their EPS protocol as that of an "exercise-like" treatment on HSkMCs (26)(27)(28)(29)(30)(31)(32)(33), investigating exercise-mediated signaling mechanisms (26,(28)(29)(30)32,33), expression of mitochondrial-related genes (21,27,31), substrate metabolism (27,28,30,33,56), and measurements of mitochondrial quantity (27,28,(30)(31)(32)(33)) (Table ). No studies have investigated the impact of EPS on HSkMC mitochondrial respiratory capacity.…”