Electrical pulse stimulation protects C2C12 myotubes against H2O2-induced injury via activating Nrf2/antioxidant pathway

Abstract

Background: In response to various physiological stimuli during exercise, skeletal muscle (SkM), a highly plastic tissue, is capable of functional and structural remodeling. The present study aimed to elucidate antioxidant adaptation of SkM to contraction by exposing C2C12 myotubes to electrical pulse stimulation (EPS). Methods: C2C12 myoblasts were differentiated to myotubes by changing the media from complete media (DMEM/High Glucose, 10% fetal bovine serum, and 1% antibiotics) to differentiation media (DMEM/High Glucose, 5% horse serum, and 1% antibiotics). The myotubes were seeded at 50,000 cells/well in a 6-well plate where they underwent EPS at 10 V, 10 ms, 50 Hz, 0.3 sec/3 sec for 1 hour/day for 4 days. Results: (1) By completing experiments for time-course (0 – 6 days) and dose-effect (0 – 10 voltage) tests, we found that NQO1 and GSTA2 proteins, two well-known targets of Nrf2, were maximally upregulated with 3.3 and 12.1 fold increase, respectively, by an EPS at 10 voltage for 4 days; therefore, these parameters were used in the following experiments. (2) MitoSOX-Red, CM-H2DCFDA, confocal microscopy, and EPR assay revealed significantly higher ROS levels in mitochondria, cytosol, and whole cell of EPS-cells than control-cells. Western blot data suggests that EPS-cells express significantly higher Nrf2 and a broad array of downstream antioxidant enzymes (SOD1, SOD2, Cat, GPX, NQO1, GSTA2, GSTA4, PRX1, and TRX). This phenomenon was completely abolished by pretreatment with N-acetylcysteine, a ROS scavenger. These data suggest that contraction enhances muscle antioxidant defense through ROS-dependent, Nrf2-activated multi-antioxidant mechanisms, including the first line of antioxidant components, glutathione system, thioredoxin system, and peroxiredoxin system. (3) We further found that EPS significantly upregulated expression of mitochondrial complexes I/V, mitofusin-1, and mitochondrial fission factor, and significantly increased mitochondrial basal oxygen consumption rate and ATP-linked respiration, suggesting an enhanced mitochondrial function and biogenesis. (4) A post-stimulation time-course experiment demonstrated that the upregulated NQO1 and GSTA2 protein expression lasts at least 24 hours after EPS, suggesting that EPS induces antioxidant preconditioning in the cells. (5) A cell viability study using CCK-8 assay demonstrated that EPS-cells have a significantly higher survival rate than control cells in response to H2O2 treatment. Conclusion: We found that EPS dramatically upregulated a large group of antioxidant enzymes in C2C12 myotubes via activating Nrf2. These cells display an increased mitochondrial function and enhanced tolerance to oxidative challenge. These data suggest that contraction can evoke antioxidant adaptation in muscle via mitochondrion-ROS-Nrf2 pathway, which provides myocytes with protection against oxidative stress-associated injury. This project is funded by NIH R01 HL 160820. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.