Hydrogen sulfide mitigates skeletal muscle mitophagy-led tissue remodeling via epigenetic regulation of the gene writer and eraser function

Abstract

Ketone bodies (KB) serve as the food for mitochondrial biogenetics. Interestingly, probiotics are known to promote KB formation in the gut (especially those that belong to the Lactobacillus genus). Furthermore, Lactobacillus helps produce folate that lowers the levels of homocysteine (Hcy); a hallmark non-proteinogenic amino acid that defines the importance of epigenetics, and its landscape. In this study, we decided to test whether hydrogen sulfide (H₂ S), another Hcy lowering agent regulates the epigenetic gene writer DNA methyltransferase (DNMT), eraser FTO and TET2, and thus mitigates the skeletal muscle remodeling. We treated hyperhomocysteinemic (HHcy, cystathionine beta-synthase heterozygote knockout; CBS^+/- ) mice with NaHS (the H₂ S donor). The results suggested multi-organ damage by HHcy in the CBS^+/- mouse strain compared with WT control mice (CBS^+/+ ). H₂ S treatment abrogated most of the HHcy-induced damage. The levels of gene writer (DNMT2) and H3K9 (methylation) were higher in the CBS^+/- mice, and the H₂ S treatment normalized their levels. More importantly, the levels of eraser FTO, TET, and associated GADD45, and MMP-13 were decreased in the CBS^+/- mice; however, H₂ S treatment mitigated their respective decrease. These events were associated with mitochondrial fission, i.e., an increase in DRP1, and mitophagy. Although the MMP-2 level was lower in CBS^+/- compared to WT but H₂ S could further lower it in the CBS^+/- mice. The MMPs levels were associated with an increase in interstitial fibrosis in the CBS^+/- skeletal muscle. Due to fibrosis, the femoral artery blood flow was reduced in the CBS^+/- mice, and that was normalized by H₂ S. The bone and muscle strengths were found to be decreased in the CBS^+/- mice but the H₂ S treatment normalized skeletal muscle strength in the CBS^+/- mice. Our findings suggest that H₂ S mitigates the mitophagy-led skeletal muscle remodeling via epigenetic regulation of the gene writer and eraser function.

Keywords: 1-carbon metabolism; cystathionine β synthase; homocysteine; mitochondria.

FIGURE 1

We hypothesize that “1‐carbon metabolism” controls epigenetic information by gene writer, and eraser, and its dysregulation leads to hyperhomocysteinemia (HHcy) in the CBS^+/−mice. The H₂S treatment helps mitigate this dysfunction.

FIGURE 2

CBS^+/−mice are hypertensive due to hyperhomocysteinemia (HHcy). Thus, the tail vein mean arterial pressure (MAP) is higher in these CBS^+/− mice as compared to the WT (CBS^+/+) mice (a). The H₂S treatment normalizes the MAP in the CBS^+/− mice. The levels of creatinine kinase (CK; the injury marker), in the muscle (CKMM) were found to be significantly higher (p < 0.01; n = 3) in the CBS^+/−mice as compared to the WT controls (b). The H₂S treatment mitigated the CK levels in the CBS^+/−mice. The bar graph shows the respective quantitative values of the CK (b).

FIGURE 3

The levels of gene writer (DNMT2), skeletal muscle musclin, and the histone 3 lysine 9 methylation (H3K9) were measured. The levels of gene eraser, fat obese associated protein (FTO), ten‐eleven translocate (TET2), growth arrest and DNA damage‐45 protein (GADD45b), associated matrix metalloproteinase‐13 (MMP13) were determined. The levels of mitochondrial mitophagy were also determined by calculating the ratios of the fission protein (DRP1), and fusion protein (MuRF1), *p < 0.05; n = 5–12.

FIGURE 4

The skeletal muscle remodeling was measured by in‐gel zymography for matrix metalloproteinase‐2 (MMP2). The levels of MMP2 were mitigated by the H₂S treatment. The lower levels of MMP2 activity in CBS^+/− may be due to inhibition of MMP‐zymographic‐activity by sulfur‐containing agents which may not be the case in the WT controls, *p < 0.05; n = 5–12.

FIGURE 5

The skeletal muscle fibrosis was measured by trichome‐blue Masson staining methods. The results suggest robust interstitial fibrosis in the CBS^+/− mice muscle. The H₂S treatment mitigated this fibrosis in the muscle, *p < 0.05; n = 5–12.

FIGURE 6

To determine whether fibrosis and skeletal muscle remodeling compromise limb blood flow, we measured the perfusion via a full‐field laser perfusion imager procedure. The flow was decreased in the CBS^+/− mice as compared to the WT controls. H₂S treatment normalized the blood flow in the limb of CBS^+/− mice, *p < 0.05; n = 5–12.

FIGURE 7

The muscle strength was measured by the grip strength test. The grip strength of the CBS^+/− mice was significantly decreased as compared to the WT control. The H₂S treatment reversed the decrease in the grip strength of CBS^+/− mice. Representative tibia, fibula, and the femur bone photographs are shown, *p < 0.05; n = 5–12.

FIGURE 8

The conclusion supports the mitochondrial mitophagy (i.e., increase in DRP1/MuRF1 ratio) in the CBS^+/− mice skeletal muscle. The H₂S treatment normalized the mitochondrial bioenergetics, successfully.