Hyperhomocysteinemia associated skeletal muscle weakness involves mitochondrial dysfunction and epigenetic modifications

Abstract

HHcy has been implicated in elderly frailty, but the underlying mechanisms are poorly understood. Using C57 and CBS+/- mice and C2C12 cell line, we investigated mechanisms behind HHcy induced skeletal muscle weakness and fatigability. Possible alterations in metabolic capacity (levels of LDH, CS, MM-CK and COX-IV), in structural proteins (levels of dystrophin) and in mitochondrial function (ATP production) were examined. An exercise regimen was employed to reverse HHcy induced changes. CBS+/- mice exhibited more fatigability, and generated less contraction force. No significant changes in muscle morphology were observed. However, there is corresponding reduction in large muscle fiber number in CBS+/- mice. Excess fatigability was not due to changes in key enzymes involved in metabolism, but was due to reduced ATP levels. A marginal reduction in dystrophin levels along with a decrease in mitochondrial transcription factor A (mtTFA) were observed. There was also an increase in the mir-31, and mir-494 quantities that were implicated in dystrophin and mtTFA regulation respectively. The molecular changes elevated during HHcy, with the exception of dystrophin levels, were reversed after exercise. In addition, amount of NRF-1, one of the transcriptional regulators of mtTFA, was significantly decreased. Furthermore, there was enhancement in mir-494 levels and a concomitant decline in mtTFA protein quantity in homocysteine treated cells. These changes in C2C12 cells were also accompanied by an increase in DNMT3a and DNMT3b proteins and global DNA methylation levels. Together, these results suggest that HHcy plays a causal role in enhanced fatigability through mitochondrial dysfunction which involves epigenetic changes.

Hyperhomocysteinemia inhibits satellite cell regenerative capacity through p38 alpha/beta MAPK signaling

“…These findings together strengthen the current finding that HHcy-associated sarcopenia could be due to compromised regenerative potential and may not be due to excessive muscle atrophy (through activation of atrogin and MuRF) or cell death. However, the previous study also indicated that there was significant reduction in the muscle fiber size in the CBSϪ/ϩ mouse muscles as evidenced by the presence of relatively more intermediate-size muscle fibers instead of large-size muscle fibers (26). The overall reduction in muscle mass and muscle fiber size could be due to other types of atrophy independent of FOXO transcription as well as defective satellite function as described in the current report.…”

“…However, our previous study ruled out the induction of atrophic genes such as atrogin-1 and MuRF-1 in the intact muscles from CBSϪ/ϩ mice (23). Furthermore, histological sections from CBSϪ/ϩ mice neither exhibited abnormal cellular infiltration nor showed centronuclear muscle fibers that are suggestive of cell death or inflammation (26). These findings together strengthen the current finding that HHcy-associated sarcopenia could be due to compromised regenerative potential and may not be due to excessive muscle atrophy (through activation of atrogin and MuRF) or cell death.…”

“…Although it needs to be further established in the case of human scenario with the HHcy condition, our study identified several similarities that indicate compromised satellite cell function in humans as well. 1) Association between HHcy and muscle weakness was reported both in the humans and in mouse models (24,26). 2) HHcy was associated with progressive decline in body weights both in humans and in mouse models (12,14,29).…”

“…All the animal studies were approved by the Institutional Animal Care and Use Committee and are in conformity with the prescribed institutional standards. Standard procedures were followed for collection of tissues after induction of general anesthesia using tribromoethanol as described before (26). We used male mice of around 5-6 mo in our study.…”

“…The C2C12 cell culture was maintained as reported before (26). When the myoblasts were at the confluence of 80%, they were subjected to Hcy treatment for 24 h. At the end of the treatment period, cells were lysed and the cell lysates were prepared using RIPA lysis buffer supplemented with protease and phosphatase inhibitors.…”