CRISPR/Cas9-mediated MSTN gene editing induced mitochondrial alterations in C2C12 myoblast cells

Background: Myostatin (MSTN) negatively regulates muscle mass and is a potent regulator of energy metabolism. However, MSTN knockout have affect mitochondrial function. This research assessed the mitochondrial energy metabolism of Mstn−/+ KO cells, and wondered whether the mitochondria biogenesis are affected. Results: In this study, we successfully achieved Mstn knockout in skeletal muscle C2C12 cells using a CRISPR/Cas9 system and measured proliferation and differentiation using the Cell-Counting Kit-8 assay and qPCR, respectively. We found that MSTN dysfunction could promote proliferation and differentiation compared with the behaviour of wild-type cells. Moreover, Mstn KO induced an increase in KIF5B expression. The mitochondrial content was significantly increased in Mstn KO C2C12 cells, apparently associated with the increases in PGC-1α, Cox1, Cox2, ND1 and ND2 expression. However, no differences were observed in glucose consumption and lactate production. Interestingly, Mstn KO C2C12 cells showed an increase in IL6 and a decrease in TNF-1α levels. Conclusion: These findings indicate that MSTN regulates mitochondrial biogenesis and metabolism. This gene-editing cells provided favourable evidence for animal breeding and metabolic diseases.

Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions

Vacuolar H+-ATPase is a large multi-subunit protein that mediates ATP-driven vectorial H+ transport across the membranes. It is widely distributed and present in virtually all eukaryotic cells in intracellular membranes or in the plasma membrane of specialized cells. In subcellular organelles, ATPase is responsible for the acidification of the vesicular interior, which requires an intraorganellar acidic pH to maintain optimal enzyme activity. Control of vacuolar H+-ATPase depends on the potential difference across the membrane in which the proton ATPase is inserted. Since the transport performed by H+-ATPase is electrogenic, translocation of H+-ions across the membranes by the pump creates a lumen-positive voltage in the absence of a neutralizing current, generating an electrochemical potential gradient that limits the activity of H+-ATPase. In many intracellular organelles and cell plasma membranes, this potential difference established by the ATPase gradient is normally dissipated by a parallel and passive Cl- movement, which provides an electric shunt compensating for the positive charge transferred by the pump. The underlying mechanisms for the differences in the requirement for chloride by different tissues have not yet been adequately identified, and there is still some controversy as to the molecular identity of the associated Cl--conducting proteins. Several candidates have been identified: the ClC family members, which may or may not mediate nCl-/H+ exchange, and the cystic fibrosis transmembrane conductance regulator. In this review, we discuss some tissues where the association between H+-ATPase and chloride channels has been demonstrated and plays a relevant physiologic role.