Mitochondrial dysfunction-associated OPA1 cleavage contributes to muscle degeneration: preventative effect of hydroxytyrosol acetate

Abstract

Mitochondrial dysfunction contributes to the development of muscle disorders, including muscle wasting, muscle atrophy and degeneration. Despite the knowledge that oxidative stress closely interacts with mitochondrial dysfunction, the detailed mechanisms remain obscure. In this study, tert-butylhydroperoxide (t-BHP) was used to induce oxidative stress on differentiated C2C12 myotubes. t-BHP induced significant mitochondrial dysfunction in a time-dependent manner, accompanied by decreased myosin heavy chain (MyHC) expression at both the mRNA and protein levels. Consistently, endogenous reactive oxygen species (ROS) overproduction triggered by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a mitochondrial oxidative phosphorylation inhibitor, was accompanied by decreased membrane potential and decreased MyHC protein content. However, the free radical scavenger N-acetyl-L-cysteine (NAC) efficiently reduced the ROS level and restored MyHC content, suggesting a close association between ROS and MyHC expression. Meanwhile, we found that both t-BHP and FCCP promoted the cleavage of optic atrophy 1 (OPA1) from the long form into short form during the early stages. In addition, the ATPase family gene 3-like 2, a mitochondrial inner membrane protease, was also markedly increased. Moreover, OPA1 knockdown in myotubes was accompanied by decreased MyHC content, whereas NAC failed to prevent FCCP-induced MyHC decrease with OPA1 knockdown, suggesting that ROS might affect MyHC content by modulating OPA1 cleavage. In addition, hydroxytyrosol acetate (HT-AC), an important compound in virgin olive oil, could significantly prevent t-BHP-induced mitochondrial membrane potential and cell viability loss in myotubes. Specifically, HT-AC inhibited t-BHP-induced OPA1 cleavage and mitochondrial morphology changes, accompanied by improvement on mitochondrial oxygen consumption capacity, ATP productive potential and activities of mitochondrial complex I, II and V. Moreover, both t-BHP- and FCCP-induced MyHC decrease was sufficiently inhibited by HT-AC. Taken together, our data provide evidence indicating that mitochondrial dysfunction-associated OPA1 cleavage may contribute to muscle degeneration, and olive oil compounds could be effective nutrients for preventing the development of muscle disorders.

O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis

“…Cells were induced into myotubes after 6 days following previously reported method [27]. siRNA transfections were performed using Lipofectamine RNAiMAX (Invitrogen), and DNA transfections were performed using X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) as described in the supplier's manuals.…”

“…Mitochondrial complex activity assays Mitochondria isolation from cultured mouse C2C12 myoblasts, myotubes and skeletal muscle tissues were performed as previously described [27,28]. Mitochondrial complex activities were measured by spectrophotometer as described in previous reports [29,30].…”

“…Oxidative stress assays Cellular oxidative stress of cultured mouse C2C12 myoblasts and myotubes was assessed by analysing intracellular reactive oxygen species (ROS) with the fluorescence probe 2′,7′-dichlorofluorescein (H2DCF-DA), carbonyl protein with the OxyBlot protein oxidation detection kit (Cell Biolabs, San Diego, CA, USA), 4-hydroxynonenal (4-HNE) with ELISA kit (RD Systems, Shanghai, China), and glutathione (GSH) with 2,3-naphthalenedicarboxyaldehyde (NDA) as previously described [23,27]. See ESM Methods for further details.…”

Dihydromyricetin Attenuates Dexamethasone-Induced Muscle Atrophy by Improving Mitochondrial Function via the PGC-1α Pathway

“…Recently, several studies demonstrated that mitochondrial dysfunction precedes and activates muscle atrophy signaling in Dex-induced muscle atrophy models [12] and some natural plant extracts, including hydroxytyrosol acetate, resveratrol and quercetin, can target the mitochondria to improve mitochondrial function and prevent muscle atrophy under several wasting conditions [12][13][14]. Thus, development of drugs or nutrients that improve mitochondrial function could be beneficial in preventing Dex-induced muscle atrophy.…”

APR3 modulates oxidative stress and mitochondrial function in ARPE‐19 cells

“…10 mM antimycin A, 6 mM carbonyl cyanide-p trifluoromethoxyphenylhydrazone (FCCP), and 10 mM oligomycin were used as inhibitors, and the basal, maximal, ATP-linked and proton leakage, were determined and adjusted by protein concentration (32).…”