Mitochondrial electron transport chain (ETC) plays a central role in ATP synthesis, and its dysfunction is associated with human diseases. Recent studies revealed that individual ETC complexes are assembled into supercomplexes. The main supercomplex, respirasome composed of complexes I, III, and IV has been suggested to improve electron channeling and control ROS production, maintain the structural integrity of ETC complexes and prevent protein aggregation in the inner mitochondrial membrane. However, many questions related to the structural organization of the respirasome, particularly, a possible role of complexes I and II in respirasome formation remain unclear. Here, we investigated whether genetic and pharmacological inhibition of complexes I and II affect respirasome assembly in cardioblast cells and isolated cardiac mitochondria. Pharmacological inhibition of the enzymatic activity of complexes I and II stimulated disruption of the respirasome. Likewise, knockdown of the complex I subunit NDUFA11 stimulated dissociation of respirasome and reduced the activity of complexes I, III, and IV. However, silencing of the membrane-anchored SDHC subunit of complex II had no effect on the respirasome assembly but reduced the activity of complexes II and IV. Downregulation of NDUFA11 or SDHC reduced ATP production and increased mitochondrial ROS production. Overall, these studies, for the first time, provide biochemical evidence that the complex I activity, and the NDUFA11 subunit are important for assembly and stability of the respirasome. The SDHC subunit of complex II is not involved in the respirasome however the complex may play a regulatory role in respirasome formation.

The mechanism by which c-Jun N-terminal protein kinase (JNK) promotes tissue injury is poorly understood. Thus we aimed at studying the roles of JNK and its phospho-target proteins in mouse models of acute liver injury. Young male mice were exposed to a single dose of CCl4 (50mg/kg, IP) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other enzyme activities were measured in CCl4-exposed mice without or with the highly-specific JNK inhibitors. Phosphoproteins were purified from control or CCl4-exposed mice and analyzed by differential mass-spectrometry followed by further characterizations of immunoprecipitation and activity measurements. JNK was activated within 1h while liver damage was maximal at 24h post-CCl4 injection. Markedly increased phosphorylation of many mitochondrial proteins was observed between 1 and 8h following CCl4 exposure. Pretreatment with the selective JNK inhibitor SU3327 or the mitochondria-targeted antioxidant mito-TEMPO markedly reduced the levels of p-JNK, mitochondrial phosphoproteins and liver damage in CCl4-exposed mice. Differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in anti-oxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. Aldehyde dehydrogenase, NADH-ubiquinone oxidoreductase, and α-ketoglutarate dehydrogenase were phosphorylated in CCl4-exposed mice but dephosphorylated after SU3327 pretreatment. Consistently, the suppressed activities of these enzymes were restored by SU3327 pretreatment in CCl4-exposed mice. These data provide a novel mechanism by which JNK, rapidly activated by CCl4, promotes mitochondrial dysfunction and acute hepatotoxicity through robust phosphorylation of numerous mitochondrial proteins.

Ferroptosis is a novel iron-dependent regulated cell death mechanism that affects cell metabolism; however, a detailed metabolomic analysis of ferroptotic cells is not yet available. Here, we elucidated the metabolome of H9c2 cardioblasts by gas chromatography-mass spectrometry during ferroptosis induced by RSL3, a GPX4 inhibitor, in the presence of ferrostatin-1 (a ferroptosis inhibitor), XJB-5-131 (a mitochondrial-targeted ROS scavenger), or TSM-1005-44 (a newly developed cellular ROS scavenger). Results demonstrated that RSL3 decreased the levels of amino acids involved in glutathione synthesis more than two-fold. In contrast, saturated fatty acids levels were markedly increased in RSL3-challenged cells, with no effects on unsaturated fatty acids. RSL3 significantly altered the levels of mitochondrial tricarboxylic acid cycle intermediates; isocitrate and 2-oxoglutarate were found to increase, whereas succinate was significantly decreased in RSL3-challenged cells. Ferrostatin-1, XJB-5-131, and TSM-1005-44 prevented RSL3-induced cell death and conserved the metabolomic profile of the cells. Since 2-oxoglutarate is involved in the regulation of ferroptosis, particularly through glutamine metabolism, we further assessed the role of glutaminolysis in ferroptosis in H9c2 cardioblasts. Genetic silencing of GLS1, which encodes the K-type mitochondrial glutaminase (glutaminase C), protected against ferroptosis in the early stage. In conclusion, our study demonstrates that RSL3-induced ferroptosis impairs the metabolome of H9c2 cardioblasts.

Alzheimer’s disease (AD) includes the formation of extracellular deposits comprising aggregated β-amyloid (Aβ) fibers associated with oxidative stress, inflammation, mitochondrial abnormalities, and neuronal loss. There is an associative link between AD and cardiac diseases; however, the mechanisms underlying the potential role of AD, particularly Aβ in cardiac cells, remain unknown. Here, we investigated the role of mitochondria in mediating the effects of Aβ_1-40 and Aβ_1-42 in cultured cardiomyocytes and primary coronary endothelial cells. Our results demonstrated that Aβ_1-40 and Aβ_1-42 are differently accumulated in cardiomyocytes and coronary endothelial cells. Aβ_1-42 had more adverse effects than Aβ_1-40 on cell viability and mitochondrial function in both types of cells. Mitochondrial and cellular ROS were significantly increased, whereas mitochondrial membrane potential and calcium retention capacity decreased in both types of cells in response to Aβ_1-42. Mitochondrial dysfunction induced by Aβ was associated with apoptosis of the cells. The effects of Aβ_1-42 on mitochondria and cell death were more evident in coronary endothelial cells. In addition, Aβ_1-40 and Aβ_1-42 significantly increased Ca²⁺ -induced swelling in mitochondria isolated from the intact rat hearts. In conclusion, this study demonstrates the toxic effects of Aβ on cell survival and mitochondria function in cardiac cells.

Individual electron transport chain complexes have been shown to assemble into the supramolecular structures known as the respiratory chain supercomplexes (RCS). Several studies reported an associative link between RCS disintegration and human diseases, although the physiological role, structural integrity, and mechanisms of RCS formation remain unknown. Our previous studies suggested that the adenine nucleotide translocase (ANT), the most abundant protein of the inner mitochondrial membrane, can be involved in RCS assembly. In this study, we sought to elucidate whether ANT knockdown (KD) affects RCS formation in H9c2 cardiomyoblasts. Results showed that genetic silencing of ANT1, the main ANT isoform in cardiac cells, stimulated proliferation of H9c2 cardiomyoblasts with no effect on cell viability. ANT1 KD reduced the ΔΨ_m but increased total cellular ATP levels and stimulated the production of total, but not mitochondrial, reactive oxygen species. Importantly, downregulation of ANT1 had no significant effects on the enzymatic activity of individual ETC complexes I–IV; however, RCS disintegration was stimulated in ANT1 KD cells as evidenced by reduced levels of respirasome, the main RCS. The effects of ANT1 KD to induce RCS disassembly was not associated with acetylation of the exchanger. In conclusion, our study demonstrates that ANT is involved in RCS assembly.

Mitochondrial respiratory chain supercomplexes (RCS), particularly, the respirasome, which contains complexes I, III, and IV, have been suggested to participate in facilitating electron transport, reducing the production of reactive oxygen species (ROS), and maintaining the structural integrity of individual electron transport chain (ETC) complexes. Disassembly of the RCS has been observed in Barth syndrome, neurodegenerative and cardiovascular diseases, diabetes mellitus, and aging. However, the physiological role of RCS in high energy-demanding tissues such as the heart remains unknown. This study elucidates the relationship between RCS assembly and cardiac function. Adult male Sprague Dawley rats underwent Langendorff retrograde perfusion in the presence and absence of ethanol, isopropanol, or rotenone (an ETC complex I inhibitor). We found that ethanol had no effects on cardiac function, whereas rotenone reduced heart contractility, which was not recovered when rotenone was excluded from the perfusion medium. Blue native polyacrylamide gel electrophoresis revealed significant reductions of respirasome levels in ethanol- or rotenone-treated groups compared to the control group. In addition, rotenone significantly increased while ethanol had no effect on mitochondrial ROS production. In isolated intact mitochondria in vitro, ethanol did not affect respirasome assembly; however, acetaldehyde, a byproduct of ethanol metabolism, induced dissociation of respirasome. Isopropanol, a secondary alcohol which was used as an alternative compound, had effects similar to ethanol on heart function, respirasome levels, and ROS production. In conclusion, ethanol and isopropanol reduced respirasome levels without any noticeable effect on cardiac parameters, and cardiac function is not susceptible to moderate reductions of RCS.