Effects of inhibiting CoQ10 biosynthesis with 4-nitrobenzoate in human fibroblasts

Coenzyme Q(10) (CoQ(10)) is a potent lipophilic antioxidant in cell membranes and a carrier of electrons in the mitochondrial respiratory chain. We previously characterized the effects of varying severities of CoQ(10) deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ(10) biosynthesis. We observed a unimodal distribution of ROS production with CoQ(10) deficiency: cells with <20% of CoQ(10) and 50-70% of CoQ(10) did not generate excess ROS while cells with 30-45% of CoQ(10) showed increased ROS production and lipid peroxidation. Because our previous studies were limited to a small number of mutant cell lines with heterogeneous molecular defects, here, we treated 5 control and 2 mildly CoQ(10) deficient fibroblasts with varying doses of 4-nitrobenzoate (4-NB), an analog of 4-hydroxybenzoate (4-HB) and inhibitor of 4-para-hydroxybenzoate:polyprenyl transferase (COQ2) to induce a range of CoQ(10) deficiencies. Our results support the concept that the degree of CoQ(10) deficiency in cells dictates the extent of ATP synthesis defects and ROS production and that 40-50% residual CoQ(10) produces maximal oxidative stress and cell death.     

Altered redox status of coenzyme Q9 reflects mitochondrial electron transport chain deficiencies in Caenorhabditis elegans

Mitochondrial disorders are often associated with primary or secondary CoQ10 decrease. In clinical practice, Coenzyme Q10 (CoQ10) levels are measured to diagnose deficiencies and to direct and monitor supplemental therapy. CoQ10 is reduced by complex I or II and oxidized by complex III in the mitochondrial respiratory chain. Therefore, the ratio between the reduced (ubiquinol) and oxidized (ubiquinone) CoQ10 may provide clinically significant information in patients with mitochondrial electron transport chain (ETC) defects. Here, we exploit mutants of Caenorhabditis elegans (C. elegans) with defined defects of the ETC to demonstrate an altered redox ratio in Coenzyme Q9 (CoQ9), the native quinone in these organisms. The percentage of reduced CoQ9 is decreased in complex I (gas-1) and complex II (mev-1) deficient animals, consistent with the diminished activity of these complexes that normally reduce CoQ9. As anticipated, reduced CoQ9 is increased in the complex III deficient mutant (isp-1), since the oxidase activity of the complex is severely defective. These data provide proof of principle of our hypothesis that an altered redox status of CoQ may be present in respiratory complex deficiencies. The assessment of CoQ10 redox status in patients with mitochondrial disorders may be a simple and useful tool to uncover and monitor specific respiratory complex defects.     

Effect of mitochondrial dysfunction and oxidative stress on endogenous levels of coenzyme Q(10) in human cells

Little is known about the regulation of endogenous CoQ(10) levels in response to mitochondrial dysfunction or oxidative stress although exogenous CoQ(10) has been extensively used in humans. In this study, we first demonstrated that acute treatment of antimycin A, an inhibitor of mitochondrial complex III, and the absence of mitochondrial DNA suppressed CoQ(10) levels in human 143B cells. Because these two conditions also enhanced formation of reactive oxygen species (ROS), we further investigated whether oxidative stress or mitochondrial dysfunction primarily contributed to the decrease of CoQ(10) levels. Results showed that H(2)O(2) augmented CoQ(10) levels, but carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a chemical uncoupler, decreased CoQ(10) levels in 143B cells. However, H(2)O(2) and FCCP both increased mRNA levels of multiple COQ genes for biosynthesis of CoQ(10) . Our findings suggest that ROS induced CoQ(10) biosynthesis, whereas mitochondrial energy deficiency caused secondary suppression of CoQ(10) levels possibly due to impaired import of COQ proteins into mitochondria.     

Muscle coenzyme Q10 concentrations in patients with probable and definite diagnosis of respiratory chain disorders

Coenzyme Q(10) (CoQ) deficiency syndrome is a disorder of unknown ethiology that may cause different forms of mitochondrial encephalomyopathy. In the present study our aim was to analyse CoQ concentration and mitochondrial respiratory chain (MRC) enzyme activities in muscle biopsies of patients with clinical suspicion and/or biochemical-molecular diagnosis of a mitochondrial disorder. We studied 36 patients classified into 3 groups: 1) 14 patients without a definitive diagnosis of mitochondrial disease, 2) 13 patients with decreased CI + III and II + III activities of the MRC, and 3) 9 patients with definitive diagnosis of mitochondrial disease. Only 1 of the 14 patients of group 1 showed slightly reduced CoQ values in muscle. Six of the 13 patients from group 2 showed partial CoQ deficiency in muscle and 1 of the 9 cases from group 3 presented a slight CoQ deficiency. Significantly positive correlation was observed between CI + III and CII + III activities with CoQ concentrations in the 36 muscle homogenates from patients (r = 0.555; p = 0.001; and r = 0.460; p = 0.005, respectively). In conclusion, measurement of MRC enzyme activities is a useful tool for the detection of CoQ deficiency, which should be confirmed by CoQ quantification.     

Coenzyme Q cytoprotective mechanisms for mitochondrial complex I cytopathies involves NAD(P)H: quinone oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ1) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ1 cytoprotective mechanism required CoQ1 reduction by DT-diaphorase (NQO1). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ1 concentrations (5 microM). This suggests that the CoQ1H2 formed by NQO1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ1 concentrations (>10 microM), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ1 or menadione cytoprotection also involves the NQO1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ1H2 formed would then also act as a ROS scavenger.     

Primary Coenzyme Q deficiency Due to Novel ADCK3 Variants,Studies in Fibroblasts and Review of Literature

Primary deficiency of coenzyme Q10 (CoQ10 ubiquinone), is classified as a mitochondrial respiratory chain disorder with phenotypic variability. The clinical manifestation may involve one or multiple tissue with variable severity and presentation may range from infancy to late onset. ADCK3 gene mutations are responsible for the most frequent form of hereditary CoQ10 deficiency (Q10 deficiency-4 OMIM #612016) which is mainly associated with autosomal recessive spinocerebellar ataxia (ARCA2, SCAR9). Here we provide the clinical, biochemical and genetic investigation for unrelated three nuclear families presenting an autosomal form of Spino-Cerebellar Ataxia due to novel mutations in the ADCK3 gene. Using next generation sequence technology we identified a homozygous Gln343Ter mutation in one family with severe, early onset of the disease and compound heterozygous mutations of Gln343Ter and Ser608Phe in two other families with variable manifestations. Biochemical investigation in fibroblasts showed decreased activity of the CoQ dependent mitochondrial respiratory chain enzyme succinate cytochrome c reductase (complex II?+?III). Exogenous CoQ slightly improved enzymatic activity, ATP production and decreased oxygen free radicals in some of the patient’s cells. Our results are presented in comparison to previously reported mutations and expanding the clinical, molecular and biochemical spectrum of ADCK3 related CoQ10 deficiencies.

     

Coenzyme Q Cytoprotective Mechanisms for Mitochondrial Complex I Cytopathies Involves NAD(P)H: Quinone Oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ 1 ) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ 1 cytoprotective mechanism required CoQ 1 reduction by DT-diaphorase (NQO 1 ). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ 1 concentrations (5 &#119 M). This suggests that the CoQ 1 H 2 formed by NQO 1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ 1 concentrations (>10 &#119 M), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ 1 or menadione cytoprotection also involves the NQO 1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ 1 H 2 formed would then also act as a ROS scavenger.     

Fatal heart failure associated with CoQ10 and multiple OXPHOS deficiency in a child with propionic acidemia

The role of a secondary respiratory chain deficiency as an additional mechanism to intoxication, leading to development of long-term energy-dependent complications, has been recently suggested in patients with propionic acidemia (PA). We show for the first time a coenzyme Q(10) (CoQ(10)) functional defect accompanied by a multiple organ oxidative phosphorylation (OXPHOS) deficiency in a child who succumbed to acute heart failure in the absence of metabolic stress. Quinone-dependent activities in the liver (complex I+III, complex II+III) were reduced, suggesting a decrease in electron transfer related to the quinone pool. The restoration of complex II+III activity after addition of exogenous ubiquinone to the assay system suggests CoQ(10) deficiency. Nevertheless, we disposed of insufficient material to perform direct measurement of CoQ(10) content in the patient's liver. Death occurred before biochemical diagnosis of OXPHOS deficiency could be made. However, this case highlights the usefulness of rapidly identifying CoQ(10) defects secondary to PA since this OXPHOS disorder has a good treatment response which could improve heart complications or prevent their appearance. Nevertheless, further studies will be necessary to determine whether CoQ(10) treatment can be useful in PA complications linked to CoQ(10) deficiency.     

Human NADH:ubiquinone oxidoreductase deficiency: radical changes in mitochondrial morphology?

Malfunction of NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest complex of the mitochondrial oxidative phosphorylation system, has been implicated in a wide variety of human disorders. To demonstrate a quantitative relationship between CI amount and activity and mitochondrial shape and cellular reactive oxygen species (ROS) levels, we recently combined native electrophoresis and confocal and video microscopy of dermal fibroblasts of healthy control subjects and children with isolated CI deficiency. Individual mitochondria appeared fragmented and/or less branched in patient fibroblasts with a severely reduced CI amount and activity (class I), whereas patient cells in which these latter parameters were only moderately reduced displayed a normal mitochondrial morphology (class II). Moreover, cellular ROS levels were significantly more increased in class I compared with class II cells. We propose a mechanism in which a mutation-induced decrease in the cellular amount and activity of CI leads to enhanced ROS levels, which, in turn, induce mitochondrial fragmentation when not appropriately counterbalanced by the cell's antioxidant defense systems.     

Coenzyme Q10 reduces ethanol-induced apoptosis in corneal fibroblasts

Dilute ethanol (EtOH) is a widely used agent to remove the corneal epithelium during the modern refractive surgery. The application of EtOH may cause the underlying corneal fibroblasts to undergo apoptosis. This study was designed to investigate the protective effect and potential mechanism of the respiratory chain coenzyme Q(10) (CoQ(10)), an electron transporter of the mitochondrial respiratory chain and a ubiquitous free radical scavenger, against EtOH-induced apoptosis of corneal fibroblasts. Corneal fibroblasts were pretreated with CoQ(10) (10 μM) for 2 h, followed by exposure to different concentrations of EtOH (0.4, 2, 4, and 20%) for 20 s. After indicated incubation period (2-12 h), MTT assay was used to examine cell viability. Treated cells were further assessed by flow cytometry to identify apoptosis. Reactive oxygen species (ROS) and the change in mitochondrial membrane potential were assessed using dichlorodihydrofluorescein diacetate/2',7'-dichlorofluorescein (DCFH-DA/DCF) assays and flow-cytometric analysis of JC-1 staining, respectively. The activity and expression of caspases 2, 3, 8, and 9 were evaluated with a colorimetric assay and western blot analysis. We found that EtOH treatment significantly decreased the viability of corneal fibroblasts characterized by a higher percentage of apoptotic cells. CoQ(10) could antagonize the apoptosis inducing effect of EtOH. The inhibition of cell apoptosis by CoQ(10) was significant at 8 and 12 h after EtOH exposure. In EtOH-exposed corneal fibroblasts, CoQ(10) pretreatment significantly reduced mitochondrial depolarization and ROS production at 30, 60, 90, and 120 min and inhibited the activation and expression of caspases 2 and 3 at 2 h after EtOH exposure. In summary, pretreatment with CoQ(10) can inhibit mitochondrial depolarization, caspase activation, and cell apoptosis. These findings support the proposition that CoQ(10) plays an antiapoptotic role in corneal fibroblasts after ethanol exposure.     

The effects of dietary coenzyme Q on Drosophila life span

Reactive oxygen species (ROS), generated as by-products of aerobic metabolism, cause damage to proteins and cellular membranes, and are thus thought to influence senescence. Caenorhabditis elegans fed on diets lacking in ubiquinone coenzyme Q (CoQ), a coenzyme in the oxidative phosphorylation pathway, show increased longevity, possibly because of reduced ROS generation. We test the role of dietary CoQ in determining Drosophila melanogaster longevity by measuring survival and cytochrome c-oxidase activity (a proxy for aerobic metabolic performance) in flies fed wild-type yeast, CoQ-less yeast, or respiratory control (RC) yeast replete with CoQ but independently deficient in mitochondrial respiration. We find no evidence that dietary manipulation of CoQ in D. melanogaster increases life span or decreases age-dependent decline in cytochrome c oxidase activity. Instead, we find evidence that flies fed a diet of respiratory-deficient yeast (CoQ-less or RC) tend to have decreased longevity and increased rates of decline in cytochrome c-oxidase activity [corrected]     

Promotion of growth by Coenzyme Q10 is linked to gene expression in C. elegans

Coenzyme Q (CoQ, ubiquinone) is an essential component of the respiratory chain, a cofactor of pyrimidine biosynthesis and acts as an antioxidant in extra mitochondrial membranes. More recently CoQ has been identified as a modulator of apoptosis, inflammation and gene expression. CoQ deficient Caenorhabditis elegans clk-1 mutants show several phenotypes including a delayed postembryonic growth. Using wild type and two clk-1 mutants, here we established an experimental set-up to study the consequences of endogenous CoQ deficiency or exogenous CoQ supply on gene expression and growth. We found that a deficiency of endogenous CoQ synthesis down-regulates a cluster of genes that are important for growth (i.e., RNA polymerase II, eukaryotic initiation factor) and up-regulates oxidation reactions (i.e., cytochrome P450, superoxide dismutase) and protein interactions (i.e., F-Box proteins). Exogenous CoQ supply partially restores the expression of these genes as well as the growth retardation of CoQ deficient clk-1 mutants. On the other hand exogenous CoQ supply does not alter the expression of a further sub-set of genes. These genes are involved in metabolism (i.e., succinate dehydrogenase complex), cell signalling or synthesis of lectins. Thus, our work provides a comprehensive overview of genes which can be modulated in their expression by endogenous or exogenous CoQ. As growth retardation in CoQ deficiency is linked to the gene expression profile we suggest that CoQ promotes growth via gene expression.     

The Coenzyme Q10 analog decylubiquinone inhibits the redox-activated mitochondrial permeability transition: role of mitcohondrial [correction mitochondrial] complex III

The mitochondrial permeability transition (MPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the MPT, we used diethylmaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory complex III (cytochrome bc1), because 1). stigmatellin, a Qo site inhibitor, blocked ROS production and prevented the MPT and cell death and 2). cytochrome bc1 activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q10 analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent MPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the MPT and cell death, whereas Ub0 did not. Since we also found that dUb did not inhibit cytochrome bc1 activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc1. These results indicate that dUb, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component.     

Are mitochondrial reactive oxygen species required for autophagy?

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H₂O₂ was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.     

Parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome

Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function.In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q₁₀ (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells.To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ₁₀ (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.     

Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells

Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles. The mitochondrial permeability transition (MPT) results in mitochondrial depolarization and increased reactive oxygen species production, which can trigger autophagy. Therefore, we hypothesized that the MPT may have a role in signaling autophagy in cardiac cells. Mitochondrial membrane potential was lower in HL-1 cells subjected to starvation compared to cells maintained in full medium. Mitochondrial membrane potential was preserved in starved cells treated with cyclosporin A (CsA), suggesting the MPT pore is associated with starvation-induced depolarization. Starvation-induced autophagy in HL-1 cells, neonatal rat cardiomyocytes and adult mouse cardiomyocytes was inhibited by CsA. Starvation failed to induce autophagy in CypD-deficient murine cardiomyocytes, whereas in myocytes from mice overexpressing CypD the levels of autophagy were enhanced even under fed conditions. Collectively, these results demonstrate a role for CypD and the MPT in the initiation of autophagy. We also analyzed the role of the MPT in the degradation of mitochondria by biochemical analysis and electron microscopy. HL-1 cells subjected to starvation in the presence of CsA had higher levels of mitochondrial proteins (by Western blot), more mitochondria and less autophagosomes (by electron microscopy) than cells starved in the absence of CsA. Our results suggest a physiologic function for CypD and the MPT in the regulation of starvation-induced autophagy. Starvation-induced autophagy regulated by CypD and the MPT may represent a homeostatic mechanism for cellular and mitochondrial quality control.     

Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N(2)A cells (EC(50) in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca(2+) in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.     

Activation of Class I histone deacetylases contributes to mitochondrial dysfunction in cardiomyocytes with altered complex activities

Histone deacetylases (HDACs) play vital roles in the pathophysiology of heart failure, which is associated with mitochondrial dysfunction. Tumor necrosis factor-α (TNF-α) contributes to the genesis of heart failure and impairs mitochondria. This study evaluated the role of HDACs in TNF-α-induced mitochondrial dysfunction and investigated their therapeutic potential and underlying mechanisms. We measured mitochondrial oxygen consumption rate (OCR) and ATP production using Seahorse XF24 extracellular flux analyzer and bioluminescent assay in control and TNF-α (10 ng/ml, 24 h)-treated HL-1 cells with or without HDAC inhibition. TNF-α increased Class I and II (but not Class IIa) HDAC activities (assessed by Luminescent) with enhanced expressions of Class I (HDAC1, HDAC2, HDAC3, and HDAC8) but not Class IIb HDAC (HDAC6 and HDAC10) proteins in HL-1 cells. TNF-α induced mitochondrial dysfunction with impaired basal, ATP-linked, and maximal respiration, decreased cellular ATP synthesis, and increased mitochondrial superoxide production (measured by MitoSOX red fluorescence), which were rescued by inhibiting HDACs with MPT0E014 (1 μM, a Class I and IIb inhibitor), or MS-275 (1 μM, a Class I inhibitor). MPT0E014 reduced TNF-α-decreased complex I and II enzyme (but not III or IV) activities (by enzyme activity microplate assays). Our results suggest that Class I HDAC actions contribute to TNF-α-induced mitochondrial dysfunction in cardiomyocytes with altered complex I and II enzyme regulation. HDAC inhibition improves dysfunctional mitochondrial bioenergetics with attenuation of TNF-α-induced oxidative stress, suggesting the therapeutic potential of HDAC inhibition in cardiac dysfunction.