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.

Changes in mitochondrial DNA (mtDNA) integrity have been reported in many cancers; however, the contribution of mtDNA integrity to tumorigenesis is not well understood. We used a transgenic mouse model that is haploinsufficient for the apurinic/apyrimidinic endonuclease 1 (Apex1+/−) gene, which encodes the base excision repair (BER) enzyme APE1, to determine its role in protecting mtDNA from the effects of azoxymethane (AOM), a carcinogen used to induce colorectal cancer. Repair kinetics of AOM-induced mtDNA damage was evaluated using qPCR after a single AOM dose and a significant induction in mtDNA lesions in colonic crypts from both wild-type (WT) and Apex1+/−animals were observed. However, Apex1+/− mice had slower repair kinetics in addition to decreased mtDNA abundance. Tumors were also induced using multiple AOM doses, and both WT and Apex1+/−animals exhibited significant loss in mtDNA abundance. Surprisingly, no major differences in mtDNA lesions were observed in tumors from WT and Apex1+/− animals, whereas a significant increase in nuclear DNA lesions was detected in tumors from Apex1+/− mice. Finally, tumors from Apex1+/− mice displayed an increased proliferative index and histologic abnormalities. Taken together, these results demonstrate that APE1 is important for preventing changes in mtDNA integrity during AOM-induced colorectal cancer.Implications: AOM, a colorectal cancer carcinogen, generates damage to the mitochondrial genome, and the BER enzyme APE1 is required to maintain its integrity. Mol Cancer Res; 15(7); 831–41. ©2017 AACR.

Much of the Caribbean has been devastated by two major hurricanes that struck last month. Hurricane Irma first rampaged through the eastern Caribbean, British and the U.S. Virgin Islands, Puerto Rico, and Cuba, leaving islands such as Barbuda and St. Martin nearly uninhabitable. Less than 2 weeks later, Hurricane Maria tore through Dominica and pummeled the Puerto Rican archipelago with great fury.

Mitochondrial dysfunction and loss of mitochondrial DNA (mtDNA) integrity are increasingly recognized as key contributors to liver diseases such as cirrhosis and hepatocellular carcinoma. However, the role of mtDNA repair in maintaining mitochondrial homeostasis during liver injury remains poorly understood. Apurinic/apyrimidinic endonuclease 1 (APE1), encoded by the Apex1 gene, is the primary endonuclease mediating base excision repair of mtDNA. We hypothesize that APE1 is required to preserve mtDNA integrity in response to genotoxic stress in the liver. To test this, wild-type (WT) and Apex1 haploinsufficient mice (Apex1^+/−) were treated with the alkylating agent azoxymethane (AOM), a carcinogen bioactivated in the liver, and tissues were collected 20 weeks after the last exposure. Apex1^+/− mice exhibited a 3.2-fold increase in mtDNA lesions and a 55% reduction in mtDNA abundance, changes not observed in WT mice. Bioenergetics profiling revealed a 1.5-fold increase in the ATP5β/GAPDH ratio in WT mice and a 2.5-fold increase in Apex1^+/− mice, indicating a more pronounced shift toward oxidative phosphorylation in the absence of full APE1 function. Histological analysis indicated increased nuclear inclusions and ductular proliferation in both strains, whereas fibrosis was attenuated in Apex1^+/− mice. Collectively, these findings show that APE1 is essential for preserving mtDNA integrity and regulating bioenergetics and histopathological responses to alkylation-induced liver injury, highlighting its dual role in mitochondrial maintenance and modulating inflammatory outcomes.

Reperfusion injury is a paradoxical phenomenon involving mitochondrial dysfunction and cell death that reduces cardiac performance once blood flow is restored after myocardial infarction (MI). While several cell death mechanisms have been implicated, their specific contributions at different stages of reperfusion in the ex vivo ischemia–reperfusion (IRI) model remain unclear. This study assessed apoptosis, necroptosis, ferroptosis, autophagy, and pyroptosis at multiple reperfusion time points in rat hearts. Isolated hearts from adult male Sprague Dawley rats were perfused via the Langendorff retrograde method using Krebs–Henseleit solution, subjected to 30 min of equilibration, 25 min of global ischemia, and then 5, 30, or 60 min of reperfusion. Cardiac functional parameters were measured during perfusion, and mitochondria were isolated and analyzed for respiration, Ca ²⁺ -induced swelling, and ROS production. Cell death pathway markers were quantified via SDS-PAGE and western blotting. Cardiac physiological parameters, measured by LVDP and RPP, decreased significantly during reperfusion compared to pre-ischemic values. Mitochondrial State 3 oxygen consumption declined significantly by 38.7% as reperfusion progressed, while ROS production and Ca ²⁺ -induced swelling significantly increased 2.30- and 2.44-fold, respectively. Apoptosis showed a time-dependent 1.35-fold increase, as evidenced by Caspase-3 cleavage. Glutathione peroxidase 4, an endogenous ferroptosis inhibitor, decreased significantly by 43% during early reperfusion. Autophagy markers p62 and LC3B showed significant time-dependent increases of 2.08- and 1.37-fold, respectively. Necroptosis and pyroptosis markers showed no significant change during 60 min of reperfusion. Our results show an association between cardiac dysfunction and mitochondrial alterations, as well as differential activation of RCD markers during the early reperfusion phase. This suggests that specific mechanisms may predominate over others at different stages of reperfusion, indicating a time-sensitive interaction between these pathways that could influence future therapeutic strategies for MI patients.