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Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers., Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting., Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca 2+ signaling., Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca 2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed., Impact and Implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication., Competing Interests: None of the authors have a conflict of interest to declare that pertains to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Authors.)

Ischemia-reperfusion (I/R) injury is a promising therapeutic target to improve clinical outcomes after acute myocardial infarction. Ferroptosis, triggered by iron overload and excessive lipid peroxides, is reportedly involved in I/R injury. However, its significance and mechanistic basis remain unclear. Here, we show that glutathione peroxidase 4 (GPx4), a key endogenous suppressor of ferroptosis, determines the susceptibility to myocardial I/R injury. Importantly, ferroptosis is a major mode of cell death in I/R injury, distinct from mitochondrial permeability transition (MPT)-driven necrosis. This suggests that the use of therapeutics targeting both modes is an effective strategy to further reduce the infarct size and thereby ameliorate cardiac remodeling after I/R injury. Furthermore, we demonstrate that heme oxygenase 1 up-regulation in response to hypoxia and hypoxia/reoxygenation degrades heme and thereby induces iron overload and ferroptosis in the endoplasmic reticulum (ER) of cardiomyocytes. Collectively, ferroptosis triggered by GPx4 reduction and iron overload in the ER is distinct from MPT-driven necrosis in both in vivo phenotype and in vitro mechanism for I/R injury. The use of therapeutics targeting ferroptosis in conjunction with cyclosporine A can be a promising strategy for I/R injury., Competing Interests: This work was supported by JSPS KAKENHI (grants JP16H07049, JP18K15892, and JP21K16090 to Dr Ikeda; JP20K08426 to Dr Ide; and JP18K19405 and JP20H00493 to Dr Yamada), Uehara Memorial Foundation (to Dr Ikeda), the Japan Foundation for Applied Enzymology (VBIC: Vascular Biology of Innovation; to Dr Ikeda), MSD Life Science Foundation, the Public Interest Incorporated Foundation (to Dr Ikeda); Novartis Pharma Grants for Basic Research 2020 (to Dr Ikeda), Kowa Life Science Foundation (to Dr Ikeda); Center for Clinical and Translational Research of Kyushu University Hospital (to Dr Ide), and AMED-CREST (grant JP21gm0910013 to Drs Yamada and Imai). Dr Tsutsui has received remunerations from Kowa, Teijin Pharma, Nippon Boehringer Ingelheim, Mitsubishi Tanabe Pharma, Pfizer Japan, Ono Pharmaceutical, Daiichi Sankyo, Novartis Pharma, Bayer Yakuhin, Otsuka Pharmaceutical, and AstraZeneca; manuscript fee from Nippon Rinsho; research funding from Mitsubishi Tanabe Pharma, Nippon Boehringer Ingelheim, IQVIA Services Japan, MEDINET, Medical Innovation Kyushu, Kowa, Daiichi Sankyo, Johnson & Johnson, and NEC Corp; and scholarship funds or donations from Abbott Medical Japan, Otsuka Pharmaceutical, Boston Scientific Japan, Ono Pharmaceutical, Bayer Yakuhin, Nippon Boehringer Ingelheim, St Mary’s Hospital, Teijin Pharma, Daiichi Sankyo, and Mitsubishi Tanabe Pharma. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2022 The Authors.)

Cyclosporin A (CsA) inhibits the mitochondrial permeability transition (MPT), but not always. To characterize the CsA-sensitive and -insensitive MPT, rat liver mitochondria were exposed to low and high doses of various MPT inducers. Mitochondrial swelling, cyclophilin D membrane binding and permeability transition (PT) pore diameter were measured. The results indicate two conductance modes for the PT pore: one activated by Ca2+ and inhibited by CsA and Mg2+ and the other unregulated. We propose a new model of pore formation and gating in which PT pores form by aggregation of misfolded integral membrane proteins damaged by oxidant and other stresses. Chaperone-like proteins initially block conductance through these misfolded protein clusters; however, increased Ca2+ opens these regulated PT pores, an effect blocked by CsA. When protein clusters exceed chaperones available to block conductance, unregulated pore opening occurs. [Copyright &y& Elsevier]

Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.