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We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6-phosphate at pH 6.3. Prior HK2 dissociation from pre- or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre- and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca2+]., Competing Interests: The authors have declared that no competing interests exist.

Doxorubicin (DOX) is an anticancer drug widely used to treat human and nonhuman tumors but the late and persistent cardio-toxicity reduces the therapeutic utility of the drug. The full mechanism(s) of DOX-induced acute, subchronic and delayed toxicity, which has a preponderant mitochondrial component, remains unclear; therefore, it is clinically relevant to identify early markers to identify patients who are predisposed to DOX-related cardiovascular toxicity. To address this, Wistar rats (16 weeks old) were treated with a single DOX dose (20 mg/kg, i.p.); then, mRNA, protein levels and functional analysis of mitochondrial endpoints were assessed 24 h later in the heart, liver, and kidney. Using an exploratory data analysis, we observed cardiac-specific alterations after DOX treatment for mitochondrial complexes III, IV, and preferentially for complex I. Conversely, the same analysis revealed complex II alterations are associated with DOX response in the liver and kidney. Interestingly, H2O2 production by the mitochondrial respiratory chain as well as loss of calcium-loading capacity, markers of subchronic toxicity, were not reliable indicators of acute DOX cardiotoxicity in this animal model. By using sequential principal component analysis and feature correlation analysis, we demonstrated for the first time alterations in sets of transcripts and proteins, but not functional measurements, that might serve as potential early acute markers of cardiac-specific mitochondrial toxicity, contributing to explain the trajectory of DOX cardiac toxicity and to develop novel interventions to minimize DOX cardiac liabilities., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in cardiac reperfusion injury and its prevention is cardioprotective. Tumour cell mitochondria usually have high levels of hexokinase isoform 2 (HK2) bound to their outer mitochondrial membranes (OMM) and HK2 binding to heart mitochondria has also been implicated in resistance to reperfusion injury. HK2 dissociates from heart mitochondria during ischaemia, and the extent of this correlates with the infarct size on reperfusion. Here we review the mechanisms and regulations of HK2 binding to mitochondria and how this inhibits mPTP opening and consequent reperfusion injury. Major determinants of HK2 dissociation are the elevated glucose-6-phosphate concentrations and decreased pH in ischaemia. These are modulated by the myriad of signalling pathways implicated in preconditioning protocols as a result of a decrease in pre-ischaemic glycogen content. Loss of mitochondrial HK2 during ischaemia is associated with permeabilization of the OMM to cytochrome c, which leads to greater reactive oxygen species production and mPTP opening during reperfusion. Potential interactions between HK2 and OMM proteins associated with mitochondrial fission (e.g. Drp1) and apoptosis (B-cell lymphoma 2 family members) in these processes are examined. Also considered is the role of HK2 binding in stabilizing contact sites between the OMM and the inner membrane. Breakage of these during ischaemia is proposed to facilitate cytochrome c loss during ischaemia while increasing mPTP opening and compromising cellular bioenergetics during reperfusion. We end by highlighting the many unanswered questions and discussing the potential of modulating mitochondrial HK2 binding as a pharmacological target., (© 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Although doxorubicin (DOX) is a very effective antineoplastic agent, its clinical use is limited by a dose-dependent, persistent and cumulative cardiotoxicity, whose mechanism remains to be elucidated. Previous works in animal models have failed to use a multi-organ approach to demonstrate that DOX-associated toxicity is selective to the cardiac tissue. In this context, the present work aims to investigate in vivo DOX cardiac, hepatic and renal toxicity in the same animal model, with special relevance on alterations of mitochondrial bioenergetics. To this end, male Wistar rats were sub-chronically (7 wks, 2 mg/Kg) or acutely (20 mg/Kg) treated with DOX and sacrificed one week or 24 hours after the last injection, respectively. Alterations of mitochondrial bioenergetics showed treatment-dependent differences between tissues. No alterations were observed for cardiac mitochondria in the acute model but decreased ADP-stimulated respiration was detected in the sub-chronic treatment. In the acute treatment model, ADP-stimulated respiration was increased in liver and decreased in kidney mitochondria. Aconitase activity, a marker of oxidative stress, was decreased in renal mitochondria in the acute and in heart in the sub-chronic model. Interestingly, alterations of cardiac mitochondrial bioenergetics co-existed with an absence of echocardiograph, histopathological or ultra-structural alterations. Besides, no plasma markers of cardiac injury were found in any of the time points studied. The results confirm that alterations of mitochondrial function, which are more evident in the heart, are an early marker of DOX-induced toxicity, existing even in the absence of cardiac functional alterations.

Mitochondria have long been involved in several cellular processes beyond its role in energy production. The importance of this organelle for cardiac tissue homeostasis has been greatly investigated and its impairment can lead to cell death and consequent organ failure. Several compounds have been described in the literature as having direct effects on cardiac mitochondria which can provide a mechanistic explanation for their toxicological or pharmacological effects. The present review describes one classic example of drug-induced cardiac mitochondrial toxicity and another case of drug-induced mitochondrial protection. For the former, we present the case for doxorubicin, an anticancer agent whose treatment is associated with a cumulative and dose-dependent cardiomyopathy with a mitochondrial etiology. Following this, we present the case of carvedilol, a β-blocker with intrinsic antioxidant activity, which has been described to protect cardiac mitochondria from oxidative injury. The final part of the review integrates information from the previous chapters, demonstrating how carvedilol can contribute to reduce doxorubicin toxicity on cardiac mitochondria. The two referred examples result in important take-home messages: a) drug-induced cardiac mitochondrial dysfunction is an important contributor for drug-associated organ failure, b) protection of mitochondrial function is involved in the beneficial impact of some clinically-used drugs and c) a more accurate prediction of toxic vs. beneficial effects should be an important component of drug development by the pharmaceutical industry.