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  • Mitochondrial binding by HK prevents mortality

    2022-01-04

    Mitochondrial binding by HK prevents mortality by inhibiting pro-apoptotic factor (Bcl-2 family)-mediated opening of the mitochondrial permeability transition pore (mPTP) (Arora and Pedersen, 1988, Calmettes et al., 2016, Kodde et al., 2007, Pedersen et al., 2002, Rosano, 2011, Schindler and Foley, 2013, Sun et al., 2008, Zhuo et al., 2015). Furthermore, mtHK also increases metabolic efficiency by providing a local source of ADP for the mitochondria (Calmettes et al., 2016, Da-Silva et al., 2004, Meyer et al., 2006, Pastorino et al., 2005, Rosano, 2011, Schindler and Foley, 2013, Sun et al., 2008). Little characterization of the mPTP and its associated proteins has been undertaken in fish, although the available evidence suggests that the teleost mPTP is similar in structure and function to its mammalian homologue (reviewed by Vianello et al., 2012). This readily-available ADP decreases the proton motive force and prevents excess mitochondrial superoxide production (Banh et al., 2016). Superoxide, like other reactive oxygen species (ROS), induces oxidative stress, which has a wide range of potential physiological effects including cardiac arrhythmogenesis (Tse et al., 2016), which is a likely a major cause of high temperature mortality in fish (Brijs et al., 2015, Chen et al., 2015, Clark et al., 2008, Ekström et al., 2016, Farrell, 2002). The importance of mtHK is especially evident in rainbow trout cardiomyocytes given their reliance on HK to maintain Carbamazepine ADP return to the mitochondria (Karro et al., 2017). Mitochondria in fish muscle also show significantly higher intrinsic rates of ROS leakage than those in mammalian tissue (Banh et al., 2016), further necessitating mtHK-mediated cardioprotection. Both hyperoxic and hyperthermic exposures can increase production of ROS (Abele et al., 1998, Abele and Puntarulo, 2004, Buchner et al., 2001). Consistent with a cardioprotective role of mtHK against oxidative stress, a higher cardiac mtHK fraction was implicated in a rapid response to in vitro hypoxia in the Carbamazepine of an Amazonian armored catfish (Treberg et al., 2007). Several recent studies have investigated the effect of environmental hyperoxia compounded with acute hyperthermia on various physiological and cardiac performance metrics (Brijs et al., 2015, Devor et al., 2015, Ekström et al., 2016). Based on the functions of HK described above, it is possible that the compounded induction of oxidative stress via hyperoxia and hyperthermia in the salmonid heart could be at least partially countered by increased mtHK density. Furthermore, as physiological responses governing cardiorespiratory function could be affected by the presence of ROS before oxidative damage is evident, reversible HK localization could provide a sensitive indication of disrupted cellular homeostasis. In this study, we aimed to characterize mitochondrial hexokinase I (mtHKI) binding in the ventricles of rainbow trout (Oncorhynchus mykiss) exposed to both aerated and hyperoxic thermal episodes, with the hypothesis that mtHKI binding would increase under a hyperoxic high-temperature event and ameliorate oxidative stress relative to a normally-aerated thermal ramp. We additionally performed in vitro experiments examining the effect of mtHK binding on ventricular muscle function and cardiomyocyte respiration and mortality, by comparing the effect of lonidamine (LND)-mediated blocking of mtHKI association with the ionophore-mediated mitochondrial uncoupling of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). We hypothesized that LND-mediated mtHKI dissociation would increase CM oxygen consumption due to mitochondrial uncoupling, as the accumulated proton gradient escapes through the open mPTP. Furthermore, we expected that the LND-affected rate of oxygen consumption would mirror that of FCCP treatment, as the proton gradient would be dissipated similarly in both treatments albeit through different mechanisms. Finally, we hypothesized that dissociation of mtHKI using LND would prove more lethal to cardiomyocytes than FCCP treatment due to the induction of apoptotic pathways from an unprotected mPTP. Our results suggest that ventricular mtHK binding is not a factor in the cardiac response to acute thermal exposures alone, but that rainbow trout increase the ventricular mtHKI fraction when an acute thermal challenge is compounded with hyperoxia. This finding, when combined with a transient decrease in plasma lactate levels and an increase in ventricular ubiquitinylated conjugates, suggests a synergy in responses, or else that a certain environmental stimulus threshold is required to mount a cardioprotective response. This regulation may represent part of a response to counter incipient oxidative stress, although further work is necessary to fully characterize the physiological implications of this exposure.