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  • It is not established if ONOO also induces tyrosine nitratio


    It is not established if ONOO− also induces tyrosine nitration of ANT and affects VDAC1 and ANT interaction, as well as the interaction of VDAC1 with HK II, all of which are events that may lead to altered mitochondrial and cellular function. Thus, in this study we examined tyrosine nitration of both ANT and VDAC1, and investigated the impact of ONOO− on these two proteins and their functional coupling as well as the effect of VDAC1 on an interacting with HK II. We hypothesized that ONOO− induces ANT tyrosine nitration, alters its interaction with VDAC1, and impairs the association of HK II with mitochondria, specifically VDAC1. We found that exposure of mitochondria to ONOO− hinders the ability of ANT to interact with VDAC1, and VDAC1 to interact with HK II. Moreover, mitochondrial function is altered by ONOO−. Consequently, mitochondrial exposure to ONOO− triggered disassembly of the HK II-VDAC1-ANT complex, which likely contributes to mitochondrial dysfunction as an underlying factor in cardiac IR injury.
    Materials and Methods
    Discussion ONOO− levels increase during cardiac IR injury (Novalija et al., 2002; Yang et al., 2012; Zweier et al., 2001) and a reduction in injury is associated with a decrease in its release (Novalija et al., 2002; Yang et al., 2012; Zweier et al., 2001). ONOO− is a strong oxidant of mitochondrial proteins and 6063 (Burwell and Brookes, 2008). Thus, mitochondria are both a site for ONOO− production and a target of ONOO−-induced dPTMs (Burwell and Brookes, 2008). Irreversible dPTMs of specific mitochondrial proteins, notably nitration of tyrosine residues by ONOO−, likely enhance mitochondrial dysfunction during IR injury. Our prior study (Yang et al., 2012) showed tyrosine nitration of VDAC1 induced after IR injury. In the present study, we found that ex vivo cardiac IR injury also led to an increase in tyrosine nitration of ANT (Fig. 2). During oxidative/nitrosative stress after IR injury, an increase in ONOO− production (Yang et al., 2012; Zweier et al., 2001) could, in part, be responsible for the dissociation of VDAC1 from ANT, and dissociation of HK II from VDAC1 (Fig. 3, Fig. 4). In vitro treatment of mitochondria with ONOO− led to ANT tyrosine nitration (Fig. 1), dissociation of both ANT (Fig. 3A) and HK II (Fig. 4A, C) from VDAC1, induced cyt c release from mitochondria (Fig. 5), reduced RCI, and delayed ΔΨm repolarization after transient depolarization with ADP (state 3 respiration) (Fig. 6). The ONOO−-induced cyt c release from mitochondria was attenuated by MCGR, BKA and DIDS (Fig. 5B), indicating that oxidative stress was involved, and that ONOO− might have altered ANT and VDAC1. As in isolated mitochondria from guinea pig hearts, H9c2 cells treated with ONOO− or hypoxia also displayed dissociation of HK II from mitochondria (Fig. 4C). In the ex vivo isolated hearts, when compared to time controls (TC), 35 min ischemia and 20 min reperfusion significantly increased diastolic LVP and decreased coronary flow rate (Fig. 2D), which coincided with indicators of mitochondrial dysfunction, i.e., reduction of RCI (Fig. 6A,B), delayed ΔΨm repolarization (Fig. 6E,F), VDAC1 dissociation from ANT (Fig. 3), and HK II dissociation from VDAC1 (Fig. 4). Altogether, these data indicate that mitochondrial dysfunctions induced by ONOO− produced during IR injury, likely resulted in alteration of metabolite fluxes across the IMM and OMM, which led to compromise of cardiac function on reperfusion (Fig. 2D). Although we have focused on the important roles of VDAC1 and ANT nitration on their associations with each other and with other proteins, it must be noted that cardiac IR injury can lead to nitration of many other mitochondrial and non-mitochondrial proteins.
    Summary and conclusions In summary, we detected tyrosine nitration of ANT and VDAC1 following exposure of isolated mitochondria or H9c2 cells to ONOO−. Similar results were also obtained following IR injury in isolated hearts. Furthermore, exposure to ONOO− resulted in the dissociation of HK II from VDAC1, and a reduction in the interaction of VDAC1 with ANT. Isolated mitochondria exposed to ONOO− exhibited compromised bioenergetics. Based on these findings, ONOO− leads to mitochondrial dysfunction and also induces cyt c release. These events may be associated with the impact of ONOO− on VDAC1 and ANT, although further studies will be required to confirm this notion. Additionally, nitration of VDAC1 and ANT may be contributors to mitochondrial dysfunction, though other potential modifications induced by ONOO− exposure should also be considered. MCGR was cardioprotective against IR injury in an isolated heart setting and also preserved HK II binding to mitochondria. This suggests that MCGR-preserved HK II association to mitochondria contributes to this cardioprotection. Therefore, preventing ONOO− generation and preserving the integrity of these mitochondrial proteins and their interactions with each other and with cytosolic proteins, e.g. HK II, could represent novel strategies in mitigating cardiac IR injury.