The main findings of this study are: (i) SNO-MPG protects hearts against IR injury in vivo and the magnitude of protection is similar to IPC; (ii) SNO-MPG and IPC induce S-nitrosation of similar mitochondrial proteins, and reversible complex I inhibition; (iii) NDUFS4+/− mice are refractory to the cardioprotective effects of IPC or SNO-MPG.
Previously we showed that SNO-MPG was cardioprotective in isolated perfused hearts and cardiomyocytes [9
]. In addition we and others have shown that S-nitrosation of mitochondrial proteins occurs during IPC, and that the reversible inhibition of respiratory complex I appears to be an important event which may contribute to cardioprotection [4
]. The studies performed herein suggest that the paradigm of cardioprotective mitochondrial S-nitrosation and reversible complex I inhibition [11
] also translates to the in vivo situation. The mechanism of protection appears to be similar in vivo and in vitro, since complex I activity measurements () showed that intraperitoneal administration of SNO-MPG was indeed capable of eliciting reversible cardiac complex I inhibition.
The molecular mechanisms linking mitochondrial S-nitrosation and complex I inhibition to cardioprotection remain to be elucidated, but several interesting candidate mechanisms have been proposed. First, S-nitrosation might protect critical thiol groups in mitochondrial proteins from irreversible oxidative damage [18
]. Complex I contains many critical thiols, and the enhanced post-IR recovery of complex I enzymatic activity in SNO-MPG treated animals () supports this hypothesis. Second, elevation of NADH due to SNO-induced complex I inhibition may enhance the mitochondrial GSH/GSSG ratio and thus also the ability of mitochondria to detoxify peroxides via glutathione dependent pathways [45
]. Third, a greater NADH/NAD+
ratio is a potent inhibitor of the mitochondrial permeability transition pore, an important contributor to the pathology of IR injury [47
]. Fourth, we recently proposed that reversible inhibition of the respiratory chain may facilitate a slower re-activation of mitochondrial metabolism at reperfusion (the “gradual wake up” hypothesis [4
]) preventing a surge of electrons into the respiratory chain and thus preventing a burst of ROS generation and Ca2+
]. Fifth, it has been proposed that S-nitrosation may serve as a “gateway” modification to further thiol adduction by for example glutathione (i.e., glutathionylation) [50
]. The role of glutathionylation in IPC overall is currently poorly understood, but ROS generation from complex I has recently been shown to be regulated by this modification, suggesting possible relevance [51
While overall the importance of each of these mechanisms is unclear, it is notable that several reversible inhibitors of complex I are protective in models of IR injury, including rotenone [44
], amobarbital [22
], volatile anesthetics [24
], ranolazine [52
], and capsaicin [54
] (for review see [4
]). Thus, complex I inhibition is emerging as an important paradigm for cardioprotection.
Surprisingly, SNO-MPG administration at the moment of reperfusion also caused a decrease in post-IR infarct size. This protection was concurrent with a small increase in mitochondrial S-nitrosation, and while the mechanism of protection may also include transient inhibition of complex I during reperfusion, such inhibition was not significant (). Similar protection was seen with administration of respiratory inhibitors at reperfusion [56
], but it is perhaps more reasonable to propose that other non-mitochondrial mechanisms may prevail under such conditions. In support of this hypothesis, we showed that EKG parameters returned to normal after reperfusion in both the pre- and post- SNO treated groups (B
), suggesting S-nitrosation may regulate the function of cardiac ion channels. Indeed, the inhibition of L-type Ca2+
channels due to S-nitrosation of the α1 subunit has been demonstrated in both IPC- and GSNO-treated hearts [10
]. This event decreases cardiomyocyte Ca2+
influx during the plateau phase of action potential, resulting in a shorter QTcorr
interval, diminishing the risk of post-ischemic arrhythmias [57
]. shows that QTcorr
duration was significantly reduced in SNO-MPG compared to vehicle-treated hearts during reperfusion. In addition to L-type Ca2+
channels, several other Ca2+
handling proteins are also regulated by S-nitrosation, including SERCA2a [10
] and RyR2 [14
]. S-nitrosation increases the activity of SERCA2a, which would stimulate sarcolemmal Ca2+
uptake and decrease the availability of Ca2+
for post-IR mitochondrial Ca2+
]. The role of SNO in the regulation of other important events of cardiac conductance, including Na+
channels and action potential transduction, is still unclear, but the effect of SNO-MPG on QRS duration at reperfusion () suggests such regulation may exist. In support of a preconditioned S-nitrosation phenotype being induced by SNO-MPG, significant overlap was observed in the proteomic pattern of S-nitrosated proteins between IPC and SNO-MPG treated hearts (online data supplement).
A requirement for complex I in either IPC or SNO-MPG mediated cardioprotection was established herein via the use of a novel mouse model in which the NDUFS4+/−
subunit of complex I was knocked out. As already mentioned, these mice exhibited elevated lactate levels, consistent with a mitochondrial metabolic defect [26
]. Several important processes relevant to IR injury and cardioprotection are pH-sensitive, including the activity of the Na+
], the mitochondrial PT pore [47
], and ischemic post-conditioning [59
]. Furthermore, another recently developed NDUFS4 knockout mouse exhibited an encephalomyopathy phenotype [61
]. Thus, to avoid the confounding effects of systemic lactate levels and neuronal effects (e.g. altered sympathetic/parasympathetic activity) on cardiac function, isolated perfused hearts from these mice were analyzed.
The NDUFS4+/− and WT perfused heart studies showed that a 30% permanent inhibition of complex I activity did not protect against IR injury, measured by myocardial infarct size. Therefore, while reversible complex I inhibition is associated with cardioprotection () it appears that irreversible complex I inhibition does not confer any cardioprotective benefit.
Most strikingly, NDUFS4+/− mouse hearts were refractory to cardioprotection by IPC and SNO-MPG (). Thus, functional complex I seems to be critically important for those cardioprotective stimuli which use complex I inhibition as a mechanism of protection. However, we cannot exclude the possibility that resistance to cardioprotection in the NDUFS4+/− hearts may be due to other alterations which accompany the genetic manipulation of complex I in this model.
From a clinical perspective, it is known that cardiac complex I activity is decreased in many pathological situations, including diabetes and aging [62
]. In addition, these same pathologies manifest a loss of cardioprotection in response to stimuli such as IPC or volatile anesthetics [65
]. Thus, it may be speculated that the loss of cardioprotection in these situations may arise at least in part from the loss of complex I activity. In future, it will be interesting to investigate whether various cardiotoxic drugs such as doxorubicin, or diseases such as Parkinson’s, both of which are known to impact cardiac complex I activity [72
], may also result in loss of IPC mediated cardioprotection.
Naturally, the current findings also have implications for the human clinical applicability of reagents such as SNO-MPG. It would be expected that such agents may be ineffective in diabetics and aged populations, or other individuals with diminished complex I function. Another important aspect of S-nitrosothiol use in humans is toxicity, and in this regard we found that high doses of SNO-MPG (>10mg/kg), as well as decomposition of S-nitrosothiols promoted by direct high-intensity white lighting of the surgical site, both resulted in ventricular fibrillation and death during early reperfusion. Thus, the dosing of S-nitrosothiols as cardioprotective agents requires careful attention.
Furthermore, the studies herein only examined short-term SNO-MPG administration. It has been suggested that SNO accumulation in the brain is related to the development of Parkinson’s disease [75
], and in this regard S-nitrosothiols are similar to 3-nitropropionic acid, which is known to protect the heart against IR injury, but also causes striatal lesions related to Huntington’s disease [23
]. We did not find any SNO in brain homogenates 30 min. after intraperitoneal injection of 20 mg/kg SNO-MPG (limit of detection 1 pmol SNO/mg protein, data not shown), suggesting that SNO-MPG is unable to penetrate the hematoencephalic barrier. These data also suggest that S-nitrosothiols may be metabolized or carried in the blood. Indeed, hemoglobin (Hb) has been widely reported as a target of S-nitrosation [16
], and it has been proposed that SNO-Hb is essential for hypoxic vasodilatation [78
] (although this concept has recently been challenged by the generation of a Hb-β93-Cys/Ala
mutant mouse [80
]). Furthermore, it has been shown that administration of SNO-Hb is cardioprotective in IR injury [81
]. Thus, the degree to which low-molecular weight SNOs such as SNO-MPG afford additional protection over that afforded by high-molecular weight blood-borne SNOs such as SNO-Hb, requires further investigation.
In conclusion, the results of the present study are consistent with the emerging paradigm that mitochondrial S-nitrosation and complex I inhibition are important in the cardioprotection afforded by IPC, and this paradigm extends to the in vivo situation. The forthcoming development of a variety of molecules which seek to exploit this cardioprotective mechanism (e.g. nitrite [25
], which is about to enter clinical trials for myocardial infarction), may afford novel therapeutics for IR injury in humans.