Original articleIn vivo cardioprotection by S-nitroso-2-mercaptopropionyl glycine
Introduction
Ischemic preconditioning (IPC) is a cardioprotective event in which intermittent sub-lethal ischemia recruits several intracellular signaling mechanisms to protect against subsequent ischemia–reperfusion (IR) injury [1], [2], [3]. Mitochondria are key players in both the initiation of IPC signaling cascades, as well as the end-targets of protection [1], [4], [5], [6]. Post-translational modifications to mitochondrial proteins (e.g. kinase mediated phosphorylation) have been widely recognized as playing an important role in IPC signaling [7], and more recently the mitochondrial formation of S-nitrosothiols (SNOs) has been proposed as a novel protective signaling mechanism in IPC [4], [8], [9], [10], [11].
S-nitrosation is a reversible nitric oxide (NO) dependent modification of thiols (e.g. protein cysteines) resulting in formation of SNOs [12]. Many proteins with important roles in energy metabolism and Ca2+ homeostasis (both critical cardiomyocyte functions) have been identified as targets of S-nitrosation, including: L-type Ca2+ channels [13], ryanodine receptor [14], sarco/endoplasmic reticulum Ca2+ ATPase [10], caspases [15], hemoglobin [16], the 75 kDa subunit of complex I [8], [10], [17], [18], thioredoxin [19], and the β-subunit of the F1-ATPase [10].
In agreement with the extensive literature suggesting a cardioprotective role for NO in IR injury [4], [20], [21], we previously showed that in Langendorff perfused hearts and isolated cardiomyocytes exposed to IPC, an elevation of mitochondrial SNOs was associated with the preservation of mitochondrial function during subsequent IR injury [9]. In order to exploit the potentially beneficial cardioprotective effects of mitochondrial S-nitrosation, the mitochondrial S-nitrosating agent S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was developed [9] and was shown to mimic IPC-mediated protection against IR injury in both perfused hearts and cardiomyocytes, concurrent with S-nitrosation of mitochondrial proteins and reversible inhibition of complex I activity. These data are also in agreement with an emerging consensus that reversible inhibition of the mitochondrial respiratory chain is cardioprotective, as evidenced by the protective efficacy of a wide range of respiratory inhibitors [22], [23], [24]. Furthermore, it has recently been suggested that mitochondrial S-nitrosation and inhibition of complex I may underlie the cardioprotective efficacy of nitrite [25].
To date, cardioprotection by SNO-MPG has only been investigated in vitro, and the role of complex I remains unclear. Thus, the current study sought to investigate the effectiveness of SNO-MPG in vivo, using the popular murine left anterior descending coronary artery (LAD) occlusion model of IR injury. In addition the role of complex I was studied by employing a genetic model of complex I deficiency, the NDUFS4+/− mouse, which is deficient in the 18 kDa subunit of complex I, resulting a permanent 30% inhibition of cardiac complex I activity [26]. Summarizing the results, intraperitoneal delivery of SNO-MPG at 1 mg/kg, either prior to ischemia or at reperfusion, was cardioprotective in vivo. Both IPC and SNO-mediated cardioprotection were abrogated in perfused hearts from NDUFS4+/− mice. Together these data support a role for mitochondrial S-nitrosation and complex I inhibition in cardioprotection in vivo by IPC, S-nitrosothiols, and related therapies.
Section snippets
Animals, chemicals and reagents
Male C57BL6 mice (30–35 g) were from Harlan (Indianapolis, IN). Mice with germ-line knockout of exon 5 of the complex I NDUFS4 gene were generated to investigate the pathology of Leigh syndrome (an inherited metabolic disorder), as described elsewhere [26]. Animals were genotyped by tail biopsy PCR. Homozygous NDUFS4−/− mice exhibited embryonic lethality, whereas heterozygous NDUFS4+/− mice were viable with no overt cardiac phenotype at 8 weeks (the age used in this study). These mice and their
SNO-MPG and IPC protect vs. IR injury in vivo
To test the in vivo cardioprotective efficacy of SNO-MPG, the popular murine LAD artery occlusion model of IR injury was employed. The data in Fig. 2B show that under baseline IR conditions (group A), after 2 h reperfusion a significant infarct developed (54 ± 4% of the area at risk). Intraperitoneal injection of SNO-MPG (1 mg/kg) 30 min prior to occlusion (group B) significantly decreased infarct size to 7 ± 1%. From a more clinically relevant perspective, SNO-MPG (1 mg/kg) was also
Discussion
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
Acknowledgements
PSB is funded by NIH grant HL071158. CAP is funded by NIH grants HD053037 and RR16286. LSB acknowledges receipt of a 2007 Elon Huntington Hooker fellowship from the University of Rochester. AEF acknowledges support from the Clinical Translational Sciences Institute of the University of Rochester.
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Current address: Department of Molecular and Cellular Biology, Cornell University, Ithaca, NY 14853, USA.