Original article
In vivo cardioprotection by S-nitroso-2-mercaptopropionyl glycine

https://doi.org/10.1016/j.yjmcc.2009.01.012Get rights and content

Abstract

The reversible S-nitrosation and inhibition of mitochondrial complex I is a potential mechanism of cardioprotection, recruited by ischemic preconditioning (IPC), S-nitrosothiols, and nitrite. Previously, to exploit this mechanism, the mitochondrial S-nitrosating agent S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was developed, and protected perfused hearts and isolated cardiomyocytes against ischemia–reperfusion (IR) injury. In the present study, the murine left anterior descending coronary artery (LAD) occlusion model of IR injury was employed, to determine the protective efficacy of SNO-MPG in vivo. Intraperitoneal administration of 1 mg/kg SNO-MPG, 30 min prior to occlusion, significantly reduced myocardial infarction and improved EKG parameters, following 30 min occlusion plus 2 or 24 h reperfusion. SNO-MPG protected to the same degree as IPC, and notably was also protective when administered at reperfusion. Cardioprotection was accompanied by increased mitochondrial protein S-nitrosothiol content, and inhibition of complex I, both of which were reversed after 2 h reperfusion. Finally, hearts from mice harboring a heterozygous mutation in the complex I NDUSF4 subunit were refractory to protection by either SNO-MPG or IPC, suggesting that a fully functional complex I, capable of reversible inhibition is critical for cardioprotection. Overall, these results are consistent with a role for mitochondrial S-nitrosation and complex I inhibition in the cardioprotective mechanism of IPC and SNO-MPG in vivo.

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 (NOradical dot) 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 NOradical dot 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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