The recent discovery that hydrogen sulfide (H ₂ S) is an endogenously produced gaseous second messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential of H ₂ S as a cardioprotective agent. In the current study, we demonstrate that the delivery of H ₂ S at the time of reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of myocardial ischemia-reperfusion (MI-R). This observed cytoprotection is associated with an inhibition of myocardial inflammation and a preservation of both mitochondrial structure and function after I-R injury. Additionally, we show that modulation of endogenously produced H ₂ S by cardiac-specific overexpression of cystathionine γ-lyase (α-MHC-CGL-Tg mouse) significantly limits the extent of injury. These findings demonstrate that H ₂ S may be of value in cytoprotection during the evolution of myocardial infarction and that either administration of H ₂ S or the modulation of endogenous production may be of clinical benefit in ischemic disorders.

Rationale: The recent emergence of hydrogen sulfide (H ₂ S) as a potent cardioprotective signaling molecule necessitates the elucidation of its cytoprotective mechanisms. Objective: The present study evaluated potential mechanisms of H ₂ S-mediated cardioprotection using an in vivo model of pharmacological preconditioning. Methods and Results: H ₂ S (100 μg/kg) or vehicle was administered to mice via an intravenous injection 24 hours before myocardial ischemia. Treated and untreated mice were then subjected to 45 minutes of myocardial ischemia followed by reperfusion for up to 24 hours, during which time the extent of myocardial infarction was evaluated, circulating troponin I levels were measured, and the degree of oxidative stress was evaluated. In separate studies, myocardial tissue was collected from treated and untreated mice during the early (30 minutes and 2 hours) and late (24 hours) preconditioning periods to evaluate potential cellular targets of H ₂ S. Initial studies revealed that H ₂ S provided profound protection against ischemic injury as evidenced by significant decreases in infarct size, circulating troponin I levels, and oxidative stress. During the early preconditioning period, H ₂ S increased the nuclear localization of Nrf2, a transcription factor that regulates the gene expression of a number of antioxidants and increased the phosphorylation of protein kinase Cε and STAT-3. During the late preconditioning period, H ₂ S increased the expression of antioxidants (heme oxygenase-1 and thioredoxin 1), increased the expression of heat shock protein 90, heat shock protein 70, Bcl-2, Bcl-xL, and cyclooxygenase-2 and also inactivated the proapoptogen Bad. Conclusions: These results reveal that the cardioprotective effects of H ₂ S are mediated in large part by a combination of antioxidant and antiapoptotic signaling.

Nitrite (NO2−) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II–IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.

Clinical studies have reported that the widely used antihyperglycemic drug metformin significantly reduces cardiac risk factors and improves clinical outcomes in patients with heart failure. The mechanisms by which metformin exerts these cardioprotective effects remain unclear and may be independent of antihyperglycemic effects. We tested the hypothesis that chronic activation of AMP-activated protein kinase (AMPK) with low-dose metformin exerts beneficial effects on cardiac function and survival in in vivo murine models of heart failure. Mice were subjected to permanent left coronary artery occlusion or to 60 minutes left coronary artery occlusion followed by reperfusion for 4 weeks. High-resolution, 2D echocardiography was performed at baseline and 4 weeks after myocardial infarction to assess left ventricular dimensions and function. Metformin (125 μg/kg) administered to mice at ischemia and then daily improved survival by 47% ( P <0.05 versus vehicle) at 4 weeks following permanent left coronary artery occlusion. Additionally, metformin given at reperfusion and then daily preserved left ventricular dimensions and left ventricular ejection fraction ( P <0.01 versus vehicle) at 4 weeks. The improvement in cardiac structure and function was associated with increases in AMPK and endothelial nitric oxide synthase (eNOS) phosphorylation, as well as increased peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression in cardiac myocytes. Furthermore, metformin significantly improved myocardial cell mitochondrial respiration and ATP synthesis compared to vehicle. The cardioprotective effects of metformin were ablated in mice lacking functional AMPK or eNOS. This study demonstrates that metformin significantly improves left ventricular function and survival via activation of AMPK and its downstream mediators, eNOS and PGC-1α, in a murine model of heart failure.

OBJECTIVE—Clinical studies have reported that metformin reduces cardiovascular end points of type 2 diabetic subjects by actions that cannot solely be attributed to glucose-lowering effects. The therapeutic effects of metformin have been reported to be mediated by its activation of AMP-activated protein kinase (AMPK), a metabolite sensing protein kinase whose activation following myocardial ischemia has been suggested to be an endogenous protective signaling mechanism. We investigated the potential cardioprotective effects of a single, low-dose metformin treatment (i.e., 286-fold less than the maximum antihyperglycemic dose) in a murine model of myocardial ischemia-reperfusion (I/R) injury.

Significance: Physiological concentrations of hydrogen sulfide (H ₂ S) exert potent prosurvival actions. We demonstrate that the cytoprotective actions of H ₂ S are mediated in part via a second gaseous signaling molecule, nitric oxide (NO). We found that cystathionine γ-lyase (CSE) KO mice with reduced H ₂ S levels exhibit increased oxidative stress and an exacerbated response to myocardial ischemia/reperfusion injury. CSE KO mice also exhibit reduced levels of NO and reduced NO synthesis via endothelial NO synthase (eNOS). Both oxidative stress and myocardial injury in CSE KO mice were attenuated by exogenous H ₂ S therapy, with increased eNOS function and restoration of NO levels. These findings provide insight into H ₂ S-mediated cytoprotetion and important information regarding the translation of H ₂ S therapy to the clinic.