Hydrogen sulfide mediates cardioprotection through Nrf2 signaling

Abstract

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 microg/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 Cepsilon 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.

Figure 1

Hydrogen sulfide preconditioning (H₂S PC) reduced the extent of injury and improved left ventricular function in mice following myocardial ischemia and reperfusion. (A) Representative mid-ventricular photomicrographs of hearts treated with vehicle and H₂S. (B) Area-at-risk (AAR) with respect to the left ventricle (LV) was similar between all groups. H₂S PC significantly attenuated myocardial infarct size with respect to the area-at-risk (Inf/AAR) and with respective to the LV (Inf/LV). (C) Circulating levels of troponin-I (ng/mL) were measured 24 hr after reperfusion. (D) Ejection fraction (%) was calculated in separate groups of mice using high-resolution, two-dimensional B-mode echocardiography images at baseline (BASE) and 7 days following myocardial ischemia. Values are means ± S.E.M. Numbers inside bars indicate the number of animals that were investigated in each group. ***p<0.001 vs. Sham or BASE. H₂S denotes Na₂S.

Figure 2

H₂S PC reduced oxidative stress and apoptotic cell death following myocardial ischemia and reperfusion. Cardiac (A) redox state (E_h) for GSH and GSSG and (B) lipid hydroperoxide levels (μM) from Sham controls, Vehicle (Veh), and H₂S PC treated mice at 1–24 hr of reperfusion following myocardial ischemia. (C) Representative immunoblots of uncleaved caspase-3, cleaved caspase-3, cytosolic cytochrome C, and mitochondrial cytochrome C from the hearts of Sham controls, Veh, and H₂S PC treated mice at 4 hr of reperfusion following myocardial ischemia. (D) Ratio of cleaved caspase-3 to uncleaved caspase-3, (E) ratio of cytosolic cytochrome C to mitochondrial cytochrome C, and (F) the number of TUNEL positive cells (% of total nuclei) from the hearts of Sham controls, Veh, and H₂S PC treated mice at 4 hr of reperfusion following myocardial ischemia. Values are means ± S.E.M. Numbers inside bars indicate the number of animals that were investigated in each group. *p<0.05, **p<0.01, ***p<0.001 vs. Sham.

Figure 3

H₂S upregulated cellular antioxidant defenses. (A) Representative immunoblots and (B) densitometric analysis of cardiac Nrf2 in the cytosolic and nuclear fractions 30 min and 2 hr following the administration of H₂S. (C) Representative immunoblots and (D) densitometric analysis of cardiac thioredoxin-1 (Trx1), heme oxygenase-1 (HO-1), copper zinc superoxide dismutase (CuZnSOD), and manganese SOD (MnSOD) 24 hr following the administration of H₂S. Values are means ± S.E.M. for an n of 4–5 animals for each group. *p<0.05, **p<0.01 vs. Sham.

Figure 4

Nrf2 mediates the cardioprotective effects of H₂S. (A) Representative immunoblots and (B) densitometric analysis of cardiac Trx1 and HO-1 from the hearts of Nrf2 deficient (Nrf2 KO) mice ± H₂S. (C) Myocardial infarct size and (D) circulating levels of troponin-I were measured 24 hr after LCA ischemia in Wild-Type (WT) and Nrf2 KO mice receiving either vehicle or H₂S PC (100 μg/kg) treatment. Nrf2 KO mice experienced exacerbated myocardial injury when compared to wild-type mice. However, no differences in myocardial infarct size or circulating troponin-I levels were observed in Nrf2 KO following H₂S PC treatment. Numbers inside bars indicate the number of animals that were investigated in each group. *p<0.05, **p<0.01 vs. Sham or WT.

Figure 5

H₂S activated PKC_ε-p/44/42-STAT-3 signaling. Representative immunoblots and densitometric analysis of (A) phosphorylated PKCε at serine residue 729 (PKC_ε^Ser729) and total PKCε (cytosolic and membranous fractions), (B) phosphorylated p44/42 and total p44/42 (cytosolic fraction), and (C) phosphorylated STAT-3 at serine residue 727 (STAT-3^Ser727) and total STAT-3 (cytosolic and nuclear fractions) 30 min and 2 hr following the administration of H₂S. Values are means ± S.E.M. for an n of 4–5 animals for each group. *p<0.05, **p<0.01 vs. Sham.

Figure 6

H₂S increased the expression of anti-apoptogens. (A–B) Representative immunoblots and densitometric analysis of phosphorylated Bad at serine residue 112 (Bad-P^Ser112) and total Bad (cytosolic and mitochondrial fractions) and (C–D) HSP90, HSP70, Bcl-2, Bcl-xL, and COX-2 24 hr following the administration of H₂S. Values are means ± S.E.M. for an n of 4–5 animals for each group. *p<0.05, **p<0.01 vs. Sham.

Figure 7

Cardiac-specific overexpression of CGL increased Nrf2 nuclear accumulation and PKC_ε-STAT-3 signaling. Representative immunoblots and densitometric analysis of (A) Nrf-2 (cytosolic and nuclear fractions), (B) phosphorylated PKC_ε^Ser729 and total PKC_ε (cytosolic and membranous fractions), (C) phosphorylated STAT-3^Ser727 and total STAT-3 (cytosolic and nuclear fractions), (D) Trx1, HO-1, HSP90, and Bcl-2 from the hearts of CGL-Tg+ (n=4) and Non-Tg (n=4) mice. Values are means ± S.E.M. *p<0.05, **p<0.01 vs. Non-Tg.