Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent

Abstract

Previous studies have demonstrated that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H2S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H2S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H2S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H2S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H2S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H2S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.

Keywords: Cth; cystathionase; eNOS uncoupling; myocardial infarction; nitrite.

Conflict of interest statement

Conflict of interest statement: D.J.L. is a participant in a pending US patent, filed through the National Institutes of Health (patent no. 60/511, 244), regarding the use of sodium nitrite in cardiovascular disease. D.J.L. and J.W.E. are founders of and scientific advisors for Sulfagenix, a biotechnology company that is currently developing hydrogen sulfide-based therapeutics for human disease conditions. The conflicts are considered significant.

Figures

Fig. 1.

Mice lacking the H2S-producing enzyme CSE display diminished free H2S and sulfur stores. (A–D) Circulating free H2S and sulfane sulfur levels (A and B) and cardiac free H2S and sulfane sulfur levels (C and D) measured in WT and CSE KO mice. (E–G) Cardiac mRNA expression of CSE (E), CBS (F), and 3-MST (G) standardized to 18s rRNA. Circles inside bars denote the number of animals per group.

Fig. 2.

Oxidative stress is exacerbated in CSE KO mice. (A and B) Heart and liver MDA levels. (C and D) Heart and liver carbonyl protein content. (E and G) Representative photomicrographs of DMPO-stained heart (E) and liver (G) depicting immunodetectable DMPO-adducted biomolecules indicative of oxidative stress and resultant biomolecular free radical formation. (F and H) Quantification of heart (F) and liver (H) tissue images. Circles inside bars denote number of animals per group. MFI, mean fluorescence intensity.

Fig. 3.

CSE KO mice exhibit altered eNOS phosphorylation status. (A) Representative immnoblots of eNOS from either WT or CSE KO hearts. (B–D) Relative intensity of P-eNOSS1177 (B), P-eNOST495 (C), and total eNOS (D) protein expression. (E–G) Levels of eNOS cofactors BH4 (E) and BH2 (F), and their ratio (G), in WT and CSE KO cardiac tissue. Circles inside bars denote the number of animals per group.

Fig. 4.

NO bioavailability and signaling is mitigated in CSE KO mice. (A) Plasma nitrite. (B) Cardiac nitrite. (C) Plasma RXNO. (D) Cardiac RXNO. (E) Plasma cGMP. (F) Cardiac cGMP. Circles inside bars denote the number of animals per group.

Fig. 5.

H2S therapy activates eNOS and augments NO bioavailability in CSE KO mice. (A) Representative immnoblots of eNOS. (B–D) Relative intensity of P-eNOSS1177 (B), P-eNOST495 (C), and total eNOS (D) protein expression in CSE KO hearts ± H2S donor. (E–H) Levels of plasma nitrite (E), cardiac nitrite (F), plasma RXNO (G), and cardiac RXNO (H). Circles inside bars denote the number of animals studied per group.

Fig. 6.

Ischemia/reperfusion injury is exacerbated in H2S-deficient mice, but rescued with H2S therapy. (A) Bar graphs of myocardial AAR/LV, INF/AAR, and INF/LV. (B) Cardiac troponin-I levels after 24 h of reperfusion. (C and D) Circulating ALT and AST and hepatic 8-isoprostane levels after 45 min of hepatic ischemia and 5 h of reperfusion. Circles inside bars denote the number of animals per group. LV, left ventricle.

Fig. 7.

H2S therapy fails to protect against myocardial I/R injury in eNOS mutant mice. Bar graphs of myocardial AAR/LV, INF/AAR, and INF/LV for eNOS KO mice with or without an H2S donor (A) and eNOSS1179A phosphomutant mice with or without an H2S donor (B). Circles inside bars denote the number of animals per group.