S-Nitrosoglutathione Reductase Is Essential for Protecting the Female Heart From Ischemia-Reperfusion Injury

Abstract

Rationale: Protein S-nitros(yl)ation (SNO) has been implicated as an essential mediator of nitric oxide-dependent cardioprotection. Compared with males, female hearts exhibit higher baseline levels of protein SNO and associated with this, reduced susceptibility to myocardial ischemia-reperfusion injury. Female hearts also exhibit enhanced S-nitrosoglutathione reductase (GSNO-R) activity, which would typically favor decreased SNO levels as GSNO-R mediates SNO catabolism.

Objective: Because female hearts exhibit higher SNO levels, we hypothesized that GSNO-R is an essential component of sex-dependent cardioprotection in females.

Methods and results: Male and female wild-type mouse hearts were subjected to ex vivo ischemia-reperfusion injury with or without GSNO-R inhibition (N6022). Control female hearts exhibited enhanced functional recovery and decreased infarct size versus control males. Interestingly, GSNO-R inhibition reversed this sex disparity, significantly reducing injury in male hearts, and exacerbating injury in females. Similar results were obtained with male and female GSNO-R^-/- hearts using ex vivo and in vivo models of ischemia-reperfusion injury. Assessment of SNO levels using SNO-resin assisted capture revealed an increase in total SNO levels with GSNO-R inhibition in males, whereas total SNO levels remained unchanged in females. However, we found that although GSNO-R inhibition significantly increased SNO at the cardioprotective Cys39 residue of nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 3 in males, SNO-NADH dehydrogenase subunit 3 levels were surprisingly reduced in N6022-treated female hearts. Because GSNO-R also acts as a formaldehyde dehydrogenase, we examined postischemic formaldehyde levels and found that they were nearly 2-fold higher in N6022-treated female hearts compared with nontreated hearts. Importantly, the mitochondrial aldehyde dehydrogenase 2 activator, Alda-1, rescued the phenotype in GSNO-R^-/- female hearts, significantly reducing infarct size.

Conclusions: These striking findings point to GSNO-R as a critical sex-dependent mediator of myocardial protein SNO and formaldehyde levels and further suggest that different therapeutic strategies may be required to combat ischemic heart disease in males and females.

Keywords: formaldehyde; heart; nitric oxide; reactive oxygen species; reperfusion injury.

Figure 1.. GSNO-R inhibition induces protection in male hearts, but exacerbates injury in female hearts.

(A) Hearts were perfused with or without the GSNO-R inhibitor N6022 for 15 minutes, and then subjected to 20 minutes of ischemia and 120 minutes of reperfusion; N6022 was also present during the first five minutes of reperfusion. (B) Post-ischemic functional recovery and (C) infarct size in control male and female WT hearts (n = 12–13 hearts/group; *p<0.05 vs. control male). (D) Post-ischemic functional recovery and (E) infarct size in control and N6022-perfused male hearts (n = 5–12 hearts/group; *p<0.05 vs. control male). (F) Post-ischemic functional recovery and (G) infarct size in control and N6022-perfused female hearts (n = 5–13 hearts/group; **p<0.05 vs. control female). (H) Percent change in post-ischemic functional recovery and (I) infarct size with 10 μmol/L N6022 treatment vs. respective control male or female heart (n = 6–7 hearts/group; *p<0.05 vs. N6022-treated male).

Figure 2.. Genetic deletion of GSNO-R induces protection in male hearts, but exacerbates injury in female hearts.

(A, B) Infarct size from Langendorff-perfused male (A) and female (B) WT (+/+) and GSNO-R^−/− (−/−) hearts (n = 6–8 hearts/group; *p<0.05 vs. male WT, **p<0.05 vs. female WT). (C-J) Area-at-risk (AAR) (C, D), infarct size (% of AAR) (E, F), infarct size (% of total ventricle) (G, H), and plasma cTnI levels (I, J) from male and female WT and GSNO-R^−/− hearts subjected to LAD occlusion surgery (n = 8–9 mice/group; *p<0.05 vs. male WT, **p<0.05 vs. female WT).

Figure 3.. SNO protein levels increase in male hearts with GSNO-R inhibition, but not in female hearts.

(A-F) Total number of SNO protein identifications from control and N6022-treated male and female WT hearts at baseline (A) with Venn diagram (B), and post-I/R (D) with Venn diagram (E) as assessed via SNO-RAC in tandem with LC-MS/MS (n = 3 hearts/group; FDR: 1%). Note: All SNO protein identifications were detected in at least two of three hearts/group; these numbers represent the total number of SNO proteins identified in each group, so statistics were not performed. (C, F) SNO-ND3 levels from control and N6022-treated male and female WT hearts at baseline (C) and post I/R (F) as assessed via spectral counting (n = 3 hearts/group; *p<0.05 vs. control male, **p<0.05 vs. control female).

Figure 4.. N6022 suppresses post-ischemic ROS production in male and female hearts.

(A) Hydrogen peroxide production over time and (B) the rate of hydrogen peroxide production in post-ischemic control and N6022-treated male and female WT hearts (n = 5 hearts/group; *p<0.05 vs. control male, **p<0.05 vs. control female).

Figure 5.. N6022 inhibits GSNO-R activity in male and female hearts.

(A-D) GSNO-R activity in whole heart homogenates from control and N6022-treated male and female, WT and GSNO-R^−/− hearts measured via NADH consumption with GSNO as a substrate (A-B), and via NADH production with formaldehyde as a substrate (C-D) (n = 3 hearts/group; *p<0.05 vs. control male, **p<0.05 vs. control female).

Figure 6.. GSNO-R inhibition increases post-ischemic free formaldehyde levels in female hearts, but not in males.

(A) Pre-ischemic and (B) post-ischemic free formaldehyde levels assessed in control, N6022-treated and GSNO-R^−/− male and female hearts (n = 3 hearts/group; *p<0.05 vs. WT male, **p<0.05 vs. WT female).

Figure 7.. ALDH2 activation reduces infarct size in GSNO-R^−/− female hearts.

(A) Hearts were perfused with or without the ALDH2 activator Alda-1 (20 μmol/L) for 10 minutes, and then subjected to 20 minutes of ischemia and 120 minutes of reperfusion; Alda-1 was also present for the first 10 minutes of reperfusion. (B) Infarct size in control and Alda-1-perfused male and female, WT and GSNO-R^−/− hearts (n = 3–10 hearts/group; *p<0.05 vs. control male, **p<0.05 vs. control female, ^#p<0.05 vs. GSNO-R^−/− female).

Figure 8.. Formaldehyde does not compete with SNO for the modification of common cysteines.

(A-E) Free thiols were labeled with fluorescent maleimide tags in whole heart homogenates from WT males and females before and after treatment with 30, 50, 100 μmol/L GSNO or formaldehyde (A-C) (n = 3–4 hearts/group) or 50 μmol/L GSNO for 15 minutes, then 50 μmol/L formaldehyde for 15 minutes, and vice versa (D, E) (n = 2–3 hearts/group). *p<0.05 vs. free thiol male, **p<0.05 vs. free thiol female. Note: gel-to-gel variability was normalized with free thiol male sample (MC1).