Endogenous S-nitrosothiols protect against myocardial injury

Abstract

Despite substantial evidence that nitric oxide (NO) and/or endogenous S-nitrosothiols (SNOs) exert protective effects in a variety of cardiovascular diseases, the molecular details are largely unknown. Here we show that following left coronary artery ligation, mice with a targeted deletion of the S-nitrosoglutathione reductase gene (GSNOR(-/-)) have reduced myocardial infarct size, preserved ventricular systolic and diastolic function, and maintained tissue oxygenation. These profound physiological effects are associated with increases in myocardial capillary density and S-nitrosylation of the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) under normoxic conditions. We further show that S-nitrosylated HIF-1alpha binds to the vascular endothelial growth factor (VEGF) gene, thus identifying a role for GSNO in angiogenesis and myocardial protection. These results suggest innovative approaches to modulate angiogenesis and preserve cardiac function.

Fig. 1.

GSNOR^−/− mice have smaller infarctions following coronary ligation. (A) Representative cross sections of wild type and GSNOR^−/− hearts 48 h following LAD ligation. Before harvesting the heart from the euthanized mouse, the ascending aorta was cannulated to infuse trypan blue for demarcation of the area at risk (AAR, outlined in white). Following removal of the heart and sectioning, each myocardial section was photographed and then incubated in triphenyltetrazolium chloride (TTC) for demarcation of viable versus infarcted myocardium (outlined in white). (B) For each trypan blue myocardial segment, the corresponding TTC stained segment was photographed and used to calculate infarcted percentage of the AAR. The GSNOR^−/− mice experienced an approximate 20% reduction in the infracted percentage of the AAR in comparison with the WT mice (*, P = 0.02, GSNOR^−/− vs. WT). (C) Structural determination of coronary artery anatomy was performed using silicone casts, digestion of the myocardium, and digital photography. WT and GSNOR^−/− mice exhibited similar coronary anatomy with a left main coronary artery that gives off a circumflex branch proximally and terminates in a bifurcation near the LV apex.

Fig. 2.

GSNOR^−/− mice have less pathological remodeling and preserved left ventricular function 12 weeks after myocardial infarction. (A) At 12 wks following LAD ligation, mice were euthanized and hearts were harvested for infarction size determination. The GSNOR^−/− mice displayed a markedly reduced infarction size compared to WT mice (17 ± 3% vs. 35 ± 10%, respectively; *, P = 0.03). Representative histological sections of infarcted LV myocardium in WT and GSNOR^−/− mice demonstrate the decreased infarction size in a knockout heart. (B) WT and GSNOR^−/− mice were evaluated with serial echocardiography immediately before, and for 12 wks following LAD ligation. Relative to the WT mice, which exhibited pathologic left ventricular remodeling with steady increase in LV end diastolic and end systolic dimensions (LV EDD, LV ESD), the GSNOR^−/− mice experienced little increase in chamber dimensions after MI. (C) LV function, as determined by the percentage of fractional shortening and percent change in fractional shortening, declined to a significantly greater degree in the WT mice during the 12 wks following LAD ligation (*, P ≤ 0.03; †, P < 0.01; ‡, P < 0.001).

Fig. 3.

GSNOR^−/− mice have increased baseline capillary density and preserved myocardial tissue oxygenation after ligation of the LAD artery. Myocardial immunohistochemical staining for capillary density using CD-31 antibody was performed in (A) WT and (B) GSNOR^−/− mice. These representative sections show a higher level of positive CD-31 staining (white arrows) in the GSNOR^−/− mouse heart, indicating increased capillary density. (C) Immunohistochemical analysis revealed a significantly greater number of CD-31 positive cells in the myocardium of GSNOR^−/− mice (*, P = 0.002). (D) Myocardial partial pressure oxygen (pO₂, mm Hg) measurements were recorded in WT and GSNOR^−/− mice before and 15 min after ligation of the LAD artery (WT mice n = 10; GSNOR^−/− mice, n = 8). Time-points represent averages of continuous readings taken over 10 seconds at each time interval. After 15 min of myocardial ischemia/infarct, WT mice experienced a significant decrease in myocardial pO₂ compared to GSNOR^−/− mice (†, P = 0.045).

Fig. 4.

GSNOR^−/− mice exhibit enhanced HIF-1 transcriptional activity in normoxia. HeLa cells were transfected with either GFP or HIF-1α, 24 h before infection with Ad-HRE-luciferase at multiplicities of infection (MOIs) of 0, 10, 100, and 500. (A) The following day, the reporter assay was performed for luciferase activity, which demonstrated greatly increased luciferase activity with increased MOI of Ad-HRE-luciferase. Conversely, transfection with GFP did not induce an appreciable change in luciferase activity relative to HIF-1α (*, P ≤ 0.008; †, P < 0.001, HIF-1α vs. GFP). (B) On day 0, mice underwent single LV injection of empty adenovirus or Ad-HRE-luciferase, and, on day 3, they underwent in vivo bioluminescence evaluation. These representative images depict the significantly increased bioluminescence observed in the GSNOR^−/− mice injected with Ad-HRE luciferase compared to WT mice (n = 3 per group). (C) In vivo quantification of emitted photons from these mice demonstrated a marked augmentation of luciferase activity in the GSNOR^−/− mice injected with Ad-HRE-luciferase (†, P < 0.001, Ad-HRE-luciferase GSNOR^−/− vs. WT; and GSNOR^−/− Ad-HRE-luciferase vs. AdEV). (D) Ex vivo quantification of luciferase activity on myocardial tissue harvested on day 6 following viral injection revealed substantially increased relative light units (normalized for protein concentration) in the GSNOR^−/− mice injected with Ad-HRE luciferase (*, P ≤ 0.008, Ad-HRE-luciferase GSNOR^−/− vs. WT; and GSNOR^−/− Ad-HRE-luciferase vs. AdEV).

Fig. 5.

HIF-1α protein stabilization and S-nitrosylation is increased in GSNOR^−/− mice in normoxia. (A) Nuclear extracts from GSNOR^−/− and WT myocardial tissue were analyzed for basal HIF-1α expression (n = 8) by western blot analysis. Normoxic HIF-1α expression was consistently higher in the GSNOR^−/− mice compared to WT. (B) Densitometric analysis demonstrates a significant increase in the relative ratio of HIF-1α to histone in the GSNOR^−/− mice compared to WT. The relative ratio of HIF-1α to histone in WT mice is arbitrarily presented as 1 (*, P = 0.03, relative density in GSNOR^−/− mice 1.41 ± 0.28). (C) S-Nitrosylation of HIF-1α was determined in GSNOR^−/− vs. WT mice by the biotin switch technique. S-nitrosylated HIF-1α (+ ascorbic acid, which enables labeling of SNO-protein with biotin) was consistently higher in the nuclear extracts of GSNOR^−/− mice compared to WT in normoxic environments. (D) Densitometric analysis demonstrates a significant increase in the relative ratio of S-nitrosylated-HIF-1α to histone in the GSNOR^−/− mice compared to WT. The relative ratio of S-nitrosylated HIF-1α to histone in WT mice is arbitrarily defined as 1 (†, P = 0.007, relative density in GSNOR^−/− mice 1.62 ± 0.19). (E) Chromatin immunoprecipitation (ChIP) analysis of HIF-1α binding to the promoter region of the VEGF gene in W293 cells treated with increasing concentrations of GSNO. Immunoprecipitation of the HIF-1α-DNA complex revealed increases after treatment with GSNO.