Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function

Abstract

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and β-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed β-adrenergic inotropic responses that were associated with impaired β-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.

Fig. 1.

Integrated hemodynamics reveal reduced vascular tone and increased cardiac output in female GSNOR^−/− mice. (A) Systolic (SBP) and diastolic (DBP) blood pressure of GSNOR^−/− mice is indistinguishable from WT, although the LV-end systolic pressure (LVPes) is decreased in GSNOR^−/− mice, which is a reflection of lower systemic vascular resistance. LVPed, LV-end diastolic pressure. (B) GSNOR^−/− mice have reduced afterload. Vascular elastance is markedly reduced (E_a; P < 0.05 vs. WT), a finding consistent with increased vascular NO bioactivity. The coupling of E_es/E_a and myocardial lusitropy (Tau) are similar between the strains. (C) Integrated CV function. GSNOR^−/− mice compensate by increasing chamber size (EDV), thereby increasing stroke volume (SV) and cardiac output (CO) (all P < 0.05 vs. WT). End systolic volume (ESV) is similar between strains. (D) Myocyte length is increased in GSNOR^−/− mice, consistent with the increased chamber size. *P < 0.05 vs. WT, ^†P < 0.001 vs. WT, t test.

Fig. 2.

GSNOR colocalizes with NOS1 and RyR2 in the heart. (A) Confocal fluorescent microscopy images depict colocalization of GSNOR with NOS1 but not NOS3 and colocalization of GSNOR with RyR2 along the T-tubular invaginations of the cardiac myocyte. (Scale bars: 10 μM.) (B) GSNOR binds NOS1 but not NOS3 in vitro. Aliquots of recombinant NOS1 and NOS3 were incubated with GST or GST-GSNOR fusion proteins. The first well corresponds to the positive control, being either NOS1 or NOS3 (2.5 ng). The second well corresponds to the first flow through the column after binding. Consecutive washes of the column were then performed before elution. The bottom gel shows the profile (silver staining) of elution of GST-GSNOR (∼67 kDa) in the NOS1 binding experiment. (C) GSNOR enzymatic activity in total skeletal and heart muscle homogenates and in purified SR preparations. GSNOR activity is enriched in SR of cardiac but not skeletal muscle.

Fig. 3.

β-Adrenergic inotropic response is blunted in GSNOR^−/− mice. (A) Dose–response curve for the in vivo effect of ISO on cardiac contractility in WT (n = 12) and GSNOR^−/− (n = 11) mice. *P < 0.05, two-way ANOVA. (B) Representative traces of sarcomere shortening and [Ca²⁺]_i in myocytes isolated from WT and GSNOR^−/− hearts at baseline and after ISO (100 nM). (C and D) [Ca²⁺]_i and sarcomere shortening dose–response to ISO in isolated myocytes. GSNOR^−/− myocytes (n = 11–13, 2–10 cells per heart) displayed a blunted response to ISO vs. WT (*P < 0.05, **P < 0.01, ***P < 0.001; n = 11–14, 2–4 cells per heart). (E and F) Specific inhibition of NOS1 with l-VNIO (100 μM) as well as pan-NOS inhibition with l-NMMA (100 μM) normalized the β-adrenergic responses in GSNOR^−/− myocytes (^†P < 0.05 vs. control). (G and H) Evaluation of the force–frequency response (sarcomere shortening and the amplitude of the [Ca²⁺]_i) in isolated myocytes stimulated at 1–8 Hz. The force–frequency relationship is preserved in GSNOR^−/− cardiomyocytes.

Fig. 4.

Increased diastolic Ca²⁺ leak in GSNOR^−/− cardiomyocytes. (A) Protocol used to estimate diastolic SR Ca²⁺ leak. (B) Load–leak relationship in WT (n = 12) and GSNOR^−/− myocytes (n = 10 animals, 4–9 cells per data point) in the presence or absence of ISO (100 nM) shows a leftward shift in GSNOR^−/− myocytes stimulated by ISO. (C) Comparison of the amount of diastolic Ca²⁺ leak against a matched load for both strains in the presence or absence of ISO. GSNOR^−/− myocytes manifest a substantial increase in SR Ca²⁺ leak after ISO stimulation (*P < 0.05, ANOVA). (D) SR Ca²⁺ content at different frequencies in the presence or absence of ISO (^†P < 0.05, ANOVA). GSNOR^−/− myocytes fail to augment SR Ca²⁺ load in response to ISO. (E) Sarcomere relaxation in response to ISO was similar in WT versus GSNOR^−/− cardiac myocytes. (F) The Tau constant of [Ca²⁺]_i decline, a measurement of Ca²⁺ reuptake, was similar in WT vs. GSNOR^−/− myocytes. Black, WT; green, WT treated with ISO; red, GSNOR^−/−; blue, GSNOR^−/− treated with ISO.

Fig. 5.

Effect of ISO and NOS on S-nitrosylation of proteins in the heart and cardiomyocytes. (A) S-nitrosylated protein band clusters in WT and GSNOR^−/− mice heart homogenates by Western blot analysis. Each lane represents biotin-switched homogenate of a whole heart perfused with Krebs–Henseleit solution (0), in the presence of 2.5 nM ISO (iso) or the NOS inhibitor (Inh) l-NMMA (100 μM). Elimination of sodium ascorbate (−Asc) or exposure to UV-photolysis (UV) are controls for the biotin-switch technique (30). Red asterisks indicate endogenously biotinylated proteins. The table shows densitometric quantification of SNO protein band clusters normalized to baseline (0). ISO and l-NMMA reduced S-nitrosylation levels in WT whereas this effect is blocked or attenuated in GSNOR^−/− mice (*P ≤ 0.01–0.05 and ^†P ≤ 0.001–0.01; ANOVA with Student Newman–Keuls post hoc test; WT 0 and ISO, n = 11; l-NMMA, n = 5; GSNOR^−/− 0 and ISO, n = 9; l-NMMA, n = 3). (B) Representative Western blot images of immunoprecipitated SNO-RyR2, LTCC, and SERCA2 after biotin-switch assay, with cumulative measurements displayed graphically. Each graph shows the effect of ISO and l-NMMA on SNO levels of RyR2, LTCC, and SERCA2 in WT and GSNOR^−/− mice hearts. The SNO level was detected by anti-biotin antibody normalized to the total protein load as detected with its specific antibody (*P ≤ 0.01–0.05; ANOVA with Student Newman–Keuls or Kruskal–Wallis posttest). Western blot images are representative of n = 4–6 RyR2, n = 7–9 LTCC, and n = 4–6 SERCA2. In the GSNOR^−/− heart, ISO-induced denitrosylation of RyR2 was substantially abrogated.