Nitrite therapy improves left ventricular function during heart failure via restoration of nitric oxide-mediated cytoprotective signaling

Abstract

Rationale: Nitric oxide (NO) bioavailability is reduced in the setting of heart failure. Nitrite (NO2) is a critically important NO intermediate that is metabolized to NO during pathological states. We have previously demonstrated that sodium nitrite ameliorates acute myocardial ischemia/reperfusion injury.

Objective: No evidence exists as to whether increasing NO bioavailability via nitrite therapy attenuates heart failure severity after pressure-overload-induced hypertrophy.

Methods and results: Serum from patients with heart failure exhibited significantly decreased nitrosothiol and cGMP levels. Transverse aortic constriction was performed in mice at 10 to 12 weeks. Sodium nitrite (50 mg/L) or saline vehicle was administered daily in the drinking water postoperative from day 1 for 9 weeks. Echocardiography was performed at baseline and at 1, 3, 6, and 9 weeks after transverse aortic constriction to assess left ventricular dimensions and ejection fraction. We observed increased cardiac nitrite, nitrosothiol, and cGMP levels in mice treated with nitrite. Sodium nitrite preserved left ventricular ejection fraction and improved left ventricular dimensions at 9 weeks (P<0.001 versus vehicle). In addition, circulating and cardiac brain natriuretic peptide levels were attenuated in mice receiving nitrite (P<0.05 versus vehicle). Western blot analyses revealed upregulation of Akt-endothelial nitric oxide-nitric oxide-cGMP-GS3Kβ signaling early in the progression of hypertrophy and heart failure.

Conclusions: These results support the emerging concept that nitrite therapy may be a viable clinical option for increasing NO levels and may have a practical clinical use in the treatment of heart failure.

Keywords: cardiomyopathy, hypertrophic; cyclic GMP; heart failure; nitric oxide; nitric oxide synthase; ventricular function, left.

Figure 1. Serum levels of NO metabolites and cGMP are reduced in humans with heart failure

(A) serum nitrite (μM), (B) serum RXNO (nM), and (c) serum cGMP. Results are expressed as mean ± SEM.

Figure 2. Oral sodium nitrite therapy augments NO metabolites in mice at 6 weeks following TAC

(A) Experimental protocol for transverse aortic constriction (TAC) model in mice. (B) serum nitrite (μM), (C) serum RXNO (nM), (D) Serum cGMP (pmol/ml), (E) cardiac nitrite (μM) and (F) Cardiac RXNO (nM). Results are expressed as mean ± SEM.

Figure 3. Nitrite therapy prevents cardiac dilatation and dysfunction following TAC at 9 weeks

(A) Circulating BNP levels (ng/ml). (B) The ratio of heart weight to tibia lengths. (C) The ratio of lung weight/tibia lengths. (D) Left ventricular end-diastolic diameter (LVEDD) (E) Left ventricular end-systolic diameter LVESD (F) Left ventricular ejection fraction (LVEF %) from 1 week to 9 weeks following TAC. Results are expressed as mean ± SEM.

Figure 4. Delayed nitrite therapy prevents cardiac dilatation and dysfunction following TAC at 9 weeks

Mice were subjected to TAC surgery and nitrite therapy (50 mg/L) was initiated at 3 weeks following TAC. (A) Left ventricular end-diastolic diameter (LVEDD) (B) Left ventricular end-systolic diameter LVESD (C) Left ventricular ejection fraction (LVEF %) from 1 week to 9 weeks following TAC. Results are expressed as mean ± SEM.

Figure 5. Nitrite attenuates myocardial fibrosis following TAC

(A) Representative photomicrographs of Masson’s Trichrome, Picrosirius Red, and CD31 stained heart sections from sham, TAC + vehicle, and TAC + nitrite mice at 6 weeks of TAC. (B) Summary of fibrosis area as % of the LV as calculated from Masson’s Trichrome sections. (C) Summary of fibrosis area as % of the LV calculated from the Picrosirius Red sections. (D) Summary of CD31+ vessels per area (mm2). Results are expressed as mean ± SEM.

Figure 6. Nitrite upregulates Akt and endothelial nitric oxide (eNOS)

(A) Representative immunoblots of Akt phosphorylation. (B–C) Total Akt and Akt-PSer473 (D) Representative immunoblots of eNOS phosphorylation in TAC + vehicle, and TAC + nitrite treated mice following TAC at 1 week and 6 weeks. Results are expressed as mean ± SEM.

Figure 7. Nitrite upregulates GSK3β activity

(A) Representative immunoblots of (B) total GSK3β and (C) GSK3β-PSer9 in Sham, TAC + vehicle, and TAC + nitrite mice at 1 week and 6 weeks following TAC. Results are expressed as mean ± SEM.

Figure 8. Nitrite upregulates ERK1/2 and attenuates p38 phosphorylation

(A) Representative immunoblots of (B) total ERK1/2 and (C) P-ERK1/2. (D) Representative immunoblots of (D) total p38 and P-p38 in Sham, TAC + vehicle, and TAC + nitrite treated mice following TAC at 1 week and 6 weeks. Results are expressed as mean ± SEM.