Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure

Abstract

Inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes of patients and experimental animals with congestive heart failure (CHF). Here we show that iNOS expression plays a role in pressure overload-induced myocardial chamber dilation and hypertrophy. In wild-type mice, chronic transverse aortic constriction (TAC) resulted in myocardial iNOS expression, cardiac hypertrophy, ventricular dilation and dysfunction, and fibrosis, whereas iNOS-deficient mice displayed much less hypertrophy, dilation, fibrosis, and dysfunction. Consistent with these findings, TAC resulted in marked increases of myocardial atrial natriuretic peptide 4-hydroxy-2-nonenal (a marker of lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite) in wild-type mice but not in iNOS-deficient mice. In response to TAC, myocardial endothelial NO synthase and iNOS was expressed as both monomer and dimer in wild-type mice, and this was associated with increased reactive oxygen species production, suggesting that iNOS monomer was a source for the increased oxidative stress. Moreover, systolic overload-induced Akt, mammalian target of rapamycin, and ribosomal protein S6 activation was significantly attenuated in iNOS-deficient mice. Furthermore, selective iNOS inhibition with 1400W (6 mg/kg per hour) significantly attenuated TAC induced myocardial hypertrophy and pulmonary congestion. These data implicate iNOS in the maladaptative response to systolic overload and suggest that selective iNOS inhibition or attenuation of iNOS monomer content might be effective for treatment of systolic overload-induced cardiac dysfunction.

Figure 1

iNOS deletion attenuates ventricular hypertrophy and pulmonary congestion in response to TAC-induced pressure overload. A. Representative hearts from wild type and iNOS^−/− mice 4 weeks after TAC or sham surgery (n=9 to 13). B. Tissue weights in each group; C. Ratio of tissue weight to body weight. Wt-TAC: Wild type mice after TAC for 4 weeks; KO-TAC: iNOS^−/− mice after TAC for 4 weeks. *P<0.05 as compared to corresponding control; #p<0.05 as compared to Wt-TAC.

Figure 2

Echocardiograms demonstrating that iNOS deletion attenuated TAC-induced left ventricular hypertrophy and dysfunction. A. Representative M-mode echocardiograms demonstrating greater left ventricular dilatation in wild type mice as compared to iNOS^−/− mice 4 weeks after TAC. B-G. Summary data from echocardiograms (n=9 to 12 per group) demonstrating that iNOS^−/− attenuated the TAC-induced increase of LV end systolic diameter (C), LV wall thickness (F), LV fractional shortening (D) and LV ejection fraction (E). *P<0.05 as compared to corresponding control; #p<0.05 as compared to group of Wt-TAC.

Figure 2

Echocardiograms demonstrating that iNOS deletion attenuated TAC-induced left ventricular hypertrophy and dysfunction. A. Representative M-mode echocardiograms demonstrating greater left ventricular dilatation in wild type mice as compared to iNOS^−/− mice 4 weeks after TAC. B-G. Summary data from echocardiograms (n=9 to 12 per group) demonstrating that iNOS^−/− attenuated the TAC-induced increase of LV end systolic diameter (C), LV wall thickness (F), LV fractional shortening (D) and LV ejection fraction (E). *P<0.05 as compared to corresponding control; #p<0.05 as compared to group of Wt-TAC.

Figure 3

Histological staining demonstrating that iNOS deletion attenuated TAC-induced myocardial fibrosis (A, B), cardiac myocyte hypertrophy (C, D), and the increase of myocardial MMP2 activity (E,F). MTS: Masson's trichrome staining, blue staining indicates fibrosis; WGA: Staining for wheat germ agglutinin with FITC-conjugated Flur-488 (Invitrogen), bright green staining indicates the area of the matrix and cell membrane. Summarized average data are from 4 representative mice per group. *p<0.05 compared to the corresponding control; #p<0.05 as compared to Wt-TAC.

Figure 4

Alterations of myocardial eNOS, iNOS, nNOS, DDAH-1, PRMT-1, ANP, 4-HNE, collagen-I, and NT protein content in wild type mice and iNOS^−/− mice (n=6 per group). iNOS was detected only in wild type mice after TAC. *P<0.05 as compared to the corresponding control; #p<0.05 as compared to Wt-TAC.

Figure 4

Alterations of myocardial eNOS, iNOS, nNOS, DDAH-1, PRMT-1, ANP, 4-HNE, collagen-I, and NT protein content in wild type mice and iNOS^−/− mice (n=6 per group). iNOS was detected only in wild type mice after TAC. *P<0.05 as compared to the corresponding control; #p<0.05 as compared to Wt-TAC.

Figure 5

iNOS deletion attenuated the TAC-induced increases of phospho-Akt^Ser473, phospho-mTOR^Ser2488, phospho-S6^Ser235/236 and phospho-Erk^Thr202/204. *P<0.05 as compared to the corresponding control; #p<0.05 as compared to Wt-TAC.

Figure 6

A. Western blot showing increased myocardial eNOS and iNOS expression in wild type mice in response to 8-days severe TAC. B. Both iNOS monomer and iNOS dimer were detected in wild type mice 8 days after TAC in unboiled conditions, while iNOS dimer was diminished after boiling the sample for 15 minutes. C. eNOS expressed as both monomer and dimer in wild type mice and iNOS^−/−mice 8 days after TAC in unboiled conditions. D. Attenuated ROS production in iNOS^−/− mice. #p<0.05 as compared to Wt-TAC.

Figure 6

A. Western blot showing increased myocardial eNOS and iNOS expression in wild type mice in response to 8-days severe TAC. B. Both iNOS monomer and iNOS dimer were detected in wild type mice 8 days after TAC in unboiled conditions, while iNOS dimer was diminished after boiling the sample for 15 minutes. C. eNOS expressed as both monomer and dimer in wild type mice and iNOS^−/−mice 8 days after TAC in unboiled conditions. D. Attenuated ROS production in iNOS^−/− mice. #p<0.05 as compared to Wt-TAC.

Figure-7

Myocardial eNOS was predominantly expressed in vascular endothelial cells in normal heart, and TAC increased eNOS expression in myocardial area with fibrosis and some adjacent cardiac myocytes in wild type mice. Wheat germ agglutinin staining (green) indicates areas of matrix, blood vessels and cell membrane. Blue staining indicates the cell nuclei; red staining indicates eNOS. Samples used for staining were obtained 8 days after TAC or sham surgery.

Figure 8

1400W attenuated TAC-induced cardiac death (A), ventricular hypertrophy (B), pulmonary congestion (C), LV dysfunction (D, G), ventricular dilation (E), and left ventricular fibrosis (J, K) in wild type mice.

Figure 8

1400W attenuated TAC-induced cardiac death (A), ventricular hypertrophy (B), pulmonary congestion (C), LV dysfunction (D, G), ventricular dilation (E), and left ventricular fibrosis (J, K) in wild type mice.