Endothelial nitric oxide synthase overexpression attenuates congestive heart failure in mice

Abstract

Congestive heart failure results in cardiovascular dysfunction and diminished vascular nitric oxide (NO) production. We hypothesized that overexpression of endothelial NO synthase (eNOS) within the endothelium would reduce the extent of contractile dysfunction in a murine model of infarct-induced congestive heart failure. We generated transgenic (TG) mice overexpressing the human eNOS gene. The TG mice displayed significantly enhanced eNOS protein levels and eNOS activity levels (10- to 12-fold greater) in the aorta and the coronary vasculature. Non-TG (NTg) and eNOS TG mice were subjected to permanent left anterior descending coronary artery occlusion and then observed for 1 mo. We assessed cardiac function in vivo by using echocardiography and ultraminiature ventricular pressure catheters. Myocardial infarct size was similar between study groups (approximately 70% of the risk zone). Survival was increased by 43% in the eNOS TG mice compared with NTg (P < 0.05). Fractional shortening and cardiac output were also significantly (P < 0.05) greater in the eNOS TG than in NTg. Interestingly, pulmonary edema was evident only in NTg mice, and no evidence of pulmonary edema was observed in the eNOS TG mice. Thus, targeted overexpression of the eNOS gene within the vascular endothelium in mice attenuates both cardiac and pulmonary dysfunction and dramatically improves survival during severe congestive heart failure.

Figure 1

(A) Representative Western blot from aorta of NTg and eNOS TG mice. (B) Representative photomicrograph of eNOS immunohistochemistry demonstrating increased abundance of eNOS protein in the endothelium of eNOS TG mice compared with NTg.

Figure 2

Assessment of aortic eNOS enzymatic activity in eight NTg and nine eNOS TG mice. eNOS activity was determined by measuring the conversion of l-arginine to l-citrulline (pmol/μg/min). TG mice displayed significantly (**, P < 0.01) enhanced eNOS enzyme activity compared with NTg control mice.

Figure 3

(A) Aortic and myocardial eNOS protein levels as measured by Western blot analysis in NTg and eNOS TG mice. eNOS protein expression is significantly enhanced in the aorta and myocardium of eNOS TG mice. (B) Representative immunohistochemical staining of myocardial tissue harvested from NTg and eNOS TG mice demonstrating increased eNOS protein in the myocardium of eNOS TG mice compared with the NTg control mice.

Figure 4

Myocardial infarct size in NTg and eNOS TG mice subjected to permanent myocardial occlusion. The area-at-risk (AAR) per LV was similar (P = NS) in the NTg and eNOS TG groups. In addition, the infarct size (INF) per AAR and LV was similar in the NTg and eNOS TG groups (P = NS). n = 6 per group.

Figure 5

Kaplan–Meier survival curve in sham NTg, NTg, and eNOS TG mice during the 4 wk after myocardial infarction. Both NTg and eNOS TG exhibited significant (P < 0.01) decreases in survival compared with sham mice after 1 mo of myocardial infarction. eNOS TG mice demonstrated significantly (P < 0.05) greater survival compared with NTg during the congestive heart failure protocol.

Figure 6

(A) LV chamber diameters during diastole (LVEDD) and systole (LVESD) in sham NTg, NTg, and eNOS TG mice after 4 wk of coronary artery occlusion. NTg and eNOS TG mice exhibited significant (**, P < 0.01) LV chamber dilatation in diastole and systole compared with sham-operated NTg mice. There were no significant differences in LV diameters between NTg and eNOS TG after 4 wk of heart failure. (B) Fractional shortening (%) was significantly attenuated in both groups (NTg and eNOS TG) of mice subjected to coronary artery occlusion compared with sham (**, P < 0.01). However, fractional shortening was significantly (P < 0.05) higher in the eNOS TG group compared with NTg. (C) Left ventricular maximum (+) and minimum (−) dP/dt was assessed by using pressure transduction catheters. Myocardial infarction induced significant (*, P < 0.05) depression of dP/dt in NTg and eNOS TG mice compared with sham. However, dP/dt was not significantly different between NTg and eNOS TG (P = NS). (D) By using transthoracic Doppler-assisted echocardiography, cardiac output was determined in sham NTg, NTg, and eNOS TG mice after 1 mo of coronary artery occlusion. Cardiac output was significantly (P < 0.01) reduced in the NTg compared with sham NTg. eNOS TG mice did not exhibit a significant decrement in cardiac output compared with sham. Overexpression of eNOS (TG) resulted in a significantly (P < 0.05) higher cardiac output compared with NTg mice after the 4-wk congestive heart failure protocol. Numbers of mice are located within the bars.

Figure 7

Pulmonary edema was assessed by net fluid weights in 11 sham, 15 NTg, and 16 eNOS TG mouse lungs. NTg mice exhibited significantly (P < 0.05) greater fluid accumulation compared with sham lungs. However, eNOS TG mice did not demonstrate any significant differences compared with NTg.