Neuronal nitric oxide synthase inhibition improves diastolic function and reduces oxidative stress in ovariectomized mRen2.Lewis rats

Abstract

Objective: The loss of estrogen in mRen2.Lewis rats leads to an exacerbation of diastolic dysfunction. Because specific neuronal nitric oxide synthase (nNOS) inhibition reverses renal damage in the same model, we assessed the effects of inhibiting neuronal nitric oxide on diastolic function, left ventricular remodeling, and the components of the cardiac nitric oxide system in ovariectomized (OVX) and sham-operated mRen2.Lewis rats treated with N5-(1-imino-3-butenyl)-L-ornithine (L-VNIO; 0.5 mg/kg per day for 28 d) or vehicle (saline).

Methods: Female mRen2.Lewis rats underwent either bilateral oophorectomy (OVX; n = 15) or sham operation (or surgical procedure) (sham; n = 19) at 4 weeks of age. Beginning at 11 weeks of age, the rats were randomized to receive either L-VNIO or vehicle.

Results: The surgical loss of ovarian hormones, particularly estrogen, led to exacerbated hypertension, impaired myocardial relaxation, diminished diastolic compliance, increased perivascular fibrosis, and increased relative wall thickness. The cardiac tetrahydrobiopterin-to-dihydrobiopterin levels were lower among OVX rats compared with sham-operated rats, and this altered cardiac biopterin profile was associated with enhanced myocardial superoxide production and decreased nitric oxide release. L-VNIO decreased myocardial reactive oxygen species production, increased nitrite concentrations, attenuated cardiac remodeling, and improved diastolic function.

Conclusions: Impaired relaxation, diastolic stiffness, and cardiac remodeling were found among OVX mRen2.Lewis rats. A possible mechanism for this unfavorable cardiac phenotype may have resulted from a deficiency in available tetrahydrobiopterin and subsequent increase in nNOS-derived superoxide and reduction in nitric oxide synthase metabolites within the heart. Selective nNOS inhibition with L-VNIO attenuated cardiac superoxide production and limited remodeling, leading to improved diastolic function in OVX mRen2.Lewis rats.

Figure 1

Tail-cuff systolic blood pressure in sham-operated or OVX conscious female mRen2.Lewis rats treated with L-VNIO or saline vehicle for 4 weeks. Values are means ± SEM.

Figure 2

A. Representative tissue Doppler image of intact or OVX mRen2.Lewis female receiving either vehicle or combination. E = early transmitral filling velocity; A = late transmitral filling velocity; e′ = early mitral annular descent (septal) velocity. B. Early mitral annular velocity (e′). Open bars are vehicle treated, while filled bars received L-VNIO. Data represent mean ± SEM; * P < 0.01 (Estrogen effect). C. Early transmitral filling velocity – to – mitral annular velocity ratio (E/e′). Data represent mean ± SEM; * P < 0.05 (L-VNIO effect).

Figure 3

Quantification and representative picrosirius red staining of perivascular collagen in the hearts of sham-operated, ovariectomized (OVX), and rats given the nNOS inhibitor (L-VNIO). Open bars are vehicle treated, while filled bars received L-VNIO. Data represent mean ± SEM; * P = 0.0005 (Estrogen effect), # P < 0.0001 (L-VNIO effect).

Figure 4

Quantification and representative images illustrating the effect of L-VNIO on cardiac levels of fluorescent 2-hydroxyethidium from dihydroethidium (DHE) staining in estrogen intact (sham) or OVX. Open bars represent vehicle treated rats, while filled bars received L-VNIO. Fluorescent images are shown at 400× magnification. Data represent mean ± SEM; * P < 0.0001 (Estrogen effect), # P = 0.0326 (L-VNIO effect).

Figure 5

Cardiac concentrations of biopterin, BH2, BH4, and nitrite. Open bars are vehicle treated, while filled bars received L-VNIO. Data represent mean ± SEM; * P < 0.01 (Estrogen effect).

Figure 6

Values are means ± SEM of cardiac nNOS, total eNOS, and phosphorylated eNOS protein expression in sham-operated or OVX rats treated with VEH or L-VNIO treated rats. Open bars are vehicle treated, while filled bars received L-VNIO. * P < 0.005 (Estrogen effect).