Roles of eNOS and nNOS in fatty acid-dependent Cardiac Inotropy and Calcium Handling in Healthy and Hypertensive Rats

Background: Fatty acids (FAs) are the predominant metabolic substrates for myocardial ATP. So far, the effects of FAs on myocyte contraction in normal and hypertensive hearts are unclear. Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) has been implicated to be essential in FA oxidation in mitochondria. Recently, we have shown that neuronal nitric oxide synthase (nNOS) is up-regulated in left ventricular (LV) myocytes from hypertensive hearts, whereas eNOS protein expression was reduced. Purpose: We aim to analyze palmitic acid (PA)-regulation of myocyte contraction and the roles of eNOS and nNOS in LV myocytes from sham and angiotensin II (Ang II)-induced hypertensive rats. Methods: Sarcomere length and Fura-2 ratio (Fura-2AM, 2 μM) were measured (field stimulation, 2Hz, IonOptix Corp, 37°C). Oxygen consumption rate (OCR) was measured (Instech). NO (nitrite content) was measured by NO assay kit (Griess Reagent System). Used whole cell patch clamp technique, was recording L-type Ca2+ current (ICa) and Na+ - Ca2+ exchanger activity (INCX). Results: Our results showed that PA (100 μM) increased the amplitude of sarcomere shortening and Ca2+ transients in LV myocytes from sham but not in hypertension. Etomoxir (10 μM), a selective carnitine palmitoyl transferase I inhibitor, blunted the inotropic effect of PA in sham, but not effect hypertension, suggesting the contribution of beta-oxidation to in PA-regulation of cardiac inotropy. PA increased basal oxygen consumption and mitochondrial OC capacity in cardiomyocytes from sham and HTN rats. Etomoxir was reversed PA induced basal OC and mitochondrial OC capacity in sham and HTN. Inhibition of eNOS and nNOS with Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mM, 30 min – 1hr) prevented PA-induced myocyte contraction and Ca2+ transients in sham; such an effect was not observed with nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC, 100 nM, 30 min – 1hr). Similarly, PA failed to increase myocyte contraction in LV myocytes from eNOS-/- mice, suggesting the critical role of eNOS in PA-induced myocyte contraction in sham. In hypertension, both L-NAME and SMTC restored PA-enhancement of myocyte contraction, suggesting the modulatory role of nNOS. PA tended to reduce eNOS-derived NO in sham but significantly increased nNOS-derived NO in hypertension. Indeed, PA increased OCR in sham and L-NAME but not SMTC reduced PA-induced DOCR. In hypertension, PA increased OCR. Importantly, L-NAME and SMTC abolished both basal and PA-induced DOCR. PA maintained Ca2+ influx via L-type Ca2+ channels, and nNOS inhibitor increased Ca2+ influx, in LV cardiomyocyte from sham. PA reduced Ca2+ influx, but nNOS inhibitor significantly increased Ca2+ influx via L-type Ca2+ channels in LV cardiomyocyte from hypertensive rats. Further experiments have shown that SMTC increased the amplitude of Ca2+ transients in hypertension. Conclusion: PA increases Ca2+ transients and myocyte contraction in normal heart, mediated by eNOS-dependent mitochondrial beta-oxidation. In hypertension, nNOS restricts PA-dependent cardiac inotropy by modulating Ca2+ handling. Nevertheless, nNOS is required for maintaining mitochondrial function in the myocytes from hypertensive hearts.