Involvement of cardiac nitric oxide synthase in saturated fatty acid regulation of myocyte metabolism and contraction in healthy and hypertensive rat hearts

Abstract

The heart has a high energy demand and must persistently generate ATP at a high rate to sustain contractile function and ionic homeostasis. 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. I examined the effects of FA-regulation of contraction and mitochondrial activity in left ventricular (LV) myocytes from healthy and angiotensin II (Ang II)-induced hypertensive (HTN) rat hearts. My results showed that palmitic acid (PA, 100 μM) increased the amplitudes of sarcomere shortening and intracellular ATP in sham but not in HTN despite that oxygen consumption rate (OCR) was increased by PA in both groups. Carnitine palmitoyl transferase I inhibitor, etomoxir (ETO), reduced OCR and ATP with PA in sham and HTN but prevented PA-potentiation of sarcomere shortening only in sham. Mechanistically, PA increased intracellular Ca2+ transient ([Ca2+]i) without changing Ca2+ influx via L-type Ca2+ channel (LTCC) and reduced myofilament Ca2+ sensitivity in sham. In HTN, PA reduced ICa-L without affecting [Ca2+]i or myofilament Ca2+ sensitivity. Cardiac neuronal nitric oxide synthase (nNOS) is an important molecule that regulates intracellular Ca2+ handling and contractility of healthy and diseased hearts. Accordingly, the effects of nNOS on FA-regulation of LV myocyte contraction in sham and HTN were investigated. PA increased nNOS-derived NO only in HTN. Subsequently, inhibition of nNOS with S-methyl-l-thiocitrulline (SMTC) prevented PA-induced OCR and restored PA-potentiation of myocyte contraction in HTN but exerted no effect in sham. Furthermore, nNOS inhibition increased [Ca2+]i, ICa-L and reduced myofilament Ca2+ sensitivity prior to PA supplementation

as such, normalized PA-increment of [Ca2+]i in sham. In contrast, nNOS inhibition increased ICa-L, reduced [Ca2+]i and increased myofilament Ca2+ sensitivity prior to PA supplementation. However, PA increased [Ca2+]i and reduced myofilament Ca2+ sensitivity following nNOS inhibition in HTN. PA reduced intracellular pH similarly in sham and in HTN. Myocardial FA uptake in vivo with PET/CT (18F-fluoro-6-thia-heptadecanoidc acid, 18F-FTHA) was comparable between two groups but nNOS inhibition increased it only in HTN. These results suggest that nNOS modulation of intracellular Ca2+ handling overrides fatty acid-potentiation of cardiac inotropy in hypertensive rats. On the other hand, since the activity of endothelial nitric oxide synthase (eNOS) is essential in FA-dependent β-oxidation in cardiac muscle, the effect of eNOS on PA-regulation of myocyte contraction was analyzed. Our results showed that Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) or eNOS gene deletion prevented PA-regulation of the myocyte contraction or OCR, indicating the pivotal role of eNOS in mediating the effects of PA in cardiac myocytes. S-palmitoylation is an important post-translational modification that affects the translocation and the activity of target proteins in a variety of cell types including cardiomyocytes. Since eNOS is known to be palmitoylated, the effect of eNOS palmitoylation by PA and its involvement in PA-regulation of myocyte contraction was examined in sham and HTN. As expected, PA increased the palmitoylation of eNOS in LV myocytes and de-palmitoylation with 2 bromopalmitate (2BP, 100 μM) abolished the increment. Furthermore, although PA did not increase eNOS-Ser1177, 2BP reduced eNOS-Ser1177 with and without PA. Intriguingly, 2BP did not affect PA-induced increases in contraction and OCR in sham. In HTN, PA did not affect eNOS palmitoylation, eNOS-Ser1177 or myocyte contraction. However, 2BP diminished basal eNOS palmitoylation and eNOS-Ser1177 in the presence and absence of PA but did not change myocyte contraction. Collectively, PA increases myocyte contraction through stimulating mitochondrial activity and [Ca2+]i, secondary to myofilament Ca2+ desensitization in healthy hearts. PA-dependent cardiac inotropy was limited in HTN by nNOS, whose activity was increased by PA in HTN, due to its modulatory effect on [Ca2+]i handling. In addition, our results confirm that eNOS is important in PA-regulation of mitochondrial activity and myocyte contraction. However, palmitoylation of eNOS in LV myocytes, which is up-regulated by PA in sham, plays a negligible role in PA-regulation of myocyte contraction and mitochondrial activity in sham and in HTN.