Palmitic acid (PA) is a predominant metabolic substrate for cardiac β-oxidation. Our recent results have shown that PA increases contraction of cardiac myocytes from normal heart but not from hypertension. S-palmitoylation is an important post-translational modification that affects the translocation in cardiomyocytes. Since endothelial nitric oxide synthase (eNOS) is known to be palmitoylated and the activity of eNOS is essential in fatty acid-dependent β-oxidation in muscle, we aim to identify whether eNOS is palmitoylated by PA and the involvement of eNOS-palmitoylation in PA-regulation of cardiomyocyte contraction.Design and Method:
Sarcomere length were measured (field stimulation, 2 Hz, IonOptix, 37 °C). Oxygen consumption rate (OCR) was measured (Instech, 37 °C). NO (nitrite content) was measured by NO assay kit (Griess Reagent System). S-palmitoylation was measured with resin-assisted capture of S-acylated proteins.Results:
Our results showed that palmitic acid (PA, 100 μM) significantly increased eNOS palmitoylation but not in hypertension. Pre-treatment of LV myocytes with an inhibitor of palmitoylation, 2-bromopalmitic acid (2BP, 100 μM) significantly reduced eNOS palmitoylation in both groups, suggesting that residual eNOS in hypertension was palmitoylated. Functionally, 2BP induced slight increase of PA-induced myocyte contraction in sham but almost doubled contraction in hypertension. PA increased OCR in cardiomyocytes from both groups. 2BP reduced OCR only in hypertension, suggesting that eNOS-depalmitoylation affects PA-dependent myocyte contraction in hypertension via a mechanism different from mitochondrial function. PA increased nitric oxide (NO) production in LV myocytes from hypertension, whereas tended to reduce it in sham. Inhibition of NOS with Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mM, 30min-1hr) or nNOS with S-methyl-l-thiocitrulline (SMTC, 100 nM, 30min-1hr) reduced NO production in hypertension, whereas 2BP increased PA-dependent NO production in both groups in the presence or absence of L-NAME or SMTC.Conclusions:
PA induces eNOS-palmitoylation and attenuates eNOS-dependent NO production. eNOS-palmitoylation does not seem to mediate PA-induced cardiac inotropy in normal rat cardiac myocyte.