Objective: Metabolic syndrome is the precursor for cardiovascular complications, such as ventricular arrhythmias (sudden cardiac death), heart failure and stroke. Here, we test metabolic substrates’ regulation of cardiac excitation-contraction coupling and the roles of endothelial and neuronal nitric oxide synthases (eNOS, nNOS) in left ventricular (LV) myocytes from sham and angiotensin (Ang II)-induced hypertensive rats. Design and Method: LV myocyte contraction and Ca2+ transients were assessed by using a video-sarcomere detection system or with Fura-2AM (2 mM, IonOptix Cop, field stimulation at 2 Hz, 361). Fatty acids and other metabolic substrates (oleic acid 200 mM, palmitic acid 100 mM, linoleic acid 100 mM, lactate 1 mM, pyruvate 100 mM and carnitine 50 mM) were supplemented (termed nutrition full, NF solution) to the normal tyrode (NT) solution. Results: NF increased LV myocyte contraction and Ca2+ transients in sham and hypertension. B-adrenergic stimulation with isoprenaline (ISO) increased myocyte contraction in both groups. Notably, ISO induced delayed-aftercontraction (DAC) and spontaneous Ca2+ transients (sCa) in NF and the frequency of DAC was ∼3 fold higher in hypertension. Diastolic and peak [Ca2+]i were greater in NF in hypertension, suggesting the role of intracellular Ca2+. eNOS phosphorylation was significantly reduced by NF in both groups, therefore, reduced NO availability might be responsible for the arrhythmogenic effects of NF. Inhibition of NOS with L-NAME increased DAC and sCa in sham but reduced these parameters in hypertension. Such effects were mimicked by nNOS inhibitor, SMTC, suggesting the role of nNOS. Mechanistically, NF significantly reduced myofilament Ca2+ sensitivity (Myo-Ca-Sen) in both groups. SMTC increased Myo-Ca-Sen in sham with NF + ISO but significantly reduced it with NF + ISO in hypertension. Conclusions: Our results demonstrate that nNOS prevents NF-induced arrhythmias in normal heart but assists arrhythmogenesis in hypertension via its contrasting roles in Myo-Ca-Sen.