RATIONALE: Although >10% of cardiac gene expression displays diurnal variations, little is known of their impact on excitation-contraction coupling. OBJECTIVE: To determine whether the time of day affects excitation-contraction coupling in rat ventricles. METHODS AND RESULTS: Left ventricular myocytes were isolated from rat hearts at 2 opposing time points, corresponding to the animals resting or active periods. Basal contraction and Ca(2+) was significantly greater in myocytes isolated during the resting versus active periods (cell shortening 12.4+/-0.3 versus 11.0+/-0.2%; P<0.05 and systolic Ca(2+) 422+/-12 versus 341+/-9 nmol/L; P<0.01. This corresponded to a greater sarcoplasmic reticulum (SR) Ca(2+) load (672+/-20 versus 551+/-13 nmol/L P<0.001). The increase in systolic Ca(2+) in response to isoproterenol (>3 nmol/L) was also significantly greater in resting versus active period myocytes, reflecting a greater SR Ca(2+) load at this time. This diurnal variation in response of Ca(2+)-homeostasis to isoproterenol translated to a greater incidence of arrhythmic activity in resting period myocytes. Inhibition of neuronal NO synthase during stimulation with isoproterenol, further increased systolic Ca(2+) and the percentage of arrhythmic myocytes, but this effect was significantly greater in active period versus resting period myocytes. Quantitative RT-PCR analysis revealed a 2.65-fold increase in neuronal NO synthase mRNA levels in active over resting period myocytes (P<0.05). CONCLUSIONS: The threshold for the development of arrhythmic activity in response to isoproterenol is higher during the active period of the rat. We suggest this reflects a reduction in SR Ca(2+) loading and a diurnal variation in neuronal NO synthase signaling.