Diurnal variation in excitation-contraction coupling in rat ventricular myocytes

Diumal variation has been reported in many cardiovascular haemodynamics parameters such as heart rate and blood pressure and the cardiac action potential. This variation may result from the diurnal variation in sympathetic activity or in cardiac gene expression. However, it is unknown whether these time-of-day dependent changes impact on excitation-contraction (EC) coupling. There is also a morning peak in the onset of ventricular arrhythmias and associated sudden cardiac death in man, which appear linked to the increase in sympathetic activity. Therefore, the aims of this investigation were to determine whether there was a time-of-day dependent variation in EC-coupling and its modulation by sympathetic stimulation. Left ventricular myocytes were isolated during either the resting period (ZT3) or the active period (ZT15) o f the adult Wistar rat. [Ca[superscript 2+]][subscript i] was determined using Fura-2 and contraction strength was determined using cell-edge detection in response to electrical field stimulation, and gene expression was determined using quantitative real-time RT-PCR. To determine the effects of hypertension-induced hypertrophy, myocytes were isolated from pre- and post-hypertensive spontaneously hypertensive rats (SHR). The basal Ca[superscript 2+] transient, contraction strength and SR Ca[superscript 2+] content were significantly greater in resting period (ZT3) myocytes than active period (ZT15) myocytes. Systolic [Ca[superscript 2+]], amplitude of Ca[superscript 2+] transient and SR Ca[superscript 2+] content in response to isoproterenol (> 3nM) were significantly greater in resting period (ZT3) myocytes. The percentage of myocytes developing arrhythmic activity in response to isoproterenol was greater in resting period (ZT3) myocytes. Nitric oxide synthase (NOS) inhibition using L-NNA significantly increased systolic [Ca[superscript 2+]], amplitude of Ca[superscript 2+] transient, SR Ca[superscript 2+] content and the percentage of myocytes developing arrhythmic activity in active period (ZT15) myocytes thereby depressing time-of-day dependent variation in these parameters. In addition, expression of NOS1 was significantly greater in active period (ZT15) myocytes. Diurnal variation in the Ca[superscript 2+] transient and its responsiveness to isoproterenol were depressed in adult SHR, however, this did not reflect a depression of diurnal cycling in NOS1 expression. This shows for the first time a time-of-day dependent variation in the Ca[superscript 2+]-transient and resulting contraction strength, reflecting levels of SR Ca[superscript 2+]-loading, due to a NOS-signalling pathway. There was also a reduction in sympathetic-induced arrhythmic activity in active period (ZT15) myocytes which was associated with increased NOS activity. Therefore, variation in NOS may be a means of protecting against arrhythmias during severe sympathetic stimulation. Loss of protection through disruption to the circadian clock resulting from cardiomyopathies such as hypertension-induced hypertrophy may result in a decreased threshold for sympathetic-induced arrhythmias, however; this requires further work to elucidate the underlying molecular mechanisms.