Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC137
The time-of-day variation in the response of ventricular myocytes to isoproterenol is lost in spontaneously hypertensive rats
G. Rodrigo1, H. E. Collins1, T. E. Helen1
1. Cardiovascular Sciences, Leicester University, Leicester, United Kingdom.
We have demonstrated a time-of-day variation in many key elements of E-C coupling in rat ventricular myocytes, including L-type Ca2+ current (LTCC), systolic [Ca2+]i and SR Ca2+-loading, which appears to reduce the incidence of arrhythmias in response to isoproterenol (ISO) (1). In a pressure-overload induced model of left ventricular hypertrophy in rats, the normal cycling of the circadian clock controlled genes are severely blunted (2). We have looked at the impact of hypertension on the time-of-day variation in E-C coupling. Adult male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) and Wistar rats were housed with a 12 hour light/dark cycle. Single left ventricular myocytes were isolated at ZT3 the resting period or ZT15 the active period, where ZT0 refers to “lights on”, by enzymatic digestion of hearts excised at <6 weeks and >24 weeks. [Ca2+]i was measured using Fura-2, LTCC using whole-cell patch clamp electrophysiology and NOS protein using Western blot analysis. (Data are mean±SE; n=number of cells, hearts; one-way ANOVA with Bonferonni post-hoc test). The data show a time-of-day variation in systolic [Ca2+]i in ventricular myocytes from younger animals (<6 weeks) at 636±40nM vs. 498±19nM (n=21,3; p<0.01) in WKY and 759±29nM vs. 554±20nM (n=23,3; p<0.01) in SHR, for ZT3 versus ZT15 respectively. This variation persisted in older WKYs (>24 weeks) at 579±28 vs. 459±17nM (23,3; p<0.001) but was absent in older SHRs at 666±33 vs. 669±31nM (21,3; ns). We also found a time-of-day variation in response to β-adrenoreceptor stimulation with ISO, with a significant reduction in maximal systolic [Ca2+]i recorded in 100nM ISO at ZT15 compared to ZT3 in ventricular myocytes isolated from younger WKY and SHR animals. However, this time-of-day variation whilst present in older WKY animals at 2,024±163 vs. 1,334±90nM (17,5; p<0.001) was absent in the older SHRs at 1,984±175 vs. 1,742±104nM (20,5; ns). We have previously suggested that this time-of-day variation reflects higher levels of NOS-signalling in ZT15 animals, which depresses the response to ISO (1). Our data show low levels of nNOS expression in adult Wistar animals, which is up-regulated in SHR hearts (>24 weeks), but with no significant time-of-day variation. However, eNOS was detected at high levels in the adult Wistar hearts and showed significant time-of-day variation at 1.06±0.09 vs. 1.69±0.16 (eNOS/β-Tubulin, n=6; p<0.05) for ZT3 vs. ZT15 respectively. This variation was absent in older SHR hearts at 1.08±0.23 vs. 1.07±0.11 (n=4; ns). Our data show that the time-of-day variation in Ca2+-regulation during E-C coupling and modulation by ISO, is absent in older SHR hearts, which may have implications for sympathetic-induced arrhythmic activity.
Where applicable, experiments conform with Society ethical requirements