We have previously shown a time-of-day variation in the response of systolic [Ca2+]i to the non-specific ÃŸ-a drenergic (ÃŸ-ADR) agonist isoproterenol (ISO), linked to a variation in Nitric Oxide (NO) signalling . This may reflect stimulation of ÃŸ3-ADRs, which induces a NO-dependent negative inotropic response . As the action potential duration (APD) regulates systolic [Ca2+]i, and a time-of-day variation exists in the cardiac action potential , we set out to investigate the effect of β3-ADR stimulation and NO-signalling on the APD and systolic [Ca2+]i.
Ventricular myocytes were isolated by enzymatic digestion at time points corresponding to 3 hours into the male Wistar rats rest (ZT3) and active-period (ZT15). Measurement of systolic [Ca2+]i was made in myocytes loaded with Fura-2 and APD using the whole-cell patch clamp technique.
A significant time-of-day variation was found in systolic [Ca2+]i following stimulation with ISO (10nM), a non-specific ÃŸ-ADR agonist, which was higher in ZT3 (1040.0+116.9nM) compared to ZT15 myocytes (428.0+63.1nM) (n=3-5, S.E.M., 2-way ANOVA, P<0.001). The difference in systolic [Ca2+]i during ISO stimulation was abolished following inhibition of NOS with L-NNA (500 µM) (2-way ANOVA, P<0.001). To determine whether this time-of-day variation in response to ISO can be explained by a variation in AP configuration in response to ÃŸ-ADR stimulation, APD at 30% (APD30) and 50% (APD50) were recorded. ISO stimulation increased APD30 and APD50 significantly more in ZT15 than ZT3 myocytes, with % increase in APD30 of 120.3+14.9% in ZT15 compared to 10.6+8.2% in ZT3 myocytes (n=3, S.E.M., students t-test, P<0.001), and APD50 of 95.9+13.2% in ZT15 compared to 11.6+7.4% in ZT3 myocytes (n=3, S.EM., students t-test, P<0.001). We also investigated systolic [Ca2+]i and APD in ZT3 and ZT15 myocytes in response to the specific ÃŸ3-ADR agonist BRL37344 (200nM), to determine if time-of-day variation in systolic [Ca2+]i following ISO-stimulation could be explained by variation in ÃŸ3-ADR signalling. A significant reduction in systolic [Ca2+]i in ZT3 myocytes was found following BRL37344 stimulation, from 458.5+41.2nM to 361.2+18.0nM (n=4, 2-way ANOVA, P<0.001) but no effect on ZT15 myocytes. BRL37344 also significantly reduced APD30, (18.3+2.2ms to 14.4+1.6ms) (n=5, 2-way ANOVA, P<0.001), and APD50 (32.9+4.3ms to 26.5+3.1ms)(n=5, 2-way ANOVA, P<0.001) in ZT3 myocytes with no significant change in ZT15 myocytes.
Our data shows a reduction in systolic [Ca2+]i in rest-period myocytes (ZT3) in response to ÃŸ3-ADR stimulation, which may reflect the reduction in APD. This suggests that the reduced response of systolic [Ca2+]i to ISO-stimulation in active-period myocytes is not due to a strong negative inotropic action of ÃŸ3-ADR activation during the active period.