Sympathetic activation of L-type Ca2+ current and Na+/Ca2+ exchange current in canine ventricular cardiomyocytes

Abstract

Sympathetic activation is known to increase L-type Ca2+ current (ICa-L) and the Na+/Ca2+ exchange current (INCX) in mammalian ventricular cardiomyocytes, however, exact downstream pathways involved in this β-adrenergic regulation are not fully elucidated.

This study was designed to determine the relative contribution of the protein kinase A (PKA) and the calcium/calmodulin-dependent protein kinase II (CaMKII) pathways in regulating ICa,L and INCX during β-adrenergic receptor activation, mimicked by 10 nM isoproterenol (ISO), in isolated canine ventricular myocytes. Also, the relationship between ICa,L and INCX was examined under various experimental conditions. ICa,L and INCX were dissected by 1 µM nisoldipine and 0.5 µM ORM10962, respectively, using action potential voltage clamp (APVC) with physiological intracellular Ca2+ cycling.

Peak current density, mid-plateau current density and the current integral for both ICa,L and INCX were significantly increased by 10 nM ISO. The ISO induced augmentation of the mid-plateau current density and the current integral for both ICa,L and INCX were significantly reduced by pretreatment with the PKA inhibitor H-89 (3 µM), or the CaMKII-inhibitor KN-93 (1 µM), or the combination of them, before the application of ISO. The reduction of ISO-induced augmentation by KN-93 and H-89 was approximately similar in size and was not additive, i.e. the simultaneous application of H-89 and KN-93 failed to increase the inhibition. All actions induced by ISO, KN-93, H-89 and their combinations caused proportional changes in ICa,L and INCX. Moreover, the ratio of mid-plateau ICa,L and INCX density as well as the ratio of ICa,L and INCX integral was 2:1 in all experimental groups.

It is concluded that both ICa,L and INCX are dominantly controlled via a PKA- and CaMKII-dependent pathway being coupled in series. The proportional changes observed between ICa,L and INCX strongly suggest that there is an additional regulatory mechanism based on changes in intracellular Ca2+ concentration in the case of INCX.