Effect of activation sequence on transmural patterns of repolarization and action potential duration in rabbit ventricular myocardium

Although transmural heterogeneity of action potential duration (APD) is established in single cells isolated from different tissue layers, the extent to which it produces transmural gradients of repolarization in electrotonically coupled ventricular myocardium remains controversial. The purpose of this study was to examine the relative contribution of intrinsic cellular gradients of APD and electrotonic influences to transmural repolarization in rabbit ventricular myocardium. Transmural optical mapping was performed in left ventricular wedge preparations from eight rabbits. Transmural patterns of activation, repolarization, and APD were recorded during endocardial and epicardial stimulation. Experimental results were compared with modeled data during variations in electrotonic coupling. A transmural gradient of APD was evident during endocardial stimulation, which reflected differences previously seen in isolated cells, with the longest APD at the endocardium and the shortest at the epicardium (endo: 165 ± 5 vs. epi: 147 ± 4 ms; P < 0.05). During epicardial stimulation, this gradient reversed (epi: 162 ± 4 vs. endo: 148 ± 6 ms; P < 0.05). In both activation sequences, transmural repolarization followed activation and APD shortened along the activation path such that significant transmural gradients of repolarization did not occur. This correlation between transmural activation time and APD was recapitulated in simulations and varied with changes in intercellular coupling, confirming that it is mediated by electrotonic current flow between cells. These data suggest that electrotonic influences are important in determining the transmural repolarization sequence in rabbit ventricular myocardium and that they are sufficient to overcome intrinsic differences in the electrophysiological properties of the cells across the ventricular wall.

β2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes

Selective stimulation of β2-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of β2-AR stimulation on L-type Ca2+ current ( ICa,L) during postnatal development. ICa,L was measured (37°C; either Ca2+ or Ba2+ as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca2+ transients were measured concomitantly by dialyzing the cell with indo-1. Activation of β2-ARs (with either 100 nM zinterol or 1 μM isoproterenol in the presence of the β1-AR antagonist, CGP20712A) stimulated ICa,L twofold in newborns but not in adults. The β2-AR-mediated increase in Ca2+ transient amplitude in newborns was due exclusively to the augmentation of ICa,L. Zinterol increased the rate of inactivation of ICa,L and increased the Ca2+ flux integral. The β2-AR inverse agonist, ICI-118551 (500 nM), but not the β1-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 μM), PKI 6-22 amide (10 μM), and Rp-cAMP (100 μM), all failed to prevent the response to zinterol but completely blocked responses to selective β1-AR stimulation of ICa,L in newborns. Our results demonstrate that in addition to the conventional β1-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel β2-AR-mediated, PKA-insensitive pathway that stimulates ICa,L. This striking developmental difference plays a major role in the age-related differences in inotropic responses to β2-AR agonists.