Cardiac pacemaker function of HCN4 channels in mice is confined to embryonic development and requires cyclic AMP.

Important targets for cAMP signalling in the heart are hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels that underlie the depolarizing 'pacemaker' current, I(f). We studied the role of I(f) in mice, in which binding of cAMP to HCN4 channels was abolished by a single amino-acid exchange (R669Q). Homozygous HCN4(R669Q/R669Q) mice die during embryonic development. Prior to E12, homozygous and heterozygous embryos display reduced heart rates and show no or attenuated responses to catecholaminergic stimulation. Adult heterozygous mice display normal heart rates at rest and during exercise. However, following beta-adrenergic stimulation, hearts exhibit pauses and sino-atrial node block. Our results demonstrate that in the embryo, HCN4 is a true cardiac pacemaker and elevation of HCN4 channel activity by cAMP is essential for viability. In adult mice, an important function of HCN4 channels is to prevent sinus pauses during and after stress while their role as a pacemaker of the murine heart is put into question. Most importantly, our results indicate that HCN4 channels can fulfil their physiological function only when cAMP is bound.

Increased InsP3Rs in the junctional sarcoplasmic reticulum augment Ca2+ transients and arrhythmias associated with cardiac hypertrophy.

Cardiac hypertrophy is a growth response of the heart to increased hemodynamic demand or damage. Accompanying this heart enlargement is a remodeling of Ca(2+) signaling. Due to its fundamental role in controlling cardiomyocyte contraction during every heartbeat, modifications in Ca(2+) fluxes significantly impact on cardiac output and facilitate the development of arrhythmias. Using cardiomyocytes from spontaneously hypertensive rats (SHRs), we demonstrate that an increase in Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) contributes to the larger excitation contraction coupling (ECC)-mediated Ca(2+) transients characteristic of hypertrophic myocytes and underlies the more potent enhancement of ECC-mediated Ca(2+) transients and contraction elicited by InsP(3) or endothelin-1 (ET-1). Responsible for this is an increase in InsP(3)R expression in the junctional sarcoplasmic reticulum. Due to their close proximity to ryanodine receptors (RyRs) in this region, enhanced Ca(2+) release through InsP(3)Rs served to sensitize RyRs, thereby increasing diastolic Ca(2+) levels, the incidence of extra-systolic Ca(2+) transients, and the induction of ECC-mediated Ca(2+) elevations. Unlike the increase in InsP(3)R expression and Ca(2+) transient amplitude in the cytosol, InsP(3)R expression and ECC-mediated Ca(2+) transients in the nucleus were not altered during hypertrophy. Elevated InsP(3)R2 expression was also detected in hearts from human patients with heart failure after ischemic dilated cardiomyopathy, as well as in aortic-banded hypertrophic mouse hearts. Our data establish that increased InsP(3)R expression is a general mechanism that underlies remodeling of Ca(2+) signaling during heart disease, and in particular, in triggering ventricular arrhythmia during hypertrophy.

Temporal changes in atrial EC-coupling during prolonged stimulation with endothelin-1.

Endothelin-1 (ET-1) is a potent G(q)-coupled agonist with important physiological effects on the heart. In the present study, we characterised the effect of prolonged ET-1 stimulation on Ca(2+) signalling within acutely isolated atrial myocytes. ET-1 induced a reproducible and complex sequence of effects, including negative inotropy, positive inotropy and pro-arrhythmic spontaneous Ca(2+) transients (SCTs). The negative and positive inotropic effects correlated with the ability of Ca(2+) to propagate from the subsarcolemmal sites where EC-coupling initiates into the centre of the atrial cells. We examined the spatial and temporal properties of the SCTs and observed them to range from elementary Ca(2+) sparks, flurries of Ca(2+) sparks, to Ca(2+) waves and action potential-evoked global Ca(2+) transients. The positive inotropic effect of ET-1 and its ability to trigger SCTs were mimicked by direct stimulation of InsP(3)Rs. An antagonist of InsP(3)Rs prevented the generation of SCTs and partially reduced the positive inotropy evoked by ET-1. Our data suggest that ET-1 engages multiple signal transduction pathways to provoke a plethora of different responses within an atrial myocyte. Some of the actions of ET-1 appear to be due to stimulation of InsP(3)Rs.

Comparison of the T-tubule system in adult rat ventricular and atrial myocytes, and its role in excitation-contraction coupling and inotropic stimulation.

Narrow, tubular, inward projections of the sarcolemma ('T-tubules') are an established feature of adult mammalian ventricular myocytes that enables them to generate the whole-cell Ca2+ transients and produce coordinated contraction. Loss of T-tubules can occur during ageing and under pathological conditions, leading to altered cardiac excitation-contraction coupling. In contrast to adult ventricular cells, atrial myocytes do not generally express an extensive T-tubule system at any stage of development, and therefore rely on Ca2+ channels around their periphery for the induction of Ca2+ signalling and excitation-contraction coupling. Consequently, the characteristics of systolic Ca2+ signals in adult ventricular and atrial myocytes are temporally and spatially distinct. However, although atrial myocytes do not have the same regularly spaced convoluted T-tubule structures as adult ventricular cells, it has been suggested that a proportion of adult atrial cells have a more rudimentary tubule system. We examined the structure and function of these atrial tubules, and explored their impact on the initiation and recovery of Ca2+ signalling in electrically paced myocytes. The atrial responses were compared to those in adult ventricular cells that had intact T-tubules, or that had been chemically detubulated. We found that tubular structures were present in a significant minority of adult atrial myocytes, and were unlike the T-tubules in adult ventricular cells. In those cells where they were present, the atrial tubules significantly altered the on-set, amplitude, homogeneity and recovery of Ca2+ transients. The properties of adult atrial myocyte Ca2+ signals were different from those in adult ventricular cells, whether intact or detubulated. Excitation-contraction coupling in detubulated adult ventricular myocytes, therefore, does not approximate to atrial signalling, even though Ca2+ signals are initiated in the periphery of the cells in both of these situations. Furthermore, inotropic responses to endothelin-1 were entirely dependent on T-tubules in adult ventricular myocytes, but not in atrial cells. Our data reveal that that the T-tubules in atrial cells impart significant functional properties, but loss of these tubular membranes does not affect Ca2+ signalling as dramatically as detubulation in ventricular myocytes.

Elevated InsP3R expression underlies enhanced calcium fluxes and spontaneous extra-systolic calcium release events in hypertrophic cardiac myocytes.

Cardiac hypertrophy is associated with profound remodeling of Ca(2+) signaling pathways. During the early, compensated stages of hypertrophy, Ca(2+) fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca(2+) homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca(2+) release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca(2+) store are primarily responsible for the Ca(2+) flux that induces myocyte contraction, a role for Ca(2+) release via the inositol 1,4,5-trisphosphate receptor (InsP(3)R) in cardiac physiology has also emerged. Specifically, InsP(3)-induced Ca(2+) signals generated following myocyte stimulation with an InsP(3)-generating agonist (e.g., endothelin, ET-1), lead to modulation of Ca(2+) signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca(2+) release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP(3)R (InsP(3)R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca(2+) release via InsP(3)Rs serves to sensitize neighboring RyRs causing an augmentation of Ca(2+) fluxes during ECC as well as an increase in non-triggered Ca(2+) release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP(3)R expression during hypertrophy, the spontaneous Ca(2+) release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP(3)R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP(3)R expression may be a general phenomenon that underlies Ca(2+) changes during hypertrophy.