The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration.

Previous observations show that β-adrenergic modulation of pacemaker current (I(f)) in sinoatrial node (SAN) cells is impaired by disruption of normal Ca(2+)-homeostasis with ryanodine or BAPTA. Recently, the presence of Ca(2+)-activated adenylyl cyclase (AC) 1 was reported in SAN, and was proposed as a possible mechanism of Ca(2+)-dependence of β-adrenergic modulation. However, direct evidence that pacemaker (HCN) channels can be regulated by Ca(2+)-activated AC and that such regulation introduces Ca(2+) dependence, is lacking. Here we co-expressed AC1 or AC6 with HCN2 in neonatal rat ventricular myocytes, which lack AC1. Although both isoforms have equivalent expression level and ability to interact with HCN2, only AC1 increases intracellular cAMP content, accelerates spontaneous beating rate and modifies HCN2 biophysics. Measured HCN2 current in the AC1 group activated ~10mV more positive than in GFP or AC6. The β-adrenergic agonist isoproterenol induced a further positive shift under control conditions, but failed to do so after pretreatment with the Ca(2+) chelator BAPTA. In the AC6 group, isoproterenol shifted the HCN2 activation relation to a similar extent in the absence and presence of BAPTA. Thus, AC1 but not AC6 over-expression introduces Ca(2+)-sensitivity to the β-adrenergic response of HCN2. These results demonstrate physical and functional interaction between AC isoforms and the HCN2 pacemaker channel and support a key role of Ca(2+) activated AC1 as a molecular mechanism in Ca(2+)-dependent modulation of β-adrenergic response of heart rate.

In rabbit, sodium current (I(Na)) contributes to newborn sinoatrial node (SAN) automaticity but is absent in adult SAN, where heart rate is slower. In contrast, heart rate is high and I(Na) is functional in adult mouse SAN. Given the slower heart rates of large mammals, we asked if I(Na) is functionally active in SAN of newborn or adult canine heart. SAN cells were isolated from newborn (6-10 days), young (40-43 days) and adult mongrels. I(Na) was observed in >80% of cells from each age. However, current density was markedly greater in newborn, decreasing with age. At all ages, I(Na) was sensitive to nanomolar tetrodotoxin (TTX); 100 nmol/L inhibited I(Na) by 46.7%, 59.9% and 90.7% in newborn, young and adult cells, respectively. While high TTX sensitivity suggested the presence of non-cardiac isoforms, steady-state inactivation was relatively negative (midpoints -89.7+/-0.7 mV, -95.1+/-1.2 mV and -93.4+/-1.9 mV from newborn to adult). Consequently, I(Na) should be unavailable at physiological potentials under normal conditions, and 100 nmol/L TTX did not change cycle length or action potential parameters of spontaneous adult SAN cells. However, computer modeling predicts the large newborn I(Na) protects against excess rate slowing from strong vagal stimulation. The results show that canine SAN cells have TTX-sensitive I(Na) which decreases with post-natal age. The current does not contribute to normal automaticity in isolated adult cells but can be recruited to sustain excitability if nodal cells are hyperpolarized. This is particularly relevant in newborn, where I(Na) is large and parasympathetic/sympathetic balance favors vagal tone.

AIMS Reentrant arrhythmias often develop in the setting of myocardial infarction and ensuing slow propagation. Increased Na(+) channel expression could prevent or disrupt reentrant circuits by speeding conduction if channel availability is not limited by membrane depolarization within the diseased myocardium. We therefore asked if, in the setting of membrane depolarization, action potential (AP) upstroke and normal conduction can be better preserved by the expression of a Na(+) channel isoform with altered biophysical properties compared to the native cardiac Na(+) channel isoform, namely having a positively shifted, voltage-dependent inactivation. METHODS AND RESULTS The skeletal Na(+) channel isoform (SkM1) and the cardiac Na(+) channel isoform (Nav1.5) were expressed in newborn rat ventricular myocyte cultures with a point mutation introduced in Nav1.5 to increase tetrodotoxin (TTX) sensitivity so native and expressed currents could be distinguished. External K(+) was increased from 5.4 to 10 mmol/L to induce membrane depolarization. APs, Na(+) currents, and conduction velocity (CV) were measured. In control cultures, elevated K(+) significantly reduced AP upstroke ( approximately 75%) and CV ( approximately 25%). Expression of Nav1.5 did not protect AP upstroke from K(+) depolarization. In contrast, in SkM1 expressing cultures, high K(+) reduced AP upstroke <50% and conduction was not significantly reduced. In a simulated anatomical reentry setting (using a void), the angular velocity (AV) of induced reentry was faster and the excitable gap shorter in SkM1 cultures compared to control for both normal and high K(+). CONCLUSION Expression of SkM1 but not Nav1.5 preserves AP upstroke and CV in a K(+)-depolarized syncytium. The higher AV and shorter excitable gap observed during reentry excitation around a void in SkM1 cultures would be expected to facilitate reentry self-termination. SkM1 Na(+) channel expression represents a novel gene therapy for the treatment of reentrant arrhythmias.

The antagonism between parasympathetic and sympathetic control of the heart and other organs is reflected not only in the opposite effect of their respective neurotransmitters acetylcholine (ACh) and norepinephrine (NE) on their target tissue, but also through the action of each nerve type to inhibit the ability of the other to release neurotransmitters. The vagus nerve can inhibit sympathetic neurotransmission by interaction of ACh with muscarinic receptors located on noradrenergic nerve terminals [ [1] Vanhoutte P. Levy M. Prejunctional cholinergic modulation of adrenergic neurotransmission in the cardiovascular system. Am J Physiol. 1980; 238: H275-H281 PubMed Google Scholar ]. Similarly, stimulation of sympathetic nerves produces strong and long lasting inhibition of the chronotropic effect of the vagus [ [2] Potter E. Prolonged non-adrenergic inhibition of cardiac vagal action following sympathetic stimulation: neuromodulation by neuropeptide Y?. Neurosci Lett. 1985; 54: 117-121 Crossref PubMed Scopus (154) Google Scholar ]. As a result of extensive studies by a number of groups (see below), we now know that in many mammalian hearts the latter effect is evoked by neuropeptide Y (NPY), which is colocalised with NE in sympathetic nerve endings.

We studied the role of beta-adrenergic receptor subtype signaling to cAMP and calcium in the genesis of catecholamine-dependent arrhythmias in German shepherd dogs that develop lethal arrhythmias at similar to5 mo of age. There were three major findings in this study: 1) isoproterenol induces similar increases in cAMP in afflicted and control dogs exclusively through beta(1)-receptors (not beta(2)), 2) cells from afflicted dogs display prolonged relaxation kinetics at long cycle lengths and large frequent spontaneous calcium oscillations (and aftercontractions) with little increase in calcium transient amplitude in response to beta(1)-receptor agonists, and 3) beta(2)-receptor agonists induce a similar marked increases in calcium transient and twitch amplitude, with only rare spontaneous calcium oscillations in afflicted and control cells. These results indicate that catecholamines provide inotropic support to canine cardiomyocytes through distinct beta(1)- and beta(2)-receptor pathways with differing requirements for cAMP. The propensity to develop arrhythmias is not induced by beta(2)-receptors (or a rise in calcium alone), but rather occurs in the context of beta(1)- receptor activation of the cAMP-dependent pathway.

The developmental increase in L-type Ca current ( I ) density in the rat ventricle is reproduced in vitro by culturing neonatal myocytes with sympathetic neurons. We tested whether this effect of sympathetic innervation results from a chronic or sustained action of neurally released neuropeptide Y (NPY). Ventricular myocytes from newborn rats were cultured in serum-free medium with or without sympathetic neurons, NPY, or NPY analogs. Ca currents were measured in single myocytes at room temperature using the perforated patch clamp. In all cell groups (control, innervated, or NPY treated), the current-voltage relation for I was represented by a bell-shaped curve with maximal value near 0 mV. The current density at 0 mV normalized to that of corresponding mean control values was 1.63 ± 0.12 and 1.52 ± 0.16 for innervated and NPY-treated myocytes, respectively. Both groups differed significantly from control ( P < 0.05). NPY analogs exhibited the following rank order of effectiveness: NPY ≥ NPY-(13-36) ≥ PYY >> [LeuPro]NPY, suggesting that the NPY effect occurs via a Y-receptor subtype. In confirmation, chronic treatment of innervated cultures with a Y-selective NPY antagonist prevented the innervation-dependent increase in I . These results indicate that sympathetic innervation contributes to the developmental increase in I via neurally released NPY acting at Yreceptors on the ventricular myocytes.