Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes.

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.

Ca2+-activated adenylyl cyclase 1 introduces Ca2+-dependence to beta-adrenergic stimulation of HCN2 current.

Previous observations show that ß-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 ß-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 ß-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 ß-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 ß-adrenergic response of heart rate.

Age-dependent changes in Na current magnitude and TTX-sensitivity in the canine sinoatrial node.

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.

Expression of skeletal but not cardiac Na+ channel isoform preserves normal conduction in a depolarized cardiac syncytium.

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.

Novel cell lines derived from adult human ventricular cardiomyocytes.

Background. - We have established proliferating human cardiomyocyte cell lines derived from non-proliferating primary cultures of adult ventricular heart tissue, using a novel method that may be applicable to many post-mitotic primary cultures. Methods and results. - Primary cells from human ventricular tissue, were fused with SV40 transformed, uridine auxotroph human fibroblasts, devoid of mitochondrial DNA. This was followed by selection in uridine-free medium to eliminate unfused fibroblasts. The fused cells were subcloned and screened for cell type-specific markers. Four clones (AC1, AC10, AC12, AC16) that express markers characteristic of cardiomyocytes were studied. Clones were homogeneous morphologically, and expressed transcription factors (GATA4, MYCD, NFATc4), contractile proteins such as alpha- and beta-myosin heavy chain, alpha-cardiac actin, troponin I, desmoplakin, alpha actinin, the muscle-specific intermediate filament protein, desmin, the cardiomyocyte-specific peptide hormones, BNP, the L-type calcium channel alpha1C subunit and gap junction proteins, connexin-43 and connexin-40. Furthermore, dye-coupling studies confirmed the presence of functional gap junctions. EM ultra structural analysis revealed the presence of myofibrils in the subsarcolemmal region, indicating a precontractile developmental stage. When grown in mitogen-depleted medium, the AC cells stopped proliferating and formed a multinucleated syncytium. When the SV40 oncogene was silenced using the RNAi technique, AC16 cells switched from a proliferating to a more differentiated quiescent state, with the formation of multinucleated syncyntium. Concurrently, the cells expressed BMP2, an important signaling molecule for induction of cardiac-specific markers, that was not expressed by the proliferating cells. The presence of the combination of transcription factors in addition to muscle-specific markers is a good indication for the presence of a cardiac transcription program in these cells. CONCLUSIONS. - Based on the expression of myogenic markers and a fully functional respiratory chain, the AC cells have retained the nuclear DNA and the mitochondrial DNA of the primary cardiomyocytes. They can be frozen and thawed repeatedly and can differentiate when grown in mitogen-free medium. These cell lines are potentially useful in vitro models to study developmental regulation of cardiomyocytes in normal and pathological states.

Regional dispersion of L-type calcium current in ventricular myocytes of German shepherd dogs with lethal cardiac arrhythmias.

OBJECTIVES: The purpose of this study was to determine if regional differences in L-type Ca(2+) current (I(Ca,L)) are altered in a German shepherd model of sudden death. BACKGROUND: German shepherd dogs with inherited sudden cardiac death have reduced sympathetic innervation in the anteroseptal left ventricle that may contribute to arrhythmias in afflicted animals compared to control unafflicted animals. Differences in a number of repolarizing K(+) currents have been identified in this model, but I(Ca,L) has not been studied. METHODS: We measured action potentials in intact tissue and I(Ca,L) in isolated myocytes from anteroseptal and anterobasal left ventricle. RESULTS: Action potential plateau level and I(Ca,L) density were significantly lower in unafflicted anteroseptal than in afflicted anteroseptal, afflicted anterobasal, or unafflicted anterobasal. Isoproterenol increased I(Ca,L) density more in the unafflicted anteroseptal group than in the other groups. CONCLUSIONS: Differences in I(Ca,L) between afflicted and control animals, combined with our earlier finding of regional reductions in I(Kr), provide a likely substrate for the occurrence of pause-dependent arrhythmias in afflicted animals and for the T-wave abnormalities characterizing them.

Neuropeptide Y is an essential in vivo developmental regulator of cardiac ICa,L.

Cell culture studies demonstrate an increase in cardiac L-type Ca2+ current (ICa,L) density on sympathetic innervation in vitro and suggest the effect depends on neurally released neuropeptide Y (NPY). To determine if a similar mechanism contributes to the postnatal increase in ICa,L in vivo, we prepared isolated ventricular myocytes from neonatal and adult mice with targeted deletion of the NPY gene (Npy-/-) and matched controls (Npy+/+). Whole-cell voltage clamp demonstrates ICa,L density increases postnatally in Npy+/+ (by 56%), but is unchanged in Npy-/-. Both ICa,L density and action potential duration are significantly greater in adult Npy+/+ than Npy-/- myocytes, whereas ICa,L density is equivalent in neonatal Npy+/+ and Npy-/- myocytes. These data indicate NPY does not influence ICa,L prenatally, but the postnatal increase in ICa,L density is entirely NPY-dependent. In contrast, there is a similar postnatal negative voltage shift in the I-V relation in Npy+/+ and Npy-/-, indicating NPY does not influence the developmental change in ICa,L voltage-dependence. Immunoblot analyses and measurements of maximally activated ICa,L (in presence of forskolin or BayK 8644) show that the differences in current density between Npy+/+ and Npy-/- cannot be attributed to altered Ca2+ channel alpha1C subunit protein expression. Rather, these results suggest that the in vivo NPY-dependent postnatal increase in ICa,L density in cardiac myocytes results from regulation ICa,L properties by NPY.

beta(1)-Receptors increase cAMP and induce abnormal Ca(i) cycling in the German shepherd sudden death model.

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 ~5 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.