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.
Abstract-Ventricular pacemaker current (I f ) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular I f , exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V 1/2 Ϫ95.9Ϯ1.9 in adult; Ϫ77.6Ϯ1.6 mV in neonate), comparable to the 22-mV difference between native I f in adult and neonatal cultures (V 1/2 Ϫ98.7 versus Ϫ77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88Ϯ5 versus 48Ϯ4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149Ϯ266 versus 3140Ϯ279 mV ⅐ ms).
Although the neonatal sinus node beats at a faster rate than the adult, when a sodium current (I(Na)) present in the newborn is blocked, the spontaneous rate is slower in neonatal myocytes than in adult myocytes. This suggests a possible functional substitution of I(Na) by another current during development. We used ruptured [T-type calcium current (I(Ca,T))] and perforated [L-type calcium current (I(Ca,L))] patch clamps to study developmental changes in calcium currents in sinus node cells from adult and newborn rabbits. I(Ca,T) density did not differ with age, and no significant differences were found in the voltage dependence of activation or inactivation. I(Ca,L) density was lower in the adult than newborn (12.1 +/- 1.4 vs. 17.6 +/- 2.5 pA/pF, P = 0.049). However, activation and inactivation midpoints were shifted in opposite directions, reducing the potential contribution during late diastolic depolarization in the newborn (activation midpoints -17.3 +/- 0.8 and -22.3 +/- 1.4 mV in the newborn and adult, respectively, P = 0.001; inactivation midpoints -33.4 +/- 1.4 and -28.3 +/- 1.7 mV for the newborn and adult, respectively, P = 0.038). Recovery of I(Ca,L) from inactivation was also slower in the newborn. The results suggest that a smaller but more negatively activating and rapidly recovering I(Ca,L) in the adult sinus node may contribute to the enhanced impulse initiation at this age in the absence of I(Na).
In rabbit, sodium current (INa) 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 INa is functional in adult mouse SAN. Given the slower heart rates of large mammals, we asked if INa 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. INa was observed in >80% of cells from each age. However, current density was markedly greater in newborn, decreasing with age. At all ages, INa was sensitive to nanomolar tetrodotoxin (TTX); 100 nmol/L inhibited INa 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 isofoms, 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, INa 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 INa protects against excess rate slowing from strong vagal stimulation. The results show that canine SAN cells have TTX–sensitive INa 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 INa is large and parasympathetic/sympathetic balance favors vagal tone.
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.
Neuropeptide Y (NPY) is released from sympathetic neurons and exerts short-term (acute) effects on prejunctional nerve terminals and postjunctional cardiac ion channels. However, NPY also exerts long-term (trophic) effects on angiogenesis, cardiac hypertrophy, autonomic signaling, and cardiac ion channels, including effects on L-type Ca2+ and pacemaker channels.
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