Disorder in Ca2+ Release Unit Locations Confers Robustness but Cuts Flexibility of Heart Pacemaking

Excitation-contraction coupling kinetics are dictated by the rate and rhythm of the excitations generated by sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the rate and rhythm of the excitations. The LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While the spatial CRU distribution in pacemaker cells exhibits substantial heterogeneity, it remains unknown if it has any functional importance. Using numerical modeling, here we showed that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad lower range of realistic ICaL densities. Allowing each CRU to deviate from its original lattice position fundamentally changes the model behavior: during diastolic depolarization sparks propagate, forming LCRs observed experimentally. As disorder in the CRU positions increases, the CRU distribution exhibits larger empty spaces but simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing AP firing rate and reviving pacemaker function within lower ICaL densities at which cells with lattice CRU distribution were dormant/non-firing. However, cells with fully disordered CRU positions cannot reach low firing rates and their β-adrenergic receptor stimulation effect was substantially decreased. Thus, order/disorder in CRU locations regulates Ca release propagation and could be harnessed by pacemaker cells to regulate their function. Excessive disorder is expected to limit heart rate range that may contribute to heart rate range decline with age and in disease.

Desarrollo de un simulador de la corriente funny del nodo sinusal para la enseñanza-aprendizaje

Las células del nodo sinusal generan la principal actividad eléctrica marcapaso del corazón. La actividad marcapaso se debe a la presencia de la corriente funny (If). Esta corriente iónica es entrante y se activa con la hiperpolarización en rangos de voltaje presentes durante la fase de despolarización diastólica; contrario a la mayoría de las corrientes iónicas que se activan con la despolarización. El canal funny es permeable a iones Na+ y K+. Generalmente, en el curso de biofísica a nivel de licenciatura los alumnos conocen algunos canales dependientes de voltaje del tipo de Hodgkin y Huxley que se activan con la despolarización. Sin embargo, no se estudia ningún canal que se active con una hiperpolarización. En este trabajo se diseñó y desarrolló un simulador para el estudio y comprensión de la corriente funny presente en el nodo sinusal del conejo. El simulador fue programado en lenguaje Visual Basic ver. 6.0 para ambiente Windows® de XP a Windows® 10. El usuario puede realizar los experimentos con la técnica de fijación de voltaje, modificar las variables y concentraciones de Na+ y K+ externos y observar el efecto en la amplitud de If y la cinética de la curva I-V. Se recomienda su uso como material didáctico de apoyo durante los cursos de biofísica y fisiología en una sala de cómputo o a distancia en cualquier computadora personal con recursos mínimos. The sinus node cells generate the main electric pacemaker activity of the heart. The pacemaker activity is due to the presence of the current funny (If). This ionic current is incoming and is activated with hypolarization in voltage ranges present during the diastolic depolarization phase, contrary to most ionic currents that are activated by depolarization. The funny channel is permeable to Na+ and K+ ions. Generally, students enrolled in the biophysics course at the undergraduate level are familiar with some voltage-gated channels of the Hodgkin and Huxley type. However, no channel that is activated by hiperpolarization is studied. In this work, a simulator was designed and developed for the study and understanding of the funny current present in the sinus node of rabbits. The simulator was programmed in Visual Basic Language ver. 6.0 for Windows® environment from XP to Windows® 10. The user can perform the experiments with the voltage clamp technique, modify the variables and concentrations of external Na+ and K+ and observe the effect on the amplitude of If and the kinetics of the curve I-V. It is recommended to be used as a support didactic material during biophysics and physiology courses in a computer room or remotely on any personal computer with minimal resources.

The Functional Role of Hyperpolarization Activated Current (If) on Cardiac Pacemaking in Human vs. in the Rabbit Sinoatrial Node: A Simulation and Theoretical Study

The cardiac hyperpolarization-activated “funny” current (If), which contributes to sinoatrial node (SAN) pacemaking, has a more negative half-maximal activation voltage and smaller fully-activated macroscopic conductance in human than in rabbit SAN cells. The consequences of these differences for the relative roles of If in the two species, and for their responses to the specific bradycardic agent ivabradine at clinical doses have not been systematically explored. This study aims to address these issues, through incorporating rabbit and human If formulations developed by Fabbri et al. into the Severi et al. model of rabbit SAN cells. A theory was developed to correlate the effect of If reduction with the total inward depolarising current (Itotal) during diastolic depolarization. Replacing the rabbit If formulation with the human one increased the pacemaking cycle length (CL) from 355 to 1,139 ms. With up to 20% If reduction (a level close to the inhibition of If by ivabradine at clinical concentrations), a modest increase (~5%) in the pacemaking CL was observed with the rabbit If formulation; however, the effect was doubled (~12.4%) for the human If formulation, even though the latter has smaller If density. When the action of acetylcholine (ACh, 0.1 nM) was considered, a 20% If reduction markedly increased the pacemaking CL by 37.5% (~27.3% reduction in the pacing rate), which is similar to the ivabradine effect at clinical concentrations. Theoretical analysis showed that the resultant increase of the pacemaking CL is inversely proportional to the magnitude of Itotal during diastolic depolarization phase: a smaller If in the model resulted in a smaller Itotal amplitude, resulting in a slower pacemaking rate; and the same reduction in If resulted in a more significant change of CL in the cell model with a smaller Itotal. This explained the mechanism by which a low dose of ivabradine slows pacemaking rate more in humans than in the rabbit. Similar results were seen in the Fabbri et al. model of human SAN cells, suggesting our observations are model-independent. Collectively, the results of study explain why low dose ivabradine at clinically relevant concentrations acts as an effective bradycardic agent in modulating human SAN pacemaking.

Speeding Up the Heart? Traditional and New Perspectives on HCN4 Function

The sinoatrial node (SAN) is the primary pacemaker of the heart and is responsible for generating the intrinsic heartbeat. Within the SAN, spontaneously active pacemaker cells initiate the electrical activity that causes the contraction of all cardiomyocytes. The firing rate of pacemaker cells depends on the slow diastolic depolarization (SDD) and determines the intrinsic heart rate (HR). To adapt cardiac output to varying physical demands, HR is regulated by the autonomic nervous system (ANS). The sympathetic and parasympathetic branches of the ANS innervate the SAN and regulate the firing rate of pacemaker cells by accelerating or decelerating SDD–a process well-known as the chronotropic effect. Although this process is of fundamental physiological relevance, it is still incompletely understood how it is mediated at the subcellular level. Over the past 20 years, most of the work to resolve the underlying cellular mechanisms has made use of genetically engineered mouse models. In this review, we focus on the findings from these mouse studies regarding the cellular mechanisms involved in the generation and regulation of the heartbeat, with particular focus on the highly debated role of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 in mediating the chronotropic effect. By focusing on experimental data obtained in mice and humans, but not in other species, we outline how findings obtained in mice relate to human physiology and pathophysiology and provide specific information on how dysfunction or loss of HCN4 channels leads to human SAN disease.

cAMP-Dependent Signaling Restores AP Firing in Dormant SA Node Cells via Enhancement of Surface Membrane Currents and Calcium Coupling

Action potential (AP) firing rate and rhythm of sinoatrial nodal cells (SANC) are controlled by synergy between intracellular rhythmic local Ca2+ releases (LCRs) (“Ca2+ clock”) and sarcolemmal electrogenic mechanisms (“membrane clock”). However, some SANC do not fire APs (dormant SANC). Prior studies have shown that β-adrenoceptor stimulation can restore AP firing in these cells. Here we tested whether this relates to improvement of synchronization of clock coupling. We characterized membrane potential, ion currents, Ca2+ dynamics, and phospholamban (PLB) phosphorylation, regulating Ca2+ pump in enzymatically isolated single guinea pig SANC prior to, during, and following β-adrenoceptor stimulation (isoproterenol) or application of cell-permeant cAMP (CPT-cAMP). Phosphorylation of PLB (Serine 16) was quantified in the same cells following Ca2+ measurement. In dormant SANC LCRs were small and disorganized at baseline, membrane potential was depolarized (−38 ± 1 mV, n = 46), and ICaL, If, and IK densities were smaller vs SANC firing APs. β-adrenoceptor stimulation or application of CPT-cAMP led to de novo spontaneous AP generation in 44 and 46% of dormant SANC, respectively. The initial response was an increase in size, rhythmicity and synchronization of LCRs, paralleled with membrane hyperpolarization and small amplitude APs (rate ∼1 Hz). During the transition to steady-state AP firing, LCR size further increased, while LCR period shortened. LCRs became more synchronized resulting in the growth of an ensemble LCR signal peaked in late diastole, culminating in AP ignition; the rate of diastolic depolarization, AP amplitude, and AP firing rate increased. ICaL, IK, and If amplitudes in dormant SANC increased in response to β-adrenoceptor stimulation. During washout, all changes reversed in order. Total PLB was higher, but the ratio of phosphorylated PLB (Serine 16) to total PLB was lower in dormant SANC. β-adrenoceptor stimulation increased this ratio in AP-firing cells. Thus, transition of dormant SANC to AP firing is linked to the increased functional coupling of membrane and Ca2+ clock proteins. The transition occurs via (i) an increase in cAMP-mediated phosphorylation of PLB accelerating Ca2+ pumping, (ii) increased spatiotemporal LCR synchronization, yielding a larger diastolic LCR ensemble signal resulting in an earlier increase in diastolic INCX; and (iii) increased current densities of If, ICaL, and IK.

Pharmacology of Ivabradine and the Effect on Chronic Heart Failure

Chronic Heart Failure (CHF) is a complex clinical syndrome with a high incidence worldwide. Although various types of pharmacological and device therapies are available for CHF, the prognosis is not ideal, for which, the control of increased Heart Rate (HR) is critical. Recently, a bradycardic agent, ivabradine, is found to reduce HR by inhibiting the funny current (If). The underlying mechanism states that ivabradine can enter the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels and bind to the intracellular side, subsequently inhibiting the If. This phenomenon can prolong the slow spontaneous phase in the diastolic depolarization, and thus, reduce HR. The clinical trials demonstrated the significant effects of the drug on reducing HR and improving the symptoms of CHF with fewer adverse effects. This review primarily introduces the chemical features and pharmacological characteristics of ivabradine and the mechanism of treating CHF. Also, some expected therapeutic effects on different diseases were also concluded. However, ivabradine, as a typical If channel inhibitor, necessitates additional research to verify its pharmacological functions.

Chronic intermittent tachypacing by an optogenetic approach induces arrhythmia vulnerability in human engineered heart tissue

Aims Chronic tachypacing is commonly used in animals to induce cardiac dysfunction and to study mechanisms of heart failure and arrhythmogenesis. Human induced pluripotent stem cells (hiPSC) may replace animal models to overcome species differences and ethical problems. Here, 3D engineered heart tissue (EHT) was used to investigate the effect of chronic tachypacing on hiPSC-cardiomyocytes (hiPSC-CMs). Methods and results To avoid cell toxicity by electrical pacing, we developed an optogenetic approach. EHTs were transduced with lentivirus expressing channelrhodopsin-2 (H134R) and stimulated by 15 s bursts of blue light pulses (0.3 mW/mm2, 30 ms, 3 Hz) separated by 15 s without pacing for 3 weeks. Chronic optical tachypacing did not affect contractile peak force, but induced faster contraction kinetics, shorter action potentials, and shorter effective refractory periods. This electrical remodelling increased vulnerability to tachycardia episodes upon electrical burst pacing. Lower calsequestrin 2 protein levels, faster diastolic depolarization (DD) and efficacy of JTV-519 (46% at 1 µmol/L) to terminate tachycardia indicate alterations of Ca2+ handling being part of the underlying mechanism. However, other antiarrhythmic compounds like flecainide (69% at 1 µmol/L) and E-4031 (100% at 1 µmol/L) were also effective, but not ivabradine (1 µmol/L) or SEA0400 (10 µmol/L). Conclusion We demonstrated a high vulnerability to tachycardia of optically tachypaced hiPSC-CMs in EHT and the effective termination by ryanodine receptor stabilization, sodium or hERG potassium channel inhibition. This new model might serve as a preclinical tool to test antiarrhythmic drugs increasing the insight in treating ventricular tachycardia.

Regulation of heart rate and the pacemaker current by phosphoinositide 3-kinase signaling

Heart rate in physiological conditions is set by the sinoatrial node (SN), the primary cardiac pacing tissue. Phosphoinositide 3-kinase (PI3K) signaling is a major regulatory pathway in all normal cells, and its dysregulation is prominent in diabetes, cancer, and heart failure. Here, we show that inhibition of PI3K slows the pacing rate of the SN in situ and in vitro and reduces the early slope of diastolic depolarization. Furthermore, inhibition of PI3K causes a negative shift in the voltage dependence of activation of the pacemaker current, IF, while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of IF are independent of, and larger than, those induced by the autonomic nervous system. These results suggest that PI3K is an important regulator of heart rate, and perturbations in this signaling pathway may contribute to the development of arrhythmias.

Angiotensin II Induces Automatic Activity of the Isolated Guinea Pig Pulmonary Vein Myocardium through Activation of the IP3 Receptor and the Na+-Ca2+ Exchanger

The automaticity of the pulmonary vein myocardium is known to be the major cause of atrial fibrillation. We examined the involvement of angiotensin II in the automatic activity of isolated guinea pig pulmonary vein preparations. In tissue preparations, application of angiotensin II induced an automatic contractile activity; this effect was mimicked by angiotensin I and blocked by losartan, but not by PD123,319 or carvedilol. In cardiomyocytes, application of angiotensin II induced an increase in the frequency of spontaneous Ca2+ sparks and the generation of Ca2+ transients; these effects were inhibited by losartan or xestospongin C. In tissue preparations, angiotensin II caused membrane potential oscillations, which lead to repetitive generation of action potentials. Angiotensin II increased the diastolic depolarization slope of the spontaneous or evoked action potentials. These effects of angiotensin II were inhibited by SEA0400. In tissue preparations showing spontaneous firing of action potentials, losartan, xestospongin C or SEA0400 decreased the slope of the diastolic depolarization and inhibited the firing of action potentials. In conclusion, in the guinea pig pulmonary vein myocardium, angiotensin II induces the generation of automatic activity through activation of the IP3 receptor and the Na+-Ca2+ exchanger.