Maximum Diastolic Potential Research Articles

Cardiac automaticity is modulated by IKACh in sinoatrial node during pregnancy.

Pregnant women have a significantly elevated resting heart rate (HR), which makes cardiac arrhythmias more likely to occur. Although electrical remodeling of the sinoatrial node (SAN) has been documented, the underlying mechanism is not fully understood. The acetylcholine-activated potassium current (IKACh), one of the major repolarizing currents in the SAN, plays a critical role in HR control by hyperpolarizing the maximal diastolic potential (MDP) of the SAN action potential (AP), thereby reducing SAN automaticity and HR. Thus, considering its essential role in cardiac automaticity, this study aims to determine whether changes in IKACh are potentially involved in the increased HR associated with pregnancy. Experiments were conducted on non-pregnant (NP, 2-3 months old) and pregnant (P, 17-18 gestation days) female CD-1 mice. IKACh was recorded on spontaneously beating SAN cells using the muscarinic agonist carbachol (CCh). Voltage-clamp data showed a reduction in IKACh density during pregnancy, which returned to control values shortly after delivery. The reduction in IKACh was explained by a decrease in protein expression of Kir3.1 channel subunit and the muscarinic type 2 receptor. In agreement with these findings, current-clamp data shows that the MDP of SAN cells from P mice were less hyperpolarized following CCh administration. Surface electrocardiograms (ECGs) recorded on anesthetized mice revealed that the cholinergic antagonist atropine and the selective KACh channel blocker tertiapin-Q increased HR in NP mice and had only a minimal effect on P mice. AP and ECG data also showed that pregnancy is associated with a decrease in beating and heart rate variability, respectively. IKACh function and expression are decreased in the mouse SAN during pregnancy, strongly suggesting that, in addition to other electrical remodeling of the SAN, reduced IKACh also plays an important role in the pregnancy-induced increased HR.

Single-cell ionic current phenotyping explains stem cell-derived cardiomyocyte action potential morphology.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising tool to study arrhythmia-related factors, but the variability of action potential (AP) recordings from these cells limits their use as an in vitro model. In this study, we use recently published brief (10 s), dynamic voltage-clamp (VC) data to provide mechanistic insights into the ionic currents contributing to AP heterogeneity; we call this approach rapid ionic current phenotyping (RICP). Features of this VC data were correlated to AP recordings from the same cells, and we used computational models to generate mechanistic insights into cellular heterogeneity. This analysis uncovered several interesting links between AP morphology and ionic current density: both L-type calcium and sodium currents contribute to upstroke velocity, rapid delayed rectifier K+ current is the main determinant of the maximal diastolic potential, and an outward current in the activation range of slow delayed rectifier K+ is the main determinant of AP duration. Our analysis also identified an outward current in several cells at 6 mV that is not reproduced by iPSC-CM mathematical models but contributes to determining AP duration. RICP can be used to explain how cell-to-cell variability in ionic currents gives rise to AP heterogeneity. Because of its brief duration (10 s) and ease of data interpretation, we recommend the use of RICP for single-cell patch-clamp experiments that include the acquisition of APs.NEW & NOTEWORTHY We present rapid ionic current phenotyping (RICP), a current quantification approach based on an optimized voltage-clamp protocol. The method captures a rich snapshot of the ionic current dynamics, providing quantitative information about multiple currents (e.g., ICa,L, IKr) in the same cell. The protocol helped to identify key ionic determinants of cellular action potential heterogeneity in iPSC-CMs. This included unexpected results, such as the critical role of IKr in establishing the maximum diastolic potential.

Analysis of changes in the action potential morphology of the mouse sinoatrial node true pacemaker cells during ontogenetic development in vitro and in silico.

Maturation of the mouse is accompanied by the increase in heart rate. However, the mechanisms underlying this process remain unclear. We performed an action potentials (APs) recordings in mouse sinoatrial node (SAN) true pacemaker cells and in silico analysis to clarify the mechanisms underlying pre-postnatal period heart rate changes. The APs of true pacemaker cells at different stages had similar configurations and dV/dtmax values. The cycle length, action potential duration (APD90), maximal diastolic potential (MDP), and AP amplitude decreased, meanwhile the velocity of diastolic depolarization (DDR) increased from E12.5 stage to adult. Using a pharmacological approach we found that in SAN true pacemaker cells ivabradine reduces the DDR and the cycle length significantly stronger in E12.5 than in newborn and adult mice, whereas the effects of Ni2+ and nifedipine were significantly stronger in adult mice. Computer simulations further suggested that the density of the hyperpolarization-activated pacemaker сurrent (If) decreased during development, whereas transmembrane and intracellular Ca2+ flows increased. The ontogenetic decrease in IK1 density from E12.5 to adult leads to depolarization of MDP to the voltage range in which calcium currents are activated, thereby shifting the balance from the "membrane-clock" to the "calcium-clock."

The W101C KCNJ5 Mutation Induces Slower Pacing by Constitutively Active GIRK Channels in hiPSC-Derived Cardiomyocytes.

Mutations in the KCNJ5 gene, encoding one of the major subunits of cardiac G-protein-gated inwardly rectifying K+ (GIRK) channels, have been recently linked to inherited forms of sinus node dysfunction. Here, the pathogenic mechanism of the W101C KCNJ5 mutation underlying sinus bradycardia in a patient-derived cellular disease model of sinus node dysfunction (SND) was investigated. A human-induced pluripotent stem cell (hiPSCs) line of a mutation carrier was generated, and CRISPR/Cas9-based gene targeting was used to correct the familial mutation as a control line. Both cell lines were further differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed GIRK channels which underly the acetylcholine-regulated K+ current (IK,ACh). hiPSC-CMs with the W101C KCNJ5 mutation (hiPSCW101C-CM) had a constitutively active IK,ACh under baseline conditions; the application of carbachol was able to increase IK,ACh, further indicating that not all available cardiac GIRK channels were open at baseline. Additionally, hiPSCW101C-CM had a more negative maximal diastolic potential (MDP) and a slower pacing frequency confirming the bradycardic phenotype. Of note, the blockade of the constitutively active GIRK channel with XAF-1407 rescued the phenotype. These results provide further mechanistic insights and may pave the way for the treatment of SND patients with GIRK channel dysfunction.

Abstract P1102: Organic Semiconductor Photomodulation Enhances Maturation Of Pluripotent Stem Cell-derived Cardiomyocytes

Cardiomyocytes differentiated from pluripotent stem cells (PSC-CMs) hold a great potential for the study and the cure of cardiovascular disease; indeed they have been largely used as platform for disease modeling and drugs testing, and represent a promising source of cells for regenerative therapies. However, their immature phenotype represents a major hurdle for their full application; indeed, PSC-CMs differ from adult ones for molecular, metabolic and morpho-functional properties. Recently, a new technology based on optical excitation of organic semiconductors (OS) has been shown to be able to modulate the behavior of many cell types by targeting different cellular processes, as proliferation, angiogenesis and calcium signalling. Here, we adopted a multidisciplinary approach based on morpho-functional, metabolic and transcriptional analyses to study the effect of OS-photoexcitation on PSC-CMs, by the use of a red-light sensitive OS polymer, namely Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta [2,1-b;3,4-b']dithiophene)-alt-4,7(2,1,3-benzothiadiazole)] (PCPDTBT), in the form of a thin film. Our results revealed a significant modulation of markers of maturation in PSC-CMs exposed to PCPDTBT and photomodulation, showing increased size, a shift of their maximal diastolic potential toward more negative values, and augmented Ca 2+ transient amplitude. Moreover, by Scanning ElectroChemical Microscopy (SECM) we found a decrease in glucose uptake and lactate release upon PCPDTBT light stimulation, potentially indicating a switch toward a more adult-like metabolism in stimulated PSC-CMs. Recently obtained RNA sequencing data are in line with these results, and will provide us with hints on the underlying molecular mechanisms. In conclusion, although not definitive, our data are in support of a potential effect of polymer-mediated optical photoexcitation in boosting PSC-CMs toward a more mature phenotype. The demonstration of a relevant effect of OS-photomodulation on PSC-CM maturation and functionality will significantly promote their full applications toward personalized medicine.This research was supported by EU Horizon 2020 FETOPEN- 2018-2020 Programme ‘LION- HEARTED’-GA #828984

Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes

BackgroundAlthough human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking.MethodsiPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs).ResultsWe showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction.ConclusionsOverexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

PITX2 Knockout Induces Key Findings of Electrical Remodeling as Seen in Persistent Atrial Fibrillation.

Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2-/-) in human induced pluripotent stem cell-derived atrial cardiomyocytes. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell-derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell-derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements. PITX2-/- atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2-/- than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2-/- versus isogenic control; P<0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca2+ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P<0.0001), and maximum diastolic potential was more negative (-78.3±3.1 versus -69.7±0.6 mV, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P=0.001), despite normal inward rectifier currents (both IK1 and IK,ACh) and carbachol-induced shortening of action potential duration. Complete PITX2 deficiency in human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels.

Adenosine reduces sinoatrial node cell action potential firing rate by uncoupling its membrane and calcium clocks.

The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A1, A3) that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gβγ signaling that activates IKAch,Ado. Adenosine also inhibits adenylyl cyclase activity via Gαi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to IKAch,Ado activation, adenosine impacts also Ca2+ via Gαi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%, n = 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca2+ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca2+releases (LCRs) (Ca2+-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate Gβγ and IKACh,Ado and to activate Gαi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.

Deciphering the pathogenic role of a variant with uncertain significance for short QT and Brugada syndromes using gene‐edited human‐induced pluripotent stem cell‐derived cardiomyocytes and preclinical drug screening

Deciphering the pathogenic role of a variant with uncertain significance for short QT and Brugada syndromes using gene‐edited human‐induced pluripotent stem cell‐derived cardiomyocytes and preclinical drug screening

Yohimbine Directly Induces Cardiotoxicity on Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Yohimbine is a highly selective and potent α2-adrenoceptor antagonist, which is usually treated as an adjunction for impotence, as well for weight loss and natural bodybuilding aids. However, it was recently reported that Yohimbine causes myocardial injury and controversial results were reported in the setting of cardiac diseases. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to explore electrophysiologic characterization after exposure to Yohimbine. HiPSC-CMs were differentiated by employment of inhibitory Wnt compounds. For analysis of electrophysiological properties, conventional whole-cell patch-clamp recording was used. Specifically, spontaneous action potentials, pacemaker currents (If), sodium (Na+) channel (INa), and calcium (Ca++) channel currents (ICa) were assessed in hiPSC-CMs after exposure to Yohimbine. HiPSC-CMs expressed sarcomeric-α-actinin and MLC2V proteins, as well as exhibited ventricular-like spontaneous action potential waveform. Yohimbine inhibited frequency of hiPSC-CMs spontaneous action potentials and significantly prolonged action potential duration in a dose-dependent manner. In addition, rest potential, threshold potential, amplitude, and maximal diastolic potential were decreased, whereas APD50/APD90 was prolonged. Yohimbine inhibited the amplitude of INa in low doses (IC50 = 14.2μM, n = 5) and inhibited ICa in high doses (IC50 = 139.7μM, n = 5). Whereas Yohimbine did not affect the activation curves, treatment resulted in left shifts in inactivation curves of both Na+ and Ca++ channels. Here, we show that Yohimbine induces direct cardiotoxic effects on spontaneous action potentials of INa and ICa in hiPSC-CMs. Importantly, these effects were not mediated by α2-adrenoceptor signaling. Our results strongly suggest that Yohimbine directly and negatively affects electrophysiological properties of human cardiomyocytes. These findings are highly relevant for potential application of Yohimbine in patients with atrioventricular conduction disorder.

An in silico hiPSC-Derived Cardiomyocyte Model Built With Genetic Algorithm.

The formulation of in silico biophysical models generally requires optimization strategies for reproducing experimentally observed phenomena. In electrophysiological modeling, robust nonlinear regressive methods are often crucial for guaranteeing high fidelity models. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), though nascent, have proven to be useful in cardiac safety pharmacology, regenerative medicine, and in the implementation of patient-specific test benches for investigating inherited cardiac disorders. This study demonstrates the potency of heuristic techniques at formulating biophysical models, with emphasis on a hiPSC-CM model using a novel genetic algorithm (GA) recipe we proposed. The proposed GA protocol was used to develop a hiPSC-CM biophysical computer model by fitting mathematical formulations to experimental data for five ionic currents recorded in hiPSC-CMs. The maximum conductances of the remaining ionic channels were scaled based on recommendations from literature to accurately reproduce the experimentally observed hiPSC-CM action potential (AP) metrics. Near-optimal parameter fitting was achieved for the GA-fitted ionic currents. The resulting model recapitulated experimental AP parameters such as AP durations (APD50, APD75, and APD90), maximum diastolic potential, and frequency of automaticity. The outcome of this work has implications for validating the biophysics of hiPSC-CMs in their use as viable substitutes for human cardiomyocytes, particularly in cardiac safety pharmacology and in the study of inherited cardiac disorders. This study presents a novel GA protocol useful for formulating robust numerical biophysical models. The proposed protocol is used to develop a hiPSC-CM model with implications for cardiac safety pharmacology.

Impaired regulation of heart rate and sinoatrial node function by the parasympathetic nervous system in type 2 diabetic mice

Heart rate (HR) and sinoatrial node (SAN) function are modulated by the autonomic nervous system. HR regulation by the parasympathetic nervous system (PNS) is impaired in diabetes mellitus (DM), which is denoted cardiovascular autonomic neuropathy. Whether blunted PNS effects on HR in type 2 DM are related to impaired responsiveness of the SAN to PNS agonists is unknown. This was investigated in type 2 diabetic db/db mice in vivo and in isolated SAN myocytes. The PNS agonist carbachol (CCh) had a smaller inhibitory effect on HR, while HR recovery time after CCh removal was accelerated in db/db mice. In isolated SAN myocytes CCh reduced spontaneous action potential firing frequency but this effect was reduced in db/db mice due to blunted effects on diastolic depolarization slope and maximum diastolic potential. Impaired effects of CCh occurred due to enhanced desensitization of the acetylcholine-activated K+ current (IKACh) and faster IKACh deactivation. IKACh alterations were reversed by inhibition of regulator of G-protein signaling 4 (RGS4) and by the phospholipid PIP3. SAN expression of RGS4 was increased in db/db mice. Impaired PNS regulation of HR in db/db mice occurs due to reduced responsiveness of SAN myocytes to PNS agonists in association with enhanced RGS4 activity.

Triggered Ca2+ Waves Induce Depolarization of Maximum Diastolic Potential and Action Potential Prolongation in Dog Atrial Myocytes.

We have identified a novel form of abnormal Ca2+ wave activity in normal and failing dog atrial myocytes which occurs during the action potential (AP) and is absent during diastole. The goal of this study was to determine if triggered Ca2+ waves affect cellular electrophysiological properties. Simultaneous recordings of intracellular Ca2+ and APs allowed measurements of maximum diastolic potential and AP duration during triggered calcium waves (TCWs) in isolated dog atrial myocytes. Computer simulations then explored electrophysiological behavior arising from TCWs at the tissue scale. At 3.3 to 5 Hz, TCWs occurred during the AP and often outlasted several AP cycles. Maximum diastolic potential was reduced, and AP duration was significantly prolonged during TCWs. All electrophysiological responses to TCWs were abolished by SEA0400 and ORM10103, indicating that Na-Ca exchange current caused depolarization. The time constant of recovery from inactivation of Ca2+ current was 40 to 70 ms in atrial myocytes (depending on holding potential) so this current could be responsible for AP activation during depolarization induced by TCWs. Modeling studies demonstrated that the characteristic properties of TCWs are potentially arrhythmogenic by promoting both conduction block and reentry arising from the depolarization induced by TCWs. Triggered Ca2+ waves activate inward NCX and dramatically reduce atrial maximum diastolic potential and prolong AP duration, establishing the substrate for reentry which could contribute to the initiation and maintenance of atrial arrhythmias.

Cardiac specific transcription factor Csx/Nkx2.5 regulates transient-outward K+ channel expression in pluripotent P19 cell-derived cardiomyocytes

The homeobox-containing gene Csx/Nkx2.5 codes several cardiac transcription factors and plays a critical role in early cardiogenesis. We investigated the effect of Csx/Nkx2.5 on the expression of cardiac ion channels using P19-derived cardiomyocytes. P19CL6 cells and P19CL6 cells with Csx/Nkx2.5 overexpression (P19CL6-Csx cells) were induced to differentiate into cardiomyocytes by treatment with dimethyl sulfoxide. Action potentials and membrane currents were measured by whole cell patch clamp at different differentiation stage: the early stage (1–5 days after beating had begun) and the late stage (10–15 days after beating). Expression of Csx/Nkx2.5 mRNA was increased as the differentiation stages advanced in both P19CL6 and P19CL6-Csx cells. In action potential configuration, maximal diastolic potentials in P19CL6-Csx cells exhibited more hyperpolarized potential (‒ 64.2 mV) than those in P19CL6 cells (‒ 54.8 mV, p < 0.01) in the early stage. In P19CL6 cells, among 6 different voltage-gated and ligand-operated K+ channels expressed during the early stage, the transient-outward K+ channel was most predominant. By overexpression of Csx/Nkx2.5, developmental decrease in the transient-outward K+ channel was suppressed. Homeobox-containing gene Csx/Nkx2.5 modifies the amount of distinct ionic channels, during differentiation periods, predominantly changing the expression of the transient-outward K+ channel.

The Hyperpolarization-Activated Cyclic-Nucleotide-Gated Channel Blocker Ivabradine Does Not Prevent Arrhythmias in Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited, stressed-provoked ventricular arrhythmia. CPVT is treated by β-adrenergic receptor blockers, Na+ channel inhibitors, sympathetic denervation, or by implanting a defibrillator. We showed recently that blockers of SK4 Ca2+-activated K+ channels depolarize the maximal diastolic potential, reduce the heart rate, and attenuate ventricular arrhythmias in CPVT. The aim of the present study was to examine whether the pacemaker channel inhibitor, ivabradine could demonstrate anti-arrhythmic properties in CPVT like other bradycardic agents used in this disease and to compare them with those of the SK4 channel blocker, TRAM-34. The effects of ivabradine were examined on the arrhythmic beating of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) from CPVT patients, on sinoatrial node (SAN) calcium transients, and on ECG measurements obtained from transgenic mice model of CPVT. Ivabradine did neither prevent the arrhythmic pacing of hiPSC-CMs derived from CPVT patients, nor preclude the aberrant SAN calcium transients. In contrast to TRAM-34, ivabradine was unable to reduce in vivo the ventricular premature complexes and ventricular tachyarrhythmias in transgenic CPVT mice. In conclusion, ivabradine does not exhibit anti-arrhythmic properties in CPVT, which indicates that this blocker cannot be used as a plausible treatment for CPVT ventricular arrhythmias.

HCN2 and TBX3 Reprogram Human-Induced Pluripotent Stem Cells-Derived Cardiomyocytes into Pacemaker-Like Cells.

TBX3 reprograms cardiac myocytes into cells that possess sinoatrial node phenotype, but no specific funny current (If) was detected. We explore whether overexpression of TBX3 alone or combined with HCN2 can reprogram human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) into pacemaker-like cells. HiPSC-CMs were transfected with TBX3 and/or HCN2 in this study. Expression analysis showed that overexpression of TBX3 induces a reduced reduction expression profile of working cardiomyocytes into that of pacemaker cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and electrophysiological analyses showed a reduced expression of connexins subunits (CX40, CX43), the sodium current (SCN5A, INa), the inward rectified potassium channels (Kir2.1, IK1), and an increased expression of connexins subunits (CX30.2, CX45). No If was detected. The reduction of IK1 resulted in a more depolarized maximum diastolic potential together with an expression of If (generated by HCN2), which they work in synergy to generate spontaneous diastolic depolarization that was the most typical characteristic of pacemaker cells. In conclusion, overexpression of TBX3 and HCN2 could reprogram hiPSC-CMs into pacemaker-like cells. The ability to enable diastolic depolarization formation provides a new strategy for the construction of a biological pacemaker.

Chronic heart failure increases negative chronotropic effects of adenosine in canine sinoatrial cells via A1R stimulation and GIRK-mediated IKado

AimsBradycardia contributes to tachy-brady arrhythmias or sinus arrest during heart failure (HF). Sinoatrial node (SAN) adenosine A1 receptors (ADO A1Rs) are upregulated in HF, and adenosine is known to exert negative chronotropic effects on the SAN. Here, we investigated the role of A1R signaling at physiologically relevant ADO concentrations on HF SAN pacemaker cells. Main methodsDogs with tachypacing-induced chronic HF and normal controls (CTL) were studied. SAN tissue was collected for A1R and GIRK mRNA quantification. SAN cells were isolated for perforated patch clamp recordings and firing rate (bpm), slope of slow diastolic depolarization (SDD), and maximum diastolic potential (MDP) were measured. Action potentials (APs) and currents were recorded before and after addition of 1 and 10 μM ADO. To assess contributions of A1R and G protein-coupled Inward Rectifier Potassium Current (GIRK) to ADO effects, APs were measured after the addition of DPCPX (selective A1R antagonist) or TPQ (selective GIRK blocker). Key findingsA1R and GIRK mRNA expression were significantly increased in HF. In addition, ADO induced greater rate slowing and membrane hyperpolarization in HF vs CTL (p < 0.05). DPCPX prevented ADO-induced rate slowing in CTL and HF cells. The ADO-induced inward rectifying current, IKado, was observed significantly more frequently in HF than in CTL. TPQ prevented ADO-induced rate slowing in HF. SignificanceAn increase in A1R and GIRK expression enhances IKAdo, causing hyperpolarization, and subsequent negative chronotropic effects in canine chronic HF at relevant [ADO]. GIRK blockade may be a useful strategy to mitigate bradycardia in HF.

Long Term Perinatal Deltamethrin Exposure Alters Electrophysiological Properties of Embryonic Ventricular Cardiomyocyte.

Increased use of pyrethroids and the exposure to pyrethroids for pregnant women and children have raised the concerns over the potential effect of pyrethroids on developmental cardiotoxicity and other abnormalities. The purpose of this study was to investigate whether long term perinatal deltamethrin exposure altered embryonic cardiac electrophysiology in mice. Pregnant mice were administered with 0 or 3 mg/kg of deltamethrin by gavage daily from gestational day (gd) 10.5 to gd 17. 5. Whole cell patch-clamp technique was used in electrophysiological study, and real time RT-PCR was applied to analyze the molecular changes for the electrophysiological properties. Deltamethrin exposure resulted in increased mortality of pregnant mice and decreased viability of embryos. Moreover, deltamethrin slowed the maximum depolarization velocity (Vmax), prolonged the action potential duration (APD) and depolarized the maximum diastolic potential (MDP) of embryonic cardiomyocytes. Additionally, perinatal deltamethrin exposure decreased the mRNA expression of Na+ channel regulatory subunit Navβ1, inward rectifier K+ channel subunit Kir2.1, and delayed rectifier K+ channel subunit MERG while the L-type Ca2+ channel subunit, Cav1.2 expression was increased. On the contrary, deltamethrin administration did not significantly alter the regulation of β-adrenergic or muscarinic receptor on embryonic cardiomyocytes. In conclusion, deltamethrin exposure at perinatal stage significantly alters mRNA expression of embryonic cardiac ion channels and therefore influences embryonic cardiac electrophysiological properties. This highlights the need to understand the persistent effects of pyrethroid exposure on cardiac function during embryonic development due to potential for cardiac arrhythmogenicity.

Why and When Your Next Heartbeat Will Occur

Cardiac pacemaker cell automaticity is regulated by molecular self-organization within a coupled system of a chemical oscillators (“Ca2+ clock”), comprised of Ca2+ and other ion cycling molecules, and ion current oscillators, comprised of an ensemble of electrogenic molecules (surface membrane or “M clock”). Both Ca2+ and M clock molecules are activated by Ca2+ and voltage cues, that also oscillate within an AP cycle. CAMP, cAMP-mediated PKA and CAMKII-dependent phosphorylation modulate the availability of clock molecules to respond these activation cues. A crucial Ca2+ clock member, the sarcoplasmic reticulum (SR), functions as a Ca2+ capacitor, pumping Ca2+ via SERCA2 and releasing Ca via ryanodine receptors, (RyR). Spontaneous RyR activation becomes evident near the time at which maximum diastolic membrane potential (MDP) is achieved, and generates sub-sarcolemmel local Ca2+ releases that organize to activate INCX. The resultant induction of slow membrane depolarization activates low-voltage-activated Ca2+ channels that continues the feed-forward driven self-organization of coupled-clock functions to further accelerate the rate of diastolic VM depolarization. When the extent of depolarization of the system reaches a critical level, L-Type Ca2+ channels activate, effecting an abrupt and marked acceleration of VM depolarization that forms the rapid upstroke of the spontaneous AP. Concurrent influx via Ca replenishes intracellular Ca2+ to balance Ca efflux via NCX; VM depolarization and an increase in intracellular Ca2+ during the AP activate a family of K channels, leading to VM repolarization, which, in turn increases availability of NCX and HCN channel molecules for subsequent reactivation. Following re-achievement of the MDP, self-organization of functions within the coupled-clock system is reiterated to drive ensuing AP cycles. Thus, your heartbeat occurs when self-organization of a system of chemical and current oscillators that slowly depolarized VM achieves criticality, i.e. the VM required to launch the next AP.

Cardiac Kir2.1 and NaV1.5 Channels Traffic Together to the Sarcolemma to Control Excitability.

In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. The objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. The NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.