Spontaneous Diastolic Depolarization Research Articles

A synthetic cardiac episode generator for explainable, pathology based action potential using heuristic polynomial signatures

Synthetic generation of biophysical signals caters to diverse applications of test vectors generation, exploration, data augmentation and machine learning training. Arrhythmiator, our contribution is a Python framework to generate synthetically, action potential for explain-ability and explore-ability of pathology (Arrhythmia) influenced cardiac episodes. In this work, we address the problem of large cardiac data-sets need, for deep learning models which are hindered by privacy laws by synthetic generation. We have addressed the problem of explainable computations, multi-scale, by virtue of novel stack sliced polynomial signatures, piece-wise linear and application context driven fitting/filtering in algorithmic framework. To account the problem of lack of standardised pathology specifications we developed PDL (Pathology descriptive language) portal which can be customised. We also addressed the challenge of high turnaround development cycle, inter-operate-ability computation issue by developing a single unified Python framework. To address computational costs, we leverage domain-specific heuristics through sliced state space control algorithms and a constant temporal resolution. We demonstrate the utility of our framework by generating two cases of Tachycardia, one influenced by sodium pump inhibition of spontaneous diastolic depolarisation (SDD) slope and other by ischaemic episode causing maximum diastolic depolarisation (MDP) shift. The concept of the pacemaker hierarchy is encompassed by developing pathology explainable computation as a structure (PECS). We showcase the flexibility and explore-ability of our framework through ease of filter connect profile plug-ins for action potential states upstroke and plateau. We validated the generated waveforms by developing trace and debug algorithms and a reference deviation score which marks the pathology influence. The validator module is a vectored attribute of action potential based on state space control algorithms.

The Action Potential Clamp Technique as a Tool for Risk Stratification of Sinus Bradycardia Due to Loss-of-Function Mutations in HCN4: An In Silico Exploration Based on In Vitro and In Vivo Data.

These days, in vitro functional analysis of gene variants is becoming increasingly important for risk stratification of cardiac ion channelopathies. So far, such risk stratification has been applied to SCN5A, KCNQ1, and KCNH2 gene variants associated with Brugada syndrome and long QT syndrome types 1 and 2, respectively, but risk stratification of HCN4 gene variants related to sick sinus syndrome has not yet been performed. HCN4 is the gene responsible for the hyperpolarization-activated 'funny' current If, which is an important modulator of the spontaneous diastolic depolarization underlying the sinus node pacemaker activity. In the present study, we carried out a risk classification assay on those loss-of-function mutations in HCN4 for which in vivo as well as in vitro data have been published. We used the in vitro data to compute the charge carried by If (Qf) during the diastolic depolarization phase of a prerecorded human sinus node action potential waveform and assessed the extent to which this Qf predicts (1) the beating rate of the comprehensive Fabbri-Severi model of a human sinus node cell with mutation-induced changes in If and (2) the heart rate observed in patients carrying the associated mutation in HCN4. The beating rate of the model cell showed a very strong correlation with Qf from the simulated action potential clamp experiments (R2 = 0.95 under vagal tone). The clinically observed minimum or resting heart rates showed a strong correlation with Qf (R2 = 0.73 and R2 = 0.71, respectively). While a translational perspective remains to be seen, we conclude that action potential clamp on transfected cells, without the need for further voltage clamp experiments and data analysis to determine individual biophysical parameters of If, is a promising tool for risk stratification of sinus bradycardia due to loss-of-function mutations in HCN4. In combination with an If blocker, this tool may also prove useful when applied to human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from mutation carriers and non-carriers.

Insulin signaling is critical for sinoatrial node maintenance and function

Insulin and insulin-like growth factor 1 (IGF-1) signaling regulate cellular growth and glucose metabolism in the myocardium. However, their physiological role in the cells of the cardiac conduction system has never been explored. Therefore, we sought to determine the spatiotemporal function of insulin/IGF-1 receptors in the sinoatrial node (SAN). We generated cardiac conduction cell-specific inducible IGF-1 receptor (IGF-1R) knockout (KO) (CSIGF1RKO), insulin receptor (IR) KO (CSIRKO), and IR/IGF-1R double-KO (CSDIRKO) mice and evaluated their phenotypes. Telemetric electrocardiography revealed regular sinus rhythm in CSIGF1RKO mice, indicating that IGF-1R is dispensable for normal pacemaking. In contrast, CSIRKO and CSDIRKO mice exhibited profound sinus bradycardia. CSDIRKO mice showed typical sinus node dysfunction characterized by junctional rhythm and sinus pauses on electrocardiography. Interestingly, the lack of an insulin receptor in the SAN cells of CSIRKO and CSDIRKO mice caused sinus nodal fibrosis. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) protein expression significantly decreased in the CSIRKO and CSDIRKO mice relative to the controls. A patch-clamp study of the SAN cells of CSIRKO mice revealed a significant decrease in the funny current, which is responsible for spontaneous diastolic depolarization in the SAN. This result suggested that insulin receptor loss reduces the heart rate via downregulation of the HCN4 channel. Additionally, HCN1 expression was decreased in CSDIRKO mice, explaining their sinus node dysfunction. Our results reveal a previously unrecognized role of insulin/IGF-1 signaling in sinus node structural maintenance and pacemaker function.

The paradigm shift: Heartbeat initiation without "the pacemaker cell".

The current dogma about the heartbeat origin is based on "the pacemaker cell," a specialized cell residing in the sinoatrial node (SAN) that exhibits spontaneous diastolic depolarization triggering rhythmic action potentials (APs). Recent high-resolution imaging, however, demonstrated that Ca signals and APs in the SAN are heterogeneous, with many cells generating APs of different rates and rhythms or even remaining non-firing (dormant cells), i.e., generating only subthreshold signals. Here we numerically tested a hypothesis that a community of dormant cells can generate normal automaticity, i.e., "the pacemaker cell" is not required to initiate rhythmic cardiac impulses. Our model includes 1) non-excitable cells generating oscillatory local Ca releases and 2) an excitable cell lacking automaticity. While each cell in isolation was not "the pacemaker cell", the cell system generated rhythmic APs: The subthreshold signals of non-excitable cells were transformed into respective membrane potential oscillations via electrogenic Na/Ca exchange and further transferred and integrated (computed) by the excitable cells to reach its AP threshold, generating rhythmic pacemaking. Cardiac impulse is an emergent property of the SAN cellular network and can be initiated by cells lacking intrinsic automaticity. Cell heterogeneity, weak coupling, subthreshold signals, and their summation are critical properties of the new pacemaker mechanism, i.e., cardiac pacemaker can operate via a signaling process basically similar to that of "temporal summation" happening in a neuron with input from multiple presynaptic cells. The new mechanism, however, does not refute the classical pacemaker cell-based mechanism: both mechanisms can co-exist and interact within SAN tissue.

Mama and papa of the early spontaneous electrical activity of the embryo sinoatrial node

In adult mammalian hearts the spontaneous diastolic depolarization of the sinoatrial node (SAN) cells is generated by two interacting systems, a so‐called “membrane‐clock” and “calcium‐clock”. Hyperpolarization‐activated pacemaker current (If) plays a key role in the “membrane‐clock”, while the “calcium‐clock” is based on rhythmic spontaneous Ca2+ release from sarcoplasmic reticulum via ryanodine receptors (RyRs). These complementing systems provide regular pacemaking even in case of damage of one of them. The aim of this study was to evaluate a role of If current and ryanodine receptors in the electrical activity of the developing mouse SAN.MethodsWe used embryos of mice, aged of E9.5, E12.5 and newborn to perform our experiments. After release from the amniotic membrane, the embryos were transferred with a pipette to a clean Petri dish with oxygenated physiological solution containing (in mM/L) 140 NaCl, 5.4 KCl, 0.1 CaCl2, 0.33 NaH2PO4, 1 MgCl2, 10 HEPES, 10 Glucose, (pH 7.38) at room temperature. To record spontaneous action potential (AP) we used conventional microelectodes technique at 37◦C. Differences were considered significant at p < 0.05ResultsMicroelectrode mapping at E9.5 revealed that the SAN cells are located in the inflow tract of the venous pole. At E12 the SAN cells are located in the cavo‐auricular junction. In neonatal mice, SAN cells were detected at intercaval area near to the crista terminalis. At E9.5, in 6 experiments, on 25‐ 30 minute exposure of 3 mkM ivabradine (Iva) an irregularity in AP generation by SAN cells was occurred. The same effects were obtained in 4 experiments on 20‐30 minutes exposure of 3 mkM ryanodine. At E12.5 exposure of Iva 3 mkM for 30 minutes reduced the pacemaker firing rate by 34% (p < 0.05) but did not break the rhythmic generation. In contrast, the exposure of 3 mkM ryanodine for 30 minutes, in 4 experiments, still caused an irregularity in AP generation by SAN cells. In neonatal isolated SAN preparations, exposure of Iva 3 mkM and 3 mkM ryanodine for 30 minutes reduced the pacemaker firing rate equally (on 32%; p < 0.05). However, co‐application of Iva and ryanodine induced an irregularity in AP generation by SAN cells (Figure).Numerical experiments within the framework of mouse SANC model (Kharche, AJP, 11) adopted for embryonic cells (Markov, IEEE, 21) have shown the decrease in the frequency of the AP generation with decreasing of the “funny” currents permeability caused by Iva action.The supposed complete RyRs opening, caused by a relatively low ryanodine concentration, led to a decrease of the AP generation frequency, but not to the rhythm disturbances. However a decrease in RyRs permeability by an order of magnitude led to the appearance of “bursts‐like” irregularity in AP generation.ConclusionThus, the membrane‐clock and the Ca2+‐clock mechanisms is critical for spontaneous electrical activity E9.5 mouse embryos, whereas the Ca2+‐clock mechanisms is critical for increase rate AP generation in development SAN cells.

The effective use of blebbistatin to study the action potential of cardiac pacemaker cells of zebrafish (Danio rerio) during incremental warming.

Blebbistatin potently inhibits actin-myosin interaction, preventing contractile activity of excitable cells including cardiac myocytes, despite electrical excitation of an action potential (AP). We collected intracellular microelectrode recordings of pacemaker cells located in the sinoatrial region (SAR) of the zebrafish heart at room temperature and during acute warming to investigate whether or not blebbistatin inhibition of contraction significantly alters pacemaker cell electrophysiology. Changes were evaluated based on 16 variables that characterized the AP waveform. None of these AP variables nor the spontaneous heart rate were significantly modified with the application of 10 μM blebbistatin when recordings were made at room temperature. Compared with the control group, the blebbistatin-treated group showed minor changes in the rate of spontaneous diastolic depolarization (P = 0.027) and the 50% and 80% repolarization (P = 0.008 and 0.010, respectively) in the 26°C–29°C temperature bin, but not at higher temperatures. These findings suggest that blebbistatin is an effective excitation-contraction uncoupler that does not appreciably affect APs generated in pacemaking cells of the SAR and can, therefore, be used in zebrafish cardiac studies.

Identification of Key Small Non-Coding MicroRNAs Controlling Pacemaker Mechanisms in the Human Sinus Node.

BackgroundThe sinus node (SN) is the primary pacemaker of the heart. SN myocytes possess distinctive action potential morphology with spontaneous diastolic depolarization because of a unique expression of ion channels and Ca2+‐handling proteins. MicroRNAs (miRs) inhibit gene expression. The role of miRs in controlling the expression of genes responsible for human SN pacemaking and conduction has not been explored. The aim of this study was to determine miR expression profile of the human SN as compared with that of non‐pacemaker atrial muscle.Methods and ResultsSN and atrial muscle biopsies were obtained from donor or post‐mortem hearts (n=10), histology/immunolabeling were used to characterize the tissues, TaqMan Human MicroRNA Arrays were used to measure 754 miRs, Ingenuity Pathway Analysis was used to identify miRs controlling SN pacemaker gene expression. Eighteen miRs were significantly more and 48 significantly less abundant in the SN than atrial muscle. The most interesting miR was miR‐486‐3p predicted to inhibit expression of pacemaking channels: HCN1 (hyperpolarization‐activated cyclic nucleotide‐gated 1), HCN4, voltage‐gated calcium channel (Cav)1.3, and Cav3.1. A luciferase reporter gene assay confirmed that miR‐486‐3p can control HCN4 expression via its 3′ untranslated region. In ex vivo SN preparations, transfection with miR‐486‐3p reduced the beating rate by ≈35±5% (P<0.05) and HCN4 expression (P<0.05).ConclusionsThe human SN possesses a unique pattern of expression of miRs predicted to target functionally important genes. miR‐486‐3p has an important role in SN pacemaker activity by targeting HCN4, making it a potential target for therapeutic treatment of SN disease such as sinus tachycardia.

Heart rhythm and conduction disorders as manifestations of cardiotoxicity of anticancer treatment: myth or reality?

Oncologic diseases are currently one of the leading causes of death. Modern anticancer therapy allows preserving life and social adaptation of cancer patients for many years. However, the use of anticancer drugs is limited due to their adverse and, in some cases, severe cardiotoxic effects such as coronary artery disease, toxic cardiomyopathy, chronic heart failure, arterial hypertension, and others. Heart rhythm and conduction disorders occur, on average, in 16–36% chemotherapy patients and atrial fibrillation is one of the most common arrhythmogenic manifestations of cardiotoxicity. Anthracyclines, alkylating agents, and monoclonal antibodies disrupt the ion pumps function, contribute to the excess release of calcium from the sarcoplasmic reticulum, cause more rapid development of spontaneous diastolic depolarization, and ultimately provoke the occurrence of atrial fibrillation. Some chemotherapy drugs, in particular, anthracyclines, tyrosine kinase inhibitors, and histone deacetylases disrupt the functioning of potassium channels, which leads to an increase in the action potential and prolongation of QT interval. Data on the effects of other classes of chemotherapy drugs on the heart conduction system are scarce and contradictory. Heart rhythm and conduction disorders caused by chemotherapy can lead to a dose reduction or discontinuation of anticancer drugs and require careful monitoring and a joint approach by doctors of several specialties in the management of these patients.

Cardiac Overexpression of Adenylyl Cyclase Type VIII Augments Function of the Coupled Oscillatory System and Action Potential Firing Rate of Sinoatrial Nodal Cells

We have shown recently that the intrinsic heart rate (IHR) in vivo (measured during double autonomic blockade) in mice with cardiac-specific overexpression of adenylyl cyclase type VIII(AC VIII) is 30% higher than in wild type littermates (WT). We studied single sinoatrial nodal cell (SANC)to elucidate how AC VIII overexpression affects a coupled-clock system that drives spontaneous action potential (AP) firing in these cells. Spontaneous local Ca2+ releases (LCRs, confocal line-scan microscopy), generated via spontaneous local activation of ryanodine receptors (RyR) of the intracellular Ca2+ oscillator-sarcoplasmic reticulum (SR), occurred earlier in diastole in TgAC8 SANC, accelerating the diastolic AP ignition process that governs the rate of spontaneous diastolic depolarization. This led to an increased rate of AP generation by the ensemble of cell surface membrane ionic current oscillators. In permeabilized (saponin) SANC, Ca2+ signal of individual LCR's, and that of the LCR ensemble were increased in TgAC8 vs WT. Double immunostaining of phosphorylated and total RyR revealed increased phosphorylation of RyR at Ser2808 and Ser2030, and of PLB at Ser16 sites in TgAC8 vs WT SANC. RNA-seq analysis showed an upregulation of several PDEs (including PDE1a, PDE3a, PDE4b, PDE4c, PDE4d, PDE7b and PDE8a), ion channels (Cacna1c, Cacnb2, Kcnb1, Kcnd3, Kcnn1, Kcnq1, Scn1b, Scn4a; Kcnj3 however was downregulated), ATPase Na+/K+ transporters (ATP1a1 and ATP2b1) and calcium cycling proteins (STIM1 and RYR2) were upregulated in TgAC8 vs WT (notably, SIN and PLM were downregulated in TgAC8 vs WT). Thus, cardiac-specific overexpression of AC VIII in SANC increases IHR in vivo via impacting upon both transcriptional and posttranslational proteins modification of the Ca2+-cAMP-PKA-dependent components of the coupled-oscillator system intrinsic to SANC that controls their spontaneous AP firing rate.

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.

Hypokalaemia: Clinical Signifi cance and Role in the Mechanisms Underlying Cardiac Arrhythmogenesis

Hypokalaemia is the most common electrolyte abnormality seen in cardiac patients, which represents a side effect of diuretic therapy or results from the overactivation of the renin-angiotensinaldosterone system and the sympathetic nervous system in heart failure. Hypokalaemia is known to contribute to an increased risk of ventricular arrhythmias whose mechanism is based on the dynamic interplay of the provocative trigger and the vulnerable substrate. A premature ectopic impulse acts as a provocative trigger, whereas the vulnerable substrate is created by the structural and functional myocardial changes that favour the impulse circulation within a closed conducting pathway, thus perpetuating the re-entrant activation. The premature ectopic impulse that serves as the initiating event for arrhythmia can be generated due to abnormal automaticity or triggered activity. Hypokalaemia facilitates abnormal automaticity by increasing the rate of spontaneous diastolic depolarisation in Purkinje fi bres, which then start to exhibit pacemaker activity that interferes with the regular activations set by the sinoatrial node. The triggered activity is attributable to the early and delayed afterdepolarisations in cardiac myocytes. The early afterdepolarisations are typically precipitated by an excessive lengthening of the cardiac action potential duration that results from the inhibition of the repolarising K+ currents in the setting of hypokalaemia. The delayed afterdepolarisations are related to calcium overload in cardiac cells, which is provoked by hypokalaemia via inhibition of the Na+-K+ ATPase on myocyte sarcolemma. This translates to the increased intracellular Na+ levels, which in turn activate the reverse mode of the Na+-Ca2+ exchange, leading to increased cytosolic Ca2+ concentration. With regard to creating a vulnerable substrate for re-entry, hypokalaemia is known to induce a non-uniform increase in the action potential duration in different myocardial regions, which amplifi es spatial heterogeneities in the recovery of ventricular excitability during the fi nal repolarisation phase. This sets favourable conditions for a unidirectional conduction block upon premature ectopic activation, which initiates the impulse propagation around a small area of refractory cardiac tissue. In addition, hypokalaemia slows cardiac conduction by inducing hyperpolarisation of the myocyte sarcolemma that results in markedly increased excitation threshold. The induced conduction delay in the re-entrant circuit then allows suffi cient time for recovery from refractoriness in the cardiac cells ahead of the excitation wavefront, which sustains re-entrant activation. The risk of ventricular tachyarrhythmia becomes particularly high when hypokalaemia is combined with the administration of cardiac glycosides or class III antiarrhythmic agents.

The Zebrafish Heart: an Archetype for Neurocardiology

The intracardiac nervous system (ICNS) is the final common pathway for reflex control of cardiac output by the autonomic nervous system (ANS). The normal operation of this system is key to supporting cardiovascular homeostasis, and ANS dysfunction or remodelling can contribute to the development of multiple cardiopathies. Thus the role of the ICNS in cardiac control is essential, however our current understanding of this role is incomplete. The majority of work on the ICNS to date has been in mammalian models, but because the neural elements comprising this network are embedded in cardiac tissues, visualization and experimental access to the entire network for integrative studies of neuronal function has been limiting. To address these issues, we have developed the zebrafish, a cyprinid teleost, as an alternate model for cardiac anatomical and neurophysiological studies. In this ancestral vertebrate the two‐chambered heart, although anatomically distinct from the mammalian four chamber arrangement, is dually innervated by sympathetic and parasympathetic limbs of the ANS and has a well‐developed ICNS which is functional in spontaneously beating isolated preparations. Activation of extrinsic cardiac nerves evokes tachycardia (via B2‐adrenoceptors) and bradycardia (M2 muscarinic receptors). Thus the essential features of ANS control of the heart are conserved across the vertebrates. We have mapped the general innervation patterns of the sinoatrial valve region (SAR), atrium and ventricle. A ganglionated plexus in the SAR surrounds the valve and contains most of the intracardiac neuronal somata; the majority is cholinergic with the remainder adrenergic, nitrergic and serotonergic. Furthermore, peptidergic axon terminals contact plexal somata. Optical mapping of the isolated heart confirms the site of origin of normal primary pacemaker activity within the SAR, where cells in the base of the valve leaflets express Islet‐1 (Isl‐1) and hyperpolarization‐activated, cyclic nucleotide‐gated channel 4 (HCN4), known markers for mammalian pacemaker cells. In addition, intracellular recordings from these cells display spontaneous diastolic membrane depolarization patterns typical of pacemaker cells. Extrinsic cardiac nerve stimulation, in addition to evoking bradycardia, can displace the pacemaker site to a secondary pacemaker in the atrioventricular region (AVR), where a population of cells also expresses Isl‐1 and HCN4. A small ganglion containing cholinergic somata is present in the AVR, suggesting local neural control of atrioventricular pacemaker activity. We have thus established the zebrafish heart as a viable model for studies of integrative neural control of pacemaker function. In particular, this model provides a platform to use optogenetic techniques to target intracardiac neurons and pacemaker cells, in combination with traditional electrophysiological methods, to perform detailed analyses of the intracardiac neuronal circuitry involved in cardiac control. This work will provide a solid foundation for further studies of ANS‐related cardiac pathophysiologies in zebrafish analogs of human cardiac disease states.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (FMS, TAQ, RPC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ionic and cellular mechanisms underlying TBX5/PITX2 insufficiency-induced atrial fibrillation: Insights from mathematical models of human atrial cells

Transcription factors TBX5 and PITX2 involve in the regulation of gene expression of ion channels and are closely associated with atrial fibrillation (AF), the most common cardiac arrhythmia in developed countries. The exact cellular and molecular mechanisms underlying the increased susceptibility to AF in patients with TBX5/PITX2 insufficiency remain unclear. In this study, we have developed and validated a novel human left atrial cellular model (TPA) based on the ten Tusscher-Panfilov ventricular cell model to systematically investigate how electrical remodeling induced by TBX5/PITX2 insufficiency leads to AF. Using our TPA model, we have demonstrated that spontaneous diastolic depolarization observed in atrial myocytes with TBX5-deletion can be explained by altered intracellular calcium handling and suppression of inward-rectifier potassium current (IK1). Additionally, our computer simulation results shed new light on the novel cellular mechanism underlying AF by indicating that the imbalance between suppressed outward current IK1 and increased inward sodium-calcium exchanger current (INCX) resulted from SR calcium leak leads to spontaneous depolarizations. Furthermore, our simulation results suggest that these arrhythmogenic triggers can be potentially suppressed by inhibiting sarcoplasmic reticulum (SR) calcium leak and reversing remodeled IK1. More importantly, this study has clinically significant implications on the drugs used for maintaining SR calcium homeostasis, whereby drugs such as dantrolene may confer significant improvement for the treatment of AF patients with TBX5/PITX2 insufficiency.

Action Potential Heterogeneity in Murine Sinoatrial Node Myocytes

Sinoatrial node myocytes (SAMs) act as cardiac pacemakers by generating spontaneous action potentials (APs). SAMs are characterized by the expression of the “funny current” (If) that contributes to the spontaneous diastolic depolarization phase of the sinoatrial AP. SAMs exhibit a large heterogeneity of morphological and electrophysiological properties. However, limited information is available about the intrinsic variability in AP waveforms in SAMs. In this study, we co-recorded APs and If from acutely isolated SAMs from mice using voltage and current clamp recordings in the same cells. We refined the definitions of commonly used AP waveform parameters to document the heterogeneity of APs in both perforated patch (PP) and whole-cell (WC) recordings. These definitions were then implemented in an open-source data analysis program (“paramAP”) written in Python 3 using NumPy, SciPy, and Matplotlib. ParamAP was utilized to characterize APs from more than 100 SAMs. Correlation analysis was used to evaluate relationships among AP waveform parameters and to identify parameters that are strongly correlated with If and AP firing rate. Furthermore, we document time-dependent changes in AP waveform parameters during both PP and WC recordings. In summary, paramAP is a powerful tool to help standardize AP waveform analysis and parameter definitions in murine SAMs. Our data suggest that commonly-held assumptions about sinoatrial node APs should be extended to accommodate a wider range of AP waveforms. In addition, the time-dependent changes in AP waveform parameters should be considered when comparing data from different studies.

Biological pacemakers: Concepts and techniques.

The sinoatrial (SA) node is the dominant pacemaker of the heart which initiates the process of impulse generation in the cardiac tissue, thereby defining the rate and rhythm of cardiac contraction. The automaticity of the conduction cells in the SA node is due to ion channels which are inter-linked by molecular, histological and electrophysiological mechanisms causing spontaneous diastolic depolarization and generation of an impulse. The SA nodal action potentials are then transmitted to the ventricles by electrical coupling of the myocytes in different areas of the heart. Regulatory pathways overseeing cardiac impulse generation and conduction provide effective and safe pacing, and help maintain the rate according to the physiological demands of the individual's body. Failure of physiological pacing due to any pathology in the SA or atrioventricular node necessitates implantation of a permanent pacemaker. Implantable pacemakers, despite technological advances, are not without practical limitations including a defined battery life leading to lead and/or generator replacement at periodic intervals, vascular complications, occasional component failure, electronic interference from external/ internal sources, e.g. myopotentials, electromechanical interference, etc., inadequate or incomplete physiological rate response to autonomic influences (devices have certain algorithms to address these issues) and most importantly the risk of infection. A biological pacemaker is therefore emerging as a promising technique to counter these challenges.

Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells.

The ‘funny’ current, also known as the If current, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The If current is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) gene. The functional If channel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouse HCN4 (mHCN4). Biomimetic studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, whether EPCS is able to influence the properties of the If current reconstructed in mHCN4-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the If current reconstructed in mHCN4-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-mHCN4-GFP. Following transfection, these cells were divided into two groups: mHCN4-transfected cMSCs (group A), and mHCN4-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the If current was recorded, and group A cMSCs showed significant time and voltage dependencies and sensitivity to extracellular Cs+. The half-maximal activation (V1/2) value was −101.2±4.6 mV and the time constant of activation was 324±41 msec under −160 mV. In the group B cells the If current increased obviously and activation curve moved to right. The absolute value of V1/2 increased significantly to −92.4±4.8 mV (P<0.05), and the time constant of activation diminished under the same command voltage (251±44 vs. 324±41, P<0.05). In addition, the mRNA and protein expression levels of HCN4, connexin 43 (Cx43) and Cx45 were upregulated in group B compared with group A, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Transmission electron micrographs also confirmed the increased gap junctions in group B. Collectively, these results indicated that reconstructed If channels may have a positive regulatory role in EPCS induction. The cMSCs transfected with mHCN4 induced by EPCS may be a source of effective biological pacemaker cells.

The importance of Ca(2+)-dependent mechanisms for the initiation of the heartbeat.

Mechanisms underlying pacemaker activity in the sinus node remain controversial, with some ascribing a dominant role to timing events in the surface membrane (“membrane clock”) and others to uptake and release of calcium from the sarcoplasmic reticulum (SR) (“calcium clock”). Here we discuss recent evidence on mechanisms underlying pacemaker activity with a particular emphasis on the many roles of calcium. There are particular areas of controversy concerning the contribution of calcium spark-like events and the importance of I(f) to spontaneous diastolic depolarisation, though it will be suggested that neither of these is essential for pacemaking. Sodium-calcium exchange (NCX) is most often considered in the context of mediating membrane depolarisation after spark-like events. We present evidence for a broader role of this electrogenic exchanger which need not always depend upon these spark-like events. Short (milliseconds or seconds) and long (minutes) term influences of calcium are discussed including direct regulation of ion channels and NCX, and control of the activity of calcium-dependent enzymes (including CaMKII, AC1, and AC8). The balance between the many contributory factors to pacemaker activity may well alter with experimental and clinical conditions, and potentially redundant mechanisms are desirable to ensure the regular spontaneous heart rate that is essential for life. This review presents evidence that calcium is central to the normal control of pacemaking across a range of temporal scales and seeks to broaden the accepted description of the “calcium clock” to cover these important influences.

Restoring the resting potential brings maturity to induced stem cell–derived human cardiac myocytes

Reprogramming of mature human cells to produce the equivalent of embryonic stem cells offers exciting possibilities for therapeutic tissue repair and artificial organs. A more immediate benefit is the possibility of testing potential new drugs and other therapies in human cells of any type that can easily be created in vitro without the use of human blastocysts and with a minimum of invasive procedures in patients. Fundamental research in embryology lead to the identification of growth factors that could be used to force cultured embryonic stem cells to differentiate into mature adult cells including atrial and ventricular cardiac myocytes. This work grew out of the initial approach of allowing the harvesting and subculturing regions of embryos growing in vitro that showed spontaneous beating. A spectacular advance came in 2006 when Takahashi and Yamanaka 1 showed that mouse or human adult fibroblasts could revert to pluripotent stem cells when exposed to 4 factors: Oct3/4, Sox2, c-Myc, and Klf4 under embryonic stem cell culture conditions. In 2012, Yamanaka received the Nobel Prize in Medicine or Physiology for this work, which he shared with Gurdon, who showed in l962 that the specialization of mature cells can be reversed by transplantation of a frog intestinal cell nucleus into a frog egg. By the time of this award, substantial work by others had shown what additional factors or conditions are needed to cause the so-called induced pluripotent stem (IPS) cells to differentiate into adult cardiac myocytes or other cellular components including vascular and endothelial cells. 2 In the heart, embryonic development and tissue differentiation is accompanied by important changes in the electrical activity that alter the cardiac action potential and underlying ionic currents. While the genome must contain sequence information for all ion channels in the heart and other tissues, the expression of the ion channels varies with the stage of embryonic development and with anatomic location. In the embryonic hearts of vertebrates, the action potential of the immature ventricle is similar to that found in the adult sinoatrial node. Specifically, there is spontaneous diastolic depolarization (the pacemaker potential) with a relatively low maximum diastolic potential in immature atrial and ventricular cells, which disappears during development. The upstroke of the action potential is mediated by calcium channels in the early embryo and by sodium channels in the late embryo and adult. The action potential upstroke becomes increasingly rapid during embryonic development, as demonstrated by Nathan and DeHaan 3 in the chick heart. Mature atrial and ventricular cells have a resting potential that is due to predominant permeability to potassium and is approximately � 90 mV, which is the potassium equilibrium

Genetic isolation of stem cell-derived pacemaker-nodal cardiac myocytes

Dysfunction of the cardiac pacemaker tissues due to genetic defects, acquired diseases, or aging results in arrhythmias. When arrhythmias occur, artificial pacemaker implants are used for treatment. However, the numerous limitations of electronic implants have prompted studies of biological pacemakers that can integrate into the myocardium providing a permanent cure. Embryonic stem (ES) cells cultured as three-dimensional (3D) spheroid aggregates termed embryoid bodies possess the ability to generate all cardiac myocyte subtypes. Here, we report the use of a SHOX2 promoter and a Cx30.2 enhancer to genetically identify and isolate ES cell-derived sinoatrial node (SAN) and atrioventricular node (AVN) cells, respectively. The ES cell-derived Shox2 and Cx30.2 cardiac myocytes exhibit a spider cell morphology and high intracellular calcium loading characteristic of pacemaker-nodal myocytes. These cells express abundant levels of pacemaker genes such as endogenous HCN4, Cx45, Cx30.2, Tbx2, and Tbx3. These cells were passaged, frozen, and thawed multiple times while maintaining their pacemaker-nodal phenotype. When cultured as 3D aggregates in an attempt to create a critical mass that simulates in vivo architecture, these cell lines exhibited an increase in the expression level of key regulators of cardiovascular development, such as GATA4 and GATA6 transcription factors. In addition, the aggregate culture system resulted in an increase in the expression level of several ion channels that play a major role in the spontaneous diastolic depolarization characteristic of pacemaker cells. We have isolated pure populations of SAN and AVN cells that will be useful tools for generating biological pacemakers.

The arrhythmogenic consequences of increasing late INa in the cardiomyocyte

This review presents the roles of cardiac sodium channel NaV1.5 late current (late INa) in generation of arrhythmic activity. The assumption of the authors is that proper Na(+) channel function is necessary to the maintenance of the transmembrane electrochemical gradient of Na(+) and regulation of cardiac electrical activity. Myocyte Na(+) channels' openings during the brief action potential upstroke contribute to peak INa and initiate excitation-contraction coupling. Openings of Na(+) channels outside the upstroke contribute to late INa, a depolarizing current that persists throughout the action potential plateau. The small, physiological late INa does not appear to be critical for normal electrical or contractile function in the heart. Late INa does, however, reduce the net repolarizing current, prolongs action potential duration, and increases cellular Na(+) loading. An increase of late INa, due to acquired conditions (e.g. heart failure) or inherited Na(+) channelopathies, facilitates the formation of early and delayed afterpolarizations and triggered arrhythmias, spontaneous diastolic depolarization, and cellular Ca(2+) loading. These in turn increase the spatial and temporal dispersion of repolarization time and may lead to reentrant arrhythmias.