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Models based on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are proposed in almost any field of physiology and pharmacology. The development of human induced pluripotent stem cell-derived cardiomyocytes is expected to become a step forward to increase the translational power of cardiovascular research. Importantly they should allow to study genetic effects on an electrophysiological background close to the human situation. However, biological and methodological issues revealed when human induced pluripotent stem cell-derived cardiomyocytes were used in experimental electrophysiology. We will discuss some of the challenges that should be considered when human induced pluripotent stem cell-derived cardiomyocytes will be used as a physiological model.

The physiological importance of NCX in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is not well characterized but may depend on the relative strength of the current, compared to adult cardiomyocytes, and on the exact spatial arrangement of proteins involved in Ca2+ extrusion. Here, we determined NCX currents and its contribution to action potential and force in hiPSC-CMs cultured in engineered heart tissue (EHT). The results were compared with data from rat and human left ventricular tissue. The NCX currents in hiPSC-CMs were larger than in ventricular cardiomyocytes isolated from human left ventricles (1.3 ± 0.2 pA/pF and 3.2 ± 0.2 pA/pF for human ventricle and EHT, respectively, p < 0.05). SEA0400 (10 µM) markedly shortened the APD90 in EHT (by 26.6 ± 5%, p < 0.05) and, to a lesser extent, in rat ventricular tissue (by 10.7 ± 1.6%, p < 0.05). Shortening in human left ventricular preparations was small and not different from time-matched controls (TMCs; p > 0.05). Force was increased by the NCX block in rat ventricle (by 31 ± 5.4%, p < 0.05) and EHT (by 20.8 ± 3.9%, p < 0.05), but not in human left ventricular preparations. In conclusion, hiPSC-CMs possess NCX currents not smaller than human left ventricular tissue. Robust NCX block-induced APD shortening and inotropy makes EHT an attractive pharmacological model.

Human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) are becoming instrumental in cardiac research, human‐based cell level cardiotoxicity tests, and developing patient‐specific care. As one of the principal functional readouts is contractility, we propose a novel electromechanical hiPSC‐CM computational model named the hiPSC‐CM‐CE. This model comprises a reparametrized version of contractile element (CE) by Rice et al., 2008, with a new passive force formulation, integrated into a hiPSC‐CM electrophysiology formalism by Paci et al. in 2020. Our simulated results were validated against in vitro data reported for hiPSC‐CMs at matching conditions from different labs. Specifically, key action potential (AP) and calcium transient (CaT) biomarkers simulated by the hiPSC‐CM‐CE model were within the experimental ranges. On the mechanical side, simulated cell shortening, contraction–relaxation kinetic indices (RT50 and RT25), and the amplitude of tension fell within the experimental intervals. Markedly, as an inter‐scale analysis, correct classification of the inotropic effects due to non‐cardiomyocytes in hiPSC‐CM tissues was predicted on account of the passive force expression introduced to the CE. Finally, the physiological inotropic effects caused by Verapamil and Bay‐K 8644 and the aftercontractions due to the early afterdepolarizations (EADs) were simulated and validated against experimental data. In the future, the presented model can be readily expanded to take in pharmacological trials and genetic mutations, such as those involved in hypertrophic cardiomyopathy, and study arrhythmia trigger mechanisms., We present the first hiPSC‐CM computational model that accounts for essential AP, CaT, and mechanical biomarkers incorporating experimental variability. The introduced passive force handling enables the model to capture the inotropic effect of non‐cardiomyocytes in hiPSC‐CM tissues. Simulated cell shortening and contraction–relaxation indices fall within experimental ranges, and EAD‐based aftercontractions predicted by the model are in accord with experimental observations. Predicted Verapamil and Bay‐K 8644 inotropic effects agree with in vitro data.

Objectives: Improvements in human stem cell-derived cardiomyocyte (hSC-CM) technology have promoted their use for drug testing and disease investigations. Several in silico hSC-CM models have been proposed to augment interpretation of experimental findings through simulations. This work aims to assess the response of three hSC-CM in silico models (Koivumäki2018, Kernik2019, and Paci2020) to simulated drug action, and compare simulation results against in vitro data for 15 drugs.Methods: First, simulations were conducted considering 15 drugs, using a simple pore-block model and experimental data for seven ion channels. Similarities and differences were analyzed in the in silico responses of the three models to drugs, in terms of Ca2+ transient duration (CTD90) and occurrence of arrhythmic events. Then, the sensitivity of each model to different degrees of blockage of Na+ (INa), L-type Ca2+ (ICaL), and rapid delayed rectifying K+ (IKr) currents was quantified. Finally, we compared the drug-induced effects on CTD90 against the corresponding in vitro experiments.Results: The observed CTD90 changes were overall consistent among the in silico models, all three showing changes of smaller magnitudes compared to the ones measured in vitro. For example, sparfloxacin 10 µM induced +42% CTD90 prolongation in vitro, and +17% (Koivumäki2018), +6% (Kernik2019), and +9% (Paci2020) in silico. Different arrhythmic events were observed following drug application, mainly for drugs affecting IKr. Paci2020 and Kernik2019 showed only repolarization failure, while Koivumäki2018 also displayed early and delayed afterdepolarizations. The spontaneous activity was suppressed by Na+ blockers and by drugs with similar effects on ICaL and IKr in Koivumäki2018 and Paci2020, while only by strong ICaL blockers, e.g. nisoldipine, in Kernik2019. These results were confirmed by the sensitivity analysis.Conclusion: To conclude, The CTD90 changes observed in silico are qualitatively consistent with our in vitro data, although our simulations show differences in drug responses across the hSC-CM models, which could stem from variability in the experimental data used in their construction.

Hypertrophic cardiomyopathy (HCM) patients are at increased risk of ventricular arrhythmias and sudden cardiac death, which can occur even in the absence of structural changes of the heart. HCM mouse models suggest mutations in myofilament components to affect Ca2+ homeostasis and thereby favor arrhythmia development. Additionally, some of them show indications of pro-arrhythmic changes in cardiac electrophysiology. In this study, we explored arrhythmia mechanisms in mice carrying a HCM mutation in Mybpc3 (Mybpc3-KI) and tested the translatability of our findings in human engineered heart tissues (EHTs) derived from CRISPR/Cas9-generated homozygous MYBPC3 mutant (MYBPC3hom) in induced pluripotent stem cells (iPSC) and to left ventricular septum samples obtained from HCM patients. We observed higher arrhythmia susceptibility in contractility measurements of field-stimulated intact cardiomyocytes and ventricular muscle strips as well as in electromyogram recordings of Langendorff-perfused hearts from adult Mybpc3-KI mice than in wild-type (WT) controls. The latter only occurred in homozygous (Hom-KI) but not in heterozygous (Het-KI) mouse hearts. Both Het- and Hom-KI are known to display pro-arrhythmic increased Ca2+ myofilament sensitivity as a direct consequence of the mutation. In the electrophysiological characterization of the model, we observed smaller repolarizing K+ currents in single cell patch clamp, longer ventricular action potentials in sharp microelectrode recordings and longer ventricular refractory periods in Langendorff-perfused hearts in Hom-KI, but not Het-KI. Interestingly, reduced K+ channel subunit transcript levels and prolonged action potentials were already detectable in newborn, pre-hypertrophic Hom-KI mice. Human iPSC-derived MYBPC3hom EHTs, which genetically mimicked the Hom-KI mice, did exhibit lower mutant mRNA and protein levels, lower force, beating frequency and relaxation time, but no significant alteration of the force-Ca2+ relation in skinned EHTs. Furthermore, MYBPC3hom EHTs did show higher spontaneous arrhythmic behavior, whereas action potentials measured by sharp microelectrode did not differ to isogenic controls. Action potentials measured in septal myectomy samples did not differ between patients with HCM and patients with aortic stenosis, except for the only sample with a MYBPC3 mutation. The data demonstrate that increased myofilament Ca2+ sensitivity is not sufficient to induce arrhythmias in the Mybpc3-KI mouse model and suggest that reduced K+ currents can be a pro-arrhythmic trigger in Hom-KI mice, probably already in early disease stages. However, neither data from EHTs nor from left ventricular samples indicate relevant reduction of K+ currents in human HCM. Therefore, our study highlights the species difference between mouse and human and emphasizes the importance of research in human samples and human-like models., Graphical abstract Unlabelled Image, Highlights • Sudden cardiac death is threatening hypertrophic cardiomyopathy (HCM) patients. • Arrhythmia mechanisms are not well understood. • Mouse HCM models showed relevant reduction in K+ currents. • Human iPSC-EHT model and HCM patient septal myectomies did not display this mechanism.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an unlimited source of human CMs that could be a standard tool in drug research. However, there is concern whether hiPSC-CMs express all cardiac ion channels at physiological level and whether they might express non-cardiac ion channels. In a control hiPSC line, we found large, &ldquo, noisy&rdquo, outward K+ currents, when we measured outward potassium currents in isolated hiPSC-CMs. Currents were sensitive to iberiotoxin, the selective blocker of big conductance Ca2+-activated K+ current (IBK,Ca). Seven of 16 individual differentiation batches showed a strong initial repolarization in the action potentials (AP) recorded from engineered heart tissue (EHT) followed by very early afterdepolarizations, sometimes even with consecutive oscillations. Iberiotoxin stopped oscillations and normalized AP shape, but had no effect in other EHTs without oscillations or in human left ventricular tissue (LV). Expression levels of the alpha-subunit (KCa1.1) of the BKCa correlated with the presence of oscillations in hiPSC-CMs and was not detectable in LV. Taken together, individual batches of hiPSC-CMs can express sarcolemmal ion channels that are otherwise not found in the human heart, resulting in oscillating afterdepolarizations in the AP. HiPSC-CMs should be screened for expression of non-cardiac ion channels before being applied to drug research.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α‐actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient‐derived human‐induced pluripotent stem cells (hiPSCs) and show that hiPSC‐derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L‐type Ca2+ current. The L‐type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM‐affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease‐causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof‐of‐principle for the use of hiPSC for personalized treatment of cardiomyopathies., Disease modeling of a rare ACTN2 mutation in iPSC‐derived cardiomyocytes & heart tissues engineering revealed typical features of hypertrophic cardiomyopathy & electrophysiological anomalies. Diltiazem reversed the in vitro phenotypes & guided clinical therapy in the family, reducing QTc intervals.

The pathophysiology of atrial fibrillation (AF) is broad, with components related to the unique and diverse cellular electrophysiology of atrial myocytes, structural complexity, and heterogeneity of atrial tissue, and pronounced disease-associated remodeling of both cells and tissue. A major challenge for rational design of AF therapy, particularly pharmacotherapy, is integrating these multiscale characteristics to identify approaches that are both efficacious and independent of ventricular contraindications. Computational modeling has long been touted as a basis for achieving such integration in a rapid, economical, and scalable manner. However, computational pipelines for AF-specific drug screening are in their infancy, and while the field is progressing quite rapidly, major challenges remain before computational approaches can fill the role of workhorse in rational design of AF pharmacotherapies. In this review, we briefly detail the unique aspects of AF pathophysiology that determine requirements for compounds targeting AF rhythm control, with emphasis on delimiting mechanisms that promote AF triggers from those providing substrate or supporting reentry. We then describe modeling approaches that have been used to assess the outcomes of drugs acting on established AF targets, as well as on novel promising targets including the ultra-rapidly activating delayed rectifier potassium current, the acetylcholine-activated potassium current and the small conductance calcium-activated potassium channel. Finally, we describe how heterogeneity and variability are being incorporated into AF-specific models, and how these approaches are yielding novel insights into the basic physiology of disease, as well as aiding identification of the important molecular players in the complex AF etiology.

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K+ current ( Ito,f) and with CaV1.2 to mediate the inward L-type Ca2+ current ( ICa,L). Anesthetized KChIP2−/− mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVβ2 protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca2+ entry into the cell, thereby augmenting Ca2+-induced Ca2+ release. Ca2+ transients in disaggregated KChIP2−/− cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca2+-induced Ca2+ release event. We next functionally probed dyad structure, ryanodine receptor Ca2+ sensitivity, and sarcoplasmic reticulum Ca2+ load and found that increased temporal synchronicity of the Ca2+ release in KChIP2−/− cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2−/− mice.

Background: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising experimental tool for translational heart research and drug development. However, their usability as a human adult cardiomyocyte model is limited by their functional immaturity. Our aim is to analyse quantitatively those characteristics and how they differ from adult CMs. Methods and Results: We have developed a novel in silico model with all essential functional electrophysiology and calcium handling features of hiPSC-CMs. Importantly, the virtual cell recapitulates the immature intracellular ion dynamics that are characteristic for hiPSC-CMs, as quantified based our in vitro imaging data. The strong “calcium clock” is a source for a dual function of excitation-contraction coupling in hiPSC-CMs: action potential and calcium transient morphology vary substantially depending on the activation sequence of underlying ionic currents and fluxes that is altered in spontaneous vs. paced mode. Furthermore, parallel simulations with hiPSC-CM and adult cardiomyocyte models demonstrate the central differences. Results indicate that hiPSC-CMs translate poorly the disease specific phenotypes of Brugada syndrome, long QT Syndrome and catecholaminergic polymorphic ventricular tachycardia, showing less robustness and greater tendency for arrhythmic events than adult CMs. Based on a comparative sensitivity analysis, hiPSC-CMs share some features with adult CMs, but are still functionally closer to prenatal CMs than adult CMs. A database analysis of 3000 hiPSC-CM model variants suggests that hiPSC-CMs recapitulate poorly fundamental physiological properties of adult CMs. Single modifications do not appear to solve this problem, which is mostly contributed by the immaturity of intracellular calcium handling. Conclusion: Our data indicates that translation of findings from hiPSC-CMs to human disease should be made with great caution. Furthermore, we established a mathematical platform that can be used to improve the translation from hiPSC-CMs to human, and to quantitatively evaluate hiPSC-CMs development toward more general and valuable model for human cardiac diseases., published version, peerReviewed

Background: Cardiac repolarization abnormalities in drug-induced and genetic long-QT syndrome may lead to afterdepolarizations and life-threatening ventricular arrhythmias. Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) should help to overcome the limitations of animal models based on species differences in repolarization reserve. Here, we compared head-to-head the contribution of I Ks (long QT1) and I Kr (long QT2) on action potentials (APs) in human left ventricular (LV) tissue and hiPSC-CM–derived engineered heart tissue (EHT). Methods: APs were measured with sharp microelectrodes in EHT from 3 different control hiPSC-CM lines and in tissue preparations from failing LV. Results: EHT from hiPSC-CMs showed spontaneous diastolic depolarization and AP generation that were sensitive to low concentrations of ivabradine. I Kr block by E-4031 prolonged AP duration at 90% repolarization with similar half-effective concentration in EHT and LV but larger effect size in EHT (+281 versus +110 ms in LV). Although I Kr block alone evoked early afterdepolarizations and triggered activity in 50% of EHTs, slow pacing, reduced extracellular K + , and blocking of I Kr , I Ks , and I K1 were necessary to induce early afterdepolarizations in LV. In accordance with their clinical safety, moxifloxacin and verapamil did not induce early afterdepolarizations in EHT. In both EHT and LV, I Ks block by HMR-1556 prolonged AP duration at 90% repolarization slightly in the combined presence of E-4031 and isoprenaline. Conclusions: EHT from hiPSC-CMs shows a lower repolarization reserve than human LV working myocardium and could thereby serve as a sensitive and specific human-based model for repolarization studies and arrhythmia, similar to Purkinje fibers. In both human LV and EHT, I Ks only contributed to repolarization under adrenergic stimulation.

Vascular endothelial growth factor B (VEGF-B) is a potent mediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60–70). These changes were partly caused by downregulation of sodium current (INa) due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K+ currents, namely decreased density of transient outward current (Ito) and total K+ current (Ipeak). At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2), a known modulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG., published version, peerReviewed

Although t-tubules have traditionally been thought to be absent in atrial cardiomyocytes, recent studies have suggested that t-tubules exist in the atria of large mammals. However, it is unclear whether regional differences in t-tubule organization exist that define cardiomyocyte function across the atria. We sought to investigate regional t-tubule density in pig and rat atria and the consequences for cardiomyocyte Ca2+ homeostasis. We observed t-tubules in approximately one-third of rat atrial cardiomyocytes, in both tissue cryosections and isolated cardiomyocytes. In a minority (≈10%) of atrial cardiomyocytes, the t-tubular network was well organized, with a transverse structure resembling that of ventricular cardiomyocytes. In both rat and pig atrial tissue, we observed higher t-tubule density in the epicardium than in the endocardium. Consistent with high variability in the distribution of t-tubules and Ca2+ channels among cells, L-type Ca2+ current amplitude was also highly variable and steeply dependent on capacitance and t-tubule density. Accordingly, Ca2+ transients showed great variability in Ca2+ release synchrony. Simultaneous imaging of the cell membrane and Ca2+ transients confirmed t-tubule functionality. Results from mathematical modeling indicated that a transmural gradient in t-tubule organization and Ca2+ release kinetics supports synchronization of contraction across the atrial wall and may underlie transmural differences in the refractory period. In conclusion, our results indicate that t-tubule density is highly variable across the atria. We propose that higher t-tubule density in cells localized in the epicardium may promote synchronization of contraction across the atrial wall.

Recent studies have demonstrated that changes in the activity of calcium-calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation-contraction (E-C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM-GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

The ability to generate homogeneous intracellular Ca2+ oscillations at high frequency is the basis of the rhythmic contractions of mammalian cardiac myocytes. While the specific mechanisms and structures enabling homogeneous high-frequency Ca2+ signals in adult cardiomyocytes are well characterized, it is not known how these kind of Ca2+ signals are produced in developing cardiomyocytes. Here we investigated the mechanisms reducing spatial and temporal heterogeneity of cytosolic Ca2+ signals in mouse embryonic ventricular cardiomyocytes. We show that in developing cardiomyocytes the propagating Ca2+ signals are amplified in cytosol by local Ca2+ releases. Local releases are based on regular 3-D sarcoplasmic reticulum (SR) structures containing SR Ca2+ uptake ATPases (SERCA) and Ca2+ release channels (ryanodine receptors, RyRs) at regular intervals throughout the cytosol. By evoking [Ca2+]i-induced Ca2+ sparks, the local release sites promote a 3-fold increase in the cytosolic Ca2+ propagation speed. We further demonstrate by mathematical modelling that without these local release sites the developing cardiomyocytes lose their ability to generate homogeneous global Ca2+ signals at a sufficiently high frequency. The mechanism described here is robust and indispensable for normal mammalian cardiomyocyte function from the first heartbeats during the early embryonic phase till terminal differentiation after birth. These results suggest that local cytosolic Ca2+ releases are indispensable for normal cardiomyocyte development and function of developing heart.

Adult cardiomyocytes exhibit complex Ca(2+) homeostasis, enabling tight control of contraction and relaxation. This intricate regulatory system develops gradually, with progressive maturation of specialized structures and increasing capacity of Ca(2+) sources and sinks. In this review, we outline current understanding of these developmental processes, and draw parallels to pathophysiological conditions where cardiomyocytes exhibit a striking regression to an immature state of Ca(2+) homeostasis. We further highlight the importance of understanding developmental physiology when employing immature cardiomyocyte models such as cultured neonatal cells and stem cells.

AIMS The study investigates how increased Ito, as mediated by the activator NS5806, affects excitation-contraction coupling in chronic heart failure (HF). We hypothesized that restoring spike-and-dome morphology of the action potential (AP) to a healthy phenotype would be insufficient to restore the intracellular Ca(2) (+) transient (CaT), due to HF-induced remodelling of Ca(2+) handling. METHODS AND RESULTS An existing mathematical model of the canine ventricular myocyte was modified to incorporate recent experimental data from healthy and failing myocytes, resulting in models of both healthy and HF epicardial, midmyocardial, and endocardial cell variants. Affects of NS5806 were also included in HF models through its direct interaction with Kv4.3 and Kv1.4. Single-cell simulations performed in all models (control, HF, and HF + drug) and variants (epi, mid, and endo) assessed AP morphology and underlying ionic processes with a focus on calcium transients (CaT), how these were altered in HF across the ventricular wall, and the subsequent effects of varying compound concentration in HF. Heart failure model variants recapitulated a characteristic increase in AP duration (APD) in the disease. The qualitative effects of application of half-maximal effective concentration (EC50) of NS5806 on APs and CaT are heterogeneous and non-linear. Deepening in the AP notch with drug is a direct effect of the activation of Ito; both Ito and consequent alteration of IK1 kinetics cause decrease in AP plateau potential. Decreased APD50 and APD90 are both due to altered IK1. Analysis revealed that drug effects depend on transmurality. Ca(2+) transient morphology changes-increased amplitude and shorter time to peak-are due to direct increase in ICa,L and indirect larger SR Ca(2+) release subsequent to Ito activation. CONCLUSIONS Downstream effects of a compound acting exclusively on sarcolemmal ion channels are difficult to predict. Remediation of APD to pre-failing state does not ameliorate dysfunction in CaT; however, restoration of notch depth appears to impart modest benefit and a likelihood of therapeutic value in modulating early repolarization.

In the mammalian heart fibroblasts have important functional roles in both healthy conditions and diseased states. During pathophysiological challenges, a closely related myofibroblast cell population emerges, and can have distinct, significant roles. Recently, it has been reported that human atrial myofibroblasts can express a Na(+) current, INa. Some of the biophysical properties and molecular features suggest that this INa is due to expression of Nav 1.5, the same Na(+) channel α subunit that generates the predominant INa in myocytes from adult mammalian heart. In principle, expression of Nav 1.5 could give rise to regenerative action potentials in the fibroblasts/myofibroblasts. This would suggest an active as opposed to passive role for fibroblasts/myofibroblasts in both the "trigger" and the "substrate" components of cardiac rhythm disturbances. Our goals in this preliminary study were: (i) to confirm and extend the electrophysiological characterization of INa in a human atrial fibroblast/myofibroblast cell population maintained in conventional 2-D tissue culture; (ii) to identify key molecular properties of the α and β subunits of these Na(+) channel(s); (iii) to define the biophysical and pharmacological properties of this INa; (iv) to integrate the available multi-disciplinary data, and attempt to illustrate its functional consequences, using a mathematical model in which the human atrial myocyte is coupled via connexins to fixed numbers of fibroblasts/myofibroblasts in a syncytial arrangement. Our experimental findings confirm that a significant fraction (approximately 40-50%) of these human atrial myofibroblasts can express INa. However, our data suggest that INa may be generated by a combination of Nav 1.9, Nav 1.2, and Nav 1.5. Our results, when complemented with mathematical modeling, provide a background for re-evaluating pharmacological management of supraventricular rhythm disorders, e.g., persistent atrial fibrillation.

Brugada syndrome (BrS) is a rare inherited disease that can give rise to ventricular arrhythmia and ultimately sudden cardiac death. Numerous loss-of-function mutations in the cardiac sodium channel Nav1.5 have been associated with BrS. However, few mutations in the auxiliary Navβ1–4 subunits have been linked to this disease. Here we investigated differences in expression and function between Navβ1 and Navβ1b and whether the H162P/Navβ1b mutation found in a BrS patient is likely to be the underlying cause of disease. The impact of Navβ subunits was investigated by patch-clamp electrophysiology, and the obtained in vitro values were used for subsequent in silico modeling. We found that Navβ1b transcripts were expressed at higher levels than Navβ1 transcripts in the human heart. Navβ1 and Navβ1b coexpressed with Nav1.5 induced a negative shift on steady state of activation and inactivation compared with Nav1.5 alone. Furthermore, Navβ1b was found to increase the current level when coexpressed with Nav1.5, Navβ1b/H162P mutated subunit peak current density was reduced by 48% (−645 ± 151 vs. −334 ± 71 pA/pF), V1/2 steady-state inactivation shifted by −6.7 mV (−70.3 ± 1.5 vs. −77.0 ± 2.8 mV), and time-dependent recovery from inactivation slowed by >50% compared with coexpression with Navβ1b wild type. Computer simulations revealed that these electrophysiological changes resulted in a reduction in both action potential amplitude and maximum upstroke velocity. The experimental data thereby indicate that Navβ1b/H162P results in reduced sodium channel activity functionally affecting the ventricular action potential. This result is an important replication to support the notion that BrS can be linked to the function of Navβ1b and is associated with loss-of-function of the cardiac sodium channel.

Chronic atrial fibrillation (AF) is a complex disease with underlying changes in electrophysiology, calcium signaling and the structure of atrial myocytes. How these individual remodeling targets and their emergent interactions contribute to cell physiology in chronic AF is not well understood. To approach this problem, we performed in silico experiments in a computational model of the human atrial myocyte. The remodeled function of cellular components was based on a broad literature review of in vitro findings in chronic AF, and these were integrated into the model to define a cohort of virtual cells. Simulation results indicate that while the altered function of calcium and potassium ion channels alone causes a pronounced decrease in action potential duration, remodeling of intracellular calcium handling also has a substantial impact on the chronic AF phenotype. We additionally found that the reduction in amplitude of the calcium transient in chronic AF as compared to normal sinus rhythm is primarily due to the remodeling of calcium channel function, calcium handling and cellular geometry. Finally, we found that decreased electrical resistance of the membrane together with remodeled calcium handling synergistically decreased cellular excitability and the subsequent inducibility of repolarization abnormalities in the human atrial myocyte in chronic AF. We conclude that the presented results highlight the complexity of both intrinsic cellular interactions and emergent properties of human atrial myocytes in chronic AF. Therefore, reversing remodeling for a single remodeled component does little to restore the normal sinus rhythm phenotype. These findings may have important implications for developing novel therapeutic approaches for chronic AF., Author Summary Atrial fibrillation is a complex disease which, at the level of individual atrial muscle cells, is a result of changes in a number of ion channels and transporters, as well as in cellular structure. How these alterations, together and separately, affect electrical and contractile function of the atrial cells is not well understood. In this study, we evaluated the effect of these changes using a computational approach. Our results show that abnormal function of both calcium and potassium ion channels at the sarcolemma has the largest impact on the electrical properties of the human atrial myocyte. Changes in intracellular calcium handling and cellular geometry are also significant for cellular function. Finally, our results highlight the interactions and additive effect of these abnormalities, in that a hypothetical restoration of any single modification does not result in recovery of function to a healthy phenotype. These findings have potentially important implications for developing novel treatment options for atrial fibrillation.

Mathematical modeling of cardiac electrophysiology is an insightful method to investigate the underlying mechanisms responsible for arrhythmias such as atrial fibrillation (AF). In past years, five models of human atrial electrophysiology with different formulations of ionic currents, and consequently diverging properties, have been published. The aim of this work is to give an overview of strengths and weaknesses of these models depending on the purpose and the general requirements of simulations. Therefore, these models were systematically benchmarked with respect to general mathematical properties and their ability to reproduce certain electrophysiological phenomena, such as action potential (AP) alternans. To assess the models' ability to replicate modified properties of human myocytes and tissue in cardiac disease, electrical remodeling in chronic atrial fibrillation (cAF) was chosen as test case. The healthy and remodeled model variants were compared with experimental results in single-cell, 1D and 2D tissue simulations to investigate AP and restitution properties, as well as the initiation of reentrant circuits.

Recent studies have demonstrated that changes in the activity of calcium-calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation-contraction (E-C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM-GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca2+ dynamics, which differ crucially from that of ventricular cells, in the modulation of excitation-contraction coupling. Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL) ion currents, accounts for the heterogeneity of intracellular Ca2+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR). Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca2+ dynamics: 1) the biphasic increment during the upstroke of the Ca2+ transient resulting from the delay between the peripheral and central SR Ca2+ release, and 2) the relative contribution of SL Ca2+ current and SR Ca2+ release to the Ca2+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca2+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca2+ release sites define the interface between Ca2+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca2+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca2+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca2+ signaling. Thus, the model provides a useful framework for future studies of excitation-contraction coupling in human atrial myocytes., Author Summary In the human heart, the contraction of atrial and ventricular muscle cells is based largely on common mechanisms. There is, however, a fundamental difference in the cellular calcium dynamics that underlie the contractile function. Here, we have developed a computational model of the human atrial cell that convincingly reproduces the experimentally observed characteristics of the electrical activity and the cyclic fluctuations of the intracellular calcium concentration. With the model, we evaluate the relative roles of the most important cellular calcium transport mechanisms and their impact on the electrical behavior of the cell. Our simulations predict that the amount of calcium released from the cellular stores during each electrical cycle crucially regulates the excitability of the human atrial cell. Furthermore, the results indicate that the cellular sodium accumulation related to faster heart rates is one of the main mechanisms driving the adaptation of cardiac electrical activity. Finally, we conclude that the presented model also provides a useful framework for future studies of human atrial cells.

The ability to generate homogeneous intracellular Ca(2+) oscillations at high frequency is the basis of the rhythmic contractions of mammalian cardiac myocytes. While the specific mechanisms and structures enabling homogeneous high-frequency Ca(2+) signals in adult cardiomyocytes are well characterized, it is not known how these kind of Ca(2+) signals are produced in developing cardiomyocytes. Here we investigated the mechanisms reducing spatial and temporal heterogeneity of cytosolic Ca(2+) signals in mouse embryonic ventricular cardiomyocytes. We show that in developing cardiomyocytes the propagating Ca(2+) signals are amplified in cytosol by local Ca(2+) releases. Local releases are based on regular 3-D sarcoplasmic reticulum (SR) structures containing SR Ca(2+) uptake ATPases (SERCA) and Ca(2+) release channels (ryanodine receptors, RyRs) at regular intervals throughout the cytosol. By evoking [Ca(2+)](i)-induced Ca(2+) sparks, the local release sites promote a 3-fold increase in the cytosolic Ca(2+) propagation speed. We further demonstrate by mathematical modelling that without these local release sites the developing cardiomyocytes lose their ability to generate homogeneous global Ca(2+) signals at a sufficiently high frequency. The mechanism described here is robust and indispensable for normal mammalian cardiomyocyte function from the first heartbeats during the early embryonic phase till terminal differentiation after birth. These results suggest that local cytosolic Ca(2+) releases are indispensable for normal cardiomyocyte development and function of developing heart.

In this manuscript we review the state of cardiac cell modelling in the context of international initiatives such as the IUPS Physiome and Virtual Physiological Human Projects, which aim to integrate computational models across scales and physics. In particular we focus on the relationship between experimental data and model parameterisation across a range of model types and cellular physiological systems. Finally, in the context of parameter identification and model reuse within the Cardiac Physiome, we suggest some future priority areas for this field.

Background The cardiomyocyte is a prime example of inherently complex biological system with inter- and cross-connected feedback loops in signalling, forming the basic properties of intracellular homeostasis. Functional properties of cells and tissues have been studied e.g. with powerful tools of genetic engineering, combined with extensive experimentation. While this approach provides accurate information about the physiology at the endpoint, complementary methods, such as mathematical modelling, can provide more detailed information about the processes that have lead to the endpoint phenotype. Results In order to gain novel mechanistic information of the excitation-contraction coupling in normal myocytes and to analyze sophisticated genetically engineered heart models, we have built a mathematical model of a mouse ventricular myocyte. In addition to the fundamental components of membrane excitation, calcium signalling and contraction, our integrated model includes the calcium-calmodulin-dependent enzyme cascade and the regulation it imposes on the proteins involved in excitation-contraction coupling. With the model, we investigate the effects of three genetic modifications that interfere with calcium signalling: 1) ablation of phospholamban, 2) disruption of the regulation of L-type calcium channels by calcium-calmodulin-dependent kinase II (CaMK) and 3) overexpression of CaMK. We show that the key features of the experimental phenotypes involve physiological compensatory and autoregulatory mechanisms that bring the system to a state closer to the original wild-type phenotype in all transgenic models. A drastic phenotype was found when the genetic modification disrupts the regulatory signalling system itself, i.e. the CaMK overexpression model. Conclusion The novel features of the presented cardiomyocyte model enable accurate description of excitation-contraction coupling. The model is thus an applicable tool for further studies of both normal and defective cellular physiology. We propose that integrative modelling as in the present work is a valuable complement to experiments in understanding the causality within complex biological systems such as cardiac myocytes.

When developing large-scale mathematical models of physiology, some reduction in complexity is necessarily required to maintain computational efficiency. A prime example of such an intricate cell is the cardiac myocyte. For the predictive power of the cardiomyocyte models, it is vital to accurately describe the calcium transport mechanisms, since they essentially link the electrical activation to contractility. The removal of calcium from the cytoplasm takes place mainly by the Na + /Ca 2+ exchanger, and the sarcoplasmic reticulum Ca 2+ ATPase (SERCA). In the present study, we review the properties of SERCA, its frequency-dependent and β -adrenergic regulation, and the approaches of mathematical modelling that have been used to investigate its function. Furthermore, we present novel theoretical considerations that might prove useful for the elucidation of the role of SERCA in cardiac function, achieving a reduction in model complexity, but at the same time retaining the central aspects of its function. Our results indicate that to faithfully predict the physiological properties of SERCA, we should take into account the calcium-buffering effect and reversible function of the pump. This ‘uncomplicated’ modelling approach could be useful to other similar transport mechanisms as well.

Background and Aim: Given higher abundance in atria vs. ventricles, and a suggested important role in atrial fibrillation (AF), small-conductance Ca2+-activated K+ channels (SKs) are a promising atrial-selective pharmacological target for AF. As their role in atrial physiology and arrhythmia is not yet fully understood, this study aims to investigate the role of the SK current (IK,Ca) in human atrial myocytes during both sinus rhythm and rapid pacing.Methods and Results: A new mathematical formulation of IK,Ca, replicating characteristic Ca2+-dependent activation and mild voltage rectification, was developed using a broad set of experimental observations in rodent and human myocytes, and incorporated into our human atrial cell model.We validated our updated model by assessing the consequences of IK,Ca inhibition on action potential (AP) properties. When paced at 1 Hz, IK,Ca block prolongs simulated AP by ∼18% (enhancing Ca2+ influx, and consequently intracellular Ca2+ load), increases the effective refractory period (ERP) by ∼16%, depolarizes the resting membrane potential by ∼1.5 mV, and decreases maximum upstroke velocity by ∼15%, all in agreement with ranges observed in human atrial experiments.Next, we used this comprehensive model to investigate the effect of hyper-activation/block of IK,Ca in several arrhythmia-provoking scenarios. Our analysis suggests that IK,Ca is protective against focal arrhythmia (phase-2 and phase-3 early afterdepolarizations, delayed-afterdepolarizations, and triggered activity), but contributes to a reentrant substrate (ERP shortening and alternans).Future directions: We will study the effect of administration of IK,Ca modulators to determine if a therapeutic window for SKs in AF exists. We will also evaluate the synergistic consequences of IK,Ca modulation and (atrial-selective) Na+ current inhibition, to test the hypothesis that a combination of low dose drugs may have higher efficacy than monotherapy.