We identified two different inherited mutations in KCNH2 gene, or human ether-a-go-go related gene (hERG), which are linked to Long QT Syndrome. The first mutation was in a 1-day-old infant, whereas the second was in a 14-year-old girl. The two KCNH2 mutations were transiently transfected into either human embryonic kidney (HEK) cells or human induced pluripotent stem-cell derived cardiomyocytes. We performed associated multiscale computer simulations to elucidate the arrhythmogenic potentials of the KCNH2 mutations. Genetic screening of the first and second index patients revealed a heterozygous missense mutation in KCNH2, resulting in an amino acid change (P632L) in the outer loop of the channel and substitution at position 428 from serine to proline (S428P), respectively. Heterologous expression of P632L and S428P into HEK cells produced no hERG current compared to the wild type (WT). Moreover, the co-transfection of WT and P632L yielded no hERG current; however, the co-transfection of WT and S428P yielded partial hERG current. Action potentials were prolonged in a complete or partial blockade of hERG current from computer simulations which was more severe in Purkinje than ventricular myocytes. Three dimensional simulations revealed a higher susceptibility to reentry in the presence of hERG current blockade. Our experimental findings suggest that both P632L and S428P mutations may impair the KCNH2 gene. The Purkinje cells exhibit a more severe phenotype than ventricular myocytes, and the hERG current blockade renders the ventricles an arrhythmogenic substrate from computer modeling.

Human induced pluripotent stem cells directed to the cardiac lineage (hiPSC-CMs) are used in many platforms such as generating models of human genetic diseases and cardiac safety pharmacology whereby compounds bound for regulatory submission are tested on hiPSC-CMs to determine the drug effect on ion channels and action potentials. The cardiac action potential (AP) is an important physiological parameter: (1) the AP initiates excitation in the heart, (2) it modulates the refractory period due to a long AP duration, and (3) associated with each AP is a contraction. Alterations in the expression or gating of ion channels will change the time- and/or voltage-dependent properties and can have marked effects on the AP waveform. The electrophysiological immaturity of hiPSC-CM suggests caution when translating the results to the adult phenotype. This chapter will highlight the electrophysiological similarities and differences of the hiPSC myocyte compared to adult myocytes. We will first contrast AP waveform in hiPSC-CMs and adult ventricle and explore the underlying ionic and molecular basis for these differences. Finally, we will explore strategies employed by various laboratories to potentially improve the maturity of hiPSC-CMs.

AIMS:Variants in SCN5A encoding Nav1.5 are associated with cardiac arrhythmias. We aimed to determine the mechanism by which c.638G>A in SCNA5 resulting in p.Gly213Asp (G213D) in Nav1.5 altered Na+ channel function and how flecainide corrected the defect in a family with multifocal ectopic Purkinje-related premature contractions (MEPPC)-like syndrome.METHODS AND RESULTS:Five patients carrying the G213D variant were treated with flecainide. Gating pore currents were evaluated in Xenopus laevis oocytes. The 638G>A SCN5A variant was introduced to human-induced pluripotent stem cell (hiPSC) by CRISPR-Cas9 gene editing and subsequently differentiated to cardiomyocytes (hiPSC-CM). Action potentials and sodium currents were measured in the absence and presence of flecainide. Ca2+ transients were measured by confocal microscopy. The five patients exhibited premature atrial and ventricular contractions which were suppressed by flecainide treatment. G213D induced gating pore current at potentials negative to -50 mV. Voltage-clamp analysis in hiPSC-CM revealed the activation threshold of INa was shifted in the hyperpolarizing direction resulting in a larger INa window current. The G213D hiPSC-CMs had faster beating rates compared with wild-type and frequently showed Ca2+ waves and alternans. Flecainide applied to G213D hiPSC-CMs decreased window current by shifting the steady-state inactivation curve and slowed the beating rate.CONCLUSION:The G213D variant in Nav1.5 induced gating pore currents and increased window current. The changes in INa resulted in a faster beating rate and Ca2+ transient dysfunction. Flecainide decreased window current and inhibited INa, which is likely responsible for the therapeutic effectiveness of flecainide in MEPPC patients carrying the G213D variant.

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.

Background: Mutations in the gene KCNH2 have been associated with both Short and Long QT syndrome. In this study, we identified a 1-day old infant that exhibited T-wave alternans coupled with Long QT Syndrome leading to aborted sudden infant death. Methods: Genetic analysis of the patient revealed a mutation in KCNH2 resulting in a proline to leucine substitution at position 632 (P632L). Functional electrophysiological studies were performed with the hERG mutation transiently transfected into either Human Embryonic Kidney (HEK) cells or human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The functional effects of P632L were incorporated into biophysical computational models of hiPSC-CM as well as adult human cardiomyocytes to further assess its arrhythmogenic potential. Results: The male infant displayed multiple repolarization disorders including T-wave alternans and a QTc =510 ms that appeared 1 day after birth. Genetic screening revealed a heterozygous missense mutation (P632L) in KCNH2 in the infant. Patch clamp analysis of HEK cells transiently transfected with P632L mutation showed a complete loss of function of HERG current compared to WT HERG. Transient transfection of P632L into WT hiPSC myocytes resulted in prolongation of the hiPSC action potential compared to untransfected hiPSC myocytes. Co-transfection of WT and non-functional P632L channels into HEK cells resulted in a dramatic loss of current suggesting a dominant negative effect. Implementing the effects of a complete block of HERG current in the hiPSC-CM computer model prolonged the action potential by 45% and depolarized the resting membrane potentials. In adult human myocyte models, the effects of HERG blockade were more severe in Purkinje cells than that in ventricular myocytes (71% vs. 16% APD prolongation, respectively). Conclusions: The mutation P632L causes a complete loss of HERG current and results in QT prolongation. Numerical simulations suggest a more severe phenotype in Purkinje cells than in ventricular myocytes which could be proarrhythmic.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a “pseudo” QRS complex suggesting reduced inward current(s). Voltage clamp analysis of ICa showed no difference in the magnitude of current. Measurements of INa reveal a 60% reduction in INa density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of INa was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of INa due to a mutation in PKP2 coupled with and a gain of function in IK,ATP due to a mutation in ABCC9.

Background: We have identified a novel form of abnormal Ca 2+ 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 Ca 2+ waves affect cellular electrophysiological properties. Methods: Simultaneous recordings of intracellular Ca 2+ 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. Results: 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 Ca 2+ 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. Conclusions: Triggered Ca 2+ 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.

Modeling cardiac cell electrophysiology relies on fitting model equations to experimental data obtained under voltage/current clamping conditions. The fitting procedure for these often-nonlinear ionic current equations are mostly executed by trial-and-error by hand or by gradient-based optimization approaches. These methods, though sometimes sufficient at converging at optimal solutions is based on the premise that the characteristic objective function is convex, which often does not apply to cardiac model equations. Meta-heuristic methods, such as evolutionary algorithms and particle swarm algorithms, have proven resilient against early convergence to local optima and saddle-point parameter solutions. This work presents a genetic algorithm-based approach for fitting the adult cardiomyocyte biophysical model formulations to the experimental data obtained in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM). Specifically, whole-cell patch clamp ionic current data of rapid delayed rectifier potassium current, IKr, transient outward potassium current, Ito and hyperpolarization-activated current, If, was used for fitting. Using a two-point crossover scheme along with initial population and mutation constraints randomly selected from a uniformly distributed constrained parameter space, near-optimal fitting was achieved with R2 values (n = 5) of 0.9960±0.0007, 0.9995±0.0002, and 0.9974±0.0014 for IKr, Ito and If respectively.

Computational modeling of cardiac electrophysiology (EP) has recently transitioned from a scientific research tool to clinical applications. To ensure reliability of clinical or regulatory decisions made using cardiac EP models, it is vital to evaluate the uncertainty in model predictions. Model predictions are uncertain because there is typically substantial uncertainty in model input parameters, due to measurement error or natural variability. While there has been much recent uncertainty quantification (UQ) research for cardiac EP models, all previous work has been limited by either: (i) considering uncertainty in only a subset of the full set of parameters; and/or (ii) assigning arbitrary variation to parameters (e.g., ±10 or 50% around mean value) rather than basing the parameter uncertainty on experimental data. In our recent work we overcame the first limitation by performing UQ and sensitivity analysis using a novel canine action potential model, allowing all parameters to be uncertain, but with arbitrary variation. Here, we address the second limitation by extending our previous work to use data-driven estimates of parameter uncertainty. Overall, we estimated uncertainty due to population variability in all parameters in five currents active during repolarization: inward potassium rectifier, transient outward potassium, L-type calcium, rapidly and slowly activating delayed potassium rectifier; 25 parameters in total (all model parameters except fast sodium current parameters). A variety of methods was used to estimate the variability in these parameters. We then propagated the uncertainties through the model to determine their impact on predictions of action potential shape, action potential duration (APD) prolongation due to drug block, and spiral wave dynamics. Parameter uncertainty had a significant effect on model predictions, especially L-type calcium current parameters. Correlation between physiological parameters was determined to play a role in physiological realism of action potentials. Surprisingly, even model outputs that were relative differences, specifically drug-induced APD prolongation, were heavily impacted by the underlying uncertainty. This is the first data-driven end-to-end UQ analysis in cardiac EP accounting for uncertainty in the vast majority of parameters, including first in tissue, and demonstrates how future UQ could be used to ensure model-based decisions are robust to all underlying parameter uncertainties.

The action potential (AP) in cardiac tissue is important for initiating and coordinating contractions in the heart. In addition, the long refractory period minimizes the potential for developing extrasystoles and arrhythmias [1]. The AP is generated by coordinate changes in different ionic currents. In human (or canine) adult ventricular cells, the depolarization phase of the AP is mainly through the influx of Na+ and Ca2+ through specific voltage gated channels [2]. Repolarization of the AP is regulated by activation of a number of different K+ currents which play important roles in regulating the AP. These K+ currents include: (i) a Ca2+-independent transient outward K+ current (Ito), (ii) an inwardly rectifying K+ current (IK1), and (iii) the rapidly and slowly activating delayed rectifier K+ channel currents (IKr and IKs, respectively). Previous studies have demonstrated that there is an excess of several K+ currents necessary for cardiac repolarization such that a net outward current remains available for repolarization if one or more currents are reduced (repolarization reserve) [3– 5]. Therefore, cardiac tissue with a lower repolarization reserve is associated with a prolonged ventricular action potential and an increased incidence of developing arrhythmias [6]. Mutations in KCNH2 (the gene which encodes IKr) cause a decrease in the magnitude of IKr and are associated with Long QT syndrome [7,8]. Patients afflicted with Long QT have episodes of fainting, irregular heartbeats and an increased incidence of developing ventricular arrhythmias. Interestingly, many non-cardiac medications have also been shown to block IKr [9,10] which has resulted in drug companies extensively testing potential therapeutic compounds for IKr block prior to introduction to the market.

Background. We report an inherited cardiac arrhythmia syndrome consisting of Brugada and Early Repolarization Syndrome associated with variants in SCN9A, PXDNL, and FKBP1B. The proband inherited the 3 mutations and exhibited palpitations and arrhythmia-mediated syncope, whereas the parents and sister, who carried one or two of the mutations, were asymptomatic. Methods and Results. We assessed the functional impact of these mutations in induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) derived from the proband and an unaffected family member. Current and voltage clamp recordings, as well as confocal microscopy analysis of Ca2+ transients, were evaluated in hiPSC-CMs from the proband and compared these results with hiPSC-CMs from undiseased controls. Genetic analysis using next-generation DNA sequencing revealed heterozygous mutations in SCN9A, PXDNL, and FKBP1B in the proband. The proband displayed right bundle branch block and exhibited episodes of syncope. The father carried a mutation in FKBP1B, whereas the mother and sister carried the SCN9A mutation. None of the 3 family members screened developed cardiac events. Action potential recordings from control hiPSC-CM showed spontaneous activity and a low upstroke velocity. In contrast, the hiPSC-CM from the proband showed irregular spontaneous activity. Confocal microscopy of the hiPSC-CM of the proband revealed low fluorescence intensity Ca2+ transients that were episodic in nature. Patch-clamp measurements in hiPSC-CM showed no difference in INa but reduced ICa in the proband compared with control. Coexpression of PXDNL-R391Q with SCN5A-WT displayed lower INa density compared to PXDNL-WT. In addition, coexpression of PXDNL-R391Q with KCND3-WT displayed significantly higher Ito density compared to PXDNL-WT. Conclusion. SCN9A, PXDNL, and FKBP1B variants appeared to alter spontaneous activity in hiPSC-CM. Only the proband carrying all 3 mutations displayed the ERS/BrS phenotype, whereas one nor two mutations alone did not produce the clinical phenotype. Our results suggest a polygenic cause of the BrS/ERS arrhythmic phenotype due to mutations in these three gene variants caused a very significant loss of function of INa and ICa and gain of function of Ito.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used for safety pharmacology and to investigate genetic diseases affecting cardiac ion channels. We have previously shown that the maximum diastolic potential (MDP) in hiPSC-CMs depends on the rapid delayed rectifier K+ current (IKr). We examined the effect of a dual Ito and IKr agonist (NS3623) on hiPSC-CMs and determined its contribution to action potentials (APs), extracellular field potentials (EFPs) and ionic currents. hiPSC-CM monolayers were used to record EFPs using CardioExcyte 96, and AP recordings were made using high resistance electrodes. Whole cell patch clamp was used to record IKr in single hiPSC-CMs. All measurements were made at 36° C. EFP signals from hiPSC-CMs monolayers showed a pseudo-QRS complex and T-wave similar to that observed in native ventricular tissue. Application of NS3623 (5 μM) resulted in a small shortening of the QT interval. Similarly, AP recordings in the presence of NS3623 resulted in hyperpolarization of the MDP (from −70.4±1.9 to −73.9±1.9 mV) and shortening of APD (from 196.6±28.0 to 176.6±25.7 ms). Voltage clamp analysis of IKr tail currents revealed a 47±9% increase in IKr following application of NS3623. A minor inhibition of Ito was observed following NS3623. AP clamp experiments revealed the IKr transient was largest during repolarization with some contribution during phase 4 depolarization. A robust IKr is present in hiPSC-CMs which can be augmented by the agonist NS3623. Application of the agonist resulted a slowing of the spontaneous rate and shortening of the APD but minimal effect on MDP. Although we have previously shown that the MDP in hiPSC-CMs critically depends on IKr, increasing the magnitude of IKr resulted only in a small hyperpolarization of the MDP.

BACKGROUND:Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for many applications including safety pharmacology. However, a deficiency or complete absence of several K+ currents suggests repolarization reserve is low in hiPSC-CMs. We determined whether a dual Ito and IKr activator can improve repolarization reserve in hiPSC-CMs resulting in a more electrophysiologically mature phenotype.METHODS AND RESULTS:Human iPSC were maintained on growth factor and differentiated into the cardiac phenotype by addition of selective Wnt molecules. Current and voltage clamp recordings in single cells were made using patch electrodes. Extracellular field potentials were made using a microelectrode array on hiPSC monolayers. Action potential recordings from hiPSC-CMs following application of an IKr inhibitor resulted in depolarization of the membrane potential and prolongation of the APD. A flattening of the T-wave was noted on the pseudo-ECG. In contrast, application of the IKr and Ito agonist, NS3623, resulted in hyperpolarization of the membrane, slowing of the spontaneous rate and shortening of the APD. Voltage clamp recording showed a significant increase in IKr; no enhancement of Ito in hiPSC-CMs was noted. AP clamp experiments revealed that IKr plays a role in both phase 3 repolarization and phase 4 depolarization. mRNA analysis revealed that KCNH2 is abundantly expressed in hiPSC-CM, consistent with electrophysiological recordings.CONCLUSIONS:Although NS3623 is a dual Ito and IKr activator in ventricular myocytes, application of this compound to hiPSC-CMs enhanced only IKr and no effect on Ito was noted. Our results suggest IKr enhancement can improve repolarization reserve in this cell type. The disconnect between a dramatic increase in Ito in adult myocytes versus the lack of effect in hiPSC-CMs suggest that the translation of pharmacological effects in hiPSC-CM to adult myocytes should be viewed with caution.

Recent efforts to ensure the reliability of computational model-based predictions in healthcare, such as the ASME V&V40 Standard, emphasize the importance of uncertainty quantification (UQ) and sensitivity analysis (SA) when evaluating computational models. UQ involves empirically determining the uncertainty in model inputs-typically resulting from natural variability or measurement error-and then calculating the resultant uncertainty in model outputs. SA involves calculating how uncertainty in model outputs can be apportioned to input uncertainty. Rigorous comprehensive UQ/SA provides confidence that model-based decisions are robust to underlying uncertainties. However, comprehensive UQ/SA is not currently feasible for whole heart models, due to numerous factors including model complexity and difficulty in measuring variability in the many parameters. Here, we present a significant step to developing a framework to overcome these limitations. We: (i) developed a novel action potential (AP) model of moderate complexity (six currents, seven variables, 36 parameters); (ii) prescribed input variability for all parameters (not empirically derived); (iii) used a single "hyper-parameter" to study increasing levels of parameter uncertainty; (iv) performed UQ and SA for a range of model-derived quantities with physiological relevance; and (v) present quantitative and qualitative ways to analyze different behaviors that occur under parameter uncertainty, including "model failure". This is the first time uncertainty in every parameter (including conductances, steady-state parameters, and time constant parameters) of every ionic current in a cardiac model has been studied. This approach allowed us to demonstrate that, for this model, the simulated AP is fully robust to low levels of parameter uncertainty - to our knowledge the first time this has been shown of any cardiac model. A range of dynamics was observed at larger parameter uncertainty (e.g., oscillatory dynamics); analysis revealed that five parameters were highly influential in these dynamics. Overall, we demonstrate feasibility of performing comprehensive UQ/SA for cardiac cell models and demonstrate how to assess robustness and overcome model failure when performing cardiac UQ analyses. The approach presented here represents an important and significant step toward the development of model-based clinical tools which are demonstrably robust to all underlying uncertainties and therefore more reliable in safety-critical decision-making.

Background: A loss of t-tubules and alterations in ultrastructure occur in cultured ventricular myocytes. A similar alteration in t-tubules and ultrastructure is well documented under certain pathological conditions such as heart failure. We examined the ultrastructural changes in cultured canine cardiac cells and determined the functional impact these changes have on excitation-contraction coupling and Ca 2+ transients. Materials, Methods and Results: Atrial, ventricle, and Purkinje myocytes were isolated by enzymatic dispersion. Atrial and ventricular myocytes were cultured for up to 48 h. Voltage clamp recordings were made with patch electrodes. Ca 2+ transient was recorded as a laser scanning confocal microscope in myocytes loaded with Ca 2+ fluorescent dyes. Membrane ultrastructure was imaged in myocytes stained with the membrane selective dye, di-8-ANEPP. Freshly isolated ventricular myocytes had a well-developed t-tubule system, while Purkinje cells had no t-tubules; some atrial cells exhibited a primitive t-tubule system. In atrial and Purkinje cells, the Ca 2+ transient had a U-shaped profile with the fluorescence highest at the edge of the cell. In contrast, ventricular myocytes showed a homogeneous rise in Ca 2+ at the edge and center of cells. Ventricular myocytes cultured for 2 days showed a nearly complete loss of t-tubules. The Ca 2+ transients revealed a phenotypic switch from a homogeneous profile to a “U”-shaped profile. Interestingly, atrial cells in culture maintained their primitive t-tubule system. Conclusions: Our results show that atrial and ventricular myocytes respond differently to being placed in culture. Ventricular myocytes, but not atrial myocytes, quickly lose their t-tubules accompanied by a Ca 2+ transient profile suggestive of a phenotypic switch in Ca 2+ handling. The differential response also suggests that the various cardiac tissue types would respond differently to pathophysiological stresses.

Downregulation of ion currents as well as a loss of T-tubules is documented during heart failure. A similar reduction of both has been observed in cultured ventricular myocytes. We further assessed the functional impact of cell-culture on excitation-contraction (EC) coupling in dog ventricular, atrial and Purkinje myocytes. Ventricular, atrial and Purkinje myocytes were isolated and cultured for up to 48 hours. Myocytes were stained with di-8-ANEPPS to visualize ultrastructure and Ca2+ transients (CaTs) were recorded by confocal microscopy. Ion channel currents were assessed via patch electrode voltage clamp. Membrane staining with di-8-ANEPPS indicated dog ventricular myocytes having an extensive T-tubular network; atrial myocytes having a rudimentary T-tubule system at best, and Purkinje myocytes having no T-tubules. Transverse X-t lines scans of electrically stimulated ventricular myocytes showed CaTs rising synchronously across the cell, whereas in Purkinje myocytes the CaT rise showed a U-shaped profile. CaT rise in atrial myocytes typically showed a U-shaped profile, but less pronounced than that in Purkinje myocytes. Ventricular myocytes cultured for 48 hours indicated a dramatic loss of T-tubules and a switch from a synchronous CaT rise to a U-shaped profile. In cultured atrial cells, no loss of T-tubules nor alterations in CaTs was noted. ICa in ventricular myocytes cultured for 48 hours was reduced by 21%. Comparative assessment of culture-induced changes in ion channel currents in atrial myocytes is ongoing. While cultured ventricular myocytes show a loss of T-tubules and phenotypic switch from a homogeneous CaT rise to a ‘U’-shaped profile, atrial myocytes cultured identically show little change in ultrastructure and CaT profile. These results suggest that character and degree of change in EC coupling induced by similar pathological insults markedly depends on cardiac myocyte type.

Introduction: The use of physiological computational models in biomedical applications is rapidly increasing. Physiological models are used in the design and evaluation of medical devices, as well as integrated into devices, or may even be the device itself [1]. Moreover, in silico clinical trials, as advanced by CDRH, require the development of reliable models of human physiology. Recent efforts to ensure the reliability of computational modeling in biomedical applications, such as the new ASME V&V40 Standard, emphasize the importance of uncertainty quantification (UQ) when assessing models. UQ involves determining the uncertainty in model inputs and then calculating the resultant uncertainty in model outputs. However, there is a disconnect between expectations in the V&V40 Standard/related documents, and what is feasible for physiological models, which typically have large numbers of parameters (often hundreds). One challenge is the experimental difficulty to characterizing uncertainty (including biological variability) in physiological parameters. Another is the high likelihood that many physiological models will fail in some way when uncertainty is integrated into the model, and the path forward if that occurs is unclear. The first aim of this work was to develop a novel model of cardiac electrophysiology and apply analytic methods not commonly used in cardiac/medical device modeling to demonstrate feasibility of comprehensive UQ for physiological models. A second aim to investigate how failure occurs in such models upon integration of uncertainty, and how can it can be handled.

Brugada syndrome (BrS) is an inherited disease associated with ST elevation in the right precordial leads, polymorphic ventricular tachycardia (PVT), and sudden cardiac death in adults. Mutations in the cardiac sodium channel account for a large fraction of BrS cases. BrS manifests in the right ventricle (RV), which led us to examine the biophysical and molecular properties of sodium channel in myocytes isolated from the left (LV) and right ventricle. Patch clamp was used to record sodium current (INa ) in single canine RV and LV epicardial (epi) and endocardial (endo) myocytes. Action potentials were recorded from multicellular preparations and single cells. mRNA and proteins were determined using quantitative RT-PCR and Western blot. Although LV wedge preparations were thicker than RV wedges, transmural ECG recordings showed no difference in the width of the QRS complex or transmural conduction time. Action potential characteristics showed RV epi and endo had a lower Vmax compared with LV epi and endo cells. Peak INa density was significantly lower in epi and endo RV cells compared with epi and endo LV cells. Recovery from inactivation of INa in RV cells was slightly faster and half maximal steady-state inactivation was more positive. β2 and β4 mRNA was detected at very low levels in both ventricles, which was confirmed at the protein level. Our observations demonstrate that Vmax and Na+ current are smaller in RV, presumably due to differential Nav 1.5/β subunit expression. These results provide a potential mechanism for the right ventricular manifestation of BrS.