Ravichandra Venkateshappa

All Publications

• Clinical trials in a dish: cardiometabolic drug development with biological and digital twins. The Journal of clinical investigation Patra, D., Sayed, I. M., Venkateshappa, R., Palaniappan, L., McLaughlin, T., Wu, J. C. 2026; 136 (19)

  Abstract

  Preclinical drug development has long relied on animal models to predict safety and efficacy before agents enter human trials, despite critical differences between human and animal model physiology. The withdrawal of rosiglitazone, rofecoxib, and terfenadine due to cardiovascular toxicity exemplifies the translational cost of this mismatch. Alternative, human-based systems enable more accurate modeling of cardiometabolic diseases in a dish; in 2025, the US FDA's new approach methodologies (NAMs) roadmap authorized the submission of results from human-relevant models. The roadmap encourages utilizing biological and digital twins as part of an integrated, context-specific, fit-for-purpose strategy. A "biological twin" is a human-derived in vitro system that captures the physiology of a patient and can be used to assess potential cardiotoxicity by drug metabolites. A "digital twin" is the computational counterpart trained on clinical drug response results that can further interpret biological twin data at the patient scale and predict pharmacological parameters. NAMs are no longer experimental but are not yet fully validated as replacements for animal models; major challenges remain before they can be effectively incorporated into the cardiometabolic disease drug discovery pipeline. Addressing these challenges head-on is essential for improving drug development and prediction of their cardiovascular safety.

  View details for DOI 10.1172/JCI209668

  View details for PubMedID 42820291

• Modeling Heart Failure With Preserved Ejection Fraction Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circulation Yu, B., Ren, L., Liu, Y., Tu, C., Kojic, A., Dai, Y., Venkateshappa, R., Shen, M., Zhao, S. R., Wu, J. C. 2026; 154 (13): 1261-1264

  View details for DOI 10.1161/CIRCULATIONAHA.126.081881

  View details for PubMedID 42804568

• RUNX1-driven endothelial-to-mesenchymal transition contributes to remodelling in LMNA cardiomyopathy. European heart journal Wu, D., Tripathi, D., Manhas, A., Noishiki, C., Liu, L., Wu, C. A., Venkateshappa, R., Zhang, H., Ren, L., Thomas, D., Nalbandian, M., Limbu, L., DaValle, C. C., Rangan, E. S., Nallamshetty, S., Boyd, J. H., Malik, S. B., Woo, Y. J., Sayed, D., Sallam, K., Blau, H. M., Wu, J. C., Sayed, N. 2026

  Abstract

  LMNA-related dilated cardiomyopathy (LMNA-DCM) is a progressive genetic disorder characterized by conduction disease, malignant arrhythmias, myocardial fibrosis, and heart failure. Although LMNA mutations have traditionally been associated with cardiomyocyte-intrinsic defects, the mechanisms driving fibrotic remodelling remain incompletely understood.Spatial transcriptomics and integrated single-nuclei multiomics were performed on explanted human LMNA-DCM hearts to define endothelial transcriptional and epigenomic states associated with fibrosis. Patient-specific induced pluripotent stem cell-derived endothelial cells, engineered cardiac organoids, and the LMNAH222P/H222P mouse model were used to investigate RUNX1-mediated endothelial-to-mesenchymal transition (EndoMT). Genetic and pharmacological RUNX1 inhibition strategies were evaluated in vitro and in vivo.Endothelial populations exhibiting EndoMT-associated transcriptional and epigenomic signatures were identified in human LMNA-DCM hearts. LMNA induced pluripotent stem cell-derived endothelial cells demonstrated endothelial dysfunction, mesenchymal gene activation, and epigenetic activation of RUNX1 following loss of LMNA-mediated repression. Genetic RUNX1 deletion restored endothelial identity, reversed EndoMT-associated transcriptional programmes, and normalized chromatin accessibility at endothelial regulatory loci. In multicellular cardiac organoids, endothelial RUNX1 activation impaired endothelial-cardiomyocyte signalling and cardiomyocyte contractile function, whereas endothelial-specific RUNX1 deletion restored endothelial and myocardial function. Pharmacological RUNX1 inhibition with Ro24-7429 similarly improved endothelial and cardiomyocyte function in vitro and reduced myocardial fibrosis while preserving cardiac function in LMNAH222P/H222P mice, including after disease onset.RUNX1-driven EndoMT represents a central mechanism linking LMNA mutations to fibrotic remodelling in LMNA cardiomyopathy. These findings support endothelial transcriptional reprogramming and RUNX1 signalling as potential therapeutic targets in fibrotic cardiomyopathy.

  View details for DOI 10.1093/eurheartj/ehag619

  View details for PubMedID 42578927

• Profiling Immune-Independent Response to Immune Checkpoint Inhibitors on Stem Cell-Derived Cardiomyocytes, Organoids, and Mouse Models. Circulation Thomas, D., Manhas, A., Liu, Y., Venkateshappa, R., Belbachir, N., Zhao, S. R., Juguilon, C., Chen, I. Y., Moslehi, J., Sayed, N., Wu, J. C. 2026; 153 (2): 132-135

  View details for DOI 10.1161/CIRCULATIONAHA.125.076926

  View details for PubMedID 41525439

• Generation of two induced pluripotent stem cell lines from patients with Facioscapulohumeral muscular dystrophy. Stem cell research Venkateshappa, R., Vacante, F., Xu, L., Canel-Rivero, G., Day, J. W., Wu, J. C. 2025; 87: 103794

  Abstract

  Facioscapulohumeral muscular dystrophy (FSHD) is a genetically complex condition marked by progressive skeletal muscle weakness, primarily affecting the face, shoulders, and upper arms. Here, we generated human induced pluripotent stem cell (iPSC) lines from two clinically diagnosed FSHD patients and characterized their pluripotency and germline markers. Both lines exhibited pluripotency markers, maintained normal karyotypes, and were capable of differentiating into all three germ layers. These iPSC lines are valuable resources for studying FSHD mechanisms and potential drug discovery applications.

  View details for DOI 10.1016/j.scr.2025.103794

  View details for PubMedID 40773824

• Gastruloids enable modeling of the earliest stages of human cardiac and hepatic vascularization. Science (New York, N.Y.) Abilez, O. J., Yang, H., Guan, Y., Shen, M., Yildirim, Z., Zhuge, Y., Venkateshappa, R., Zhao, S. R., Gomez, A. H., El-Mokahal, M., Dunkenberger, L., Ono, Y., Shibata, M., Nwokoye, P. N., Tian, L., Wilson, K. D., Lyall, E. H., Jia, F., Wo, H. T., Zhou, G., Aldana, B., Karakikes, I., Obal, D., Peltz, G., Zarins, C. K., Wu, J. C. 2025; 388 (6751): eadu9375

  Abstract

  Although model organisms have provided insight into the earliest stages of cardiac and hepatic vascularization, we know very little about this process in humans because of ethical restrictions and the technical difficulty of obtaining embryos during very early development. In this study, we demonstrate that micropatterned human pluripotent stem cell-derived gastruloids enable in vitro modeling of the earliest stages of vascularization. We identify a combination of vascular-inducing factors that give rise to cardiac vascularized organoids with a spatially organized and branched vascular network. To show the broader utility of our vascularization strategy, we use the same vascular-inducing factors to produce hepatic vascularized organoids. Our results suggest that a conserved developmental program generates the vasculature within different types of organs.

  View details for DOI 10.1126/science.adu9375

  View details for PubMedID 40472086

• Protocol to study electrophysiological properties of hPSC-derived 3D cardiac organoids using MEA and sharp electrode techniques. STAR protocols Venkateshappa, R., Yildirim, Z., Zhao, S. R., Wu, M. A., Vacante, F., Abilez, O. J., Wu, J. C. 2024; 5 (4): 103406

  Abstract

  Continuing advancements in human pluripotent stem cell (hPSC)-derived complex three-dimensional (3D) cardiac tissues require the development of novel technologies or adaptation of existing technologies to understand the physiology of the derived 3D cardiac tissues. In this protocol, we describe the use of multielectrode array (MEA) and sharp electrode electrophysiology techniques to investigate the electrical properties of 3D cardiac organoids. This protocol deciphers the electrical behavior of 3D cardiac organoids at both the single-cell level and tissue level.

  View details for DOI 10.1016/j.xpro.2024.103406

  View details for PubMedID 39514393

• Generation of Marfan syndrome-specific induced pluripotent stem cells harboring FBN1 mutations. Stem cell research Vacante, F., Venkateshappa, R., Htet, M., Yan, C., Wu, J. C. 2024; 80: 103518

  Abstract

  Marfan syndrome (MFS) is a hereditary condition caused by mutations in the FBN1 gene. Genetic mutations in the FBN1 locus impact the function of the encoded protein, Fibrillin 1, a structural molecule forming microfibrils found in the connective tissue. MFS patients develop severe cardiovascular complications including thoracic aortic aneurysm and aortic dissection, which predispose them to an enhanced risk of premature death. Here, we generated two induced pluripotent stem cell (iPSC) lines harboring mutations in the FBN1 gene (p.C1942C>A and c.1954 T>C), directly derived from MFS patients. We have shown that both iPSC lines displayed expression of pluripotency markers, normal karyotype and ability of trilineage differentiation, representing a valuable tool for the identification of new therapeutic strategies for intervening in this disease.

  View details for DOI 10.1016/j.scr.2024.103518

  View details for PubMedID 39096853

• Development of an Absolute Quantification Method for hERG Using PRM with Single Isotopologue in-Sample Calibration ACS OMEGA Chang, G., Aroge, F. A., Venkateshappa, R., Claydon, T. W., Sun, B. 2024; 9 (31): 33972-33982

  Abstract

  The human ether-à-go-go-related gene (KCNH2)-encoded protein hERG constitutes the α subunit of the Kv11.1 channel and contributes to the I kr current, which plays an important role in the cardiac action potential. Genetically and xenobiotically triggered malfunctions of hERG can cause arrhythmia. The expression of hERG in various study systems was assessed mainly as the fold change relative to the corresponding control. Here, we developed a simple and sensitive quantitation method using targeted mass spectrometry, i.e., the parallel reaction monitoring approach, to measure the absolute quantity of hERG in copy number. Such measurements do not require controls, and the obtained values can be compared with similar results for any other protein. To effectively avoid matrix effects, we used the heavy-match-light (HML) in-sample calibration approach that requires only a single isotopologue to achieve copy-number quantitation. No significant difference was observed in the results obtained by HML and by the classic standard addition in-sample calibration approach. Using four proteotypic peptides, we quantified the average number of copies of hERG in the HEK293T heterologous expression system as 3.6 ± 0.5 × 106 copies/cell, i.e., 1 million copies/cell for the fully assembled Kv11.1 channel.

  View details for DOI 10.1021/acsomega.4c04541

  View details for Web of Science ID 001277914100001

  View details for PubMedID 39130540

  View details for PubMedCentralID PMC11308013

• Generation and characterization of induced pluripotent stem cells from breast cancer patients carrying ATM mutations. Stem cell research Zhang, M., Venkateshappa, R., Li, A., Fowler, M. B., Telli, M. L., Wu, J. C. 2023; 73: 103246

  Abstract

  We generated two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of breast cancer patients carrying germline ATM mutations, a gene associated with a 7% prevalence in breast cancer. These iPSC lines displayed typical morphology, expressed pluripotency markers, maintained a stable karyotype, and retained the ability to differentiate into the three germ layers. These patient-specific iPSC lines hold great potential for mechanistic investigations and the development of drug screening strategies aimed at addressing ATM-related cancer.

  View details for DOI 10.1016/j.scr.2023.103246

  View details for PubMedID 37951143

• Targeted activation of hERG channels rescues electrical instability induced by the hERG R56Q+/- Long QT Syndrome variant. Cardiovascular research Venkateshappa, R., Hunter, D. V., Muralidharan, P., Nagalingam, R. S., Huen, G., Faizi, S., Luthra, S., Lin, E., Cheng, Y. M., Hughes, J., Khelifi, R., Dhunna, P., Johal, R., Sergeev, V., Shafaattalab, S., Julian, L. M., Poburko, D. T., Laksman, Z., Tibbits, G. F., Claydon, T. W. 2023

  Abstract

  Long QT Syndrome Type 2 (LQTS2) is associated with inherited variants in the cardiac hERG K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant, and its phenotypic rescue by the type 1 hERG activator, RPR260243.These approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean APD90. However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of action potential duration (APD90) during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness.Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs.

  View details for DOI 10.1093/cvr/cvad155

  View details for PubMedID 37739930

• CRISPR-Cas9-mediated Precise Knock-in Edits in Zebrafish Hearts. Journal of visualized experiments : JoVE Simpson, K. E., Faizi, S., Venkateshappa, R., Yip, M., Johal, R., Poburko, D., Cheng, Y. M., Hunter, D., Lin, E., Tibbits, G. F., Claydon, T. W. 2022

  Abstract

  Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

  View details for DOI 10.3791/64209

  View details for PubMedID 36190280

• Ion channel model reduction using manifold boundaries. Journal of the Royal Society, Interface Whittaker, D. G., Wang, J., Shuttleworth, J. G., Venkateshappa, R., Kemp, J. M., Claydon, T. W., Mirams, G. R. 2022; 19 (193): 20220193

  Abstract

  Mathematical models of voltage-gated ion channels are used in basic research, industrial and clinical settings. These models range in complexity, but typically contain numerous variables representing the proportion of channels in a given state, and parameters describing the voltage-dependent rates of transition between states. An open problem is selecting the appropriate degree of complexity and structure for an ion channel model given data availability. Here, we simplify a model of the cardiac human Ether-à-go-go related gene (hERG) potassium ion channel, which carries cardiac IKr, using the manifold boundary approximation method (MBAM). The MBAM approximates high-dimensional model-output manifolds by reduced models describing their boundaries, resulting in models with fewer parameters (and often variables). We produced a series of models of reducing complexity starting from an established five-state hERG model with 15 parameters. Models with up to three fewer states and eight fewer parameters were shown to retain much of the predictive capability of the full model and were validated using experimental hERG1a data collected in HEK293 cells at 37°C. The method provides a way to simplify complex models of ion channels that improves parameter identifiability and will aid in future model development.

  View details for DOI 10.1098/rsif.2022.0193

  View details for PubMedID 35946166

  View details for PubMedCentralID PMC9363999

• Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243. The Journal of general physiology Kemp, J. M., Whittaker, D. G., Venkateshappa, R., Pang, Z., Johal, R., Sergeev, V., Tibbits, G. F., Mirams, G. R., Claydon, T. W. 2021; 153 (10)

  Abstract

  Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.

  View details for DOI 10.1085/jgp.202112923

  View details for PubMedID 34398210

  View details for PubMedCentralID PMC8493834

• Utility of Zebrafish Models of Acquired and Inherited Long QT Syndrome FRONTIERS IN PHYSIOLOGY Simpson, K. E., Venkateshappa, R., Pang, Z., Faizi, S., Tibbits, G. F., Claydon, T. W. 2021; 11: 624129

  Abstract

  Long-QT Syndrome (LQTS) is a cardiac electrical disorder, distinguished by irregular heart rates and sudden death. Accounting for ∼40% of cases, LQTS Type 2 (LQTS2), is caused by defects in the Kv11.1 (hERG) potassium channel that is critical for cardiac repolarization. Drug block of hERG channels or dysfunctional channel variants can result in acquired or inherited LQTS2, respectively, which are typified by delayed repolarization and predisposition to lethal arrhythmia. As such, there is significant interest in clear identification of drugs and channel variants that produce clinically meaningful perturbation of hERG channel function. While toxicological screening of hERG channels, and phenotypic assessment of inherited channel variants in heterologous systems is now commonplace, affordable, efficient, and insightful whole organ models for acquired and inherited LQTS2 are lacking. Recent work has shown that zebrafish provide a viable in vivo or whole organ model of cardiac electrophysiology. Characterization of cardiac ion currents and toxicological screening work in intact embryos, as well as adult whole hearts, has demonstrated the utility of the zebrafish model to contribute to the development of therapeutics that lack hERG-blocking off-target effects. Moreover, forward and reverse genetic approaches show zebrafish as a tractable model in which LQTS2 can be studied. With the development of new tools and technologies, zebrafish lines carrying precise channel variants associated with LQTS2 have recently begun to be generated and explored. In this review, we discuss the present knowledge and questions raised related to the use of zebrafish as models of acquired and inherited LQTS2. We focus discussion, in particular, on developments in precise gene-editing approaches in zebrafish to create whole heart inherited LQTS2 models and evidence that zebrafish hearts can be used to study arrhythmogenicity and to identify potential anti-arrhythmic compounds.

  View details for DOI 10.3389/fphys.2020.624129

  View details for Web of Science ID 000612656700001

  View details for PubMedID 33519527

  View details for PubMedCentralID PMC7844309

• The hERG channel activator, RPR260243, enhances protective IKr current early in the refractory period reducing arrhythmogenicity in zebrafish hearts. American journal of physiology. Heart and circulatory physiology Shi, Y. P., Pang, Z., Venkateshappa, R., Gunawan, M., Kemp, J., Truong, E., Chang, C., Lin, E., Shafaattalab, S., Faizi, S., Rayani, K., Tibbits, G. F., Claydon, V. E., Claydon, T. W. 2020; 319 (2): H251-H261

  Abstract

  Human ether-à-go-go related gene (hERG) K+ channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach.NEW & NOTEWORTHY hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.

  View details for DOI 10.1152/ajpheart.00038.2020

  View details for PubMedID 32559136