Bipolar Patient-Specific In Vitro Diagnostic Test Reveals Underlying Cardiac Arrhythmia Phenotype Caused by Calcium Channel Genetic Risk Factor.

A common genetic risk factor for bipolar disorder is CACNA1C, a gene that is also critical for cardiac rhythm. The impact of CACNA1C mutations on bipolar patient cardiac rhythm is unknown. Here, we report the cardiac electrophysiological implications of a bipolar disorder-associated genetic risk factor in CACNA1C using patient induced pluripotent stem cell-derived cardiomyocytes. Results indicate that the CACNA1C bipolar disorder-related mutation causes cardiac electrical impulse conduction slowing mediated by impaired intercellular coupling via connexin 43 gap junctions. In vitro gene therapy to restore connexin 43 expression increased cardiac electrical impulse conduction velocity and protected against thioridazine-induced QT prolongation. Patients positive for bipolar disorder CACNA1C genetic risk factors may have elevated proarrhythmic risk for adverse events in response to psychiatric medications that slow conduction or prolong the QT interval. This in vitro diagnostic tool enables cardiac testing specific to patients with psychiatric disorders to determine their sensitivity to off-target effects of psychiatric medications.

Bipolar disorder (BD) is associated with genetic risk factors that present as mutations in specific genes. One gene commonly associated with BD is the calcium channel gene CACNA1C, found in the brain and the heart. The impact of CACNA1C mutation on cardiac function in patients with BD is unclear. Here, we created a BD CACNA1C mutant patient "heart in a dish" using patient-specific stem cells. Gene editing was also used to correct the mutation to create an isogenic control cell line. We found that the BD calcium gene mutation caused slow electrical impulse propagation, reduced the function of the calcium channel, and was associated with low intercellular communication channels called connexin. Using connexin gene therapy in vitro, the the cardiac dysfunction could be corrected and cured. This new approach offers patient-specific hearts-in-a-dish that can be used to ensure that medications will not cause heart racing or arrhythmias.

High-throughput longitudinal electrophysiology screening of mature chamber-specific hiPSC-CMs using optical mapping.

hiPSC-CMs are being considered by the Food and Drug Administration and other regulatory agencies for in vitro cardiotoxicity screening to provide human-relevant safety data. Widespread adoption of hiPSC-CMs in regulatory and academic science is limited by the immature, fetal-like phenotype of the cells. Here, to advance the maturation state of hiPSC-CMs, we developed and validated a human perinatal stem cell-derived extracellular matrix coating applied to high-throughput cell culture plates. We also present and validate a cardiac optical mapping device designed for high-throughput functional assessment of mature hiPSC-CM action potentials using voltage-sensitive dye and calcium transients using calcium-sensitive dyes or genetically encoded calcium indicators (GECI, GCaMP6). We utilize the optical mapping device to provide new biological insight into mature chamber-specific hiPSC-CMs, responsiveness to cardioactive drugs, the effect of GCaMP6 genetic variants on electrophysiological function, and the effect of daily ß-receptor stimulation on hiPSC-CM monolayer function and SERCA2a expression.

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers.

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins.

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

In vitro model of ischemic heart failure using human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here, our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated into functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the nonmyocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures that had more than 94% cTnT+ cells. After purification, hiPSC-CMs were replated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. The phenotype of metabolic stress-selected hiPSC-CM monolayers recapitulated many of the functional and structural hallmarks of ischemic CMs, including elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy-induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate, and abnormal response to ß1-adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generated cell populations with phenotype similar to what is well known to occur in the setting of ischemic heart failure and thus provide a opportunity for study of human ischemic heart disease.

Human perinatal stem cell derived extracellular matrix enables rapid maturation of hiPSC-CM structural and functional phenotypes.

The immature phenotype of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is a major limitation to the use of these valuable cells for pre-clinical toxicity testing and for disease modeling. Here we tested the hypothesis that human perinatal stem cell derived extracellular matrix (ECM) promotes hiPSC-CM maturation to a greater extent than mouse cell derived ECM. We refer to the human ECM as Matrix Plus (Matrix Plus) and compare effects to commercially available mouse ECM (Matrigel). hiPSC-CMs cultured on Matrix Plus mature functionally and structurally seven days after thaw from cryopreservation. Mature hiPSC-CMs showed rod-shaped morphology, highly organized sarcomeres, elevated cTnI expression and mitochondrial distribution and function like adult cardiomyocytes. Matrix Plus also promoted mature hiPSC-CM electrophysiological function and monolayers' response to hERG ion channel specific blocker was Torsades de Pointes (TdP) reentrant arrhythmia activations in 100% of tested monolayers. Importantly, Matrix Plus enabled high throughput cardiotoxicity screening using mature human cardiomyocytes with validation utilizing reference compounds recommended for the evolving Comprehensive In Vitro Proarrhythmia Assay (CiPA) coordinated by the Health and Environmental Sciences Institute (HESI). Matrix Plus offers a solution to the commonly encountered problem of hiPSC-CM immaturity that has hindered implementation of these human based cell assays for pre-clinical drug discovery.

Detection of Drug-Induced Torsades de Pointes Arrhythmia Mechanisms Using hiPSC-CM Syncytial Monolayers in a High-Throughput Screening Voltage Sensitive Dye Assay.

We validated 3 distinct hiPSC-CM cell lines-each of different purity and a voltage sensitive dye (VSD)-based high-throughput proarrhythmia screening assay as a noncore site in the recently completed CiPA Myocyte Phase II Validation Study. Blinded validation was performed using 12 drugs linked to low, intermediate, or high risk for causing Torsades de Pointes (TdP). Commercially sourced hiPSC-CMs were obtained either from Cellular Dynamics International (CDI, Madison, Wisconsin, iCell Cardiomyoyctes2) or Takara Bio (CLS, Cellartis Cardiomyocytes). A third hiPSC-CM cell line (MCH, Michigan) was generated in house. Each cell type had distinct baseline electrophysiological function (spontaneous beat rate, action potential duration, and conduction velocity) and drug responsiveness. Use of VSD and optical mapping enabled the detection of conduction slowing of sodium channel blockers (quinidine, disopyramide, and mexiletine) and drug-induced TdP-like activation patterns (rotors) for some high- and intermediate-risk compounds. Low-risk compounds did not induce rotors in any cell type tested. These results further validate the utility of hiPSC-CMs for predictive proarrhythmia screening and the utility of VSD technology to detect drug-induced APD prolongation, arrhythmias (rotors), and conduction slowing. Importantly, results indicate that different ratios of cardiomyocytes and noncardiomyocytes have important impact on drug response that may be considered during risk assessment of new drugs. Finally, we present the first blinded CiPA hiPSC-CM validation results to simultaneously detect drug-induced conduction slowing, action potential duration prolongation, action potential triangulation, and drug-induced rotors in a proarrhythmia assay.

HDAC inhibitor valproic acid protects heart function through Foxm1 pathway after acute myocardial infarction.

BACKGROUND: Epigenetic histone acetylation is a major event controlling cell functions, such as metabolism, differentiation and repair. Here, we aim to determine whether Valproic acid (VPA), a FDA approved inhibitor of histone deacetylation for bipolar disease, could protect heart against myocardial infarction (MI) injury and elucidate key molecular pathways. METHODS: VPA was administrated to MI rats at different time points, onset and after MI injury. Echocardiography, histology, serum biology assays, and gene expression, inhibition, and over-expression were performed to characterize the systolic function, infarct size, gene and signaling pathways. FINDINGS: VPA treatment reduced the infarct size by ~50% and preserved the systolic function of heart after acute MI in rats. Even 60 min after infarction, VPA treatment significantly decreased infarct size. Furthermore, long-term treatment of VPA markedly improved myocardial performance. VPA regulated gene expression essential for cell survival and anti-inflammatory response. Consequently, oxidative stress and cell death were notably reduced after VPA treatment. Moreover, Foxm1 was identified as a potential key target of VPA. Overexpression of Foxm1 provided similar heart protective effect to VPA treatment. Particularly, both VPA treatment and Foxm1 over-expression repressed inflammatory response after MI for heart protection. In contrast, inhibition of Foxm1 activity abolished the cardiac protective effect of VPA. VPA mediated CM protection through Foxm1 upregulation was also identified in a human ESC derived CM hypoxia/reperfusion system. INTERPRETATION: VPA treatments significantly reduce cardiac damage after MI and the cardioprotective effect of VPA is likely mediated via Foxm1 pathway. FUND: This work was mainly supported by 1R01HL109054.