Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays.

BACKGROUND: Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities. METHODS AND RESULTS: Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls. CONCLUSIONS: We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells.

Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca(2+)) imaging indicated dysregulation of Ca(2+) cycling and elevation in intracellular Ca(2+) ([Ca(2+)](i)) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca(2+) homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy.

Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a ß-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with ß-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

On the effect of prestin on the electrical breakdown of cell membranes.

The voltage-dependent activity of prestin, the outer hair cell (OHC) motor protein essential for its electromotility, enhances the mammalian inner ear's auditory sensitivity. We investigated the effect of prestin's activity on the plasma membrane's (PM) susceptibility to electroporation (EP) via cell-attached patch-clamping. Guinea pig OHCs, TSA201 cells, and prestin-transfected TSA cells were subjected to incremental 50 mus and/or 50 ms voltage pulse trains, or ramps, at rates from 10 V/s to 1 kV/s, to a maximum transmembrane potential of +/-1000 mV. EP was determined by an increase in capacitance to whole-cell levels. OHCs were probed at the prestin-rich lateral PM or prestin-devoid basal portion; TSA cells were patched at random points. OHCs were consistently electroporated with 50 ms pulses, with significant resistance to depolarizing pulses. Although EP rarely occurred with 50 mus pulses, prior stimulation with this protocol had a significant effect on the sensitivity to EP with 50 ms pulses, regardless of polarity or PM domain. Consistent with these results, resistance to EP with depolarizing 10-V/s ramps was also found. Our findings with TSA cells were comparable, showing resistance to EP with both depolarizing 50-ms pulses and 10 V/s ramps. We conclude prestin significantly affects susceptibility to EP, possibly via known biophysical influences on specific membrane capacitance and/or membrane stiffness.