Frequency-dependent drug screening using optogenetic stimulation of human iPSC-derived cardiomyocytes.

Side effects on cardiac ion channels are one major reason for new drugs to fail during preclinical evaluation. Herein we propose a simple optogenetic screening tool measuring extracellular field potentials (FP) from paced cardiomyocytes to identify drug effects over the whole physiological heart range, which is essential given the rate-dependency of ion channel function and drug action. Human induced pluripotent stem cell-derived cardiomyocytes were transduced with an adeno-associated virus to express Channelrhodopsin2 and plated on micro-electrode arrays. Global pulsed illumination (470 nm, 1 ms, 0.9 mW/mm2) was applied at frequencies from 1 to 2.5 Hz, which evoked FP simultaneously in all cardiomyocytes. This synchronized activation allowed averaging of FP from all electrodes resulting in one robust FP signal for analysis. Field potential duration (FPD) was ~25% shorter at 2.5 Hz compared to 1 Hz. Inhibition of hERG channels prolonged FPD only at low heart rates whereas Ca2+ channel block shortened FPD at all heart rates. Optogenetic pacing also allowed analysis of the maximum downstroke velocity of the FP to detect drug effects on Na+ channel availability. In principle, the presented method is well scalable for high content cardiac toxicity screening or personalized medicine for inherited cardiac channelopathies.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

Modeling long QT syndromes using induced pluripotent stem cells: current progress and future challenges.

Long QT syndromes (LQTS) are a family of inherited monogenetic disorders caused by gain or loss-of-function mutations of cardiac ion channels and are characterized by a prolonged QT interval in the ECG. The disease-specific mutations lead to prolonged action potential durations and early after-depolarizations in cardiomyocytes potentially giving rise to triggered extrabeats and life-threatening arrhythmias in patients. The generation of induced pluripotent stem cells from somatic cells of patients and their differentiation into cardiomyocytes represents a powerful method enabling the investigation of disease-specific cardiomyocytes. In this review we highlight the latest progress in the generation of long QT syndrome-specific induced pluripotent stem cells and cardiomyocytes to investigate the disease in vitro. We also point out future challenges that need to be addressed to allow drug screening using patient-specific cardiomyocytes.

Cardiomyocytes obtained from induced pluripotent stem cells with long-QT syndrome 3 recapitulate typical disease-specific features in vitro.

RATIONALE: Current approaches for the investigation of long-QT syndromes (LQTS) are mainly focused on identification of the mutation and its characterization in heterologous expression systems. However, it would be extremely helpful to be able to characterize the pathophysiological effects of mutations and to screen drugs in cardiomyocytes. OBJECTIVE: The aim of this study was to establish as a proof of principle the disease-specific cardiomyocytes from a mouse model with LQTS 3 by use of induced pluripotent stem (iPS) cells and to demonstrate that the mutant cardiomyocytes display the characteristic pathophysiological features in vitro. METHODS AND RESULTS: We generated disease-specific iPS cells from a mouse model with a human mutation of the cardiac Na(+) channel that causes LQTS 3. The control and LQTS 3-specific iPS cell lines were pluripotent and could be differentiated into spontaneously beating cardiomyocytes. Patch-clamp measurements of LQTS 3-specific cardiomyocytes showed the biophysical effects of the mutation on the Na(+) current, with faster recovery from inactivation and larger late currents than observed in controls. Moreover, LQTS 3-specific cardiomyocytes had prolonged action potential durations and early afterdepolarizations at low pacing rates, both of which are classic features of the LQTS 3 mutation. CONCLUSIONS: We demonstrate that disease-specific iPS cell-derived cardiomyocytes from an LQTS 3 mouse model with a human mutation recapitulate the typical pathophysiological phenotype in vitro. Thus, this method is a powerful tool to investigate disease mechanisms in vitro and to perform patient-specific drug screening.