Abstract
Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are increasingly recognized as valuable for determining the effects of drugs on ion channels but they do not always accurately predict contractile responses of the human heart. This is in part attributable to their immaturity but the sensitivity of measurement tools may also be limiting. Measuring action potential, calcium flux or contraction individually misses critical information that is captured when interrogating the complete excitation-contraction coupling cascade simultaneously. Here, we develop an hypothesis-based statistical algorithm that identifies mechanisms of action. We design and build a high-speed optical system to measure action potential, cytosolic calcium and contraction simultaneously using fluorescent sensors. These measurements are automatically processed, quantified and then assessed by the algorithm. Multiplexing these three critical physical features of hiPSC-CMs allows identification of all major drug classes affecting contractility with detection sensitivities higher than individual measurement of action potential, cytosolic calcium or contraction.
Highlights
Cardiomyocytes from human induced pluripotent stem cells are increasingly recognized as valuable for determining the effects of drugs on ion channels but they do not always accurately predict contractile responses of the human heart
This is a complex cascade of ion channel activities, internal calcium storage and contractile filament availability with the action potential (AP), cytosolic calcium flux (Ca) and the actual contraction (Co) being the most important physical transients[6]
The effect of some of these cardioactive drugs would not be expected in hiPSC-CM cultures while often the effect of other drugs would be expected but is undetectable because of hiPSC-CM immaturity[7]
Summary
Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are increasingly recognized as valuable for determining the effects of drugs on ion channels but they do not always accurately predict contractile responses of the human heart. These measurements are automatically processed, quantified and assessed by the algorithm Multiplexing these three critical physical features of hiPSC-CMs allows identification of all major drug classes affecting contractility with detection sensitivities higher than individual measurement of action potential, cytosolic calcium or contraction. We engineer a high-speed system capable of measuring and quantifying AP, Ca and Co simultaneously by means of fluorescent sensors ANNINE6plus, Rhod-3 and CellMask Deep Red in combination with our recently developed optical contraction software tool MUSCLEMOTION12 Using this Triple Transient Measurement (TTM) system we measure and quantify the response of commercial hiPSC-CMs to a set of drugs that covers the complete range of potentially affected contractile mechanisms. We find that simultaneous TTM in combination with our algorithm allows automatic and unbiased identification of the MOAs affected by the test set of compounds and increased the sensitivity of detecting contractile MOAs compared to separate assessment of AP, Ca and Co
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