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Single-Cell Mechanical Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Testing and Pathophysiological Studies.

ABSTRACT: Current platforms for studying the mechanical properties of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as single cells do not measure forces directly, require numerous assumptions, and cannot study cell mechanics at different loading conditions. We present a method for directly measuring the active and passive forces generated by single-cell hPSC-CMs at different stretch levels. Utilizing this technique, single hPSC-CMs exhibited positive length-tension relationship and appropriate inotropic, klinotropic, and lusitropic changes in response to pharmacological treatments (isoproterenol and verapamil). The unique potential of the approach for drug testing and disease modeling was exemplified by doxorubicin and omecamtiv mecarbil drug studies revealing their known actions to suppress (doxorubicin) or augment (omecamtiv mecarbil at low dose) cardiomyocyte contractility, respectively. Finally, mechanistic insights were gained regarding the cellular effects of these drugs as doxorubicin treatment led to cellular mechanical alternans and high doses of omecamtiv mecarbil suppressed contractility and worsened the cellular diastolic properties.

SUBMITTER: Ballan N

PROVIDER: S-EPMC7486198 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Single-Cell Mechanical Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Testing and Pathophysiological Studies.

Ballan Nimer N Shaheen Naim N Keller Gordon M GM Gepstein Lior L

Stem cell reports 20200806 3

Current platforms for studying the mechanical properties of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as single cells do not measure forces directly, require numerous assumptions, and cannot study cell mechanics at different loading conditions. We present a method for directly measuring the active and passive forces generated by single-cell hPSC-CMs at different stretch levels. Utilizing this technique, single hPSC-CMs exhibited positive length-tension relationship and approp ...[more]

PMID: 32763158

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Project description:Cardiotoxicity is a major cause of high attrition rates among newly developed drugs. Moreover, anti-cancer treatment-induced cardiotoxicity is one of the leading reasons of mortality in cancer survivors. Cardiotoxicity screening in vitro may improve predictivity of cardiotoxicity by novel drugs, using human pluripotent stem cell (hPSC)-derived-cardiomyocytes. Anthracyclines, including Doxorubicin, are widely used and highly effective chemotherapeutic agents for the treatment of different forms of malignancies. Unfortunately, anthracyclines cause many cardiac complications early or late after therapy. Anthracyclines exhibit their potent anti-cancer effect primarily via induction of DNA damage during the DNA replication phase in proliferative cells. In contrast, studies in animals and hPSC-cardiomyocytes have revealed that cardiotoxic effects particularly arise from (1) the generation of oxidative stress inducing mitochondrial dysfunction, (2) disruption of calcium homeostasis, and (3) changes in transcriptome and proteome, triggering apoptotic cell death. To increase the therapeutic index of chemotherapeutic Doxorubicin therapy several protective strategies have been developed or are under development, such as (1) reducing toxicity through modification of Doxorubicin (analogs), (2) targeted delivery of anthracyclines specifically to the tumor tissue or (3) cardioprotective agents that can be used in combination with Doxorubicin. Despite continuous progress in the field of cardio-oncology, cardiotoxicity is still one of the major complications of anti-cancer therapy. In this review, we focus on current hPSC-cardiomyocyte models for assessing anthracycline-induced cardiotoxicity and strategies for cardioprotection. In addition, we discuss latest developments toward personalized advanced pre-clinical models that are more closely recapitulating the human heart, which are necessary to support in vitro screening platforms with higher predictivity. These advanced models have the potential to reduce the time from bench-to-bedside of novel antineoplastic drugs with reduced cardiotoxicity.

Project description:RationaleCardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling.ObjectiveWe investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs).Methods and resultsWe differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs.ConclusionsThe results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts.

Dataset Information

Single-Cell Mechanical Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Testing and Pathophysiological Studies.

Publications

Single-Cell Mechanical Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Testing and Pathophysiological Studies.

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