high-throughput-cardiotoxicity-screening-using-mature-human-induced

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 ...

University of Michigan, Ann Arbor

Increased relative expression of the slow molecular motor of the heart (beta-myosin heavy chain [MyHC]) is well known to occur in many rodent models of cardiovascular disease and in human heart failure. The direct effect of increased relative beta-MyHC expression on intact cardiac myocyte contractility, however, is unclear. To determine the direct effects of increased relative beta-MyHC expression on cardiac contractility, we used acute genetic engineering with a recombinant adenoviral vector (AdMYH7) to genetically titrate beta-MyHC protein expression in isolated rodent ventricular cardiac myocytes that predominantly expressed alpha-MyHC (fast molecular motor). AdMYH7-directed beta-MyHC protein expression and sarcomeric incorporation was observed as soon as 1 day after gene transfer. Effects of beta-MyHC expression on myocyte contractility were determined in electrically paced single myocytes (0.2 Hz, 37 degrees C) by measuring sarcomere shortening and intracellular calcium cycling. Gene transfer-based replacement of alpha-MyHC with beta-MyHC attenuated contractility in a dose-dependent manner, whereas calcium transients were unaffected. For example, when beta-MyHC expression accounted for approximately 18% of the total sarcomeric myosin, the amplitude of sarcomere-length shortening (nanometers, nm) was depressed by 42% (151.0+/-10.7 [control] versus 87.0+/-5.4 nm [AdMYH7 transduced]); and genetic titration of beta-MyHC, leading to 38% beta-MyHC content, attenuated shortening by 57% (138.9+/-13.0 versus 59.7+/-7.1 nm). Maximal isometric cross-bridge cycling rate was also slower in AdMYH7-transduced myocytes. Results indicate that small increases of beta-MyHC expression (18%) have Ca2+ transient-independent physiologically relevant effects to decrease intact cardiac myocyte function. We conclude that beta-MyHC is a negative inotrope among the cardiac myofilament proteins.

Calcium-independent negative inotropy by beta-myosin heavy chain gene transfer in cardiac myocytes.

Inherited arrhythmias can be caused by mutations in the cardiac ryanodine receptor (RyR2). The cellular source of these arrhythmias is unknown. Isolated RyR2(R4496C) mouse ventricular myocytes display arrhythmogenic activity related to spontaneous Ca(2+) release during diastole. On the other hand, recent whole-heart epicardial and endocardial optical mapping data demonstrate that ventricular arrhythmias in the RyR2(R4496C) mouse model of catecholaminergic polymorphic ventricular tachycardia (CPVT) originate in the His-Purkinje system, suggesting that Purkinje cells, and not ventricular myocytes, may be the cellular source of arrhythmogenic activity. The relative effect of the RyR2(R4496C) mutation on calcium homeostasis in ventricular myocytes versus Purkinje cells is unknown.

Purkinje cell calcium dysregulation is the cellular mechanism that underlies catecholaminergic polymorphic ventricular tachycardia.

The advent of induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) has revolutionized the cardiovascular research field. Now it is possible to generate a virtually unlimited supply of patient specific pluripotent stem cells and cardiomyocytes that can be used for research purposes, drug toxicity testing and/or regenerative medicine therapies. The most immediate application for this technology is in vitro disease modeling and in vitro drug toxicity testing. To date the majority of disease modeling and drug toxicity testing has been performed on single hiPSC-CMs in culture. However, the study of complex cardiac arrhythmia mechanisms requires a more physiological model system of electrically and mechanically connected hiPSC-CMs that function as a syncytium-like the cardiomyocytes of the adult heart. This review focuses on the work that has been performed recently using hiPSC-CM 2D monolayers for the study of cardiac electrical impulse propagation.

Calcium and voltage mapping in hiPSC-CM monolayers.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles.

Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

Mutations of cardiac sarcomere genes have been identified to cause HCM, but the molecular mechanisms that lead to cardiomyocyte hypertrophy and risk for sudden death are uncertain. The aim of this study was to examine HCM disease mechanisms at play during cardiac differentiation of human HCM specific pluripotent stem cells.

Deficient cMyBP-C protein expression during cardiomyocyte differentiation underlies human hypertrophic cardiomyopathy cellular phenotypes in disease specific human ES cell derived cardiomyocytes.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer a novel in vitro platform for pre-clinical cardiotoxicity and pro-arrhythmia screening of drugs in development. To date hiPSC-CMs used for cardiotoxicity testing display an immature, fetal-like cardiomyocyte structural and electrophysiological phenotype which has called into question the applicability of hiPSC-CM findings to the adult heart. The aim of the current work was to determine the effect of cardiomyocyte maturation state on hiPSC-CM drug responsiveness. To this end, here we developed a high content pro-arrhythmia screening platform consisting of either fetal-like or mature hiPSC-CM monolayers. Compounds tested in the screen were selected based on the pro-arrhythmia risk classification (Low risk, Intermediate risk, or High risk) established recently by the FDA and major stakeholders in the Drug Discovery field for the validation of the Comprehensive In vitro Pro-Arrhythmia Assay (CiPA). Here we show that maturation state of hiPSC-CMs determines the absolute pro-arrhythmia risk score calculated for these compounds. Thus, the maturation state of hiPSC-CMs should be considered prior to pro-arrhythmia and cardiotoxicity screening in drug discovery programs.

hiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen.

Stiffness triggers non-coding RNA pathology.

Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and "cellrhythmias" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define "fit for purpose" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable "fit for purpose" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.

Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations.

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.

Article	Year
Calcium-independent negative inotropy by beta-myosin heavy chain gene transfer in cardiac myocytes. Circulation research\| PubMed ID: 17363698	2007
Purkinje cell calcium dysregulation is the cellular mechanism that underlies catecholaminergic polymorphic ventricular tachycardia. Heart rhythm\| PubMed ID: 20538074	2010
Calcium and voltage mapping in hiPSC-CM monolayers. Cell calcium\| PubMed ID: 26922095	2016
Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function. Circulation. Arrhythmia and electrophysiology\| PubMed ID: 27069088	2016
Deficient cMyBP-C protein expression during cardiomyocyte differentiation underlies human hypertrophic cardiomyopathy cellular phenotypes in disease specific human ES cell derived cardiomyoc... Journal of molecular and cellular cardiology\| PubMed ID: 27620334	2016
hiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen. Scientific reports\| PubMed ID: 29061979	2017
Stiffness triggers non-coding RNA pathology. Nature biomedical engineering\| PubMed ID: 30944436	2019
Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations. Regulatory toxicology and pharmacology : RTP\| PubMed ID: 32822771
Human perinatal stem cell derived extracellular matrix enables rapid maturation of hiPSC-CM structural and functional phenotypes. Scientific reports\| PubMed ID: 33149250	2020

Todd J. Herron

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