Elsevier

Advanced Drug Delivery Reviews

Volume 96, 15 January 2016, Pages 214-224
Advanced Drug Delivery Reviews

Human engineered heart tissue as a model system for drug testing

https://doi.org/10.1016/j.addr.2015.05.010Get rights and content

Abstract

Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional engineered heart tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening.

Introduction

Drug development is a long and costly process with high attrition rates. With costs rising exponentially particularly in late stages of development involving clinical studies, the failure of drugs in late development or after approval is a worst-case scenario. Many pharmaceutical companies therefore follow a “fail early, fail cheap” approach, bearing the risk of wasting potentially valuable drugs. Cardiac side effects such as arrhythmias are the most common reason for the withdrawal of drugs [1], [2]. An estimated ~ 45% of all withdrawals and ~ 30% of restrictions to drug application are due to unwanted cardiovascular effects [2]. Estimates suggest that 70% of the toxicity seen in clinical trials could have been detected in the preclinical phase [3] and that a 10% improvement of predictive value of preclinical tests could save averaged development costs of 100 million dollars per drug [4]. This situation has prompted regulatory agencies in the US and the EU (Federal Drug and Food Administration, FDA, and European Medicines Agency, EMA) a decade ago to establish guidance for industry, e.g., for the preclinical evaluation of proarrhythmic drug effects [5]. Though the currently approved test systems increased the sensitivity for the detection of certain side effects, they still have major shortcomings, including the lack of consideration of species differences and complex interactions between more than one ion channel [6], [7]. A reliable human-based cost-effective preclinical screening tool may circumvent some of them.

The ideal test-bed for the prediction of cardiac side effects would be the human heart, but the limited availability of these preparations (e.g., atrial appendages as a by-product of cardiac surgery or ventricular muscle strips from explanted failing human hearts in the course of heart transplantation) precludes this possibility. The prospects of hiPSC-derived cardiomyocytes (hiPSC-CM) readily available (e.g., from commercial sources such as Cellular Dynamics International (CDI), Axiogenesis, Pluriomics) and devoid of the ethical concerns related to embryonic stem cells, have raised hopes in the field. HiPSC-CM express most, if not all, structural and regulatory elements present in a human CM [8], [9], [10], [11], display a cross-striated ultrastructure, contract spontaneously and show basic functional properties of the human heart, including typical responses to drugs (Table 1). This unambiguously establishes hiPSC-CM as bona fide CM. Yet, most parameters differ quantitatively between hiPSC-CM and adult CM. HiPSC-CM are smaller, beat spontaneously (which is a clear sign of immaturity), show a lower degree of ultrastructural organization (which is almost crystalline in adult CM), no t-tubules, less negative diastolic membrane potential, slower action potential upstroke velocity and lower contractile force, and smaller responses to β-adrenergic stimulation (Table 2) or the Ca2 +-channel agonist Bay K 8644 [12]. Their maturation state has been estimated to resemble 16-week old human fetal CM [13], raising the question whether testing in hiPSC-CM has indeed a higher validity than classical animal experiments. Thus, much effort is currently directed towards means to maturate CM and improve the functional readout. This article reviews the potential of hydrogel-based engineered heart tissue (EHT) for this purpose.

Section snippets

Established systems (FDA and EMA guidelines)

Cardiac side effects such as arrhythmias are common and potentially life threatening [2]. Many drugs causing arrhythmias interact with the human ether-á-go-go (hERG) related potassium channel [14], [15]. The hERG-dependent current IKr plays a major role in human cardiac repolarization and its inhibition is a common cause of severe ventricular arrhythmias, such as “Torsades-des-Pointes” (TdP) arrhythmias [16]. Thus, screening for hERG-interaction is obligatory in the preclinical phase of drug

General aspects of EHTs

Engineered heart tissues (EHTs) are three-dimensional, hydrogel-based muscle constructs that can be generated from isolated heart cells of chicken, rat, mouse, hESC and hiPSC [41], [42], [43], [44]. The method for the generation of EHTs was introduced in 1997 [41] and has not been principally changed since then. It requires (i) heart cells, (ii) a liquid hydrogel that solidifies and promotes tissue formation, (iii) a casting mold that determines the 3D shape of the developing tissue and (iv) a

Disease modeling

Besides drug screening, EHTs may prove valuable as a robust, simple and high content assay for hiPSC-mediated disease modeling with patient-specific hiPSC-CM (review in [81]). The principal idea to detect consequences of individual mutations in patient-derived hiPSC-CM has been validated in several studies for cardiac diseases such as long QT syndrome type 1 (LQT1, [82]), long QT syndrome type 2 (LQT2, [83]), Timothy syndrome [84], catecholaminergic polymorphic ventricular tachycardia (CPVT,

Conclusion & outlook

Preclinical drug testing with EHTs offers a number of advantages compared to standard 2D culture formats, including a more physiological 3D muscle environment, longitudinal alignment, increased maturity, directed contractions and simple access to measurements of force, the most important parameter of heart function. Though drug testing has already been formulated as the major goal 20 years ago [41], the EHT technology just now starts to enter wider practice. The two major reasons are the design

Funding

This work is supported by grants from the European Union (Biodesign, FP7-NMP-2010-LARGE-4 #262948), the European Research Council (ERC-AG IndivuHeart erca.c(2014)1813543), the German Research Foundation (DFG Es 88/12-1, DFG Ha 3/1), the British National Centre for the Replacement Refinement & Reduction of Animals in Research (NC3Rs, CRACK IT challenge), the German Centre for Cardiovascular Research (DZHK) and the German Ministry of Research (BMBF), the German Heart Foundation and the Freie and

Conflict of interest

The authors have founded a company (EHT Technologies GmbH) based on a patent TE is holding for the method (patent number: PCT/EP0100856).

References (105)

  • N.H.L. van den Heuvel et al.

    Lessons from the heart: mirroring electrophysiological characteristics during cardiac development to in vitro differentiation of stem cell derived cardiomyocytes

    J. Mol. Cell. Cardiol.

    (2014)
  • M.N. Hirt et al.

    Deciphering the microRNA signature of pathological cardiac hypertrophy by engineered heart tissue- and sequencing-technology

    J. Mol. Cell. Cardiol.

    (2015)
  • H. Nogawa et al.

    Effects of probucol, a typical hERG expression inhibitor, on in vivo QT interval prolongation in conscious dogs

    Eur. J. Pharmacol.

    (2013)
  • H. Kempf et al.

    Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture

    Stem Cell Rep.

    (2014)
  • M.N. Hirt et al.

    Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation

    J. Mol. Cell. Cardiol.

    (2014)
  • W. Bian et al.

    Robust T-tubulation and maturation of cardiomyocytes using tissue-engineered epicardial mimetics

    Biomaterials

    (2014)
  • R.H. Schwinger et al.

    Evidence for a sustained effectiveness of sodium-channel activators in failing human myocardium

    J. Mol. Cell. Cardiol.

    (1991)
  • D. Ma et al.

    Modeling type 3 long QT syndrome with cardiomyocytes derived from patient-specific induced pluripotent stem cells

    Int. J. Cardiol.

    (2013)
  • X. Yang et al.

    Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells

    J. Mol. Cell. Cardiol.

    (2014)
  • J.M. Cordeiro et al.

    Identification and characterization of a transient outward K+ current in human induced pluripotent stem cell-derived cardiomyocytes

    J. Mol. Cell. Cardiol.

    (2013)
  • M. Honda et al.

    Electrophysiological characterization of cardiomyocytes derived from human induced pluripotent stem cells

    J. Pharmacol. Sci.

    (2011)
  • T. Tanaka et al.

    In vitro pharmacologic testing using human induced pluripotent stem cell-derived cardiomyocytes

    Biochem. Biophys. Res. Commun.

    (2009)
  • J. Bowes et al.

    Reducing safety-related drug attrition: the use of in vitro pharmacological profiling

    Nat. Rev. Drug Discov.

    (2012)
  • H. Laverty et al.

    How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines?

    Br. J. Pharmacol.

    (2011)
  • FDA

    Guidance for Industry S7B Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals

    (2005)
  • R.L. Martin et al.

    The utility of hERG and repolarization assays in evaluating delayed cardiac repolarization: influence of multi-channel block

    J. Cardiovasc. Pharmacol.

    (2004)
  • I. Kehat et al.

    Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes

    J. Clin. Invest.

    (2001)
  • E.G. Navarrete et al.

    Screening drug-induced arrhythmia events using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays

    Circulation

    (2013)
  • C.Y. Ivashchenko et al.

    Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype

    Am. J. Physiol. Heart Circ. Physiol.

    (2013)
  • J. Kang et al.

    Ca2 + channel activators reveal differential L-type Ca2 + channel pharmacology between native and stem cell-derived cardiomyocytes

    J. Pharmacol. Exp. Ther.

    (2012)
  • L. Chen et al.

    Unitary current analysis of L-type Ca2 + channels in human fetal ventricular myocytes

    J. Cardiovasc. Electrophysiol.

    (1999)
  • J.S. Mitcheson et al.

    A structural basis for drug-induced long QT syndrome

    Proc. Natl. Acad. Sci. U. S. A.

    (2000)
  • M.E. Del Rosario et al.

    Drug-induced QT prolongation and sudden death

    Mo. Med.

    (2010)
  • I.D. Wakefield et al.

    The application of in vitro methods to safety pharmacology

    Fundam. Clin. Pharmacol.

    (2002)
  • W.S. Redfern et al.

    Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development

    Cardiovasc. Res.

    (2003)
  • T. Meyer et al.

    New cell models and assays in cardiac safety profiling

    Expert Opin. Drug Metab. Toxicol.

    (2007)
  • P. Kannankeril et al.

    Drug-induced long QT syndrome

    Pharmacol. Rev.

    (2010)
  • A.R. Gelzer et al.

    Validation of a telemetry system for measurement of blood pressure, electrocardiogram and locomotor activity in beagle dogs

    Clin. Exp. Hypertens.

    (1997)
  • S.D. Lamore et al.

    Cellular impedance assays for predictive preclinical drug screening of kinase inhibitor cardiovascular toxicity

    Toxicol. Sci.

    (2013)
  • J. Kramer et al.

    MICE models: superior to the HERG model in predicting Torsade de Pointes

    Sci. Rep.

    (2013)
  • V. Ahuja et al.

    Drug safety testing paradigm, current progress and future challenges: an overview

    J. Appl. Toxicol.

    (2014)
  • G.O. On et al.

    ECVAM Test Method Submissions 2008–2014

    (2014)
  • ECVAM
  • X. Yang et al.

    Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes

    Circ. Res.

    (2014)
  • X. Sheng et al.

    Human pluripotent stem cell-derived cardiomyocytes: response to TTX and lidocaine reveals strong cell to cell variability

    PLoS One

    (2012)
  • A.M. Gerdes et al.

    Structural remodeling of cardiac myocytes in patients with ischemic cardiomyopathy

    Circulation

    (1992)
  • D.K. Lieu et al.

    Absence of transverse tubules contributes to non-uniform Ca(2 +) wavefronts in mouse and human embryonic stem cell-derived cardiomyocytes

    Stem Cells Dev.

    (2009)
  • M. Gherghiceanu et al.

    Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure

    J. Cell. Mol. Med.

    (2011)
  • J. Ma et al.

    High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents

    AJP Hear. Circ. Physiol.

    (2011)
  • S.D. Lundy et al.

    Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells

    Stem Cells Dev.

    (2013)
  • Cited by (133)

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    This review is part of the Advanced Drug Delivery Reviews theme issue on “Tissue engineering of the heart: from in vitro models to regenerative solutions.”

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