Human Engineered Heart Tissue: Analysis of Contractile Force

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe advent of human induced pluripotent stem cell (hiPSC) technology and protocols to efficiently differentiate cardiomyocytes (CM) () have opened the perspective to use hiPSC-CM for cardiac research or drug development. This biotechnology advancement also boosted the development of test systems to evaluate hiPSC-CM electrophysiology (reviewed in ), impedance (, ), field potentials (, , , , , ), action potentials and calcium transients with fluorescent dyes, and cellular shortening video-optically (, , , ). These assays may improve preclinical drug development and safety toxicology, because current systems are based either on recombinant cell lines or animal cells, both susceptible to typical shortcomings. HiPSC-CM promise an intact, human cardiomyocyte context in which a drug and/or principle is tested. Moreover, patient-specific cell lines offer (1) the perspective of testing drugs in a wide spectrum of genetic backgrounds and (2) individualized risk prediction and testing of adverse drug effects. Different technologies have been employed for predictive toxicology application (, href="#bib45" rid="bib45" class=" bibr popnode">Pointon et al., 2015), and many studies have successfully demonstrated disease-specific phenotypes in hiPSC-CM from patients with inherited cardiac diseases (reviewed in href="#bib38" rid="bib38" class=" bibr popnode">Moretti et al., 2013, href="#bib27" rid="bib27" class=" bibr popnode">Karakikes et al., 2015). Most test systems use hiPSC-CM as 2D layers on rigid plastic cell-culture dishes that do not allow the cells to perform physiological auxotonic contractions (href="#bib41" rid="bib41" class=" bibr popnode">Nishimura et al., 2004). Contractile function, the main feature of the heart, can only be analyzed in a very restricted manner.We have developed protocols to form 3D force-generating engineered heart tissues (EHTs; href="#bib10" rid="bib10" class=" bibr popnode">Eschenhagen et al., 2012, href="#bib21" rid="bib21" class=" bibr popnode">Hirt et al., 2014). This follows the principle of hydrogel formation with dissociated cardiomyocytes in casting molds and maintenance with a defined preload. Cardiomyocytes remodel the hydrogel, align along force lines, increase in size, and form a coherently beating syncytium. Potential advantages of the EHT system for preclinical drug development and safety toxicology are 2-fold. (1) It allows monitoring effects of drugs on all major parameters of heart function: force, pacemaking activity and contraction, and relaxation kinetics. (2) The analysis is done under stable conditions that resemble cardiac physiology, i.e., 3D heart-like muscle strips that contract under auxotonic, work-performing, steady-state conditions.First publications with EHTs from human embryonic stem cells or hiPSC-CM demonstrated the principal feasibility to generate human constructs and some basic characterization (href="#bib49" rid="bib49" class=" bibr popnode">Schaaf et al., 2011, href="#bib56" rid="bib56" class=" bibr popnode">Tulloch et al., 2011, href="#bib29" rid="bib29" class=" bibr popnode">Kensah et al., 2013, href="#bib42" rid="bib42" class=" bibr popnode">Nunes et al., 2013, href="#bib55" rid="bib55" class=" bibr popnode">Thavandiran et al., 2013). However, neither we nor others have yet shown that EHTs from hiPSC-CM are indeed suitable for the proposed purpose, i.e., whether they faithfully replicate effects on indicator compounds affecting rate, force, and contraction kinetics. This is particularly important for inotropes, since modulators of inotropy are a mainstay of cardiac drug development and new concepts are urgently needed (href="#bib15" rid="bib15" class=" bibr popnode">Francis et al., 2014). The present study therefore set out to answer the following questions. (1) Do drugs known to interfere with pacemaking mechanisms in the sinoatrial node (SAN) affect the spontaneous beating rate of hiPSC-EHTs? (2) Do positive and negative inotropic drugs affect contraction force of hiPSC-EHTs in a manner similar to that of human heart muscle strips? (3) Do drugs with known and supposedly specific effects on individual ion currents affect contractile function? (4) Is prolongation of relaxation time a surrogate for prolongation of repolarization and proarrhythmic risk?