Search

Background: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. Methods: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. Results: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN , the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient’s condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. Conclusions: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format. The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Irreversible Electroporation (IRE) has gained significant interest as an alternative treatment for atrial fibrillation (AF). Typically, to terminate AF, thermal energy is delivered to the arrhythmic tissue causing rare but serious damage to surrounding areas. IRE is a non-thermal approach that has the potential to be faster yet more targeted. We are developing a novel, species relevant, standard laboratory test to evaluate IRE cardiac ablation using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model. hiPSC-CMs were cultured in monolayers. Pulses were delivered by a AVOZ D7B generator through contact electrodes. Electric field (E) distribution maps were obtained with numerical simulations, Sim4Life v3.2. Electroporation (EP) was quantified by florescence with YO-PRO-1 10 minutes after exposure. hiPSC-CMs were exposed to a range of electrical parameters including 120 unipolar pulses of 5 μs duration, at 1 kHz frequency (Fig. 1). We found that the EP threshold was ~1.5 kV/cm in line with previous reports on single adult ventricular mouse cardiomyocytes (Semenov et al. 2018). The treated area increased with the voltage (V) applied. These data suggest that this assay can delineate the extent of EP. In this work we will vary different pulse parameters (durations, amplitude, shape, repetition rate and number) to evaluate their effects on different human cell lines anatomically adjacent to the heart. Fig.1: (A) Representation of the EP threshold determined by overlaying the E map (B) with a fluorescence image of pulse-treated hiPSC-CMs (C). To define the E threshold the distributions values obtained by modeling 1 V applied was scaled by the experimental V.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

SUMMARY To assess the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro proarrhythmia model, we evaluated the concentration dependence and sources of variability of electrophysiologic responses to 28 drugs linked to low, intermediate, and high torsades de pointes (TdP) risk categories using two commercial cell lines and standardized protocols in a blinded multisite study using multielectrode array or voltage-sensing optical approaches. Logistical and ordinal linear regression models were constructed using drug responses as predictors and TdP risk categories as outcomes. Three of seven predictors (drug-induced arrhythmia-like events and prolongation of repolarization at either maximum tested or maximal clinical exposures) categorized drugs with reasonable accuracy (area under the curve values of receiver operator curves ~0.8). hiPSC-CM line, test site, and platform had minimal influence on drug categorization. These results demonstrate the utility of hiPSCCMs to detect drug-induced proarrhythmic effects as part of the evolving Comprehensive In Vitro Proarrhythmia Assay paradigm., In Brief Blinova et al. tested human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for improving torsades de pointes arrhythmia risk prediction of drugs in the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative. This validation study confirms their utility based on electrophysiologic responses to 28 blinded drugs, with minimal influence from cell lines, test sites, and electrophysiological platforms., Graphical Abstract

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo , we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro . In this study, we employed a library of combinatorial polymers comprising of three functional subunits – poly-e-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) – as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-96%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro . Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are being increasingly used to model human heart diseases. hiPSC-CMs generated by earlier aggregation-based methods (i.e., embryoid body) often lack functional sarcoplasmic reticulum (SR) Ca stores characteristic of mature mammalian CMs. Newer monolayer-based cardiac differentiation methods (i.e., Matrigel sandwich or small molecule-based differentiation) produce hiPSC-CMs of high purity and yield, but their Ca handling has not been comprehensively investigated. Here, we studied Ca handling and cytosolic Ca buffering properties of hiPSC-CMs generated independently from multiple hiPSC lines at Stanford University, Vanderbilt University and University of Wisconsin-Madison. hiPSC-CMs were cryopreserved at each university. Frozen aliquots were shipped, recovered from cryopreservation, plated at low density and compared 3–5days after plating with acutely-isolated adult rabbit and mouse ventricular CMs. Although hiPSC-CM cell volume was significantly smaller, cell capacitance to cell volume ratio and cytoplasmic Ca buffering were not different from rabbit-CMs. hiPSC-CMs from all three laboratories exhibited robust L-type Ca currents, twitch Ca transients and caffeine-releasable SR Ca stores comparable to adult CMs. Ca transport by sarcoendoplasmic reticulum Ca ATPase (SERCA) and Na/Ca exchanger (NCX) was similar in all hiPSC-CM lines, but slower compared to rabbit-CMs. However, the relative contribution of SERCA and NCX to Ca transport of hiPSC-CMs was comparable to rabbit-CMs. Ca handling maturity of hiPSC-CMs increased from 15 to 21days post-induction. We conclude that hiPSC-CMs generated independently from multiple iPSC lines using monolayer-based methods can be reproducibly recovered from cryopreservation and exhibit comparable and functional SR Ca handling.

This unit describes the published Matrigel mattress method. Briefly, we describe the preparation of the mattress, replating of the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) on the Matrigel mattress, and hiPSC-CM mattress maintenance. Adherence to this protocol will yield individual, robustly shortening hiPSC-CMs, which can be used for downstream applications. © 2017 by John Wiley & Sons, Inc.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have great potential as tools for cell based therapy, disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as methods to do so are cumbersome and not easily translated to standard laboratories. We sought to develop a more efficient protocol to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the hydrogel mattress, and their cellular contractile properties were evaluated using video based edge detection (Figure A). We found individual hiPSC-CMs maintained for 5 days on the mattress assumed an elongated shape (Figure B) and exhibited robust, reproducible cell shortening (~10% versus

Rationale: The lack of measurable single-cell contractility of human-induced pluripotent stem cell–derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. Objective: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Methods and Results: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (I Na ). Conclusions: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

Utilizing an unbiased in vivo phenotypic chemical screening platform in zebrafish embryos, our laboratory has identified a number of novel compounds with high selectivity for a wide range of cellular targets, including kinases (CK2a, DRAK2, DYRK2 and bone morphogenetic protein receptors), GPCRs (lysophosphatidic acid receptor 1, and extracellular proton sensor), p300 histone acetyltransferase, and phosphodiesterase-4 (PDE4). While the compounds we discovered have therapeutic implications for a wide range of diseases, our translational work has focused on addressing the cardiovascular diseases. Heart failure (HF) is a leading cause of disability and mortality in US, affecting about 6 million Americans, and the incidence of heart failure is anticipated to increase substantially in the coming decades. Yet, current HF pharmaceuticals are palliative, and the outlook for HF drug pipeline is uncertain. Within this backdrop, we recently discovered eggmanone, an extraordinarily selective PDE4 inhibitor which has no known off-target. An usual feature of eggmanone is that it increases cAMP levels specifically in distinct cellular microdomains, without raising the total cellular cAMP content. In isolated mouse cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), eggmanone increases cardiac contractility by targeting a discrete myocyte microdomain without causing significant changes in myocyte calcium cycling. Importantly, eggmanone enhances systolic function in mice with failing hearts without increasing the heart rate. These results raise the exciting possibility that a microdomain-specific PDE4 inhibitor like eggmanone may be useful as an inotropic therapy for HF which avoids the pitfalls of traditional PDE inhibitors, whose utility has been limited by proarrhythmia, tachyphylaxis and cardiotoxicity.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have great potential as tools for human heart disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as current methods are not accessible for most laboratories. We sought to develop a more efficient method to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the “hydrogel mattress,” and their cellular contractile properties evaluated using video-based edge detection. We found that hiPSC-CMs maintained on the mattress reproducibly exhibited robust cell shortening, in dramatic contrast to hiPSC-CMs maintained in a standard manner. We further found that contraction and peak cell shortening amplitude of hiPSC-CMs on mattress was comparable to that of freshly isolated adult ventricular mouse CM. Importantly, hiPSC-CMs maintained on the mattress exhibited several characteristics of a native CM, in terms of myocyte elongation, calcium handling and pharmacological response. Finally, using this platform, we could calculate the traction force generated by individual CMs. In summary, the Hydrogel mattress platform is a simple and reliable in vitro platform that not only enables the quantification of contractile performance of isolated hiPSC-CMs, but also enhances CM maturation. This flexible platform can be extended to in vitro disease modeling, drug discovery and cardiotoxicity testing.

Congenital dilated cardiomyopathy (cDCM) is a rare but often fatal disease. In most cases, there is no family history, and its etiology is unknown. A major hurdle to elucidating a mechanistic understanding of congenital cardiomyopathy, and primary cardiomyopathies in general, has been a lack of access to diseased human cardiac tissues. Recent advances in patient-derived induced pluripotent stem cells (iPSCs) now enable production of human cardiomyocytes (iPSC-CMs) and allows for a systematic study of normal and diseased cardiomyocytes. We hypothesize that cardiomyocytes generated from iPSCs derived from cDCM patients will exhibit cellular and molecular differences from those generated from healthy donor iPSCs and that a rare genetic mutation, or a collection of mutations, plays a critical role in cDCM pathogenesis. To test these hypotheses, we generated cardiomyocytes from iPSCs derived from a 7-month old male with cDCM using a robust cardiac induction protocol based on the “matrigel sandwich” method of Kamp and colleagues. With this remarkably robust induction method, iPSC-CMs from the cDCM patient and a healthy control donor exhibited proteomic profiles that were 99.7% superimposable. Despite the close similarity at the global proteome level, iPSC-CMs from the cDCM patient showed greatly reduced contractility and dramatic structural defects in the sarcomere and the mitochondria. Finally, bioinformatics analyses of the RNAseq data of the patient’s iPSC-CMs discovered a putative causal mutation in an evolutionarily conserved site in a X-linked gene with unknown function. In summary, our work demonstrates that iPSC-based approaches are particularly useful for the study of human congenital heart diseases. We plan to confirm the causality of this mutation using gene editing techniques such as CRISPR/Cas9 and explore the role of this novel gene in cardiomyocyte structure and function.

There is a tremendous interest in human cardiomyocytes generated from patient-derived induced pluripotent stem cells (iPSC-CMs) for the study and possible treatment of human heart diseases. Despite their vast potential, a significant impediment to a broader application of iPSC-CMs to study human myocyte biology is the structural and functional immaturity of iPSC-CMs. Growing evidence indicates that synthetic polymers utilized as extracellular substrates can exert significant effects on in vitro tissue generation, although the underlying mechanisms remain largely unknown. Based on the profound impact of the extracellular matrix of developing embryos on in vivo organogenesis, we hypothesize that engineered polymer substrates will likewise influence in vitro maturation of iPSC-CMs. A subset of combinatorial polymers was synthesized by polymerizing poly(ε-caprolacton) (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL), abbreviated as x%PEG-y%PCL-z%cPCL (x, y, and z: molar %). We investigated effects of the polymer composition on maturation of iPSC-CMs with respect to the beating behavior, mitochondrial function and molecular profiles after 30 days in culture on polymer scaffolds. Results showed the 4%PEG-96%PCL scaffold promoted the most active beating in iPSC-CMs at 30 days and further, that the mitochondrial function, as assessed by tetramethyl rhodamine methylester (TMRM) was significantly increased in the iPSC-CMs cultured on 4%PEG-96%PCL over other polymers. Molecular profiling analysis indicates 4%PEG-96%PCL scaffolds enhanced the expression of MYL2 (a commonly accepted marker of mature ventricular myocytes) as well as of components of the intermediate filaments linking the plasma membrane to the myofilament. In summary, although the polymers we used here exhibit similar physicochemical properties, they have divergent effects on iPSC-CM differentiation. Thus, specific chemical compositions of synthetic substrates can exert profound influence on in vitro maturation of hiPSC-CMs. Our work exploring the effects of synthetic biomaterials on human stem cell differentiation could pave the way for a successful translation of ongoing advances in tissue engineering to new treatments for human heart diseases.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a unique tool to evaluate cellular properties in disease models, investigate underlying mechanisms and optimize cell-based therapies. We recently reported that after plating 30 day old single hiPSC-CMs on undiluted Matrigel “Mattress” (thickness ∼0.5 mm) for 5 days, mattress hiPSC-CMs developed a rod-like cell shape, aligned myofilament structure, increased sarcomere length and exhibited robust cell shortening. Here, we compared the electrophysiological properties of mattress hiPSC-CMs to hiPSC-CMs cultured on standard substrates (control) and acutely-isolated adult rabbit CMs. The action potentials of mattress hiPSC-CMs were comparable to rabbit CMs. Compared to control, mattress hiPSC-CMs had significantly increased the action potential upstroke velocity due to two-fold increase in sodium current (INa) (−68.61±11.63 pA·pF-1 in mattress vs. −29.66±7.50 pA·pF-1 in control at −30mV). The inward rectifier current (IK1) is responsible for the maintenance of resting membrane potential and contributes to phase 3 of the action potential. Although resting potential and action potential duration was not different from rabbit CMs, IK1 density in mattress hiPSC-CMs was nine-time smaller than in rabbit CMs, but there was no difference between mattress and control hiPSC-CMs (−4.86±0.66 pA·pF-1 in mattress vs. −43.75±4.13 pA·pF-1 in rabbit vs. −3.98±0.83 pA·pF-1 in control at −120 mV). The funny current (If) is an inward current activating at hyperpolarized membrane potential, which is detectable in the developing heart but absent in the adult ventricle and contributes to the spontaneous beating of hiPSC-CMs. Mattress hiPSC-CMs exhibit robust If that was not significantly different from control (−6.22±1.31 pA·pF-1 in mattress vs.-5.60±1.43 pA·pF-1 in control at −120 mV). In summary, the matrigel mattress method enables the rapid generation of robustly contracting single hiPSC-CMs and increases INa, with negligible effect on IK1 or If.

Background Chromosomal segmental copy number variation (CNV) has been recently recognized as a very important source of genetic variability. Some CNV loci involve genes or conserved regulatory elements. Compelling evidence indicates that CNVs impact genome functions. The chicken is a very important farm animal species which has also served as a model for biological and biomedical research for hundreds of years. A map of CNVs in chickens could facilitate the identification of chromosomal regions that segregate for important agricultural and disease phenotypes. Results Ninety six CNVs were identified in three lines of chickens (Cornish Rock broiler, Leghorn and Rhode Island Red) using whole genome tiling array. These CNVs encompass 16 Mb (1.3%) of the chicken genome. Twenty six CNVs were found in two or more animals. Whereas most small sized CNVs reside in none coding sequences, larger CNV regions involve genes (for example prolactin receptor, aldose reductase and zinc finger proteins). These results suggest that chicken CNVs potentially affect agricultural or disease related traits. Conclusion An initial map of CNVs for the chicken has been described. Although chicken genome is approximately one third the size of a typical mammalian genome, the pattern of chicken CNVs is similar to that of mammals. The number of CNVs detected per individual was also similar to that found in dogs, mice, rats and macaques. A map of chicken CNVs provides new information on genetic variations for the understanding of important agricultural traits and disease.