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Dilated cardiomyopathy (DCM) is a primary disorder of the cardiac muscle, characterized by dilatation of the left ventricle and contractile dysfunction. About 50% of DCM cases can be attributed to monogenic causes, whereas the etiology in the remaining patients remains unexplained. We report a family with two brothers affected by severe DCM with onset in the adolescent period. Using exome sequencing, we identified a homozygous premature termination variant in the MYZAP gene in both affected sibs. MYZAP encodes for myocardial zonula adherens protein—a conserved cardiac protein in the intercalated disc structure of cardiomyocytes. The effect of the variant was demonstrated by light and electron microscopy of the heart muscle and immunohistochemical and western blot analysis of the MYZAP protein in the heart tissue of the proband. Functional characterization using patient-derived induced pluripotent stem cell cardiomyocytes revealed significantly lower force and longer time to peak contraction and relaxation consistent with severe contractile dysfunction. Weprovide independent support for the role of biallelic loss-of-function MYZAP variants in dilated cardiomyopathy. This report extends the spectrum of cardiac disease associated with dysfunction of cardiac intercalated disc junction and sheds light on the mechanisms leading to DCM. [ABSTRACT FROM AUTHOR]

In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease. [ABSTRACT FROM AUTHOR]

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) may serve as a new assay for drug testing in a human context, but their validity particularly for the evaluation of inotropic drug effects remains unclear. In this blinded analysis, we compared the effects of 10 indicator compounds with known inotropic effects in electrically stimulated (1.5Hz) hiPSC-CM-derived 3-dimensional engineered heart tissue (EHT) and human atrial trabeculae (hAT). Human EHTs were prepared from iCell hiPSCCM, hAT obtained at routine heart surgery. Mean intra-batch variation coefficient in baseline force measurement was 17% for EHT and 49% for hAT. The PDE-inhibitor milrinone did not affect EHT contraction force, but increased force in hAT. Citalopram (selective serotonin reuptake inhibitor), nifedipine (LTCC-blocker) and lidocaine (Na+ channel-blocker) had negative inotropic effects on EHT and hAT. Formoterol (beta-2 agonist) had positive lusitropic but no inotropic effect in EHT, and positive clinotropic, lusitropic, and inotropic effects in hAT. Tacrolimus (calcineurin-inhibitor) had a negative inotropic effect in EHTs, but no effect in hAT. Digoxin (Na+-K+-ATPase-inhibitor) showed a positive inotropic effect only in EHTs, but no effect in hAT probably due to short incubation time. Ryanodine (ryanodine receptor-inhibitor) reduced contraction force in both models. Rolipram and acetylsalicylic acid showed noninterpretable results in hAT. Contraction amplitude and kinetics were more stable over time and less variable in hiPSC-EHTs than hAT. HiPSC-EHT faithfully detected cAMP-dependent and -independent positive and negative inotropic effects, but limited beta-2 adrenergic or PDE3 effects, compatible with an immature CM phenotype. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSC) constitute a powerful tool to study cardiac physiology and represents a promising treatment strategy to tackle cardiac disease. However, iPSCs remain relatively immature after differentiation. Additionally, engineered heart tissue (EHT) has been investigated as a therapy option in preclinical disease models with promising results, although their vascularization and functionality leave room for improvement. Thymosin β4 (Tβ4) has been shown to promote the differentiation of progenitor cell lines to cardiomyocytes while it also induces angiogenic sprouting and vascular maturation. We examined the potential impact of Tβ4 to enhance maturation of cardiomyocytes from iPSCs. Assessing the expression of transcription factors associated with cardiac differentiation, we were able to demonstrate the increased generation of cells displaying cardiomyocyte characteristics in vitro. Furthermore, we demonstrated, in a zebrafish model of embryonic vascular development, that Tβ4 is crucial for the proper execution of lymphatic and angiogenic vessel sprouting. Finally, utilizing Tβ4-transduced EHTs generated from mice genetically engineered to label endothelial cells in vitro, we show that treatment with Tβ4 promotes vascularization and contractility in EHTs, highlighting Tβ4 as a growth factor improving the formation of cardiomyocytes from iPSC and enhancing the performance of EHTs generated from neonatal cardiomyocytes. [ABSTRACT FROM AUTHOR]

The article discusses a study which examined the results of the transplantation of human pluripotent stem cell-derived cardiomyocytes (hiPSC). The study created human engineered heart tissue (hEHT) strips from hiPSC-derived cardiomyocytes and hiPSC-derived endothelial cells. It demonstrated that the hearts repaired with hEHT strips 28 days after their transplantation.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM). Here we investigated whether a 3D engineered heart tissue (EHT) culture format favors maturation and addressed the L-type Ca2+-current (ICa,L) as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML) and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were two- to three-fold smaller than adult CM, independently of culture format [capacitance ML 45 ± 1 pF (n = 289), EHT 45 ± 1 pF (n = 460), atrial CM 87 ± 3 pF (n = 196), ventricular CM 126 ± 8 pF (n = 50)]. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10 ± 1 pA/pF (n = 207) for ML and 12 ± 1 pA/pF (n = 361) for EHT and was larger than in adult CM [7 ± 1 pA/pF (p < 0.05, n = 196) for atrial CM and 6 ± 1 pA/pF (p < 0.05, n = 47) for ventricular CM]. However, ML and EHT showed robust T-type Ca2+-currents (ICa,T). While (-)-Bay K 8644, that activates ICa,L directly, increased ICa,Lto the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (-)-Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (-logEC50: 5.9 and 6.1 for norepinephrine (NE) and epinephrine (Epi), respectively), but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (-logEC50: 5.3 and 5.3 respectively for NE and Epi). Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation. [ABSTRACT FROM AUTHOR]

Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca2+ concentration. The analysis of contractile function and Ca2+ transients is therefore important to discriminate between myofilament responsiveness and changes in Ca2+ homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca2+ transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded engineered heart tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca2+ and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca2+ concentration- response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca2+ (EC50: 0.15, 0.39, and 1.05 mM Ca2+ in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca2+ sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca2+ concentration is due to changes in Ca2+ handling proteins. Finally, this study confirms that fura-2 has Ca2+ buffering effects and is thereby changing the force response to extracellular Ca2+. [ABSTRACT FROM AUTHOR]

Hypertrophic cardiomyopathy (HCM) is a myocardial disease associated with mutations in sarcomeric genes. Three mutations were found in ANKRD1, encoding ankyrin repeat domain 1 (ANKRD1), a transcriptional co-factor located in the sarcomere. In the present study, we investigated whether expression of HCM-associated ANKRD1 mutations affects contraction parameters after gene transfer in engineered heart tissues (EHTs). EHTs were generated from neonatal rat heart cells and were transduced with adeno-associated virus encoding GFP or myc-tagged wild-type (WT) or mutant (P52A, T123M, or I280V) ANKRD1. Contraction parameters were analyzed from day 8 to day 16 of culture, and evaluated in the absence or presence of the proteasome inhibitor epoxomicin for 24 h. Under standard conditions, only WT- and T123M-ANKRD1 were correctly incorporated in the sarcomere. T123M- ANKRD1-transduced EHTs exhibited higher force and velocities of contraction and relaxation than WT- P52A- and I280V-ANKRD1 were highly unstable, not incorporated into the sarcomere, and did not induce contractile alterations. After epoxomicin treatment, P52A and I280V were both stabilized and incorporated into the sarcomere. I280V-transduced EHTs showed prolonged relaxation. These data suggest different impacts of ANKRD1 mutations on cardiomyocyte function: gain-of-function for T123M mutation under all conditions and dominant-negative effect for the I280V mutation which may come into play only when the proteasome is impaired. [ABSTRACT FROM AUTHOR]

Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30-40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5-10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 μN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the b-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research. [ABSTRACT FROM AUTHOR]

The article deals with the identification of myomasp (Myosin-interacting, M-band-associated stress-responsive protein) as a component of an M-band-associated signaling pathway using a bioinformatic approach. It relates the expression of myomasp in heart and skeletal muscle. It describes the interaction of myomasp with myosin heavy chains. It discusses the role of myomasp in cardiomyocyte function.

Background: Cardiac disease modelling using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) requires thorough insight into cardiac cell type differentiation processes. However, current methods to discriminate different cardiac cell types are mostly time-consuming, are costly and often provide imprecise phenotypic evaluation. DNA methylation plays a critical role during early heart development and cardiac cellular specification. We therefore investigated the DNA methylation pattern in different cardiac tissues to identify CpG loci for further cardiac cell type characterization. Results: An array-based genome-wide DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChips led to the identification of 168 differentially methylated CpG loci in atrial and ventricular human heart tissue samples (n = 49) from different patients with congenital heart defects (CHD). Systematic evaluation of atrial-ventricular DNA methylation pattern in cardiac tissues in an independent sample cohort of non-failing donor hearts and cardiac patients using bisulfite pyrosequencing helped us to define a subset of 16 differentially methylated CpG loci enabling precise characterization of human atrial and ventricular cardiac tissue samples. This defined set of reproducible cardiac tissue-specific DNA methylation sites allowed us to consistently detect the cellular identity of hiPSC-CM subtypes. Conclusion: Testing DNA methylation of only a small set of defined CpG sites thus makes it possible to distinguish atrial and ventricular cardiac tissues and cardiac atrial and ventricular subtypes of hiPSC-CMs. This method represents a rapid and reliable system for phenotypic characterization of in vitro-generated cardiomyocytes and opens new opportunities for cardiovascular research and patient-specific therapy. [ABSTRACT FROM AUTHOR]

Background: Human induced pluripotent stem (iPS) cells have revolutionised research and spark hopes for future tissue replacement therapies. To obtain high cell numbers, iPS cells can be expanded indefinitely. However, as long-term expansion can compromise cell integrity and quality, we set out to assess potential reduction of clonal diversity by inherent growth imbalances. Methods: Using red, green, blue marking as a lentiviral multi-colour clonal cell tracking technology, we marked three different iPS cell lines as well as three other cell lines, assigning a unique fluorescent colour to each cell at one point in culture. Subsequently, we followed the sub-clonal distribution over time by flow cytometry and fluorescence microscopy analysis in regular intervals. Results: In three human iPS cell lines as well as primary human fibroblasts and two widely used human cell lines as controls (K562 and HEK 293 T), we observed a marked reduction in sub-clonal diversity over time of culture (weeks). After 38 passages, all iPS cultures consisted of less than 10 residual clones. Karyotype and function, the latter assessed by cardiomyocyte differentiation and tissue engineering, did not reveal obvious differences. Conclusions: Our results argue for a quick selection of sub-clones with a growth advantage and flag a normally invisible and potentially undesired behaviour of cultured iPS cells, especially when using long-term cultured iPS cells for experiments or even in-vivo applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca2+-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT. [ABSTRACT FROM AUTHOR]

Introduction: We have previously shown that engineered heart tissue (EHT) from neonatal rat heart cells responds to sustained mechanical or pharmacological stress stimuli similarly as hearts in vivo. Inter alia , EHTs showed a decline in contractile force, prolongation of relaxation, cardiomyocyte hypertrophy, reactivation of fetal genes (e.g. natriuretic peptides) and fibrotic changes. However, human EHTs containing cardiomyocytes obtained by differentiating human induced pluripotent stem cells (hiPSCs) did not respond to stress stimuli. We hypothesized that non-cardiomyocytes might account for this difference and sought a method to produce human EHTs containing all major cardiac cell types. Methods and Results: HiPSCs produced from fibroblasts of a healthy donor were expanded and split into 4 groups. Each group was transduced with lentiviruses encoding different fluorescent proteins. Mesodermal induction was initiated for all groups, but then blue (BFP) cells were differentiated to cardiomyocytes via inhibition of Wnt-signaling, green (Venus) cells were differentiated via VEGF to endothelial cells, orange (mOrange2) cells via TGFβ, bFGF and additional activation of Wnt-signaling to smooth muscle cells, and red (Katushka2) cells via bFGF and additional activation of Wnt-signaling to fibroblasts. All 4 differentiated cell types showed bright fluorescence and were clearly discernible by mRNA-markers and functional or histological parameters: cardiomyocytes (alpha actinin, spontaneous beating), endothelial cells (CD31, tube formation), smooth muscle cells (alpha smooth muscle actin, stress fibers), fibroblasts (periostin, collagen-production). Beating multicellular human EHTs could be successfully produced and the survival of all cell types for several weeks was verified, quantified and visualized by flow cytometry and confocal microscopy. The first multicellular EHTs produced did not develop higher contractile forces than unicellular EHTs (containing only cardiomyocytes). Whether multicellular human EHTs respond differently to stress stimuli than unicellular EHTs, is still to be investigated. Conclusion: Multicellular human EHTs can be produced from color-coded hiPSCs and will enable the investigation of different cell compositions on contractility under basal and stressed conditions. Cell-type specific analyses will be possible following simple FACS-separation by color. [ABSTRACT FROM AUTHOR]