Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice.

Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant.

Increased afterload induces pathological cardiac hypertrophy: a new in vitro model.

Increased afterload results in 'pathological' cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2 weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4 % under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6 %), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner.

Thymosin ß4 Improves Differentiation and Vascularization of EHTs.

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 ß4 (Tß4) 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 Tß4 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 Tß4 is crucial for the proper execution of lymphatic and angiogenic vessel sprouting. Finally, utilizing Tß4-transduced EHTs generated from mice genetically engineered to label endothelial cells in vitro, we show that treatment with Tß4 promotes vascularization and contractility in EHTs, highlighting Tß4 as a growth factor improving the formation of cardiomyocytes from iPSC and enhancing the performance of EHTs generated from neonatal cardiomyocytes.

Generation of strip-format fibrin-based engineered heart tissue (EHT).

This protocol describes a method for casting fibrin-based engineered heart tissue (EHT) in standard 24-well culture dishes. In principle, a hydrogel tissue engineering method requires cardiomyocytes, a liquid matrix that forms a gel, a casting mold, and a device that keeps the developing tissue in place. This protocol refers to neonatal rat heart cells as the cell source; the matrix of choice is fibrin, and the tissues are generated in rectangular agarose-casting molds (12 × 3 × 3 mm) prepared in standard 24-well cell culture dishes, in which a pair of flexible silicone posts is suspended from above. A master mix of freshly isolated cells, medium, fibrinogen, and thrombin is pipetted into the casting mold and, over a period of 2 h, polymerizes and forms a fibrin cell block around two silicone posts. Silicone racks holding four pairs of silicone posts each are used to transfer the fresh fibrin cell blocks into new 24-well dishes with culture medium. Without further handling, the cells start to remodel the fibrin gel, form contacts with each other, elongate, and condense the gel to approximately » of the initial volume. Spontaneous and rhythmic contractions start after 1 week. EHTs are viable and relatively stable for several weeks in this format and can be subjected to repeated measurements of contractile function and final morphological and molecular analyses.

Functional microRNA library screening identifies the hypoxamir miR-24 as a potent regulator of smooth muscle cell proliferation and vascularization.

UNLABELLED: Smooth muscle cells (SMCs) are key components within the vasculature. Dependent on the stimulus, SMC can either be in a proliferative (synthetic) or differentiated state. Alterations of SMC phenotype also appear in several disease settings, further contributing to disease progression. AIMS: Here, we asked whether microRNAs (miRNAs, miRs), which are strong posttranscriptional regulators of gene expression, could alter SMC proliferation. Results and Innovation: Employing a robotic-assisted high-throughput screening method using miRNA libraries, we identified hypoxia-regulated miR-24 as a master regulator of SMC proliferation. Proteome profiling showed a strong miR-24-dependent impact on cellular stress-associated factors, overall resulting in reduced stress resistance. In vitro, synthetic miR-24 overexpression had detrimental effects on SMC functional capacity inducing apoptosis, migration defects, enhanced autophagy, and loss of contractile marker genes. Impaired SMC function was mediated in part by the herein identified direct target gene heme oxygenase 1. Ex vivo, miR-24 was shown to inhibit the development of vasculature in a model of engineered heart tissue. CONCLUSION: Collectively, we report the identification of the hypoxamir-24 as an inhibitor of SMC proliferation, contributing to loss of vascularization.

Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology.

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 ß-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.