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.

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.

Development of a drug screening platform based on engineered heart tissue.

RATIONALE: Tissue engineering may provide advanced in vitro models for drug testing and, in combination with recent induced pluripotent stem cell technology, disease modeling, but available techniques are unsuitable for higher throughput. OBJECTIVE: Here, we present a new miniaturized and automated method based on engineered heart tissue (EHT). METHODS AND RESULTS: Neonatal rat heart cells are mixed with fibrinogen/Matrigel plus thrombin and pipetted into rectangular casting molds in which two flexible silicone posts are positioned from above. Contractile activity is monitored video-optically by a camera and evaluated by a custom-made software program. Fibrin-based mini-EHTs (FBMEs) (150 microL, 600 000 cells) were transferred from molds to a standard 24-well plate two hours after casting. Over time FBMEs condensed from a 12x3x3 mm gel to a muscle strip of 8 mm length and, depending on conditions, 0.2 to 1.3 mm diameter. After 8 to 10 days, FBMEs started to rhythmically deflect the posts. Post properties and the extent of post deflection allowed calculation of rate, force (0.1 to 0.3 mN), and kinetics which was validated in organ baths experiments. FBMEs exhibited a well-developed, longitudinally aligned actinin-positive cardiac muscle network and lectin-positive vascular structures interspersed homogeneously throughout the construct. Analysis of a large series of FBME (n=192) revealed high yield and reproducibility and stability for weeks. Chromanol, quinidine, and erythromycin exerted concentration-dependent increases in relaxation time, doxorubicin decreases in contractile force. CONCLUSIONS: We developed a simple technique to construct large series of EHT and automatically evaluate contractile activity. The method shall be useful for drug screening and disease modeling.