Identification of biophysical and paracrine factors governing electrical integration of cardiomyocytes into a functional syncytium

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Syncytial organization of cardiomyocytes requires intimate cell-cell-contacts through connexin-containing gap junctions (GJ). Constitution of the latter is commonly altered in developing and diseased hearts causing physiological and pathological changes of myocardial conduction. Factors controlling GJ-assembly are not well defined, but likely include biophysical stimuli and growth factors. Assessing mechanisms of GJ-assembly and -function in vivo and in standard monolayer cultures in vitro is confounded by physiological complexity of the former and low cardiomyocyte maturation in the latter model. In engineered heart tissue (EHT) myocytes regain a characteristic rod-shaped morphology and form apparently regular end-to-end contacts containing GJs, essentially generating an anisotropically organized syncytium. Thus, we will use EHT as a simplified model of heart muscle development to identify the role of mechanical, electrical, and paracrine stimuli on syncytial organization of cardiomyocytes. In addition, we hypothesize that enhanced syncytial arrangement will improve contractile performance, electrical stability, and in vivo integration of EHT. After identification of GJ-constitution in rat EHT, we will take advantage of a novel embryonic stem cell (ESC)-based tissue engineering concept and of stably expressed calcium- as well as voltage-sensor proteins to gain detailed insight into mechanisms of GJ-assembly, -maintenance, and -function. High resolution optical imaging of genetically engineered ESC-EHTs will allow time-course analyses of electrical modelling and conduction properties as well as direct identification of factor-cause relationships in vitro. We ultimately aim at generating human calcium/voltage-sensor ESC-EHTs for potential applications in drug screening in vitro and cardiac repair in vivo.