In vitro cellular models for cardiac development and pharmacotoxicology.

Permanent cultures of cardiac cells described so far have limited value for studying cell biology and pharmacology of the developing heart because of the loss of proliferative capacity and cardiac-specific properties of cardiomyocytes during long-term cultivation. Pluripotent embryonic carcinoma (EC) and embryonic stem (ES) cells cultivated as permanent lines offer a new approach for studying cardiogenic differentiation in vitro. We describe cardiogenesis in vitro by differentiating EC and ES cells by way of embryo-like aggregates (embryoid bodies) into spontaneously beating cardiomyocytes. During cardiomyocyte differentiation three distinct developmental stages were defined by expression of specific action potentials and ionic currents measured by the whole-cell patch-clamp technique. Whereas early differentiated cardiomyocytes are characterized by action potentials and ionic currents typical for early pacemaker cells, terminally differentiated cardiomyocytes show action potentials and ionic currents inherent to ventricular-, atrial- or sinus nodal-like cells. These functional characteristics are in accordance with the expression of alpha- and beta-cardiac myosin heavy chain at early differentiation stages and the additional expression of ventricular-specific MLC-2V and atrial-specific ANF genes at terminal stages demonstrated by reverse transcription polymerase chain reaction (RT-PCR) analysis. Pharmacological studies performed by measuring chronotropic responses and by analysing the Ca(2+) channel activity correspond to data obtained with cardiac cells from living organisms. For testing the influence of exogenous compounds on cardiac differentiation the teratogenic compound retinoic acid (RA) was applied during distinct stages of embryoid body development. A temporally controlled influence of RA on cardiac differentiation and expression of cardiac-specific genes was found. We conclude that ES cell-derived cardiomyocytes provide an excellent cellular model to study early cardiac development and to perform pharmacological and embryotoxicological investigations.