CARDIOMYOCYTES DIFFERENTIATED IN-VITRO FROM EMBRYONIC STEM-CELLS DEVELOPMENTALLY EXPRESS CARDIAC-SPECIFIC GENES AND IONIC CURRENTS

Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) we re characterized by the whole-cell patch-clamp technique during the en tire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowt hs of early, intermediate, and terminal differentiation stages. The ea rly differentiated cardiomyocytes exhibited an outwardly rectifying, t ransient K+ current sensitive to 4-aminopyridine and an inward Ca2+ cu rrent but no Na+ current. The Ca2+ current showed all features of L-ty pe Ca2+ current, being highly sensitive to 1,4-dihydropyridines but no t to omega-conotoxin. Cardiomyocytes of intermediate stage were charac terized by the additional expression of cardiac-specific Na+ current, the delayed K+ current, and I, current. Terminally differentiated card iomyocytes expressed a Ca2+ channel density about three times higher t han that of early stage. In addition, two types of inwardly rectifying K+ currents (I-K1 and I-K,I-Ach) and the ATP-modulated K+ current wer e found. During cardiomyocyte differentiation, several distinct cell p opulations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcriptio n polymerase chain reaction revealed the transcription of alpha- and b eta-cardiac myosin heavy chain (MHC) genes synchronously with the firs t spontaneous contractions. Transcription of embryonic skeletal MHC ge ne at intermediate and terminal differentiation stages correlated with the expression of Na+ channels. The selective expression of alpha-car diac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of the LacZ construct driven by the alpha-cardia c MHC promotor region followed by ES cell differentiation and beta-gal actosidase staining. In conclusion, our data demonstrate that ES cell- derived cardiomyocytes represent a unique model to investigate the ear ly cardiac development and permit pharmacological/toxicological studie s in vitro.