We developed and explored an adult rat ventricular myocytes

(AVM) culture system with minimised de-differentiation and stable physiological parameters allowing the ectopic expression of a Ca2+ sensor. AVM were cultured under various conditions such as medium supplement, substrate coating and electrical pacing for one week. The cells were probed for (i) viability, (ii) morphology, (iii) frequency-dependence of contractions and (iv) Ca2+ transients as well as for (v) their tolerance towards adenovirus-mediated expression of the Ca2+ -sensor inverse pericam. Fetal calf serum dedifferentiated the myocytes into flat cells within 3 days. In serum-free medium this process was reduced but cell viability and morphology was impaired. Medium supplemented with an insulin-transferrin-selenite mixture and extracellular matrix proteins coating increased cell survival while cross-striation was conserved. Furthermore, these myocytes displayed preservation of their contractility and Ca2+ signalling even under continuous electrical pacing. Expression of inverse pericam did not alter the myocyte functions tested and allowed high frequency imaging (50–60 Hz) of electrically paced myocytes exceeding 1.5 h without a loss in signal quality. Our model allows the genetically long-term manipulation of AVM

(e.g. Ca2+ sensor), a prerequisite for high-content screening of adult cardiac cells. Fundings: HOMFOR and the DFG: SFB 530, and Graduiertenkolleg "Cellular Regulation and Growth".