title: A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes.
abstract: For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.
authors.affiliation: Institute for Molecular Cell Biology, Medical Faculty, Saarland University, Building 61, 66421 Homburg/Saar, Germany.
authors.forename: Cedric
authors.initials: C
authors.lastname: Viero
authors.forename: Udo
authors.initials: U
authors.lastname: Kraushaar
authors.forename: Sandra
authors.initials: S
authors.lastname: Ruppenthal
authors.forename: Lars
authors.initials: L
authors.lastname: Kaestner
authors.forename: Peter
authors.initials: P
authors.lastname: Lipp
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis*; D020013:Calcium Signaling; D018929:Cell Culture Techniques; D002470:Cell Survival; D002478:Cells, Cultured; D004558:Electric Stimulation; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats