Alterations of Ca signaling and Ca release sites in cultured ventricular myocytes with intact internal Ca storage.

Although cultured adult cardiac myocytes in combination with cell-level genetic modifications have been adopted for the study of protein function, the cellular alterations caused by the culture conditions themselves need to be clarified before we can interpret the effects of genetically altered proteins. We systematically compared the cellular morphology, global Ca signaling, elementary Ca release (sparks), and arrangement of ryanodine receptor (RyR) clusters in short-term (2 days)-cultured adult rat ventricular myocytes with those of freshly isolated myocytes. The transverse (t)-tubules were remarkably decreased (to ∼25%) by culture, and whole-cell capacitance was reduced by ∼35%. The magnitude of depolarization-induced Ca transients decreased to ∼50%, and Ca transient decay was slowed by culture. The culture did not affect sarcoplasmic reticulum (SR) Ca loading. Therefore, fractional Ca release was attenuated by culture. In the cultured cells, the L-type Ca current (I) was smaller (∼50% of controls) and its inactivation was slower. In cultured myocytes, there were significantly fewer (∼50% of control) Ca sparks, the local Ca releases through RyR clusters, compared with in freshly isolated cells. Amplitude and kinetics (duration and time-to-peak) of individual sparks were similar, but they showed greater width in cultured cells. Immunolocalization analysis revealed that the cross-striation of RyRs distribution became weaker and less organized, and that the density of RyR clusters decreased in cultured myocytes. Our data suggest that the loss of t-tubules and generation of compromised Ca transients and I in short-term adult ventricular cell culture are independent of SR Ca loading status. In addition, the deteriorated arrangement of the RyR-clusters and their decreased density after short-term culture may be partly responsible for fewer Ca sparks and a decrease in global Ca release.