Impaired Local Calcium Signaling in Primary Cultured Adult Rat Ventricular Myocytes

Abstract

Although cultured adult cardiac myocytes have been adopted in studying protein functions in combination with cell-level genetic modifications, cellular alterations by culturing itself need to be clarified to understand real function of the protein genetically altered. We systematically compared contractile properties, calcium ion (Ca2+) signaling, transverse (t)-tubules, ryanodine receptor distributions between freshly isolated and two-days cultured adult rat ventricular myocytes. Density of t-tubules was remarkably decreased by culture. In cultured myocytes, cell shortenings were attenuated by ∼60% and relaxation was slowed. Consistently, magnitudes of action potential-induced Ca2+ transients were decreased to ∼50% and decays of the Ca2+ transients were retarded by culture. In cultured cells, density of L-type Ca2+ current was reduced to ∼40% and its inactivation was retarded. The latter is consistent with smaller Ca2+ transients in cultured group. However, sarcoplasmic reticulum Ca2+ contents were not different between two groups. To know the mechanism for smaller Ca2+ transient in cultured cells we examined Ca2+ sparks in these two groups of cells. The frequency of spontaneous Ca2+ sparks was significantly decreased by culturing. The amplitude, duration, and time-to-peak of individual Ca2+ sparks were not different between the two groups. Mean spark width was two-fold larger in cultured cells compared with freshly isolated cells. Quantitative analysis of immunofluorescence revealed shortening of longitudinal spacing between RyR2 clusters, and less dense and disorganized distributions of RyR2 clusters in cultured cells, which may be related to lower frequency of sparks in these cells. These results provide evidence on significant difference in Ca2+ sparks as well as excitation-contraction coupling in primary cultured adult ventricular myocytes. (This work was supported by National Research Foundation of Korea grants funded by the Ministry of Education, Science and Technolgy (2010-0000070).)