Ca2+ sparks and cellular distribution of ryanodine receptors in developing cardiomyocytes from rat

Although abundant ryanodine receptors (RyRs) exist in cardiomyocytes from newborn (NB) rat and despite the maturity of their single-channel properties, the RyR contribution to excitation–contraction (E-C) coupling is minimal. Immature arrangement of RyRs in the Ca2+ release site of the sarcoplasmic reticulum and/or distant RyRs location from the sarcolemmal Ca2+ signal could explain this quiescence. Consequently, Ca2+ sparks and their cellular distribution were studied in NB myocytes and correlated with the formation of dyads and transverse (T) tubules. Ca2+ sparks were recorded in fluo-4-loaded intact ventricular myocytes acutely dissociated from adult and NB rats (0–9 days old). Sparks were defined/compared in the center and periphery of the cell. Co-immunolocalization of RyRs with dihydropyridine receptors (DHPR) was used to estimate dyad formation, while the development of T tubules was studied using di-8-ANEPPS and diIC12. Our results indicate that in NB cells, Ca2+ sparks exhibited lower amplitude (1.7 ± 0.5 vs. 3.6 ± 1.7 F/F0), shorter duration (47 ± 3.2 vs. 54.1 ± 3 ms), and larger width (1.7 ± 0.8 vs. 1.2 ± 0.4 μm) than in adult. Although no significant changes were observed in the overall frequency, central sparks increased from ~ 60% at 0–1 day to 82% at 7–9 days. While immunolocalization revealed many central release sites at 7–8 days, fluorescence labeling of the plasma membrane showed less abundant internal T tubules. This could imply that although during the first week, release sites emerge forming dyads with DHPR-containing T tubules; some of these T tubules may not be connected to the surface, explaining the RyR quiescence during E-C coupling in NB.