Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes

Objective: The aim of this study was to characterize the spatio-temporal dynamics of [Ca2+](i) in rat heart in the fetal and neonatal periods. Methods: Using confocal scanning laser microscopy and the Ca2+ indicator fluo-3, we investigated Ca2+ transients and Ca2+ sparks in single ventricular myocytes freshly isolated from rat fetuses and neonates. T-tubules were labeled with a membrane-selective dye (di-8-ANEPPS). Spatial association of dihydropyridine receptors (DHPR) and ryanodine receptors (RyR) was also examined by double-labeling immunofluorescence. Results: Ca2+ transients in the fetal myocytes were characterized by slower upstroke and decay of [Ca2+](i) compared to those in adult myocytes. The magnitude of fetal Ca2+ transients was decreased after application of ryanodine (1 muM) or thapsigargin (1 muM). However, Ca2+ sparks were rarely detected in the fetal myocytes. Frequent ignition of Ca2+ sparks was established in the 6-9-day neonatal period, and was predominantly observed in the subsarcolemmal region. The developmental change in Ca2+ sparks coincided with development of the t-tubule network. The immunofluorescence study revealed colocalization of DHPR and RyR in the postnatal period, which was, however, not observed in the fetal period. In the adult myocytes, Ca2+ sparks disappeared after disruption of t-tubules by glycerol incubation (840 mM). Conclusions: The sarcoplasmic reticulum (SR) of rat ventricular myocytes already functions early in the fetal period. However, ignition of Ca2+ sparks depends on postnatal t-tubule formation and resultant colocalization of DHPR and RyR. (C) 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.