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Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes
Sumihiko Seki1, Masato Nagashima, Yoichi Yamada
1Department of Cellular Physiology and Signal Transduction, Sapporo Medical University School of Medicine, Sapporo 060, Japan. sseki@kanazawa-med.ac.jp
Objective:
The aim of this study was to characterize the spatio-temporal dynamics of [Ca(2+)](i) in rat heart in the fetal and neonatal periods.
Methods:
Using confocal scanning laser microscopy and the Ca(2+) indicator fluo-3, we investigated Ca(2+) transients and Ca(2+) 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:
Ca(2+) transients in the fetal myocytes were characterized by slower upstroke and decay of [Ca(2+)](i) compared to those in adult myocytes. The magnitude of fetal Ca(2+) transients was decreased after application of ryanodine (1 microM) or thapsigargin (1 microM). However, Ca(2+) sparks were rarely detected in the fetal myocytes. Frequent ignition of Ca(2+) sparks was established in the 6-9-day neonatal period, and was predominantly observed in the subsarcolemmal region. The developmental change in Ca(2+) 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, Ca(2+) 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 Ca(2+) sparks depends on postnatal t-tubule formation and resultant colocalization of DHPR and RyR.