Masaaki Tsutsuura

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 [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.

Contribution of KChIP2 to the developmental increase in transient outward current of rat cardiomyocytes

The Ca(2+)-independent, voltage-gated transient outward current (I(to)) displays a marked increase during development of cardiomyocytes. However, the molecular mechanism remained unclear. In rat adult ventricular myocytes, I(to) can be divided into a fast (I(to,f)) and a slow (I(to,s)) component by recovery process from inactivation. Voltage-gated K(+) channel-interacting proteins 2 (KChIP2) has recently been shown to modify membrane expressions and current densities of I(to,f). Here we examined the developmental change of I(to) and the putative molecular correlates of I(to,f) (Kv4.2 and Kv4.3) and KChIP2 in rat ventricular myocytes. Even in rat embryonic day 12 (E12) myocytes, we detected I(to). However, I(to) in E12 was solely composed of I(to,s). In postnatal day 10 (P10), we recorded much increased I(to) composed of two components (I(to,f) and I(to,s)), and I(to,f) was dominant. Thus, the developmental increase of I(to) from E12 to P10 can be explained by the dramatic appearance of I(to,f). Real-time RT-PCR revealed that Kv4.2 and Kv4.3 mRNA levels were slightly changed. By contrast, KChIP2 mRNA level increased from E12 to P10 by 731-fold. Therefore, the huge increase of KChIP2 expression was likely to be the cause of the great increase of I(to,f). In order to confirm that KChIP2 is crucial to induce I(to,f), we used adenoviral gene transfer technique. When KChIP2 was over-expressed in E12 myocytes, a great amplitude of I(to,f) appeared. Immunocytochemical experiments also demonstrated that KChIP2 enhanced the trafficking of Kv4.2 channels to cell surface. These results indicate that KChIP2 plays an important role in the generation of functional I(to,f) channels during development.

A truncated splice variant of KCNQ1 cloned from rat heart.

KCNQ1 encodes a pore-forming subunit of potassium channels. Mutations in this gene cause inherited diseases, i.e., Romano-Ward syndrome and Jervell and Lange-Nielsen syndrome. A truncated isoform of KCNQ1 was reported to be expressed physiologically and to suppress a delayed rectifier potassium current dominant-negatively in human heart. However, it is not known whether this way of modulation occurs in other species. We cloned another truncated splice variant of KCNQ1 (tr-rKCNQ1) from rat heart. Judging from the deleted sequence of the tr-rKCNQ1, the genomic structure of rat in this portion might be different from those of human and mouse. Otherwise, an unknown exon might exist. RT-PCR analysis demonstrated that the tr-rKCNQ1 was expressed in fetal and neonatal hearts. When this gene was expressed along with a full-length KCNQ1, it suppressed potassium currents, whether a regulatory subunit minK was co-expressed or not.