Fluctuations in the Concentration of Extracellular ATP Synchronized with Intracellular Ca²⁺Oscillatory Rhythm in Cultured Cardiac Myocytes

Abstract

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca(2+) also changes rhythmically in association with the rhythmic contraction of myocytes (Ca(2+) oscillation). Both the contraction and Ca(2+) oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP-purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca(2+) oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca(2+) oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca(2+) oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca(2+) revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca(2+) oscillation. In addition, power spectrum and cross-correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca(2+) oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca(2+) and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca(2+), contributing to the intercellular synchronization of Ca(2+) oscillation among cultured cardiac myocytes.

Oxidative stress-induced formation of a positive-feedback loop for the sustained activation of p38 MAPK leading to the loss of cell division in cardiomyocytes soon after birth

“…The method of culturing cardiomyocytes is described in detail in our previous papers [24][25][26]31,46].…”

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia

“…ATP concentrations of the cultures were analyzed with a CheckLite TM 250 (Kikkoman, Chiba, Japan) or ATP-assay kit (Toyo B-net, Tokyo, Japan) according to each manufacturer's instructions, based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin [3,11]. For the measurement of the concentration of intracellular ATP, the cells were placed in 400 l of Tris-HCl buffer, and lysis reagent was added 20 min later.…”

“…We have recently revealed in cultured cardiomyocytes that the extracellular ATP-purinoceptor system is responsible for the intercellular synchronization of Ca 2+ oscillation probably via the activation of G-protein-coupled P2Y 1 receptors [2,3]. ATP is known to be released into the interstitial spaces within the heart in response to a variety of physiological and pathological stimuli [4], including hypoxia or ischemia [5,6].…”