P2Y2 receptor-mediated Ca2+ signaling and spontaneous Ca2+ releases in human valvular myofibroblasts

Valvular myofibroblasts (VMFs), being the most predominant cells in the cardiac valve, perform a variety of functions to maintain normal valvular physiology. These functions, such as contraction, proliferation, and wound repair, are all directly or indirectly mediated by intracellular Ca2+ concentrations ([Ca2+](i)). Knowing how [Ca2+](i) is regulated by vasoactive agents in VMFs enriches the understanding of valvular biology in both health and diseases. In this study we examined the characteristics of purinergic agonist-induced [Ca2+](i) responses and observed spontaneous Ca2+ releases in cultured human VMFs. Secondary cultures of human mitral VMFs were incubated with the Ca2+-sensitive fluorescent indicator fura-2 or fluo-4 and visualized with fluorescence microscopy. Both ATP and UTP activated P-2Y2 receptors and induced endoplasmic reticulum (ER) Ca2+ release and Ca2+ influx. The lack of [Ca2+](i) responses in VMFs challenged with the selective P-2Y1, agonists ADP beta S and 2-Me-S-ATP further supported that functional P-2Y2 receptors are responsible for the Ca2+ signals. Finally, in a small number of VMFs spontaneous Ca2+ releases in localized areas were observed. Blockade of the RyR elongated the latency period between each Ca2+ releasing event, demonstrating the presence of functional RyRs in VMFs.