P_2Y2 Receptor-Mediated Ca²⁺ Signaling and Spontaneous Ca²⁺ Releases in Human Valvular Myofibroblasts

Abstract

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 ([Ca ²⁺]_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 ADPβS 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.