<p>Piezo1 channels play important roles in physiological processes such as tactile sensation, blood pressure (BP) control, cardiac development, inflammatory responses as well as in disease processes. Sarco-endoplasmic reticulum Ca²⁺-transporting ATPase (SERCA) is the only active protein in the SR that orchestrates calcium homeostasis by translocation of Ca²⁺ from the cytoplasm to the sarcoplasmic reticulum. It has been shown that SERCA2 inhibits Piezo1 function in mammals by directly acting on the Piezo1 mechano-transduction module of mechanosensitive ion channels. In this study, we investigated whether SERCA2 regulates Piezo1 activation indirectly by modulating Ca²⁺ homeostasis. We showed that treatment with a Piezo1 agonist Yoda1 (5 µM) markedly increased the viability and ATP synthesis of primary cardiomyocytes as well as intracellular Ca²⁺ content through activation of Piezo1, and upregulated the expression of Piezo1 and SERCA2 in the cardiomyocytes. However, si-Piezo1 transfection resulted in downregulation of SERCA2 expression with opposite effects on viability and ATP synthesis and intracellular Ca²⁺ content that could not be reversed by application of Yoda1. Interestingly, application of a SERCA2 channel inhibitor paxilline (Pax, 10 µM) reversed the inhibitory effect of si-Piezo1 transfection on cardiomyocyte function. Using patch clamping and Ca²⁺ transient analyses in cardiomyocytes, we demonstrated that application of Pax inhibited Yoda1-mediated Ca²⁺ currents and APD₅₀, confirming that Piezo1 activation by Yoda1 was significantly inhibited by Pax. Furthermore, application of Yoda1 was able to reverse si-SERCA2 transfection-induced impairment of myocardial function. Microinjection of Yoda1 and Pax into nodose ganglion (NG) in HFD-HTN model rats also demonstrated that the effect of&#xa0;Yoda1 was inhibited in the presence of Pax, thus confirming that Pax inhibited intracellular Ca²⁺ recycling by SERCA2. These results demonstrate for the first time that the application of Pax inhibits the recycling of intracellular Ca²⁺ by SERCA2 and reverses the reduction in cardiomyocyte function caused by downregulation of Piezo1 expression.</p>