Project Summary/Abstract Transient receptor potential (Trp) channels are involved in many fundamental cell functions and associated with many disease states (65). Trpm channels are a subgroup of Trp channel superfamily. Trpm2 is expressed in many tissues including heart, vasculature, hematopoietic cells and brain. Trpm2 is activated by ADP-ribose (ADPR) and H2O2 and mediates Ca2+ influx into the cell. Trpm2 has an essential role in susceptibility to oxidative stress. The existing paradigm is that activation of Trpm2 induces cell death by sustained increases in [Ca2+]i, or mediates enhanced chemokine production in hematopoietic cells thereby aggravating inflammatory response and tissue injury. Quite unexpectedly, our recent data demonstrated that Trpm2 is essential in cellular bioenergetics maintenance in both the heart and neuroblastoma; and that Trpm2 actually protects the heart and neuroblastoma from oxidative-stress induced injury. Trpm2 is overexpressed in many cancers and the level of Trpm2 overexpression correlated with decreased patient survival and increased propensity for metastasis in some tumors. Based on these observations, Trpm2 has increasingly become a rational target for cancer therapy. In support of this concept, we showed that neuroblastoma xenografts over- expressing the dominant-negative Trpm2-S had significantly suppressed growth and enhanced sensitivity to doxorubicin (Doxo). In addition, targeting Trpm2 was recently shown to promote cell death in T cell leukemia. Therefore, although Trpm2 inhibition can enhance the therapeutic effect of chemotherapy (e.g., Doxo), it may inadvertently disturb mitochondrial energy metabolism and redox balance and as such, aggravate existing ischemic heart disease and Doxo-induced cardiomyopathy. The study of mechanisms by which Trpm2 protects the heart is thus timely and will significantly contribute to the nascent field of onco-cardiology. We are the first to demonstrate that Trpm2 channels are expressed at the sarcolemma and t-tubules and measure Trpm2 channel activity in adult mouse LV myocytes. We have obtained preliminary data indicating Trpm2 activation phosphorylates proline-rich tyrosine kinase 2 (Pyk2), one of Ca2+- and redox- sensitive non-receptor tyrosine kinase in the heart, that subsequently translocates to mitochondrial matrix. We have shown that phosphorylated Pyk2 (pPyk2) can enhance mitochondrial Ca2+ uptake through mitochondrial Ca2+ uniporter (MCU). To explore the function of Trpm2 in cardiac myocytes, we have generated both global Trpm2 knockout (gKO) and cardiac-specific Trpm2 KO (cKO) mice. Compared to WT myocytes, gKO myocytes had decreased expression of proteins involved in mitochondrial function and oxidative defense. Functionally, gKO myocytes had lower mitochondrial membrane potential (∆ψm), reduced MCU activity, decreased mitochondrial Ca2+ uptake, increased mitochondrial superoxide (O2-.) levels, decreased oxygen consumption rate (OCR) and lower ATP levels: ind...