PROJECT SUMMARY Junctophilin 2 (JP2) is an essential structural protein required for the formation of junctional couplings (i.e., cardiac dyads) between the transverse (T)-tubule membrane and the sarcoplasmic reticulum (SR). JP2 function is therefore fundamental for the local control of Ca2+-induced Ca2+ release and efficient contraction in ventricular myocytes during cardiac excitation-contraction (E-C) coupling. JP2 protein levels progressively decline in failing human hearts and in animal models of heart failure leading to T-tubule remodeling and loss of E-C coupling function. The downregulation of JP2 at E-C coupling sites is in part due to specific cleavage by the Ca2+-activated protease calpain that is implicated in a variety of heart diseases. During the previous funding period, we demonstrated that stress- and calpain-dependent cleavage of JP2 liberates a novel, nuclear translocating, N- terminal fragment (JP2NT) that represses maladaptive transcriptional reprogramming in diseased hearts, thus transducing E-C uncoupling information into a unique cardio-protective excitation-transcription (E-T) coupling signal to the nucleus. However, how JP2-mediated E-C and E-T coupling phenomena are mechanistically regulated remains to be determined. Our new preliminary results show that JP2 is reproducibly phosphorylated in stressed hearts near regions responsible for JP2 cleavage and the subcellular localization of JP2NT. In this competitive renewal application, we aim to define how stress-induced post-translational modifications regulate the structure, localization, and function of JP2/JP2NT. We hypothesize that JP2NT-mediated E-T coupling is tightly regulated by cardiac stress-dependent phosphorylation of JP2 that determines JP2 sensitivity to calpain and JP2NT nuclear translocation and transcriptional activity. To test our hypothesis, in Aim 1, we will use mutation analysis and cell models to determine how JP2 phosphorylation regulates E-C coupling and cleavage-induced JP2NT generation, nuclear translocation and transcriptional regulation. In Aim 2, we will utilize our novel JP2 calpain resistant mice in combination with JP2NT overexpression to determine how these targeted approaches modulate cardiac responses to stress in vivo. We will determine how E-C coupling structure/function and cardiac gene transcription are altered in these mice in response to pressure overload and myocardial infarction. We expect our studies will provide significant insights into the regulatory mechanisms governing JP2/JP2NT function and their salutary contribution toward heart disease pathogenesis.