This MPI proposal has the overarching theme that mitochondrial Ca2+ uptake plays dual “ying-yang” roles when the heart is under stress: it ensures that extra energy demands are met under workload stress, while it is the route for Ca2+ overload/toxicity under oxidative stress. The project uses a new conditional knock- out rat model for EMRE/Smdt1, an essential subunit of the main mitochondrial Ca2+ uptake system, the mito- chondrial Ca2+ uniporter channel complex (MCUC). The excitation-bioenergetics (EB) coupling is a signaling loop that uses the Ca2+ released by sarcoplasmic reticulum (SR) to activate MCUC and generate mitochon- drial matrix Ca2+ signals to upregulate ATP production. Our long-term goal is to translate the fundamental mechanism of EB coupling to health and disease of the heart. Our previous work demonstrated that MCUC is recruited to hotspots at the mitochondria-SR (Mito-SR) contacts to effectively mediate EB coupling by a yet unknown mechanism. This proposal aims to address the following gaps in our knowledge about EB coupling: i). The role of MCUC in cardiac stress tolerance, diastolic dysfunction and heart failure with preserved ejection fraction (HFpEF). ii). Molecular mechanisms, protein-protein interactions of MCUC recruitment to hotspots at Mito-SR contacts. iii). How are organelle dynamics (contact formation, fusion/fission) integrated with the dy- namics of Ca2+ transport distribution? iv). The role of a dominant negative subunit MCUB in the MCUC hotspot formation, control of the inactive and activatable MCUC channel pool distribution to attain balance between EB coupling and Ca2+ toxicity. The Central Hypothesis is that the dichotomy of mitochondrial Ca2+ in controlling health and disease of cardiomyocytes hinges on the crucial role of EMRE and MCUB in regulating the location and quantity of func- tional MCUC; this elaborate regulation is critical in cardiac adaptation to “fight or flight” and oxidative stress re- sponses. Three specific aims are set up to test this hypothesis: Aim 1. Assess the regulation of EB coupling by MCUC during “fight or flight” sympathetic stress and determine if cessation of this function could lead to diastolic dysfunction or HFpEF. Aim 2. Elucidate the molecular mechanism of the MCUC hotspot recruitment in the rat cardiomyocytes. Aim 3. Study the dynamic interactions between EMRE, MCU and MCUB in regulating the MCUC localization to maintain EB coupling efficiency while preventing Ca2+ toxicity under pathological stresses. Completion of the proposed studies will generate a new paradigm for the regulatory mechanisms of mi- tochondrial Ca2+ in cardiac energetics. The new findings will provide mechanistic basis for a new therapeutic strategy to treat heart failure.