Role of ER-mitochondria contact sites in right ventricular fibrosis - endoplasmic reticulum (ER)-mitochondrial (ER-MITO) microdomains play significant roles in the maintenance of bioenergetics and…

Role of ER-mitochondria contact sites in right ventricular fibrosis - endoplasmic reticulum (ER)-mitochondrial (ER-MITO) microdomains play significant roles in the maintenance of bioenergetics and basal cell functions via the exchange of lipids, CA2+, and reactive oxygen species (ROS). Genetic inhibition of mitofusin 2 (MFN2), one of the key components of ER-MITO tethering, in cardiomyocytes (CMS) revealed the importance of the microdomains between mitochondria and sarcoplasmic reticulum (SR), a differentiated form of ER in muscle cells, for maintaining normal mitochondrial CA2+ (MTCA2+) handling and bioenergetics in the heart. However, IT is still unclear which cellular signaling mechanism modulates CA2+ handling at the ER/SR-MITO microdomains in the heart during cardiac stress, and how this alters MTCA2+ and mitochondrial ROS (MROS) levels in cardiac pathology. Our preliminary studies show that 1) MFN2 is likely a C-SRC substrate in mitochondria; 2) C-SRC can phosphorylate the C-terminal tail of MFN2 at the outer mitochondrial membrane (OMM), a domain critical for MFN2 dimerization and redox sensing; and 3) C-SRC-dependent tyrosine phosphorylation (P-TYR) of MFN2 decreases the ER-MITO distance and facilitates ER-to-MITO CA2+ transfer, followed by increases in MROS. Importantly, using a preclinical RAT model of pulmonary arterial hypertension (PAH) with right ventricular (RV) hypertrophy, fibrosis, and failure, we found significant C-SRC activation occurs only in cardiac fibroblasts (CFS) but not in CMS in the RV under PAH, which subsequently causes a C-SRC-dependent P-TYR of MFN2, decreased ER-MITO distance, increased MTCA2+ uptake and MROS, CF activation, and RV fibrosis. Similar preliminary results were obtained in RV tissue samples from a pulmonary hypertension patient. Lastly, we found that MTCA2+ uptake via MTCA2+ uniporter (MCU) is required for MROS elevation and subsequent activation of proliferative signaling in CFS. Based on these preliminary data, we hypothesize that 1) C-SRC-dependent P-TYR of MFN2 alters the ER-MITO tethering structure and causes increases in MTCA2+ and MROS that promote CF activation, thereby acting as a molecular "switch" for the activation of RV-CFS in PAH; and 2) CF-specific inhibition of C-SRC at the OMM in vivo can be leveraged as a novel therapeutic strategy to attenuate cardiac fibrosis in response to stress/injury such as PAH. The long-term goals of our study are to precisely understand 1) the molecular basis of ER-MITO microdomain-mediated regulation of CF functions under pathological conditions including PAH; and 2) develop novel therapeutic approaches targeting cardiac fibrosis-specific molecular mechanisms. In aim 1, we will establish MFN2 as a novel C-SRC substrate in mitochondria and assess the impact of C-SRC-dependent P-TYR of MFN2 on ER-mitochondria tethering, CA2+ transport to mitochondria, MROS generation, and downstream CF signaling activation using cellular systems. In aim 2, we will specifically inhibit mitochondrial C- SRC only in the quiescent CFS by CF-specifically expressing OMM-targeted dominant-negative C-SRC (MT-C-SRC-DN) in a preclinical RAT PAH to evaluate the therapeutic potential of mitochondrial C-SRC inhibition in vivo. The expected outcomes of this project will provide fundamental new insights into the molecular interaction between ER-MITO association, MTCA2+ uptake, and MROS and its functional relevance in CF activation and fibrosis.