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 another 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 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 artery 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. We also 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-Mfn2 on ER-mitochondria tethering, Ca2+ transport to mitochondria, mROS generation, and downstream CF signaling activation using cell 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.