Project Grant R01HL171710

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.