Project Grant F30HL179946

NEPRILYSIN MEDIATED PHOSPHOLAMBAN CLEAVAGE IN HEART FAILURE - PROJECT SUMMARY/ABSTRACT THE GOAL OF THIS THREE-AIM PROJECT IS TO EXPLORE DOWNSTREAM CONSEQUENCES OF NEPRILYSIN CLEAVAGE OF PHOSPHOLAMBAN (PLB). NEPRILYSIN IS A PROMISCUOUS, MEMBRANE-BOUND/CIRCULATING METALLOPROTEASE WHICH WE HAVE SHOWN TO BE SIGNIFICANTLY UPREGULATED IN HEART FAILURE (HF). I HAVE FOUND THAT NEPRILYSIN IS ABLE TO CLEAVE THE C-TERMINAL SIDE OF PLB, WHICH MAY SIGNIFICANTLY DISRUPT ITS ABILITY TO REGULATE THE SARCOPLASMIC/ENDOPLASMIC RETICULUM CALCIUM ATPASE (SERCA) IN FAILING HEARTS AND OTHER CARDIOVASCULAR CONDITIONS. MY PRELIMINARY DATA SUGGESTS THAT THIS CLEAVAGE CAN CAUSE SIGNIFICANT ALTERATIONS IN PLB MEMBRANE ANCHORING AND SR CALCIUM HANDLING. AIM 1 WILL FURTHER INVESTIGATE HOW THE C-TERMINAL CLEAVAGE OF PLB WILL AFFECT ITS MEMBRANE LOCALIZATION AND DYNAMICS WITHIN THE MEMBRANE. THIS AIM WILL ALSO USE TOP DOWN PROTEOMICS TO UNDERSTAND THE SPECIFIC AMINO ACID LOCATION PLB IS CLEAVED, AND THE AMOUNT OF NEPRILYSIN REQUIRED FOR CLEAVAGE. AIM 2 SEEKS TO UNDERSTAND HOW THIS DYSREGULATION OF SERCA WILL AFFECT IMPORTANT PHYSIOLOGICAL PARAMETERS, SUCH AS SR CALCIUM HANDLING AND [CA2+]-FREQUENCY RELATIONSHIP. THESE ARE CRITICAL PARAMETERS IN HEALTHY CARDIOMYOCYTE CONTRACTILITY, AND IMPORTANT FOR THE HEART'S INCREASED CONTRACTILITY IN RESPONSE TO A FASTER HEARTBEAT, WHICH IS NOTABLY ABSENT IN HF. THUS, MY EXPERIMENTS TO EXAMINE HOW CALCIUM HANDLING IS DISRUPTED WITH NEPRILYSIN TREATMENT WOULD PROVE CRITICAL IN UNDERSTANDING MECHANISMS BEHIND ALTERATIONS IN CALCIUM HANDLING IN HF USING HUMAN INDUCED PLURIPOTENT STEM- CELL CARDIOMYOCYTES. FINALLY, AIM 3 SEEKS TO UNDERSTAND THE ROLE OF NEPRILYSIN IN HUMAN HF, AND WHETHER NEPRILYSIN INHIBITORS PLAY A ROLE ON PLB CLEAVAGE. USING OUR TISSUE BANK, WE CAN ALSO ANALYZE THE PORTION OF PATIENTS WITH ARRYTHMIAS AND WHETHER NEPRILYSIN INHIBITORS HAD A GREATER IMPACT ON IMPROVING MORTALITY AND HEALTH. UNDERSTANDING NEPRILYSIN'S LOCAL ACTIONS ON PLB CLEAVAGE IN CARDIOMYOCYTES COULD REVEAL NOVEL THERAPEUTIC STRATEGIES FOR HF AND ARRHYTHMIAS. THESE PROPOSED EXPERIMENTS WILL PROVIDE CRITICAL INSIGHTS INTO NEPRILYSIN'S ROLE IN DISEASE AND SHIFT CLASSIC PARADIGMS OF HOW NEPRILYSIN INHIBITORS ARE USED TO TREAT HF AND ARRHYTHMIAS.