Cardiomyocyte Phenotype and a Perinuclear Phospholamban Compartment

About This Grant

Recent studies have revealed the existence of distinct, non-membranous Ca2+ signaling compartments within the myocyte, which independently control gene expression involved in pathological cardiac remodeling. However, the precise mechanisms underlying such compartmentation remain poorly understood. Elucidation of the mechanisms conferring compartmentalized Ca2+ signaling in remodeling will inform the development of targeted therapies for heart failure, including non-ischemic Dilated Cardiomyopathy (DCM). We have defined a Ca2+ compartment organized by the scaffold protein A-Kinase Anchoring Protein 6β (AKAP6β, mAKAPβ) at the myocyte outer nuclear membrane (ONM), where AKAP6β is required for the induction of pathological gene transcription and myocyte hypertrophy by the Ca2+/calmodulin-dependent phosphatase calcineurin (CaN). In this application we present new preliminary data that phospholamban (PLN) interacts with AKAP6β and regulates Ca2+ efflux from the AKAP6β compartment into the lumen of the nuclear envelope. In addition, a pathogenic mutation in PLN (p.R14del) increases perinuclear CaN signaling in patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs). These findings suggest a novel, non-canonical role for PLN in perinuclear Ca2+ homeostasis that regulates gene transcription. We propose the central hypothesis PLN is a critical regulator of perinuclear Ca2+ signaling responsible for pathological gene expression, such that targeting of PLN within this compartment comprises a new therapeutic strategy for DCM. Specific Aim 1: Defining the role of PLN in regulating AKAP6β Ca2+ signaling and myocyte hypertrophy. Preliminary data suggest that AKAP6β-bound PLN at the ONM plays a critical role in regulating Ca2+ efflux from a nanometer-scale perinuclear Ca2+ compartment. Using live cell imaging and biochemical and cytochemical assays in primary rat ventricular myocytes and human iCMs, we will elucidate how ONM-localized PLN modulates AKAP6β-associated Ca2+ signaling. Specific Aim 2: Targeting of Perinuclear PLN in Dilated Cardiomyopathy. To test the hypothesis that dysregulation of local Ca2+ efflux from the AKAP6β compartment contributes to DCM pathogenesis, we will use adeno-associated virus (AAV) vectors to confer gain- and loss-of perinuclear PLN function in wildtype and DCM mice. Specific Aim 3: Therapeutic targeting of perinuclear Ca2+ signaling in PLN R14del Cardiomyopathy. We hypothesize that increased AKAP6β-associated Ca2+ signaling contributes to PLN R14del DCM. We will use patient-specific iCM monolayers and engineered heart tissues to study transcriptional and contractile defects associated with PLN R14del DCM and test whether altered AKAP6β signaling will be beneficial in this disease. Together, these aims will define the function of ONM PLN in controlling Ca2+ efflux from the AKAP6β perinuclear compartment. As AKAP6β signalosomes regulate gene expression promoting pathological remodeling, these studies will establish a new paradigm for treating heart failure, including PLN DCM, based upon the targeting of AKAP6β-PLN complexes.