Gene therapy targeting of AKAP6β-CaMKII signalosomes improves myocardial inflammation and heart failure in a swine model of cardiometabolic syndrome.

BACKGROUND: Cardiometabolic heart failure with preserved ejection fraction (HFpEF) is associated with systemic and cardiac inflammation and diastolic dysfunction. A-kinase anchoring protein 6β (AKAP6β) is a scaffold protein located at the cardiomyocyte outer nuclear membrane that promotes pathological cardiac remodeling via the recruitment of multiple regulatory proteins including protein kinases. In mice, adeno-associated virus (AAV) mediated expression of a peptide based upon a kinase binding domain (KBD) within AKAP6β inhibited the development of heart failure due to chronic pressure overload. Whether KBD expression can also inhibit the development of cardiometabolic heart failure is unknown, and if so, the mechanism of KBD action in HFpEF has yet to be explored. METHODS: The efficacy of a cardiotropic self-complementary AAV gene therapy that expresses the AKAP6β KBD peptide (AAV9sc.KBD) was tested in a female Ossabaw swine model of cardiometabolic syndrome and HFpEF. Single nucleus and bulk RNA sequencing of swine heart tissue and immunoprecipitation-mass spectrometry, live cell imaging, and biochemical assays using primary rat cardiomyocytes were employed to study KBD mechanism of action. RESULTS: AAV9sc.KBD inhibited the development of diastolic dysfunction and heart failure in the Ossabaw model, without negatively impacting systolic function. The improvement in cardiac phenotype was associated with decreased T-cell myocardial infiltrates and partial reversal of pathological gene expression. An unbiased interactome study revealed that the KBD peptide binds Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), identifying CaMKII as a new AKAP6β binding partner. Perinuclear CaMKII activity detected by live cell imaging required AKAP6β expression and was inhibited by KBD expression. In addition, the CaMKII substrate Inhibitor of NF-κB Kinase β (IKKβ) bound AKAP6β. IKK phosphorylation in the Ossabaw model and in myocytes was inhibited by KBD expression, and NF-κB nuclear translocation in myocytes was dependent upon AKAP6β-CaMKII protein complex formation. AAV9sc.KBD treatment inhibited cardiomyocyte NF-κB-dependent gene expression in the Ossabaw model. CONCLUSIONS: Regulated by perinuclear AKAP6β-CaMKII signalosomes, NF-κB pro-inflammatory gene expression in cardiomyocytes participates in a positive feedback loop with cardiac inflammation promoting HFpEF. Proof-of-concept is provided in a large animal model that gene therapy-based cardiomyocyte expression of the KBD peptide will prevent cardiac dysfunction in cardiometabolic syndrome. CLINICAL PERSPECTIVE: What is new: The cardiomyocyte-selective gene therapy AAV9sc.KBD, which targets signalosomes organized by the scaffold protein AKAP6β, is shown to inhibit myocardial T-cell infiltration and improve cardiac structure and function in a large animal model of cardiometabolic HFpEF.The AKAP6β KBD peptide is shown to bind and inhibit the function of CaMKII.CaMKII and IKKβ are shown to participate in perinuclear AKAP6β signalosomes, where they regulate activation of the NF-κB pro-inflammatory gene regulatory pathway.Clinical implications:: Proof-of-concept for a novel strategy for the treatment of HFpEF is provided, intracellular expression by a cardiomyocyte-selective gene therapy vector of an inhibitory peptide, which will inhibit compartmentalized intracellular signal transduction.In conjunction with previous studies in small rodents, the new data obtained in Ossabaw swine support clinical translation of the AAV9sc.KBD gene therapy.