Intracellular Angiotensin II Interacts with the Intercalated Disc Protein Xinα in Cardiomyocytes: A Novel Mechanism of Intracrine Angiotensin II Action in Cardiac Disease
Authors: Kumar Rajesh , Chen Wen , Baker Kenneth M 
Published: November 2023

The intracellular renin‐angiotensin system (RAS), defined by synthesis of angiotensin (Ang) II inside the cell and its actions from an intracellular location, has emerged as a pathologically relevant sub‐component of the RAS. Studies in our laboratory have indicated that the cardiac intracellular RAS, which is not inhibited by ACE inhibitors and AT1 blockers, might be the reason for incomplete RAS inhibition in certain pathological conditions, such as diabetes. Using AT1 knockout mice, which lack extracellular Ang II effects, we have shown that intracellular Ang II has a significant role in the development of diabetic cardiomyopathy. The AT1‐independent mechanism(s) of intracellular Ang II in cardiac myocytes is not known. Our hypothesis is that these mechanisms involve novel protein: protein interactions. We overexpressed FLAG‐tagged Ang II (FA) and in FLAG‐scrambled Ang II (FSA) in HL‐1 cells (mouse cardiac myocyte cell line). Ang II interacting proteins were pulled down by co‐immunoprecipitation using anti‐FLAG (M2) affinity gel and their identity determined by mass spectrometry. Based on total number of discreet peptides (19) identified in MS analysis, presence in repeated screens, and a clean negative control, we identified Xinα as a novel Ang II interacting protein with high confidence. Ang II‐Xinα interaction was further confirmed by Western analysis using an anti‐Xinα antibody in HL‐1 as well as cultured adult rat cardiac myocytes. A receptor‐ligand binding assay showed that Ang II binds with Xinα in a saturable, reversible, and specific manner with a Kd of 44 nM. Ang I and Ang 1–7 did not bind Xinα. Xinα is a muscle‐specific, actin‐binding, intercalated disc (ICD) protein. It is required for the maintenance of ICDs and gap junctions and thus, cellular conductance and muscle contraction. We further observed that Ang II causes downregulation and nuclear translocation of Xinα, which likely contributes to conduction defects and increased cell growth/proliferation in response to Ang II. A significant downregulation of Xinα was also observed in the hearts of diabetic mice, which have high intracellular Ang II levels. We measured cell proliferation in HL‐1 cells and HL‐1 cells stably expressing intracellular Ang II and scrambled Ang II, with or without Xinα knock‐down by siRNA. Intracellular Ang II expressing cells and those transfected with siRNA to knockdown Xinα showed increased cell proliferation compared to native HL‐1 cells. Xinα levels are reduced in heart failure patients and knockdown of this protein in animal models is associated with cardiomyopathies with conduction defects, suggesting an essential role in cardiac disease. Our studies have suggested a strong correlation between increased intracellular Ang II, decreased Xinα levels, and cardiac failure. Ang II‐Xinα interaction represents a novel heart specific mechanism of intracellular or intracrine actions of Ang II.Support or Funding InformationThis work was supported by funding by National Heart, Lung, and Blood Institute Grant 5‐R01‐HL‐090817. This material is the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System (Temple, TX).