Proteomic and Immunohistological Characterization of Cytoskeletal Alterations during the Progression of Human Cardiomyopathy

Abstract

Background: Intermediate filaments (IFs) and microtubules (MTs) play diverse structural and functional roles in the cardiomyocyte, including newly defined regulatory roles in cardiac mechanics and mechanosignaling. Defects in cytoskeleton may contribute to cardiomyopathies through a combination of structural and signaling mechanisms. Previously, we demonstrated that detyrosination of α-tubulin increases MT-IF interaction to regulate the stiffness of cardiomyocytes and impede contractility. Levels of detyrosination are inversely correlated with left ventricular ejection fraction in patients with heart disease. Aim: To broadly characterize cytoskeletal alterations in diverse human cardiac disease. To this end we performed immunohistological and proteomic analysis of left ventricle tissue samples from patients with heart disease of varying origin and severity. Results: Our analysis reveals alterations in a range of cytoskeletal proteins, including different tubulin isotypes, microtubule associated proteins (MAPs), IFs, and sarcomeric proteins. Among these, IFs (such as desmin and synemin) and MAP4 are strikingly and progressively upregulated in hypertrophy and hypertrophic cardiomyopathy, as well as dilated and ischemic cardiomyopathy. This suggests a broadly conserved upregulation and stabilization of IFs and MTs during the progression of cardiomyopathy. Protein expression changes were confirmed by western blot, and quantitative immunofluorescence was used to characterize the density and distribution of cytoskeletal networks in diseased myocardium. Through these studies we aim to provide the most complete characterization to date of cytoskeletal changes associated with human heart disease, in an effort to better understand how pathological changes in cytoskeletal structure correlate with declining function.