Specific activation of ANKRD1 in Dilated Cardiomyopathy

Abstract

Introduction and Aims: Cardiomyopathies are a heterogeneous group of diseases encompassing all pathologies that affect the heart and its ability to maintain the needed cardiac output (CO). Much research has been devoted to pinpoint muscle specific signaling pathways that result in the development of myopathies, such as dilated cardiomyopathy (DCM). Animal models, such as the muscle LIM protein (MLP/Csrp3) knockout that develops DCM have been used to discover and identify specific pathways and pathway components involved in disease development. ANKRD1, a muscle specific protein, is proposed to act as a biomechanical stretch sensor in muscle cells. To investigate how ANKRD1 may modulate biomechanical signaling pathways on the molecular level we analyzed posttranslational modifications of the protein in healthy and hearts of cardiomyopathic mice. The goal of this research proposal is to investigate cell-biological effects of ANKRD1 phosphorylation. This was investigated by measuring levels of total ANKRD1 (totANKRD1) and phosphorylated ANKRD1 (pT102-ANKRD1) and characterizing the subcellular localization of ANKRD1 by immunofluorescence in healthy and cardiomyopathic mouse hearts. Methods: Our experiments used three different populations of mice, two of which were genetically modified: muscle lim protein (MLP) knockout mice as a model of DCM, and Gq-transgenic mice, which develop a type of hypertrophic cardiomyopathy (HCM). Data from immunoprecipitation and gel electrophoresis/immunoblot as well as immunofluorescence experiments were collected and analyzed. Data obtained were protein levels and subcellular localizations of ANKRD1, both total non-phosphorylated ANKRD1, as well as ANKRD1 phosphorylated at T102. Sex as a biological variable was disregarded, as both males and females have been demonstrated to develop DCM or HCM to a similar amount. Results: In total the cardiac tissue of 6 mice were analyzed, two in each of the three groups: healthy wildtype (WT) controls, Gq-transgenic and MLP-ko mice. Using immunoprecipitation and immunoblot analysis we determined that MLP-ko samples contained 170 times the amount of pT102-ANKRD1 compared to the WT-population (p = 0.05) or compared with Gq transgenic mice. In our immunofluorescence analyses we found that pT102-ANKRD1 coincides with the location of the intercalated disk (ID) while ANKRD1 not phosphorylated at T102 was exclusively in sarcomeres, and that MLP-ko showed a disorganized cytoskeletal architecture. Conclusions: pT102-ANKRD1 levels were increased in MLP-ko mice compared to all other mouse populations in the study. The subcellular localization of pT102-ANKRD1 coincides with the location of the ID, a result that has also been described in preliminary studies on human cardiac biopsies from DCM patients. The experiment would need to be run again with a larger number of animals to obtain a significant statistical basis for all results.