Abstract P2039: Alteration In Intercalated Disc Composition Impacts Cardiac Disease Progression

Abstract

Intercalated discs (IDs) connect cardiomyocytes electro-mechanically and ID dysfunction has been associated with the onset of cardiomyopathies and arrhythmias. The ID- and myofibril-associated protein nebulin-related anchoring protein (NRAP) was identified as a putative novel interaction partner of protein kinase D1 (PKD). PKD1 phosphorylates class IIa histone deacetylases (HDAC), leading to their cytosolic export. This causes a de-repression of HDAC-mediated inhibition of myocyte enhancer factor 2 (MEF2)-dependent fetal and pro-hypertrophic gene expression. We investigated the regulation of NRAP by PKD1 in the developing and diseased heart. PKD activity was modulated by using the pharmacological PKD inhibitor BPKDi and/or the pro-hypertrophic stimulus phenylephrine (PE) in neonatal rat ventricular myocytes (NRVMs) and -derived engineered heart tissues (EHTs). NRVMs were analyzed by immunofluorescence, western blotting and stable isotope labelling with amino acids in cell culture/mass spectrometry. The outcome was studied by exposure to an autophagy (bafilomycin A1) or a proteasomal (MG132) inhibitor or performing proteome analysis from hearts of MuRF-1/3 double-knock-out (DKO) mice. Exposure of NRVMs to PE enhanced myofibril maturation and NRAP translocation towards IDs while BPKDi led to perinuclear accumulation of NRAP and improved force in EHTs. Combined BPKDi and PE disturbed sarcomeric structure and prevented NRAP localization at the ID. Proteomic analysis revealed that BPKDi reduced NRAP abundance and enhanced the abundance of proteins involved in proteasomal, lysosomal and spliceosomal pathways. Inhibition of proteasomal protein degradation by MG132 increased NRAP levels, whereas NRAP was 42-fold more abundant in hearts of MuRF-1/3-DKO mice that show a protein surplus cardiomyopathy. In conclusion, PKD1 impacts NRAP expression and translocation, and negatively affects EHT force development while proteasomal inhibition increased NRAP abundance suggesting a regulation via the PKD1-HDAC-MEF2 axis. Further investigations aim to study pathways involving PKD1 to regulate myofibril assembly and maintenance in the development and progression of cardiac disease.