Abstract 18550: Impaired Microtubule Mediated Protein Trafficking as a Mechanism of PLN R14del Dilated Cardiomyopathy

Abstract

Introduction: Phospholamban (PLN) regulates calcium handling and contractility in the heart by altering its phosphorylation status, thereby activating or inhibiting the calcium ATPase SERCA2a. A pathogenic mutation in PLN at the Arginine 14 codon (R14del) results in dilated cardiomyopathy characterized by ventricular arrythmias and sudden cardiac death. Cytoplasmic aggregation of PLN is a hallmark of the disease. However, exactly how the R14del mutation causes DCM remains largely unknown. As PLN is trafficked to the cell membrane before it localizes to the t-tubules, we hypothesize that PLN protein trafficking may be impaired, contributing to the PLN aggregation and DCM. Methods: Induced pluripotent stem cell derived cardiomyocytes (iPSC- iPSC-CMs) from patients carrying the PLN R14del mutation and isogenic controls generated by CRISPR-Cas9 genome editing were tagged with biotin to quantify membrane-bound protein expression. Western blotting and mass spectrometry was used to identify differences membrane protein expression, and immunocytochemistry was used to image microtubule layout, PLN and junctional protein expression at the cell borderintercalated disc (do you mean the intercalated disk?) in micropatterned iPSC-CMs. Results: Quantification of membrane-bound proteins showed a significant decrease of PLN at the cell membrane and connexin 43 expression in the intercalated disk of R14del iPSC-CMs compared to isogenic controls. Additionally, we observed significantly reduced levels of EB1, the microtubule tip protein, at the cell border of PLN R14del iPSC-CMs by immunofluorescence imaging and western blot analysis. Finally, disruption of microtube polymerization by Nocodazole treatment, induced PLN aggregate formation in iPSC-CMs. Conclusion: Our data suggest that the transportation of PLN and Cx43 proteins by microtubules is impaired in PLN R14del iPSC-CMs, which results in PLN accumulation in the cytoplasm and a decrease of Cx43 expression in the intercalated disc. These findings provide a novel mechanism for the pathogenesis of PLN R14del cardiomyopathy and a possible therapeutic target.