Abstract 14011: Myomegalin, a 250 Kilodalton Phosphodiesterase Interacting Protein (PDE4DIP), Shows a Novel Protein Interaction With Cardiac Myosin Binding Protein C

Abstract

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein important in the regulation of cardiac contraction through its phosphorylation-dependent interactions with myosin heavy chain and actin. MYBPC3, the gene encoding cMyBP-C, contains the largest number of hypertrophic cardiomyopathy (HCM)-causing mutations. Interestingly, the region of MYBPC3 containing the greatest number of HCM-causing missense mutations, the central domains, have an unknown physiological relevance to the overall function of the protein. Intriguingly, central domain C3 contains a positively charged, putative surface binding interface for an as-yet unidentified ligand. Two of the most common HCM-causing mutations, R495Q and R502W, located in C3, appear to alter this charged interface. We have previously shown that these two mutations lead to altered contractile kinetics and hypophosphorylation of cMyBP-C. We hypothesize that this region of C3 contains an important binding region that is disrupted by both the R495Q and R502W mutations. To reveal candidate binding proteins, we performed a pull-down assay using mouse cardiac homogenate and a GST-tagged C2-C4 fragment of cMyBP-C, with or without the R495Q mutation. Mass spectrometry revealed that myomegalin, a 250 kDa phosphodiesterase-4D-interacting protein (PDE4DIP), bound the WT, but not the R495Q C2-C4 fragment. To confirm the interaction between cMyBP-C and myomegalin in situ , we performed Duolink proximity ligase assay (PLA) in cultured neonatal mouse cardiomyocytes which revealed a positive interaction in cMyBP-C +/+ but not in cMyBP-C -/- cells (n>20) (Figure). Our data strongly suggest that a large isoform of myomegalin, separately known to form a protein complex with PKA/AKAP, forms a novel interaction with cMyBP-C. We hypothesize that perturbations in this interaction lead to altered PKA localization and the observed changes in R495Q and R502W cMyBP-C phosphorylation, and may contribute to the HCM phenotype.