Abstract 19104: Molecular Insights into Pathological Functions of the Mutant Pk69r Muscle Lim Protein That Led to the Development of Dilated Cardiomyopathy In Vivo
Introduction: The Cysteine and Glycine-Rich Protein 3 (CSRP3) gene encoding muscle LIM protein (MLP) is a master regulator of cardiac muscle myogenesis and mechanosensing. The p.K69R mutant MLP lacking interactions with the Z-disk α-actinin and affecting the MLP acetylation at the K69-residue by HDAC4 is associated with dilated cardiomyopathy (DCM) and endocardial fibroelastosis (EFE).
Hypothesis: The K69R-MLP causes DCM leading to heart failure.
Methods: The stable C2C12 myoblasts expressing wild-type (WT-MLP) and mutant K69R-MLP were created. Nuclear export inhibition, proliferation and differentiation assays were performed. Comparative analysis of embryonic, neonatal and adult mouse hearts from Csrp3 knock-in (heterozygous/MLPWT/K69R and homozygous/MLPK69R/K69R) mutants and WT littermates were performed.
Results: Localization of WT-MLP was detected throughout the cell, while K69R-MLP was only detected in the cytoplasm of HL-1 or C2C12 cells. While some nuclear K69R-MLP was detected, most remained in the cytoplasm after leptomycin B treatment, indicating lack of nuclear import of mutant compared to WT-MLP. Myogenesis of K69R-MLP C2C12 myoblasts was blocked compared to WT-MLP, forming polynucleated myoblasts. At 12-weeks of age, in vivo echocardiography demonstrated significant (P<0.01) ventricular dilation and decreased cardiac function (LVvol;d=83.9±18.1 ul; LVvol;s=43.6±11.4 ul; EF=48.1±7.4%; FS=24.2±4.5%) in CSRP3K69R/K69R mutant mice compared to WT (LVvol;d= 57.3±16.3 ul; LVvol;s= 22.1±7.6 ul; EF=61.9±6.1%; FS=32.8±4.4%) and CSRP3WT/K69R (LVvol;d= 58.6±13.1 ul; LVvol;s= 23.7±11.2 ul; EF=61.2±12.5%; FS=32.9±9.1%) littermates. Histology of CSRP3K69R/K69R embryonic hearts indicated abnormal ventricular wall development consistent with noncompaction with DCM evident at E16.5.
Conclusions: The K69R mutation blocks nuclear import/shuttling of MLP and the entry of myoblasts into myogenesis suggesting a novel paradigm for the development of DCM and noncompaction. Effects of K69R-MLP mutation on expression of binding partner proteins, signaling pathways and myocardial remodeling are being further delineated.
Author Disclosures: R. Hiltenbrand: None. J.I. Lin: None. J.F. James: None. T. Hollingsworth: None. W. Shou: None. Z. Khuchua: None. J.A. Towbin: None. E. Purevjav: None.
- © 2016 by American Heart Association, Inc.