Dysregulated Cofilin 1/2 and Actin Depolymerization in Mouse Models of Cardiac Laminopathies and Heterozygous Lamin A/C Deficiency

Abstract

Background Lamin A/C (LMNA) gene mutations can cause LMNA cardiomyopathy which accounts for 6-8% dilated cardiomyopathy. Alternations in cardiomyocyte contractile structure especially in actin filaments might underlie the mechanisms of LMNA cardiomyopathy. As actin-depolymerizing factors, cofilins are essential for the dynamics of actin filaments and both cofilin 1 and 2 are reported involving in the pathogenesis of certain LMNA cardiomyopathy. Objectives This study sought to investigate how cofilins and actin polymerization are dysregulated in mouse models of homozygous and heterozygous LMNA deficiency. We further explored related upstream signaling pathways. Methods Heterozygous mice harboring the targeted Lmna null mutation, Lmnatm1Stw, were inbred. Homozygous Lmna-/- mice were selected by genotype. We homogenized mouse hearts from 1-month-old homozygous Lmna-/- (KO), heterozygous Lmna+/- (hets) and wild type (WT) mice, and compared their levels of cofilin 1/2, phospho-cofilin 1/2, slingshot protein phosphatase 1 (SSH1), ERK1/2, phospho-ERK1/2 and LIM Domain Kinase 1 (LIMK1) by western blot. F-actin/G-actin ratio was tested using an in vivo assay kit. Fluorescent staining for F-actin and G-actin was performed in paraffin-embedded mouse heart tissue sections. Results Compared to WT, KO mice had minor decrease in total cofilin 1/2 and significant decrease in phospho-cofilin 1/2, suggesting more non-phosphorylated cofilins and more actin-depolymerizing activity. This explained the decreased F/G-actin ratio in KO mice. Hets demonstrated decreases in both total cofilin 1/ 2 and phospho-cofilin 1/2, suggesting they might not have more non-phosphorylated cofilins although cofilin phosphorylation had minor decrease compared to that of WT. This might be the reason why hets had similar F/G-actin ratio as WT. Fluorescent staining showed less F-actin and G-actin in KO heart tissue sections compared to those in WT and hets sections. In upstream study, we found significant decrease of SSH1 in hets and increase of SSH1 in KO mice. We also observed remarkable decrease of ERK2 in hets and increase of ERK1/2 in KO mice. In addition, we found notable decrease of LIMK1 in both hets and KO mice compared to WT. Conclusions Significant decrease in cofilin phosphorylation and increased actin depolymerization was observed in Lmna KO mice, which might be driven by higher SSH1, ERK1/2 and lower LIMK1 levels. Lmna hets also had decreased cofilin phosphorylation which might be due to less LIMK1. But their total cofilin level deceased as well and some mechanisms might keep cofilin phosphorylated (lower SSH1 and ERK2 levels), which might have prevented actin over-depolymerization.