Deletion of the Z-disc Protein Enigma Homolog Depresses Cross-bridge Cycling Kinetics in Mouse Myocardium

Abstract

Background: The Enigma Homolog (ENH) protein is a member of the Enigma subfamily of the PDZ/LIM family of proteins. ENH isoforms are abundantly expressed in the heart and loss of protein expression results in cardiac dysfunction and the development of dilated cardiomyopathy. Despite the fact that ENH isoforms have been shown to bind and modulate the activity of a variety of kinases involved in signalling to contractile proteins, the function of ENH in cardiac muscle remains unclear. In this study, we sought to determine whether ENH can modulate cardiac contractile function, possibly by influencing contractile protein phosphorylation. Methods: Right ventricular trabeculae were isolated from the hearts of ENH+/+, ENH+/-, and ENH-/- mice, skinned, and subjected to mechanical measurements. SDS-PAGE analysis was used to assess the expression of myosin heavy chain (MHC) isoforms and other major contractile proteins. Top-down proteomics was utilized to analyze contractile protein phosphorylation. Results: The steady-state mechanical properties of trabeculae isolated from ENH+/+, ENH+/-, and ENH-/- mice did not differ, but a significant depression in cross-bridge cycling kinetics was observed in ENH+/- and ENH-/- trabeculae. While increased expression of β-MHC partially explains decreased cross-bridge cycling kinetics in ENH-/- trabeculae, the relative expression of β-MHC was not different between ENH+/+ and ENH+/- trabeculae despite depressed cross-bridge cycling kinetics in the latter. The expression of other major contractile proteins were similar between trabeculae from ENH+/+, ENH+/-, and ENH-/- animals. Although the phosphorylation of cardiac troponins and the myosin regulatory light chain were significantly decreased in ENH-/- mouse myocardium, contractile protein phosphorylation in ENH+/- mouse myocardium was not significantly different from that in ENH+/+ mouse myocardium. Conclusions: Collectively, this information suggests that loss of ENH protein expression depresses cross-bridge cycling kinetics in mouse myocardium independent of changes in contractile protein expression or phosphorylation.