Structural and protein interaction effects of hypertrophic and dilated cardiomyopathic mutations in alpha-tropomyosin.

Audrey N Chang,Norma J Greenfield,Abhishek Singh,James D Potter,Jose R Pinto

doi:10.3389/fphys.2014.00460

Abstract

The potential alterations to structure and associations with thin filament proteins caused by the dilated cardiomyopathy (DCM) associated tropomyosin (Tm) mutants E40K and E54K, and the hypertrophic cardiomyopathy (HCM) associated Tm mutants E62Q and L185R, were investigated. In order to ascertain what the cause of the known functional effects may be, structural and protein-protein interaction studies were conducted utilizing actomyosin ATPase activity measurements and spectroscopy. In actomyosin ATPase measurements, both HCM mutants and the DCM mutant E54K caused increases in Ca2+-induced maximal ATPase activities, while E40K caused a decrease. Investigation of Tm's ability to inhibit actomyosin ATPase in the absence of troponin showed that HCM-associated mutant Tms did not inhibit as well as wildtype, whereas the DCM associated mutant E40K inhibited better. E54K did not inhibit the actomyosin ATPase activity at any concentration of Tm tested. Thermal denaturation studies by circular dichroism and molecular modeling of the mutations in Tm showed that in general, the DCM mutants caused localized destabilization of the Tm dimers, while the HCM mutants resulted in increased stability. These findings demonstrate that the structural alterations in Tm observed here may affect the regulatory function of Tm on actin, thereby directly altering the ATPase rates of myosin.

Highlights

Numerous mutations in the proteins of the sarcomere have been associated with cardiomyopathies (Chang and Potter, 2005; Force et al, 2010; Seidman and Seidman, 2011; Hershberger et al, 2013)
As a control for the assay, a well-characterized hypertrophic cardiomyopathic (HCM) mutation E180G was included in the actomyosin ATPase assays
The results of these studies show that the two dilated cardiomyopathic (DCM) mutants gave strikingly different results where E40K causes a significant decrease in maximal ATPase activity and E54K causes an increase (Figure 1A)

Summary

Introduction

Numerous mutations in the proteins of the sarcomere have been associated with cardiomyopathies (Chang and Potter, 2005; Force et al, 2010; Seidman and Seidman, 2011; Hershberger et al, 2013). Various studies of cardiomyopathic mutations suggest distinct mechanisms for the progression of the dilated cardiomyopathic (DCM) and hypertrophic cardiomyopathic (HCM) phenotypes (Chang et al, 2005; Robinson et al, 2007; Willott et al, 2010). An understanding of the disease progression necessitates the study of the effects of the mutations on protein structure and function (Force et al, 2010; Tardiff, 2011). One of the sarcomeric proteins found to have mutations associated with cardiomyopathies is cardiac α-tropomyosin (Tm) (Redwood and Robinson, 2013). Putative actin on/off regions along the Abbreviations: wild-type human α-striated tropomyosin with and N-terminal Ala-Ser dipeptide replacing the acetyl group (ASWT); DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; Tm, tropomyosin; Tn, troponin

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