Dilated cardiomyopathy myosin mutants have reduced force-generating capacity

Zoltan Ujfalusi,Carlos D Vera,Srbolujub M Mijailovich,Marina Svicevic,Elizabeth Choe Yu,Masataka Kawana,Kathleen M Ruppel,James A Spudich,Michael A Geeves,Leslie A Leinwand

doi:10.1074/jbc.ra118.001938

Abstract

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human β-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M·D complex in the steady state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force-holding capacity due to the reduced occupancy of the force-holding state.

Highlights

Our analysis suggests that Dilated cardiomyopathy (DCM) mutations use less ATP than WT in generating high force or high velocities
The five DCM mutations studied here were selected because they are located in quite distinct regions of the motor domain and are highly conserved across myosins, and yet all cause DCM (Fig. 1, A and B)
Ile-201 is at the end of a helix that links the P-loop to the start of loop 1, a surface loop known in many myosins as a variable loop that can influence the affinity of ADP for myosin and actin1⁄7myosin. (For a review of structures, see Refs. 23–25.) Ala-223 is a buried residue near the ATP-binding site, in a helix just after loop 1, and the helix has potential interactions with major structural elements in the upper 50-kDa domain, such as helix O and the central ␤-sheet

Summary

Introduction

These include S532P and F764L [9]. We completed detailed characterization of the biochemical and chemo-mechanical properties of human ␤-cardiac myosin motor domains carrying either R403Q or R453C HCM mutations [13,14,15]. We published a modeling analysis of the WT ␤-cardiac S1 and the HCM mutation, R453C [16], which demonstrated that such detailed kinetic information on the individual molecular events in the ATPase cycle can be used to model the complete mechanochemical cycle and predict some of the properties of the motor, such as maximum shortening velocity, and the load dependence of force-holding states. The same approach should allow us to define how missense mutations alter the mechanochemical cycle and may provide insights into how mutations can cause different cardiac phenotypes

Results

Discussion

Conclusion