Dilated cardiomyopathy myosin mutants have reduced force-generating capacity

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.

Myosin motor domains carrying mutations implicated in early or late onset Hypertrophic Cardiomyopathy have similar properties

“…All other rate constants had variable changes or none at all. This highly variable pattern was also seen for the set of DCM mutations we previously reported (Ujfalusi et al, 2018).…”

“…We previously performed kinetic analysis of five adult-onset DCM mutations in β-MyHC (Ujfalusi, 2018). Overall, our analysis did not reveal a pattern of common defects in individual steps of the cycle, other than a few altered properties specific to the myosin subdomain where the mutation is localized.…”

“…Considerable amounts of pre-steady state kinetic data have been collected for a number of DCM and HCM mutations in the β-MyHC motor domain. The descriptions of methods, analysis tools, model assumptions, data quality and details of the measurements have been set out in our earlier work (Bloemink et al, 2014;Deacon, Bloemink, Rezavandi, Geeves, & Leinwand, 2012;Srbolujub M. Mijailovich et al, 2017;Nag et al, 2015;Ujfalusi et al, 2018). We have taken the same approach to understand the impact of this set of HCM mutations and to compare to the results of previous studies.…”

“…We modeled the complete cycle using all of our kinetic data following the approach outlined in recent papers comparing different myosin isoforms and different DCM-causing mutations in MYH7 (Srbolujub M. Mijailovich et al, 2017;Ujfalusi et al, 2018). The Table 2), and ATP-economy ( Fig.…”

“…However, a pattern did emerge when the full cycle was modeled using all the data from ATPase and stopped-flow experiments (Srbolujub M. Mijailovich et al, 2017). Modeling predicted that the DCM mutations altered the steady-state motor function and the state occupancies in the minimal 8-step crossbridge cycle in a manner consistent with a loss of the ability to generate steady-state force (Ujfalusi et al 2018). Here, for the early-and late-onset HCM mutations, we show that the kinetic parameters of mutations also do not have a "unifying" disruption of the cycle.…”