Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties

Abstract

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β-cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin–actin interaction

“…For the control sample of actin and S1, we determined K 1 k +2 , 1/ K 1 , and k +2 values of 3.83 ± 0.25 μM −1 s −1 , 421 ± 34 μM, and 1621 ± 78 s −1 ( Table 1 ), respectively, which are consistent with that observed previously for cardiac β-S1 where K 1 k + 2, 1/ K 1 , and k +2 were 4.4 ± 0.3 μM −1 s −1 , 328 ± 53 μM, and 1543 ± 100 s −1 ( 8 ). All the compounds except Car and Hon induced modest increases in the range of 1.34–1.76-fold in the second-order rate constant K 1 k +2 for ATP binding ( Table 1 ).…”

“…11 ). This is consistent with previous reports of cardiac S1 from various species; for tissue-purified bovine cardiac β-S1 K A was 6.3 nM ( 40 ), for tissue-purified human cardiac (HC) S1 K A was 8 ± 2 nM or 10 ± 1.8 nM ( 8 ), and for bovine masseter β-S1 K A was 7 nM ( 41 ). The dissociation equilibrium constant, K A, for actin binding to S1 in the presence of Mit and Dan, was 3-fold and 3.8-fold higher, respectively ( Fig.…”

“…At least eight compounds (Phn, Flp, Flu, Thi, Mef, Teg, Dan, and Mit) increased K 1 and decreased k +2 by at least a factor of 2 (up to a factor of 4 in some cases), thus increasing the affinity of ATP and slowing the rate of isomerization, respectively. Similar effects have been observed for myosin HCM mutations that showed little or no change in K 1 k +2 or k cat , but showed significant decreases in k +2 or 1/ K 1 ( 8 ). For several of those HCM mutations, these were among the most significant effects, and it was pointed out that changes greater than 20% can be important ( 8 ).…”

“…Similar effects have been observed for myosin HCM mutations that showed little or no change in K 1 k +2 or k cat , but showed significant decreases in k +2 or 1/ K 1 ( 8 ). For several of those HCM mutations, these were among the most significant effects, and it was pointed out that changes greater than 20% can be important ( 8 ). We observed changes greater than 20% for K 1 k +2 , 1/ K 1 , and k +2 ( Table 1 ).…”

“…The release of ADP to form the A.M complex completes the actin–myosin ATPase cycle ( 4 , 5 ). Thus, the key actin–myosin interaction during force production, involving the weak-to-strong transition (W→S), can be fine-tuned by changes in either LCD structure (flexibility or axial rotations) or equilibrium/rate constants leading to this transition ( 6 , 7 , 8 ).

Figure 1 Actin (PFA) binding to and dissociation from myosin S1.

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