How to Measure Load-Dependent Kinetics of Individual Motor Molecules Without a Force-Clamp

Bhowmik

Dawood

et al. 2021

Preprint

Mutations in the lever arm of β-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy (HCM), a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin S2 tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of β-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

Section: Resultsmentioning

confidence: 99%

Section: Harmonic Force Spectroscopy Reveals Altered Detachment Kinetics and Step Sizes Due To Pliant Region Mutationsmentioning

confidence: 99%

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human β-cardiac myosin have divergent effects on myosin function

Bhowmik

Dawood

et al. 2021

Preprint

“…Recombinant human β-cardiac myosin protein constructs (short subfragment 1 [sS1], short-tailed [2-hep], and long-tailed [25hep]) were expressed in C2C12 mouse myoblast cells and purified as previously described (13,20). The load-dependent detachment rates of WT and P710R sS1-eGFP molecules were measured in a dual-beam optical trap using the HFS method previously described (14,51,78) with slight modifications. The step sizes of myosins were determined from the same HFS data by adapting the ensemble averaging method (79) to HFS's oscillatory data.…”

Section: Methodsmentioning

confidence: 99%

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state

Roest

Liu

Proc. Natl. Acad. Sci. U.S.A.

et al. 2021

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)–cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.

“…The load-dependent detachment rates of WT and P710R sS1-eGFP molecules were measured in a dual-beam optical trap using the harmonic force spectroscopy (HFS) method previously described [10, 11, 58] with slight modifications. The sample chamber consisted of two double-sided tapes between a glass slide and a coverslip spin-coated with 1.6-um-diameter silica beads (Bang Laboratories) and a solution of 0.1% nitrocellulose/0.1% collodion in amyl acetate.…”

Section: Methodsmentioning

confidence: 99%

Hypertrophic cardiomyopathy ß-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super-relaxed state

Roest

Liu

et al. 2020

Preprint

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreases in vitro motility and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load-sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super-relaxed state in larger, two-headed myosin constructs, freeing more heads to generate force. Micropatterned hiPSC-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling, as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrates measured molecular changes to demonstrate that closely predict the measured traction forces. These results confirm a key role for regulation of the super-relaxed state in driving hypercontractility in HCM and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.Significance StatementHeart disease is the leading cause of death world wide, and hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, affecting over 1 in 200 people. Mutations in myosin, the motor protein responsible for contraction of the heart, are a common cause of HCM but have diverse effects on the biomechanics of the myosin protein that can make it difficult to predict the combined effects of each mutation. We demonstrate that complex biomechanical effects of mutations associated with heart disease can be effectively studied and understood using a multi-scale experimental and computational modeling approach. This work can be extended to aid in the development of new targeted therapies for patients with different mutations.