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
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Evaluation Summary:
This work is of broad interest to readers in the fields of cytoskeletal research, muscle biology and heart disease. By utilizing a combination of quantitative biochemical and biophysical experimental approaches, this work provides critical new insights into the molecular mechanisms of understudied mutations in myosin that cause heart disease. The data are rigorously controlled and analyzed and support the claims of the work.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Mutations in the lever arm of β-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, 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 subfragment 2 (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.
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Author Response
Reviewer #1 (Public Review):
Morck et al. report the effect of five HCM causing lever arm mutations - two in the light chain binding region and three in the pliant region - on beta-cardiac myosin motor function and autoinhibition. Overall, this is a strong and very interesting work, especially since the functional consequences of mutations in the lever arm are understudied. The authors carefully compared light chain binding stoichiometries to the myosin heavy chain, steady-state ATPases, in vitro gliding velocities, and single ATP turnover kinetics of lever arm mutants in the context of short-tailed and long-tailed double-headed myosin constructs to investigate their effect on motor function and autoinhibition. They additionally used harmonic force spectroscopy to measure load-dependent detachment rates and step sizes …
Author Response
Reviewer #1 (Public Review):
Morck et al. report the effect of five HCM causing lever arm mutations - two in the light chain binding region and three in the pliant region - on beta-cardiac myosin motor function and autoinhibition. Overall, this is a strong and very interesting work, especially since the functional consequences of mutations in the lever arm are understudied. The authors carefully compared light chain binding stoichiometries to the myosin heavy chain, steady-state ATPases, in vitro gliding velocities, and single ATP turnover kinetics of lever arm mutants in the context of short-tailed and long-tailed double-headed myosin constructs to investigate their effect on motor function and autoinhibition. They additionally used harmonic force spectroscopy to measure load-dependent detachment rates and step sizes of single-headed pliant region mutants and then calculate parameters including ensemble force, power output, and duty ratio. Finally, the authors discuss their findings with a structural model of the autoinhibited state of beta-cardiac myosin and conclude that mutations in the light chain binding region lead to changes in myosin motor activity and the formation of the autoinhibited state whereas mutations in the pliant region impact the ability of myosin to form the autoinhibited state. In summary, this work makes a significant contribution to the mechanisms of disease-causing mutations in beta-cardiac myosin and SRX myosin biology and will be of wide general interest.
We thank the reviewer for their kind comments and wish to point out a minor typo in the above public review–the reviewer states, “the authors…conclude that mutations in the light chain binding region lead to changes in myosin motor activity and the formation of the autoinhibited state whereas mutations in the pliant region impact the ability of myosin to form the autoinhibited state.” The statement should be reversed to read, “the authors…conclude that mutations in the light chain binding pliant region lead to changes in myosin motor activity and the formation of the autoinhibited state whereas mutations in the pliant light chain binding region impact the ability of myosin to form the autoinhibited state.”
The strengths of this work are the rigorous and well controlled experimental design and data analysis in addition to the use of human proteins to study human disease-causing mutations.
A weakness of this work is that the interpretation/discussion of the experimental results heavily relies on previous homology models of beta-cardiac myosin (e.g. Fig. S3) rather than the relevant parts of the recent high-resolution structures of smooth muscle myosin in the autoinhibited state (PMIDs: 34936462, 33268893, 33268888). For example, one of these studies showed a previously unknown conformation of the RLC bound to the lever arms of autoinhibited myosin. The same study also showed that the C-terminus of the RLC interacts with the hook to stabilize the autoinhibited state and that the RLC interacts with the ELC. It would be insightful to analyze or comment if the studied lever arm mutations may change these interactions and possibly alter an allosteric pathway that operates between the light chain bound lever arm and the motor domain.
While we have cited and discussed the structures from PMIDs 33268893 and 33268888 (references 42 and 43), we are grateful to the reviewer for bringing to our attention our omission of the structure from PMID 34936462 and apologize for this oversight. We have now included this citation whenever we refer to smooth muscle myosin structures and have added a comment on the interaction of the RLC with the hook (pg. 25 line 7 - 9). We thank the reviewer for this comment.
We have also added these three experimentally solved structures to figure S7 (previously fig. S3) and added commentary on how these structures differ from the homology-modeled structures. We thank the reviewer for this comment.
We wish to point out that the reason homology-modeled structures are also included in figure S7 (previously S3) is because the sequence of the smooth muscle myosin differs from the sequence of human β-cardiac myosin; thus, assessing the impact of an individual point mutation in the background of many baseline mutations becomes difficult. Ideally, a new modeled structure of human β-cardiac myosin should be created based on the newly available structures of the smooth muscle myosin in the autoinhibited state or an atomic structure of human β-cardiac myosin in the off state should be determined experimentally, but we believe that this is outside the scope of the present work. Additionally, it is possible that the autoinhibited structure of human β-cardiac myosin will differ from the autoinhibited structure of smooth muscle myosin in meaningful ways because smooth muscle myosin forms the autoinhibited state outside of sarcomeres, whereas human β-cardiac myosin would experience autoinhibition within the context of sarcomeres. The goal of including the modeled structures is, in essence, to show that they are all likely incorrect with regards to the real lever arm conformation and to highlight how they differ from the lever arm structures in the aforementioned smooth muscle myosin structures. This point has been clarified in the figure legend and text (pg. 25 line 19 - 22).
Reviewer #3 (Public Review):
The paper by Morck et al. explores the functional consequences of a group of single mutations in the lever arm of myh7 that are associated with hypertrophic cardiomyopathy (HCM). The underlying hypothesis is that these mutations affect the population of the super-relaxed state of myosin. The investigators use range of biochemical and biophysical techniques to explore the activities of these myosins. They conclude that the mutations have a range of effects on the motors, and there is not a single mechanism that can account for hypercontractility that leads to HCM. Although there is not a straightforward connection between the mutations and HCM, the study is important in that it reveals the range of functional effects of the mutations.
A strength of the paper is the range of techniques used to examine the functional consequences of a range of myh7 mutations. Using single-molecule and ensemble techniques, they conclude the lever-arm mutations affect SRX to various extents, in addition to affecting force-dependent actin detachments, actin-activated ATPase activities, and power output. The effort required to express, purify, and characterize the six constructs (WT + 5 mutants) is considerable. A unified mechanism is not proposed as to how these mutants drive HCM, but the work remains significant in showing the range of functional effects that should be considered when modeling SRX, thin-filament activation, and interaction with other sarcomeric proteins.
A weakness in the paper is the variability in the reported ATPase activities as outlined below. This variability leads one to question the validity of the conclusions about actin-activation of the ATPase activity. Additionally, the paper does not show primary single-molecule data, and it does not adequately discuss limitations of the harmonic force spectroscopy method. To be clear, this method is appropriate for this study, but its model-dependent limitations need to be stated.
Specific Points:
The authors ability to conclude that there are differences in the ATPase activities among the isoforms is not convincing. The authors are to be commended for providing the detailed data summary in Table S1, but it is these data that raise concerns. For example, the ranges in the values of kcat's (3.8 - 6.1 s-1) and Km's (1.5 - 13.6 uM) in Table S1 obtained from the different WT-control experiments are very large. In a well-controlled ATPase assay, these numbers should be very similar. It makes one question the health of the proteins and the ability to know the active site concentrations. Normalizing each mutant to the paired WT protein provides a control for assay variability, but it does not control for variability in the health of the proteins. The reader is left to wonder if the percent differences reported for the mutants are meaningful.
We hope that the analysis and adjustments described in the “essential revisions” section help to alleviate these concerns.
Readers need to see primary optical trapping data. Only the results of analyses are shown. It would be helpful to see single interactions, and it would be useful to see displacement distributions. Given the mutations are in the myosin lever, one might expect changes in average displacements or changes in the width of the displacement. These data are not provided.
We thank the reviewer for this comment. We have now added a figure (Figure S3) that includes representative raw optical trapping data (S3 A, B, C, and D) and the process of identification of the respective stroking events from the changes in phase and amplitudes of oscillation of the trapped beads. We have also added displacement distribution for one representative single molecule (Figure S3 Q, R, S, and T).
It is surprising that the authors do not show lifetimes of attachment durations from the optical trapping in the absence of force. Figure 3B is from the model-dependent fitting of the harmonic force spectroscopy experiment.
We have now added representative raw data as obtained from HFS experiments (Figure S3 A, B, C, D) and the analysis of the HFS data for one representative molecule for each myosin type (Figure S3 E, F, G, H, I, J, K, L). In the HFS experiments, due to oscillation of the sample stage, an actin interacting myosin molecule experiences a sinusoidally oscillating load with a definite mean force for each stroking event. In this manner, some of the stroking events occur against zero or near zero mean external force (3rd force bin from the left in Figure S3 I, J, K, L). We thank the reviewer for pointing this out. We hope with the inclusion of the raw data, this concern is now addressed.
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Evaluation Summary:
This work is of broad interest to readers in the fields of cytoskeletal research, muscle biology and heart disease. By utilizing a combination of quantitative biochemical and biophysical experimental approaches, this work provides critical new insights into the molecular mechanisms of understudied mutations in myosin that cause heart disease. The data are rigorously controlled and analyzed and support the claims of the work.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Morck et al. report the effect of five HCM causing lever arm mutations - two in the light chain binding region and three in the pliant region - on beta-cardiac myosin motor function and autoinhibition. Overall, this is a strong and very interesting work, especially since the functional consequences of mutations in the lever arm are understudied. The authors carefully compared light chain binding stoichiometries to the myosin heavy chain, steady-state ATPases, in vitro gliding velocities, and single ATP turnover kinetics of lever arm mutants in the context of short-tailed and long-tailed double-headed myosin constructs to investigate their effect on motor function and autoinhibition. They additionally used harmonic force spectroscopy to measure load-dependent detachment rates and step sizes of single-headed …
Reviewer #1 (Public Review):
Morck et al. report the effect of five HCM causing lever arm mutations - two in the light chain binding region and three in the pliant region - on beta-cardiac myosin motor function and autoinhibition. Overall, this is a strong and very interesting work, especially since the functional consequences of mutations in the lever arm are understudied. The authors carefully compared light chain binding stoichiometries to the myosin heavy chain, steady-state ATPases, in vitro gliding velocities, and single ATP turnover kinetics of lever arm mutants in the context of short-tailed and long-tailed double-headed myosin constructs to investigate their effect on motor function and autoinhibition. They additionally used harmonic force spectroscopy to measure load-dependent detachment rates and step sizes of single-headed pliant region mutants and then calculate parameters including ensemble force, power output, and duty ratio. Finally, the authors discuss their findings with a structural model of the autoinhibited state of beta-cardiac myosin and conclude that mutations in the light chain binding region lead to changes in myosin motor activity and the formation of the autoinhibited state whereas mutations in the pliant region impact the ability of myosin to form the autoinhibited state. In summary, this work makes a significant contribution to the mechanisms of disease-causing mutations in beta-cardiac myosin and SRX myosin biology and will be of wide general interest.
The strengths of this work are the rigorous and well controlled experimental design and data analysis in addition to the use of human proteins to study human disease-causing mutations.
A weakness of this work is that the interpretation/discussion of the experimental results heavily relies on previous homology models of beta-cardiac myosin (e.g. Fig. S3) rather than the relevant parts of the recent high-resolution structures of smooth muscle myosin in the autoinhibited state (PMIDs: 34936462, 33268893, 33268888). For example, one of these studies showed a previously unknown conformation of the RLC bound to the lever arms of autoinhibited myosin. The same study also showed that the C-terminus of the RLC interacts with the hook to stabilize the autoinhibited state and that the RLC interacts with the ELC. It would be insightful to analyze or comment if the studied lever arm mutations may change these interactions and possibly alter an allosteric pathway that operates between the light chain bound lever arm and the motor domain.
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Reviewer #2 (Public Review):
The manuscript by Morck et al examines five HCM-causing mutations in the lever arm of beta-cardiac myosin using biochemical and biophysical assays to assess their impact on myosin function. A range of effects were seen in the various assays with a common defect in the force sensitivity of the detachment rate constant.
The authors have selected six of the many mutations known to occur in β-cardiac myosin heavy chain in cases of hypertrophic cardiomyopathy. The common feature of these mutations is that all are located in the lever arm of the myosin. Three are in the pliant region and two are in the region that binds the ELC and the RLC. A strength of the manuscript is that multiple assays are used to access the effect of these mutations on myosin function. These include measurement of the extent of light chain …
Reviewer #2 (Public Review):
The manuscript by Morck et al examines five HCM-causing mutations in the lever arm of beta-cardiac myosin using biochemical and biophysical assays to assess their impact on myosin function. A range of effects were seen in the various assays with a common defect in the force sensitivity of the detachment rate constant.
The authors have selected six of the many mutations known to occur in β-cardiac myosin heavy chain in cases of hypertrophic cardiomyopathy. The common feature of these mutations is that all are located in the lever arm of the myosin. Three are in the pliant region and two are in the region that binds the ELC and the RLC. A strength of the manuscript is that multiple assays are used to access the effect of these mutations on myosin function. These include measurement of the extent of light chain binding, steady state actin-activated ATPase assays to determine the kcat and the dependence on actin concentration, in vitro motility assays, single turnover assays to access the amount of myosin in the SRX state in the absence of actin, comparison of a short tailed vs a longer tailed HMM fragments to access the fraction of myosin in the SRX state in the presence of actin and optical trapping to extract numerous mechanical and kinetic parameters. The investigation of HCM-causing mutations is a very active area of research and this manuscript adheres to a high standard in terms of examining a number of possibly related mutations using a number of assays. In doing so, it sets a high standard.
A weakness of the study is that it is not always easy to interpret the in vitro assays in the context of how the mutation would lead to a hypertrophic heart disease and the authors acknowledge that future studies will likely have to done in a cellular or tissue model where an intact sarcomere along with other relevant proteins such as myosin binding protein-C are present in order to appreciate the full impact of the mutations. -
Reviewer #3 (Public Review):
The paper by Morck et al. explores the functional consequences of a group of single mutations in the lever arm of myh7 that are associated with hypertrophic cardiomyopathy (HCM). The underlying hypothesis is that these mutations affect the population of the super-relaxed state of myosin. The investigators use range of biochemical and biophysical techniques to explore the activities of these myosins. They conclude that the mutations have a range of effects on the motors, and there is not a single mechanism that can account for hypercontractility that leads to HCM. Although there is not a straightforward connection between the mutations and HCM, the study is important in that it reveals the range of functional effects of the mutations.
A strength of the paper is the range of techniques used to examine the …
Reviewer #3 (Public Review):
The paper by Morck et al. explores the functional consequences of a group of single mutations in the lever arm of myh7 that are associated with hypertrophic cardiomyopathy (HCM). The underlying hypothesis is that these mutations affect the population of the super-relaxed state of myosin. The investigators use range of biochemical and biophysical techniques to explore the activities of these myosins. They conclude that the mutations have a range of effects on the motors, and there is not a single mechanism that can account for hypercontractility that leads to HCM. Although there is not a straightforward connection between the mutations and HCM, the study is important in that it reveals the range of functional effects of the mutations.
A strength of the paper is the range of techniques used to examine the functional consequences of a range of myh7 mutations. Using single-molecule and ensemble techniques, they conclude the lever-arm mutations affect SRX to various extents, in addition to affecting force-dependent actin detachments, actin-activated ATPase activities, and power output. The effort required to express, purify, and characterize the six constructs (WT + 5 mutants) is considerable. A unified mechanism is not proposed as to how these mutants drive HCM, but the work remains significant in showing the range of functional effects that should be considered when modeling SRX, thin-filament activation, and interaction with other sarcomeric proteins.
A weakness in the paper is the variability in the reported ATPase activities as outlined below. This variability leads one to question the validity of the conclusions about actin-activation of the ATPase activity. Additionally, the paper does not show primary single-molecule data, and it does not adequately discuss limitations of the harmonic force spectroscopy method. To be clear, this method is appropriate for this study, but its model-dependent limitations need to be stated.
Specific Points:
The authors ability to conclude that there are differences in the ATPase activities among the isoforms is not convincing. The authors are to be commended for providing the detailed data summary in Table S1, but it is these data that raise concerns. For example, the ranges in the values of kcat's (3.8 - 6.1 s-1) and Km's (1.5 - 13.6 uM) in Table S1 obtained from the different WT-control experiments are very large. In a well-controlled ATPase assay, these numbers should be very similar. It makes one question the health of the proteins and the ability to know the active site concentrations. Normalizing each mutant to the paired WT protein provides a control for assay variability, but it does not control for variability in the health of the proteins. The reader is left to wonder if the percent differences reported for the mutants are meaningful.
Readers need to see primary optical trapping data. Only the results of analyses are shown. It would be helpful to see single interactions, and it would be useful to see displacement distributions. Given the mutations are in the myosin lever, one might expect changes in average displacements or changes in the width of the displacement. These data are not provided.
It is surprising that the authors do not show lifetimes of attachment durations from the optical trapping in the absence of force. Figure 3B is from the model-dependent fitting of the harmonic force spectroscopy experiment.
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Reviewer #4 (Public Review):
This manuscript describes studies using measurements of steady-state actin-activated ATPase activity, in vitro actin filament motility, single molecule force spectroscopy, and single ATP turnover, to investigate the biochemical and mechanical impact of Hypertrophic Cardiomyopathy associated amino acid changes in the pliant and light chain-binding region of human beta cardiac myosin. The study is motivated by the aim of understanding how nonsynonymous mutations in the beta cardiac myosin gene impact disease associated cardiac physiology. Studies that determine the molecular impact of disease associated amino acid changes in cardiac myosin will inform the development of new therapies to treat associated heart disease. They will also inform a general understanding of the molecular basis of cardiac myosin …
Reviewer #4 (Public Review):
This manuscript describes studies using measurements of steady-state actin-activated ATPase activity, in vitro actin filament motility, single molecule force spectroscopy, and single ATP turnover, to investigate the biochemical and mechanical impact of Hypertrophic Cardiomyopathy associated amino acid changes in the pliant and light chain-binding region of human beta cardiac myosin. The study is motivated by the aim of understanding how nonsynonymous mutations in the beta cardiac myosin gene impact disease associated cardiac physiology. Studies that determine the molecular impact of disease associated amino acid changes in cardiac myosin will inform the development of new therapies to treat associated heart disease. They will also inform a general understanding of the molecular basis of cardiac myosin function. This paper seeks both goals, providing mechanistic insights into how changes in the pliant and light chain-binding region of soluble, recombinant human beta cardiac myosin fragments, isolated in solution, impact the protein's mechanochemistry in isolation. In the context of well cited previous publications, these results suggest molecular underpinnings for how these amino acid changes might drive disease in the heart.
The manuscript presents a clear, and concise summary of the work. The experiments that the authors perform, are well designed and appropriate and those experiments have the ability to inform on the hypothesis that the authors seek to test.
The primary criticism of the paper is the potential lack of statistical power. For each experiment in the study, the authors perform 2 biological replicates (independent expression, purification, and testing of each protein examined). Within each experiment, the authors include 3 technical replicates (replicate samples). The authors justify performing only two biological replicates by their claim that their previous experience shows that if two replicates produce the same result, then additional replicates will as well. The quality of the paper would be greatly enhanced with an additional biological replicate for each experiment and the determination of effect size and statistical significance performed across all (more than 2) biological replicates.
The results in the paper are consistent with the papers conclusions, but the statistical power of these conclusions is limited by the studies reliance on only two biological replicates.
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