Peer review of: Orthophosphate increases the efficiency of slow muscle-myosin isoform in the presence of (2020)

Reviewers' comments:
Reviewer #1 (Remarks to the Author):
Summary: The authors of the submitted manuscript present data investigating the mechanism by which the systolic heart failure (HF) drug, Omecamtiv Mecarbil (OM), affects the force output and cycling kinetic of rabbit soleus muscle in situ. Mechanical experiments confirmed previously published results that OM increases force at submaximal [Ca¬2+], decreases force near maximal [Ca2+], increases calcium sensitivity, and decreases cooperativity. Using a simplified spring-model of the sarcomere, the authors show that the OM-induced reduction in force at max [Ca2+] can be attributed to the reduction in the average strain experienced by each attached motor, as opposed to a reduction in the number of bound motors. At submaximal [Ca2+], OM-induced force increases were attributed to an increase in the number of bound motors, which supports previous findings. Interestingly, the addition of physiological concentrations of inorganic phosphate (Pi) significantly mitigates the OM-induced force reduction at maximal [Ca2+]. The addition of Pi also had significant dose dependent effects on the rate of force redevelopment and the average motor strain. As a second source of kinetic insight, the authors measured the overall ATPase rate (kcat) in isometrically contracting muscle to find that OM did not significantly impact the Pi dependence of kcat but did reduce the muscle’s tension cost. As the authors mention, this finding is specific to the context of high load contraction. To explain the observed effects of OM and Pi, the authors extend a previously published kinetic model of muscle myosin. The main feature of the model is the addition of an OM pathway that inhibits the power stroke and introduces an isomerization step following P¬¬i where the new state has an increased Pi affinity. The new state allows for competitive interactions between OM and Pi¬ in which P¬i can allosterically displace OM, thus allowing the motor to reenter the usual force producing ATPase cycle. Finally, the authors suggest that their experimental findings and model predictions may identify an energy-efficient response to OM during exercise when intercellular Pi increases within the heart.
Major Comments:
1. While it is understandable from the experimental perspective why OM’s effects were studied in soleus skeletal muscle instead of cardiac muscle, the authors translate results between the two systems without much discussion of potential limitations and caveats. Such translation may be appropriate for single molecule results as the dominant myosin isoform in soleus and cardiac muscle are so similar (ref 1) but this is harder to justify at the fiber level where other important sarcomeric proteins differ in isoform and functional role. Furthermore, considering that the vast majority of fiber level studies on OM are in cardiac muscle and that little has been previously published on OM’s effect on soleus skeletal muscle, a detailed limitations section and/or additional experimental evidence is needed to contextualize/justify the translation from soleus to cardiac muscle.
2. In the introduction (manuscript lines 63-69) the authors use the fact that intracellular [Ca2+] remains below saturating levels during left ventricular systole in their proposed mechanism explaining how OM and Pi jointly impact muscle function. Submaximal [Ca2+] data is reported for OM in the absence of presence of Pi in figure 3, but data for the joint effect of OM and Pi is only reported for maximal [Ca2+]. It would greatly strengthen the proposed mechanism if the authors would repeat the Pi experiments reported in figures 4 and 5 at submaximal [Ca2+]. This would ensure that cooperative mechanisms prevalent at submaximal [Ca2+]2, whose impacts may be difficult to predict in the context of a drug, do not significantly impact the results.
3. There are important details missing about the development and implementation of the kinetic model shown in figure 6. First, each rate in table 3 should be accompanied with a reference for which the value was obtained from. If the value had to be inferred, then a similar indication should be made in the table with a written or mathematical justification for why or how the parameter was assigned a specific value. Second, the force generating states used to predict the OM/Pi dependencies of the mechanical and kinetic parameters in figures 2, 4, 5 need to be clearly marked in figure 6. Third, at a minimum, the general form of the differential equations underlying the kinetic scheme should be stated in the supplemental information. Ideally, however, the whole system of equations would be written down. Finally, what numerical methods were used to solve the system or what software was used? The inclusion of each of these would significantly add credibility and transparency to the model and following conclusions.
4. As the manuscript mentions (manuscript lines 41-42), it is well known that OM increases the rate of phosphate release (refs 3, 4). However, the kinetic rates found in table 3 do not reflect this; the rate of phosphate release normal XB cycle (k+4) is given a value of 25s-1, while the rate of phosphate release in the OM affected cycle (k+10) is only 7s-1. Please comment on why this is.
5. While it is true that this is the first study to examine OM’s effects in situ by combining fast sarcomere-level mechanics and in situ enzyme kinetics, the individual methodologies are well established (refs 5-7). Furthermore, previous studies using different fiber-level techniques have arrived at very similar conclusions as the ones in this study. For instance, OM’s mechanism of force enhancement and its potential effect on the muscle’s tension cost had both been previously predicted (ref 8). Finally, the manuscript’s primary novel contribution, the joint effect of Pi and OM on contractile performance, was not obtained in the most relevant muscle system (cardiac muscle), which limits its usefulness in understanding OM’s mechanism. The authors would well be served by making a stronger case for the unique contribution of the data within the context of the substantial literature on OM that already exists.
Minor Comments:
1. In figures 1D and the solid trace in 4B that display the rates of force redevelopment (rTD) as functions of [OM] and [Pi], the points closest to the y-axis are presumably measured under the same experimental conditions: [OM]=0mM and [Pi]=1mM. However the furthest left value in 1D is (3.7±2)s-1 while the furthest left value in figure 4B is significantly lower, approximately (2.7±2) s-1, but increases with [Pi] to a value close to 3.7s-1. Should this be the case?
2. The methods section explains the experimental apparatus used at the Amsterdam lab to measure ATPase rate. However, it is not clear how the force was measured and recorded during the ATPase experiments. Please clarify this process in the methods section and include any relevant instrument specifications.
3. Please report the temperature at which ATPase rates were measured at the Amsterdam lab.
References
1. Schiaffino, S. & Reggiani, C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol. Rev. 76, 371–423 (1996).
2. Gordon, A. M., Homsher, E. & Regnier, M. Regulation of contraction in striated muscle. Physiol. Rev. 80, 853–924 (2000).
3. Malik, F. I. et al. Cardiac myosin activation: A potential therapeutic approach for systolic heart failure. Science (80-. ). 331, 1439–1443 (2011).
4. Woody, M. S. et al. Positive cardiac inotrope omecamtiv mecarbil activates muscle despite suppressing the myosin working stroke. Nat. Commun. 9, 1–11 (2018).
5. Goldman, Y. Kinetics Of The Actomyosin ATPase In Muscle Fibers. Annu. Rev. Physiol. 49, 637–654 (1987).
6. Caremani, M., Melli, L., Dolfi, M., Lombardi, V. & Linari, M. Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products. J. Physiol. 593, 3313–3332 (2015).
7. Percario, V. et al. Mechanical parameters of the molecular motor myosin II determined in permeabilised fibres from slow and fast skeletal muscles of the rabbit. J. Physiol. 596, 1243–1257 (2018).
8. Kieu, T. T., Awinda, P. O. & Tanner, B. C. W. Omecamtiv Mecarbil Slows Myosin Kinetics in Skinned Rat Myocardium at Physiological Temperature. Biophys. J. 116, 2149–2160 (2019).
Reviewer #2 (Remarks to the Author):
Major Points
The authors explore the effect of phosphate on the action of omecamtiv mecarbil – which surprisingly has a significant effect on muscle mechanics. Overall, this is a thoughtful set of experiments that grow the understanding of how omecamtiv mecarbil works to increase muscle contractility and the energetics of its action. Many will be interested in the conclusions of the paper. However, overall the paper is complex for the reader not already an expert in muscle mechanics. I would suggest the authors simplify the text substantially. The key findings of the paper reviewed in the discussion are lost because of the complexity of the discussion.
Cardiac contraction is more of a twitch (the time of systole ~300 msec is approximately the same as a single turnover of ATP by myosin). Do the results for an isometric contraction differ from what might happen during a short isokinetic contraction? Is there an experiment the authors can do to understand if there is a difference?
In regard to the following sentence and central to the conclusions of the paper:
“The inhibitory effect of OM on force generation is reversed by Pi, which acts as an allosteric competitor that allows the OM-bound no-force-generating motors to release OM and re-enter the force-generating cycle without any change in the rate of ATP hydrolysis.”
How is the alternate model - that of OM stabilizing the lever arm in the pre-powerstroke position, engaging the thin filament, releasing Pi and OM together, and then myosin proceeding to generate force – excluded? Or should it be considered as an alternate model?
I would suggest a table in the text of the paper that describes the various parameters as an easy reference for the reader working through the paper.
Minor Comments
Abstract
The statement below seems to also apply to resting conditions – why the focus on exercise? Suggest you end sentence after the word “patients”.
“This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM- treated patients under exercise, when [Pi] increases with heart-beat frequency.”
Introduction
Misspellings like disfunction (dysfunction) and some other typos and grammatical errors – authors please correct.
Omecamtiv mecarbil not Omecamtiv Mecarbil
This statement - “while the velocity of actin sliding in the in vitro motility assay (IVMA) is reduced15- 17”- is not needed and distracts from the main relevant description of the mechanism of action.
Long sentence – please revise.
“Here we determine the effect of OM on the number and force of the attached myosin motors and on the rate of ATP hydrolysis and their modulation by physiological concentrations of inorganic phosphate (Pi), by applying fast sarcomere level mechanics to Ca2+-activated demembranated fibres from rabbit soleus muscle, which contains the slow myosin isoform.”
Suggest use of the work “contractile” rather than “inotropic” in the sentence below. Inotropic generally references an increase in the rate and extent of contractility. OM doesn’t appear to increase the rate of cardiac contractility.
“This effect of Pi on the OM-bound motors represents an energetically efficient inotropic response in patients under OM treatment during exercise and increased workload, when [Pi] increases23,24 with the increase of heart beat frequency.”
Results
The authors are applauded for examining the concentration effect relationship of omecamtiv mecarbil in Figure 1. They do focus on the changes at 1 uM. The target range of concentration of omecamtiv mecarbil in humans is 300-750 ng/mL and the free fraction of OM is only 20%. So a plasma concentration of 400 ng/mL translates to a free concentration of 0.2 uM, 5x lower than 1 uM, where the effects are more modest and probably more clinically relevant. The authors should include some text in the results section about the magnitude of the effects in the clinically relevant range of omecamtiv mecarbil concentrations and not just focus on 1 uM.
Unusual to reference to Amsterdam or Florence experiments. The paper’s results usually are described without reference to location.
Generally, the authors should try to simplify the text in the results section if possible. It is difficult to follow – a table of measured parameters would be helpful as mentioned above so that the reader can easily figure out the meaning of the many different variables used.
Discussion
The paper discussion should focus on how these findings contribute to our understanding how OM works in conditions that mimic living, beating cardiac muscle. The relevance of a mechanism of action for omecamtiv mecarbil described previously in the absence of phosphate should be questioned since there is always mM levels of phosphate in working muscle. The authors should compare/contrast their findings with prior findings that did not examine the effects of physiological phosphate and focus on what are the implications to the energetics of cardiac contraction in the presence of omecamtiv mecarbil.
Reviewer #3 (Remarks to the Author):
This contribution from the Lombardi group provides critical insights into the mechanism of Omecamtiv mercarbil (OM), an activator of cardiac myosin that is in Phase 3 clinical trials for heart failure. The data from this laboratory is as always of excellent quality - it combines sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus.
A major finding from this paper is the impact of Pi ions on OM-bound myosin heads. In the absence of Pi, OM leads to an increase in ATP consumption due to the inability of OM-bound heads to complete the force-generating power stroke, and thus detaching without completing the powerstoke but having consumed ATP. The addition of physiological levels of ATP reverses the excess ATP consumption and brings it back to the consumption in the absence of drug. This is of great importance since it shows a previously unappreciated effect of OM – its ability to reduce the energetic cost of systolic tension when Pi concentrations increases with heart-beat frequency. Without knowledge this Pi effect, it has been difficult to explain the impact of the drug on the working heart from the experimental data published to date.
While the data is well presented and of very high quality, I have reservations concerning the modeling and its justification. The authors should at least indicate that the model proposed is only one possible model. There is for example no evidence that rebinding of Pi would lead to release of OM as indicated in step 14. In fact there is no basis for suggesting the transition (11) to a state that has a higher affinity for Pi exists. If it is based on other data in the literature, this should be cited. However, it seems simpler to propose that OM increases the occupancy of the state formed by Pi release, allowing exogenous Pi to increase the population of the same state that detaches in the model (green) in the absence of OM. But that OM inhibits its detachment and futile loss of ADP and Pi. In any event, it is important that the authors acknowledge that they have chosen one of the possible models to account for the data since controversy exists as to the states sensitive to Pi rebinding to the myosin head during the powerstroke.
Overall, this is a very strong and well written paper that provides a critical new insight into how a cardiac/slow myosin stimulator can achieve positive results in the clinic. The data is state of the art and the findings will be of wide interest.