Probing relaxed myosin states in hypertrophic cardiomyopathy by second harmonic-generation microscopy

Reviewer #1 (Public review):

Summary:

This study utilizes polarized second-harmonic generation (pSHG) microscopy to investigate myosin conformation in the relaxed state, distinguishing between the disordered, actin-accessible ON state and the ordered, energy-conserving OFF state. By pharmacologically modulating the ON/OFF equilibrium with a myosin activator (2-deoxyATP) and inhibitor (Mavacamten), the authors demonstrate that pSHG can sensitively quantify the ON/OFF ratio in both skeletal and cardiac muscle. Validation with X-ray diffraction supports the accuracy of the method. Applying this approach to a hypertrophic cardiomyopathy model, the study shows that R403Q/MYH7-mutated minipigs exhibit an increased ON state fraction relative to controls. This difference is eliminated under saturating concentrations of myosin modulators, indicating that the ON/OFF balance can be pharmacologically shifted to its extremes. Additionally, ATPase assays reveal elevated resting ATPase activity in R403Q samples, which persists even when the ON state is saturated, suggesting that increased energy consumption in this mutation is driven by both a shift toward the ON state and inherently higher myosin ATPase activity.

Strengths:

This is a well-written and well-conducted study that clearly reveals the power of SHG microscopy. The study clearly establishes the great utility of SHG to study thick filament regulation.

Weaknesses:

(1) Several studies have shown that the ON state of the thick filament is sensitive to both temperature and filament lattice spacing, with a common recommendation to conduct skinned fiber experiments at temperatures above 27{degree sign}C and in the presence of dextran to better preserve physiological conditions. The authors should clarify the experimental temperature used in their skinned fiber studies, indicate whether dextran was included, and discuss whether adherence to these recommended conditions would have impacted their results.

(2) On page 13, the authors report the proportion of disordered heads as approximately 30% in wild-type and 65% in R403Q fibers. They should clarify whether these values represent the percentage of total myosin heads, or rather the percentage of heads that are responsive to Mavacamten and dATP.

(3) In Figure 5, regarding ATPase measurements, the content of contractile material per unit volume of muscle preparation will influence the results. Did the authors account for this variable, and if not, how might it have affected the conclusions?

(4) For readers primarily interested in assessing the ON/OFF state of thick filaments, could the authors list the specific advantages of polarized second harmonic generation (pSHG) microscopy compared to X-ray diffraction?

(5) Given that many data points were derived from the same fiber or myocyte, how did the authors address the risk of type I errors due to non-independence of measurements? Was a nested or hierarchical statistical approach used?

Reviewer #2 (Public review):

Summary:

In striated muscle, myosin motors can dynamically switch between an energy-conserving OFF state and an activated ON state. This switching is important for meeting the body's needs under different physiological conditions, and previous studies have shown that disease-causing mutations associated with cardiomyopathies can affect the population of these states, leading to aberrant contractility. Studying these structural states in muscle has previously only been possible via X-ray diffraction, which requires access to a beam line. Here, Arecchi et al. demonstrate that polarized second-harmonic generation microscopy (pSGH), a technique that is more accessible, can be used to probe the ON/OFF states of myosin in both permeabilized and intact muscle.

Strengths:

(1) There is an outstanding need in the field to better understand the regulation of the ON/OFF states of myosin. Currently, this is studied using X-ray diffraction, meaning that it is accessible to only a few labs. The authors demonstrate that pSGH can be used to probe the ON/OFF states of myosin both in intact and permeabilized muscle. This is a significant advance, since it makes it possible to study these states in a standard research laboratory.

(2) The authors demonstrate that this approach can be employed in both skeletal and cardiac muscle. Importantly, it works with both porcine and mouse cardiac muscle, which are two of the most important animal models for preclinical studies.

(3) The authors manipulate the ON/OFF equilibrium using both drugs and a genetic model of hypertrophic cardiomyopathy that has been shown to modulate the ON/OFF equilibrium. Their results generally agree with previous studies conducted using X-ray diffraction as well as biochemical measurements of myosin autoinhibition.

Weaknesses:

(1) While the application of pSGH to the ON/OFF equilibrium is an important advance, there are limited new biological insights since the perturbations used here have been extensively characterized in previous studies.

(2) SGH has previously been applied to study the nucleotide-dependent orientation of myosin motors in the sarcomere (PMID: 20385845). The authors have previously interpreted the value of gamma as being a readout of lever arm position, but here, it is interpreted as a measure of ON/OFF equilibrium. When this technique is applied to intact muscle, it is not clear how to deconvolve the contributions of lever arm angle from the ON/OFF population (especially where there is a mix of states that give rise to the gamma value). This is an important limitation that is not discussed in the manuscript.

(3) The R403Q mutation has previously been shown to cause an increase in ATP usage. Here, the authors measure an elevated basal ATPase rate under relaxing conditions, and they interpret this as showing increased myosin ATPase activity intrinsic to the motors; however, care should be used in interpreting these results. Work from the Spudich lab has shown that the R403Q mutation can appear as increasing motor function in some assays but depressing motor function in others (see PMID: 32284968, 26601291). Moreover, the actin-activated ATPase rate is an order of magnitude higher than the basal ATPase rate, and thus, small changes in the basal ATPase rate are unlikely to be important for physiology.

(4) The authors interpret some of their data based on the assumption that the high concentrations of drugs cause the myosin to either adopt 100% OFF or ON states. This assumption is not validated, limiting the ability to interpret the fraction of myosins in the ON/OFF states.

(5) The ATPase measurements are innovative but hard to interpret. dATP and ATP do not have identical ATPase kinetics, meaning that it is hard to deconvolve whether the elevated ATPase rate with dATP is due to changes in the ON/OFF population and/or intrinsic ATPase activity. Similarly, mavacamten reduces the rate of phosphate release from myosin, and this effect is not strictly coupled to the formation of the OFF state (e.g., see PMID: 40118457). As such, it is difficult to deconvolve drug-based changes in the inherent ATPase kinetics of the myosin from changes in the OFF-state population.

Reviewer #3 (Public review):

Summary:

This is a very interesting paper extending the use of SHG to the study of relaxed muscle and its use to assess the order-disorder (and on /off) states of myosin heads in the thick filament. The work convincingly shows that SHG and the parameter gamma provide a reliable measure of the state of the myosin heads in a range of different relaxed muscle fibres, both intact and skinned, and in myofibrils. In mini pig cardiac fibres, the use of dATP and mavacamten increased or decreased the number of heads in the disordered state, respectively. On the assumption that these treatments push myosins fully into the disordered or ordered state, then this allows the fraction of ordered heads to be assessed under a wide variety of conditions. It is unfortunate that dATP treatment was not used (as mavacmten was) on rabbit psoas and mouse samples to further test this hypothesis.

The results with the myosin mutant R403Q support the idea that this mutation reduces the fraction of myosin heads in the ordered state and that mavacamten can recover the WT situation.

The results from SHG were compared with parallel studies using X-rays to validate the conclusions. Independent fibre ATPase data further support the conclusions.

The work is solid and provides a novel approach to assessing the activity state of muscle thick filaments. The authors point out some of the potential uses of this approach in the future, including time-resolved SHG measurements. Indeed, jumps in mavacamten or dATP concentration with time-resolved SHG could measure the rates of entry and exit from the ordered, off state of the filament. A measurement is urgently needed in the field.

Strengths:

(1) The SHG signal is convincingly shown to assess the fraction of ordered/disordered myosin heads in the thick filament of a variety of muscle fibres.

(2) The results are similar for rabbit psoas, mouse, and minipig cardiac fibres. Skinning the fibres and production of myofibrils do not change the SHG signal.

(3) Use of myosin R403Q mutant in mini pig confirms a loss of ordered myosin heads, and the ordered heads can be recovered by mavacamten.

(4) Parallel X-ray scattering and ATPase data support the conclusions.

(5) Assuming that dATP and mavacamten generate 100% disordered vs ordered myosin heads respectively, then the percentage of ordered heads can be calculated for a variety of conditions.

Weaknesses:

(1) Issues like the effect of fibre disarray and lattice spacing on the SHG signal are not well defined.

(2) The, now well-defined heterogeneity of thick filament structure is not acknowledged.

(3) dATP was only used on minipig cardiac fibres. The effect of dATP on rabbit psoas and mouse cardiac fibres would be a useful comparison and would help validate the calculation of % ordered heads.