Musculoskeletal defects associated with myosin heavy chain‐embryonic loss of function are mediated by the YAP signaling pathway

Abstract Mutations in MYH3, the gene encoding the developmental myosin heavy chain‐embryonic (MyHC‐embryonic) skeletal muscle‐specific contractile protein, cause several congenital contracture syndromes. Among these, recessive loss‐of‐function MYH3 mutations lead to spondylocarpotarsal synostosis (SCTS), characterized by vertebral fusions and scoliosis. We find that Myh3 germline knockout adult mice display SCTS phenotypes such as scoliosis and vertebral fusion, in addition to reduced body weight, muscle weight, myofiber size, and grip strength. Myh3 knockout mice also exhibit changes in muscle fiber type, altered satellite cell numbers and increased muscle fibrosis. A mass spectrometric analysis of embryonic skeletal muscle from Myh3 knockouts identified integrin signaling and cytoskeletal regulation as the most affected pathways. These pathways are closely connected to the mechanosensing Yes‐associated protein (YAP) transcriptional regulator, which we found to be significantly activated in the skeletal muscle of Myh3 knockout mice. To test whether increased YAP signaling might underlie the musculoskeletal defects in Myh3 knockout mice, we treated these mice with CA3, a small molecule inhibitor of YAP signaling. This led to increased muscle fiber size, rescue of most muscle fiber type alterations, normalization of the satellite cell marker Pax7 levels, increased grip strength, reduced fibrosis, and decline in scoliosis in Myh3 knockout mice. Thus, increased YAP activation underlies the musculoskeletal defects seen in Myh3 knockout mice, indicating its significance as a key pathway to target in SCTS and other MYH3‐related congenital syndromes.

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Please note: When submitting your revision you will be prompted to enter your funding and payment information.This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance.This quote takes into account any reduction or fee waivers that you may be eligible for.Authors do not need to pay any fees before their manuscript is accepted and transferred to the publisher.EMBO Press participates in many Publish and Read agreements that allow authors to publish Open Access with reduced/no publication charges.Check your eligibility: https://authorservices.wiley.com/author-resources/Journal-Authors/openaccess/affiliation-policies-payments/index.html***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System for Author): Novelty -This model system was previously published by this group's 2020 Development paper, but without a focus on it as a disease model for SCTS.Recent reports on MYH3 loss of function in this and other syndromes motivated this study and gave the MYH3 null animal a disease application.The results do suggest an exciting new medical approach to treating these rare MYH3 syndromes, but the model system first needs to better reflect the genetics of the disease as it occurs.As the authors point out MYH3 seems to be a difficult target for generating transgenic animals, so the system they have created is a reasonable starting point.Future study is necessary with the mutations to MYH3 observed in humans in addition to this engineered null condition.
Referee #1 (Remarks for Author): This study from Bharadwaj, Sharma, and colleagues presents new findings regarding the role of the embryonic myosin heavy chain isoform (MYH3) in muscle development and disease.They use a null MYH3 mouse as a model of SCTS and show 1) YAP signaling is perturbed in the absence of MYH3 and 2) signaling can be normalized by treatment with a small molecule inhibitor of YAP.This is the first application of this transgenic model as a potential model of disease, but the study is incremental There are several major and minor concerns/comments, including: Major 1) Lines 145 -148 ("Although Myh3 germline knockout mice (Myh3Δ/Δ) exhibited severe muscle defects affecting myofiber number, area, fiber type and satellite cell numbers in embryonic and early postnatal stages, this did not result in lethality (Agarwal et al., 2020)") seem at odds with a statement in their previous paper: "Myh3Δ/Δ homozygous pups were not obtained at the expected 25% frequency but at about 14% (17 out of 126), suggesting that approximately half of Myh3Δ/Δ genotype animals die in utero during embryonic or fetal stages".The authors need to explain these apparently conflicting statements?
2) The timing of the C2C12 MYH3 knockdown experiment should be clarified (Line 296), and greater detailed methods provided to improve its interpretation.Based on the methods, the cells were transfected with siRNA for 24 hours, then transfected with the luciferase construct for 24 hours, allowed to grow for 24 hours, and finally cultured in low serum differentiation media for 24 hours before assay.After 24 hours in differentiation media did either the control or treated cells have assembled sarcomeric structures?If not, do the authors have any insight into how MYH3 contributes to mechano-transduction in the cell prior to assembly?Are the traction forces different in myoblasts with knockdown?Is the same effect observed if the cells are treated with inhibitors of contraction/cell tension?
3) The grip strength data presented in Figures 1 and 6 seems inconsistent for the same genotype.The 12 -14 week old / in Figure 1G range from 5 -8 gf/gms, but the vehicle control 12 -14 week old / in Figure 6A appear stronger, ~8 -9.5 gf/gms.The authors should comment on this?The grip strength data for the +/+ control and treated mice used for Fig EV4B , C should also be presented as it seems like a natural comparator given the phenotypic rescue claims in this section.Figures EV4B and EV4C are not currently referenced in-text.4) In Figure 1, was the muscle weight as a fraction of body weight also different between genotypes?
5) The authors should provide quantitative data on any differences in activity level observed between null and normal animals.Could activity differences explain some of the observed isoform shifts?6) Do the myosin isoform shifts observed in the TA and GAS have functional consequences?Are there differences in specific force or contractile kinetics?This manuscript describes the adult phenotype of Myh3 knockout mice, along with identification of YAP as a crucial signaling molecule that, when inhibited, can partially rescue the knockout phenotype.The description of the adult mouse skeletal muscle is comprehensive, in terms of its characterization, including change in muscle fiber predominance over time.Remarkably, the molecular defects that were detected in RNA-seq analysis of skeletal muscle were then used to treat P15 mice with a YAP small molecule inhibitor and partially reversed the phenotype.The experimental approach and execution are all high quality with appropriate controls for the immunohistochemistry and Western blots.Overall, this is an exciting manuscript that has translational implications because it identifies a molecular pathway that could potentially be harnessed for therapy in human disease.

Major concerns:
(1) The scoliosis phenotype is incompletely described.Were spinal fusions seen?Were there fusions of other skeletal elements as seen in patients with ?Did the authors use proper technique to measure the Cobb angle (as described in Chen et al., BMC Musculoskeletal Disord, Proper positioning of mice for Cobb angle radiographic measurements, 2021), as these can be misleading if performed without proper technique.
(2) Additional discussion should address the time sensitivity of treatment with the YAP inhibitor.This was initiated at P15, but how does this timing correspond with reductions in Myh3 expression?Would initiation of treatment at later stages be expected to rescue the phenotype?
Minor points: (1) The y-axis for Figure 2 W and X could be similar for consistency.
(2) Figure 4D shows effects of knockout on other myosins.What is the authors interpretation of these results?This should be added to the discussion.
Referee #3 (Comments on Novelty/Model System for Author): This is a fairly novel manuscript.The analysis of the Myh3 KO defect in combination with a pharmacological manipulation of the Yap inhibitor (CA3) is highly novel for a mechanism of mechanotransduction.There just needs to be some clarification as highlighted in my review.

Referee #3 (Remarks for Author):
The manuscript by Bharadwaj and colleagues is centered on the musculoskeletal defects associated with Myh3-deficiency in skeletal muscle.Recessive loss-of-function mutations in MYH3 cause spondylocarpotarsal synostosis (SCTS), characterized by vertebral fusions and scoliosis, which is also observed in the Myh3 knockout (KO) mouse model.The authors further characterize these and demonstrate them to exhibit changes in muscle fiber type, altered satellite cell numbers and increased skeletal muscle fibrosis.Their mass spectrometric analysis of embryonic skeletal muscle from Myh3 KO muscles revealed integrin signaling and cytoskeletal regulation as the most affected pathways.YAP signaling is predominantly a regulator of these pathways.The authors evaluated the YAP inhibitor CA3 and showed that it rescued many of the satellite cell and muscle pathophysiological defects in the Myh3 KO mice.The authors conclude that YAP-mediated defects are driving Myh3 pathologies and that modulation of this pathway may be a strategy for therapeutic intervention.
Overall, this is a well-written manuscript with logically-designed experiments.The findings are of interest to the ultra-rare disease community that might be affected with SCTC but could be adapted potentially to other similar mechanotransduction disorders affected by disrupted Yap signaling.However, there are a few questions that warrant probing into the proposed mechanism by the authors.Some examination of the Yap-Taz dynamics and TEAD activation is warranted to validate the proposed mechanism of dysregulated in the Myh3 knockout mice.
Major Comments: 1.It would be helpful to know if the decreased Pax7-positive satellite cells observed in the Myh3 KO mice are due to hyperactivation of the satellite cells resulting in their fusion and/or apoptosis?Can the authors comment or have the authors evaluated the differentiation capacity of the satellite cells from the Myh3 KO mice?If they are in the activated state early on in development (Fig. 3G) but decrease later (Fig. 3I), they are likely exhausted and would be either fusing/differentiating or undergoing apoptosis.Markers for both can be evaluated.7B/Model proposes that TEAD-mediated Yap target gene activation does not occur because of the loss of Myh3.The authors assume this based on Yap target gene activation; however there are some Yap and Taz independent activation of TEAD myogenic factors (Sun et al., Stem Cells, 2017).The authors should determine if TEAD actually does change in its localization and/or expression in the Myh3 KO muscle.This would go a long way in strengthening their proposed model.

Figure
3. Minor comment.I would fix the labeling of the protein immunoblots in Figure 4E, some extra spaces between the numbers.4. Can the authors comment on whether the other myosins (MYH8 appears to be upregulated; Fig. 4D) similarly have any impact on Yap/Hippo signaling? 5. Does TAZ/WWTR1 change in expression and/or localization in the Myh3 KO mouse muscles?It is not clear to me if the changes observed in Hippo signaling are TAZ dependent or independent?6. Yap has been proposed to signal through dystrophin/dystrophin-associated protein complex (DAPC) (Iyer et al., AJP-Cell Physiol., 2019).Did dystrophin or other DAPC proteins come up in the authors' mass-spectrometry analyses?7. Minor comment.Based on the authors' figure legends, were only male mice used?Please clarify in the methods section.
8. Similar to my first question.Supp.EV3.Are the decreased satellite cells due to increased fusion or increased apoptosis of the satellite cells themselves?

Response to Reviewer Comments
We are grateful to the Reviewers for evaluating our manuscript and providing excellent comments and suggestions.We have attempted to address every comment to the best of our ability, resulting in several changes to the revised version of the manuscript that we are submitting.Briefly, new data has been added to Figure 1, Figure 3, Figure 4, Figure EV1, Figure EV4 and Figure EV5.Old Figure EV4 data has been moved to Figure EV5 where new data has been incorporated; revised Figure EV4 has fully new data.Please find below our detailed response to the Reviewer comments inline.The line numbers in the response below refer to the highlighted version of the manuscript; we find that the line numbers are changing during word to PDF conversion on the submission portal and therefore request the Reviewers to use the line numbers with respect to the revised, highlighted manuscript which has been submitted as a Supplementary PDF.In this same manuscript, the changes that have been made to the text during revision are highlighted in yellow and in track change mode to show the parts that have been moved or deleted.We hope that the revised manuscript addresses all the queries raised, improving it substantially.

Referee #1 (Comments on Novelty/Model System for Author):
Novelty -This model system was previously published by this group's 2020 Development paper, but without a focus on it as a disease model for SCTS.Recent reports on MYH3 loss of function in this and other syndromes motivated this study and gave the MYH3 null animal a disease application.The results do suggest an exciting new medical approach to treating these rare MYH3 syndromes, but the model system first needs to better reflect the genetics of the disease as it occurs.As the authors point out MYH3 seems to be a difficult target for generating transgenic animals, so the system they have created is a reasonable starting point.Future study is necessary with the mutations to MYH3 observed in humans in addition to this engineered null condition.We thank the Reviewer for the comments and agree that this study could be the starting point for future studies on human disorders characterized by MYH3 mutations.As pointed out by the Reviewer, our results identify YAP signalling as a novel potential target in such disorders, which could be valuable in future patient-directed approaches.

Referee #1 (Remarks for Author):
This study from Bharadwaj, Sharma, and colleagues presents new findings regarding the role of the embryonic myosin heavy chain isoform (MYH3) in muscle development and disease.They use a null MYH3 mouse as a model of SCTS and show 1) YAP signaling is perturbed in the absence of MYH3 and 2) signaling can be normalized by treatment with a small molecule inhibitor of YAP.This is the first application of this transgenic model as a potential model of disease, but the study is incremental.There are several major and minor concerns/comments, including: Major 1) Lines 145 -148 ("Although Myh3 germline knockout mice (Myh3Δ/Δ) exhibited severe muscle defects affecting myofiber number, area, fiber type and satellite cell numbers in embryonic and early postnatal stages, this did not result in lethality (Agarwal et al., 2020)") seem at odds with a statement in their previous paper: "Myh3Δ/Δ homozygous pups were not 30th May 2023 1st Authors' Response to Reviewers obtained at the expected 25% frequency but at about 14% (17 out of 126), suggesting that approximately half of Myh3Δ/Δ genotype animals die in utero during embryonic or fetal stages".The authors need to explain these apparently conflicting statements?We thank the Reviewer for pointing this out.What we meant to convey was that germline knockout of Myh3 did not result in complete lethality; as explained in the earlier publication (Agarwal et al., 2020), in a cross between Myh3 Δ/+ male to Myh3 Δ/+ female, of the expected 25% Myh3 Δ/Δ pups, only 14% were born.The remaining 11% die in utero during embryonic or fetal stages of development.The surviving 14% of Myh3 Δ/Δ pups that are born were used in this study.Based on the Reviewer suggestion, we have added additional sentences and modified the section as follows: "As previously described, we observed 11% lethality of Myh3 germline knockout (Myh3 Δ/Δ ) embryos during developmental stages; the remaining 14% completed embryonic development and were born as pups (Agarwal et al., 2020).Although these surviving Myh3 germline knockout mice (Myh3 Δ/Δ ) exhibited severe muscle defects affecting myofiber number, area, fiber type and satellite cell numbers in embryonic and early postnatal stages, this did not result in increased postnatal lethality compared to controls (Agarwal et al., 2020)" lines 143-148.
2) The timing of the C2C12 MYH3 knockdown experiment should be clarified (Line 296), and greater detailed methods provided to improve its interpretation.Based on the methods, the cells were transfected with siRNA for 24 hours, then transfected with the luciferase construct for 24 hours, allowed to grow for 24 hours, and finally cultured in low serum differentiation media for 24 hours before assay.After 24 hours in differentiation media did either the control or treated cells have assembled sarcomeric structures?If not, do the authors have any insight into how MYH3 contributes to mechano-transduction in the cell prior to assembly?We have added more details for the C2C12 Myh3 knockdown experiment as suggested by the Reviewer.The sentence has been modified to "To confirm the increased YAP activity, we carried out a luciferase reporter assay by transfecting the YAP responsive 8xGTIIC-luciferase plasmid into C2C12 mouse myogenic cells treated with a control siRNA or Myh3 siRNA and allowing the cells to differentiate for 24 hours (Dupont et al., 2011)" (lines 315-318).The methodology used for the experiment is as described by the Reviewer (cells transfected with siRNA for 24 hours, then transfected with the 8xGTIIC-luciferase plasmid for 24 hours, allowed to grow for 24 hours, followed by change to differentiation media and culturing for 24 hours).The luciferase assay is carried out 96 hours after siRNA transfection.The reason to do the experiment in this manner is twofold: first, MyHC-embryonic is the earliest MyHC to be expressed and might have roles in early stages of sarcomere formation, which can be captured by observing early stages of differentiation; second, the effect of the siRNA knockdown is transient and looking at early time points of differentiation ensures that the knockdown persists during the assay.To address the Reviewer comment, we have done MyHC-embryonic blots on the samples, which clearly shows expression in control siRNA treated samples and knockdown in Myh3 siRNA treated samples (Figure 4H-I).Total and phospho-YAP expression is seen in C2C12 cells at this time point (Figure 4H-I).Differentiating myofibers can be clearly observed in these cells and alpha-actinin, a Z-disc protein in the sarcomere, is expressed in C2C12 cells treated in this manner (see image below showing alpha-actinin labelling in red, where arrows point to alpha-actinin puncta marking sarcomere formation).Since MyHC-embryonic and alpha-actinin expression are seen, sarcomere formation has initiated in C2C12 cells where the luciferase assay has been carried out.MyHC-embryonic probably contributes to mechanotransduction during sarcomere assembly, since most studies indicate that MyHC-embryonic expression occurs in differentiating muscle cells, where sarcomere assembly is underway.However, whether MyHC-embryonic has non-sarcomeric functions is unclear, especially since its expression has been reported in non-skeletal muscle cell types in some studies (Rice and Leinwand, J Cell Biol 2003;Zieba et al, Scientific Reports 2017).
Are the traction forces different in myoblasts with knockdown?Is the same effect observed if the cells are treated with inhibitors of contraction/cell tension.These are very interesting points raised by the Reviewer.For traction force, we carried out a contractility measurement assay by growing control and Myh3 siRNA treated differentiating C2C12 cells on a specialized matrix which shrinks based on the contractile forces exerted by the cells, which was quantified and found to be reduced in Myh3 depleted cells (Figure EV4E-F and lines 365-368).ATPase activity of myosin protein isolated from E16.5 control and Myh3 knockout mouse embryos clearly suggest reduced ATPase activity in Myh3 knockout embryos, indicating the requirement of MyHC-embryonic (Figure EV1M and Lines 182-183).Based on the Reviewer comment, we treated C2C12 cells with para-aminoblebbistatin, a myosin inhibitor, which led to increased levels of total YAP and reduced levels of phospho-YAP .This is similar to the effect we observed in Myh3 knockouts (Figure 4H-I and Lines 321-323).Interestingly, another contractility inhibitor, 2,3-Butanedione monoxime (BDM) had a somewhat different effect, where its treatment caused no change in total YAP levels but increase in phospho-YAP levels (Figure EV4C-D and Lines 361-363).The differences observed could be related to the specific effects of these inhibitors on different myosin isoforms and the particular activity they target.Overall, these experiments clearly indicate that inhibitors of contraction do have effects on YAP signalling in skeletal muscle cells, confirming the effects mediated by MyHC-embryonic on YAP that we observe.
3) The grip strength data presented in Figures 1 and 6 seems inconsistent for the same genotype.The 12 -14 week old / in Figure 1G range from 5 -8 gf/gms, but the vehicle control 12 -14 week old / in Figure 6A appear stronger, ~8 -9.5 gf/gms.The authors should comment on this?The grip strength data for the +/+ control and treated mice used for Fig EV4B, C should also be presented as it seems like a natural comparator given the phenotypic rescue claims in this section.Figures EV4B and EV4C are not currently referenced in-text.
There is some amount of variability observed in the grip strength, especially at the 12-week time point (Figure 1R, Figure 6A).We agree that there is a slight increase in grip strength in the Myh3 Δ/Δ vehicle controls in Figure 6A, compared to Figure 1R.The only difference between these mice is the treatment with DMSO as the vehicle control in Figure 6.Several studies report that DMSO might have effects on skeletal muscle contractility, excitation-contraction coupling and differentiation.We have not explored this further, since our direct comparisons are between the vehicle treated mice and the CA3 treated mice (where CA3 is reconstituted in DMSO).We have now also included new data on vehicle and CA3 treatment starting in adult stage (6 weeks) instead of perinatal stage, where we observe a similar, slightly increased grip strength (Figure EV5F).We have added a sentence to address this comment "A slight increase in grip strength is observed in the vehicle treated Myh3 Δ/Δ mice (Figure 6A and EV5F) compared to untreated ones (Figure 1R), which could suggest an effect of the vehicle DMSO on muscle function" (lines 387-389).As suggested by the Reviewer, we have included the Myh3 +/+ data for treated mice used (Figure EV5D).We thank the Reviewer for pointing out some panels that were not referenced in the text previously, which have now been referenced (lines 409-411).4) In Figure 1, was the muscle weight as a fraction of body weight also different between genotypes?
The muscle weight as a fraction of body weight was significantly different for the tibialis anterior (TA) and the quadriceps, but was not significantly different for the gastrocnemius.Thus, loss of MyHC-embryonic might affect specific muscles in diverse ways, possibly as a consequence of the fiber type composition of the specific muscle.The gastrocnemius has more MyHC-slow+ fibers compared to the TA and quadriceps, and loss of MyHC-embryonic reduces MyHC-slow levels drastically (Figure 2M-P, V-X and lines 195-198, 201-202, 204-206).However, the total number of myofibers are unchanged in the gastrocnemius of Myh3 Δ/Δ mice (Figure EV1A, E).This indicates that replacement of the MyHC-slow+ fibers by MyHC-IIa+ and IIb+ fibers is able to compensate for weight in the case of the gastrocnemius (Figure EV2C-G).
5) The authors should provide quantitative data on any differences in activity level observed between null and normal animals.Could activity differences explain some of the observed isoform shifts?We thank the Reviewer for this suggestion.To address this, we have carried out rotarod and treadmill assays comparing activity levels between control and Myh3 Δ/Δ mice.This data has been included in Figure 1S-T and Figure EV1J, referred to in lines 179-183 in the revised manuscript.We observe a significant reduction in activity in the Myh3 Δ/Δ mice using both techniques.It is likely that the decreased activity underlies some of the MyHC isoform shifts that are observed in the adult Myh3 Δ/Δ mice.However, we cannot rule out the possibility that the isoform shifts lead to the decreased activity.This raises the interesting conundrum as to whether the decreased activity precedes the isoform shift or vice versa, which is a difficult question to address.6) Do the myosin isoform shifts observed in the TA and GAS have functional consequences?Are there differences in specific force or contractile kinetics?Yes, there are functional consequences as seen with the effect on activity levels on rotarod and treadmill assays as described for the previous comment.We observe a reduction in the myosin ATPase activity in the E16.5 Myh3 Δ/Δ embryos compared to controls (Figure EV1M and Lines 182-183).We also observe changes in contractility in C2C12 cells where Myh3 has been depleted using siRNA (Figure EV4E-F and Lines 365-368).

Referee #2 (Remarks for Author):
This manuscript describes the adult phenotype of Myh3 knockout mice, along with identification of YAP as a crucial signaling molecule that, when inhibited, can partially rescue the knockout phenotype.The description of the adult mouse skeletal muscle is comprehensive, in terms of its characterization, including change in muscle fiber predominance over time.Remarkably, the molecular defects that were detected in RNA-seq analysis of skeletal muscle were then used to treat P15 mice with a YAP small molecule inhibitor and partially reversed the phenotype.The experimental approach and execution are all high quality with appropriate controls for the immunohistochemistry and Western blots.Overall, this is an exciting manuscript that has translational implications because it identifies a molecular pathway that could potentially be harnessed for therapy in human disease.We thank the Reviewer for the comments and agree that the YAP pathway could be of great interest as a molecular target to treat MYH3-associated human musculoskeletal disorders.

Major concerns:
(1) The scoliosis phenotype is incompletely described.Were spinal fusions seen?Were there fusions of other skeletal elements as seen in patients with ?Did the authors use proper technique to measure the Cobb angle (as described in Chen et al., BMC Musculoskeletal Disord, Proper positioning of mice for Cobb angle radiographic measurements, 2021), as these can be misleading if performed without proper technique.We thank the Reviewer for this suggestion and looked more closely for other phenotypes characteristic of MYH3 mutations.We observe severe spinal fusions in the Myh3 Δ/Δ mice, representative images and quantification of which have now been incorporated in the revised version of the manuscript (Figure 1N-Q; Figure EV1K, L,N,O).We have ensured that the mice are oriented properly for the Cobb angle measurements and followed the technique suggested by Chen et al and have now cited this paper in the materials and methods.
(2) Additional discussion should address the time sensitivity of treatment with the YAP inhibitor.This was initiated at P15, but how does this timing correspond with reductions in Myh3 expression?Would initiation of treatment at later stages be expected to rescue the phenotype?Based on this suggestion by the Reviewer, we have carried out new experiments where YAP inhibitor treatment was initiated later in postnatal life in Myh3 knockouts, starting at 6 weeks after birth.This fails to rescue most of the phenotypes exhibited by Myh3 knockouts, such as reduced grip strength, increased number of myofibers per cross sectional area, reduction in myofiber size, elevated fibrosis, altered MyHC-IIb, -IIa and -slow levels and severity of scoliosis.The only phenotypes which exhibited a rescue were decrease in Pax7 levels in the TA and reduction in expression of the YAP target gene CTGF in the TA.Therefore, we conclude that inhibiting YAP late during postnatal stages does not lead to a proper rescue of the abnormalities exhibited by Myh3 Δ/Δ mice.All of this data has been incorporated in Figure EV5E-R and is referred to in lines 415-428 and 432-436.

Minor points:
(1) The y-axis for Figure 2 W and X could be similar for consistency.We have made this change as suggested by the Reviewer (Figure 2W, X).
(2) Figure 4D shows effects of knockout on other myosins.What is the authors interpretation of these results?This should be added to the discussion.We have added a paragraph to the discussion in the revised manuscript as suggested by the Reviewer, detailing the effect of Myh3 knockout on other myosins (lines 517-539).

Referee #3 (Comments on Novelty/Model System for Author):
This is a fairly novel manuscript.The analysis of the Myh3 KO defect in combination with a pharmacological manipulation of the Yap inhibitor (CA3) is highly novel for a mechanism of mechanotransduction.There just needs to be some clarification as highlighted in my review.We are thankful to the Reviewer for the comments and agree that YAP signalling opens up a novel molecular target for MYH3-associated musculoskeletal disorders, which needs to be explored further.

Referee #3 (Remarks for Author):
The manuscript by Bharadwaj and colleagues is centered on the musculoskeletal defects associated with Myh3-deficiency in skeletal muscle.Recessive loss-of-function mutations in MYH3 cause spondylocarpotarsal synostosis (SCTS), characterized by vertebral fusions and scoliosis, which is also observed in the Myh3 knockout (KO) mouse model.The authors further characterize these and demonstrate them to exhibit changes in muscle fiber type, altered satellite cell numbers and increased skeletal muscle fibrosis.Their mass spectrometric analysis of embryonic skeletal muscle from Myh3 KO muscles revealed integrin signaling and cytoskeletal regulation as the most affected pathways.YAP signaling is predominantly a regulator of these pathways.The authors evaluated the YAP inhibitor CA3 and showed that it rescued many of the satellite cell and muscle pathophysiological defects in the Myh3 KO mice.The authors conclude that YAP-mediated defects are driving Myh3 pathologies and that modulation of this pathway may be a strategy for therapeutic intervention.
Overall, this is a well-written manuscript with logically-designed experiments.The findings are of interest to the ultra-rare disease community that might be affected with SCTC but could be adapted potentially to other similar mechanotransduction disorders affected by disrupted Yap signaling.However, there are a few questions that warrant probing into the proposed mechanism by the authors.Some examination of the Yap-Taz dynamics and TEAD activation is warranted to validate the proposed mechanism of dysregulated in the Myh3 knockout mice.

Major Comments:
1.It would be helpful to know if the decreased Pax7-positive satellite cells observed in the Myh3 KO mice are due to hyper-activation of the satellite cells resulting in their fusion and/or apoptosis?Can the authors comment or have the authors evaluated the differentiation capacity of the satellite cells from the Myh3 KO mice?If they are in the activated state early on in development (Fig. 3G) but decrease later (Fig. 3I), they are likely exhausted and would be either fusing/differentiating or undergoing apoptosis.Markers for both can be evaluated.These are all excellent questions and suggestions by the Reviewer and we have tried to address them as follows.Satellite cells in Myh3 KO mice indeed seem to be hyper-activated.We performed single muscle fiber isolation (along with satellite cells resident on the fibers) and culture experiments from 8-10-week old control and Myh3 KO mice.We find that the satellite cells of Myh3 KO mice exhibit increased activation as shown by greater proportion of satellite cells having MyoD expression.This data has been added to the manuscript (Figure 3N-P) and described (lines 252-257 and 510-513).Regarding the differentiation capability of satellite cells from Myh3 KO mice, we have already shown this data in our previous paper characterizing the embryonic and perinatal role of Myh3 (Figure 5K and Figure S5C-D in Agarwal et al, Development, 2020).We do observe a significant reduction in fusion index in satellite cells from Myh3 KO mice, most likely due to exhaustion of the satellite cell pool.To address the third part of this comment, we carried out western blots for MyoD (differentiation marker) and Caspase3 (apoptosis marker).We observe elevated MyoD levels at the early time point (8-10 weeks) and reduced MyoD levels at the later time point (6 months), indicating exhaustion of the satellite cell pool with age (Figure 3G, I, L and lines 242-252).With respect to apoptosis, Caspase3 levels are significantly reduced at 8-10 weeks and trends towards an increase which is not significant at 6 months (Figure 3G, J, M and lines 242-252).
2. Figure 7B/Model proposes that TEAD-mediated Yap target gene activation does not occur because of the loss of Myh3.The authors assume this based on Yap target gene activation; however there are some Yap and Taz independent activation of TEAD myogenic factors (Sun et al., Stem Cells, 2017).The authors should determine if TEAD actually does change in its localization and/or expression in the Myh3 KO muscle.This would go a long way in strengthening their proposed model We thank the Reviewer and agree that YAP-TAZ independent TEAD activation does occur in many instances as pointed out.To clarify this, we looked at TEAD localization in the cytoplasmic and nuclear fractions in skeletal muscle lysates from 4-month old control and Myh3 Δ/Δ mice.We were unable to detect TEAD in the cytoplasmic fraction but observed similar TEAD levels in the nuclear and total muscle lysates, indicating that TEAD localization or levels do not undergo significant changes in Myh3 KO mice (Figure EV4G-L and lines 370-374).
3. Minor comment.I would fix the labeling of the protein immunoblots in Figure 4E, some extra spaces between the numbers.We thank the Reviewer for pointing this out and have made corrections to these labels.4. Can the authors comment on whether the other myosins (MYH8 appears to be upregulated; Fig. 4D) similarly have any impact on Yap/Hippo signaling?
Based on the Reviewer comment, we performed knockdown of Myh8 which codes for MyHCperinatal, another skeletal muscle myosin heavy chain expressed during development and regeneration, in C2C12 cells.Interestingly, total YAP levels are increased and phospho-YAP (S109) levels are decreased upon Myh8 knockdown, indicating that the effect on YAP signalling might be a conserved phenomenon exhibited by developmental MyHCs (Figure 4J-K; lines 323-327 and 525-527).
5. Does TAZ/WWTR1 change in expression and/or localization in the Myh3 KO mouse muscles?It is not clear to me if the changes observed in Hippo signaling are TAZ dependent or independent?Similar to TEAD, TAZ levels do not change in Myh3 KO mice, although unlike TEAD, we do observe cytoplasmic TAZ in addition to nuclear TAZ (Figure EV4E-J and lines 369-374).
6. Yap has been proposed to signal through dystrophin/dystrophin-associated protein complex (DAPC) (Iyer et al., AJP-Cell Physiol., 2019).Did dystrophin or other DAPC proteins come up in the authors' mass-spectrometry analyses?Dystrophin or DAPC proteins did not come up in our mass spectrometry analysis but Filamin B was the top downregulated protein in Myh3 KO.Filamin B does interact with Dystrophin and mutations in Filamin B lead to spondylocarpotarsal synostosis (SCTS), the same congenital disorder.We have confirmed the Filamin B downregulation by western blots (Figure 4E-F) and added this to the text (lines 299-310 and 457-459).
7. Minor comment.Based on the authors' figure legends, were only male mice used?Please clarify in the methods section.We are grateful for this suggestion.Male and female segregation was done for body weight, muscle weight, grip strength, rotarod and treadmill assays, where there are known differences between sexes.For other parameters, such segregation was not done.We have now stated this in the methods (lines 604-606).
8. Similar to my first question.Supp.EV3.Are the decreased satellite cells due to increased fusion or increased apoptosis of the satellite cells themselves?Based on the single fiber experiments, we believe that the decreased satellite cell numbers are due to increased satellite cell activation and fusion (Figure 3N-P; lines 252-257 and 510-513).Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Please address the referee #2 minor suggestion by discussing the relationship between MYH3 and FLNB.
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Each figure should *Additional important information regarding figures and illustrations can be found at https://bit.ly/EMBOPressFigurePreparationGuideline.See also figure legend preparation guidelines: https://www.embopress.org/page/journal/17574684/authorguide#figureformat The system will prompt you to fill in your funding and payment information.This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance.This quote takes into account any reduction or fee waivers that you may be eligible for.Authors do not need to pay any fees before their manuscript is accepted and transferred to our publisher.***** Reviewer's comments ***** Referee #2 (Comments on Novelty/Model System for Author): This is an exciting manuscript linking early contractility to YAP/hippo signaling.The effects of early loss of MYH3 are striking on these pathways and the rescue of the phenotype with YAP inhibitors may lead to novel human treatments.
Referee #2 (Remarks for Author): The authors have carefully responded to all prior reviews with additional experiments that provide additional support for their conclusions, even that the hippo pathway is also altered by changes in other early expressed myosin genes.My only minor suggestion would be to add 1-3 sentences that emphasize the relationship between MYH3 and FLNB.Knockout of MYH3 caused marked reduction in FLNB and both alterations of both genes cause SCTS, which were noted by the reviewers.Is there existing data about loss of FLNB that showing similar effects on downstream targets?All prior questions have been adequately addressed in the manuscript and in the response to reviewers.
Referee #3 (Comments on Novelty/Model System for Author): The manuscript is appropriate to evaluate the roles of Myosin Heavy Chain (specifically Myh3) which has an impact on the human spondylocarpotarsal synostosis (SCTS) disorder.The ability to dissect out individual isoforms and adequately model their function is appropriate in a knockout mouse model.No concerns are noted with the model choice.

Referee #3 (Remarks for Author):
The authors have done an impressive job in response to my and the other reviewer comments.I appreciate the additional work elucidating additional satellite cell expression and acknowledge that some of their previous work focused on satellite cells in the Myh3 knockout.Additional functional testing of the Myh3 knockout mice is supportive of the model and the authors' hypotheses.Additional supportive data on TEAD localization is also well conducted.I have no additional concerns and believe that the manuscript is suitable for publication in EMBO Molecular Medicine.

Response to Editor and Reviewer Comments
Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Please address the referee #2 minor suggestion by discussing the relationship between MYH3 and FLNB.
We have now added a sentence in the discussion (line numbers 485-487) and cited two references to address this suggestion.
2) In the main manuscript file, please do the following: -Correct/answer the track changes suggested by our data editors by working from the attached document.
We have made the changes as suggested.
-Reduce number of keywords to max. 5. Done.
-Please add callouts for supplementary (Appendix) tables at appropriate places in the text.Further, all figures and panels should be called out in a sequential order.Currently Fig. 3I  -In M&M, the statistical paragraph should reflect all information that you have filled in the Authors Checklist, especially regarding randomization, blinding, replication.We have made changes as suggested.
-Please rename "Conflict of Interest" to "Disclosure Statement & Competing Interests".We updated our journal's competing interests policy in January 2022 and request authors to consider both actual and perceived competing interests.Please review the policy https://www.embopress.org/competing-interests and update your competing interests if necessary.We have made changes as suggested.
-Author contributions: Please remove it from the manuscript and specify author contributions in our submission system.CRediT has replaced the traditional author contributions section because it offers a systematic machine-readable author contributions format that allows for more effective research assessment.You are encouraged to use the free text boxes beneath each contributing author's name to add specific details on the author's contribution.More information is available in our guide to authors: https://www.embopress.org/page/journal/17574684/authorguide#authorshipguidelinesWe have made changes as suggested.
3) Appendix: Please rename "Supporting information" to "Appendix" and remove all EV Figures and their legends and leave only the 2 tables.Tables should be renamed to Appendix Table S1 and S2 and the table of content amended accordingly.EV Figure legends should be moved to the main manuscript file and placed after the main figure legends.We have made changes as suggested.4) Funding: Please make sure the information about funding are complete in both our submission systema and in the manuscript.Currently information about the Regional Centre for Biotechnology (RCB), Indian Council of Medical Research (ICMR), the University Grants We have added the funding information that was missing in the system submission.
5) The Paper Explained: Please provide "The Paper Explained" and add it to the main manuscript text.Please refer to any of our published primary research articles for an example.Check "Author Guidelines" for more information.https://www.embopress.org/page/journal/17574684/authorguide#researcharticleguide "The Paper Explained" has been added to the main manuscript text.6) Synopsis: Every published paper now includes a 'Synopsis' to further enhance discoverability.Synopses are displayed on the journal webpage and are freely accessible to all readers.They include separate synopsis image and synopsis text.
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-Please check your synopsis text and image before submission with your revised manuscript.Please be aware that in the proof stage minor corrections only are allowed (e.g., typos).Synopsis text and image have been included.7) For more information: This space should be used to list relevant web links for further consultation by our readers.Could you identify some relevant ones and provide such information as well?Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc...We have added the OMIM link for SCTS under this section.8) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329),EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts.This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication.Please note that the Authors checklist will be published at the end of the RPF.We agree for the complete RPF and authors checklist to be published with this manuscript.This is an exciting manuscript linking early contractility to YAP/hippo signaling.The effects of early loss of MYH3 are striking on these pathways and the rescue of the phenotype with YAP inhibitors may lead to novel human treatments.

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