Generating fast-twitch myotubes in vitro using an optogenetic-based, quantitative contractility assay

  1. Katharina Hennig
  2. David Hardman
  3. David Barata
  4. Inês Martins
  5. Miguel O. Bernabeu
  6. Edgar R. Gomes
  7. William Roman

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Abstract

The composition of fiber types within skeletal muscle impacts the tissue’s physiological characteristics and susceptibility to disease and ageing. In vitro systems should therefore account for fiber type composition when modelling muscle conditions. To induce fiber specification in vitro , we designed a quantitative contractility assay based on optogenetics and particle image velocimetry. We submitted cultured myotubes to long-term intermittent light stimulation patterns and characterized their structural and functional adaptations. After several days of in vitro exercise, myotubes contract faster and are more resistant to fatigue. The enhanced contractile functionality was accompanied by advanced maturation such as increased width and upregulation of neuron receptor genes. We observed an upregulation in the expression of distinct myosin heavy chain isoforms (namely, neonatal-Myh8 and fast-Myh), which induced a shift towards a fast fiber phenotype. This long-term in vitro exercise strategy can be used to study fiber specification and refine muscle disease modelling.

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    Reply to the reviewers

    Manuscript number: #RC-2022-01697

    Corresponding author(s): William Roman; Edgar R. Gomes

    [The “revision plan” should delineate the revisions that authors intend to carry out in response to the points raised by the referees. It also provides the authors with the opportunity to explain their view of the paper and of the referee reports.

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    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We would like to thank the reviewers for their careful evaluation of our study. The goal of this work is to demonstrate that fiber type composition can be altered with exercise of in vitro muscle cultures. These findings provide an additional strategy to better mimic muscle in vitro for biological investigation and disease modelling. The reviewers’ comments will strengthen the conclusions of our study.

    In this point-by-point answer, we also include a statement on the feasibility of each comment based on preliminary work we have performed since receiving the reviews. We expect experiments can be achieved within 2 – 3 months.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Henning et al describes a method to induce myofiber subtype specification in vitro based on optogenetics and particle image velocimetry. The work is well performed and the manuscript is clear. The findings might be useful to the muscle community, but there are some issues which should be addressed in order to improve the quality and impact of the manuscript.

    My main concern is that the whole work is performed in murine cells. Although I appreciate that the authors have used primary myoblasts rather than cell lines, I also think that the key advantage of such in vitro platforms is the possibility to "humanise" the experiments as much as possible. In this context, the key findings of this work should be reproduced using human myoblasts. This will significantly enhance the relevance of the work. *

    Point 1.1) We thank the reviewer for his suggestion and have already performed some pilot experiments to “humanize” experiments. We infected hiPSC-derived myotubes (van der Wal et al., 2018) and human immortalized myotubes (Mamchaoui et al., 2011) with AAV9-pACAGW-ChR2-Venus-AAV. After infection, human immortalized myotubes did not express ChR2, not permitting optogenetic training on these cultures. For hiPSC-derived myotubes, the infection rate was very low and insufficient to perform a bulk analysis to evaluate the effect of long term intermittent light stimulation. Moreover, the contractile behavior of hiPSC-derived myotubes expressing ChR2 significantly differed from primary mouse myotubes. They underwent a single and slow contraction when compared to the cyclic contractions observed in mouse myotubes. This suggests that the maturation of the contractile apparatus of 2D hiPSC-derived myotubes is insufficient to perform consistent in vitro training studies.

    As such, we agree with the reviewer that reproducing our key findings with human cells would improve the relevance of this work. However, due to the experimental limitations described above, significant improvements in human myotube maturation in vitro are required to perform such experiments. We will attempt to increase infection efficiency by using another AAV serotype in hiPSC-derived myotubes but this has a low probability of solving all the technical limitations. Our work is a proof of principal that fiber type composition can be influenced in vitro through contraction stimulation. We expect these findings to be the translated to human cultures when the field has discovered the necessary protocols to push human myotube maturation.

    Feasibility: run additional tests but probability of success is low due to technical limitations.

    *Other issues: *

    1) From a methodological perspective, I think some clarifications are needed on the western blots shown in Fig 4K-L, as the pattern of Myh3 and Myh8 in both panels appear very similar. This could easily be ruled out by providing raw data/images. Please accept my apologies if this is simply caused by similar migration patterns in the gels (worth checking).

    Point 1.2) The very similar appearance of both patterns is due to the same molecular weight (220 kDA) of distinct myh isoforms. After an initial staining of western blot membranes, primary and secondary antibodies were stripped off and the membrane was subsequently re-probed using a primary and secondary antibody. We incubated stripped membranes with secondary antibodies only and observed no signal, confirming the stripping was efficient. We have updated the representative images of the Western Blot membranes in Figure 4 and included the α-actinin loading controls on which the bands are normalized to account for sarcomerogenesis (Figure 4 K-M).

    Feasibility: Accomplished

    *2) Figure 3K-L (BTX): better imaging should be performed to assess morphology of NMJ (eg. pretzel-shaped as in mature/adult NMJ?) *

    Point 1.3) We agree with the point raised by the reviewer. However, a morphological assessment of the NMJ is difficult in this in vitro system due to our inability to generate mature muscle end plates as seen in i*n vivo *adult NMJs. We will nevertheless perform a more quantitative evaluation of BTX stainings imaged with high spatial resolution by measuring the size and shape of the AChR clusters. The technical pipeline to do this quantitative approach is already established.

    Feasibility: will be accomplished

    *3) Figure 3 N-P: Why did the authors used a relatively complex techniques such as smFISH to answer a question more simply addressable with more conventional (and perhaps less operator dependent) techniques such quantitative PCR?

    Point 1.4) We agree with the reviewer that the more conventional qPCR technique would highlight similar results to the smFISH quantifications. Due to the heterogeneity of our primary myotube cultures (presence of non-muscle cell types and varying degrees of muscle cell maturation), we opted to monitor AChR expression by conserving a spatial dimension. This allows us to observe ChrnE and ChrnG expression in mature muscle cells selected to perform the contraction analysis. Nevertheless, performing a bulk RNA expression analysis would be informative to show a significant increase in AChR expression across the culture. This point will be fully addressed by qPCR assays of ChrnE and ChrnG.

    Feasibility: will be accomplished

    *Reviewer #1 (Significance (Required)):

    Nature and significance: as mentioned in the previous section, the work can be very significant if expanded to human myoblasts/myotubes, which can have different slow/fast myosin expression pattern. The work is clearly methodological/descriptive, so showing an application of this technique using diseased/mutant cells may increase its relevance even more (but I do not believe it is a key barrier to publication). *

    We thank the reviewer for his comments as the “other issues” raised will significantly improve the manuscript and will all be tackled. With regards to using human myotubes, we will attempt a few more strategies to translate our findings to human cultures, but our preliminary data suggests that many technical barriers need to be overcome to perform such experiments. Nevertheless, it is our opinion that the main contribution of this manuscript is to show that fiber switching can be achieved in vitro and that this will be routinely used in the next generation of human in vitro muscle systems.

    *Comparison with other methods: Similar methods have been published but not with this level of resolution.

    Expertise: muscle disease and regeneration, in vitro and in vivo models.*

    *Reviewer #2 (Evidence, reproducibility and clarity (Required)): *

    The work presented shows that muscle stem cells isolated from 5-day-old mice can be transduced with a DNA coding for a Channelrhodopsin2-Venus which will allow the muscle cell to be excited by a light beam (475nm) and to induce the contraction of myotubes. The authors measure the speed of contraction, relaxation and fatigability of such cells as a function of a more or less long excitation time. In particular, they show that myotubes in culture, excited at a frequency of 5 Hz, 8 hours per day for 7 days are larger than unstimulated myotubes and are more resistant to fatigue. Surprisingly, they show that myotubes stimulated at the low frequency of 5Hz express the neonatal Myosin heavy chain more than the slow Myh whose expression is known in adult muscle to be specifically strong in muscle fibers stimulated at low frequency. As the authors do not apply a high stimulation frequency (100Hz) to their culture, it is difficult to conclude whether the stimulation frequency applied in the study induces a specific phenotypic specialization of the myofiber, or a more general role. In this respect, the size of the myotubes obtained after training seems to be increased, showing a hypertrophic effect on the cultured myotubes. This study does not allow us to conclude, beyond the expression of the Myh8 gene, on the “gain” of the fast-twitch specialization of the myofiber by repeated stimulation over several days. A complementary study would certainly provide elements to better understand the role of muscle fiber stimulation, apart from the trophic contribution provided in vivo by the motoneuron. If the study is well conducted, some points are nevertheless important to address before publication.*

    *Reviewer #2 (Significance (Required)): *

    • Figures 4F/G are difficult to understand: the Myh7 signal seems much higher in trained myonuclei (F), but the histogram shows the opposite (G).*

    __Point 2.1) __We apologize for the confusion. The apparent higher Myh7 signal in trained cells in Figure 4F is due to background noise in the image. When mRNA is expressed, the smFISH probes are visible as small round dots. For clarity, we updated the representative images for the smFISH probes and highlighted the smFISH dots with arrows. We also adapted the y-axis of each graph to better represent the analysis of mRNA counts per myonuclei.

    Feasibility: Accomplished

    *- Figures 4L, the western blot shows the same increase in Myh3 and Myh8 at day 4, while the graph shows an increase at d4 only in Myh8, why? *

    Point 2.2) We have chosen another western blot to better reflect the quantification. It is important to note that we have normalized the band intensity to a-actinin instead of a house keeping gene to account for changes in sarcomerogenesis over the lifetime of the cultures. As such, although we observe an increase in Myh3 intensity, it is counter balanced by an increase in a-actinin expression. We have now added the a-actinin bands.

    - For immunocytochemistry against fMyh (Fig4 H, I) as well as for Western blots (Fig 4M, N), the authors have to provide arguments regarding the specificity of the antibodies used: some fMyh-specific antibodies recognize, Myh 3, 8, 1, 2, and 4, some only Myh 8, 1, 2, and 4, so it is quite difficult to conclude on the experiments using sc-32732 antibodies, (clone F59) which Myh are actually recognized in Western blot or immunocytochemistry.

    Point 2.3) According to the manufacturer, the sc-32732 antibody is specific for fast Myh (Myh1, 2, 4 and 6). Nevertheless, we will ensure the specificity of the sc-32732 antibody against fast Myosins by staining neonatal and adult TA/EDL muscle sections with anti-Myh3 (embryonic), anti-Myh8 (neonatal) and anti-fMyh antibodies.

    Feasibility: will be accomplished

    *While 10Hz stimulation is known in vivo to increase the slow program, and Myh7 expression in adult muscles, the authors show that ex vivo this is not the case with primary myotubes, with Myh7 protein level not being upregulated in the 7 day stimulation paradigm, while on the contrary Myh8 expression is upregulated. I think it would be important to quantify the mRNA of each of the Myh genes to be sure that there is no problem with the antibodies, which could recognize several Myh proteins, in the absence of a resolving acrylamide gel allowing visualization and relative level of each isoform according to its migration. Nevertheless, this is an interesting observation that could be related to the early phases of muscle contraction in vivo. Indeed, it has been shown in rats that early postnatal development animals are essentially sedentary and whose muscles (Sol and EDL) are stimulated by short intermittent bursts similar to 10Hz (doi: 10.1111/j.0953-816X.2004.03418.x) during the first 2-3 weeks of life. This should be compatible with Myh8 expression. It would be relevant in this idea to verify that the paradigm presented leads to myotubes with a "neonatal" phenotype. Quantification of the expression level of **genes specifically expressed during the neonatal period, compared with those expressed in adult slow or fast myofibers, would enhance the conclusions drawn by the authors. *

    Point 2.4) The reviewer raises an important technical limitation of observing Myh proteins to identify fiber types due to the cross-reactivity of antibodies. Despite our best efforts to select the appropriate antibodies, we agree that investigating mRNA expression of individual Myh isoforms would strengthen the conclusion of our study. We will design specific primers and perform qPCR for distinct Myh isoforms on untrained and trained cultures.

    With regards to the “neonatal” phenotype of these in vitro cultures, this does indeed seem to be the case as the cultures transition from embryonic and neonatal myosins to adult myosins during the lifetime of the cultures.

    Feasibility: will be accomplished

    *Should we also be cautious about bulk analysis since, as shown in Figure S1, not all myotubes express ChR2? *

    Point 2.5) Although 10% of myotubes do not express ChR2, we believe that 90% of infected myotubes is sufficient for bulk analysis. We nevertheless combine in our study bulk analysis with single cell assays such as smFISH and immunofluorescence, which are in line with the bulk analyses.

    Feasibility: Accomplished

    May the authors correlate the ex vivo neonatal phenotype observed with the neonatal muscles they used to prepare myogenic stem cells?

    Point 2.6) We understand from this that the reviewer would like us to check the expression of distinct Myh isoforms in our in vitro system and compare it to neonatal muscle. We will perform Myh staining of muscle sections from 6-day old mouse pups (time of myogenic stem cell isolation) and compare the expression of Myosin heavy chains with what we observe in our in vitro cultures.

    Feasibility: will be accomplished

    Overall, we will address all the points of the reviewer. Those ensuring the specificity of antibodies used are particularly relevant. With regards to the comparison between our in vitro cultures with neonatal muscle, we believe this will help contextualize our findings with the literature.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    *Summary: *

    *In this work, the authors propose an in vitro model describing a strategy to alter fiber type composition of myotubes with a long-term, intermittent mechanical training. The authors present a model of myotubes transfected with an adenovirus, which makes them photosensitive; in this way, fibers contraction can be induced upon stimulation with blue LEDs. *

    *Even though ChR2 expressing myotubes have previously been used by other groups (Asano T, Ishizua T, Yawo H. Optically controlled contraction of photosensitive skeletal muscle cells. Biotechnol Bioeng. 2012 Jan;109(1):199-204), no one has ever used it in the way proposed by the authors. For this reason, this work opens new perspectives on the possible use for clinical and therapeutic purposes for this in vitro muscle system. *

    *Major comments: *

    *I believe that the authors have presented their results, conclusion and methods in a fair and clear way, so that the experiment could also be reproduced. *

    *However, I think there are some adjustments that could be done in order to improve and strengthen the quality of this work: *

    *- The authors have analysed the expression of different myosin heavy chain isoforms, both regarding the slow and fast twitch fibers. Though, I think it would be interesting to investigate also the expression of Myh4, which is mainly expressed in type IIB fast twitch fibers; *

    Point 3.1) We agree with the reviewer’s comment. We will add the analysis for Myh 4 (western blots and qPCR) to our manuscript.

    Feasibility: will be accomplished

    The authors have observed a switch in the fiber type upon prolonged intermittent stimulation with blue LEDs, which translates into a higher number of type II fibers. It is known that exercise helps rescuing the loss of type II fibers, which is typical of age-related physiological processes, such as sarcopenia (Brunner F, Schmid A, Sheikhzadeh A, Nordin M, Yoon J, Frankel V. Effects of aging on Type II muscle fibers: a systematic review of the literature. J Aging Phys Act. 2007 Jul;15(3):336-48). However, I believe that providing a deeper analysis of the metabolism of the type II fibers (i.e. oxidative or glycolytic) could be helpful in order to have a clearer view on the specific subset of fibers that are generated with the given experimental conditions;

    Point 3.2) We agree with the reviewer's suggestion that an additional metabolic analysis would strengthen our observation. We propose to perform lactate measurements in cell lysate and supernatant to monitor a switch from oxidative to glycolytic metabolism. Specific inhibitors of the glycolytic pathway (2-DG, UK5099, Rotenone and AntimycinA) will be used as a control to prevent trained cells to shift towards a fast fiber type.

    Alternatively, we will assess the protein expression levels of key metabolic proteins involved in oxidative phosphorylation and in pyruvate and lactate production (e.g. OxPhos, …). All these techniques are routinely performed in an adjacent laboratory and we foresee no technical limitations.

    Feasibility: will be accomplished

    *Minor comments: *

    *The text and the figures are clear and well written, and help to explain better the experimental setup and procedures. Still, I would suggest some minor adjustments: *

    - I would suggest providing more information on the pH used for the experiments, since it plays a pivotal role in regulating myosin ATPase activity and, thus, muscular contractility. This would improve the replicability of your experiment.

    We thank the reviewer for this comment. We will provide information regarding the pH and add it in the method and materials section.

    Feasibility: will be accomplished

    The caption of Figure 1 is missing a description of panel E, even if it has been addressed in the text.

    Point 3.3.) We apologize for this mistake. We added the missing description of Fig. 1E.

    Feasibility: Accomplished

    *Reviewer #3 (Significance (Required)): *

    *This model opens new perspectives on in vitro muscle systems for the study of pathologies. The authors have been able to assess that myofibers contraction is able to induce a shift towards type II fibers, reproducing in vitro what is also known in vivo. For this reason, I believe that this model could be useful for further clinical approaches. It is important, though, to keep in mind that muscular disorders are not all characterized by a loss of type II fibers; for instance, myotonic dystrophies type I and type 2 exhibit similar phenotypes, even if different types of muscle fibers are affected. *

    *For this reason, it would be interesting to investigate the versatility of this model in terms of giving rise to different fiber types. *

    Point 3.4.) We added a sentence in the introduction that highlights an example of muscle disorders in which slow muscle fibers are predominately affected. Concerning the versatility of the model, we will add a paragraph to the discussion elaborating on how different stimulus frequency and durations could influence the specialization of fiber types.

    Feasibility: Accomplished

    Overall, we will address all major and minor comments from the reviewer. We have identified the experiments required for the metabolic analysis and agree that it will bolster our findings.

    Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    We have already carried out the following changes in the manuscript, which were proposed by the reviewers:

    __Point 1.2: __*pattern of Myh3 and Myh8 in both panels appear very similar - *We updated the representative images of Myh 3 and Myh8 in __Figure 4 K-N __and included the loading controls Myh 8 and fMyh images in __Figure 4K-N __and to supplementary Figure 4 A, B.

    Point 2.1: *Figures 4F/G: representative images of Myh7 smFISH probe and the graph showing opposite trends – *We have updated the representative images of Figure 4F and we have changed the x-axis of the graph in Figure 4E and G.

    __Point 2.5: __caution around bulk analysis – we consider that based on the high percentage of contracting cells in response to blue light (~90%), this concern is not warranted.

    Point 3.3: *caption of Figure 1 is missing a description of panel E – *We have added the missing description to the manuscript (Figure 1E).

    Point 3.4: *muscular disorders are not all characterized by a loss of type II fibers *– we have added an example of a muscle disorder, in which slow fibers are predominantly affected, to the introduction (line 42-44) of the manuscript.

    *investigate the versatility of this model in terms of giving rise to different fiber types *– we added a paragraph to the discussion elaborating on how different stimulus frequency can lead to different fiber types (line 264-275).

    3. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Point 1.1: *Reproducing our key findings with human cells – *we ran pilot experiments on immortalized human cell lines and human iPSC-derived myotubes but were not able to mature these cells sufficiently nor infect them to allow long-term in vitro training. Increased maturation of myotubes derived from hiPSCs is an endeavor currently undertaken by many laboratories. Although we will attempt a few more trials, we believe the technical limitations are too important to address this point.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this work, the authors propose an in vitro model describing a strategy to alter fiber type composition of myotubes with a long-term, intermittent mechanical training. The authors present a model of myotubes transfected with an adenovirus, which makes them photosensitive; in this way, fibers contraction can be induced upon stimulation with blue LEDs. Even though ChR2 expressing myotubes have previously been used by other groups (Asano T, Ishizua T, Yawo H. Optically controlled contraction of photosensitive skeletal muscle cells. Biotechnol Bioeng. 2012 Jan;109(1):199-204), no one has ever used it in the way proposed by the authors. For this reason, this work opens new perspectives on the possible use for clinical and therapeutic purposes for this in vitro muscle system.

    Major comments:

    I believe that the authors have presented their results, conclusion and methods in a fair and clear way, so that the experiment could also be reproduced.

    However, I think there are some adjustments that could be done in order to improve and strengthen the quality of this work:

    • The authors have analysed the expression of different myosin heavy chain isoforms, both regarding the slow and fast twitch fibers. Though, I think it would be interesting to investigate also the expression of Myh4, which is mainly expressed in type IIB fast twitch fibers;
    • The authors have observed a switch in the fiber type upon prolonged intermittent stimulation with blue LEDs, which translates into a higher number of type II fibers. It is known that exercise helps rescuing the loss of type II fibers, which is typical of age-related physiological processes, such as sarcopenia (Brunner F, Schmid A, Sheikhzadeh A, Nordin M, Yoon J, Frankel V. Effects of aging on Type II muscle fibers: a systematic review of the literature. J Aging Phys Act. 2007 Jul;15(3):336-48). However, I believe that providing a deeper analysis of the metabolism of the type II fibers (i.e. oxidative or glycolytic) could be helpful in order to have a clearer view on the specific subset of fibers that are generated with the given experimental conditions;

    Minor comments:

    The text and the figures are clear and well written, and help to explain better the experimental setup and procedures. Still, I would suggest some minor adjustments:

    • I would suggest providing more information on the pH used for the experiments, since it plays a pivotal role in regulating myosin ATPase activity and, thus, muscular contractility. This would improve the replicability of your experiment;
    • The caption of Figure 1 is missing a description of panel E, even if it has been addressed in the text.

    Significance

    This model opens new perspectives on in vitro muscle systems for the study of pathologies. The authors have been able to assess that myofibers contraction is able to induce a shift towards type II fibers, reproducing in vitro what is also known in vivo. For this reason, I believe that this model could be useful for further clinical approaches. It is important, though, to keep in mind that muscular disorders are not all characterised by a loss of type II fibers; for instance, myotonic dystrophies type I and type 2 exhibit similar phenotypes, even if different types of muscle fibers are affected.

    For this reason, it would be interesting to investigate the versatility of this model in terms of giving rise to different fiber types.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The work presented shows that muscle stem cells isolated from 5-day-old mice can be transduced with a DNA coding for a Channelrhodopsin2-Venus which will allow the muscle cell to be excited by a light beam (475nm) and to induce the contraction of myotubes. The authors measure the speed of contraction, relaxation and fatigability of such cells as a function of a more or less long excitation time. In particular, they show that myotubes in culture, excited at a frequency of 5 Hz, 8 hours per day for 7 days are larger than unstimulated myotubes and are more resistant to fatigue. Surprisingly, they show that myotubes stimulated at the low frequency of 5Hz express the neonatal Myosin heavy chain more than the slow Myh whose expression is known in adult muscle to be specifically strong in muscle fibers stimulated at low frequency. As the authors do not apply a high stimulation frequency (100Hz) to their culture, it is difficult to conclude whether the stimulation frequency applied in the study induces a specific phenotypic specialization of the myofiber, or a more general role. In this respect, the size of the myotubes obtained after training seems to be increased, showing a hypertrophic effect on the cultured myotubes. This study does not allow us to conclude, beyond the expression of the Myh8 gene, on the "gain" of the fast-twitch specialization of the myofiber by repeated stimulation over several days. A complementary study would certainly provide elements to better understand the role of muscle fiber stimulation, apart from the trophic contribution provided in vivo by the motoneuron.

    If the study is well conducted, some points are nevertheless important to address before publication.

    Significance

    • Figures 4F/G are difficult to understand: the Myh7 signal seems much higher in trained myonuclei (F), but the histogram shows the opposite (G).
    • Figures 4L, the western blot shows the same increase in Myh3 and Myh8 at day 4, while the graph shows an increase at d4 only in Myh8, why?
    • For immunocytochemistry against fMyh (Fig4 H, I) as well as for Western blots (Fig 4M, N), the authors have to provide arguments regarding the specificity of the antibodies used: some fMyh-specific antibodies recognize, Myh 3, 8, 1, 2, and 4, some only Myh 8, 1, 2, and 4, so it is quite difficult to conclude on the experiments using sc-32732 antibodies, (clone F59) which Myh are actually recognized in Western blot or immunocytochemistry.
    • While 10Hz stimulation is known in vivo to increase the slow program, and Myh7 expression in adult muscles, the authors show that ex vivo this is not the case with primary myotubes, with Myh7 protein level not being upregulated in the 7 day stimulation paradigm, while on the contrary Myh8 expression is upregulated. I think it would be important to quantify the mRNA of each of the Myh genes to be sure that there is no problem with the antibodies, which could recognize several Myh proteins, in the absence of a resolving acrylamide gel allowing visualization and relative level of each isoform according to its migration. Nevertheless, this is an interesting observation that could be related to the early phases of muscle contraction in vivo. Indeed, it has been shown in rats that early postnatal development animals are essentially sedentary and whose muscles (Sol and EDL) are stimulated by short intermittent bursts similar to 10Hz (doi: 10.1111/j.0953-816X.2004.03418.x) during the first 2-3 weeks of life. This should be compatible with Myh8 expression. It would be relevant in this idea to verify that the paradigm presented leads to myotubes with a "neonatal" phenotype. Quantification of the expression level of genes specifically expressed during the neonatal period, compared with those expressed in adult slow or fast myofibers, would enhance the conclusions drawn by the authors.
    • Should we also be cautious about bulk analysis since, as shown in Figure S1, not all myotubes express ChR2?
    • May the authors correlate the ex vivo neonatal phenotype observed with the neonatal muscles they used to prepare myogenic stem cells?
  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Henning et al describes a method to induce myofiber subtype specification in vitro based on optogenetics and particle image velocimetry. The work is well performed and the manuscript is clear. The findings might be useful to the muscle community, but there are some issues which should be addressed in order to improve the quality and impact of the manuscript.

    My main concern is that the whole work is performed in murine cells. Although I appreciate that the authors have used primary myoblasts rather than cell lines, I also think that the key advantage of such in vitro platforms is the possibility to "humanise" the experiments as much as possible. In this context, the key findings of this work should be reproduced using human myoblasts. This will significantly enhance the relevance of the work.

    Other issues:

    1. From a methodological perspective, I think some clarifications are needed on the western blots shown in Fig 4K-L, as the pattern of Myh3 and Myh8 in both panels appear very similar. This could easily be ruled out by providing raw data/images. Please accept my apologies if this is simply caused by similar migration patterns in the gels (worth checking).
    2. Figure 3K-L (BTX): better imaging should be performed to assess morphology of NMJ (eg. pretzel-shaped as in mature/adult NMJ?)
    3. Figure 3 N-P: Why did the authors used a relatively complex techniques such as snFISH to answer a question more simply addressable with more conventional (and perhaps less operator dependent) techniques such quantitative PCR?

    Significance

    Nature and significance: as mentioned in the previous section, the work can be very significant if expanded to human myoblasts/myotubes, which can have different slow/fast myosin expression pattern. The work is clearly methodological/descriptive, so showing an application of this technique using diseased/mutant cells may increase its relevance even more (but I do not believe it is a key barrier to publication).

    Comparison with other methods: Similar methods have been published but not with this level of resolution.

    Expertise: muscle disease and regeneration, in vitro and in vivo models.

  5. Version published to 10.1101/2022.10.04.510824v1 on bioRxiv