Fine tuning of Piezo1 Expression and Activity Ensures Efficient Myoblast Fusion during Skeletal Myogenesis

  1. Huascar Pedro Ortuste Quiroga
  2. Massimo Ganassi
  3. Shingo Yokoyama
  4. Kodai Nakamura
  5. Tomohiro Yamashita
  6. Daniel Raimbach
  7. Arisa Hagiwara
  8. Oscar Harrington
  9. Jodie Breach-Teji
  10. Atsushi Asakura
  11. Yoshiro Suzuki
  12. Makoto Tominaga
  13. Peter S. Zammit
  14. Katsumasa Goto

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Abstract

Mechanical stimuli such as stretch and resistance training are essential to regulate growth and function of skeletal muscle. However, the molecular mechanisms involved in sensing mechanical stress during muscle formation remain unclear. Here, we investigate the role of the mechano-sensitive ion channel Piezo1 during myogenic progression. Direct manipulation of Piezo1 in muscle stem cells alters their myogenic progression. Indeed, Piezo1 knockdown suppresses myoblast fusion leading to smaller myotubes. Such event is accompanied by significant downregulation of the fusogenic protein Myomaker . In parallel, while Piezo1 knockdown also lowers Ca 2+ influx in response to stretch, Piezo1 activation increases Ca 2+ influx in response to stretch and enhances myoblasts fusion. We believe these findings may help understand molecular defects present in some muscle diseases. Altogether our study shows that Piezo1 is essential for terminal muscle differentiation acting on myoblast fusion, suggesting that Piezo1 deregulation may have implications in muscle aging and degenerative diseases including muscular dystrophies.

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  1. Version published to 10.1101/2020.09.27.315242v3 on bioRxiv
  2. eLife

    Reviewer #3:

    Quiroga et al. studied the molecular function of mechanosensitive ion channel protein Piezo1 during mouse primary myoblast differentiation in culture condition. The authors measured myoblast proliferation and differentiation after either knockdown of Piezo1 or chemical activation of Piezo1 protein. In overall, the study is significant given its conclusion directly contradicts with a recent study by Masaki Tsuchiya et al. Nature Communications (2018) by which knockout of Piezo1 produced opposite effects. However, major concerns were identified and need to be addressed to strengthen their claim.

    1. It is unfortunate that the authors have confused "fusion index" with "differentiation index". By the description in Method, they actually measured differentiation index though claimed as "fusion index". The commonly used fusion index is the ratio of nuclei in myocytes with {greater than or equal to} 3 nuclei normalized with total number of nuclei in MyHC+ myocytes. Therefore, it appears that what the author claimed about "fusion defect" was actually a differentiation defect. These errors need to be corrected.

    2. Following comment 1, the authors need to evaluate whether or not the differentiation is affected when Piezo1 is knocked-down or activated. It is suggested to run a panel of qPCR assay for myogenic markers including myosin genes (Myh3, Myh8). Western blots of myosin by MF20 antibody will also need to be performed and quantified.

    3. The author discussed the potential off-target effects for siRNA from the previous study. Although it is comparatively more convincing that this manuscript tested 4 siRNA, for the scientific rigor, the authors still need to clarify whether the study by Tsuchiya et al is reproducible. As such, the authors should measure myoblast fusion by using the same siRNAs as listed in Tsuchiya et al. In addition, the authors should also characterize the myoblast fusion phenotype of Piezo1 gene-KO from CRISPR treatment of primary myoblast.

    4. To rule out any off-target effects of the chemical activator of Piezo1, the authors should test whether this drug's effect on myoblast fusion /differentiation can be negated when Piezo1 is knocked down.

    5. Concerning the role of myomixer gene in Piezo1 KD phenotype, the authors should use another set of primers for qPCR. The current forward primer only detects a predicted longer transcript isoform of Mymx but not its predominant isoform (NM_001177468).

    6. For Fig.6, the details of experiment procedure, e.g. the timing of drug treatment in relation to differentiation timing, needs to be provided.

    7. The authors should cite the correct references as being consistent with their description. For instance, line# 528, 1011. In addition, the writing needs to be improved for better readability.

  3. eLife

    Reviewer #2:

    In this study, Ortuste Quiroga et al. showed that the mechanosensitive ion channel Piezo1 promotes myoblast fusion during the formation of multinucleated, mature myotubes. The authors show that Piezo1 knockdown suppressed myoblast fusion during myotube formation and maturation. This was accompanied by a decrease in Myomaker expression. In addition, Piezo1 knockdown lowered Ca2+ influx in response to stretch. In contrast, the agonist (Yoda1)-mediated activation of Piezo1 increased Ca2+ influx and enhanced myoblast fusion, but only under certain conditions. Over-activation of Piezo1 resulted in the loss of myotube integrity. Surprisingly, the myotubes were thinner in Yoda1-treated cells compared to the control. Furthermore, the authors showed that Piezo1 activation enhanced Ca2+ influx in cultured myotubes and the influx of Ca2+ increased in response to stretch. However, it is unclear how this is related to myoblast fusion.

    Overall, the authors made several interesting observations in this study, such as Piezo1's role in myoblast fusion and Piezo1-mediated Ca2+ influx, etc. However, how these phenomena are linked and what is causal remain largely unclear. Another issue is the discrepancy between this study and Tsuchiya et al. Nature Communication (2018) on the function of Piezo in myoblast fusion.

    Major comments:

    1. In this study, the authors uncovered a positive role for Piezo1 in myoblast fusion. This is in contrast to Tsuchiya et al., which demonstrated an inhibitory role of Piezo1 in this process. While this study used an RNAi approach to knock down Piezo1 and found a decrease in myoblast fusion, Tsuchiya et al. used CRISPR/Cas9 to knock out Piezo1 in muscle cells and observed a significant increase in myoblast fusion. These two opposite results are difficult to interpret and made the role of Piezo1 in myoblast fusion confusing. It is necessary that the authors make some effort to bring clarity to this issue. First, the authors need to perform rescue experiments in their RNAi cells to make sure that the fusion defect is not due to off-target effects caused by the siRNAs. Second, the authors should design an siRNA that causes a more significant knockdown of Piezo1 than the current siRNAs and test if myoblast fusion is enhanced as in the knockout cells (Tsuchiya et al.). Third, the authors could make their own CRISPR/Cas9 knockout cells and examine the resulting fusion index.

    2. How does Ca2+ influx regulate fusion? Tsuchiya et al. provided evidence that Piezo1-mediated Ca2+ influx activates actomyosin activity and inhibits myoblast fusion. This current study suggests that Ca2+ influx increases fusion, but without providing mechanistic explanations. What are the effects of Ca2+ influx that lead to an increase in myoblast fusion? Does it cause more IL4 secretion? Or transcription upregulation of Myomaker? How? Does the Ca2+ influx level correlate with Myomaker expression level? If Ca2+ influx indeed leads to upregulation of Myomaker, why would Piezo1 knockout cells (low Ca2+ influx) show increased levels of fusion (Tsuchiya et al.)?

    3. Is Piezo1 required in myoblasts or myotubes or both cell types for fusion? Is it localized to the fusion sites?

  4. eLife

    Reviewer #1:

    The manuscript from Quiroga and colleagues reports a function for the mechanosensor Piezo 1 in myocyte fusion. The manuscript concludes via a series of in vitro experiments that Piezo 1 knockdown results in decreased myotube formation.

    While overall the manuscript reports some potentially interesting observations, the main conclusion seems preliminary and the work would benefit from substantial additional validations in multiple models to strengthen the tie between myomaker and Piezo1 functions.

    Major Comments:

    1. siRNA reduces gene expression in a transient manner and it is unclear for how long there is significant silencing of Piezo1 RNA during differentiation. Therefore, a more consistent model that expresses consistent amounts of Piezo1 might be beneficial. Importantly, a more stable mutant form of Piezo 1 (generated with CRISPR/Cas9) was generated in a previous study (Tsuchiya et al, 2018, ref. 17). The long-term consequences of differentiation/fusion of myogenic cells following loss of Piezo 1 expression in the Tsuchiya study reached opposite conclusions to the current study. These findings raise concerns that are not clearly addressed in the present study. While the authors attempt to explain the opposite findings by the use of a different Piezo 1 silencing model, it is difficult to reconcile with the present data the very opposite findings.

    2. Figure 3A and C have duplicated images showing siRNA of Piezo 1 in EDL and Soleus. The correct images need to be inserted.

    3. Quantification of proteins levels downstream of Piezo silencing should be corroborated by western blot analyses. These include data presented in Figures 2 and 3.

    4. In Figure 4, it would be helpful to include a graph illustrating the amount of Piezo1 silencing and the corresponding decrease in Myomaker expression.

    5. In Figure 6, expression of myomaker and myomixer should be monitored following administration of Yoda1. If Yoda1 increases fusion at low concentrations, the fusion genes should be upregulated in expression.

    6. In Figure 7 the myotube width should also be accompanied by quantifications of numbers of nuclei fused in the myotubes. This data will address whether cell fusion changes following Yoda1 treatment.

    7. While the present work explores the function of Piezo 1 in myogenesis in vitro, no experiments address a potential parallel function of Piezo1 in vivo. Supporting data using injured/regenerating muscle should strengthen the overall message.

    8. Figure 9 proposes an interesting hypothesis linking Piezo 1 to FSHD. However, the hypothesis is not supported by experimental data and remains rather exploratory in its current form.

  5. Version published to 10.1101/2020.09.27.315242v2 on bioRxiv
  6. Version published to 10.1101/2020.09.27.315242v1 on bioRxiv