Heat Shock Proteins: Important Helpers for the Development, Maintenance and Regeneration of Skeletal Muscles

Round 1

Reviewer 1 Report

The review by Pomella S. et al summarized the function and targets of heat shock proteins in muscle differentiation and homeostasis, as well as their potential role in the development of neuromyopathies and rhabdomyosarcoma (RMS). Overall, the review is well-structured and have included the recent findings and techniques in the research of heat shock proteins. In the section of “Heat shock protein muscle-specific targets”, which have focused on muscle structural genes. Since many heat shock proteins are involved in promoting muscle differentiation, it is necessary to summarize the targets, such as myogenic factors, regulated by heat shock proteins in muscle differentiation.

Other points,

“Boosting HSP function may also help to combat the development of rhabdomyosarcoma (RMS), a highly aggressive type of cancer that develops from mesenchymal cells that fail to fully differentiate into striated muscle cells.” This sentence is misleading. First, rhabdomyosarcoma might be derived from mesenchymal cells or myogenic precursor cells. Second, striated muscle cells are not derived from mesenchymal cells in normal myogenesis.

Line 130, (Bar-Lavan 2016 PLoS Genet), reference is not consistent.

Line 152, “induce the differentiation of rhabdomyosarcoma cells” It is necessary to point out the myogenic program.

Line 159, HSPS should be corrected to HSPs.

Line 200, animal model should be corrected to c. elegans model.

Line 254, reference is not consistent.

Line 464, more information on HSF1 should be provided.

Line 465, c-for should be corrected to c-fos.

Author Response

Review Report (Reviewer 1)

Comments and Suggestions for Authors

The review by Pomella S. et al summarized the function and targets of heat shock proteins in muscle differentiation and homeostasis, as well as their potential role in the development of neuromyopathies and rhabdomyosarcoma (RMS). Overall, the review is well-structured and have included the recent findings and techniques in the research of heat shock proteins. In the section of “Heat shock protein muscle-specific targets”, which have focused on muscle structural genes. Since many heat shock proteins are involved in promoting muscle differentiation, it is necessary to summarize the targets, such as myogenic factors, regulated by heat shock proteins in muscle differentiation.

Reply to Reviewer 1: As requested we have included a new paragraph describing the how HSPs can regulate myogenic factors. “Finally, myogenic transcription factors are targeted by the two main master chaperones in cells: HSP70s and HSP90s. For example, Hsc70 and HSP70 interact with MK2, a substrate of p38 mitogen-activated protein kinase (p38MAPK), ultimately stabilizing it. This, in turn, promotes the expression of E47, a splice product of the E2A gene that interacts with MyoD to activate transcription, as well as myocyte enhancer binding factors MEF2A and MEF2C, and BAF60, a subunit of the chromatin-modifying enzyme SWI/SNF that participates in muscle determination and renewal (Fan, Gao et al. 2018). Instead, HSP90 stabilizes MyoD and MyoG and promotes the conformational change of MyoD from an inactive to an active state (Shaknovich, Shue et al. 1992, Wagatsuma, Shiozuka et al. 2011). Concerning sHSPs, αB-crystallin/HSPB5 affects cell cycle exit and decreases MyoD levels, promoting its ubiquitination and degradation (Singh, Rao et al. 2010), while HSPB3 indirectly stabilizes myogenin, enhancing myogenesis (Tiago, Hummel et al. 2021)”.

We also included in the section entitled “Heat shock proteins, muscle activity and resistance training” the following sentence: “Of note, heat shock activates the muscle transcriptional program by enhancing the expression of key myogenic transcription factors such as myocyte enhancer factor 2D (MEF2D), myogenic differentiation factor 1 (MYOD1), myogenic factor 5 (MYF5) and myogenic factor 6 (MYF6), increasing myotube formation (Obi, Nakajima et al. 2019), thus providing a direct link between HSPs and muscle build-up and maintenance”.

Other points,

“Boosting HSP function may also help to combat the development of rhabdomyosarcoma (RMS), a highly aggressive type of cancer that develops from mesenchymal cells that fail to fully differentiate into striated muscle cells.” This sentence is misleading. First, rhabdomyosarcoma might be derived from mesenchymal cells or myogenic precursor cells. Second, striated muscle cells are not derived from mesenchymal cells in normal myogenesis.

Reply to Reviewer 1: We agree with the Reviewer that the cell of origin of rhabdomyosarcoma is still debated. Indeed, rhabdomyosarcoma cells have features of skeletal muscle cells arrested at different stages of myogenic differentiation. Indeed, as reported by very recent papers, these stages include an immature stem cell-like/mesoderm/mesenchymal state (expressing PAX3/PAX7/CD44), a highly proliferative myoblast state (expressing MYF5/CDC20/CCNB1) and a more differentiated myocyte state (expressing MYF4/ MYH3/ MYH8) (Patel, Chen et al. 2022, Wei, Qin et al. 2022, Danielli, Porpiglia et al. 2023). We, therefore, corrected as follows “Boosting HSP function may also help to combat the development of rhabdomyosarcoma (RMS), a highly aggressive type of pediatric soft tissue sarcoma whose cells have skeletal muscle features but are unable to fully differentiate into skeletal muscle cells.”

Line 130, (Bar-Lavan 2016 PLoS Genet), reference is not consistent.

Reply to Reviewer 1: reference removed.

Line 152, “induce the differentiation of rhabdomyosarcoma cells” It is necessary to point out the myogenic program.

Reply to Reviewer 1: we integrated as follows ““In fact, forced expression of an exogenous HSPB3 in a RMS cell line resulted in fiber-like multinucleated fused structures characterized by increased expression of MYOG and de novo expression of the late marker of skeletal muscle differentiation MyH2 (Tiago, Hummel et al. 2021)”.

Line 159, HSPS should be corrected to HSPs.

Reply to Reviewer 1: corrected.

Line 200, animal model should be corrected to c. elegans model.

Reply to Reviewer 1: corrected.

Line 254, reference is not consistent.

Reply to Reviewer 1: corrected.

Line 464, more information on HSF1 should be provided.

Reply to Reviewer 1:  We included the description of HSF1: “For example, HSP70 acts as a corepressor with Heat Shock Factor 1 (HSF1), a stress-inducible transcription factor that induces the expression of HSPs and represents the core component of the heat shock response (PMID: 28890254)”.

Line 465, c-for should be corrected to c-fos.

Reply to Reviewer 1: corrected.

Reviewer 2 Report

Dear Authors,

The manuscript “Heat shock proteins: important helpers for the development, maintenance and regeneration of the motor units” reviews current understanding on the role of HSP regulating to general muscle plasticity.

The manuscript is well written and structured giving an introduction on HSP, their role on skeletal muscle differentiation, muscle activity and resistance training, function in muscle disease, and action on specific muscle proteins. There is a flavour of neuromuscular junctions (NMJ) throughout the manuscript, but the manuscript in not focused on NMJ. Therefore, the title is not representative of the content of the manuscript. The NMJ should be down played in the title as the focus of the manuscript is the role of HSP on the skeletal muscle differentiation, muscle activity, muscle disease and muscle proteins. The title should change removing the focus on NMJ. 

Minor

Line 130 Amend Reference Bar-Lavan 2016 PLoS Genet to a number.

Author Response

Review Report (Reviewer 2)

Dear Authors,

The manuscript “Heat shock proteins: important helpers for the development, maintenance and regeneration of the motor units” reviews current understanding on the role of HSP regulating to general muscle plasticity.

The manuscript is well written and structured giving an introduction on HSP, their role on skeletal muscle differentiation, muscle activity and resistance training, function in muscle disease, and action on specific muscle proteins. There is a flavour of neuromuscular junctions (NMJ) throughout the manuscript, but the manuscript in not focused on NMJ. Therefore, the title is not representative of the content of the manuscript. The NMJ should be down played in the title as the focus of the manuscript is the role of HSP on the skeletal muscle differentiation, muscle activity, muscle disease and muscle proteins. The title should change removing the focus on NMJ.

Reply to Reviewer 2: We changed the title as requested. The tile is now “Heat shock proteins: important helpers for the development, maintenance and regeneration of skeletal muscles”.

Minor

Line 130 Amend Reference Bar-Lavan 2016 PLoS Genet to a number.

Reply to Reviewer 2: corrected.

Reviewer 3 Report

Comments to the authors

The manuscript by Silvia Pomella and others, entitled “Heat shock proteins: important helpers for the development, maintenance and regeneration of the motor units” summarize key findings supporting the notion that HSPs are important components required to maintain skeletal muscle integrity and functionality. The following comments need to be address by the authors:

1-     In page 4, line 185-187, the authors reported that “A large body of evidence demonstrates that HSP70 upregulation in the skeletal muscle occurs not only after exercise, but also following muscle injury and upon muscle regeneration.”, without supporting and citing any literature. The authors should reinforce this paragraph with citations and examples or add a new section discussing examples of upregulation of HSPs upon muscle injury and regeneration.

2-    It will be beneficial to summarize the entire review in a graphical illustration/diagram to help the reader.

3-    Overexpression of certain HSPs, such as HSP27, has been associated with muscle atrophy in some studies. Thus, it is recommended that the authors provide new section to cover the effects of HSPs overexpression in muscle.

Author Response

Review Report (Reviewer 3)

The manuscript by Silvia Pomella and others, entitled “Heat shock proteins: important helpers for the development, maintenance and regeneration of the motor units” summarize key findings supporting the notion that HSPs are important components required to maintain skeletal muscle integrity and functionality. The following comments need to be address by the authors:

1-     In page 4, line 185-187, the authors reported that “A large body of evidence demonstrates that HSP70 upregulation in the skeletal muscle occurs not only after exercise, but also following muscle injury and upon muscle regeneration.”, without supporting and citing any literature. The authors should reinforce this paragraph with citations and examples or add a new section discussing examples of upregulation of HSPs upon muscle injury and regeneration.

Reply to Reviewer 3: We included the following references:

PMID: 30730806; PMID: 24273516; PMID: 19864323; PMID: 23626847

2-    It will be beneficial to summarize the entire review in a graphical illustration/diagram to help the reader.

Reply to Reviewer 3: We included “muscle stress response and repair” and “stabilization of sarcomeric proteins” in revised Figure 1 in order to highlight these important aspects that were not initially shown. The following sentence has been added to the Figure legend: “HSPs are key players of the cellular stress response that may promote muscle repair, also by stabilizing sarcomeric proteins, protecting them from unfolding and aggregation due to mechanical stress and exercise”.

3-    Overexpression of certain HSPs, such as HSP27, has been associated with muscle atrophy in some studies. Thus, it is recommended that the authors provide new section to cover the effects of HSPs overexpression in muscle.

Reply to Reviewer 3: We included in the section entitled “Heat shock proteins and muscle disease” the following paragraph: “The protective effects exerted by HSPs at the level of muscles is further supported by overexpression studies. Briefly, overexpression of HSP70 in mice attenuated skeletal muscle damage induced by cryolesioning (Miyabara, Martin et al. 2006), as well as from exercise-induced skeletal muscle damage (Liu, Lin et al. 2013), attenuated sarcopenia and improved structural and functional recovery of skeletal muscles from atrophy (Miyabara, Nascimento et al. 2012). In addition, overexpression of Hsp27 (via electrotransfer into the soleus muscle of rats) attenuated skeletal muscle disuse atrophy (Dodd, Hain et al. 2009), and overexpression of mitochondrial HSP10 in transgenic mice prevented against contraction-induced damage and preserved muscle force generation (Kayani, Close et al. 2010)”.

Author Response File: Author Response.pdf