The Myokine Landscape: Sources, Signaling, and Context-Dependent Actions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, congratulations on a professionally and objectively written review!

Author Response

We sincerely thank the reviewer for this kind and encouraging comment. We greatly appreciate their positive assessment of our review.

Reviewer 2 Report

Comments and Suggestions for Authors

In this review, the authors summarize the current knowledge on the biology and functions of myokines in skeletal muscle. Although the manuscript is comprehensive and well organized, it is primarily descriptive and lacks a sufficiently critical evaluation of the available evidence. Therefore, I believe that the manuscript requires major revision before it can be considered for publication.

1. The section Main Myokines and Their Actions follows an almost identical structure for each myokine, resulting in considerable redundancy. The authors should reduce repetitive descriptions and instead emphasize similarities, differences, and common signaling mechanisms among the different myokines.
2. Although the review repeatedly emphasizes the context-dependent effects of myokines, the discussion remains largely descriptive. A more critical comparison of conflicting findings, particularly between in vitro studies, animal models, and human evidence, would substantially strengthen the manuscript.
3. The manuscript would benefit from a summary table reporting, for each myokine, the principal cellular source, biological functions, signaling pathway(s), level of experimental evidence (cellular, animal, or human), and potential clinical relevance. Such a table would improve readability given the large number of molecules discussed throughout the review.
4. The translational implications are only briefly addressed in the Introduction and Conclusions. A dedicated section discussing the current limitations and future perspectives of myokines as biomarkers and therapeutic targets would increase the impact of the review.

Author Response

In this review, the authors summarize the current knowledge on the biology and functions of myokines in skeletal muscle. Although the manuscript is comprehensive and well organized, it is primarily descriptive and lacks a sufficiently critical evaluation of the available evidence. Therefore, I believe that the manuscript requires major revision before it can be considered for publication.

1. The section Main Myokines and Their Actions follows an almost identical structure for each myokine, resulting in considerable redundancy. The authors should reduce repetitive descriptions and instead emphasize similarities, differences, and common signaling mechanisms among the different myokines.

   We thank the reviewer for this important comment. We agree that the use of a largely parallel structure for the individual myokines may result in some degree of repetition. However, this organization was deliberate and reflects the specific purpose of the present Review within this Special Issue of the journal.

   The primary aim of this article is to provide potential readers with a solid, comprehensive, and easily accessible framework for understanding the myokine field and, in particular, to facilitate the interpretation of the more focused contributions included in this Special Issue. This is especially relevant because interest in myokines and related muscle-derived factors now extends well beyond researchers primarily working in skeletal muscle biology. These mediators are increasingly being considered in a wide variety of conditions in which skeletal muscle may represent either a primary or a secondary target organ, including muscular, neurological, cardiovascular, rheumatological, respiratory, and immune-mediated diseases. Many readers approaching these areas may therefore not be familiar with the individual myokines, their cellular sources, receptors, signaling mechanisms, or biological actions.

   For this reason, we believe that retaining an individual, relatively systematic description of the major myokines is important. The repeated consideration of their source, regulation, signaling, biological effects, and strength of evidence is intended to provide a consistent reference framework rather than to serve primarily as a synthesis of mechanistic controversies across the field. In this sense, the somewhat encyclopedic organization of the section is a deliberate feature of the Review and, in our view, contributes to its usefulness as an introductory and reference article.

   Nevertheless, we agree with the reviewer that the comparative and integrative dimension can be strengthened without altering this overall objective. We have therefore revised the section to reduce unnecessary repetitive wording where possible and have increased emphasis on shared and divergent signaling mechanisms, overlapping biological actions, and important differences in cellular source, receptor usage, temporal regulation, and physiological versus pathological context. We have also reinforced the cross-comparison of myokines in the integrative elements of the Review, including the figures, the expanded Table 1, and the integrative sections of the Review.

   We believe that these changes improve the balance between the reference-oriented purpose of the article and the synthesis of common concepts across the myokine field, while preserving the structure that we consider most appropriate for the intended readership of this Special Issue.

Changes in the revised manuscript:

   The Abstract has been revised (page 1 line 36), and the final paragraph of the Introduction has been expanded to clarify the primary purpose and intended readership of the Review (page 3, lines 75-80).

   A new section entitled “Definition and Scope of Myokines” has been added (pages 3-4).

   The concluding “Overall” paragraphs for the individual myokines have been shortened and streamlined to reduce redundancy while retaining the systematic molecule-by-molecule organization of the Review.

   Manuscript changes to reduce repeated caveats and unnecessary repetition, and to clarify specific biological actions: Page 6, lines 161-165, tracked changes version); page 9, lines 234-238; page 12, lines 304-307; page 19-20, lines 494-502; page 24, lines 605-611; page 30, lines 763-768; page 55, lines 1377-1380 of the tracked changes version.

   In addition, a new integrative paragraph has been added at the end of the molecule-by-molecule discussion (pages 65-66, lines 1641-1669) to provide an overall perspective on shared intracellular signaling modules, differences in receptor usage and cellular targets, and the context-dependent biological actions of myokines.

   The Figure 1 legend has been expanded to clarify the purpose of the figure.

2. Although the review repeatedly emphasizes the context-dependent effects of myokines, the discussion remains largely descriptive. A more critical comparison of conflicting findings, particularly between in vitro studies, animal models, and human evidence, would substantially strengthen the manuscript.

   We thank the reviewer for this important observation. We agree that emphasizing context dependence should be accompanied by a critical appraisal of the level of evidence supporting individual myokine actions, particularly when findings obtained in cellular or animal models are extrapolated to human physiology.

   The manuscript already distinguished these levels of evidence in several individual sections. For example, we discuss divergent experimental findings regarding the role of IL-15 in muscle regeneration, the limitations of translating IL-15 overexpression models to physiological human exposure, the marked methodological and translational uncertainties surrounding irisin, and several instances in which mechanisms demonstrated in cultured cells or animal models have not yet been confirmed in human skeletal muscle. Nevertheless, we agree that this distinction was not sufficiently synthesized at the conceptual level.

   We have therefore strengthened the revised manuscript by explicitly comparing the evidential value and limitations of in vitro, animal, and human studies. A new integrative paragraph now emphasizes that cellular models are particularly informative for identifying receptors and signaling mechanisms but may involve simplified systems or non-physiological concentrations; that animal loss- and gain-of-function approaches provide stronger causal evidence but may introduce species-specific, compensatory, or exposure-related effects; and that human studies provide the greatest physiological relevance but frequently remain observational and may not establish tissue origin or causality. We further emphasize that confidence is greatest when findings converge across these experimental levels, whereas effects demonstrated only in a single model should be regarded as provisional.

   Together with the revisions made throughout the individual myokine sections, the expanded Table 1, and the new integrative discussion, these changes provide a clearer distinction between mechanistic plausibility, experimental causality, and demonstrated physiological relevance in humans, while preserving the reference-oriented purpose and structure of the Review.

Manuscript changes: Additional revisions throughout the individual myokine subsections further distinguish cellular, animal, and human evidence, as described in our response to Comment 1 above.

3. The manuscript would benefit from a summary table reporting, for each myokine, the principal cellular source, biological functions, signaling pathway(s), level of experimental evidence (cellular, animal, or human), and potential clinical relevance. Such a table would improve readability given the large number of molecules discussed throughout the review.

   We thank the reviewer for this helpful suggestion. We agree that, given the number and heterogeneity of the myokines discussed, a molecule-by-molecule summary table can provide a useful complementary reference for the reader.

   The original manuscript already included two integrative figures: Figure 1 groups myokines according to their predominant biological actions within skeletal muscle, whereas Figure 2 summarizes proposed inter-organ communication and emphasizes differences in the strength of the supporting evidence. However, we agree that these figures do not provide, in a single location, the principal cellular source, signaling pathway, level of experimental evidence, and potential clinical relevance of each individual factor.

   We have therefore expanded Table 1 to provide a concise molecule-by-molecule summary including the principal muscle-associated source, major biological actions, main receptor/signaling pathways, level and nature of the supporting evidence (cellular, animal, and/or human), and potential clinical relevance. Particular care has been taken to distinguish established human evidence from findings derived predominantly from cellular or animal models and to avoid implying clinical applicability when this remains speculative.

   We believe that the expanded table complements, rather than duplicates, Figures 1 and 2: the figures provide functional and inter-organ perspectives, whereas the table allows rapid comparison of the individual factors discussed throughout the Review.

Changes in the revised manuscript: Table 1 has been substantially expanded.

4. The translational implications are only briefly addressed in the Introduction and Conclusions. A dedicated section discussing the current limitations and future perspectives of myokines as biomarkers and therapeutic targets would increase the impact of the review.

   We thank the reviewer for this important suggestion and fully agree that the translational implications of myokine biology deserved a more explicit and comprehensive discussion. In the original manuscript, these aspects were addressed primarily within individual myokine sections and briefly in the Introduction and Conclusions. We have therefore added a dedicated section entitled “Translational Implications: Myokines as Biomarkers and Therapeutic Targets.”.

   The new section discusses the opportunities and current limitations of using myokines as circulating or tissue biomarkers, including uncertainties regarding tissue source, molecular form, assay standardization, temporal variability, and the distinction between circulating concentration and biological activity. It also considers therapeutic strategies aimed at inhibiting maladaptive myokine signaling or enhancing adaptive and regenerative pathways, while emphasizing the challenges imposed by pleiotropy, receptor distribution, dose, timing, and local versus systemic exposure. Recent examples of clinical translation, including myostatin-targeted therapy and GDF15-directed treatment in cancer cachexia, have been incorporated to illustrate the increasing clinical relevance of this field. Finally, we outline methodological priorities for future studies, including longitudinal human designs, improved source attribution, standardized assays, integrated molecular profiling, and clinically meaningful functional endpoints.

   We believe that this new section substantially strengthens the translational perspective of the Review while remaining consistent with its central emphasis on source attribution, strength of evidence, and context-dependent myokine biology.

Changes in the revised manuscript: A new section entitled “Translational Implications: Myokines as Biomarkers and Therapeutic Targets” has been added.

Reviewer 3 Report

Comments and Suggestions for Authors

A well composed review focusing on myokines and their context dependent action in various metabolic and immune pathways

Several comments are inserted below

In the abstract, please revise muscle being introduced as a secretory organ. While muscle (as well as adipose tissue and perhaps, the majority of connective tissues) have as well a signaling molecule release function, their role is not that of a secretory organ stricto senso and should not be mistaken for one.

Introduction dives abruptly into description of myokine types. Here reader should have a manuscript overview, current state of the art in the perception and understanding of myokines and most of all, a statement about the purpose of the review. Why did authros engaged in composing such a manuscript, what do they consider is still missing in the literature to which they want to add/clarify, trace a future direction for research. Here it is the place where the authors should state as well the type of the review (narrative)  and only after that delineating a separate chapter dedicated to defining and describing myokines

Here, again, the same confusion with muscle being a secretory organ pointed out previously.

Figure 1 (actualy and infographic) and 2 could be redesigned to appear 1. More readable (for 1) and more visually expressive (for 2) Maybe icons representing the respective “targets” instead of thick black arrows dominating the picture and  reducing the size of muscle in the middle could more serve the graphical representation (but authros should not take this opinions as instructions but use their creative understanding to more suggestive rending the concept )

Maybe the listing of myokines could benefit categorizing them into cytokines with myokine activity (such as the IL inserted) growth factors with and “true”  myokines

Even if not the purpose of the review but in strict parallel with the intended purpose of demonstrating the “context dependent role” authros could briefly mention IL-6 context dependent role not only as a myokine but as well as a  pleiotropic cytokine with pro or anti inflammatory activity  depending on the tissue environment, the duration of exposure, and the specific molecular pathway—classic signaling versus trans-signaling. Similar remarks for the following described  cytokines since man y of them exert a context dependent role

The long listing of myokine types is  missing a structure regarding level o f evidence presented in the respective subchapter to supporting it. Experimental only based evidence (in vitro, cell based), in vivo animal studies and clinical observational studies need to be clearly identified in arguing evidence regarding the context dependent role of each molecule introduced. Otherwise the list risks to read like yet another unstructured search result.

Likewise the final Myokines in aging and disease” while with an ambitious title only presents and partially repeat seme notions The authors have  very well described why interpretation is difficult, but not what experiments or evidence would resolve those difficulties. Therefore this what would supposedly be the most important part of the whole review,  remains predominantly descriptive. In particular, the repeated recommendation for “caution” should be translated into specific interpretative limitations, including uncertainty regarding tissue of origin, distinction between local and circulating signaling, temporal dependence, receptor availability, and the inability of circulating associations alone to establish causality.

For example authors could identify what researcher need to be cautious on (and again, only take this as a suggestion not a reviewer requirement)

  Deducing muscle origin from circulating concentrations (since a rise in IL-6, FGF21, GDF15, etc. does not demonstrate skeletal-muscle secretion because many are multi-organ signals.

Modified  concentration does not obligatory equal   biological activity. Circulating abundance does not establish receptor engagement, tissue exposure, or downstream signaling.

A very classic but much neglected association is not causation  A changed myokine may be a driver, compensatory response, consequence of tissue injury, or s  disease-severity marker.

Tissue origin and timing of release matter . Local intramuscular/paracrine signaling may differ fundamentally from sustained systemic exposure; acute exercise-associated secretion cannot simply be extrapolated to chronic disease.

There is always a disease-specific context. The same myokine may behave differently according to inflammation, hypoxia, insulin resistance, denervation, cancer, aging, medication, or muscle mass.

 “myokines”  should not be perceived as a  homogeneous functional class. The designation identifies a possible source, not necessarily a shared biological role.

Cross-sectional blood measurements are not  evidence of a muscle secretory phenotype without muscle expression, arteriovenous gradients, tissue sampling, tracer approaches, or other source-attribution method

Moreover  this chapter  would benefit from identifying a trajectory for future investigation—for example, defining the evidence required to progress from altered circulating myokine concentrations to demonstration of skeletal-muscle origin, target engagement, mechanistic relevance, biomarker validity, and ultimately therapeutic applicability.

Same remarks regarding the “summary” which is basically yet another abstract correctly identifying the challenge of environment dependent activity but not offering a path for further approach, investigation, conceptual perspective

Author Response

A well composed review focusing on myokines and their context dependent action in various metabolic and immune pathways. Several comments are inserted below.

1. In the abstract, please revise muscle being introduced as a secretory organ. While muscle (as well as adipose tissue and perhaps, the majority of connective tissues) have as well a signaling molecule release function, their role is not that of a secretory organ stricto senso and should not be mistaken for one.

   We thank the reviewer for this precise and helpful comment. We agree that referring to skeletal muscle as a “secretory organ” may be interpreted too literally and could imply that secretion represents its primary defining function. Our intention was to emphasize the now well-established secretory and signaling capacity of skeletal muscle rather than to classify it as a secretory organ stricto sensu. We have therefore revised the opening sentence of the Abstract to state that, in addition to its contractile role, skeletal muscle has important secretory functions and communicates with local and distant tissues through myokines. For consistency, we have also replaced the corresponding wording in the main text with language emphasizing the secretory and signaling functions of skeletal muscle.

Changes in the revised manuscript: The relevant terminology has been revised in the Abstract (page 1, line 27) and in the section “Definition and Scope of Myokines” (page 2, lines 52-54), with corresponding changes throughout the manuscript.

2. Introduction dives abruptly into description of myokine types. Here reader should have a manuscript overview, current state of the art in the perception and understanding of myokines and most of all, a statement about the purpose of the review. Why did authors engaged in composing such a manuscript, what do they consider is still missing in the literature to which they want to add/clarify, trace a future direction for research. Here it is the place where the authors should state as well the type of the review (narrative) and only after that delineating a separate chapter dedicated to defining and describing myokines. Here, again, the same confusion with muscle being a secretory organ pointed out previously.

   We thank the reviewer for this thoughtful comment. We agree that the opening of the manuscript should provide the reader with a clearer overview of the current state of the field, the rationale and objectives of the Review, and its overall organization before entering into the definition and description of individual myokines.

   We have therefore reorganized and expanded the opening of the manuscript. The Introduction now briefly describes the evolution of the myokine concept and highlights major unresolved issues, including differences in the strength of evidence supporting muscle-cell origin and secretion, uncertainties regarding the tissue origin of circulating factors, discrepancies between cellular, animal, and human evidence, and the context-dependent nature of myokine actions. We also now explicitly identify the manuscript as a narrative Review.

   The statement of the primary aim of the Review has been retained and further refined. The revised Introduction now makes clear that the principal objective is to provide a structured and accessible reference framework for readers from diverse biomedical and clinical backgrounds, while secondarily identifying areas of mechanistic convergence, uncertainty, conflicting evidence, and ongoing debate. The translational implications and future perspectives of myokines as biomarkers and therapeutic targets are also explicitly included in the roadmap of the Review.

   In addition, the material defining and broadly describing myokines has been separated from the general Introduction and is now presented under a dedicated section entitled “Definition and Scope of Myokines.” In accordance with the reviewer’s preceding comment, we have also replaced the description of skeletal muscle as a “secretory organ” with wording emphasizing its important secretory and signaling capabilities in addition to its primary contractile functions.

   We believe that this reorganization provides the reader with a clearer rationale and roadmap for the Review before entering the molecule-by-molecule discussion.

Changes in the revised manuscript: Introduction and “Definition and Scope of Myokines” sections.

3. Figure 1 (actually and infographic) and 2 could be redesigned to appear 1. More readable (for 1) and more visually expressive (for 2) Maybe icons representing the respective “targets” instead of thick black arrows dominating the picture and reducing the size of muscle in the middle could more serve the graphical representation (but authors should not take these opinions as instructions but use their creative understanding to more suggestive rending the concept)

Maybe the listing of myokines could benefit categorizing them into cytokines with myokine activity (such as the IL inserted) growth factors with and “true” myokines

   We thank the reviewer for these thoughtful suggestions regarding the graphical presentation of Figures 1 and 2. We agree that conceptual figures should remain readily interpretable and visually accessible, particularly in a Review encompassing a large number of molecules.

After reconsidering the figures in light of the reviewer’s comments, we have elected to retain their current schematic design. This was a deliberate choice because the figures are intended to provide simple conceptual summaries rather than comprehensive molecular maps. Figure 1 groups selected factors according to their principal biological actions in skeletal muscle and is specifically intended to highlight functional overlap among otherwise different molecules. Figure 2, in turn, provides a simplified representation of proposed muscle-to-organ communication; the arrows indicate directionality rather than the magnitude or relative importance of individual pathways. Increasing the number of graphical elements or introducing additional molecular classifications within these figures would, in our view, increase visual density and could reduce their immediate interpretability.

   Importantly, in response to another reviewer’s suggestion, Table 1 has now been substantially expanded and provides a molecule-by-molecule summary of the principal muscle-associated source, biological functions, receptor/signaling pathways, level of experimental evidence, and potential clinical relevance. It also retains the structural classification of the factors into cytokines and chemokines, growth factors and growth-related proteins, and peptides/neurotrophic or extracellular-matrix-associated proteins.      We therefore believe that the revised Table 1 provides the detailed molecular categorization suggested by the reviewer, while the figures can remain focused on functional and inter-organ concepts.

   We have also preferred not to introduce a separate category of “true myokines.” In our usage, the term myokine is defined primarily by demonstrated production and release from skeletal muscle cells and by its signaling action, rather than by belonging to a particular molecular family. Cytokines and growth factors may therefore represent bona fide myokines when these criteria are fulfilled. We considered that distinguishing them from a separate group of “true myokines” could imply a hierarchy that is not inherent in the current functional definition of the term.

   We hope that the complementary roles of the expanded Table 1 and the deliberately simplified schematic figures provide an appropriate balance between molecular detail and visual clarity.

Changes in the revised manuscript: Figure 1 legend has been clarified and Table 1 substantially expanded; the schematic design of Figures 1 and 2 has otherwise been retained.

4. Even if not the purpose of the review but in strict parallel with the intended purpose of demonstrating the “context dependent role” authors could briefly mention IL-6 context dependent role not only as a myokine but as well as a pleiotropic cytokine with pro or anti-inflammatory activity depending on the tissue environment, the duration of exposure, and the specific molecular pathway—classic signaling versus trans-signaling. Similar remarks for the following described cytokines since man y of them exert a context dependent role

   We thank the reviewer for this useful clarification. We agree that the context-dependent biology of IL-6 extends beyond its role as a myokine and also reflects its broader pleiotropic activity as a cytokine. The manuscript already distinguished classical IL-6 signaling through membrane-associated IL-6R from trans-signaling through soluble IL-6R and emphasized the importance of receptor availability, cellular context, and duration of exposure. We have now made this distinction more explicit by noting that classical signaling is frequently associated with homeostatic, regenerative, or anti-inflammatory responses, whereas trans-signaling is more commonly linked to pro-inflammatory effects, while emphasizing that this distinction is not absolute and remains tissue- and context-dependent.

   We have also reviewed the corresponding sections on the other cytokines to ensure that their context-dependent actions are sufficiently clear. In several cases, including IL-1β, TNF-α, LIF, and other inflammatory mediators, the manuscript already distinguishes transient or locally regulated actions that may contribute to adaptation or repair from sustained or excessive signaling associated with inflammation, impaired regeneration, or catabolism. We have therefore limited additional changes to targeted clarifications where appropriate, in order to avoid unnecessary repetition.

Changes in the revised manuscript: IL-6 section (page 6, lines 161-165).

5. The long listing of myokine types is missing a structure regarding level of evidence presented in the respective subchapter to supporting it. Experimental only based evidence (in vitro, cell based), in vivo animal studies and clinical observational studies need to be clearly identified in arguing evidence regarding the context dependent role of each molecule introduced. Otherwise the list risks to read like yet another unstructured search result.

   We thank the reviewer for this important observation. We agree that, in a Review encompassing a large number of molecules, the experimental level supporting individual claims should be readily identifiable so that mechanistic findings from cellular models are not inadvertently presented with the same weight as animal or human evidence.

   The manuscript already distinguished these levels of evidence within many individual sections, for example by specifying when findings derive from cultured myogenic cells, loss- or gain-of-function animal models, human muscle studies, arteriovenous measurements, or clinical observations. Nevertheless, we agree that this evidence hierarchy was not sufficiently explicit as an organizing principle of the molecule-by-molecule section.

   We have therefore revised the introductory paragraph of the section “Main Myokines and Their Actions” to state explicitly that each factor is evaluated by distinguishing evidence derived from in vitro/cellular models, in vivo animal studies, and human observational or interventional studies whenever such evidence is available. We have also reviewed the individual subsections to ensure that the experimental level supporting the principal context-dependent actions is clearly identified.

   Importantly, the substantially expanded Table 1 now provides a concise molecule-by-molecule summary of the level and nature of the supporting evidence, alongside the principal cellular source, biological functions, signaling pathways, and potential clinical relevance. In addition, the revised manuscript includes an integrative discussion of the different inferential strengths and limitations of cellular, animal, and human studies.

  We believe that these changes provide a clearer evidence-based structure to the molecule-by-molecule discussion and reduce the risk that the section is perceived as a simple descriptive listing of reported effects.

6. Likewise, the final Myokines in aging and disease” while with an ambitious title only presents and partially repeat same notions. The authors have very well described why interpretation is difficult, but not what experiments or evidence would resolve those difficulties. Therefore, this what would supposedly be the most important part of the whole review, remains predominantly descriptive. In particular, the repeated recommendation for “caution” should be translated into specific interpretative limitations, including uncertainty regarding tissue of origin, distinction between local and circulating signaling, temporal dependence, receptor availability, and the inability of circulating associations alone to establish causality.

For example, authors could identify what researcher need to be cautious on (and again, only take this as a suggestion not a reviewer requirement)

   We thank the reviewer for this important observation. We agree that the previous version of the section “Myokines in Disease and Aging” identified several reasons why disease-associated changes in myokines are difficult to interpret, but did not sufficiently specify what experimental evidence would be required to resolve these uncertainties.

   We have therefore revised and expanded this section and renamed it “Myokines in Disease and Aging: An Interpretative Framework” to better reflect its purpose. Rather than repeatedly recommending general caution, the revised text now identifies specific interpretative limitations: uncertainty regarding tissue of origin; the distinction between local autocrine/paracrine and circulating endocrine signaling; temporal dependence of    myokine responses; cell-specific receptor availability; and the inability of circulating associations alone to establish causality.

   Importantly, we now link each of these limitations to experimental approaches that could strengthen inference. These include combined tissue and circulating measurements, arteriovenous assessment of net muscle release, tissue- and cell-specific approaches, including single-cell and spatial analyses, serial and longitudinal sampling, assessment of receptor expression together with downstream pathway activation, and interventional studies that directly perturb ligand-receptor signaling and evaluate functional outcomes. We also emphasize that cross-sectional associations between circulating myokines and clinical phenotypes should generally be regarded as hypothesis-generating rather than causal evidence.

   This revised section is now followed by the newly added section “Translational Implications: Myokines as Biomarkers and Therapeutic Targets,” which extends these methodological considerations to biomarker development and therapeutic translation. We believe that the two sections now have complementary functions: the first provides an evidence-based framework for interpreting myokine alterations in disease and aging, whereas the second addresses how sufficiently supported findings may be translated into clinical applications and identifies the remaining barriers to such translation.

   We believe that these revisions make the final part of the Review more analytical and prospective rather than predominantly descriptive.

   Changes in the revised manuscript: The section “Myokines in Disease and Aging” has been substantially revised and retitled “Myokines in Disease and Aging: An Interpretative Framework” (pages 67–69, lines 1686-1733). A new section entitled “Translational Implications: Myokines as Biomarkers and Therapeutic Targets” has also been added (pages 69-73).

7. Deducing muscle origin from circulating concentrations (since a rise in IL-6, FGF21, GDF15, etc. does not demonstrate skeletal-muscle secretion because many are multi-organ signals.

   We thank the reviewer for emphasizing this important point. We fully agree that increased circulating concentrations of IL-6, FGF21, GDF15, or other multi-organ signaling factors cannot, by themselves, be taken as evidence of skeletal-muscle secretion. This concern substantially overlaps with issues raised in the preceding comments and has therefore been addressed throughout the revised manuscript rather than by adding a further repetitive discussion.

   In the revised manuscript, we now explicitly state that changes in muscle expression or circulating concentrations are not considered sufficient evidence of muscle-cell-derived secretion unless supported by appropriate cellular, tissue-specific, arteriovenous, or other source-attribution data. The individual myokine sections have also been reviewed to distinguish direct evidence of muscular production or release from circulating associations of uncertain tissue origin. This distinction is additionally summarized in the expanded Table 1, which reports the principal muscle-associated source and nature of the supporting evidence for each factor.

   The revised section “Myokines in Disease and Aging: An Interpretative Framework” further identifies uncertainty regarding tissue of origin as a specific interpretative limitation and discusses the experimental approaches required to address it, including paired tissue and circulating measurements, arteriovenous assessment, and tissue- and cell-specific approaches such as single-cell and spatial analyses. The new translational section similarly emphasizes that circulating abundance and skeletal-muscle production should not be regarded as interchangeable when evaluating candidate biomarkers or therapeutic targets.

   Finally, we have reviewed the wording throughout the manuscript to avoid inferring skeletal-muscle secretion solely from changes in circulating concentrations.

8. Modified concentration does not obligatory equal   biological activity. Circulating abundance does not establish receptor engagement, tissue exposure, or downstream signaling.

   We thank the reviewer and fully agree with this distinction. Changes in circulating concentration should not be equated with biological activity, since effective signaling additionally depends on target-tissue exposure, receptor availability and engagement, molecular form, and activation of the relevant downstream pathways.

   This principle is now emphasized at several levels throughout the revised manuscript. Individual sections distinguish circulating abundance from demonstrated biological activity; for example, the myostatin section explicitly notes that circulating concentrations do not necessarily reflect biologically active ligand within skeletal muscle and that biological relevance requires evidence of local pathway activation, receptor engagement, and downstream signaling. The revised interpretative framework for disease and aging likewise emphasizes receptor availability and local versus systemic exposure as determinants of biological effects.

   In addition, the new section “Translational Implications: Myokines as Biomarkers and Therapeutic Targets” now explicitly states that tissue expression, circulating abundance, receptor engagement, and biological activity should not be regarded as interchangeable readouts, and that a change in circulating concentration cannot by itself establish target-tissue exposure or downstream pathway activation.

   We believe that these revisions make this important distinction sufficiently explicit without introducing further repetitive discussion.

9. A very classic but much neglected association is not causation.A changed myokine may be a driver, compensatory response, consequence of tissue injury, or a disease-severity marker.

   We thank the reviewer and fully agree that association should not be interpreted as causation. This distinction is central to the revised manuscript and has been reinforced throughout the text. In the section on disease and aging, we now explicitly state that altered myokine profiles may represent causal mediators, compensatory responses, consequences of tissue injury or systemic stress, or biomarkers reflecting disease severity.

   In addition, the revised manuscript repeatedly distinguishes circulating associations from evidence of tissue origin, biological activity, receptor engagement, and downstream signaling, and emphasizes that cross-sectional associations alone cannot establish causal involvement. The expanded discussion of experimental evidence further clarifies that causal inference is strengthened by longitudinal observations and, particularly, by interventions that directly modify the ligand, receptor, or downstream pathway and demonstrate corresponding functional effects.

   We therefore believe that the revised manuscript now makes the distinction between association, biological relevance, and causality explicit without requiring further repetitive discussion.

10. Tissue origin and timing of release matter. Local intramuscular/paracrine signaling may differ fundamentally from sustained systemic exposure; acute exercise-associated secretion cannot simply be extrapolated to chronic disease.

We thank the reviewer and fully agree that the temporal context of myokine release is critical for interpretation. In the revised interpretative framework, we now explicitly state that an acute increase associated with exercise, injury, or regeneration cannot be directly extrapolated to chronic disease, where sustained exposure may have different biological consequences. This distinction complements the separate considerations regarding tissue origin, local versus circulating signaling, receptor availability, and causality.

Similar temporal distinctions are also emphasized within several individual myokine sections, particularly for mediators such as IL-6, for which transient physiological signaling and sustained exposure in chronic inflammatory or catabolic conditions may have substantially different consequences.

11. There is always a disease-specific context. The same myokine may behave differently according to inflammation, hypoxia, insulin resistance, denervation, cancer, aging, medication, or muscle mass.

   We thank the reviewer and fully agree that myokine biology must be interpreted within a disease-specific context. The revised manuscript already emphasizes that skeletal muscle may be exposed to markedly different biological environments, including inflammation, metabolic dysfunction, hypoxia, denervation, inactivity, malnutrition, aging, malignancy, and treatment-related effects, and that these conditions may alter both myokine production and signaling.

   To make this point more explicit, we have added a statement emphasizing that the biological significance of a given myokine cannot be extrapolated uniformly across diseases, since factors such as inflammation, hypoxia, insulin resistance, denervation, cancer, aging, treatment exposure, and the amount and functional state of skeletal muscle may modify its source, concentration, receptor context, and downstream effects.

   We believe that this clarification reinforces the disease-specific interpretation of myokine changes without requiring an exhaustive disease-by-disease discussion.

Changes in the revised manuscript: “Myokines in Disease and Aging: An Interpretative Framework”.

12. “myokines” should not be perceived as a homogeneous functional class. The designation identifies a possible source, not necessarily a shared biological role.

   We thank the reviewer and fully agree that “myokines” should not be interpreted as a homogeneous functional class. The designation primarily reflects production and release by skeletal muscle cells together with signaling activity, rather than a shared molecular identity or biological function.

   To make this distinction explicit, we have revised the definition in the manuscript to state that the term “myokine” refers primarily to a factor’s production and release by skeletal muscle cells and its signaling capacity, rather than to membership in a specific molecular or functional class. We further emphasize that myokines constitute a heterogeneous group of molecules that may differ markedly in structure, receptor usage, target cells, and biological actions.

   This distinction is also reflected throughout the Review, including the molecular classification provided in Table 1 and the discussion showing that even when different myokines converge on common intracellular signaling pathways, this does not imply biological equivalence.

Changes in the revised manuscript: “Definition and Scope of Myokines”, with corresponding clarification in the integrative discussion and Table 1.

13. Cross-sectional blood measurements are not evidence of a muscle secretory phenotype without muscle expression, arteriovenous gradients, tissue sampling, tracer approaches, or other-source-attribution method.

   We thank the reviewer and fully agree that cross-sectional measurements of circulating myokines cannot, by themselves, establish a skeletal-muscle secretory phenotype. This issue substantially overlaps with the preceding comments regarding tissue origin and source attribution and has been addressed throughout the revised manuscript.

   The manuscript now explicitly states that changes in muscle expression or circulating concentrations are not considered sufficient evidence of muscle-cell-derived secretion unless supported by appropriate source-attribution data. Throughout the individual sections, we distinguish circulating associations from direct evidence of muscular production or release, including tissue measurements and arteriovenous studies where available.

   In addition, the revised interpretative framework now specifies the types of experimental approaches that can strengthen attribution of tissue origin, including combined muscle-tissue and circulating measurements, arteriovenous gradients across skeletal muscle, tracer-based approaches where applicable, tissue- and cell-specific analyses, and muscle-specific loss- or gain-of-function strategies in experimental models.

   We therefore agree with the reviewer that cross-sectional blood measurements should be regarded as associative evidence unless complemented by methods capable of establishing tissue origin and secretion.

14. Moreover, this chapter would benefit from identifying a trajectory for future investigation—for example, defining the evidence required to progress from altered circulating myokine concentrations to demonstration of skeletal-muscle origin, target engagement, mechanistic relevance, biomarker validity, and ultimately therapeutic applicability.

   We thank the reviewer for this constructive suggestion. We agree that, beyond identifying the limitations of the current literature, the Review should indicate how evidence can progress from an observed circulating association toward physiological and ultimately clinical relevance. Although the revised manuscript already discusses the individual requirements for source attribution, target engagement, mechanistic inference, biomarker development, and therapeutic translation, we agree that these elements were not previously presented as an explicit investigative trajectory.

   We have therefore added a stepwise framework to the section “Translational Implications: Myokines as Biomarkers and Therapeutic Targets.” The revised text now proposes that an association between a circulating myokine concentration and a physiological or clinical phenotype should first be followed by evidence of skeletal-muscle production and release, then by demonstration of target-tissue exposure, receptor engagement, and downstream signaling at physiologically relevant concentrations, and subsequently by mechanistic studies showing that perturbation of the pathway modifies the relevant phenotype. Progression toward biomarker use additionally requires analytical validation, reproducibility, longitudinal association, and added clinical value, whereas progression toward therapeutic application requires demonstration that pathway modulation produces clinically meaningful benefit with acceptable safety.

   We believe that this addition provides a clearer trajectory for future investigation and integrates the methodological and translational considerations developed throughout the revised Review.

   Changes in the revised manuscript: A stepwise framework for the investigation and translation of candidate myokines has been added to the section “Translational Implications: Myokines as Biomarkers and Therapeutic Targets.”

15. Same remarks regarding the “summary” which is basically yet another abstract correctly identifying the challenge of environment dependent activity but not offering a path for further approach, investigation, conceptual perspective.

  We thank the reviewer for this important comment. We agree that the previous “Summary” was overly repetitive and, in retrospect, reproduced several elements already presented in the Abstract and elsewhere in the Review without providing a sufficiently forward-looking conceptual perspective.

   We have therefore substantially revised the final part of the manuscript. Rather than retaining separate “Summary” and “Conclusion” sections, we have combined them into a new section entitled “Conclusions and Future Perspectives.” The revised section no longer reiterates the catalogue of myokines and their principal actions. Instead, it provides a conceptual framework for future investigation, emphasizing the need to distinguish muscle production and release, target-tissue exposure and receptor engagement, downstream biological activity, and causal or compensatory relationships with disease phenotypes.

   The revised section also outlines the methodological progression required to address these questions, including tissue- and cell-specific source attribution, arteriovenous and tracer-based approaches, serial sampling, assessment of receptor and downstream pathway activation, and longitudinal and interventional human studies. It further distinguishes the additional requirements for biomarker validation and therapeutic translation. Finally, we propose a broader conceptual shift from a molecule-centered view of individual myokines toward a network- and context-based model incorporating cellular source, temporal regulation, receptor distribution, tissue exposure, disease environment, and interactions with signals derived from other organs.

   We believe that this restructuring makes the final section more analytical and prospective and avoids its previous overlap with the Abstract.

   Changes in the revised manuscript: The previous “Summary” and “Conclusion” sections have been replaced by a single section entitled “Conclusions and Future Perspectives.”

Reviewer 4 Report

Comments and Suggestions for Authors

The authors prepared the detailed, comprehensive review article about myokiones - biologically active molecules secreted by sceletal muscles , in particular their sources, signaling mechanisms and physiological role. The manuscript is well-written and supported by huge set of adequate references. The paper would be of interest for broad research and clinical community interested in endocrine function of non-endocrine organs, sceletal muscle physiology and metabolic abnormalities.

Author Response

We sincerely thank the reviewer for their kind and encouraging comments. We greatly appreciate their positive assessment of the scope, clarity, and supporting literature of our Review, as well as their view that it may be of interest to a broad biomedical and clinical readership.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the authors’ detailed responses and the revisions made to the manuscript. However, some aspects would benefit from further consideration. In particular, the review would benefit from a more integrated and less predominantly molecule-by-molecule presentation, with greater emphasis on comparison and synthesis across the different myokines. A more explicit and systematic critical discussion of conflicting or divergent findings, particularly when comparing cellular, animal, and human evidence, could also further strengthen the manuscript.

In addition, the revisions should be incorporated into the original manuscript file while maintaining the original journal formatting.

Overall, I consider the manuscript improved, but I believe that further revision would be appropriate before it can be considered for publication.

Author Response

We sincerely thank the reviewer for the careful reassessment of our manuscript and for recognizing the improvements introduced in the revised version. We also appreciate the reviewer’s continued emphasis on the need for greater integration and critical synthesis across the different myokines.

  We agree that comparison across factors and a more explicit appraisal of the different levels of evidence are important. However, we respectfully believe that a major restructuring away from the molecule-by-molecule format would fundamentally alter the primary purpose of this Review. The manuscript was specifically designed as an accessible reference for readers from diverse clinical and biomedical fields who may not be specialists myokine biology, allowing rapid access to up-to-date information on each individual factor, including its cellular source, regulation, major biological actions, signaling pathways, and areas of uncertainty or debate. We believe that preserving this structure also facilitates interpretation of the more focused contributions included in this Special Issue.

   Nevertheless, in response to the reviewer’s concern, we have further strengthened the integrative and critical components of the manuscript without changing its overall structure. Specifically, we have added cross-myokine synthesis highlighting convergence in major signaling pathways and the importance of cellular source, receptor distribution, local versus systemic exposure, concentration, timing, and biological context. We have also included a more explicit discussion of the hierarchy and limitations of evidence derived from cellular models, animal studies, and human investigations, emphasizing that mechanistic plausibility, experimental causality, and demonstrated physiological relevance in humans should not be considered equivalent levels of evidence. In addition, the sections on disease and aging and on translational implications have been expanded to provide a broader interpretative framework, and Table 1 has been substantially revised to facilitate direct comparison across factors.

   Finally, following the Editor’s suggestion, we have now added a new summary schematic in the Conclusions and Future Perspectives section (Figure 3), which integrates the main interpretative and translational principles of the Review, from initial candidate identification through source attribution, target engagement, mechanistic validation, and potential biomarker or therapeutic development. We believe that this additional synthesis directly addresses the reviewer’s concern while preserving the intended reference-oriented structure of the manuscript.

   We have also ensured that all revisions have been incorporated into the original manuscript file while maintaining the journal’s formatting requirements.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have  modfed structure and content of ther work 

Author Response

   We sincerely thank the reviewer for this positive assessment and for recognizing the revisions made to the manuscript. We greatly appreciate the reviewer’s constructive comments.