A coordinated multiorgan metabolic response contributes to human mitochondrial myopathy

Abstract Mitochondrial diseases are a heterogeneous group of monogenic disorders that result from impaired oxidative phosphorylation (OXPHOS). As neuromuscular tissues are highly energy‐dependent, mitochondrial diseases often affect skeletal muscle. Although genetic and bioenergetic causes of OXPHOS impairment in human mitochondrial myopathies are well established, there is a limited understanding of metabolic drivers of muscle degeneration. This knowledge gap contributes to the lack of effective treatments for these disorders. Here, we discovered fundamental muscle metabolic remodeling mechanisms shared by mitochondrial disease patients and a mouse model of mitochondrial myopathy. This metabolic remodeling is triggered by a starvation‐like response that evokes accelerated oxidation of amino acids through a truncated Krebs cycle. While initially adaptive, this response evolves in an integrated multiorgan catabolic signaling, lipid store mobilization, and intramuscular lipid accumulation. We show that this multiorgan feed‐forward metabolic response involves leptin and glucocorticoid signaling. This study elucidates systemic metabolic dyshomeostasis mechanisms that underlie human mitochondrial myopathies and identifies potential new targets for metabolic intervention.

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(1) It is not clear what is the causal role of amino acid catabolism via aminotransferases into TCA cycle. Please test whether this is adaptive or maladaptive by administering aminooxy acetate (AOA) to mice. AOA has been administered to mice. AOA will inhibit aminotransferases and prevent glutamate conversion to aKG. What happens to the mice grip strength or some other functional output as well as glutamate tracing in vivo with AOA. see AOA in mice reference:PMID: 23153536 (2) The OGDH reaction is NAD+ dependent and succinate to fumarate uses SDH. COX10 KO will not allow complex I to regenerate NAD+ and SDH to function. I am struggling how oxidative TCA cycle is functioning in COX10 KO. They report a decreased NAD+/NADH ratio in COX10 KO vs. CTL muscle. Is there any reductive carboxylation going on (citrate m+5 from glutamate)? I guess proline synthesis can generate some NAD+ to keep oxidative cycle going but how does SDH functions in this setting?
The work by Southwell and colleagues uses an elegant multi-omic approach to investigate the metabolic, signalling and molecular changes associated with impaired oxidative phosphorylation in skeletal muscle samples from patients with MERRF syndrome and from a muscle-specific Cox10 knockout mouse. In keeping with previously published results, the authors found a general rewiring of energy metabolism, leading to increased glutamate oxidation, activation of the mitochondrial integrated stress response, inhibition of translation via reduced activity of mTORC1, activation of autophagy and reduced fatty acids oxidation. In addition, glutamate non-anaplerotic pathways were also increased, possibly to sustain NAD+ levels. Probably, the most original finding is the identification of a novel crosstalk pathway between defective skeletal muscles and other organs, in particular liver and white adipose tissue. This phenomenon is possibly mediated by mitokynes, such as FGF21 and GDF15, and leads to increased release of fatty acids from WAT, contributing to the increased levels in muscle and blood, and upregulation of gluconeogenesis, ureagenesis and ketogenesis in the liver. Additional findings include the dysregulation of leptin signalling Cox10 KO mice, and the presence of increased levels of glucocorticoids in mouse and patients' blood, which may contribute to the increased proteostasis found in the skeletal muscle of mice and patients.
The paper is interesting, and the experiments are elegantly performed. However, I have some concerns that the authors should address.

Major comments/concerns
The authors used skeletal muscle from both MERRF patients and muscle-specific Cox10 KO mice. However, the rationale for this comparison should be made clearer. MERRF is a complex, multisystem disease with reduced activity of several respiratory chain complexes due to impaired translation of mtDNA-encoded genes, while Cox10 is a nuclear gene encoding an enzyme involved in the biosynthesis of hemeA and its disruption in mouse skeletal muscle causes severe deficiency in cytochrome c oxidase. In addition, a great amount of work was carried out to describe changes in fat metabolism in Cox10 mice, but its relevance for human disease is unclear. I also suggest the authors to better identify the potential of their findings for the clinical management of the patients and/or for the development of new therapies. Some of the described changes are not entirely new, including those related to mtISR, 1C metabolism, trans-sulfuration, etc... I recommend the authors highlight the novelties compared to previously published data. I am not convinced by the interpretation of some results. For instance, the authors interpret the increased levels of asparagine, glutamate, and their acetylated derivatives (N-acetyl asparagine and N-acetyl glutamate as an increase in amino acid utilization. However, in my opinion, this could be seen as the result of reduced utilization leading to accumulaiton. In addition, I find incorrect the authors' interpretation of the data on autophagy. The authors say that "the levels of the autophagy biomarker proteins LC3II and p62 were significantly elevated only at the late stage of myopathy", but this is most like reflecting a block in autophagy, not an activation. However, this can be proven only by using colchicine as a blocker of muscle autophagic flux. As this would be a very time-consuming experiment, I am only suggesting the authors consider and eventually discuss this possibility.
Specific/minor comments Page 9: the sentence "the variable upregulation among the markers...suggests the operation of complex regulatory processes" sounds too vague. Page 12: the authors dissect mTORC1 signalling in Cox10 muscles. Can the authors speculate on why mTORC1 signalling is downregulated in this model? Is this the case also in the MERRF patients' muscles?
Referee #3 (Comments on Novelty/Model System for Author): Novelty: The paper from Southwell et al. aims to study the muscle metabolic remodeling mechanisms involved in severe mitochondrial myopathy conditions and understands the downstream consequences on other tissue such as the liver, WAT. The current paper highlight that the muscle metabolic adaptations are part of inter-organ cross talk. It also identifies potential new targets for metabolic intervention.
Technical qualities and adequacy of the model: The metabolic responses to OXPHOS defect was first characterised in human muscle from m.8344A>G MERRF patients, and confirmed in murine model of mitochondrial myopathy. The murine model where COX10 is excised in skeletal muscle specifically has been characterised in depth (eg Expression level of Cox1 and 4 at different time point via Western blot analysis, muscle activity tested via treadmill and grip test, visceral fat assessed, and body weight overtime reported) Different mitochondrial markers tested (involved in protein. Transport or mitochondrial metabolism), metabolites involved in different metabolic pathways assessed as well. Several Omic analysis have been used in the current paper, including transcriptome, metabolome.
Referee #3 (Remarks for Author): The authors have convincingly show that OXPHOS-defective muscle adapts metabolically by implementing alternative pathways for rewiring of redox and metabolic homeostasis.
The figures are very well organised and full of information: including in all figures a graphical abstract of the metabolic pathway being investigated is very useful. All western blots are neat, as well as the histology. The paper is reach in data and extremely complete. The discussion is very complete.
Minor comment: Page 5 Typo: "reveled" instead of "Revealed" Also the data generated via the different omics analysis should be deposited in a public database (I could not find any GEO number or any thing similar in the text). In the methods, the authors should detail a bit more the omic analysis (eg the statistical approach used, etc)

Point by point response to reviewers' comments
First, we want to thank the reviewers for their thorough analysis of the manuscript and their insightful comments. They provided important suggestions for improving the manuscript. In the revision, we have addressed their comments and concerns as follows.

Referee #1
This is an interesting paper that demonstrates elevation of oxidation of amino acids in mitochondrial disease patients and mouse model of mitochondrial myopathy (COX10 KO). The paper is largely well executed however there are two key points that need to be addressed. 1) It is not clear what is the causal role of amino acid catabolism via aminotransferases into TCA cycle. Please test whether this is adaptive or maladaptive by administering aminooxy acetate (AOA) to mice. AOA has been administered to mice. AOA will inhibit aminotransferases and prevent glutamate conversion to aKG. What happens to the mice grip strength or some other functional output as well as glutamate tracing in vivo with AOA. see AOA in mice reference: PMID: 23153536 We thank the reviewer for this valuable suggestion. In vivo treatments with Aminooxy acetate (AOA), an inhibitor of PLP-dependent transaminases (e.g., GOT, ALT), have been effective in reducing cancer growth through the inhibition of the glutamine-glutamate utilization pathway (Korangath et al, 2015;Qing et al, 2012). Therefore, we tested the effect of AOA administration in COX10 KO mice. The in vivo AOA treatment was performed following the experimental condition reported by Qing G. et al. (Qing et al, 2012) as recommended by the reviewer. Briefly, 70 days old COX10 KO and CTL mice were intraperitoneally injected daily with 10 mg/Kg of AOA (treated) or PBS (untreated). After 7 weeks of treatment, exercise endurance (measured by treadmill) was decreased in AOA treated COX10 KO vs. In summary, the worsening of exercise endurance on the treadmill by AOA suggests that upregulation of the energy-generating glutamate flux through the TCA cycle is an adaptive response in COX10 KO muscle. Of note, because of the non-selective inhibition of AOA (e.g., AOA is also inhibitor of cystathionine β synthase, CBS; cystathionine γ lyase, CSE; GABA aminotransferase, GABA-T; ornithine aminotransferase, OAT) we cannot exclude that suppression of additional pathways (trans-sulfuration, GABA, arginine metabolism) could contribute to the worsening of COX10 KO exercise endurance. Further investigation would be required to dissect specific pathway contribution and fully address this point. The results, interpretation, and limitations of this new AOA study are presented in Results (pages 12) and Discussion (page 27).
2) The OGDH reaction is NAD+ dependent and succinate to fumarate uses SDH. COX10 KO will not allow complex I to regenerate NAD+ and SDH to function. I am struggling how oxidative TCA cycle is functioning in COX10 KO. They report a decreased NAD+/NADH ratio in COX10 KO vs. CTL muscle. Is there any reductive carboxylation going on (citrate m+5 from glutamate)? I guess proline synthesis can generate some NAD+ to keep oxidative cycle going but how does SDH functions in this setting?

13th Apr 2023 1st Authors' Response to Reviewers
We agree with the reviewer that the sources of NAD + supply for the α-ketoglutarate dehydrogenase complex (OGDHC) activity in COX10 KO muscle need to be better addressed. In fact, in the presence of defective respiratory chain electron transfer, NADH is expected to accumulate and limit OGDHC activity, essential for the provision of succinyl-CoA to succinyl-CoA ligase (SUCLA). It is common knowledge that NADH generated by the TCA cycle is oxidized by complex I, which re-supply NAD + to the TCA enzymes. However, it was shown that during respiratory inhibition and anoxic conditions (functionally comparable to COX deficiency), mitochondrial diaphorases can contribute up to 80% to the NAD + matrix pool and support OGDHC-mediated substrate-level phosphorylation (Kiss et al, 2014). Diaphorases, also known as DT-diaphoresis or NAD(P)H:quinone oxidoreductase (NQO), are flavoenzymes catalyzing the oxidation of reduced pyridine nucleotides (both NADH and NADPH) by endogenous electron acceptors. Among the several diaphorases isoforms, NQO1 has been found to localize not only in the cytosol but also in mitochondria from several tissues (Dong et al, 2013). Interestingly, transcript levels of Nqo1 are upregulated in COX10 KO vs. CTL (now shown in the new Fig. S6J) suggesting that mitochondrial DTdiaphorases could contribute to the matrix NAD + pool regeneration in COX10 KO muscle. Interestingly, the re-oxidation of the reducible substrates of diaphorases is mediated by respiratory chain Complex III (Kiss et al, 2014) which would also allow SDH activity. However, the electron acceptor of reduced cytochrome c under conditions of oxygen deprivation and/or dysfunctional complex IV remains to be determined. Further identification and understanding of this electron acceptor (metabolite/protein) of the intermembrane space could provide viable therapeutic approaches for potentiating mitochondrial redox and energy state during OXPHOS impairment. As accurately suggested by the reviewer, another potential source of matrix NAD + could derive from the reverse operation of isocitrate dehydrogenase (IDH2 and IDH3 isoforms). Reductive carboxylation of glutamate-derived αKG would predictably generate M+5 forms of citrate, (i.e., with the TCA cycle running in a counter-clockwise direction). This possibility was tested by tracing muscle incorporation of 13 C into the TCA cycle intermediates 30 minutes after IP injections of labeled glutamate. Our data show that muscle citrate displays an overall greater incorporation (combination of M+1, M+2, and M+4, new Fig. S6K) demonstrating increased oxidative flux of glutamate (i.e., with the TCA cycle running in a clockwise direction) in COX10 KO vs CTL muscle. The M+5 forms of citrate were not detected, indicating that during the tracer exposure reductive carboxylation does not play a major role in NAD + regeneration in COX10 KO muscle, at least in the time frame of the experiment. Nevertheless, accumulation of glutamate and 3OH-FA/3OH-acylcarnitines, suggests that despite the utilization of alternative pathways of NAD + regeneration, such as DT-diaphorases and proline synthesis ( Fig.6D-G), the NAD + matrix pool remains inadequate to support glutamate and FA oxidation in COX10 KO and MERRF muscle. New figures (Fig. S6J and Fig S6K) and all these considerations have been added to manuscript Results (page 21) and Discussion (page 31)

Referee #2
The work by Southwell and colleagues uses an elegant multi-omic approach to investigate the metabolic, signaling and molecular changes associated with impaired oxidative phosphorylation in skeletal muscle samples from patients with MERRF syndrome and from a muscle-specific Cox10 knockout mouse. In keeping with previously published results, the authors found a general rewiring of energy metabolism, leading to increased glutamate oxidation, activation of the mitochondrial integrated stress response, inhibition of translation via reduced activity of mTORC1, activation of autophagy and reduced fatty acids oxidation. In addition, glutamate non-anaplerotic pathways were also increased, possibly to sustain NAD+ levels. Probably, the most original finding is the identification of a novel crosstalk pathway between defective skeletal muscles and other organs, in particular liver and white adipose tissue. This phenomenon is possibly mediated by mitokynes, such as FGF21 and GDF15, and leads to increased release of fatty acids from WAT, contributing to the increased levels in muscle and blood, and upregulation of gluconeogenesis, ureagenesis and ketogenesis in the liver. Additional findings include the dysregulation of leptin signaling Cox10 KO mice, and the presence of increased levels of glucocorticoids in mouse and patients' blood, which may contribute to the increased proteostasis found in the skeletal muscle of mice and patients. The paper is interesting, and the experiments are elegantly performed. However, I have some concerns that the authors should address.
1) The authors used skeletal muscle from both MERRF patients and muscle-specific Cox10 KO mice. However, the rationale for this comparison should be made clearer. MERRF is a complex, multisystem disease with reduced activity of several respiratory chain complexes due to impaired translation of mtDNA-encoded genes, while Cox10 is a nuclear gene encoding an enzyme involved in the biosynthesis of heme A and its disruption in mouse skeletal muscle causes severe deficiency in cytochrome c oxidase.
We thank the reviewer for this comment, which give us the opportunity to justify the comparison between a specific clinical-genetic subgroup of mitochondrial diseases (MERRF) and the muscle-specific COX10 KO mouse. Our working hypothesis is that in patients with and in a mouse model of mitochondrial myopathy, a common metabolic response to OXPHOS defect in muscle and a conserved systemic signaling pathway contributes to mitochondrial myopathy. The rationale behind the MERRF/COX10 KO comparison resides in the muscle bioenergetic, phenotypic, and metabolic similarities which we highlight below. The most common MERRF mutation is the m.8344A>G in the MT-TK gene encoding for transfer RNA Lysine , which affects the translation of mtDNA-encoded proteins. Although this mutation can potentially affect complex I, III, IV, and V assembly, complex IV (cytochrome c oxidase, COX) remains the most affected complex in MERRF tissues and cells as clearly shown by abundance of COX-negative fibers by COX staining (new Fig. S1E) and impaired COX activity by biochemical testing (Antonicka et al, 1999;Catteruccia et al, 2015). Interestingly, the m.3243A>G mutation in the MT-TL1 gene encoding the mitochondrial tRNA Leucine is often associated with complex I dysfunction (Sharma et al, 2021) and results in mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) suggesting that mutations in mitochondrial tRNAs can impair some respiratory chain complexes more severely than others. This can explain why MERRF and COX10 KO muscle share severe COX deficiency despite their genetic differences. We agree with the reviewer that MERRF syndrome is a complex disease, with possible multisystem involvement. However, unlike other canonical mitochondrial syndromes (such as chronic progressive external ophthalmoplegia, CPEO, and MELAS) the m.8344A>G MERRF phenotype is poorly represented by its acronym (myoclonus epilepsy with ragged-red fibers), because it is mostly characterized by myopathy (Catteruccia et al, 2015;Mancuso et al, 2013). Indeed, MERRF phenotype is among the mitochondrial syndromes belonging to the nosological group of Primary mitochondrial myopathies (PMM) (de Barcelos et al, 2019) a genetically defined group of disorders leading to defects of oxidative phosphorylation affecting predominantly skeletal muscle (Mancuso et al, 2017). Therefore, mitochondrial myopathy is a common feature of MERRF patients and COX10 KO mice. Lastly, similarities in amino acid and lipid profiles in muscle and plasma, clearly reflecting a common metabolic rewiring, suggest conserved metabolic responses to OXPHOS dysfunction in MERRF and COX10 KO muscle. We have emphasized these clinical-biochemical-metabolic aspects in the Discussion (pages 26-27) and by adding a new Fig. S1E (COX staining in MERRF muscle).
2) In addition, a great amount of work was carried out to describe changes in fat metabolism in Cox10 mice, but its relevance for human disease is unclear. I also suggest the authors to better identify the potential of their findings for the clinical management of the patients and/or for the development of new therapies.
We thank the reviewer for this comment which give us the opportunity to better clarify the relevance of lipid metabolism in mitochondrial diseases. Dysregulation of lipid metabolism has been recently documented in m.3243A>G MELAS patients by the Mootha's group (Sharma et al, 2021). In that study, the authors found that acylcarnitines, β-hydroxy acylcarnitines (BOHCAs), and β-hydroxy fatty acids (BOHFAs) are new validated plasma markers of MELAS that strongly correlate with disease severity. These metabolites have also been identified in our study, in both muscle and plasma. Similar to our interpretation, they suggested that the fatty acid (FA) derivatives increase results from β-oxidation inhibition due to reduced NAD + /NADH. In support of this hypothesis, BOHCAs were elevated in a mouse model of Ndufs4 deficiency (Leong et al, 2012) and accumulation of BOHFAs found in isolated mitochondria treated with respiratory chain inhibitors (Jin et al, 1992), further linking OXPHOS defect and lipid dysmetabolism. Therefore, FA derivatives in muscle and plasma could become valuable disease biomarkers for clinical management and testing of novel therapies for mitochondrial disorders. In addition, these findings justify therapeutic approaches targeting the NAD + /NADH imbalance (Russell et al, 2020). Increased FA levels in plasma (from WAT lipolysis) and impaired FA oxidation in muscle result in accumulation of lipids droplets in both COX10 and MERRF muscle. The ectopic accumulation of lipids in non-adipose tissue is known to be cytotoxic. Specifically, a mouse model of muscle lipid overload, the muscle-specific hLPL-mouse (Levak-Frank et al, 1995), shows skeletal muscle mass reduction, ultrastructural damage, impairment in regeneration, and severe myopathy (Tamilarasan et al, 2012). Similarly, human Lipid Storage Disorders (LSDs) lead to intramuscular lipid accumulation and impaired mitochondrial bioenergetics and progressive myopathy (Debashree et al, 2018). Moreover, lipid accumulation has been described in skeletal muscle of MERRF patients (Munoz-Malaga et al, 2000). On the other hand, recent meta-analysis of clinical data from multiple cohorts of mitochondrial patients revealed that increased energy expenditure (hypermetabolism) is the most common disease feature which prevents accumulation of body fat and contributes to shortened patient's lifespan (Sturm et al, 2023). According to this study OXPHOS-induced ISR activation with increased extracellular secretion of metabokines produces a negative energy balance which reduces adiposity. Therefore, investigating the ISR-mediated starvation like/hypermetabolic response leading to COX10 KO muscle and WAT wasting, can shed some light on the systemic muscle-WAT regulation and help in the development of treatments that preserve adipose store. We have included these considerations in the manuscript Discussion (pages [29][30] 3) Some of the described changes are not entirely new, including those related to mtISR, 1C metabolism, trans-sulfuration, etc... I recommend the authors highlight the novelties compared to previously published data.
We have highlighted the novelties as suggested from the reviewer. Briefly, we have emphasized the identification of a cross talk between defective skeletal muscle and other organs, such as liver and WAT. This inter-organ crosstalk mediated by myokines, leptin, and glucocorticoids signaling leads to increased WAT lipolysis and fatty acids (FA) release, contributing to FA accumulation in muscle and circulation (see also point 2), and upregulation of gluconeogenesis, ureagenesis and ketogenesis in the liver. In this revised manuscript, the reader can better evaluate how this study extends beyond previous work. 4) I am not convinced by the interpretation of some results. For instance, the authors interpret the increased levels of asparagine, glutamate, and their acetylated derivatives (N-acetyl asparagine and Nacetyl glutamate) as an increase in amino acid utilization. However, in my opinion, this could be seen as the result of reduced utilization leading to accumulation.
We agree with the reviewer that increased levels of glutamate and its acetylated derivative (N-acetyl glutamate) could reflect the muscle inability to fully oxidize glutamate. In fact, despite increased glutamate flux through the TCA cycle (as shown by increased alanine, Fig.1C and upregulated transaminases and TCA enzymes, Fig.1I-H), glutamate oxidation by OGDHC (rate-limiting step) is still limited by NAD + matrix pool. This important point has been fully addressed in response to point 2 of reviewer 1. Asparagine is synthetized from glutamate-derived aspartate by Asparagine synthetase (ASNS, Fig. 1A). Accumulation of asparagine/acetyl-asparagine most likely results from increased glutamate oxidation, ASNS upregulation (8.5-fold increase, target of ATF4, new Fig. S4G-H)), and reduced incorporation of asparagine in muscle proteins, due to reduced protein translation. Moreover, we speculate that the rapid conversion of aspartate to asparagine (by ATP-dependent ASNS) facilitates the glutamate to αKG oxidative influx in the TCA cycle by preventing accumulation of OAA, a potent inhibitor of succinate dehydrogenase. We have better clarified these points in manuscript Results (pages 6-7) and Discussion (page 31).

5)
In addition, I find incorrect the authors' interpretation of the data on autophagy. The authors say that "the levels of the autophagy biomarker proteins LC3II and p62 were significantly elevated only at the late stage of myopathy", but this is most like reflecting a block in autophagy, not an activation. However, this can be proven only by using colchicine as a blocker of muscle autophagic flux. As this would be a very time-consuming experiment, I am only suggesting the authors consider and eventually discuss this possibility.
We agree with the reviewer that upregulation of LC3II and p62 at 200 days of age could reflect a block in autophagy which could at least in part be explained by the re-activation of mTORC1 as shown by recovery of Ph-4EBP1 levels (Fig. 3B). In addition, the autophagy machinery may become defective at later disease stages contributing to the increase of LC3II and p62 at 200. This interpretation has been added to the Discussion (pages 28-29).
Specific/minor comments 6) Page 9: the sentence "the variable upregulation among the markers...suggests the operation of complex regulatory processes" sounds too vague.
This sentence has been modified to "the variability in the upregulation of mitochondrial proteins (from 30% to 180%) at 200 days, suggests that some pathways are more upregulated than others (e.g., TCA cycle and mitochondrial import), precluding their use as mitochondrial markers" (page 9).

7)
Page 12: the authors dissect mTORC1 signaling in Cox10 muscles. Can the authors speculate on why mTORC1 signaling is downregulated in this model? Is this the case also in the MERRF patients' muscles?

Referee #3
The authors have convincingly show that OXPHOS-defective muscle adapts metabolically by implementing alternative pathways for rewiring of redox and metabolic homeostasis. The figures are very well organized and full of information: including in all figures a graphical abstract of the metabolic pathway being investigated is very useful. All western blots are neat, as well as the histology. The paper is reach in data and extremely complete. The discussion is very complete.
We would like to thank the reviewer for the positive feedback on the manuscript and for appreciating the quality of the experiments as well as the extent of the work performed. In this revision, we have addressed all referee 3 minor comments as follows.
Minor comment: 1) Page 5 Typo: "reveled" instead of "Revealed" We thank the reviewer for catching this typo: "reveled" was replaced by "revealed" 2) Also the data generated via the different omics analysis should be deposited in a public database (I could not find any GEO number or anything similar in the text). We have provided the GEO numbers for transcriptomics data in Data Availability (page 44).

3)
In the methods, the authors should detail a bit more the omic analysis (eg the statistical approach used, etc) Metabolomics data processing, as described in metabolite profiling section of the methods, was performed using commercially available Agilent metabolomics software. We have added more details for downstream processing to the statistical analyses section (Page 43). We also add statistical details to each figure legend. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) In the main manuscript file, please do the following: -In M&M, please include statement that the informed consent was obtained from all human subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. -Data availability: Please be aware that all deposited datasets should be made freely available upon acceptance, without restriction. Use the following format to report the accession number of your data: The datasets produced in this study are available in the following databases: Please check "Author Guidelines" for more information. https://www.embopress.org/page/journal/17574684/authorguide#availabilityofpublishedmaterial 2) Author Checklist: Please check the list carefully and enter all required information. Currently information about human participants are missing. 3) Funding: Please place funding information under "Acknowledgment". 4) Synopsis: -Synopsis text: Please provide a short standfirst (maximum of 300 characters, including space) as well as 2-5 one sentence bullet points that summarise the paper as a .doc file. Please write the bullet points to summarise the key NEW findings. They should be designed to be complementary to the abstract -i.e. not repeat the same text. We encourage inclusion of key acronyms and quantitative information (maximum of 30 words / bullet point). Please use the passive voice.
-Please check your synopsis text and image before submission with your revised manuscript. Please be aware that in the proof stage minor corrections only are allowed (e.g., typos). 5) For more information: Remove information about Appendix. This space should be used to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc... 6) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF. 7) Please provide a point-by-point letter INCLUDING my comments as well as the reviewer's reports and your detailed responses (as Word file).
I look forward to reading a new revised version of your manuscript as soon as possible.

Zeljko Durdevic
Zeljko Durdevic Editor EMBO Molecular Medicine *** Instructions to submit your revised manuscript *** *** PLEASE NOTE *** As part of the EMBO Publications transparent editorial process initiative (see our Editorial at https://www.embopress.org/doi/pdf/10.1002/emmm.201000094), EMBO Molecular Medicine will publish online a Review Process File to accompany accepted manuscripts.
In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. If you do NOT want this file to be published, please inform the editorial office at contact@embomolmed.org.
When submitting your revised manuscript, please include: 1) a .docx formatted version of the manuscript text (including Figure legends and tables) 2) Separate figure files* 3) supplemental information as Expanded View and/or Appendix. Please carefully check the authors guidelines for formatting Expanded view and Appendix figures and tables at https://www.embopress.org/page/journal/17574684/authorguide#expandedview 4) a letter INCLUDING the reviewer's reports and your detailed responses to their comments (as Word file).
5) The paper explained: EMBO Molecular Medicine articles are accompanied by a summary of the articles to emphasize the major findings in the paper and their medical implications for the non-specialist reader. Please provide a draft summary of your article highlighting -the medical issue you are addressing, -the results obtained and -their clinical impact. This may be edited to ensure that readers understand the significance and context of the research. Please refer to any of our published articles for an example. 6) For more information: There is space at the end of each article to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc... 7) Author contributions: the contribution of every author must be detailed in a separate section. 8) EMBO Molecular Medicine now requires a complete author checklist (https://www.embopress.org/page/journal/17574684/authorguide) to be submitted with all revised manuscripts. Please use the checklist as guideline for the sort of information we need WITHIN the manuscript. The checklist should only be filled with page numbers were the information can be found. This is particularly important for animal reporting, antibody dilutions (missing) and exact values and n that should be indicted instead of a range. 9) Every published paper now includes a 'Synopsis' to further enhance discoverability. Synopses are displayed on the journal webpage and are freely accessible to all readers. They include a short stand first (maximum of 300 characters, including space) as well as 2-5 one sentence bullet points that summarise the paper. Please write the bullet points to summarise the key NEW findings. They should be designed to be complementary to the abstract -i.e. not repeat the same text. We encourage inclusion of key acronyms and quantitative information (maximum of 30 words / bullet point). Please use the passive voice. Please attach these in a separate file or send them by email, we will incorporate them accordingly.
You are also welcome to suggest a striking image or visual abstract to illustrate your article. If you do please provide a jpeg file 550 px-wide x 400-px high.
10) A Conflict of Interest statement should be provided in the main text 11) Please note that we now mandate that all corresponding authors list an ORCID digital identifier. This takes <90 seconds to complete. We encourage all authors to supply an ORCID identifier, which will be linked to their name for unambiguous name identification.
Currently, our records indicate that the ORCID for your account is 0000-0001-5247-3238.
Please click the link below to modify this ORCID: Link Not Available 12) The system will prompt you to fill in your funding and payment information. This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance. This quote takes into account any reduction or fee waivers that you may be eligible for. Authors do not need to pay any fees before their manuscript is accepted and transferred to our publisher.

*Additional important information regarding Figures
Each figure should be given in a separate file and should have the following resolution: Graphs 800-1,200 DPI Photos 400-800 DPI Colour (only CMYK) 300-400 DPI" Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: Editor 1) In the main manuscript file, please do the following: In M&M, please include statement that the informed consent was obtained from all human subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
The statement is now included in the Materials and Methods section.
Data availability: Please be aware that all deposited datasets should be made freely available upon acceptance, without restriction. Use the following format to report the accession number of your data: The datasets produced in this study are available in the following databases: Information regarding the dataset is available without restriction.
2) Author Checklist: Please check the list carefully and enter all required information. Currently information about human participants are missing.
Human participant information has been added to Materials and Methods and Table S1.
The funding information is now underneath the "Acknowledgment" section.
10th May 2023 2nd Authors' Response to Reviewers 4) Synopsis: Synopsis text: Please provide a short standfirst (maximum of 300 characters, including space) as well as 2-5 one sentence bullet points that summarise the paper as a .doc file. Please write the bullet points to summarise the key NEW findings. They should be designed to be complementary to the abstract -i.e. not repeat the same text. We encourage inclusion of key acronyms and quantitative information (maximum of 30 words / bullet point). Please use the passive voice.
-Please check your synopsis text and image before submission with your revised manuscript. Please be aware that in the proof stage minor corrections only are allowed (e.g., typos). 5) For more information: Remove information about Appendix. This space should be used to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc...
The appendix was removed from "For more information." 6) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF.
We agree with the publication of our anonymous referee reports, point-by-point response, and correspondence regarding the manuscript. We are pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.
Please read below for additional IMPORTANT information regarding your article, its publication and the production process.
Congratulations on your interesting work,

Zeljko Durdevic
Zeljko Durdevic Editor EMBO Molecular Medicine Follow us on Twitter @EmboMolMed Sign up for eTOCs at embopress.org/alertsfeeds *** *** *** IMPORTANT INFORMATION *** *** *** SPEED OF PUBLICATION The journal aims for rapid publication of papers, using using the advance online publication "Early View" to expedite the process: A properly copy-edited and formatted version will be published as "Early View" after the proofs have been corrected. Please help the Editors and publisher avoid delays by providing e-mail address(es), telephone and fax numbers at which author(s) can be contacted.
Should you be planning a Press Release on your article, please get in contact with embomolmed@wiley.com as early as possible, in order to coordinate publication and release dates.

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