1. Problem or question being addressed
It is a well-accepted notion that heart function reflects on skeletal muscle homeostasis and performance1. However, a large body of evidence is emerging showing that skeletal muscle operates as an endocrine organ2. In fact, muscle-derived cytokines and other peptides (i.e. myokines) are released by muscle fibers and exert paracrine, autocrine or endocrine effects, modulating the physiology of distal organs3. Among the molecules released by skeletal muscles, there are microRNAs, small oligonucleotides which control the expression of a variety of transcripts, at post-transcriptional level, and are secreted into the blood where they are stable. There is a subset of miRs that are muscle specific and are named myomiRs4. Among these, miR-206 is specifically expressed in skeletal, but not in cardiac muscle. Its expression is activated during muscle differentiation both in vitro5 and in vivo6,7, and it has been reported to play an essential role in ameliorating and delaying skeletal muscle atrophy in Duchenne muscular dystrophy (DMD)8 and Amyotrophic lateral sclerosis (ALS)9, by improving skeletal muscle fibers and NMJ regeneration8,9. In line with this, miR-206 levels have been found increased in blood of DMD and ALS patients, correlating with the severity of the disorders. For this reason, miR-206 has been proposed as a biomarker in both the pathological conditions10,11. The increased serum levels in patients of muscular and neuro/muscular disorders, perfectly fits with the presence in miR-206 sequence of the recently described releasing motif (EXOmotif)12, which leads to the emission in the bloodstream of microRNAs through extracellular vesicles (ECVs). Although several targets of miR-206 were reported, all of them are located in skeletal muscle5,13–17, while extra skeletal muscle targets remain largely unknow.
2. Rationale for your approach
The evidence describing the presence of miR-206 in the bloodstream prompted us to investigate extra skeletal muscle target of miR-206. For this purpose, we took advantage of the previously described ATG7-/- model18, which exhibits a block of autophagy specifically in the skeletal muscle. This block of autophagy leads to skeletal muscle atrophy, weakness, inflammation and NMJ destabilization18. The combination of block of autophagy, which has been reported to increase ECVs release19,20, and the neuro/muscular impairment, led the skeletal muscles of these mice to release a higher number of ECVs enriched in miR-206. Consistently, circulating miR-206 increased in ATG7-/- compared to controls. Interestingly, miR-206 was mainly taken up by the myocardium (Fig. 1). This evidence encourages us to study potential effects of the skeletal muscle specific miRNA in this district.
3. Details of suggested approach
With the purpose to investigate the effects of the ectopic presence of miR-206 in the heart, we plan to combine in vivo cardiac functional assays (i.e. echocardiography, ECG-telemetry) to ex vivo analyses (i.e. confocal IF, multiphoton imaging, histological and molecular/biochemical assays) on hearts harvested from ATG7-/- male mice and littermate controls. If ATG-/- mice will show morphological or functional cardiac impairment, we plan to administer to C57BL/6J male mice in vitro-collected ECVs loaded with synthetic miR-206 oligos via caudal vein injection. In this way, we will be able to isolate the effect of exogenous miR-206 on the heart. Moreover, we plan to predict putative miR-206 targets through bioinformatic analysis, searching not only among possible targets within cardiac muscle cells, but in addition among other cell types that compose cardiac interstitium (i.e. fibroblasts, sympathetic and parasympathetic neuronal fibers, endothelial and inflammatory cells). Indeed, other cell types have been shown to participate to cardiac remodeling21,22 and to arrhythmic propensity23,24. The targeting will be then confirmed through in vitro assays (i.e. luciferase and molecular/biochemical assays) and, possibly, in vivo in ATG7-/-, as well as miR-206-treated C57BL/6J, mouse models. Finally, the ultimate step will be treating ATG7-/- mice with GW4869 in order to rescue the cardiac phenotype. Indeed, GW4869 is a neutral, non-competitive inhibitor of sphingomyelinase which blocks the budding in multivesicular bodies25 and has been successfully used to reduce the secretion of ECVs in vivo26. Thus, by inhibiting the release of miR-206-containing ECVs from the skeletal muscle, we will hopefully demonstrate their causative role in the heart.
4. How it will affect the broader field
We aim to identify miR-206 as a player in the ‘muscle-to-heart’ communication. Such interorgan interaction may explain the cardiac dysfunction secondary to skeletal muscle defects, a common feature of neurodegenerative disorders characterized by increased miR-206 plasma levels such as DMD, ALS and unhealthy aging.
5. References
1. von Haehling S, Ebner N, dos Santos MR, Springer J, Anker SD. Muscle wasting and cachexia in heart failure: mechanisms and therapies. Nat Publ Gr [Internet]. 2017 [cited 2021 Jul 27];14. Available from: www.nature.com/nrcardio
2. Hoffmann C, Weigert C. Skeletal Muscle as an Endocrine Organ: The Role of Myokines in Exercise Adaptations. Cold Spring Harb Perspect Med [Internet]. 2017 [cited 2021 Jul 27];7. Available from: /pmc/articles/PMC5666622/
3. F N, T R, B T, AC R, CA D, F H. Proteomic identification of secreted proteins from human skeletal muscle cells and expression in response to strength training. Am J Physiol Endocrinol Metab [Internet]. 2011 [cited 2021 Jul 27];301. Available from: https://pubmed.ncbi.nlm.nih.gov/21828336/
4. J S, N K, S B. Circulating myomiRs: a new class of biomarkers to monitor skeletal muscle in physiology and medicine. J Cachexia Sarcopenia Muscle [Internet]. 2018 [cited 2021 Jul 27];9:20–27. Available from: https://pubmed.ncbi.nlm.nih.gov/29193905/
5. Hak KK, Yong SL, Sivaprasad U, Malhotra A, Dutta A. Muscle-specific microRNA miR-206 promotes muscle differentiation. J Cell Biol [Internet]. 2006 [cited 2022 Sep 11];174:677. Available from: /pmc/articles/PMC2064311/
6. Takada S, Berezikov E, Yamashita Y, Lagos-Quintana M, Kloosterman WP, Enomoto M, Hatanaka H, Fujiwara SI, Watanabe H, Soda M, Choi YL, Plasterk RHA, Cuppen E, Mano H. Mouse microRNA profiles determined with a new and sensitive cloning method. Nucleic Acids Res [Internet]. 2006 [cited 2022 Sep 11];34:e115. Available from: /pmc/articles/PMC1635289/
7. Sweetman D, Goljanek K, Rathjen T, Oustanina S, Braun T, Dalmay T, Münsterberg A. Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133. Dev Biol. 2008;321:491–499.
8. Liu N, Williams AH, Maxeiner JM, Bezprozvannaya S, Shelton JM, Richardson JA, Bassel-Duby R, Olson EN. microRNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice. J Clin Invest [Internet]. 2012 [cited 2022 Aug 24];122:2054. Available from: /pmc/articles/PMC3366415/
9. Williams AH, Valdez G, Moresi V, Qi X, McAnally J, Elliott JL, Bassel-Duby R, Sanes JR, Olson EN. MicroRNA-206 Delays ALS Progression and Promotes Regeneration of Neuromuscular Synapses in Mice. Science [Internet]. 2009 [cited 2022 Aug 24];326:1549. Available from: /pmc/articles/PMC2796560/
10. Toivonen JM, Manzano R, Oliván S, Zaragoza P, García-Redondo A, Osta R. MicroRNA-206: A Potential Circulating Biomarker Candidate for Amyotrophic Lateral Sclerosis. PLoS One [Internet]. 2014 [cited 2022 Sep 8];9. Available from: /pmc/articles/PMC3930686/
11. Hu J, Kong M, Ye Y, Hong S, Cheng L, Jiang L. Serum miR-206 and other muscle-specific microRNAs as non-invasive biomarkers for Duchenne muscular dystrophy. J Neurochem [Internet]. 2014 [cited 2022 Sep 11];129:877–883. Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/jnc.12662
12. Garcia-Martin R, Wang G, Brandão BB, Zanotto TM, Shah S, Kumar Patel S, Schilling B, Kahn CR. MicroRNA sequence codes for small extracellular vesicle release and cellular retention. Nature [Internet]. 2022 [cited 2022 Aug 23];601:446. Available from: /pmc/articles/PMC9035265/
13. Chen JF, Tao Y, Li J, Deng Z, Yan Z, Xiao X, Wang DZ. microRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7. J Cell Biol [Internet]. 2010 [cited 2022 Sep 11];190:867. Available from: /pmc/articles/PMC2935565/
14. Hirai H, Verma M, Watanabe S, Tastad C, Asakura Y, Asakura A. MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3. J Cell Biol [Internet]. 2010 [cited 2022 Sep 11];191:347. Available from: /pmc/articles/PMC2958479/
15. Anderson C, Catoe H, Werner R. MIR-206 regulates connexin43 expression during skeletal muscle development. Nucleic Acids Res [Internet]. 2006 [cited 2022 Sep 11];34:5863. Available from: /pmc/articles/PMC1635318/
16. Rosenberg MI, Georges SA, Asawachaicharn A, Analau E, Tapscott SJ. MyoD inhibits Fstl1 and Utrn expression by inducing transcription of miR-206. J Cell Biol [Internet]. 2006 [cited 2022 Sep 11];175:77. Available from: /pmc/articles/PMC2064500/
17. Winbanks CE, Wang B, Beyer C, Koh P, White L, Kantharidis P, Gregorevic P. TGF-β Regulates miR-206 and miR-29 to Control Myogenic Differentiation through Regulation of HDAC4. J Biol Chem [Internet]. 2011 [cited 2022 Sep 11];286:13805. Available from: /pmc/articles/PMC3077581/
18. Carnio S, LoVerso F, Baraibar MA, Longa E, Khan MM, Maffei M, Reischl M, Canepari M, Loefler S, Kern H, Blaauw B, Friguet B, Bottinelli R, Rudolf R, Sandri M. Autophagy Impairment in Muscle Induces Neuromuscular Junction Degeneration and Precocious Aging. Cell Rep [Internet]. 2014 [cited 2021 Jun 17];8:1509–1521. Available from: /pmc/articles/PMC4534571/
19. Fader CM, Sánchez D, Furlán M, Colombo MI. Induction of Autophagy Promotes Fusion of Multivesicular Bodies with Autophagic Vacuoles in K562 Cells. Traffic [Internet]. 2008 [cited 2022 Sep 11];9:230–250. Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1600-0854.2007.00677.x
20. Minakaki G, Menges S, Kittel A, Emmanouilidou E, Schaeffner I, Barkovits K, Bergmann A, Rockenstein E, Adame A, Marxreiter F, Mollenhauer B, Galasko D, Buzás EI, Schlötzer-Schrehardt U, Marcus K, Xiang W, Lie DC, Vekrellis K, Masliah E, Winkler J, Klucken J. Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype. Autophagy [Internet]. 2018 [cited 2022 Sep 11];14:98. Available from: /pmc/articles/PMC5846507/
21. Scalco A, Liboni C, Angioni R, Di Bona A, Albiero M, Bertoldi N, Fadini GP, Thiene G, Chelko SP, Basso C, Viola A, Mongillo M, Zaglia T. Arrhythmogenic Cardiomyopathy Is a Multicellular Disease Affecting Cardiac and Bone Marrow Mesenchymal Stromal Cells. J Clin Med [Internet]. 2021 [cited 2022 Sep 28];10. Available from: https://pubmed.ncbi.nlm.nih.gov/33925921/
22. Pianca N, Di Bona A, Lazzeri E, Costantini I, Franzoso M, Prando V, Armani A, Rizzo S, Fedrigo M, Angelini A, Basso C, Pavone FS, Rubart M, Sacconi L, Zaglia T, Mongillo M. Cardiac sympathetic innervation network shapes the myocardium by locally controlling cardiomyocyte size through the cellular proteolytic machinery. J Physiol. 2019;597:3639–3656.
23. Gardner RT, Ripplinger CM, Myles RC, Habecker BA. Molecular Mechanisms of Sympathetic Remodeling and Arrhythmias. Circ Arrhythm Electrophysiol [Internet]. 2016 [cited 2022 Sep 2];9:e001359. Available from: /pmc/articles/PMC4730917/
24. Scalco A, Moro N, Mongillo M, Zaglia T. Neurohumoral Cardiac Regulation: Optogenetics Gets Into the Groove. Front Physiol [Internet]. 2021 [cited 2022 Apr 13];12. Available from: https://pubmed.ncbi.nlm.nih.gov/34531763/
25. Essandoh K, Yang L, Wang X, Huang W, Qin D, Hao J, Wang Y, Zingarelli B, Peng T, Fan GC. Blockade of Exosome Generation with GW4869 Dampens the Sepsis-Induced Inflammation and Cardiac Dysfunction. Biochim Biophys Acta [Internet]. 2015 [cited 2022 Aug 16];1852:2362. Available from: /pmc/articles/PMC4581992/
26. Dinkins MB, Dasgupta S, Wang G, Zhu G, Bieberich E. Exosome Reduction In Vivo Is Associated with Lower Amyloid Plaque Load in the 5XFAD Mouse Model of Alzheimer’s Disease. Neurobiol Aging [Internet]. 2014 [cited 2022 Aug 16];35:1792. Available from: /pmc/articles/PMC4035236/