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.
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