miR-378a influences vascularization in skeletal muscles

Abstract

Aims MicroRNA-378a, highly expressed in skeletal muscles, was demonstrated to affect myoblasts differentiation and to promote tumour angiogenesis. We hypothesized that miR-378a could play a pro-angiogenic role in skeletal muscle and may be involved in regeneration after ischaemic injury in mice. Methods and results Silencing of miR-378a in murine C2C12 myoblasts did not affect differentiation but impaired their secretory angiogenic potential towards endothelial cells. miR-378a knockout (miR-378a−/−) in mice resulted in a decreased number of CD31-positive blood vessels and arterioles in gastrocnemius muscle. In addition, diminished endothelial sprouting from miR-378a−/− aortic rings was shown. Interestingly, although fibroblast growth factor 1 (Fgf1) expression was decreased in miR-378a−/− muscles, this growth factor did not mediate the angiogenic effects exerted by miR-378a. In vivo, miR-378a knockout did not affect the revascularization of the ischaemic muscles in both normo- and hyperglycaemic mice subjected to femoral artery ligation (FAL). No difference in regenerating muscle fibres was detected between miR-378a−/− and miR-378+/+ mice. miR-378a expression temporarily declined in ischaemic skeletal muscles of miR-378+/+ mice already on Day 3 after FAL. At the same time, in the plasma, the level of miR-378a-3p was enhanced. Similar elevation of miR-378a-3p was reported in the plasma of patients with intermittent claudication in comparison to healthy donors. Local adeno-associated viral vectors-based miR-378a overexpression was enough to improve the revascularization of the ischaemic limb of wild-type mice on Day 7 after FAL, what was not reported after systemic delivery of vectors. In addition, the number of infiltrating CD45+ cells and macrophages (CD45+ CD11b+ F4/80+ Ly6G−) was higher in the ischaemic muscles of miR-378a−/− mice, suggesting an anti-inflammatory action of miR-378a. Conclusions Data indicate miR-378a role in the pro-angiogenic effect of myoblasts and vascularization of skeletal muscle. After the ischaemic insult, the anti-angiogenic effect of miR-378a deficiency might be compensated by enhanced inflammation.

Lack of miR-378 attenuates muscular dystrophy in mdx mice

“…Our initial results demonstrate decreased levels of both mature strands of miR-378 in muscles concomitantly with the elevation of miR-378 in the serum of dystrophic animals, which is in accordance with already published data concerning also DMD patients' samples (18,(21)(22)(23). Moreover, similar regulation of miR-378 in muscles and serum was revealed by us recently in experimental hind limb ischemia, together with the elevated level of miR-378-3p in the plasma of patients with intermittent claudication (17). The differential expression pattern of miR-378 could be explained by the release of miR-378 from the damaged myofibers; however, the active secretion of miR-378 exhibiting paracrine function cannot be ruled out.…”

“…Here, we demonstrate FGF1 as one of the possible mediators of changes driven by the lack of miR-378. In accordance with this data, we have recently revealed decreased expression of FGF1 in the C2C12 myoblast cells in which miR-378 was silenced (17). Strikingly, according to the miRWalk database, FGF1 is one of the predicted -yet not validated -targets of miR-378-5p, suggesting that decreased FGF1 is due to indirect regulation of FGF1 by miR-378.…”

“…Immunofluorescent staining of different muscle fiber types was performed as described by Dyar et al Immunofluorescent staining of Pax7 or CD31/α-SMA was performed on the gastrocnemius frozen sections as described previously (17,48). The stainings were visualized under Nikon Eclipse Ti microscope or Zeiss LSM-880 meta laser scanning confocal microscope and analyzed by the investigator blind to the mice genotype using ImageJ software (NIH).…”

“…Muscle regeneration is accomplished by the formation of new blood vessels, and miR-378 was demonstrated to contribute to this process -for example, by regulating Vegfa expression (17,36). Nonetheless, although VEGFA was markedly decreased in mdx versus WT animals, indicating possible alterations of angiogenesis, no differences in mice additionally lacking miR-378 were observed (Supplemental Figure 4A).…”

“…Among miRNAs, miR-378a (miR-378) might be of particular importance in muscular dystrophy, and it was already reported to be highly abundant in muscles in comparison with other tissues (17,18). Two mature strands, miR-378-3p and miR-378-5p, originate from the first intron of the peroxisome proliferator-activated receptor-γ-coactivator 1 β gene (Ppargc1b), encoding PGC-1β, a key regulator of energy metabolism (19).…”