MIR-206 regulates connexin43 expression during skeletal muscle development

Abstract

Skeletal myoblast fusion in vitro requires the expression of connexin43 (Cx43) gap junction channels. However, gap junctions are rapidly downregulated after the initiation of myoblast fusion in vitro and in vivo. In this study we show that this downregulation is accomplished by two related microRNAs, miR-206 and miR-1, that inhibit the expression of Cx43 protein during myoblast differentiation without altering Cx43 mRNA levels. Cx43 mRNA contains two binding sites for miR-206/miR-1 in its 3'-untranslated region, both of which are required for efficient downregulation. While it has been demonstrated before that miR-1 is involved in myogenesis, in this work we show that miR-206 is also upregulated during perinatal skeletal muscle development in mice in vivo and that both miR-1 and miR-206 downregulate Cx43 expression during myoblast fusion in vitro. Proper development of singly innervated muscle fibers requires muscle contraction and NMJ terminal selection and it is hypothesized that prolonged electrical coupling via gap junctions may be detrimental to this process. This work details the mechanism by which initial downregulation of Cx43 occurs during myogenesis and highlights the tight control mechanisms that are utilized for the regulation of gap junctions during differentiation and development.

Figure 1

Conserved predicted miR-206/miR-1 binding sites in Cx43 3′-UTR. Outlined boxes demark the region that is perfectly complimentary to miR-206/miR-1 and is most important for microRNA:mRNA interaction (28). This region is 2 nt larger than the ‘seed’ region used by Lewis et al. (25) to find potential miR-206/miR-1 targets.

Figure 2

Tissue distribution of miR-206. RNase protection assay to detect miR-206 was performed on 5 μg of total RNA from various tissue samples. 5 μg of total RNA was subjected to RPA. 1 μg of total RNA was used to detect U6 snRNA by northern blotting.

Figure 3

Regulation of Cx43 by miR-206/1 in vitro. (A) HeLa cells were mock transfected or transfected with cDNA for Cx43 in addition to empty pSUPER plasmid or pSUPER 206pri. After 48 h Western blots and RNA analysis was performed as described in the Materials and Methods section. (B) Diagram of luciferase reporter construct with putative miR-206 binding sites and mutations. Binding sites were mutated in pcDNA Luc 43-3′-UTR and the 5′-UTR was individually cloned into each mutant to make the constructs containing both the 5′- and 3′-UTRs. (C) Effect of binding site mutants on luciferase expression in the presence of miR-206, miR-1 or both miR-206 and miR-1. HeLa cells were transfected with pcDNA Luc constructs along with either pSUPER or pSUPER 206pri and analysed 24 h later. Data is presented as relative firefly luciferase units (RLUs). Relative firefly luciferase values were determined by a ratio of firefly to renilla luciferase with the negative control set at 1. Error bars—standard deviation; 0 ≥ 9. *A student's t-test performed between microRNA transfected samples and the negative control yielded a P-value < 0.05 (actual P-value < 0.01).

Figure 4

miR-206 regulates Cx43 during myoblast fusion and skeletal muscle development. Total RNA and total lysates were isolated from skeletal muscle from Swiss-Webster mice at E16 (−2days), day 1, 3, or 5 after birth. RNase protection assay was performed against miR-206 (500 ng total RNA) or Cx43 mRNA and β-actin (10 μg total RNA). Western blots were performed on 10 μg total protein as described in the Materials and Methods section.

Figure 5

Connexin43 is downregulated during myoblast differentiation. Arrows denote positive immunostaining for Cx43 at membrane appositions. Cells were incubated with 1:100 dilution of anti-connexin43 rabbit polyclonal antibody (Sigma) and 1 μg of anti-myogenin mouse monoclonal antibody (Sigma) overnight at 4°C in 5% HS in PBS. Dishes were examined by fluorescence and phase contrast microscopy using FITC, rhodamine and DAPI filters.

Figure 6

(A) C2C12 cells were grown in growth medium and induced to differentiate at ∼70% confluency by switching to DMEM supplemented with 5% HS and 1× ITS. Cells were harvested prior to induction (t = 0) and at 24 h intervals thereafter. miR-206 was detected by RNase protection assay on 1 μg of total RNA. Cx43 mRNA and β-actin mRNA were detected by RNase protection assay on 10 μg of total RNA. Western blots against Cx43, myogenin and α-tubulin were performed on 10 μg of total protein. (B) C2C12 cells were grown in growth medium and induced to differentiate at ∼50% confluency by switching to DMEM supplemented with 5% HS and 1× ITS. Cells were harvested prior to induction (t = 0) or transfected with negative control microRNA inhibitor, anti-miR-206, anti-miR-1 or anti-miR-206 and anti-miR-1 and induced to differentiate. Transfected cells were harvested 48 h later. Western blots against Cx43 and α-tubulin were performed on 10 μg of total protein.