Dystrophin Dp71ab is monoclonally expressed in human satellite cells and enhances proliferation of myoblast cells

Abstract

Dystrophin Dp71 is the smallest isoform of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 has also been shown to have roles in various cellular processes. Stem cell-based therapy may be effective in treating DMD, but the inability to generate a sufficient number of stem cells remains a significant obstacle. Although Dp71 is comprised of many variants, Dp71 in satellite cells has not yet been studied. Here, the full-length Dp71 consisting of 18 exons from exons G1 to 79 was amplified by reverse transcription-PCR from total RNA of human satellite cells. The amplified product showed deletion of both exons 71 and 78 in all sequenced clones, indicating monoclonal expression of Dp71ab. Western blotting of the satellite cell lysate showed a band corresponding to over-expressed Dp71ab. Transfection of a plasmid expressing Dp71ab into human myoblasts significantly enhanced cell proliferation when compared to the cells transfected with the mock plasmid. However, transfection of the Dp71 expression plasmid encoding all 18 exons did not enhance myoblast proliferation. These findings indicated that Dp71ab, but not Dp71, is a molecular enhancer of myoblast proliferation and that transfection with Dp71ab may generate a high yield of stem cells for DMD treatment.

Figure 1

RT-PCR amplification of marker gene mRNAs in human satellite cells. Electropherograms of amplified products are shown. Fragments of the PAX3, PAX7, Myf5, MyoD, and myogenin genes were amplified from total RNA of satellite cells (S) and skeletal muscle (M). In the satellite cells, expected size product was obtained at different density from PAX3, PAX7, Myf5, and myogenin, but not from MyoD. In contrast, all 5 fragments were amplified in skeletal muscle (M). The expected size of each amplified product is shown on the right margin of each electropherogram. Mk refers to the 100 bp ladder size marker. Full gel-images are available in Supplementary Figs. 1, 2 and 3.

Figure 2

Dp71ab transcript in human satellite cells. (A) Schematic description of Dp71 mRNA. The exon structure of Dp71 is shown. Boxes indicate exons. The shaded exon indicates the Dp71-specific exon1 (G1). Numbers in boxes indicate DMD exon number. Brackets indicate the amplified fragment. Numbers over the bracket indicate exon number of amplified fragments. (B) Amplified product of Dp71 transcript: the 5′ end fragment and full-length Dp71 transcript were RT-PCR amplified from total RNA of human satellite cells. Electropherograms of amplified products are shown. The amplified fragment from exon G1 to exon 66 was of the expected size (lane S in the left panel). Amplification of full-length Dp71, from exon G1 to exon 79, yielded a single product of expected size (lane S in the right panel). In AGS cells, in contrast, both fragments were not obtained (AGS lane in left and right panels). Mk refers to size markers. (C) Schematic description of Dp71ab. The exon structure of Dp71ab is shown as in panel (A). Boxes and boxes over the bar indicate exons and skipped exons, respectively. Partial sequences of the junctions between exons 70 and 72, and between exons 77 and 79, are shown below these junctions. Full gel-images are available in Supplementary Figs. 4 and 5.

Figure 3

Western blot assay of Dp71ab in human satellite cells. Lysates of human satellite cells and AGS cells transfected with Dp71 and Dp71ab-expression plasmids were analyzed by Western blotting using antibodies ab12577 and 7A10, directed against different domains of the C-terminal region of dystrophin. The immunoblotting results are shown. Binding of ab12577 (A) and 7A10 (B) antibodies to proteins in AGS cells expressing Dp71 (Dp71) and Dp71ab (Dp71ab) was disclosed. These antibodies also bound to a protein in human satellite cells (S) located at the same position as artificially expressed Dp71ab. The blots were reacted with antibody to β-actin as a loading control (bottom). The left margins show molecular size markers. Full gel-images are available in Supplementary Figs. 6, 7, and 8.

Figure 4

Myoblast cell proliferation. (A) CCK-8 assay of the proliferation of myoblasts transfected with the mock-, Dp71- and Dp71ab- plasmids at 72 h (left). The absorbance of cells expressing Dp71ab was significantly higher than the absorbance of the Dp71- or mock-transfected cells (left). Absorbance is expressed as the mean ± standard error of the mean (SEM) of three independent experiments. Time course showing that cell absorbance began to increase significantly from baseline at 48 h (right). *p < 0.05, **p < 0.01. (B) Time course of numbers of myoblasts transfected with the mock-, Dp71- and Dp71ab-plasmids from 0 to 72 h. Cells were counted in a microscopic field of each well at 0, 24, 48 and 72 h. Data are expressed as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001.