Human Dystrophin Dp71ab Enhances the Proliferation of Myoblasts Across Species But Not Human Nonmyoblast Cells

Abstract

Dystrophin Dp71 is an isoform produced from the Dp71 promoter in intron 62 of the DMD gene, mutations in which cause Duchenne muscular dystrophy. Dp71 is involved in various cellular processes and comprises more than 10 isoforms produced by alternative splicing. Dp71ab, in which both exons 71 and 78 are deleted, has a hydrophobic C-terminus that is hydrophilic in Dp71. Therefore, Dp71ab is believed to have different roles from Dp71. Previously, we reported that Dp71ab enhanced the proliferation of human myoblasts. Here, we further characterized Dp71ab, focusing on the activation of cell proliferation. Dp71ab increased the proliferation of immortalized human myoblasts in a dose-dependent manner. In contrast, Dp71 suppressed proliferation in a dose-dependent manner. Consistent with these opposite effects, eGFP-tagged Dp71ab and mCherry-tagged Dp71 showed different cellular distributions, with Dp71ab mostly in the nucleus. Notably, human Dp71ab enhanced the proliferation of rat and mouse myoblasts. Despite these findings, human Dp71ab did not enhance the proliferation of human nonmyoblast cells, including rhabdomyosarcoma cells. We concluded that Dp71ab is a myoblast-specific proliferation enhancer. In further studies, Dp71ab will be employed for the expansion of myoblasts in clinical settings.

Keywords: DMD; Dp71; Dp71ab; cell proliferation; dystrophin; isoform; myoblast.

FIGURE 1

Characterization of human immortalied myoblasts. (A). Expression of myogenic genes. The mRNA levels of the PAX3, PAX7, Myf5, MyoD, and myogenin genes in human immortalized myoblasts were assessed by RT-PCR using GAPDH mRNA as a control. Electropherograms of the amplified products are shown with size markers on the left (Mk). All amplifications except that of PAX7 resulted in a band at the expected size (Mb) as amplified products from skeletal muscles (Sk). (B). Differentiation of human immortalized myoblasts. Human immortalized myoblasts were cultured in two types of differentiation (HSM and TISM) and one type of growth (GM) media for 10 days and analyzed. Merged results of immunostaining of myosin heavy chain and Hoechst 33342 staining are shown. In differentiated cells, spindle-shaped and multinuclear cells were observed microscopically (HSM, TISM), whereas they were not observed in the cells cultured in growth medium (GM). Myosin heavy chain signals were observed as green signals in the cells cultured in differentiation medium (TISM, HSM) but not in the cells in growth medium (GM). Squares marked by red lines in the upper panels were enlarged and are shown in the lower panels. Scale bars = 100 μm. (C). The fusion index. The fusion index (%) was calculated for each medium and is shown as bars.

FIGURE 2

Dp71ab, but not Dp71, enhances the proliferation of myoblasts. Myoblasts were transfected with Dp71ab and Dp71 plasmids at different doses and cultured for 72 h. Cell proliferation was assessed by the CCK-8 assay. The absorbance was determined in each well. Data are expressed as the mean ± SEM of three independent experiments. The absorbance increased linearly at doses from 0 to 300 ng of the Dp71ab plasmid (A) left. The EC₅₀ was calculated as 100 ng (A) right. In contrast, Dp71 decreased the absorbance linearly from 100 to 500 ng of the plasmid (B) left. The IC₅₀ was calculated as 260 ng (B) right. Glycine, glutamine, alanine and alpha-ketoglutarate were examined for their effect on myoblast proliferation. The addition of glycine (green), glutamine (brown) and alanine (yellow), but not alpha-ketoglutarate (gray), in the culture medium increased the absorbance significantly compared with that of the nontreated cells (blue) (p < 0.05 and p < 0.001 after 48 and 72 h, respectively) (C). The addition of glycine to the culture medium of the Dp71ab-transfected myoblasts (gray) did not increase the absorbance compared to those in the Dp71ab-transfected (red) and glycine-supplemented (green) cells. However, all treatments increased the absorbance significantly compared to that of the nontreated cells (blue) (D). * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

FIGURE 3

Cellular localization of Dp71ab and Dp71 in myoblasts. eGFP-tagged Dp71ab and mCherry-tagged Dp71 were expressed in myoblasts, and fluorescence images of red mCherry-Dp71 and green eGFP-Dp71a were obtained at 24, 48 and 72 h. The cellular localization of the expressed proteins was determined by visualizing the nucleus with Hoechst 33342 staining and classified into three groups: cytoplasm (C), cytoplasm + nucleus (cytoplasm/nucleus) (C/N) and nucleus (N). Representative images of the three groups of cellular localization (left of each group) and sagittal section (right of each group) results are shown (A). In the cytoplasmic group, mCherry-Dp71 was present in the cytoplasm, leaving the nucleus open (left). The distribution of signals along the bar in the top figure is shown (bottom). A red signal was present, avoiding the nucleus (blue signal). In the nuclear group, green eGFP-tagged Dp71ab was present in the nucleus (middle). In the cytoplasm/nucleus group, mCherry-Dp71 was present in both the cytosol and nucleus (right). The percentages of cells classified into the three groups were calculated at 24, 48 and 72 h and are shown as bars (B). At 24 h, 94.9% of the myoblasts transfected with Dp71 belonged to the cytoplasm group. In contrast, 82.5% of the myoblasts transfected with Dp71ab belonged to the cytoplasm/nucleus group. At 72 h, 86.3% of the myoblasts transfected with Dp71 belonged to the cytoplasm/nucleus group. In contrast, 98.4% of the myoblasts transfected with Dp71ab belonged to the nucleus group. The percentage of the major group at 24 h showed significant changes at 48 and 72 h * = p < 0.001 compared with the major group at 24 h.

FIGURE 4

Enhancement of L6 rat and C2C12 mouse myoblast proliferation by Dp71ab. L6 rat and C2C12 mouse myoblasts were transfected with Dp71ab, Dp71 and mock plasmids and incubated for 72 h. As expected, Dp71ab (red) significantly increased the number of L6 and C2C12 cells compared to that of the Dp71 (green) or mock (blue) transfected cells (134 and 132%, respectively). Human Dp71ab enhanced the proliferation of myoblasts across species. * = p < 0.05, ** = p < 0.001.

FIGURE 5

No enhancement of proliferation of human nonmyoblast cells by Dp71ab. Dp71ab, Dp71 and mock vectors were transfected into eight human cell lines, and their cell numbers were assessed microscopically at 0, 24, 48 and 72 h (red, green, and blue lines, respectively). Dp71 did not increase the cell number in all cell lines examined compared with the control (myoblast, CRL-2061, CCL-126, HeLa, HEK293, SH-SY5Y, HepG2 and AGS). As expected, Dp71ab significantly increased the number of myoblasts compared with that of the mock plasmid-transfected cells (p < 0.001). However, Dp71ab had no effect in the other seven cell lines, including two myogenic cell lines (CRL-2061 and CCL-136). * = p < 0.05, ** = p < 0.01, *** = p ≤ 0.001.