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.

Duchenne muscular dystrophy patients lacking the dystrophin isoforms Dp140 and Dp71 and mouse models lacking Dp140 have a more severe motor phenotype

“…We confirm that myotubes in culture produce high levels of Dp71, and that this capacity is retained in DMD patients with mutations upstream of the Dp71 promoter. This, and previous findings, strongly, indicate a role for Dp71 in myoblast cell proliferation and satellite cell activation [31][32][33]. This suggests that the low level of Dp71 production in DMD muscle is a secondary phenomenon related to the muscle degeneration and regeneration characteristic of DMD.…”

Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts

“…Given that Dp71 dystrophin is found in undifferentiated myogenic cells [26], and that in development shorter dystrophins have been associated with proliferation and migration, and long isoforms with terminal commitment [77], we hypothesized that eliminating expression of Dp71 may exacerbate dysfunctions in myogenic cells. Surprisingly, we found that both Dmd mdx and Dmd mdx-βgeo cells had similar abnormalities and, contrary to Dp71 overexpression increasing myoblast proliferation [64], BrdU incorporation in dystrophin-null myoblasts was still significantly increased compared to WT. Interestingly, Dmd mdx myoblasts, having a point mutation in exon 23, express a significantly lower levels of Dp71 (Figure S6), whose expression is driven by a promoted located over 40 exons downstream of the mutation.…”

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

“…Based on this structural difference, Dp71ab has been shown to have different functions from Dp71 ( Marquez et al, 2003 ; Herrera-Salazar et al, 2016 ), but the functional characterization of each isoform has been hampered by the coexpression of isoforms ( Aragon et al, 2016 ; Rani et al, 2019 ). In our previous study, human Dp71ab was shown to be exclusively expressed in human satellite cells ( Farea et al, 2020 ). Moreover, we demonstrated that Dp71ab enhances myoblast proliferation ( Farea et al, 2020 ).…”

“…In our previous study, human Dp71ab was shown to be exclusively expressed in human satellite cells ( Farea et al, 2020 ). Moreover, we demonstrated that Dp71ab enhances myoblast proliferation ( Farea et al, 2020 ). Thus, Dp71ab was shown to be involved in cell proliferation.…”