Abstract
Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene translating in lack of functional dystrophin and resulting in susceptibility of myofibers to rupture during contraction. Inflammation and fibrosis are critical hallmarks of DMD muscles, which undergo progressive degeneration leading to loss of independent ambulation in childhood and death by early adulthood. We reported that intraperitoneal injection of microencapsulated Sertoli cells (SeC) in dystrophic mice translates into recovery of muscle morphology and performance thanks to anti-inflammatory effects and induction of the dystrophin paralogue, utrophin at the muscle level, opening new avenues in the treatment of DMD. The aim of this study is to obtain information about the direct effects of SeC on myoblasts/myotubes, as a necessary step in view of a translational application of SeC-based approaches to DMD. We show that (i) SeC-derived factors stimulate cell proliferation in the early phase of differentiation in C2C12, and human healthy and DMD myoblasts; (ii) SeC delay the expression of differentiation markers in the early phase nevertheless stimulating terminal differentiation in DMD myoblasts; (iii) SeC restrain the fibrogenic potential of fibroblasts, and inhibit myoblast-myofibroblast transdifferentiation; and, (iv) SeC provide functional replacement of dystrophin in preformed DMD myotubes regardless of the mutation by inducing heregulin β1/ErbB2/ERK1/2-dependent utrophin expression. Altogether, these results show that SeC are endowed with promyogenic and antifibrotic effects on dystrophic myoblasts, further supporting their potential use in the treatment of DMD patients. Our data also suggest that SeC-based approaches might be useful in improving the early phase of muscle regeneration, during which myoblasts have to adequately proliferate to replace the damaged muscle mass.
Highlights
Duchenne muscular dystrophy (DMD) is a recessive X-linked lethal disease affecting one over 3600–5000 live male births [1,2]
The bromo-20 deoxyuridine (BrdU) incorporation assay showed a ~65% increase in the percentage of BrdU-positive cells (Figure 1C), and cytofluorimetric analysis after annexin V/propidium iodide staining showed a concomitant ~53% reduction in the apoptotic extent (Figure 1D), in the presence of Sertoli cells (SeC)-DM compared with unconditioned medium (U-DM)
Differentiating myoblasts cultured with SeC-conditioned differentiation medium (SeC-DM) were characterized by reduced oxidative stress since we found 45.0 ± 14% reduction in DCF fluorescence relative intensity, which is a measure of intracellular production of reactive oxygen species (ROS), compared with control myoblasts (Figure 1E)
Summary
Duchenne muscular dystrophy (DMD) is a recessive X-linked lethal disease affecting one over 3600–5000 live male births [1,2]. DMD is due to mutations in the dystrophin. Biomolecules 2021, 11, 1504 gene (DMD) leading to lack of a functional protein and subsequent inability to recruit the dystrophin-associated protein complex (DAPC) at the sarcolemma resulting in susceptibility of myofibers to rupture during contraction [3]. Inflammation and fibrosis are the most critical hallmarks of DMD muscles, responsible of the onset and the progression of the pathology [5,6]. A wide spectrum of therapeutic approaches to DMD have been explored in the last decades and many of them are currently under clinical evaluation, several limitations, such as unsatisfying muscle recovery, the need for immunosuppression, and relevant adverse effects encourage for further investigation [8,9,10]. The standard therapy to DMD still remains the use of glucocorticoids despite their limited efficacy and undesired side effects [10]
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