Myogenesis modelled by human pluripotent stem cells uncovers Duchenne muscular dystrophy phenotypes prior to skeletal muscle commitment

Abstract

19Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an 20 incidence of approximately 1/5,000 male births. Symptoms appear in early childhood, with a diagnosis made 21 around 4 years old, a time where the amount of muscle damage is already significant, preventing early 22 therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the 23 precise moment at which disease phenotypes arise -even asymptomatically -is still unknown. Thus, there is a 24 critical need to better define DMD onset as well as its first manifestations, which could help identify early 25 disease biomarkers and novel therapeutic targets. 26In this study, we have used human induced pluripotent stem cells (hiPSCs) from DMD patients to model 27 skeletal myogenesis, and compared their differentiation dynamics to healthy control cells by a comprehensive 28 multi-omics analysis. Transcriptome and miRnome comparisons combined with protein analyses at 7 time 29 points demonstrate that hiPSC differentiation 1) mimics described DMD phenotypes at the differentiation 30 endpoint; and 2) homogeneously and robustly recapitulates key developmental steps -mesoderm, somite, 31 skeletal muscle -which offers the possibility to explore dystrophin functions and find earlier disease 32 biomarkers. 33Starting at the somite stage, mitochondrial gene dysregulations escalate during differentiation. We also 34 describe fibrosis as an intrinsic feature of skeletal muscle cells that starts early during myogenesis. In sum, our 35 data strongly argue for an early developmental manifestation of DMD whose onset is triggered before the 36 entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin 37 functions during muscle development.Duchenne muscular dystrophy (DMD) is a rare genetic disease, but it is the most common form of myopathy 40 affecting approximately one in 5,000 male births and very rarely female. In this recessive X-linked monogenic 41 disorder, mutations in the DMD gene lead to the loss of a functional dystrophin protein, resulting in a 42 progressive -yet severe -muscle wasting phenotype (1). In patients, symptoms usually appear in early 43 childhood (2-5 years old) and worsen with age, imposing the use of wheelchair before 15 and leading to 44 premature death by cardiac and/or respiratory failure(s) mostly around 30 years of age (2). 45At the age of diagnosis, around 4 years old, muscles of DMD patients have already suffered from the pathology 46 (3,4). Several reviews pointed out the limitations of current disease biomarkers, which fail to detect the 47 development of DMD specifically and at an early age (5,6). Meanwhile, no treatment is available to stop this 48 degenerative disease yet. Developing therapies aim at restoring the expression of dystrophin in muscle cells 49 but, so far, the level stays too low to be beneficial to patients (7). The absence of both reliable biomarkers and 50 effective therapies stress the need o...

Downregulation of dystrophin expression occurs across diverse tumors, correlates with the age of onset, staging and reduced survival of patients

“…The calcium signaling pathway enriched in DEGs in tumors samples with low DMD expression from 13 out of the 15 analyzed primary tumors, and well as in sarcoma cell lines with low DMD expression agrees with the dysregulation of calcium signaling across a whole spectrum of dystrophic cells (reviewed in (53)), as do GO terms related to the regulation of the developmental mechanisms (8). Thus, the DMD gene may play similar roles in cancer and development, two processes showing biological and molecular similarities (63).…”

“…However, more recent data demonstrate that the loss of DMD expression impacts a broader spectrum of cells than those affected in DMD. In myoblasts (10,16,45,46), lymphocytes (33), endotheliocytes (32,47,48), mesodermal (8), and myogenic cells (11,15), loss of DMD expression leads to significant abnormalities. Moreover, the same abnormalities can occur in very distinct cells, e.g., calcium dys-homeostasis was found across multiple cells (49), and the damaging purinergic phenotype affects myoblasts and lymphocytes (33,45).…”

“…The specific GO Biological Process terms enriched in the DEGs in primary tumors with low vs. high DMD expression were also identified (Figure 6). The GO terms enriched in more than 50% of tumors were: extracellular matrix organization (14 out of 15 tumors), axonogenesis (12), regulation of cell migration, synapse organization and nervous system development (11), skeletal system development and regulation of ERK1 and ERK2 cascade (10) and calcium ion transmembrane import into cytosol (8). Lists of the DEGs in each comparison and results of the pathway and GO term analysis can be found in Supplementary File 2.…”

“…In fact, studies of human fetuses (2)(3)(4) and in various animal models (5)(6)(7) revealed that the pathology starts during prenatal development and continues into adulthood. The first DMD defects are detectable in developing cells even before their differentiation into muscle (8). Given that muscle regeneration replicates processes occurring in muscle development and that some developmental mechanisms are reactivated in tumors, it is intriguing that changes in DMD gene expression are increasingly being described in various malignancies (9).…”

Long Non-coding RNAs Are Differentially Expressed After Different Exercise Training Programs

“…The effects were mediated through increased expression of utrophin (homolog of dystrophin), and this effect was mediated via the upregulation of H19/miR-675 and downregulation of let-7 ( Morgoulis et al, 2019 ). DMD-induced pluripotent stem cells (DMD-iPSCs) subjected to skeletal muscle differentiation at day 10 showed downregulation of several somite markers, including H19 ( Mournetas et al, 2019 ). Another muscle-related lncRNA is LINCMD1, which plays a role in muscle differentiation by acting as a ceRNA and sponging miR-133/miR-135.…”