Temporal Proteomic Profiling During Differentiation of Normal and Dystrophin-Deficient Human Muscle Cells

Abstract

Background: Myogenesis is a dynamic process involving temporal changes in the expression of many genes. Lack of dystrophin protein such as in Duchenne muscular dystrophy might alter the natural course of gene expression dynamics during myogenesis. Objective: To gain insight into the dynamic temporal changes in protein expression during differentiation of normal and dystrophin deficient myoblasts to myotubes. Method: A super SILAC spike-in strategy in combination and LC-MS/MS was used for temporal proteome profiling of normal and dystrophin deficient myoblasts during differentiation. The acquired data was analyzed using Proteome Discoverer 2.2. and data clustering using R to define significant temporal changes in protein expression. Results: sFour major temporal protein clusters that showed sequential dynamic expression profiles during myogenesis of normal myoblasts were identified. Clusters 1 and 2, consisting mainly of proteins involved mRNA splicing and processing expression, were elevated at days 0 and 0.5 of differentiation then gradually decreased by day 7 of differentiation, then remained lower thereafter. Cluster 3 consisted of proteins involved contractile muscle and actomyosin organization. They increased in their expression reaching maximum at day 7 of differentiation then stabilized thereafter. Cluster 4 consisting of proteins involved in skeletal muscle development glucogenesis and extracellular remodeling had a lower expression during myoblast stage then gradually increased in their expression to reach a maximum at days 11–15 of differentiation. Lack of dystrophin expression in DMD muscle myoblast caused major alteration in temporal expression of proteins involved in cell adhesion, cytoskeleton, and organelle organization as well as the ubiquitination machinery. Conclusion: Time series proteome profiling using super SILAC strategy is a powerful method to assess temporal changes in protein expression during myogenesis and to define the downstream consequences of lack of dystrophin on these temporal protein expressions. Key alterations were identified in dystrophin deficient myoblast differentiation compared to normal myoblasts. These alterations could be an attractive therapeutic target.

Downregulation of Dystrophin Expression Occurs across Diverse Tumors, Correlates with the Age of Onset, Staging and Reduced Survival of Patients

“…However, more recent data demonstrate that the loss of DMD expression impacts a broader spectrum of cells than those affected in DMD. In myoblasts [10,16,64,65], lymphocytes [33], endotheliocytes [32,66,67], 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 [51], and the damaging purinergic phenotype affects myoblasts and lymphocytes [33,65].…”

Characterization of the dystrophin‐associated protein complex by mass spectrometry

“…Cavin‐1 was also identified as being a member of the cardiac DAPC using highly specific dystrophin antibody‐based immunoprecipitation from a mouse cardiac muscle extract followed by mass spectrometry. Furthermore, in a recent study using the SILAC strategy, we showed that levels of cavin‐1 protein were decreased by twofold in differentiated dystrophin‐deficient DMD muscle cells relative to normal muscle cells (Goswami et al, 2021). These data, taken together, support cavin‐1 as being a new candidate member of the DAPC.…”

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

“…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).…”