Proteomic Identification of Markers of Membrane Repair, Regeneration and Fibrosis in the Aged and Dystrophic Diaphragm

Abstract

Deficiency in the membrane cytoskeletal protein dystrophin is the underlying cause of the progressive muscle wasting disease named Duchenne muscular dystrophy. In order to detect novel disease marker candidates and confirm the complexity of the pathobiochemical signature of dystrophinopathy, mass spectrometric screening approaches represent ideal tools for comprehensive biomarker discovery studies. In this report, we describe the comparative proteomic analysis of young versus aged diaphragm muscles from wild type versus the dystrophic mdx-4cv mouse model of X-linked muscular dystrophy. The survey confirmed the drastic reduction of the dystrophin-glycoprotein complex in the mdx-4cv diaphragm muscle and concomitant age-dependent changes in key markers of muscular dystrophy, including proteins involved in cytoskeletal organization, metabolite transportation, the cellular stress response and excitation-contraction coupling. Importantly, proteomic markers of the regulation of membrane repair, tissue regeneration and reactive myofibrosis were detected by mass spectrometry and changes in key proteins were confirmed by immunoblotting. Potential disease marker candidates include various isoforms of annexin, the matricellular protein periostin and a large number of collagens. Alterations in these proteoforms can be useful to evaluate adaptive, compensatory and pathobiochemical changes in the intracellular cytoskeleton, myofiber membrane integrity and the extracellular matrix in dystrophin-deficient skeletal muscle tissues.

A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle

“…3 J–K). These results are in agreement with results obtained in the mdx mouse model and in primary muscle biopsies from human patients, whereby fibrosis is one of the main pathological features of Duchenne muscular dystrophy [ 24 ].…”

“…Like previous studies that used DMD-deficient primary myoblasts or CRISPR/Cas9-induced DMD knockouts to model Duchenne muscular dystrophy in 3D-TESMs, the knock down of DMD resulted in a significantly reduced force-generating capacity [ 39 – 41 ]. Concurrent with this reduction in contractile properties, we found a significant reduction of the expression of proteins involved in the structure and function of the sarcomere (MYHs, TTN, NEB, OBSCN, ATP1A2, ATP2A1), as well as of SGCA, SGCD, and SGCG, which are part of the sarcoglycan complex that directly connects dystrophin to the surrounding ECM [ 24 , 42 ]. In addition, we observed an upregulation of the expression of several annexin isoforms (ANXA1-5) and tubulins (TUBB2A and TUBB2B), which are commonly associated with membrane repair, a pathway that is upregulated in muscle tissues from Duchenne patients and the mdx mouse model [ 24 , 43 ].…”

“…Concurrent with this reduction in contractile properties, we found a significant reduction of the expression of proteins involved in the structure and function of the sarcomere (MYHs, TTN, NEB, OBSCN, ATP1A2, ATP2A1), as well as of SGCA, SGCD, and SGCG, which are part of the sarcoglycan complex that directly connects dystrophin to the surrounding ECM [ 24 , 42 ]. In addition, we observed an upregulation of the expression of several annexin isoforms (ANXA1-5) and tubulins (TUBB2A and TUBB2B), which are commonly associated with membrane repair, a pathway that is upregulated in muscle tissues from Duchenne patients and the mdx mouse model [ 24 , 43 ]. Another key aspect related to the pathogenicity of Duchenne muscular dystrophy is the progressive conversion of skeletal muscle tissue into fibrotic tissue [ 24 , 44 ].…”

“…In addition, we observed an upregulation of the expression of several annexin isoforms (ANXA1-5) and tubulins (TUBB2A and TUBB2B), which are commonly associated with membrane repair, a pathway that is upregulated in muscle tissues from Duchenne patients and the mdx mouse model [ 24 , 43 ]. Another key aspect related to the pathogenicity of Duchenne muscular dystrophy is the progressive conversion of skeletal muscle tissue into fibrotic tissue [ 24 , 44 ]. Immune cell infiltration is thought to play a role in this process.…”

“…Immune cell infiltration is thought to play a role in this process. Although the 3D model employed here lacks immune cells, we nevertheless found an increased expression of COL4A1, COL4A2, COL6A1, LAMA1, and LAMA2 and other ECM proteins that are commonly associated with a fibrotic phenotype, suggesting the existence of an immune cell-independent pathway underlying fibrosis in Duchenne muscular dystrophy [ 24 , 44 ].…”