New insights into the protein aggregation pathology in myotilinopathy by combined proteomic and immunolocalization analyses

Abstract

IntroductionMyofibrillar myopathies are characterized by progressive muscle weakness and impressive abnormal protein aggregation in muscle fibers. In about 10 % of patients, the disease is caused by mutations in the MYOT gene encoding myotilin. The aim of our study was to decipher the composition of protein deposits in myotilinopathy to get new information about aggregate pathology.ResultsSkeletal muscle samples from 15 myotilinopathy patients were included in the study. Aggregate and control samples were collected from muscle sections by laser microdissection and subsequently analyzed by a highly sensitive proteomic approach that enables a relative protein quantification. In total 1002 different proteins were detected. Seventy-six proteins showed a significant over-representation in aggregate samples including 66 newly identified aggregate proteins. Z-disc-associated proteins were the most abundant aggregate components, followed by sarcolemmal and extracellular matrix proteins, proteins involved in protein quality control and degradation, and proteins with a function in actin dynamics or cytoskeletal transport. Forty over-represented proteins were evaluated by immunolocalization studies. These analyses validated our mass spectrometric data and revealed different regions of protein accumulation in abnormal muscle fibers. Comparison of data from our proteomic analysis in myotilinopathy with findings in other myofibrillar myopathy subtypes indicates a characteristic basic pattern of aggregate composition and resulted in identification of a highly sensitive and specific diagnostic marker for myotilinopathy.ConclusionsOur findings i) indicate that main protein components of aggregates belong to a network of interacting proteins, ii) provide new insights into the complex regulation of protein degradation in myotilinopathy that may be relevant for new treatment strategies, iii) imply a combination of a toxic gain-of-function leading to myotilin-positive protein aggregates and a loss-of-function caused by a shift in subcellular distribution with a deficiency of myotilin at Z-discs that impairs the integrity of myofibrils, and iv) demonstrate that proteomic analysis can be helpful in differential diagnosis of protein aggregate myopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0280-0) contains supplementary material, which is available to authorized users.

Proteomics of rimmed vacuoles define new risk allele in inclusion body myositis

“…11-13 Using this technique, we identified distinct protein signatures in the muscle of myofibrillar myopathy patients. In the present study, we utilize the same proteomic approach to identify the protein composition of RVs in sIBM.…”

215th ENMC International Workshop VCP-related multi-system proteinopathy (IBMPFD) 13–15 November 2015, Heemskerk, The Netherlands

“…The authors applied a proteomic approach to decipher the composition of these aggregates in muscle samples from two patients. Protein aggregates and intraindividual control samples (from aggregate-free muscle fibres) were collected by laser microdissection and analysed by a label-free mass spectrometric approach for identification and relative quantification of proteins [21–23]. These analyses showed an over-representation of several proteins including Z-disc (-associated) proteins, chaperones, and proteins involved in proteasomal and autophagic protein degradation in aggregate samples (compared to control samples).…”

FHL1‐mutated reducing body myopathy

“…Defects in sarcomere proteins of the skeletal muscle can result in myofibrillar myopathy (MFM), a rare muscular disorder . The four and a half LIM domain protein 1 (FHL1) is a part of a family of proteins related to MFM, in addition to well‐known Z‐line associated proteins, including desmin, αB‐crystallin, myotilin, Z‐band alternatively spliced PDZ‐motif (ZASP), Filamin C, Bag3, slow myosin heavy chain, fast myosin heavy chain IIA, and titin …”

“…Childhood‐onset and mutations in the LIM 2 domain of FHL1 are associated with more severe phenotypes and the presence of reducing bodies (RBs), which are characteristic inclusion bodies . RBM is an extremely rare myopathy showing progressive muscle weakness and accumulation of characteristic inclusions in the muscle …”

“…Initially, the muscle weakness begins in the distal or proximal leg muscles and then spreads to other lower and upper extremity muscle groups . Cardiomyopathy has been reported while peripheral neuropathy has not been reported …”