Translocation of molecular chaperones to the titin springs is common in skeletal myopathy patients and affects sarcomere function

Abstract

SummaryMyopathies encompass a wide variety of acquired and hereditary disorders. The pathomechanisms include structural and functional changes affecting, e.g., myofiber metabolism and contractile properties. In this study, we observed increased passive tension (PT) of skinned myofibers from patients with myofibrillar myopathy (MFM) caused by FLNC mutations (MFM-filaminopathy) and limb-girdle muscular dystrophy type-2A due to CAPN3 mutations (LGMD2A), compared to healthy control myofibers. Because the giant protein titin determines myofiber PT, we measured its molecular size and the titin-to-myosin ratio, but found no differences between myopathies and controls. All-titin phosphorylation and site-specific phosphorylation in the PEVK region were reduced in myopathy, which would be predicted to lower PT. Electron microscopy revealed extensive ultrastructural changes in myofibers of various hereditary myopathies and also suggested massive binding of proteins to the sarcomeric I-band region, presumably heat shock proteins (HSPs), which can translocate to elastic titin under stress conditions. Correlative immunofluorescence and immunoelectron microscopy showed that two small HSPs (HSP27 and αB-crystallin) and the ATP-dependent chaperone HSP90 translocated to the titin springs in myopathy. The small HSPs, but not HSP90, were upregulated in myopathic versus control muscles. The titin-binding pattern of chaperones was regularly observed in Duchenne muscular dystrophy (DMD), LGMD2A, MFM-filaminopathy, MFM-myotilinopathy, titinopathy, and inclusion body myopathy due to mutations in valosin-containing protein, but not in acquired sporadic inclusion body myositis. The three HSPs also associated with elastic titin in mouse models of DMD and MFM-filaminopathy. Mechanical measurements on skinned human myofibers incubated with exogenous small HSPs suggested that the elevated PT seen in myopathy is caused, in part, by chaperone-binding to the titin springs. Whereas this interaction may be protective in that it prevents sarcomeric protein aggregation, it also has detrimental effects on sarcomere function. Thus, we identified a novel pathological phenomenon common to many hereditary muscle disorders, which involves sarcomeric alterations.Electronic supplementary materialThe online version of this article (10.1186/s40478-017-0474-0) contains supplementary material, which is available to authorized users.

Imbalances in protein homeostasis caused by mutant desmin

“…Beyond a crucial role in the organization of the three‐dimensional desmin intermediate filament cytoskeleton itself, our findings further highlight the notion that αB‐crystallin and Hsp27 display aberrant sarcomeric localization pattern in stressed as well as diseased striated muscle that clearly differs from the ones observed in normal skeletal muscle. Similar to the results observed in our desminopathy mouse models, analyses of adult zebrafish myocardium exposed to hyperthermia and mechanical stretching as well as of human skeletal muscle samples derived from patients with myopathies with or without evidence of abnormal protein aggregation revealed a translocation of both small heat shock proteins from a localization at Z‐bands to the level I‐band titin.…”

“…We recently demonstrated that αB‐crystallin, Hsp27 and Hsp90 associate with I‐band titin in various forms of human myopathies with and without evidence of abnormal protein aggregation . To address this issue in our desminopathy mouse model, we performed corresponding immunogold electron microscopic analyses in homozygous and immunofluorescence imaging analyses in hetero‐ and homozygous animals compared to wild‐type littermates.…”

“… desmin (both wild‐type and mutant desmin, ‘total’ desmin), mouse monoclonal, 1:1000 in PBS‐T for immunoblotting, 1:100 in PBS for immunofluorescence microscopy, clone D33, Dako/Agilent, Santa Clara, California, USA desmin (‘total’ desmin), rabbit monoclonal, 1:100 in PBS for immunofluorescence microscopy, #5332, Cell Signalling, Danvers, Massachusetts, USA R349P point‐mutant desmin, rabbit polyclonal, 1:1000 in PBS‐T for immunoblotting, GAPDH, rabbit polyclonal, 1:10 000 in PBS‐T for immunoblotting, G9545, Sigma‐Aldrich, St. Louis, Missouri, USA α‐actinin 2 (sarcomeric α‐actinin), mouse monoclonal, 1:700 in PBS for immunofluorescence microscopy, A7732, Sigma‐Aldrich ubiquitin, rabbit polyclonal, 1:1000 in PBS‐T for immunoblotting, ab7780, Abcam, Cambridge, UK 19S proteasome subunit 4 (Rpt2), rabbit polyclonal, 1:1000 in PBS‐T for immunoblotting, #09‐280, Upstate/Fisher Scientific, Hampton, New Hampshire, USA actin, mouse monoclonal, 1:1000 in PBS‐T for immunoblotting, A4700, Sigma‐Aldrich light chain 3B (LC3B), rabbit monoclonal, 1:100 in PBS for immunofluorescence microscopy, 1:1000 in PBS‐T for immunoblotting, #3868, Cell Signalling ULK1, rabbit polyclonal, 1:500 in PBS‐T for immunoblotting sequestosome 1 (SQSTM1)/p62, rabbit polyclonal, 1:3000 in PBS‐T for immunoblotting, P0067, Sigma‐Aldrich filamin‐C, rabbit polyclonal, 1:10 000 in PBS‐T for immunoblotting, d16‐20, kindly provided by D. Fürst, Bonn, Germany BAG‐3, rabbit polyclonal, 1:3000 in PBS‐T for immunoblotting, 10599‐1‐AP, Proteintech, Manchester, UK αB‐crystallin, rabbit polyclonal antibody, 1:1,000 in PBS‐T for immunoblotting, SPA‐223, Stressgen/Enzo Life Sciences, Lörrach, Germany αB‐crystallin, rabbit polyclonal antibody, 1:250 for immunoelectron microscopy, SMC 1653A10, StressMarq Biosciences, Victoria, British Columbia, Canada αB‐crystallin, rabbit polyclonal antibody, 1:400 for immunoelectron microscopy, SR 223F, MBL, Woburn, Massachusetts, USA Hsp27, rabbit polyclonal antibody, 1:1000 in PBS‐T for immunoblotting, ab12351, Abcam Hsp27, rabbit polyclonal antibody, 1:250 for immunoelectron microscopy, SMC 1615D12, StressMarq Biosciences Hsp27, rabbit polyclonal antibody, 1:200 for immunoelectron microscopy, SR B800, MBL Hsp90 (total), rabbit monoclonal, 1:1000 in PBS‐T for immunoblotting, 1:400 for immunoelectron microscopy, #4877, Cell Signalling Hsp90‐alpha, rabbit polyclonal, 1:500 for immunoelectron microscopy, Pineda titin (PEVK domain), mouse monoclonal, 1:200 for immunoelectron microscopy, #9D10, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA. …”

“…Sections of 5 μm were prepared with a RM 2235 microtome (Leica, Mannheim, Germany), rehydrated and blocked in peroxidase blocking buffer. A citrate‐EGTA antigen recovery protocol was performed , slides were rinsed with PBS and blocked with 5% of bovine serum albumin containing 0.5% of Triton X‐100 for 60 min. Subsequently, sections were incubated with primary and secondary antibodies each overnight at 4°C; stained samples were embedded in Mowiol supplemented with N ‐propyl‐gallate (#02370; Sigma‐Aldrich) to reduce photobleaching and analysed by confocal laser scanning microscopy (Eclipse Ti; Nikon, Minato, Tokio, Japan) using a 63× oil Plan‐Apochromat objective.…”

Editorial on EMC 2017 special issue

“…The delicate and sometimes contradicting nature of these processes has recently been demonstrated in myofibers from skeletal myopathy patients. Recently, Unger et al demonstrated that chaperone-binding to the titin springs prevents sarcomeric protein aggregation and thereby protects the structural integrity of the myofibers, but at the same time, by increasing titin-based passive tension, the chaperone association is detrimental for sarcomere function (Unger et al 2017). This study again underlines the need for more work to increase our understanding of the processes involved in sarcomere assembly and disassembly, myofilament metabolism and muscle repair in the context of striated muscle disease.…”