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Evidence for skeletal muscle fiber type-specific expressions of mechanosensors

  • Sebastian Mathes
  • Mathias Vanmunster
  • Wilhelm Bloch
  • Frank SuhrEmail author
Original Article

Abstract

Mechanosensors govern muscle tissue integrity and constitute a subcellular structure known as costameres. Costameres physically link the muscle extracellular matrix to contractile and signaling ‘hubs’ inside muscle fibers mainly via integrins and are localized beneath sarcolemmas of muscle fibers. Costameres are the main mechanosensors converting mechanical cues into biological events. However, the fiber type-specific costamere architecture in muscles is unexplored. We hypothesized that fiber types differ in the expression of genes coding for costamere components. By coupling laser microdissection to a multiplex tandem qPCR approach, we demonstrate that type 1 and type 2 fibers indeed show substantial differences in their mechanosensor complexes. We confirmed these data by fiber type population-specific protein analysis and confocal microscopy-based localization studies. We further show that knockdown of the costamere gene integrin-linked kinase (Ilk) in muscle precursor cells results in significantly increased slow-myosin-coding Myh7 gene, while the fast-myosin-coding genes Myh1, Myh2, and Myh4 are downregulated. In parallel, protein synthesis-enhancing signaling molecules (p-mTORSer2448, p < 0.05; p-P70S6KThr389, tendency with p < 0.1) were reduced upon Ilk knockdown. However, overexpression of slow type-inducing NFATc1 in muscle precursor cells did not change Ilk or other costamere gene expressions. In addition, we demonstrate fiber type-specific costamere gene regulation upon mechanical loading and unloading conditions. Our data imply that costamere genes, such as Ilk, are involved in the control of muscle fiber characteristics. Further, they identify costameres as muscle fiber type-specific loading management ‘hubs’ and may explain adaptation differences of muscle fiber types to mechanical (un)loading.

Keywords

Skeletal muscle Fiber types Costamere Integrin-linked kinase Laser microdissection Multiplex PCR 

Notes

Acknowledgements

This study was supported by the German Sport University (920122 and 920119), the Federal Institute of Sports Sciences (IIA1-070114/13), and a KU Leuven Grant (ZKD2412) to F.S. The authors thank Bianca Collins and Mojgan Ghilav (both Department of Molecular Cellular Sport Medicine, German Sport University Cologne) for their excellent technical assistance. Professor Rik Lories (Skeletal Biology and Engineering Research Center, KU Leuven) is highly acknowledged for critically reading the manuscript.

Author contributions

FS designed the study. SM, MV and FS carried out experiments, performed statistics and interpreted the data. WB contributed to data interpretation and reagents. SM and FS prepared the figures. FS wrote the manuscript. All authors approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

18_2019_3026_MOESM1_ESM.pdf (43 kb)
Figure S1. CSA and gait dysfunction scores. (A) CSA of mice of groups Control, WBV, and IML are given. At day 7, WBV induced significantly higher CSA compared to control and IML. At day 7, IML induced significantly reduced CSA compared to control and WBV. (B) Gait dysfunction scores show that control and WBV maintained full activity of the PBS-treated hindlimb over the cause of 7 days. However, the Dysport®-treated hindlimb of IML mice showed immediate declines in activity, which worsened over the course of 7 days. Respective SEM values are not visible due to their small sizes. * p<0.05, ** p<0.01, *** p<0.001 (PDF 43 kb)
18_2019_3026_MOESM2_ESM.pdf (948 kb)
Figure S2. Comparison of metachromatic ATPase staining with periodic acid-Schiff (PAS) staining in consecutive soleus cross sections. Characteristic staining pattern obtained after metachromatic ATPase staining (A) was not clearly present upon PAS staining (B) indicating polysaccharides (glycogen). The lower panels show the enlargement of the black square in the upper panels. Characteristic fiber types (1, 2A, 2B) were only identified easily after metachromatic ATPase staining (C) compared to PAS staining (D). Upper panels: scale bar =100 μm; lower panels: scale bar =20 μm (PDF 947 kb)
18_2019_3026_MOESM3_ESM.pdf (1021 kb)
Figure S3. Comparison of metachromatic ATPase staining with Oil Red O lipid staining in consecutive soleus cross sections. Characteristic staining pattern obtained upon metachromatic ATPase staining (A) was not present upon Oil Red O lipid staining (B). Characteristic fiber types (1, 2A, 2B) were only identified easily upon metachromatic ATPase staining (C) compared to Oil Red O lipid staining (D). Red arrowheads indicate stained lipid droplets predominantly present in type 2 fibers. Upper panels: scale bar =100 μm; lower panels: scale bar =20 μm (PDF 1020 kb)
18_2019_3026_MOESM4_ESM.pdf (461 kb)
Figure S4. RNA integrity check, melting and amplification curves, and amplicon gel electrophoresis upon MT-PCR. (A) 28S:18S rRNA ratio of 2.2:1.0 revealed no degradation of RNA isolated from fresh frozen soleus muscle (lane 1). In contrast, lane 2 shows full RNA degradation, if metachromatic ATPase staining of soleus cross sections was performed without RNase inhibitor. No RNA degradation was observed, if tissue was dehydrated and exposed to RT for 2 h (lane 3; ratio 2.0:1.0). RNA isolated from ATPase-stained cross sections that were exposed to RT for 2h revealed a 28S:18S rRNA ratio of 0.8:1 (lane 4); for each lane 1 μg of total RNA was loaded and separated using RNA gel electrophoresis. (B) Exemplary Rn18s amplification curves of laser-microdissected type 1 and type 2 fibers. No primer dimers were observed in the related melting curves. Comparable curves were detected for all included genes. (C) DNA gel electrophoresis showed amplification specificity of MT-PCR from type 1 (odd lane numbers) and type 2 fibers (even lane numbers). No primer dimer formation was detected in MT-PCR products. GOIs were as follows: Itgb1 201-bp; Itga7 222-bp; Ilk 85-bp; Parva 152-bp; Tln1 89-bp; Vcl 172-bp; Fermt2 107-bp; Trim63 201-bp; Lims1 130-bp; Xirp2 170-bp; Fbxo32 218-bp; Rpl41 260-bp; Rpl27 144-bp; Myh1 203-bp; Myh2 299-bp; Myh4 295-bp; Myh7 267-bp; Rn18s 130-bp; M =marker; bp =base pairs (PDF 461 kb)
18_2019_3026_MOESM5_ESM.pdf (58 kb)
Table S1. Primary and secondary antibodies used in histology and western blot analyses. WB=western blot (PDF 58 kb)
18_2019_3026_MOESM6_ESM.pdf (57 kb)
Table S2. Primer sequences used in multiplex tandem and real-time PCRs. Lims1* and Fermt2* were nested inside Fermt2 and Lims1, respectively (PDF 57 kb)
18_2019_3026_MOESM7_ESM.docx (33 kb)
Supplementary material 7 (DOCX 33 kb)

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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of Molecular and Cellular Sport MedicineGerman Sport University CologneCologneGermany
  2. 2.Exercise Physiology Research Group, Department of Movement Sciences, Biomedical Sciences GroupKU LeuvenLeuvenBelgium

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