Z-disc Titin in Sarcomere Assembly and Mechanotransduction

• Applicant: Professor Dr. Michael Gotthardt
• Subject Area: Biochemistry
• Term: from 2010 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 148688621

• Final Report Year: 2019

Final Report Abstract

Titin is the giant elastic backbone of the sarcomere. It is expressed in multiple isoforms, including some that are integrated only at the Z-disc and some that extend through the M-band. Titin acts as a molecular spring and has been linked to mechanosignaling in striated muscle. Mutations of titin lead to skeletal muscle or heart disease such as hypertrophic or dilated cardiomyopathy. To study the role of Z-disc titin and how titin is integrated into the Sarcomere, we used a three pronged approach: (1) We generated titin GFP and RFP knockin alleles that enable the visualization of titin molecules as they are moved between sarcomeres and integrate into the myofilament lattice. (2) We generated a titin BioID knockin that allows us to interrogate the protein environment at the Z- disc. (3) To study the role of Z-disc titin, we generated a striated muscle specific knockout, with either progressive postnatal loss of the complete titin protein or an M-band truncation that eliminates proper sarcomeric integration but retains all other functional domains. The latter allowed us to dissect the differences in a Z-disc integrated titin from a missing titin filament. While skeletal muscle atrophy with reduced strength and lethality from 2 weeks of age were shared between the models with and without Z-disc titin, they display diverse cardiac phenotypes: The loss of titin protein leads to the development of dilated cardiomyopathy - the absence of M-band titin to cardiac atrophy. This was also reflected in the echocardiography were in addition the systolic properties were changed only in the complete titin knockout. The passive properties of the myocytes were only reduced in the complete titin knockout, while there were unchanged in the M-band knockout at the age of 4 weeks. Using life imaging of double heterozygous animals that express titins with a red fluorophore at the Z-disc and a green fluorophore at the M-band, we show how titin is synthesized and moved: titin expression is not compartmentalized in the embryonic cardiomyocyte, but in adult working cardiomyocytes. In differentiating cardiomyocytes and upon remodelling, we find that sarcomere assembly does not proceed sequentially with titin integration from Z-disc to M-band, contrary to the premyofibril model. As Z-discs cannot be purified, our current understanding is built on a candidate approach with immunofluorescence staining and interaction screens with known Z-disc proteins to study the localized protein composition. In its current implementation, proximity proteomics had been restricted to cultured cells, which limits potential interventions and analysis of a systemic or disease context. Here we have adapted the BioID system to enzymatically modify and purify proteins localized proximal to Z-disc titin in vivo. Proteins identified by mass spec capture the majority of the known sarcomeric Z-disc proteome and biotinylation sites faithfully reflect the localization of BioID at nm resolution. We used superresolution imaging to demonstrate that biotinylated Z-disc proteins indeed flank the Z-disc. Applying proximity proteomics in vivo has the potential to interrogate the dynamic localization of proteins dependent on genetic, pharmaceutical, physiological and disease context. Taken together, our implementation of mouse genetics to understand the integration of titin into the Z-disc of the sarcomere, its local protein environment, and its function provides novel insights into basic sarcomere biology and how titin deficiency differentially affects cardiac and skeletal muscle function depending on the presence or absence or truncation of a continuous titin filament system.

Publications

• Calcium sensitivity and the Frank-Starling mechanism of the heart are increased in titin N2B region-deficient mice. JMCC 2010; 49:449-458
  Lee EJ, Peng J, Radke M, Gotthardt M, Granzier HL
  
• Titin based viscosity in ventricular physiology: an integrative investigation of PEVK-actin interactions. J Mol Cell Cardiol 2011; 51:428–434
  Chung CS, Methawasin M, Nelson OL, Radke MH, Hidalgo CG, Gotthardt M, Granzier HL
  
• Titin visualization in real time reveals an unexpected level of mobility within and between sarcomeres. J Cell Biol 2011; 193:785–798
  da Silva Lopes K, Pietas A, Radke MH, Gotthardt M
  
• RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat Med 2012; 18:766–773
  Guo W, Schafer S, Greaser ML, Radke MH, Liss M, Govindarajan T, Maatz H, Schulz H, Li S, Parrish AM, Dauksaite V, Vakeel P, Klaassen S, Gerull B, Thierfelder L, Regitz-Zagrosek V, Hacker TA, Saupe KW, Dec GW, Ellinor PT, MacRae CA, Spallek B, Fischer R, Perrot A, Özcelik C, Saar K, Hubner N, Gotthardt M
  
• Calcium sensitivity and myofilament lattice structure in titin N2B KO mice. Arch Biochem Biophys 2013; 535:76–83
  Lee E-J, Nedrud J, Schemmel P, Gotthardt M, Irving TC, Granzier HL
  
• Deletion of the titin N2B region accelerates myofibrillar force development but does not alter relaxation kinetics. J Cell Sci 2014; 127:3666–3674
  Elhamine F, Radke MH, Pfitzer G, Granzier H, Gotthardt M, Stehle R
  
• Recessive TTN truncating mutations define novel forms of core myopathy with heart disease. HMG 2014; 23:980-991
  Chauveau C, Bonnemann CG, Julien C, Kho AL, Marks H, Talim B, Maury P, Arne-Bes MC, Uro- Coste E, Alexandrovich A, Vihola A, Schafer S, Kaufmann B, Medne L, Hübner N, Foley AR, Santi M, Udd B, Topaloglu H, Moore SA, Gotthardt M, Samuels ME, Gautel M, Ferreiro A
  
• RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing. J Clin Invest 2014; 124:3419–3430
  Maatz H, Jens M, Liss M, Schafer S, Heinig M, Kirchner M, Adami E, Rintisch C, Dauksaite V, Radke MH, Selbach M, Barton PJ, Cook SA, Rajewsky N, Gotthardt M, Landthaler M, Hubner N
  
• The Src Homology and Collagen A (ShcA) Adaptor Protein Is Required for the Spatial Organization of the Costamere/Z-disk Network during Heart Development. J Biol Chem 2015; 290:2419-2430
  Mlih M, Host L, Martin S, Niederhoffer N, Monassier L, Terrand J, Messaddeq N, Radke M, Gotthardt M, Bruban V, Kober F, Bernard M, Canet-Soulas E, Abt-Jijon F, Boucher P, Matz RL
  
• Axial tubule junctions control rapid calcium signaling in atria. J Clin Invest. 2016; 126:3999-4015
  Brandenburg S, Kohl T, Williams GS, Gusev K, Wagner E, Rog-Zielinska EA, Hebisch E, Dura M, Didié M, Gotthardt M, Nikolaev VO, Hasenfuss G, Kohl P, Ward CW, Lederer WJ, Lehnart SE