The titin N2B and N2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles

Figures

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  Fig. 1. A Electron micrograph of cardiac muscle (top panel) identifying regions of the sarcomere (I-band and A-band) as well as structural cross-linkers (Z-disc and M-band) also presented in the schematic overview (middle panel) and in the sarcomeric unit (bottom panel). Identity of filament systems (titin, myosin and actin filament) and important crosslinker proteins (sarcomeric α-actinin and myomesin) are shown. B Schematics of skeletal and cardiac titin N2A (top panel) and N2BA splice isoforms (bottom panel), respectively (not to scale). The N2A region, present in both cardiac and skeletal titin, and the cardiac specific N2B region are highlighted, as are known binding partners. C Changes to passive force development by titin depending on I-band splicing of skeletal muscle titin N2A isoform (top panel) or cardiac N2B and N2BA isoforms (bottom panel)

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  Fig. 2. A Schematic representation of titin N2A region domain structure (top), encompassing Ig-domain 80 (I80) to Ig-domain 83 (I83). The titin N2Aus linker and PEVK element are shown (P = phosphorylation). Location of binding site(s) within titin for each interaction partner, their roles and linkage to other proteins are also depicted (bottom panel; TFs = transcription factors). The described functional roles for each protein are visualized by circles colorized for each category, with small white circles indicating lack of evidence, and larger circles representing strong experimental support. B Influence of direct titin I83 binding to F-actin (right panel) on I-band titin in unstretched and stretched muscle. Linkage of titin I83 to actin leads to increased stretch of distal I-band domains and the PEVK element, protecting proximal Ig-domains from unfolding and preventing stretch of the N2B region (in cardiac titin isoforms). C Schematic representation of the Ankrd-dimer linking to adjacent titin filaments at the N2Aus region, and of the Ankrd1/titin/F-actin trimeric complex that ‘locks’ titin to the actin filament. Structures shown in the representation are adapted from RCSB accession…

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  Fig. 3.. A Schematics of the titin N2B region, spanning Ig-domains Ig23 (I23) to Ig27 (I27), with positions of the N2Bus3 region and PEVK element highlighted. Location of binding site(s) within titin for each interaction partner, their roles and linkage to other proteins are also depicted (bottom panel; TFs = transcription factors). The described functional roles for each protein are visualized by circles colorized for each category, with small white circles indicating lack of evidence, and larger circles representing strong experimental support. B Representative diagram of the effect that titin modifications have on the passive force development during sarcomere stretch. C Structure of Lim domains ½-1 of FHL2. Coordination of Zinc ions into specialized Zinc-fingers by side-chains in FHL2 is highlighted in the representation. Structure adapted from RCSB accession 2MIU. D Characterized FHL1 splice isoforms and domain layout. NLS nuclear localization signal, NES nuclear export signal. Figure adapted from Domenighetti et al. (2014)

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  Fig. 4.. A–B Localization (A) and expression levels (B) of endogenous FHL2 (green in the overlay in (A)) in neonatal rodent cardiomyocyte cultures treated with Wnt-conditioned medium, 10 mM LiCl or vehicle treated controls. Arrows in (A) indicate increased nuclear localization of FHL2. Sarcomeric counterstain in (A) was sarcomeric α-actinin (red in the overlay), while cardiac actin served as loading control in (B). Dvl-1 expression levels indicated activation of Wnt signaling. Scalebar = 10 μm. C Analysis of HA-tagged androgen receptor (AR, red in the overlay) and GFP-tagged FHL2 localization (green in the overlay) in transfected rat cardiomyocyte cultures, treated with 0.5 nM of AR agonist androstan, 1 μM AR antagonist flutamide or vehicle controls. Titin-M8 served as sarcomeric counterstain (blue in the overlay). Scalebar = 10 μm. D Analysis of Flag-tagged FHL3 localization (green in the overlay) in transfected neonatal rodent cardiomyocyte cultures. Titin-M8 served as sarcomeric counterstain (red in the overlay). Scalebar = 10 μm. E Analysis of wildtype and SY-mutant titin N2B binding to FHL1 or FHL2 in co-immunoprecipitation assays shows loss of FHL interaction with mutant titin. Sup…