E3-ligase knock down revealed differential titin degradation by autopagy and the ubiquitin proteasome system

Abstract

The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination.

Figure 1

Autophagy and proteasomal inhibition in ERCs induced general protein and full-length titin ubiquitination and increased the level of total titin and titin degradation intermediates. Autophagy related marker proteins LC3 and p62 were investigated under basal conditions and after 24 h of inhibition with CQ, BAF or MG132 by immunofluorescence (A/B) and Western blot (B). (C) Protein ubiquitination in the molecular weight range of ~ 70–250 kDa using total ubiquitin and K63-polyubiquitin antibodies. (D/E) Total ubiquitination and K63-polyubiquitination of titin under control conditions and after autophagy inhibition. (F) N2B titin content and relative T2/T1 ratio of ERCs at cultivation day 3 under control conditions and 24 h inhibition of autophagy or the proteasome. (G) Relative titin/actinin ratio under control conditions and after autophagy or proteasomal inhibition. An antibody targeting titin PEVK was used in immunofluorescence as cardiomyocyte marker and in titin blot as a marker for titin loading (Total Titin). GAPDH and α-actinin antibodies were used as loading marker for standard Western blots. Data are shown as mean ± SEM (n = 6–17 for T2/T1; n = 13–14 for titin/actinin ratio). Asterisks (P < 0.05 in one-way ANOVA with Dunn`s multiple comparison) mark statistical significance. BAF = bafilomycin; Ctrl = control; MG = proteasomal inhibitor MG132; α-Ub = anti-Ubiquitin antibody; α-Ub K63 = anti-K63-polyubiquitin antibody; N2B and N2BA = cardiac titin isoforms; T2 = specific titin degradation band. Bars = 10 µm.

Figure 2

E3-ligase interaction with the titin filament. (A) Schematic overview of the titin filament including the analyzed domains, N2B, N2A, PEVK and A168-170. (B) In vitro pull-down interaction tests of muscle specific E3-ligases with several titin domains from the I-band and the A-band. Representative images shown for all combinations of interaction tests, including GST controls. Calculated molecular weights of the recombinant titin fragments are shown. Immunofluorescence analysis of E3-ligase localization in (C) embryonic and (D) adult rat cardiomyocytes under standard cultivation conditions. Insets in C and D show magnifications of E3-ligase and actinin localization. D3 or D8 = cultivation day 3 or 8. Bar = 10 µm.

Figure 3

Decreased titin ubiquitination by the knock-down of E3-ligases. Cardiomyocytes were treated with siRNAs and 72 h later with BAF or MG132 for 24 h. (A) Representative Western blot images of E3-ligase knock-down. (B) Titin/Actinin ratio in siRNA treated cells under basal conditions (n = 11–12). (C) Representative Western blot images and analysis of protein ubiquitination in the range of 70–250 kDa in siRNA treated cells after autophagy or proteasomal inhibition (n = 4–6) and. (D) Representative Western blot images of siRNA treated cardiomyocytes with or without inhibition. (E) Analysis of titin ubiquitination levels after MuRF-1, -2 and -3 or CHIP/Fbx32 siRNA treatment (n = 5–8). Either Coomassie stained membranes or an antibody targeting titin PEVK was used in titin blot as a marker for titin loading. Data are shown as mean ± SEM. Asterisks (P < 0.05 in one-way ANOVA with Bonferroni t-test) mark statistical significance. BAF = bafilomycin; Ctrl = control; MG132 = proteasome inhibitor; α-Ub = anti-Ubiquitin antibody.

Figure 4

Differential autophagy- or proteasome-related ubiquitination of titin. Cardiomyocytes were treated with siRNAs and 72 h later with MG132 or BAF. (A/C) Representative Western blot images of siRNA treated cardiomyocytes with or without inhibition. Analysis of titin ubiquitination levels after MuRF-1, -2 or -3 (B) or CHIP or Fbx32 (D) siRNA treatment. Either Coomassie stained membranes or an antibody targeting titin PEVK was used in titin blot as a marker for titin loading. Data are shown as mean ± SEM (n = 4–6). Asterisks (P < 0.05 in one-way ANOVA with Bonferroni t-test) mark statistical significance. BAF = bafilomycin; Ctrl = control; MG132 = proteasome inhibitor; α-Ub = anti-Ubiquitin antibody.

Figure 5

Schematic view of E3-ligase interaction and modification of the titin filament. The scheme illustrates a possible regulation of E3-ligase based titin ubiquitination and subsequent degradation by autophagy or the proteasome based on the in vitro pull down experiments and immunofluorescence data. Straight lines represent potential degradation via the proteasome and dashed lines degradation via autophagy after ubiquitination by the respective E3-ligase. NH2 = titin N-terminus; COOH = titin C-terminus; * = identified by in vitro interaction assay; # = identified by immunofluorescence stainings.