Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics

Figures

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  Fig. 1. Structural and evolutionary conservation of titin mechanical cysteines. A: Top: Representation of half a sarcomere (not to scale), indicating the positions of the I-band (green) and the A-band (teal) of titin. Immunoglobulin-like (Ig), random-coil (PEVK and N2Bus) and fibronectin III (Fn) domains are shown. Bottom: Under mechanical force, the serially linked Ig domains and the random coil regions extend, and Ig domains unfold. B: 3D homology model of an Ig domain containing the disulfide-competent CysB-CysF-CysG triad (I74, positions 9079-9168 from Uniprot entry Q8WZ42). The reduced contour length of the disulfide-containing unfolded state leads to domain stiffening. C: High-resolution structure of the traditionally named I27 domain of titin (also known as I91, PDB code 1tit), which contains unpaired, S-thiolation-competent cysteines 47 and 63 (positions 12674-12765 from Uniprot entry Q8WZ42). S-thiolations inhibit folding, leading to domain softening [19]. D: Map of cysteine positions along the alignment of Ig domains of human titin (breaks leave out Cys-free segments of the alignment). Structurally conserved positions B, 47, 63, F and G are indicated on top of the alignment.…

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  Fig. 2. Titin is oxidized in basal conditions. A: Left: Reaction scheme to label reversibly oxidized thiols with mBBr. Protein extracts in which reduced thiols have been initially blocked by NEM are run in an SDS-PAGE gel. Following electrophoresis, oxidized thiols are reduced by DTT and then labeled with the fluorescent probe mBBr. Right: example SDS-PAGE gel used to quantify mBBr fluorescence from titin bands. Coomassie staining (Coom) of the same gel is used for normalization. B, C: The mBBr and Coomassie signals originating from titin are linearly dependent on the amount of lysate loaded in SDS-PAGE gels (all data points are the average of duplicates) (Supplementary Fig. S2A). D: Final mBBr/Coomassie ratio is independent of the amount of lysate loaded in the gel. E: Scan to determine the concentration of NEM needed to completely block initially reduced titin thiols. Duplicates were performed for each NEM concentration. F: Quantification of the oxidation of titin and myosin in four different mouse hearts using 3.5% and 12% SDS-PAGE gels, respectively (see also Supplementary Fig. S2B). The volume of lysate analyzed was adjusted so that both titin (15 μl) and myosin (3 μl) were in the…

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  Fig. 3. Landscape of cysteine oxidation in murine and human cardiac titin. A: Fraction of identified peptides belonging to murine titin (reference sequence Uniprot A2ASS6-1, which codes for the long N2BA titin isoform) when the MS/MS spectra are searched against a mouse proteome database containing common contaminants (cRAP). B: Aggregated titin coverage obtained after searching against the mouse proteome (n = 3 mouse hearts; yellow and green, peptides detected with FDR<5% and <1%, respectively). C: Redox Identification Index (RII) map for every cysteine position in the alignment of Ig domains of mouse titin, according to the color code indicated on the left (red, oxidized; blue, reduced). Average RII (AvRII) for cysteines 47, 63 and the BFG triad across all Ig domains are indicated. Cysteines shown in white were not identified and do not contribute to RII calculation. The horizontal dashed line shows the boundary between the I- and A-bands of titin (positions 14880-14881 in Uniprot A2ASS6-1). The number and positions of Ig domains are indicated on the right. Ig domains are classified and colored as in Fig. 1. D: Violin-plot distributions of RII for cysteines belonging to the I- (n = 97)…

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  Fig. 4. Effects of reversible cysteine oxidations on titin mechanics.A: Monte Carlo simulations subject virtual titins to a 1 Hz oscillating triangular force pulse to a predefined peak force (100 pN in the example shown). B: Example of the length of reduced N2BA titin when pulled to a 100 pN peak force (10 s of simulation after reaching steady state are shown). C,D: Length of N2BA titin at peak force during simulations in which titin is reduced, or oxidized by disulfides, S-thiolation adducts or both (color code is indicated at the bottom; peak forces are indicated in the insets). The parameters of S-thiolations used in these simulations correspond to S-glutathionylations (Supplementary Note S1). Graphs show the average of 10 independent simulations and SD is indicated by shaded areas. For subsequent analyses, we considered times longer than 1 h, in which the length of titin fluctuates around steady state values (region outside the grey shading). E: Ratio of oxidized vs reduced N2BA titin length at different peak forces. F: Ratio of oxidized vs reduced N2BA unfolding events in simulations at different peak forces. G: Ratio of oxidized vs reduced N2B titin length at different peak forces.…

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  Fig. 5. Oxidations of conserved cysteines enable mechanical modulation of titin. Structurally and evolutionary conserved cysteines B, F, G, 47 and 63 of Ig domains of the I-band of titin are oxidized in basal conditions. Mean evolutionary conservation percentages for cysteines BFG (98%) and 47/63 (94%) are shown on a conservation scale from 0 (white) to 100% (dark green). Average basal redox identification indexes for each individual cysteine are indicated on a scale ranging from -1 (always reduced) to +1 (always oxidized), both for mouse (above) and for human (below), as they appear on Fig. 3. Disulfides (S–S) between cysteines belonging to the BFG triad soften titin at low forces and stiffen the protein at high forces, while S-thiolations (S–S-●) on cysteines 47 and 63 are always softening. Both types of oxidative posttranslational modifications (PTMs) increase the unfolding dynamics of Ig domains. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)