Truncated titin is structurally integrated into the human dilated cardiomyopathic sarcomere
Dalma Kellermayer, … , Béla Merkely, Miklós S.Z. Kellermayer
Dalma Kellermayer, … , Béla Merkely, Miklós S.Z. Kellermayer
Published November 14, 2023
Citation Information: J Clin Invest. 2024;134(2):e169753. https://doi.org/10.1172/JCI169753.
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Research Article Cardiology Muscle biology Article has an altmetric score of 2

Truncated titin is structurally integrated into the human dilated cardiomyopathic sarcomere

Abstract

Heterozygous (HET) truncating variant mutations in the TTN gene (TTNtvs), encoding the giant titin protein, are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here, we studied 127 clinically identified DCM human cardiac samples with next-generation sequencing (NGS), high-resolution gel electrophoresis, Western blot analysis, and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene sequence predictions were detected in the majority of the TTNtv+ samples. Full-length titin was reduced in TTNtv+ compared with TTNtv– samples. Proteomics analysis of washed myofibrils and stimulated emission depletion (STED) super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin was structurally integrated into the sarcomere. Sarcomere length–dependent anti–titin epitope position, shape, and intensity analyses pointed at possible structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres, which probably contribute, possibly via faulty mechanosensor function, to the development of manifest DCM.

Authors

Dalma Kellermayer, Hedvig Tordai, Balázs Kiss, György Török, Dániel M. Péter, Alex Ali Sayour, Miklós Pólos, István Hartyánszky, Bálint Szilveszter, Siegfried Labeit, Ambrus Gángó, Gábor Bedics, Csaba Bödör, Tamás Radovits, Béla Merkely, Miklós S.Z. Kellermayer

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Figure 8

Schematic model of the structural and mechanical changes in the cardiac sarcomere caused by the presence of truncated titin.

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Schematic model of the structural and mechanical changes in the cardiac ...
Gaussian functions above the sarcomere schemes indicate the intensity profiles of the anti-titin epitopes used in this work. The red arrowheads indicate the epitope positions in the respective titin molecules. The black double-headed arrows indicate the changes in measured parameters. (A) TTNtv_ sarcomere at slack. Full-length titin molecules (6 per half thick filament, shown as green lines) are present. The anti-titin epitopes are in register, and the corresponding STED profiles are of high intensity and have a narrow width. (B) TTNtv+ sarcomere at slack. Truncated titin (shown as blue lines) is incorporated into the sarcomere (3 per half thick filament on average, presumably randomly distributed). Because the anchorage of TTNtv in the A-band is compromised, the molecules are pulled slightly toward the Z-line (hence, the A-band titin length is increased). The MIR epitopes are slightly out of register, resulting in an increase in the STED epitope profile width. Because only about half of the titins contribute to the A170 labeling, the A170 epitope intensity is reduced. The M-line–to–A170 distance is slightly increased (by ~10 nm), suggesting that the M-band region is disarranged (indicated by a crooked M-band). (C) TTNtv– sarcomere after stretch. The apparent A-band titin length is increased (by ~236 nm per every micrometer increment of sarcomere length), but the MIR epitopes remain in register. The M-band–to–A170 epitope distance is increased upon sarcomere stretch, which is most likely due to structural changes in the TK domain related to its mechanosensory function (ref. 36). (D) TTNtv+ sarcomere after stretch. The apparent A-band titin length is increased, but to a smaller degree than in the TTNtv– sarcomere. The MIR epitopes on the normal and truncated titin molecules approach each other, resulting in a relative narrowing of the STED intensity profile (see Supplemental Figure 4A). The M-line–to–A170 epitope distance becomes reduced upon sarcomere stretch due to a mechanically driven ordering, indicated by a straightened M-band.