Targeted cleavage of titin reduces the transverse stiffness of cardiac fibers less than the longitudinal stiffness

Felix Alexander Wagner (Münster)1, W. A. Linke (Münster)1

1Universitätsklinikum Münster Institut für Physiologie II Münster, Deutschland


Background: Heart failure (HF) is frequently caused by dilated cardiomyopathy. In many of those patients, truncating variants of the giant sarcomere protein titin underlie disease development. Titin is a main determinant of myocardial passive stiffness and increased titin-based stiffness can be observed in HF, especially HF with preserved ejection fraction (HFpEF). The longitudinal stiffness contribution of titin has been well studied, whereas much less is known about titin’s contribution to transversal cardiac stiffness, although this stiffness component is equally important for the heart’s pump function. Previous studies of the titin contribution to myocardial stiffness have been hampered by the fact that there were no tools available to specifically eliminate this stiffness. We solved this problem by developing a titin-cleavage (TC) mouse model, in which the cardiac fibers contain a Tobacco Etch Virus-protease (TEVp) recognition site within elastic titin, which can be acutely cleaved by TEVp. Interestingly, the protein fragments obtained after titin cleavage are similar in size to the fragments found in patients with titin-truncation cardiomyopathy.

Objective: To quantify the transversal component of myocardial stiffness, especially the titin-based contribution, on isolated, permeabilized cardiac preparations of the TC mouse heart using atomic force microscopy nanoindentation.

Methods & Results: Vital sections of left ventricular trabecular muscle were prepared from TC hearts and permeabilized by incubation with 0.5% Triton-X for 20 minutes. The transversal stiffness of sections from genotypically homozygous TC hearts (allowing 100% titin cleavage, confirmed by protein gel electrophoresis) and wild type (wt) hearts was measured by AFM nanoindentation before and after 25-min incubation with TEVp in relaxing buffer. The same sites were indented before and after titin cleavage, and a paired analysis (before vs. after) of the indentation curves was performed.
We found that acute cleavage of the titin springs led to a ~25% decrease in the Young´s modulus (YM) of homozygous TC fibers (n= 143). This relative change is much less than that previously measured by us for the longitudinal stiffness contribution of titin, which was >50% of total myocardial stiffness  (Loescher et al., Nature Cardiovasc. Res. 2023; in press).
For comparison, we studied the effects of 1 M KCl treatment of the permeabilized vital sections, known to depolymerize mainly the sarcomeric thick filaments (myosin). We performed a 10-min incubation protocol with KCl, followed by careful washing. This treatment resulted in a ~40% decrease of the Young’s modulus  (n= 126). Interestingly, the stiffness decrease was associated with a notable shape change of the nanoindentation curves.  

Conclusions: Both titin and the thick filaments contribute to the transverse stiffness of myocardium. The relative contribution of titin (~25%) is less than half the contribution of titin to tensile stiffness (>50%). Since truncated titin proteins can be embedded in sarcomeres, they may destabilize the passive stiffness of the myocardial wall, contributing to dilated cardiomyopathy development.  It will be interesting to measure transversal stiffness changes also in failing hearts, and to perform other types of manipulation, such as disruptions of the actin filaments, microtubules, and the extracellular matrix.

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