Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

The specific cleavage of titin springs and severing of actin filaments to determine the contribution of sarcomeric elements to cardiomyocyte viscoelasticity.
J. K. Freundt1, A. Unger1, C. Loescher1, W. A. Linke1
1Institut für Physiologie II, Universitätsklinikum Münster, Münster;

Question: Pathological changes to cardiac stiffness impair the function of the diseased heart, notably in heart failure (HF) with preserved ejection fraction. The giant sarcomere protein titin bears passive load in cardiomyocytes (CMs), and increased titin-based stiffness occurs in HF. Other cytoskeletal structures, such as actin filaments, also contribute to total CM stiffness. Actin and titin interact in the sarcomeric I-band. Here, we aimed to quantify the contribution of titin and actin filaments to CM passive stiffness, using a new genetic mouse model that allows the specific and acute cleavage of the titin springs.

Methods & Results: In the mouse model, a tobacco etch virus (TEV) protease-recognition site and a HaloTag were cloned into elastic titin (HaloTag-TEV knock-in (KI)). This cassette allows for specific in-situ cleavage of titin during mechanical measurements of permeabilized mouse CMs and visualization of successful cleavage by tracking the fluorescence signal of cells incubated with fluorophore-conjugated HaloLigand (which binds covalently to HaloTag), using confocal microscopy or protein gel electrophoresis. Recombinant TEV protease caused the rapid, specific, and complete cleavage of cardiac titin in homozygous KI heart samples, and   ̴50% cleavage in heterozygous KI. In CMs stained with HaloLigand-Alexa488, HaloTag-TEV titin was equally distributed within individual cells and throughout all cells. Single CMs isolated from homozygous KI hearts were stretched and the resulting force recorded before/after cleavage of titin using TEV protease. The specific titin cleavage resulted in a 61±5% (n=10) reduction in elastic force. Actin filaments were severed using a Ca2+-independent gelsolin fragment. This treatment reduced the elastic force of single CMs by 30±6% (n=7). Cleavage of titin first and actin second decreased the total force by 67±4% (n=10) while cleavage of actin first and titin second reduced the total force by 63±7% (n=7).

Conclusions: The HaloTag-TEV mouse allows, for the first time, the direct and reliable quantitation of the titin contribution to CM stiffness. Our findings show that the intact titin springs are responsible for the majority of the elastic forces of the mouse CM. Actin filaments contribute much less to CM elastic force than titin. The order of cleavage of these cytoskeletal structures is important, suggesting the presence of a cellular tensegrity architecture.

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