1Universitätsklinikum Münster Institut für Physiologie II Münster, Deutschland
Background: The passive stiffness properties of the heart are crucial for its normal pump function. However increased myocardial stiffness is also a hallmark of heart failure with preserved ejection fraction. Various mechanical elements determine and regulate myocardial stiffness, including the extracellular matrix and cardiomyocyte proteins. Although several studies have investigated the passive stiffness properties of these elements individually, differing parameters used to study myocardial stiffness, and the interconnected nature of these elements, makes it difficult to compare their individual contributions to myocardial stiffness. In addition, although titin is recognised as a key contributor to myocardial passive stiffness, up until now, there have been no precise tools available to quantify titin’s contribution to stiffness in a direct manner, i.e. by severing the titin springs acutely in otherwise normal sarcomeres.
Objective: To dissect and compare the passive stiffness contributions of the microtubules (MTs), the sarcolemma, titin, actin, and infer contributions of the extracellular matrix (ECM) and desmin, by disrupting them one by one in healthy cardiac fibers and cardiomyocytes.
Methods & Results: To enable the specific cleavage of titin, we used non-activated cardiac fiber bundles or single cardiomyocytes from the homozygous titin cleavage-Halo mouse. This mouse contains a genetic cassette in the titin spring that is cleavable using tobacco etch virus protease (TEVp). Using a ‘musical chairs’ approach, we systematically and acutely disrupted key cytoskeletal elements and evaluated stress-strain relationships. Fibres were stepwise stretched up to a 20% strain before the MTs were disrupted with 10 µM colchicine incubation for 90 min and the measurements repeated. Similarly, the sarcolemma was disrupted with 0.5% Triton-X for 30 min. pre-permeabilized fibers and cardiomyocytes were measured before and after a 10 min incubation with TEVp to sever titin or 30 min incubation with gelsolin to disrupt actin. From this the extracellular matrix and desmin contributions were inferred. The disruption of each cytoskeletal element was confirmed biochemically and changes to the cellular substructure were examined by microscopy.
At low strain (10%) titin contributed ~55% of the total elastic force, with the MTs and actin contributing ~22% and ~23% respectively. At high strain titin still contributed ~36% elastic force with the remaining force coming from the MTs, sarcolemma, actin and ECM, contributing between ~12-18% each. At low strain the viscous forces were mostly distributed between the MTs, titin and actin (between 22-35%, each) while at high strain the viscous forces were mostly distributed between the ECM, the MTs and titin (~26% each). Desmin showed a low level contribution in all cases.
Conclusions: Titin governs myocardial elastic forces at both low and high strain with major support from the MT and actin networks. In contrast, the viscous forces are almost equally shared between the MTs, titin and the ECM/actin, depending on the strain level. However, the complex interplay between the various structural networks highlights the need to consider how changes to one network in heart failure may influence the stiffness profile of the entire cytoskeleton. Our findings answer long-standing questions about cardiac mechanical architecture and inform on therapeutic strategies that target myocardial stiffness in heart failure.