Background: A hallmark of heart failure with preserved ejection fraction is increased myocardial stiffness due to both extracellular matrix remodeling and cardiomyocyte stiffening. A major source of cardiomyocyte stiffness has been attributed to modifications to titin stiffness. However, recent evidence has suggested that changes to the microtubule network occur in heart failure and could have a larger contribution to myocardial stiffness than first thought. Two major challenges arise when trying to compare the contributions of both the microtubules and titin to cardiomyocyte stiffness. First, microtubules are very unstable and can only be studied in intact cellular preparations, while most titin studies are performed on permeabilized fibres to allow direct access to the intracellular structures. The stiffness contribution of the sarcolemma itself has not been thoroughly investigated. Second, 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 determine and compare the contribution of the microtubules, the sarcolemma and titin to myocardial passive stiffness at multiple stretch lengths.

Methods & Results: We have developed a genetic mouse model, the titin cleavage (TC) mouse, that contains a tobacco etch virus protease (TEVp) cleavage site in the elastic titin region. Adding the TEVp allows the acute, specific, and complete (in animals homozygous for the mutation) severing of the titin springs. Using the TC homozygous mouse, fresh cardiac fibres were isolated and attached to a rig with a force transducer and motor arm and perfused with O₂ supplemented normal Tyrode’s solution. Fibres were stepwise stretched from 100- 120% of slack length before the microtubules were disrupted with 10 µM colchicine incubation for 90 min and the measurements repeated. The sarcolemma was then permeabilized by incubating with a sodium-based relaxing solution containing 0.5% Triton-X for 30 min and the stretch protocol was once again repeated. In addition, pre-permeabilized fibres were measured before and after incubation with TEVp to sever titin. At short stretch lengths (105% of slack length) titin contributed ~66% of the total elastic force, with the microtubules and sarcolemma contributing ~22% and ~12% respectively. Similarly, titin contributed ~67% of the total viscous forces at short stretch lengths and the microtubules contributed ~29%. The sarcolemma did not contribute to viscous forces at all. At long stretch lengths (120% stretch), where the extracellular matrix also starts to contribute to passive forces, titin still contributed ~44% elastic and ~41% viscous forces.  The microtubule contribution was reduced to ~13% and ~23% for elastic and viscous forces respectively. The sarcolemma still only contributed to the elastic force at long stretch lengths (~15%).    

Conclusions: Titin is the major contributor to cardiomyocyte stiffness over a range of stretch lengths. This suggests that any changes to titin stiffness that occur during the development of heart failure will have major consequences on overall myocardial stiffness and cardiac output. These findings provide insight into the fundamental organizing principles of cardiomyocyte architecture and inform on the mechanism of action for therapeutic strategies that target the stiffness of myocardium in heart failure.