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 cleaving the titin springs acutely in otherwise normal sarcomeres.
Objective:
To determine and compare the contribution of the microtubules, the sarcolemma and titin to cardiomyocyte passive stiffness.
Methods & Results:
We have developed a genetic mouse model, the titin cleavage (TC) mouse, that contains a tobacco etch virus (TEV) protease cleavage site in the elastic titin region. Adding the TEV protease allows the acute, specific, and complete (in animals homozygous for the mutation) cleavage of the titin springs. Using the TC mouse model, fresh cardiac trabeculae 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 to 120% of slack length and then the microtubules were removed with a 10 µM colchicine incubation for 1.5 hrs before the measurements were repeated. This resulted in a 16 ± 4% (n=12), mean reduction in peak passive force at maximum stretch. The sarcolemma was then removed by incubating with a sodium-based relaxing solution containing 0.05% Triton-X for 30 min, and a mean reduction of 11 ± 5% (n=12) peak passive force was seen. Isolated TC homozygous fibres were also measured before and after skinning and then incubated with TEV protease to cleave titin. Titin removal resulted in a peak passive force reduction, on average by 48 ± 3% (n=12). The removal of each element was confirmed with immunofluorescence and Western blot.
Conclusions:
Titin is the major contributor to cardiomyocyte stiffness under healthy conditions. This suggests that any changes to titin stiffness that occur during the development of heart failure will have major consequences on overall myocardial stiffness. Understanding the stiffness relationship between the microtubules, the sarcolemma and titin will bridge the gap between intact and permeabilized cardiomyocyte research, enable a better definition of the sources of myocardial stiffness, and help understand better, how changes to individual cytoskeletal elements may affect the progression of heart failure and cardiomyopathy.