A ‘musical chairs’ approach to untangle the sources of myocardial passive stiffness

Passive stiffness measurements in heart samples of a ‘titin-cleavage’ mouse model reveal the elastic and viscous force contributions of individual myocardial components. Titin is the principal contributor to elastic forces, whereas the microtubules and titin, followed by actin, dominate the viscous force contributions; the extracellular matrix contributes at high strain.


A 'musical chairs' approach to untangle the sources of myocardial passive stiffness
Passive stiffness measurements in heart samples of a 'titin-cleavage' mouse model reveal the elastic and viscous force contributions of individual myocardial components.Titin is the principal contributor to elastic forces, whereas the microtubules and titin, followed by actin, dominate the viscous force contributions; the extracellular matrix contributes at high strain.

The problem
The passive stiffness of myocardium is essential for diastolic heart properties (such as filling and elastic recoil) and also influences systolic function, as implied by the Starling law of the heart (in which more filling means increased cardiac output).Heart failure often involves altered passive stiffness, which compromises diastolic function.The sources of passive stiffness are ill-defined, although cardiomyocytes and the extracellular matrix are clear contributors 1 .Cardiomyocytes have an intricate network of microtubules, intermediate filaments, actin-myosin assemblies and sarcomeric titin molecules 2 , all of which could contribute to passive stiffness and be altered in disease.Passive stiffness in cardiomyocytes has previously been studied by removing network members 2,3 , but this approach is challenging for titin, whose elastic region should ideally be cleaved in a specific manner.What is needed is the precise quantification of the contribution of each network member to passive stiffness, while considering that they may be interdependent and change with myocardial stretching.This information would help to identify the best targets to normalize myocardial passive stiffness for heart-failure therapeutic agents.

The solution
The problem of unspecific targeting of titin stiffness was solved by using a mouse model that enables the specific and acute cleavage of the titin springs.In this model, a genetic cassette is inserted into elastic I-band titin, which can be specifically cleaved by a tobacco etch virus protease (TEVp) 4 .In heart samples prepared from this titin-cleavage mouse, the contribution of titin to passive stiffness was quantified by comparing passive stress-strain relationships before and after incubation with TEVp.Consideration was given to both the elastic (velocity-insensitive) and the viscous (velocity-sensitive) force components of the stress-strain curves.Moreover, the contribution of titin was compared to that of other cardiomyocyte elements by biochemically removing them one by one, including the microtubules, the sarcolemma and the actin filaments.Along with this 'musical chairs' approach, the contributions from the extracellular matrix and the intermediate (desmin) filaments could be inferred by comparing cardiac fiber bundles and single cardiomyocytes.
The musical chairs approach revealed that titin is the dominant contributor to elastic forces in mouse myocardium (Fig. 1).
The lead contribution to viscous forces comes from the microtubules, although these forces are more uniformly distributed among the different myocardial elements.The contributions from the extracellular matrix greatly increase at high strain.Generally, the distribution of passive forces among individual elements varied with strain.For example, titin alone contributed over one-half of the elastic forces at 10% strain and just over onethird at 20% strain, 'losing' mainly to the matrix and the sarcolemma, whereas actin and microtubules contributed to a similar proportion at all strain levels.The microtubules contributed about 35% to viscous forces at low strain but their contribution at high strain (27%) was matched by titin and the extracellular matrix, with actin and desmin having lesser roles (Fig. 1).Importantly, no element truly acted in isolation and evidence suggested a tensegrity relationship.

The implications
Collectively, these experiments answer long-standing, fundamental questions about the passive stiffness contributions from key mechanical proteins of myocardium, in particular highlighting the critical role of titin -but also the roles of the microtubules, actin filaments and the collagen matrix.The findings also inform on potential avenues for therapeutic strategies aimed at treating pathologically stiffened hearts.Interestingly, the cleaved titin species generated are similar in size to those found in patients with heart failure who have a titin-truncation cardiomyopathy 5 , so that -by association -one could use the titin-cleavage model to explore disease pathomechanisms.
A limitation is that the musical chairs approach did not involve one and the same cardiac sample from start to finish, because such an experiment would probably result in sample damage (making the results less reliable).An obvious caveat is that the specific titin cleavage performed in this study requires the mouse model, and the results cannot immediately be extrapolated to human myocardium.
Finally, TEVp could be delivered to the cardiomyocytes, such as by using adenoassociated virus 9 subtype, to specifically cleave titin in vivo.Overexpression of TEVp in the living heart of the mouse model would enable us to gain more important insights into the role of titin stiffness for myocardial function.expeRt opinion "The Linke laboratory has made contributions to this topic over the past two decades and with their new titin Halo mouse they have a model system to tackle this in a more controlled way.In this paper, they tease apart the role of different elements one by one and come up with quantifications of their contributions to elastic and viscous force.This very conscientious work is definitely of interest to the field."Elisabeth Ehler, King's College London, London, UK.

Behind the papeR
Working on titin for more than three decades, it has always been a dream to simply cut the titin springs in situ, as one can cut a rubber band, and directly determine how this alters cardiomyocyte stiffness.A golden opportunity arrived with the titin-cleavage mouse, developed during a long-term collaboration with the team of J. Fernandez at Columbia University, New York 4 .It took my laboratory several years to establish how to perform and prove the specific titin cleavage in cardiac samples of this model.Another objective of the current study was to track the cleavage not only of titin but also of the other cytoskeletal structures targeted by our musical chairs approach, such as by fluorescence.This was particularly challenging for the microtubules, as their stability is sensitive to temperature and membrane permeabilization.We are excited to see how our efforts have worked out.W.A.L.

"
This work by Loescher et al. stood out to me because it shows the individual contributions from the diverse mechanical elements to the total passive stiffness of the myocardium, which has been a bottleneck in the field.The elegance and creativity they applied to solve this problem -a riddle that has long tormented the field -will be an important reference for future research."Elisa Martini, Associate Editor, Nature Cardiovascular Research.

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