Decrypting the contribution of titin and other cytoskeletal elements to cardiac force generation at the cellular level

• Applicant: Christine Loescher, Ph.D.
• Subject Area: Anatomy and Physiology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 511358296

The failing heart is characterized by multi-level remodeling, including myocardial stiffening and diastolic dysfunction. Apart from extracellular matrix stiffening, failing hearts show stiffened cardiomyocytes (CMs), notably in heart failure with preserved ejection fraction. A major source of myocardial stiffness has been attributed to the giant elastic CM protein titin. However, recent evidence has suggested that changes to the cellular microtubule network occur in heart failure and could have a larger contribution to myocardial stiffness than first thought. The microfilament (actin) network also contributes to CM mechanical strength and interactions with titin make their individual contribution to CM stiffness difficult to determine. Therefore, a critical re-evaluation of the contribution of titin to CM stiffness, in comparison to other cytoskeletal elements, is required. Two major challenges arise in this context: First, only limited consideration has been given to how titin may be involved with overall cellular architecture and mechanical strength. This issue is amplified by titin traditionally being studied in permeabilized fibers which eliminates other contributors to passive stiffness, such as interactions with the microtubules. 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. The same applies to the role of titin stiffness in regulating CM active force generation, which remains to be tested directly. We have now overcome this challenge with a titin-cleavage (TC) mouse model that allows the acute, specific, and complete cutting of the titin springs. This model also contains a HaloTag in elastic titin for specific labelling by fluorescent HaloLigand. Using the TC model, I aim to decrypt the contributions of titin, the microtubules and actin filaments to CM passive mechanics in healthy mice. I will test the hypothesis that titin is the major contributor of CM stiffness in native and permeabilized CMs, even when considering interactions with other cytoskeletal elements. I will also determine how titin cleavage in CMs modulates the production of active force. I postulate that titin stiffness is critical in regulating the development of active CM force with a reduction in active force detected after titin cleavage. The outcome of these studies will enable a better quantification of the sources of myocardial stiffness and help understand how changes to individual cytoskeletal elements may affect the progression of heart failure and cardiomyopathy. It will also aid in linking native and permeabilized studies. Crucial insight into the interplay between passive stiffness and active force generation can be gained.

• DFG Programme: Research Grants