Review
Titin-based tension in the cardiac sarcomere: Molecular origin and physiological adaptations

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Abstract

The passive stiffness of cardiac muscle plays a critical role in ventricular filling during diastole and is determined by the extracellular matrix and the sarcomeric protein titin. Titin spans from the Z-disk to the M-band of the sarcomere and also contains a large extensible region that acts as a molecular spring and develops passive force during sarcomere stretch. This extensible segment is titin's I-band region, and its force-generating mechanical properties determine titin-based passive tension. The properties of titin's I-band region can be modulated by isoform splicing and post-translational modification and are intimately linked to diastolic function. This review discusses the physical origin of titin-based passive tension, the mechanisms that alter titin stiffness, and titin's role in stress-sensing signaling pathways.

Introduction

The cardiac sarcomere contains, in addition to actin-based thin filaments and myosin-based thick filaments, the giant protein titin (also called connectin). While the proteins that compose the thin and thick filaments are responsible for generating active force in the sarcomere, titin provides passive elasticity to muscle. This elastic property of titin, in addition to the extracellular matrix, defines the passive stiffness of muscle that resists sarcomere stretch. Most of the extracellular matrix's contribution to passive tension is due to collagen, with collagen playing a larger role towards the upper limit of the physiological sarcomere length range (Wu et al., 2000, 2002). While the extracellular matrix is also important to passive stiffness, this review will focus on titin. In addition to providing elasticity to muscle, titin is also essential for maintaining the structural integrity of the sarcomere. Diastolic filling of the beating heart is largely determined by titin-based passive tension, with changes in titin stiffness associated with diastolic dysfunction and cardiac disease.

A single titin molecule spans the half sarcomere, binding to the Z-disk and to the thin filament at its N-terminus, and binding to the thick filament and M-band towards its C-terminus (Fig. 1A). Between its thin and thick filament binding domains titin contains a large segment that behaves as a molecular spring that extends during sarcomere stretch. This spring-like segment is titin's I-band region; it centers the thick filament in the middle of the sarcomere and generates a passive restoring force that resists increases in sarcomere length. However, this portion of titin is not only a molecular spring but can also interact with thin filament proteins or possibly adjacent titin molecules. Nonetheless, it primarily defines titin-based passive tension. Titin's I-band region contains three extensible elements: 1) serially-linked immunoglobulin(Ig)-like domains, 2) the PEVK element (rich in proline (P), glutamate (E), valine (V), and lysine (K) residues), and 3) the N2B element. Changes in titin's I-band region, namely isoform splicing and post-translational modifications, directly influence titin-based passive tension. Although the I-band region of titin generates most titin-based passive tension, other regions of titin (Z-disk, A-band, and M-band) are involved in numerous cellular processes including maintaining sarcomere structure and force-dependent signaling. This review focuses on the physical concepts at the core of titin's elasticity, the physiological mechanisms that tune titin-based tension, and titin's role in mechanical stress sensing.

Section snippets

Titin-based passive tension

The importance of titin-based passive tension cannot be overstated. Drastic changes in titin isoform expression occur during neonatal cardiac development as large, compliant titin isoforms are replaced by smaller, stiffer isoforms to meet the increased needs of the newborn heart (Lahmers et al., 2004; Opitz et al., 2004; Greaser et al., 2005). Certain cardiac diseases exhibit changes in titin isoform expression that can be compensatory or contribute to the disease phenotype (Neagoe et al., 2002

Entropic force

The passive tension generated when striated muscle is stretched comes from titin and the extracellular matrix (ECM) (Wu et al., 2000). The two types of resistance are different, however, as the ECM contribution is an elastic force derived primarily from collagen's high tensile strength and intertwined matrix orientation (Weber, 1989), while titin-based passive tension is entropic in nature. This entropic force is due to titin's extensible I-band region that contains three distinct spring-like

Modulating titin stiffness

The two most recognized mechanisms for tuning titin-based passive tension are changes in isoform expression and post-translational modifications of titin. Isoform changes affect titin stiffness because the cardiac titin isoforms (N2B, N2BA, FCT) contain I-band regions of different length that are generated by titin splicing pathways.

Studying physical properties of titin I-band elements

In order to gain a better fundamental understanding of how the spring-like elements of titin's I-band region contribute to titin-based passive tension and how phosphorylation of the spring-like elements leads to changes in titin stiffness, the physical properties of the PEVK, N2B, and tandem Ig domains have been studied in isolation using single molecule techniques. Although laser tweezers are an excellent tool for probing the biomechanics of single molecules, most single molecule titin studies

Viscoelasticity

Titin's I-band region has been treated as a non-linear spring so far, but sources of viscosity also exist within cardiac myocytes. The presence of viscosity creates a phase shift between stress (force) and strain (length) as the muscle is stretched and increases the force needed to stretch the myocardium because viscosity resists motion and must be overcome during sarcomere length changes (de Tombe and ter Keurs, 1992). A schematic of how viscosity is measured is shown in Fig. 5. The viscous

Titin and signaling pathways

It is clear that titin isoform composition largely defines titin-based passive stiffness and that changes in hemodynamic load affect titin isoform expression, but what is less clear, and extremely interesting, is the pathways responsible for this adaptive dynamic. Significant contributions have recently been made towards a better understanding of titin's role in stretch-sensing and mechanical signaling. Although more work is needed to fully elucidate the details of titin's involvement, it has

Future directions

Due to its immense size and multi-functional role, there is still much to be learned about titin's contribution in cardiac health and disease. The relationship between titin isoform composition and passive tension is well-understood, but the splicing pathways that respond to cardiac insult and bias expression of titin isoforms are largely unknown. Elucidation of these splice pathways will unlock the possibility of using pharmacological therapies to modulate titin isoform expression in chronic

Editors' note

Please see also related communications in this issue by Werdich et al. (2012) and Fabritz et al. (2012).

Acknowledgments

This review was supported by NIH training grant GM084905 and an award from the American Heart Association 11PRE7370083 to B.A and by NIH HL062881 to H.G.

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