More roles for the (passive) giant. Focus on “The increase in non-cross-bridge forces after stretch of activated striated muscle is related to titin isoforms”

as striated muscles, the heart and skeletal muscle both act to provide force and motion in animals; the heart rhythmically contracts and produces force to pump blood into the circulation, while skeletal muscle contracts to perform a broad array of precise and dynamic movement. The core molecular mechanism that gives rise to muscle force and movement was described in the seminal studies of A. F. Huxley and R. Niedergerke (6) and H. E. Huxley and J. Hanson, which demonstrated that muscle contraction occurs through the sliding of actin filaments past myosin. A multitude of studies have since continued to identify the force determinants and regulatory components of the striated muscle contractile machinery. In addition to delineating the precise mechanisms by which active muscle contraction occurs, an area not as well understood, but gaining considerable research interest, is the contribution and regulation of force from noncontractile proteins.

Titin (also known as “connectin”) is the largest known protein in the human body. This multifunctional muscle protein affords structural integrity and noncontractile force production. As a structural protein, titin spans half the length of a striated muscle sarcomere and acts to maintain thick filament alignment over a dynamic range of sarcomere lengths. Titin possesses a springlike quality that generates passive resistance and a restoring element in response to increases in sarcomere length. At sarcomere lengths where there is an absence of cross-bridge interaction, titin is the primary contributor of noncontractile muscle force. The contribution of muscle tension by titin is variable according to a number of key properties. The majority of titin's mass consists of immunoglobulin domains and PEVK (proline, glutamine, valine, and lysine-rich) segment repeats derived from differential splicing of isoforms. Additionally, posttranslational modifications and calcium activation can regulate the degree of titin-derived static tension (3). The importance of titin to passive tension in muscle is evident in developmental and disease states, where changes in titin isoforms lead to consequential changes in muscle elastic properties (10). Because of its dynamic function in muscle physiology and pathophysiology, a number of recent studies have attempted to elucidate titin's role in the regulation of noncontractile force.

In this issue of American Journal of Physiology-Cell Physiology, Cornachione and colleagues (1) examined the relationship between titin isoforms and non-cross-bridge-derived static tension. The authors built an elaborate single myofibril apparatus with high spatial and time resolution to detect small alterations in passive muscle force. The studies assessed calcium-activated static tension at different sarcomere lengths in three different muscle types (psoas, soleus, and cardiac ventricle) with distinct titin isoforms. The authors observed that static tension was different between muscles, as fast fiber psoas myofibrils produced more tension than slow fiber soleus myofibrils. Interestingly, cardiac myofibrils did not exhibit altered static tension under activation and stretch. This finding contrasts the role of titin in the preload-mediated increases in cardiac contractility described by the Frank-Starling mechanism (2). The absence of static tension was observed in parallel with an absence of residual force enhancement and further strengthened the case that Ca²⁺ activation of titin is responsible for both occurrences. The result also suggests that titin isoforms correlate with the physiological working range of specific muscles, as normal cardiac muscle is not stretched during active contraction.

Cornachione and colleagues also performed studies to investigate the mechanism by which titin produces noncontractile force under stretching conditions. Ca²⁺ can bind to the PEVK region of titin (8), and a prevailing proposed mechanism suggests that Ca²⁺ binds directly to titin in an activation-dependent manner, leading to increased stiffness and force. An alternative proposed mechanism suggests a dynamic regulation of actin-binding sites where titin binds to actin in an activation- and force-dependent manner that shortens its free length and increases stiffness (5, 9). In this current study, the authors demonstrated that titin is modulated by Ca²⁺ ions, and the difference in the number of PEVK segments of titin isoforms correlated to the muscle-specific differences in static tension. Conversely, static tension remained unchanged following actin filament extraction by gelsolin, providing support to refute the hypothesis that titin-actin interaction is responsible for Ca²⁺-activated tension or residual force enhancement. Thus, the Ca²⁺ dependence of the passive force-sarcomere length relationship appears to be regulated by titin stiffening and not by titin-actin interactions.

Finally, the findings from Cornachione and colleagues support the notion that titin's role as a force regulating protein in skeletal muscle contraction may be more relevant under eccentric contractions. Eccentric contractions act as a normal braking mechanism in response to greater opposing forces that can occur during daily activities and exercise. The authors put forward an interesting perspective that increased titin stiffness during eccentric contractions might be important for attenuating stretch-induced skeletal muscle injury. This concept agrees with the proposal by Herzog (4) that titin protects muscles against injuries on the descending portion of the force-length relationship, but, as mentioned above, contrasts Herzog's proposal that passive tension under stretching conditions occurs in part through titin binding directly to actin. Time will tell regarding direct titin-actin interactions, with active research ongoing. Continued research that investigates the extent of muscle damage in titin-mutant animals following eccentric contractions will further help clarify the role of titin in attenuating muscle injury. Lastly, studies investigating changes in titin stiffness and/or titin calcium sensitivity with repetitive eccentric exercise will help address titin's role in muscle adaptation to physical activity.

DISCLOSURES

D. T. Hwee is an employee of Cytokinetics, Inc. and J. R. Jasper is an employee of Revolution Medicines, Inc. and also cofounder and advisor to Altos Therapeutics, LLC.

AUTHOR CONTRIBUTIONS

D.T.H. and J.R.J. drafted the manuscript; D.T.H. and J.R.J. edited and revised manuscript; D.T.H. and J.R.J. approved final version of manuscript.