Titin-Based Force Modulates Cardiac Thick and Thin Filaments

The Frank-Starling law states that the heart’s stroke volume increases with greater preload due to increased venous return, allowing the heart to adapt to varying circulatory demands. Molecularly, increasing preload increases sarcomere length (SL), which alters sarcomere structures that are correlated to increased calcium sensitivity upon activation. The titin protein, spanning the half-sarcomere (Fig. 1A), acts as a spring in the I-band, applying an SL-dependent force suggested to pull against and alter myofilaments, critically supporting the Frank-Starling effect.¹ Studies of diseased, genetically modified, or otherwise damaged titins have made clear that titin-based forces play a crucial role in the etiology of heart failure (see references within¹). However, the disease state obscures titin’s role, impeding therapeutic advances. We solved this problem using the titin-cleavage (TC) mouse model, where a tobacco-etch virus protease (TEV_P) recognition site is inserted into distal I-band titin allowing for the study of rapid, specific cleavage of titin within the same sample. The method is state-of-the-art for immediate reductions to titin-based force and stiffness in an otherwise-healthy sarcomere.² Here, we evaluated the atomic-level structures of amyopathic permeabilized papillary myofilaments following 50% titin cleavage (100% cleavage unsuitable) under passive stretch conditions using small-angle X-ray diffraction (Fig. 1B)³.

Figure 1. Molecular changes to cardiac sarcomeres after 50% titin cleavage.

A. A half-sarcomere with myosin heads shown in ON (green) and off (gray) states. In the titin-cleavage model, I-band titin is cleavable at the TEV protease recognition site (scissors). B. An X-ray diffraction pattern of permeabilized TC papillary muscle, with markers of interest labeled. C-E. Analysis of myofilament structures at different sarcomere lengths (expressed as L_o), before (gray) and after (blue) 50% titin cleavage. For each parameter, we used a mixed-model ANOVA, with fixed effects length (L;*), treatment (T;#), interaction (I), and a repeated-measures random effect (individual). Model assumptions were assessed via residual analysis. The main effect P-values overlay panels. Regression analysis between √M3 intensity (√I_M3) and A6 spacing (S_A6). Arrows cross through treatment means. F. Experimental findings for myofilaments at short (top) and long (bottom) lengths. We directly demonstrate a relationship between titin-based forces and the myosin head OFF-to-ON transition. Methods: Diffraction patterns were collected from permeabilized papillary muscles in 25°C relaxing solution at initially slack length (100% L₀; ~1.9 μm SL)² and at 110% L₀, then incubated with TEV_P (100 units acTEV in 300 ml relaxing solution) for 20 minutes, rinsed, and the protocol repeated. Analysis described previously.³ Datasets are generated from 28 papillary muscles from 28 unique hearts, age range 4–9 months, and presented as mean±s.e.m.

The myofilament lattice spacing is influenced by titin-based forces and was evaluated via the 1,0 spacing (D₁₀; Fig. 1C).¹ Before and after TEV_P treatment, D₁₀ decreased with increasing SL, though the lattice was expanded after treatment, as was expected upon titin cleavage.³ We quantified myofibrillar and myofilament orientation (Angle σ; Fig. 1C)¹, a determinate of cardiomyopathies¹, and found greater disorientation after titin cleavage, highlighting a role in papillary-wide order.

Increasing titin-based forces at longer SLs could stretch the cardiac thick filament, accompanied by a transition of some myosin heads from an OFF to an ON conformation, as supported by X-ray¹ and evaluation of florescent probes attached to the myosin regulatory light chains.⁴ This mechanism may increase the myosin head’s ability to form crossbridges, contributing to the Frank-Starling effect.¹ Here, we show that the M6 spacing (S_M6; Fig. 1C), a measure of thick filament length, increases at the longer SL and decreases with 50% titin cleavage. We further observed myosin head OFF-to-ON transition with increasing SL before cleavage, as indicated by increasing M3 spacing (S_M3; Fig. 1D; axial periodicity of crowns), decreasing √M3 intensity (√I_M3; Fig. 1D; proportional to number of ordered heads), and increasing intensity ratio between the 1,1 and 1,0 reflections (I₁₁/I₁₀; Fig. 1D; mass distribution between thick and thin filaments). Importantly, titin cleavage decreased S_M3 and increased √I_M3, suggesting myosin head ON-to-OFF transitions; however, I₁₁/I₁₀ remained unchanged. Therefore, the radial position of myosin heads (I₁₁/I₁₀) may not change, while myosin heads still reorientate (S_M3; √I_M3). As a cautionary note, I₁₁/I₁₀ can be affected by observed lattice changes. We conclude that reducing titin-based forces leads to a myosin head ON-to-OFF transition while leaving the length-dependent OFF-to-ON transition mechanism intact.

Interestingly, thin-filament length, as measured by the A6 reflection spacing (S_A6; Fig. 1E), also increased with increasing SL, and decreased after titin cleavage, like TC skeletal muscle.³ The length change of the thin filament is perplexing, as titin is not in an ideal position to stretch it. We postulate that low-level crossbridges in passive cardiac muscle⁵ are recruited from the ON-state motors and strain the thin filament; fewer ON-state heads lead to fewer crossbridges and thus shorter thin filaments. This hypothetical mechanism is supported by a significant negative correlation between √I_M3 and S_A6 (Fig. 1E) and may also be regulated by myosin-binding protein C (MyBP-C).³ The physiological meaning of this phenomenon remains unknown.

In summary, titin-based forces in permeabilized papillary muscle regulate both thick and thin myofilament structures (Fig. 1F), clearly supporting titin’s role in the Frank-Starling mechanism. Unavoidable limitations are that permeabilization without lattice compression (we observed dextran-associated TEV_P inactivity) and experiments at 25°C naturally reduce myosin head order and partially shift heads towards the ON state, changing absolute values measured here away from intact preparations at body temperature. Nevertheless, there is no reason to believe that the directionality of the effects of reduced titin-based forces on the myofilaments is different from in vivo conditions. Identifying a strategy to rapidly cleave titin using intact TC cardiac preparations is the field’s long-term goal. A study of TC papillary muscle during contraction is the next logical step.