Titin-based force regulates cardiac myofilament structures mediating length-dependent activation

Abstract

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, acts as a spring in the I-band, applying a SL-dependent force suggested to pull against and alter myofilaments in a way that supports the Frank-Starling effect. To evaluate this, we employed the titin cleavage (TC) model, where a tobacco-etch virus protease recognition site is inserted into distal I-band titin and allows for rapid, specific cleavage of titin in an otherwise-healthy sarcomere. Here, we evaluated the atomic-level structures of amyopathic cardiac myofilaments following 50% titin cleavage under passive stretch conditions using small-angle X-ray diffraction, which measures these structures under near-physiological (functional) conditions. We report that titin-based forces in permeabilized papillary muscle regulate both thick and thin myofilament structures clearly supporting titin's role in the Frank-Starling mechanism.

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

A. Schematic of 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-I. Analysis of myofilament structures at different sarcomere lengths (expressed as length relative to slack (L_o)), before (gray) and after (blue) 50% titin cleavage. Both the technical diffraction nomenclature, as well as what they represent, are included. Overlaid are the main effects ANOVA results for length (L) and treatment (T). Interaction main effects were never significant (P>0.05). J. Regression analysis between √M3 intensity (√I_M3) and A6 spacing (S_A6), with R² and P-value included. K. A summary of our findings for sarcomeric structures at short (top) and long (bottom) lengths. We directly demonstrate a relationship between titin-based forces and the myosin head OFF-to-ON transition. Datasets are generated from papillary muscle preparations of 28 heterozygote TC hearts, age range 4–9 months, and presented as mean±s.e.m.