X-ray diffraction evidence for myosin-troponin connections and tropomyosin movement during stretch activation of insect flight muscle

Abstract

Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called "troponin bridges," by analyzing real-time X-ray diffraction "movies" from sinusoidally stretch-activated Lethocerus muscles. Observed changes in X-ray reflections arising from myosin heads, actin filaments, troponin, and tropomyosin were consistent with the hypothesis that troponin bridges are the key agent of mechanical signal transduction. The time-resolved sequence of molecular changes suggests a mechanism for stretch activation, in which troponin bridges mechanically tug tropomyosin aside to relieve tropomyosin's steric blocking of myosin-actin binding. This enables subsequent force production, with crossbridge targeting further enhanced by stretch-induced lattice compression and thick-filament twisting. Similar linkages may operate in other muscle systems, such as mammalian cardiac muscle, where stretch activation is thought to aid in cardiac ejection.S tretch activation is a striking example of mechanical signal transduction, in which stretching a partially activated muscle yields, after a delay, greater activation. It is observed to varying degrees in all striated muscles, is prominent in vertebrate cardiac muscle where it may underlie the Frank-Starling relationship (1), and is essential to flight in multiple orders of insects, which together comprise ∼75% of insect species (2) and fully half of all animal taxa (3). Since stretch activation was first reported by Pringle (4), a great deal has been learned about how muscular contraction is driven by cyclic myosin-actin interactions (5) which, in vertebrate skeletal muscles, are controlled by calcium's effect on the steric blocking action of troponin-tropomyosin (6). However, even atomic resolution models of myosin-actin (7) and the troponin complex (8, 9) fail to shed any light on the mechanism of activation by stretch. The central question of stretch activation remains: How does mechanical stress convert to myosinactin activation while the requisite [Ca 2þ ] stays constant?Recently we observed cross-bridges between thick (mainly myosin) filaments and thin (mainly actin-troponin-tropomyosin) filaments at the level of the troponin (10). These myosin-troponin connections, referred to here simply as troponin bridges, comprised about 15% of all cross-bridges identified in an insect flight muscle (IFM) quick frozen during an isometric contraction. Troponin bridges represent a newly recognized class of crossbridges, distinct from the "traditional" force-generating myosin cross-bridges that bind to actin target zones halfway between troponins in IFM (10, 11). The direct observation of troponin bridges revived a previously speculative mechanism for stretch activation (12), in which troponin bridges serve as the agent of mechanica...

Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

“…Because of the low resolution of the reconstruction (∼13 nm), it is not possible to determine the chemical identity of this bridging structure directly from our data, and caution should be exercised to not overinterpret this density. The bridging density could simply be due to some of the small number of cycling cross-bridges, known to exist even in diastole (29), special "troponin bridges" linking the thick filament directly to the troponin complex (30,31), or perhaps to myosin-binding protein C. Myosin-binding protein C is emerging as an important regulator of muscle contraction (27,(32)(33)(34). Recent evidence indicates that myosin-binding protein C may activate the thin filament via a direct interaction between its N′ domain and actin and/or tropomyosin (35,36) that may be straindependent.…”

Steric blocking mechanism explains stretch activation in insect flight muscle

“…Moreover, one can speculate about the nature of the troponin crossbridge. As Perz-Edwards et al (6) postulate, one-half of the bridge seems likely to be a standard myosin cross-bridge bound in an unusual way, perhaps involving myosin loop3 (15). Another candidate could be the N-terminal extension of the myosin essential light chain, which is present in both insect flight muscle and heart muscle and can bind to actin regulated by troponintropomyosin (19).…”

“…Flight muscles are switched on by release of Ca 2+ into the sarcoplasm; however, in insect flight muscles, Ca 2+ alone is not enough to free up the myosin-binding sites-you need stretch as well. The paper in PNAS by Perz-Edwards et al (6) shows that the stretch activation of insect flight muscle uses the same steric blocking model for controlling the actin-myosin interaction that was found in skeletal muscle. In the case of insect flight muscles, stretching the muscle causes the tropomyosin to move across the surface of actin to free up the myosin-binding sites.…”

“…Lethocerus flight muscle was an attractive model because it could be motor-driven in a precise way that facilitated stroboscopic recording. As a climax to their work, Perz-Edwards et al (6) took an oscillating flight muscle from Lethocerus indicus and placed it in a very intense and highly collimated X-ray beam at the Advanced Photon Source, Argonne National Laboratory. The muscle could be made to oscillate at 2 Hz.…”

“…Perz-Edwards et al (6) used electron microscopy to identify crossbridges between the thick (myosin) filaments and the thin (actin) filaments at the level of the troponin molecules on the actin filament. These are clearly distinct in form from the force-producing type crossbridges.…”