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

Abstract

The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca 2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA. myofilament length-dependent activation | small-angle X-ray diffraction | rat | passive force | fluorescent probes T he Frank-Starling law of the heart describes a cardiac regulatory control mechanism that operates on a beat-to-beat basis (1). There is a unique relationship between ventricular endsystolic volume and end-systolic pressure that is determined by cardiac contractility. As a result, ventricular stroke volume is directly proportional to the extent of diastolic filling. In conjunction with heart rate and contractility, the Frank-Starling mechanism constitutes a major determinant of cardiac output. Although the Frank-Starling mechanism has been well established for well over a century, the molecular mechanisms underlying this phenomenon are not resolved (1). At the cellular level, an increase in sarcomere length (SL) results in an immediate increase in twitch force development. Existing data, mostly derived from permeabilized isolated myocardium, strongly support the notion that this phenomenon is due to an increase in the Ca 2+ responsiveness of the cardiac contractile apparatus, a phenomenon termed "myofilament lengthdependent activation" (LDA) (1).The mechanism by which the mechanical strain signal is transduced by the cardiac sarcomere is not known. We have recently demonstrated that LDA develops within a few milliseconds following a change in SL (2), a finding suggestive of a mole...

Myosin filament activation in the heart is tuned to the mechanical task

“…LDA is generally attributed to the SL modulation of the sensitivity of the thin filament to cytosolic Ca 2+ concentration combined with the fact that, during heart contraction, [Ca 2+ ] i undergoes a transient increase that may not reach the full activation level (8,9,11). The molecular mechanisms underlying LDA remain largely unknown, although LDA has been shown to be modulated by several factors, such as the degree of phosphorylation of sarcomeric proteins and cardiomyopathy causing mutations in these proteins (14)(15)(16)(17)(18)(19)(20)30). X-ray diffraction provides a unique tool for in situ investigation of the structural changes undergone by the myofilaments during the cardiac contraction-relaxation cycle.…”

“…For a given [Ca 2+ ] i , the maximal force is larger at longer sarcomere lengths [SLs; length-dependent activation (LDA)], which is the cellular basis of the Frank-Starling law of the heart (10,11). LDA is the result of a chain of yet undefined events relating a mechanosensor of the SL to the number of force-generating motors and consequently the force (12, 13), and moreover, it is modulated by the degree of phosphorylation of contractile (Myosin Regulatory Light Chain), regulatory [Troponin I on the thin filament and myosin-binding protein C (MyBP-C) in the thick filament], and cytoskeletal proteins (14)(15)(16)(17)(18)(19)(20). We exploited the nanometer-micrometer-scale X-ray diffraction possible at beamline ID02 of the European Synchrotron Radiation Facility (ESRF) to record the structural changes undergone by the thick filament and the myosin motors during the systole-diastole cycle in intact trabeculae from rat cardiac ventricle under SL control.…”

Radiação de Síncrotron no Brasil UVX e Sirius

“…O alto brilho dos síncrotrons continua a contribuir para a compreensão mais profunda da regulação muscular, especialmente explorando a estrutura fina de interferência dos motores de miosina observada no padrão de difração de raios X de ângulo pequeno. Novos mecanismos foram revelados no funcionamento dos músculos esquelético (8) e cardíaco (9). Embora os métodos de dispersão e de difração sejam capazes de proporcionar uma imagem quantitativa estrutural e dinâmica da matéria macia e dos materiais biológicos não cristalinos, a informação espacial real é necessária numa série de aplicações.…”

“…Estas novas fontes são: MAX IV (3 GeV, Suécia, em operação), Sirius (3 GeV, Brasil, 2019), ESRF (6 GeV, França, 2020). O termo quarta geração também é usado para designar laser de raios X de elétrons livres, baseado em aceleradores lineares (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) GeV) e onduladores muito longos (100/150 m). XFEL é um novo tipo de fonte de raios X, complementar às fontes RS, mas com um aumento da intensidade de pico em cerca de 10 ordens de grandeza.…”

Autocorrelation Patterns in the Left-Ventricular Pressure Course of Isolated Working Hearts at Sinus Rhythm

“…up to the highest possible ventricular stretching [19,21] and responds very sensitively (e.g., eightfold twitch force by 15% fiber length increase [19]). The response curve is adjusted (shifted) by other regulatory pathways and attributed to Ca 2+ sensitivity of the myocardial filament which increases with precontraction sarcomere length (length dependent activation) [20,22,23,24]. As standing to reason, this effect has been thoroughly investigated by sudden preload changes [25,26] and in the presence of spontaneous [16] or artificially induced ventricular arrhythmia [27,28].…”