Titin-actin Interaction Research Articles

Fitting a model to multiscale data suggests thick filament activation can produce force depression in muscle fibers.

Multiscale muscle models aim to connect muscle's molecular and organ-scale function to, e.g., inform the treatment of muscle diseases. Current muscle models have not quantitatively fit data spanning the molecular to cellular scales due, in part, to a lack of self-consistent multiscale data. To address this gap, we measured the force response from single skinned rabbit psoas muscle fibers to ramp shortenings (5% amplitude) and step stretches (0.25%, 0.5%, 0.75% amplitude), performed on the plateau region of the force-length relationship. We isolated myosin from the same muscle source and, under similar conditions, performed single molecule and mini ensemble measurements of event duration and displacement using optical trapping and in vitro motility assays. We fit the fiber data by developing a partial differential equation model that includes thick filament activation, whereby an increase in force on the thick filament pulls myosin out of the superrelaxed state, a series elastic element and a parallel elastic element. This parallel elastic element models a titin-actin interaction proposed to account for the increase in isometric force following stretch (residual force enhancement). By optimizing the model fit to fiber measurements, we specified seven unknown parameters. The model then successfully predicted our molecular measurements from the optical trap and in vitro motility at different ATP concentrations. The success of the model suggests that our multiscale data are self-consistent and can serve as a testbed for other multiscale modeling efforts. Moreover, the model captures the decrease in isometric force observed in our muscle fibers after active shortening (force depression), suggesting a molecular mechanism for force depression, whereby a parallel elastic element combined with thick filament activation serve to decrease the number of cycling cross-bridges.

Cross-Bridges and Sarcomeric Non-cross-bridge Structures Contribute to Increased Work in Stretch-Shortening Cycles.

Stretch-shortening cycles (SSCs) refer to the muscle action when an active muscle stretch is immediately followed by active muscle shortening. This combination of eccentric and concentric contractions is the most important type of daily muscle action and plays a significant role in natural locomotion such as walking, running or jumping. SSCs are used in human and animal movements especially when a high movement speed or economy is required. A key feature of SSCs is the increase in muscular force and work during the concentric phase of a SSC by more than 50% compared with concentric muscle actions without prior stretch (SSC-effect). This improved muscle capability is related to various mechanisms, including pre-activation, stretch-reflex responses and elastic recoil from serial elastic tissues. Moreover, it is assumed that a significant contribution to enhanced muscle capability lies in the sarcomeres itself. Thus, we investigated the force output and work produced by single skinned fibers of rat soleus muscles during and after ramp contractions at a constant velocity. Shortening, lengthening, and SSCs were performed under physiological boundary conditions with 85% of the maximum shortening velocity and stretch-shortening magnitudes of 18% of the optimum muscle length. The different contributions of cross-bridge (XB) and non-cross-bridge (non-XB) structures to the total muscle force were identified by using Blebbistatin. The experiments revealed three main results: (i) partial detachment of XBs during the eccentric phase of a SSC, (ii) significantly enhanced forces and mechanical work during the concentric phase of SSCs compared with shortening contractions with and without XB-inhibition, and (iii) no residual force depression after SSCs. The results obtained by administering Blebbistatin propose a titin-actin interaction that depends on XB-binding or active XB-based force production. The findings of this study further suggest that enhanced forces generated during the active lengthening phase of SSCs persist during the subsequent shortening phase, thereby contributing to enhanced work. Accordingly, our data support the hypothesis that sarcomeric mechanisms related to residual force enhancement also contribute to the SSC-effect. The preload of the titin molecule, acting as molecular spring, might be part of that mechanism by increasing the mechanical efficiency of work during physiological SSCs.

I-Band Titin Interaction with Myosin in the Muscle Sarcomere during Eccentric Contraction: The Titin Entanglement Hypothesis

The mechanisms underlying muscle contraction have largely been defined within a two filament, actin and myosin based model. The discovery of a third filament decades later called titin, has yet to be adequately incorporated into contemporary thinking, despite mounting evidence for the significance of this molecular spring. Upon muscle stretch, this titin spring works to restore muscles to their resting length by developing a passive force. The magnitude of this passive force can be quite variable due to titin's dynamic nature, but one of the most significant changes to force development may arise from a reversible titin truncation through transient binding to neighboring actin filaments. When titin was fluorescently labelled within the muscle myofibril, segments of I-band titin were tracked during passive and active stretch in order to evaluate a titin-actin interaction. Interestingly, a titin-actin binding was not observed, but unexpectedly, a titin-myosin entanglement ensued whereby parts of I-band titin overlapped spatially with myosin filaments. When muscles were activated, the distal Ig and part of the PEVK region became firmly anchored to the myosin thick filament, while the proximal Ig and PEVK regions were forced to elongate dramatically to accommodate the muscle lengthening. This pattern remained until a threshold stress was achieved, after which point titin was rapidly freed from myosin, and the elongation pattern reversed. After this threshold, the pattern of active titin elongation resembled that of the passive titin elongation. This previously unimagined mechanism has the potential to explain some of the departures from theory that the two filament model cannot, and may have great implications for muscle contraction.

A novel three-filament model of force generation in eccentric contraction of skeletal muscles.

We propose and examine a three filament model of skeletal muscle force generation, thereby extending classical cross-bridge models by involving titin-actin interaction upon active force production. In regions with optimal actin-myosin overlap, the model does not alter energy and force predictions of cross-bridge models for isometric contractions. However, in contrast to cross-bridge models, the three filament model accurately predicts history-dependent force generation in half sarcomeres for eccentric and concentric contractions, and predicts the activation-dependent forces for stretches beyond actin-myosin filament overlap.

S100A1: a major player in cardiovascular performance.

Calcium cycling is a major determinant of cardiac function. S100A1 is the most abundant member of the calcium-binding S100 protein family in myocardial tissue. S100A1 interacts with a variety of calcium regulatory proteins such as SERCA2a, ryanodine receptors, L-type calcium channels and Na(+)/Ca(2+) exchangers, thus enhancing calcium cycling. Aside from this major function, S100A1 has an important role in energy balance, myofilament sliding, myofilament calcium sensibility, titin-actin interaction, apoptosis and cardiac remodeling. Apart from its properties regarding cardiomyocytes, S100A1 is also important in vessel relaxation and angiogenesis. S100A1 potentiates cardiac function thus increasing the cardiomyocytes' functional reserve; this is an important feature in heart failure. In fact, S100A1 seems to normalize cardiac function after myocardial infarction. Also, S100A1 is essential in the acute response to adrenergic stimulation. Gene therapy experiments show promising results, although further studies are still needed to reach clinical practice. In this review, we aim to describe the molecular basis and regulatory function of S100A1, exploring its interactions with a myriad of target proteins. We also explore its functional effects on systolic and diastolic function as well as its acute actions. Finally, we discuss S100A1 gene therapy and its progression so far.

Static Stiffness in Slow and Fast Mouse Muscle Fibers Expressing Different Titin Isoforms

We showed previously (Bagni et al., 2002) that most of the increase of muscle fiber stiffness during the early phases of a tetanic contraction is due to a non-crossbridge sarcomere component whose stiffness (called static stiffness) increases after stimulation with a time course very similar to the internal Ca2+ concentration. This led us to speculate that Ca2+ concentration, in addition to promote crossbridge formation, could also leads to a stiffening of a sarcomere structure, identified with the titin filament, either directly or through a titin-actin interaction leading to the observed sarcomere stiffness increase. According to this hypothesis, it is expected that static stiffness has different properties in muscles expressing titin with different mechanical properties. Therefore we compared the static stiffness values in soleus and EDL adult mouse muscles, which express titin isoforms with long and short PEVK segment, respectively. Considering that Ca2+ binding to E-rich motifs in the PEVK segment increases its bending rigidity, the higher proportion of these motifs in EDL compared to soleus is expected to lead to a greater static stiffness in EDL. Our results showed that in agreement with the titin hypothesis, the static stiffness measured in single fibers at 25°C was more than five times greater in EDL than in soleus and about two times greater than previously reported on FDB muscle. The static stiffness time course in EDL was about the same as in FDB but slightly faster than in soleus, and it became much faster at 35°C in both EDL and soleus similarly to tension time course. These results are in agreement with the idea that static stiffness depends on the increment of titin stiffness due to the interaction between Ca2+ and E-rich motifs in PEVK segment.

Titin-Isoform Dependence of Titin-Actin Interaction and Its Regulation by S100A1/Ca2+in Skinned Myocardium

Titin, also known as connectin, is a large filamentous protein that greatly contributes to passive myocardial stiffness. In vitro evidence suggests that one of titin's spring elements, the PEVK, interacts with actin and that this adds a viscous component to passive stiffness. Differential splicing of titin gives rise to the stiff N2B and more compliant N2BA isoforms. Here we studied the titin-isoform dependence of titin-actin interaction and studied the bovine left atrium (BLA) that expresses mainly N2BA titin, and the bovine left ventricle (BLV) that expresses a mixture of both N2B and N2BA isforms. For comparison we also studied mouse left ventricular (MLV) myocardium which expresses predominately N2B titin. Using the actin-severing protein gelsolin, we obtained evidence that titin-actin interaction contributes significantly to passive myocardial stiffness in all tissue types, but most in MLV, least in BLA, and an intermediate level in BLV. We also studied whether titin-actin interaction is regulated by S100A1/calcium and found that calcium alone or S100A1 alone did not alter passive stiffness, but that combined they significantly lowered stiffness. We propose that titin-actin interaction is a “viscous break” that is on during diastole and off during systole.

Transcriptional and Posttranslational Modifications of Titin

See related article, pages 87–94 Myocardial diastolic stiffness has been variably attributed to extracellular matrix composition, cytoskeletal properties of cardiomyocytes, or residual diastolic crossbridge cycling because of incomplete relaxation or cytosolic calcium removal.1 Extracellular matrix and cardiomyocyte cytoskeleton are presumed to mediate chronic rises in myocardial diastolic stiffness, as occur during aging, pressure overload or heart failure, whereas residual diastolic crossbridge cycling accounts for acute changes, as observed during ischemia, exercise, or pharmacological interventions. The elegant study by Kruger et al, published in this issue of Circulation Research , challenges this conceptual framework.2 The study demonstrates that protein kinase (PK)G is capable of phosphorylating the giant cytoskeletal protein titin, as previously reported for PKA3,4 and that phosphorylation by PKG or PKA of a serine residue within the N2B fragment of titin leads to an acute fall in cardiomyofibrillar stiffness. An acute effect produced by a cytoskeletal protein invalidates the concept of distinct mediators for chronic or acute changes in myocardial diastolic stiffness. From these and other recent observations it becomes evident that the cytoskeletal protein titin can alter myocardial diastolic stiffness, both acutely and chronically, through multiple mechanisms such as isoform shifts, phosphorylation by PKG or PKA, and titin–actin interaction at the Z-disc (Figure). Figure. Titin alters cardiomyocyte stiffness through isoform shifts, phosphorylation, and titin–actin interaction. A, Sarcomeric structure with detailed view of I-band region of N2B titin isoform showing tandem immunoglobulin (Ig), N2B, and elastic PEVK segments. B through D, Shift from N2B to N2BA titin isoform (B) and phosphorylation by PKG or PKA at S469 (C) reduce stiffness of the elastic PEVK segment, whereas …

Matrix, Cytoskeleton, or Myofilaments: Which One to Blame for Diastolic Left Ventricular Dysfunction?

During cardiac filling, the relative contribution of distension of interstitial collagen, of distension of cytoskeletal proteins, and of low-grade diastolic cross-bridge cycling in the generation of diastolic left ventricular (LV) pressure remains unclear. In failing myocardium, interstitial collagen deposition and cross-linking are augmented. This increase in collagen deposition is accompanied by expression of a stiffer isoform of titin in the cardiomyocytes. Higher diastolic stiffness of failing myocardium is therefore not necessarily related to increased interstitial collagen content. Moreover, phosporylation of titin by protein kinase A and G, and inhibition of titin-actin interaction by cytosolic calcium allow for dynamic modulation of its diastolic tension generation and could account for acute shifts of myocardial distensibility. Acute shifts of myocardial distensibility, as occur in hypertrophy or in demand ischemia, have usually been attributed to a diastolic resurgence of cross-bridge interaction. In hypertrophied and failing myocardium, the recent demonstrations of diastolic calcium release from the ryanodine receptor, of deficient diastolic calcium removal from the cytosol, and of enhanced myofilamentary calcium sensitivity support residual diastolic cross-bridge interaction. In demand ischemia, the role of calcium overload in the reduction of diastolic LV distensibility is less clear because of correction of the reduced diastolic LV distensibility by quick stretches but not by a calcium desensitizer. Simultaneous imposition in animal models of multiple molecular changes involving interstitial, cytoskeletal, and myofilamentary proteins could elucidate their relative importance for myocardial stiffness and lead to selective correction of diastolic LV dysfunction as a novel mode of heart-failure therapy.

Titin–Actin Interaction in Mouse Myocardium: Passive Tension Modulation and Its Regulation by Calcium/S100A1

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK–actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK–actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK–actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin’s extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1–PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.

Titin-thin filament interaction and potential role in muscle function.

Titin (connectin) is a giant polypeptide that forms a single-molecule filamental structure extending from the M-line to the Z-line in the sarcomere of striated muscle. The primary structure of titin consists mainly of repeats of two types of approximately 100-amino acid modules (fibronectin type III and immunoglobulin-like motifs, respectively) and a Pro rich segment named the PEVK domain. The I-band region of titin shows an elasticity important to the passive properties of the myofibril. To investigate the biological function of titin, we cloned cDNA segments encoding single or linked structural modules of titin into expression vectors to produce non-fusion titin fragments in E. coli. High level expression of titin fragments was achieved and effective purification procedures were developed. We also developed specific monoclonal antibodies against the titin fragments and solid-phase protein-binding assays to investigate the interaction of the titin structural modules and other sarcomeric proteins. The results show that the immunoglobin-like module that are enriched in the I-band titin binds to F-actin. In contrast to the rigid association of A-band titin with the thick filament, the relatively weak titin-actin binding suggests that the I-band titin may reversibly interact with the thin filament during muscle contraction. This hypothesis is supported by an epitope similarity between the actin-binding site of caldesmon and the immunoglobulin-like module of titin, which suggests analogous functions of caldesmon and titin in organizing the contractile proteins. Together with data from mechanical studies demonstrating that the titin-actin interaction may contribute to the passive property of cardiac muscle in a Ca(2+)-dependent manner, we speculate that the Ca(2+)-mediated thin filament regulation may coordinate the function of titin during muscle contraction and relaxation.