Length-dependent Activation Research Articles

Hypertrophic cardiomyopathy mutation in cardiac troponin T (R95H) attenuates length-dependent activation in guinea pig cardiac muscle fibers.

The central region of cardiac troponin T (TnT) is important for modulating the dynamics of muscle length-mediated cross-bridge recruitment. Therefore, hypertrophic cardiomyopathy mutations in the central region may affect cross-bridge recruitment dynamics to alter myofilament Ca2+ sensitivity and length-dependent activation of cardiac myofilaments. Given the importance of the central region of TnT for cardiac contractile dynamics, we studied if hypertrophic cardiomyopathy-linked mutation (TnTR94H)-induced effects on contractile function would be differently modulated by sarcomere length (SL). Recombinant wild-type TnT (TnTWT) and the guinea pig analog of the human R94H mutation (TnTR95H) were reconstituted into detergent-skinned cardiac muscle fibers from guinea pigs. Steady-state and dynamic contractile measurements were made at short and long SLs (1.9 and 2.3 µm, respectively). Our results demonstrated that TnTR95H increased pCa50 (-log of free Ca2+ concentration) to a greater extent at short SL; TnTR95H increased pCa50 by 0.11 pCa units at short SL and 0.07 pCa units at long SL. The increase in pCa50 associated with an increase in SL from 1.9 to 2.3 µm (ΔpCa50) was attenuated nearly twofold in TnTR95H fibers; ΔpCa50 was 0.09 pCa units for TnTWT fibers but only 0.05 pCa units for TnTR95H fibers. The SL dependency of rate constants of cross-bridge distortion dynamics and tension redevelopment was also blunted by TnTR95H Collectively, our observations on the SL dependency of pCa50 and rate constants of cross-bridge distortion dynamics and tension redevelopment suggest that mechanisms underlying the length-dependent activation cardiac myofilaments are attenuated by TnTR95HNEW & NOTEWORTHY Mutant cardiac troponin T (TnTR95H) differently affects myofilament Ca2+ sensitivity at short and long sarcomere length, indicating that mechanisms underlying length-dependent activation are altered by TnTR95H TnTR95H enhances myofilament Ca2+ sensitivity to a greater extent at short sarcomere length, thus attenuating the length-dependent increase in myofilament Ca2+ sensitivity.

Omecamtiv Mecarbil Abolishes Length-Mediated Increase in Guinea Pig Cardiac Myofiber Ca2+ Sensitivity

Omecamtiv mecarbil (OM) is a pharmacological agent that augments cardiac contractile function by enhancing myofilament Ca2+ sensitivity. Given that interventions that increase myofilament Ca2+ sensitivity have the potential to alter length-dependent activation (LDA) of cardiac myofilaments, we tested the influence of OM on this fundamental property of the heart. This is significant not only because LDA is prominent in cardiac muscle but also because it contributes to the Frank-Starling law, a mechanism by which the heart increases stroke volume in response to an increase in venous return. We measured steady-state and dynamic contractile indices in detergent-skinned guinea pig (Cavia porcellus) cardiac muscle fibers in the absence and presence of 0.3 and 3.0 μM OM at two different sarcomere lengths (SLs), short SL (1.9 μm) and long SL (2.3 μm). Myofilament Ca2+ sensitivity, as measured by pCa50 (−log of [Ca2+]free concentration required for half-maximal activation), increased significantly at both short and long SLs in OM-treated fibers when compared to untreated fibers; however, the magnitude of increase in pCa50 was twofold greater at short SL than at long SL. A consequence of this greater increase in pCa50 at short SL was that pCa50 did not increase any further at long SL, suggesting that OM abolished the SL dependency of pCa50. Furthermore, the SL dependency of rate constants of cross-bridge distortion dynamics (c) and force redevelopment (ktr) was abolished in 0.3-μM-OM-treated fibers. The negative impact of OM on the SL dependency of pCa50, c, and ktr was also observed in 3.0-μM-OM-treated fibers, indicating that cooperative mechanisms linked to LDA were altered by the OM-mediated effects on cardiac myofilaments.

Abstract 226: Elucidating the Mechanism of Reduced Length-dependent Activation Due to a Dilated Cardiomyopathy-associated Mutation in Tropomyosin

At the subcellular level, the Frank-Starling law of the heart is described by an increase in calcium sensitivity and force with increased sarcomere length (SL). We examine how this relationship is affected by a dilated cardiomyopathy-associated mutation in tropomyosin (D230N, denoted Tm D230N ) by measuring contractility of intact and permeabilized cardiac muscle preparations at short (2.0 μm) and long (2.3 μm) SL. Transgenic mouse hearts containing the Tm D230N mutation have significantly dilated hearts and reduced cardiac output by ~6 months of age. Intact trabeculae were electrically stimulated and paced at 1 Hz with oxygenated solution (30°C) circulating through the experimental chamber, and permeabilized preparations were bathed in solutions (15°C) of progressively increased [Ca 2+ ] for measures of steady-state force. For intact muscle we found that the Tm D230N mutation results in significantly reduced twitch forces at SL 2.0 and 2.3 μm relative to wild-type (WT). Also, WT trabeculae displayed a significant increase in twitch force upon increase in SL (as expected) but Tm D230N trabeculae did not, demonstrating a loss of SL dependence of contraction. In permeabilized preparations, maximal activation (pCa 4.5) of both WT and Tm D230N preparations exhibited significant SL-dependent increases in force. However, at submaximal Ca 2+ (pCa 5.8), where the heart operates, WT preparations had significant increases in force with increasing length (comparing SL 2.0 to 2.3 μm), while this length-dependence of force augmentation in Tm D230N was absent. The increase in pCa 50 (pCa that produces half-maximal force) going from SL 2.0 to 2.3 μm was significantly less for Tm D230N preparations compared to WT, owing to a significantly smaller increase in pCa 50 at SL 2.3 μm (the pCa 50 at SL 2.0 μm was not significantly different between WT and Tm D230N ). These results suggest that the Tm D230N mutation limits an increase in the Ca 2+ sensitivity of contraction as the muscle lengthens by damping thin filament activation. To further examine length-dependent effects of the Tm D230N mutation, future experiments will test conditions that augment cross-bridge binding/inhibition, and other models of dilated cardiomyopathy that inhibit thin filament activation. Funding: HL111197

A model of cardiac contraction based on novel measurements of tension development in human cardiomyocytes

Experimental data from human cardiac myocytes at body temperature is crucial for a quantitative understanding of clinically relevant cardiac function and development of whole-organ computational models. However, such experimental data is currently very limited. Specifically, important measurements to characterize changes in tension development in human cardiomyocytes that occur with perturbations in cell length are not available. To address this deficiency, in this study we present an experimental data set collected from skinned human cardiac myocytes, including the passive and viscoelastic properties of isolated myocytes, the steady-state force calcium relationship at different sarcomere lengths, and changes in tension following a rapid increase or decrease in length, and after constant velocity shortening. This data set is, to our knowledge, the first characterization of length and velocity-dependence of tension generation in human skinned cardiac myocytes at body temperature.We use this data to develop a computational model of contraction and passive viscoelasticity in human myocytes. Our model includes troponin C kinetics, tropomyosin kinetics, a three-state crossbridge model that accounts for the distortion of crossbridges, and the cellular viscoelastic response. Each component is parametrized using our experimental data collected in human cardiomyocytes at body temperature. Furthermore we are able to confirm that properties of length-dependent activation at 37°C are similar to other species, with a shift in calcium sensitivity and increase in maximum tension. We revise our model of tension generation in the skinned isolated myocyte to replicate reported tension traces generated in intact muscle during isometric tension, to provide a model of human tension generation for multi-scale simulations. This process requires changes to calcium sensitivity, cooperativity, and crossbridge transition rates. We apply this model within multi-scale simulations of biventricular cardiac function and further refine the parametrization within the whole organ context, based on obtaining a healthy ejection fraction. This process reveals that crossbridge cycling rates differ between skinned myocytes and intact myocytes.

Recent developments in understanding the length dependence of contractile response of skeletal muscle.

Maximal active force of skeletal muscle contraction occurs at a sarcomere length where overlap of thick and thin filaments is optimal. However, the interaction of muscle length and active force is complicated. Active force, is the force generated by energy-requiring processes. To calculate active force, passive force provided by in-parallel structures must be subtracted from total force. Sarcomere length will change during a contraction with constant muscle-tendon length, due to tendon stretch. Passive force therefore changes during the contraction. Taking this into account, it has been demonstrated that there is less length dependence of fatigue than previously thought. The remaining difference may be associated with length dependence of activation, a property that is evident with submaximal activation. The sarcomere length at which peak contraction occurs is longer than the length that gives optimal overlap of the filaments and this shift of optimal length appears to be due to increased Ca2+ sensitivity. The increased Ca2+ sensitivity occurs because at longer lengths, the myofilaments are closer together allowing greater force than expected. However, the potential for length-dependent activation has been challenged. Submaximal contractions obtained by recruitment of fewer motor units but with maximal stimulation across different muscle lengths still demonstrate length-dependent activation. In contrast, contractions with similar absolute electromyographic signal magnitude at different lengths do not demonstrate length-dependent activation. Recent work has improved our understanding of how sarcomere length impacts the force of contraction but also reveals inadequacies in our knowledge that need to be addressed by additional research.

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

The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors.

Titin Gene and Protein Functions in Passive and Active Muscle.

The thin and thick filaments of muscle sarcomeres are interconnected by the giant protein titin, which is a scaffolding filament, signaling platform, and provider of passive tension and elasticity in myocytes. This review summarizes recent insight into the mechanisms behind how titin gene mutations cause hereditary cardiomyopathy and how titin protein is mechanically active in skeletal and cardiac myocytes. A main theme is the evolving role of titin as a modulator of contraction. Topics include strain-sensing via titin in the sarcomeric A-band as the basis for length-dependent activation, titin elastic recoil and refolding of titin domains as an energy source, and Ca2+-dependent stiffening of titin stretched during eccentric muscle contractions. Findings suggest that titin stiffness is a principal regulator of the contractile behavior of striated muscle. Physiological or pathological changes to titin stiffness therefore affect contractility. Taken together, titin emerges as a linker element between passive and active myocyte properties.

Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle

The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling. At the cellular level, heart muscle cells generate higher force when stretched, but despite intense efforts the underlying molecular mechanism remains unknown. We applied a fluorescence-based method, which reports structural changes separately in the thick and thin filaments of rat cardiac muscle, to elucidate that mechanism. The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation. Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure, but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure.

The Frank-Starling Mechansim is Attenuated by a Dilated Cardiomyopathy-Associated Tropomyosin Mutation

Sarcomere length-dependent activation (LDA) of force development in cardiac muscle is the cellular basis of the Frank-Starling mechanism, and is often blunted in heart failure. In patients with dilated cardiomyopathy (DCM), LDA may be affected differently depending on the sarcomeric protein mutation underlying the disorder, thus complicating strategies for therapeutic intervention. We investigated the effects of the DCM-associated tropomyosin mutation D230N (TpmD230N) on LDA by measuring force generation and twitch kinetics at short (∼2.0μm) and long (∼2.3μm) sarcomere lengths (SL) of intact and demembranated trabeculae/papillary muscles from hearts of a transgenic murine model (>6 months of age) containing the TpmD230N mutation. Both types of preparations were mounted between a force transducer and linear motor. Intact trabeculae were perfused with oxygenated physiological solution (30°C) and stimulated at 2Hz. Demembranated muscle preparations were bathed in physiological solutions (15°C) containing varying Ca2+ concentrations and allowed to generate steady-state force. As expected, for intact WT trabeculae, the twitch force (Tp) and maximum rate of force development increased and the time to peak tension decreased when muscles were stretched from short to long SL (p<0.02). In contrast, TpmD230N trabeculae did not show significant changes in these parameters between short and long SL. There were no significant length-dependent changes in twitch relaxation kinetics for either WT or TpmD230N trabeculae. In demembranated muscle preparations, pCa50 (the [Ca2+] required to generate half-maximal force) decreased for TpmD230N preparations at both SL compared with WT preparations (with a larger effect at long SL), and the increase in pCa50 with stretch from SL ∼2.0μm to ∼2.3μm was greater for WT compared to TpmD230N preparations (p<0.05). Together, these results demonstrate an attenuated LDA in cardiac muscle of DCM TpmD230N hearts and suggest a role for tropomyosin in modulating LDA. Funding: HL111197.

Force-Dependent Recruitment of Cross-Bridges from the Myosin Off-State can Contribute to Length-Dependent Activation

Cardiac muscle cells develop more force if they are stretched before they are activated. The Ca2+ concentration that is required to produce half-maximal force also decreases. These effects are known as length-dependent activation and are thought to contribute to the Frank-Starling Mechanism. The precise mechanisms underlying length-dependent activation remain unclear. Kampourakis et al. (PMID 27162358) recently showed that increasing the length of cardiac muscle causes myosin head domains to adopt orientations that are more perpendicular to the thick filament axis.

Abnormal Cardiac Cross-Bridge Kinetics in a Troponin T ILE79ASN Transgenic Mouse Model

The missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been associated with phenotypic outcomes of familial hypertrophic cardiomyopathy, arrhythmias, and sudden cardiac death. Previous characterization of a transgenic mouse model bearing the HcTnT-I79N mutation, which expressed 52% HcTnT-I79N of total troponin T, demonstrated increased Ca2+ sensitivity of isometric force. However, little is known about the changes in cross-bridge kinetics, if any, that result from the mutation. This study investigates isometric force, sinusoidal stiffness and rate of tension redevelopment (kTR) of skinned papillary muscle fiber bundles at two sarcomere lengths (1.9 µm and 2.1 µm); cardiac muscle was isolated from the left ventricle of non-transgenic wild-type (WT) mice or transgenic mice bearing the HcTnT-I79N mutation. Sarcomere length (SL) was set at pCa8 using HeNe laser diffraction after the attachment points of each skinned fiber bundle were fixed with glutaraldehyde to minimize end compliance. Sinusoidal stiffness and kTR were obtained after recording steady-state isometric tension by oscillating the fiber bundle 0.2% of its relaxed length or by applying a ramped shortening followed by a quick re-stretch to original fiber bundle length, respectively. As previously reported, the HcTnT-I79N mutation resulted in increased Ca2+-sensitivity of isometric force although no difference in maximum force per cross-sectional area was observed. Length-dependent activation at the longer SL resulted in increased Ca2+-sensitivity of isometric force and sinusoidal stiffness, and decreased kTR for both WT and HcTnT-I79N. However, HcTnT-I79N exhibited faster kTR and greater sinusoidal stiffness at sub-maximum pCa (-log[Ca2+]) solutions compared to WT regardless of SL. These data indicate that HcTnT-I79N mutation, and SL, markedly influence not only the dynamics of individual regulatory units, but also the kinetics of cross-bridge cycling.

Comparison of the Regulation of Contraction in Insect Flight Muscle and Vertebrate Cardiac Muscle

The indirect flight muscle of insects (IFM), such as Drosophila and Lethocerus, is activated by periodic stretches at a priming concentration of calcium. The alternate activation of opposing muscles produces the rapid wing movements needed for flight. Cardiac muscle develops more force at longer sarcomere lengths (length-dependent activation), and force is enhanced by a rapid stretch at each beat. Troponin bridges between thick and thin filaments have been identified in Lethocerus IFM (Perz-Edwards et al, 2014) and these may be stretch sensors. Cardiac TnC and the TnC-F2 isoform in IFM both bind one regulatory calcium in the N-lobe. The force-pCa curve for IFM with only the TnC-F2 isoform (pCa50 5.8 and co-operativity nH 3.2) is similar to that of cardiac trabeculae, suggesting the co-operative behaviour is produced in the same way. TnC-F1, the IFM isoform needed for stretch activation, binds one calcium in the C-lobe, which is not regulatory. The force-pCa curve for IFM fibres, in which TnC-F1 is the main isoform, shows greater calcium-sensitivity and less co-operativity (pCa50 6.2, nH 1.3). The force-pCa curve of IFM fibres is similar to that of cardiac trabeculae with bound C1-C2 fragment of MyBP-C, in which calcium-sensitivity is increased and co-operativity greatly reduced. In this case, cardiac and IFM fibres achieve the same mechanical properties by different mechanisms. In order to determine if troponin forms part of a link between thick and thin filaments in IFM, we have tested binding between a tropomyosin-troponin complex and isolated thick filaments or myosin. The subunits of tropomyosin-troponin and the conditions needed for binding will be described.

Sarcomere Lengthdependent Effects on the Ca2+-Troponin Regulation in Skinned Myocardial Fiber from Titin RBM20 Deletion Mice

The length dependent activation of the myofilaments has been considered the cellular basis underlying the Frank-Starling law of the heart trough dynamic and complex interplays between a multitude of thick- and thin-filament components. Among these components, both troponin and giant protein titin are likely key players, but the mechanism, by which the length dependent myofilament Ca2+ regulation is linked functionally to titin, is elusive. In this study we investigate the link using in situ FRET technique to monitor how the length dependent Ca2+-induced conformational change of the N-domain of cTnC within myocardium is modulated by alteration of titin compliance that directly determines the passive tension of myocardium. To achieve the objective, FRET donor and acceptor modified cTnC(13C/51C)AEDANS-DDPM was reconstituted into chemically-skinned myocardial fibers from wild-type mice and RBM20 deficient mice that expresses more compliant N2BA titin. The Ca2+-induced conformational change in cTnC under different conditions were quantitatively characterized using time-resolved FRET measurements. Our results from wild type myocardium showed that sarcomere length dependent enhancement of Ca2+-troponin regulation was found to be directly associated with strong crossbridge and it was diminished when the strong crossbridge was inhibited. When measurements were performed with skinned myocardial fibers from RBM20 deletion animals, the length dependent change was blunted. These results support that titin based passive tension development of myocardium is indeed involved in sarcomere length modulated Ca2+-troponin regulation and that the strong crossbridge plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation.

Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres

The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation.

Anisotropic engineered heart tissue made from laser-cut decellularized myocardium.

We have developed an engineered heart tissue (EHT) system that uses laser-cut sheets of decellularized myocardium as scaffolds. This material enables formation of thin muscle strips whose biomechanical characteristics are easily measured and manipulated. To create EHTs, sections of porcine myocardium were laser-cut into ribbon-like shapes, decellularized, and mounted in specialized clips for seeding and culture. Scaffolds were first tested by seeding with neonatal rat ventricular myocytes. EHTs beat synchronously by day five and exhibited robust length-dependent activation by day 21. Fiber orientation within the scaffold affected peak twitch stress, demonstrating its ability to guide cells toward physiologic contractile anisotropy. Scaffold anisotropy also made it possible to probe cellular responses to stretch as a function of fiber angle. Stretch that was aligned with the fiber direction increased expression of brain natriuretic peptide, but off-axis stretches (causing fiber shear) did not. The method also produced robust EHTs from cardiomyocytes derived from human embryonic stem cells and induced pluripotent stem cells (hiPSC). hiPSC-EHTs achieved maximum peak stress of 6.5 mN/mm2 and twitch kinetics approaching reported values from adult human trabeculae. We conclude that laser-cut EHTs are a viable platform for novel mechanotransduction experiments and characterizing the biomechanical function of patient-derived cardiomyoctyes.

Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20ΔRRM) Mice.

Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20ΔRRM) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20ΔRRM animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20ΔRRM on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca2+-activated, skinned papillary muscle strips from Rbm20ΔRRM and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca2+-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20ΔRRM vs. WT mice. As sarcomere length increased from 1.9 to 2.2 μm, length-dependent activation of contraction was eliminated in the Rbm20ΔRRM myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

Titin stiffness modifies the force-generating region of muscle sarcomeres.

The contractile units of striated muscle, the sarcomeres, comprise the thick (myosin) and thin (actin) filaments mediating active contraction and the titin filaments determining “passive” elasticity. We hypothesized that titin may be more active in muscle contraction by directly modulating thick-filament properties. We used single-myofibril mechanical measurements and atomic force microscopy of individual sarcomeres to quantify the effects of sarcomere strain and titin spring length on both the inter-filament lattice spacing and the lateral stiffness of the actin-myosin overlap zone (A-band). We found that strain reduced the lattice spacing similarly in sarcomeres with stiff (rabbit psoas) or compliant titin (rabbit diaphragm), but increased A-band lateral stiffness much more in psoas than in diaphragm. The strain-induced alterations in A-band stiffness that occur independently of lattice spacing effects may be due to titin stiffness-sensing by A-band proteins. This mechanosensitivity could play a role in the physiologically important phenomenon of length-dependent activation of striated muscle.

Sarcomere length dependent effects on the interaction between cTnC and cTnI in skinned papillary muscle strips

Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how the sarcomeres detect the length changes and convert them into altered activation of thin filament. In this study we investigated how the C-domain of cardiac troponin I (cTnI) functionally and structurally responds to the comprehensive effects of the Ca2+, crossbridge, and sarcomere length of chemically skinned myocardial preparations. Using our in situ technique which allows for simultaneous measurements of time-resolved FRET and mechanical force of the skinned myocardial preparations, we measured changes in the FRET distance between cTnI(167C) and cTnC(89C), labeled with FRET donor and acceptor, respectively, as a function of [Ca2+], crossbridge state and sarcomere length of the skinned muscle preparations. Our results show that [Ca2+], cross-bridge feedback and sarcomere length have different effects on the structural transition of the C-domain cTnI. In particular, the interplay between crossbridges and sarcomere length has significant impacts on the functional structural change of the C-domain of cTnI in the relaxed state. These novel observations suggest the importance of the C-domain of cTnI and the dynamic and complex interplay between various components of myofilament in the LDA mechanism.

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

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.

The Role of Calcium Affinity and C-I Interaction in Length-Dependent Activation

The Frank-Starling response is an intrinsic property of cardiac muscle that helps to modulate cardiac output on a beat-to-beat basis. At the cellular level an increase in pressure during ventricle loading stretches the myocytes in the ventricle increasing sarcomere length (SL) and results in increased Ca2+ sensitivity and force of contraction. This mechanism is known as length-dependent activation (LDA), and multiple components of the sarcomere are believed to be involved in modulating this response. This includes changes in myofilament lattice spacing and/or myosin crossbridge orientation, affecting the probability of myosin binding to actin at longer SLs. Previous work from our lab, using the L48Q-cTnC variant, demonstrated that the inherent properties of cardiac troponin C (cTnC) maybe important in determining LDA. LDA is measured as a difference between pCa50 in force-pCa relationship at SL=2.3 and 2.0 in demembranated rat trabecula after a passive exchange of troponin complexes containing cTnC variants. Incorporation of L48Q-cTnC variant (with higher Ca2+binding affinity and increased strength of cTnC-cTnI interaction, denoted KCa and KC-I, respectively) into rat cardiac trabeculae diminished LDA, ΔpCa50= ∼0.03 compared to ∼0.1 for WT-cTn. Alternatively, L57Q-cTnC (which decreases KCa and KC-I) increases LDA, ΔpCa50= ∼0.2. The aim of the current study is to isolate changes in Ca2+ binding affinity of cTnC vs. C-I interaction on LDA using a series of cTnC variants that affect KCa and/or KC-I, as determined using fluorescently labeled cTnC. Our initial findings suggest that salmonid cTnC (ScTnC) with higher KCa, increases LDA (ΔpCa50= ∼0.2), in contrast to effects of L48Q-cTnC. In ongoing experiments we are examining the strength of ScTnC-cTnI interaction and investigating the changes in LDA caused by L48A-cTnC, which does not alter KCa but decreases KC-I.