Sarcomere Length Dependence Research Articles

Dystrophic cardiomyopathy: role of the cardiac myofilaments.

Dystrophic cardiomyopathy arises from mutations in the dystrophin gene. Dystrophin forms part of the dystrophin glycoprotein complex and is postulated to act as a membrane stabilizer, protecting the sarcolemma from contraction-induced damage. Duchenne muscular dystrophy (DMD) is the most severe dystrophinopathy, caused by a total absence of dystrophin. Patients with DMD present with progressive skeletal muscle weakness and, because of treatment advances, a cardiac component of the disease (i.e., dystrophic cardiomyopathy) has been unmasked later in disease progression. The role that myofilaments play in dystrophic cardiomyopathy is largely unknown and, as such, this study aimed to address cardiac myofilament function in a mouse model of muscular dystrophy. To assess the effects of DMD on myofilament function, isolated permeabilized cardiomyocytes of wild-type (WT) littermates and Dmdmdx-4cv mice were attached between a force transducer and motor and subjected to contractile assays. Maximal tension and rates of force development (indexed by the rate constant, k tr) were similar between WT and Dmdmdx-4cv cardiac myocyte preparations. Interestingly, Dmdmdx-4cv cardiac myocytes exhibited greater sarcomere length dependence of peak power output compared to WT myocyte preparations. These results suggest dystrophin mitigates length dependence of activation and, in the absence of dystrophin, augmented sarcomere length dependence of myocyte contractility may accelerate ventricular myocyte contraction-induced damage and contribute to dystrophic cardiomyopathy. Next, we assessed if mavacamten, a small molecule modulator of thick filament activation, would mitigate contractile properties observed in Dmdmdx-4cv permeabilized cardiac myocyte preparations. Mavacamten decreased maximal tension and k tr in both WT and Dmdmdx-4cv cardiac myocytes, while also normalizing the length dependence of peak power between WT and Dmdmdx-4cv cardiac myocyte preparations. These results highlight potential benefits of mavacamten (i.e., reduced contractility while maintaining exquisite sarcomere length dependence of power output) as a treatment for dystrophic cardiomyopathy associated with DMD.

Uncovering cross-bridge properties that underlie the cardiac active complex modulus using model linearisation techniques

The properties underlying cardiac cross-bridge kinetics can be characterised by a muscle’s active complex modulus. While the complex modulus can be described by a series of linear transfer functions, the biophysical mechanisms underlying these components are represented inconsistently among existing cross-bridge models. To address this, we examined the properties commonly implemented in cross-bridge models using model linearisation techniques and assessed their contributions to the complex modulus. From this analysis, we developed a biophysical model of cross-bridge kinetics that captures the three components of the active complex modulus: (1) the elastic modulus at low frequencies that arises from allowing the proportion of cross-bridges in the post-power stroke state to increase with sarcomere length, (2) the increase in elastic modulus at high frequencies that arises from the dependence of cross-bridge strain on sarcomere velocity, and (3) the negative viscous modulus which signifies the production of work by cross-bridges arises from either a sarcomere length or strain dependence, or both, on the rate of change of cross-bridge proportion in the post-power stroke state. While a model that includes all these features can theoretically reproduce the cardiac complex modulus, analysis of their transfer functions reveals that the relative contributions of these components are often not taken into account. As a result, the negative viscous component that signifies work production is not visible because the complex modulus is dominated by the effects of sarcomere velocity on cross-bridge strain.

Myosin motors that cannot bind actin leave their folded OFF state on activation of skeletal muscle.

The myosin motors in resting skeletal muscle are folded back against their tails in the thick filament in a conformation that makes them unavailable for binding to actin. When muscles are activated, calcium binding to troponin leads to a rapid change in the structure of the actin-containing thin filaments that uncovers the myosin binding sites on actin. Almost as quickly, myosin motors leave the folded state and move away from the surface of the thick filament. To test whether motor unfolding is triggered by the availability of nearby actin binding sites, we measured changes in the x-ray reflections that report motor conformation when muscles are activated at longer sarcomere length, so that part of the thick filaments no longer overlaps with thin filaments. We found that the intensity of the M3 reflection from the axial repeat of the motors along the thick filaments declines almost linearly with increasing sarcomere length up to 2.8 µm, as expected if motors in the nonoverlap zone had left the folded state and become relatively disordered. In a recent article in JGP, Squire and Knupp challenged this interpretation of the data. We show here that their analysis is based on an incorrect assumption about how the interference subpeaks of the M3 reflection were reported in our previous paper. We extend previous models of mass distribution along the filaments to show that the sarcomere length dependence of the M3 reflection is consistent with <10% of no-overlap motors remaining in the folded conformation during active contraction, confirming our previous conclusion that unfolding of myosin motors on muscle activation is not due to the availability of local actin binding sites.

Cardiac MyBP-C phosphorylation regulates the Frank-Starling relationship in murine hearts.

The Frank-Starling relationship establishes that elevated end-diastolic volume progressively increases ventricular pressure and stroke volume in healthy hearts. The relationship is modulated by a number of physiological inputs and is often depressed in human heart failure. Emerging evidence suggests that cardiac myosin-binding protein-C (cMyBP-C) contributes to the Frank-Starling relationship. We measured contractile properties at multiple levels of structural organization to determine the role of cMyBP-C and its phosphorylation in regulating (1) the sarcomere length dependence of power in cardiac myofilaments and (2) the Frank-Starling relationship in vivo. We compared transgenic mice expressing wild-type cMyBP-C on the null background, which have ∼50% phosphorylated cMyBP-C (Controls), to transgenic mice lacking cMyBP-C (KO) and to mice expressing cMyBP-C that have serine-273, -282, and -302 mutated to aspartate (cMyBP-C t3SD) or alanine (cMyBP-C t3SA) on the null background to mimic either constitutive PKA phosphorylation or nonphosphorylated cMyBP-C, respectively. We observed a continuum of length dependence of power output in myocyte preparations. Sarcomere length dependence of power progressively increased with a rank ordering of cMyBP-C KO = cMyBP-C t3SA < Control < cMyBP-C t3SD. Length dependence of myofilament power translated, at least in part, to hearts, whereby Frank-Starling relationships were steepest in cMyBP-C t3SD mice. The results support the hypothesis that cMyBP-C and its phosphorylation state tune sarcomere length dependence of myofibrillar power, and these regulatory processes translate across spatial levels of myocardial organization to control beat-to-beat ventricular performance.

Effects of mavacamten on Ca2+ sensitivity of contraction as sarcomere length varied in human myocardium.

Heart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca2+ as cells are stretched. This represents a fundamental property of the myocardium that contributes to the Frank-Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten, which binds to myosin, is under investigation as a potential therapy for heart disease. We investigated how mavacamten affects the sarcomere-length dependence of Ca2+ -sensitive isometric contraction to determine how mavacamten might modulate the Frank-Starling mechanism. Multicellular preparations from the left ventricular-free wall of hearts from organ donors were chemically permeabilized and Ca2+ activated in the presence or absence of 0.5-μM mavacamten at 1.9 or 2.3-μm sarcomere length (37°C). Isometric force and frequency-dependent viscoelastic myocardial stiffness measurements were made. At both sarcomere lengths, mavacamten reduced maximal force and Ca2+ sensitivity of contraction. In the presence and absence of mavacamten, Ca2+ sensitivity of force increased as sarcomere length increased. This suggests that the length-dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca2+ sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross-bridge recruitment and speeding cross-bridge detachment under maximally activated conditions (pCa 4.5). Mavacamten did not eliminate sarcomere length-dependent increases in the Ca2+ sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Drugs that modulate myofilament function may be useful therapies for cardiomyopathies.

Regulation of Myofilament Contractile Function in Human Donor and Failing Hearts.

Heart failure (HF) often includes changes in myocardial contractile function. This study addressed the myofibrillar basis for contractile dysfunction in failing human myocardium. Regulation of contractile properties was measured in cardiac myocyte preparations isolated from frozen, left ventricular mid-wall biopsies of donor (n = 7) and failing human hearts (n = 8). Permeabilized cardiac myocyte preparations were attached between a force transducer and a position motor, and both the Ca2+ dependence and sarcomere length (SL) dependence of force, rate of force, loaded shortening, and power output were measured at 15 ± 1°C. The myocyte preparation size was similar between groups (donor: length 148 ± 10 μm, width 21 ± 2 μm, n = 13; HF: length 131 ± 9 μm, width 23 ± 1 μm, n = 16). The maximal Ca2+-activated isometric force was also similar between groups (donor: 47 ± 4 kN⋅m–2; HF: 44 ± 5 kN⋅m–2), which implicates that previously reported force declines in multi-cellular preparations reflect, at least in part, tissue remodeling. Maximal force development rates were also similar between groups (donor: ktr = 0.60 ± 0.05 s–1; HF: ktr = 0.55 ± 0.04 s–1), and both groups exhibited similar Ca2+ activation dependence of ktr values. Human cardiac myocyte preparations exhibited a Ca2+ activation dependence of loaded shortening and power output. The peak power output normalized to isometric force (PNPO) decreased by ∼12% from maximal Ca2+ to half-maximal Ca2+ activations in both groups. Interestingly, the SL dependence of PNPO was diminished in failing myocyte preparations. During sub-maximal Ca2+ activation, a reduction in SL from ∼2.25 to ∼1.95 μm caused a ∼26% decline in PNPO in donor myocytes but only an ∼11% change in failing myocytes. These results suggest that altered length-dependent regulation of myofilament function impairs ventricular performance in failing human hearts.

Regulation of Myofilament Contractile Function in Human Donor and Failing Hearts

Heart failure (HF) often includes changes in myocardial contractile function. This study addressed the myofibrillar basis for contractile dysfunction in failing human myocardium. Regulation of contractile properties was measured in cardiac myocyte preparations isolated from frozen, left ventricular mid-wall biopsies of donor (n = 7) and failing human hearts (n = 8). Permeabilized cardiac myocyte preparations were attached between a force transducer and a position motor, and both the Ca2+ dependence and sarcomere length (SL) dependence of force, rate of force, loaded shortening, and power output were measured at 15 ± 1°C. The myocyte preparation size was similar between groups (donor: length 148 ± 10 μm, width 21 ± 2 μm, n = 13; HF: length 131 ± 9 μm, width 23 ± 1 μm, n = 16). The maximal Ca2+-activated isometric force was also similar between groups (donor: 47 ± 4 kN⋅m-2; HF: 44 ± 5 kN⋅m-2), which implicates that previously reported force declines in multi-cellular preparations reflect, at least in part, tissue remodeling. Maximal force development rates were also similar between groups (donor: k tr = 0.60 ± 0.05 s-1; HF: k tr = 0.55 ± 0.04 s-1), and both groups exhibited similar Ca2+ activation dependence of k tr values. Human cardiac myocyte preparations exhibited a Ca2+ activation dependence of loaded shortening and power output. The peak power output normalized to isometric force (PNPO) decreased by ∼12% from maximal Ca2+ to half-maximal Ca2+ activations in both groups. Interestingly, the SL dependence of PNPO was diminished in failing myocyte preparations. During sub-maximal Ca2+ activation, a reduction in SL from ∼2.25 to ∼1.95 μm caused a ∼26% decline in PNPO in donor myocytes but only an ∼11% change in failing myocytes. These results suggest that altered length-dependent regulation of myofilament function impairs ventricular performance in failing human hearts.

Engineered Thin Filament Mutations to Study the Sarcomere Length Dependence of Cardiac Muscle Contractility

Engineered Thin Filament Mutations to Study the Sarcomere Length Dependence of Cardiac Muscle Contractility

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.

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

Mutation‐specific effects on thin filament length in thin filament myopathy

Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969.

Force-Sarcomere Length Relations in Patients with Thin Filament Myopathy Caused by Mutations in NEB, ACTA1, TPM2, TPM3, KBTBD13, KLHL40 and KLHL41

Background: Mutations in NEB, ACTA1, TPM2, TPM3, KBTBD13, KLHL40 and −41 lead to thin filament myopathies, such as nemaline myopathy, congenital fiber type disproportion and cap disease. A hallmark feature of these myopathies is muscle weakness. Here, we aimed to elucidate whether mutations in NEB, ACTA1, TPM2, TPM3, KBTBD13, KLHL40 and KLHL41 affect the maximal force generating capacity of muscle fibers. Subsequently, by determining the sarcomere length-dependence of force, we investigated whether changes in thin filament length contributed to muscle weakness.Methods: Biopsies from NM, CFTD, and CAP patients (n=39) with mutations in NEB, ACTA1, TPM2, TPM3, KBTBD13, KLHL40 or KLHL41 were compared to biopsies from healthy controls (n=9). Using permeabilized muscle fibers, maximal active tension was determined at incremental sarcomere lengths (range 2.0-3.5 µm) to obtain the force-sarcomere length relationship.Results: The maximal active tension (Fmax (in mN/mm2, mean±SEM)) was significantly lower in biopsies from NEB (46±5), ACTA1 (48±4), TPM2 (71±8), TPM3 (85±10), KBTBD13 (78±3), KLHL40 (2.8±0.2) and KLHL41 (63±4) patients compared to biopsies of controls (129±7).No shift in the force-sarcomere length relationship was observed in TPM3, TPM2, KBTBD 13, KLHL 40 and KLHL41 patients. In contrast, several patients with ACTA1 and NEB mutations showed a leftward shift of the force-sarcomere length relationship indicating shorter thin filaments. Furthermore, the slope of the descending limb of the force-sarcomere length relationship in these patients is less steep than the slope of the CTRL curve, suggesting heterogeneity of thin filaments.Conclusion: Our data suggest that mutations in NEB and ACTA1 result in changes in thin filament length. Insights in the mechanisms underlying weakness in patients with thin filament mutations are necessary to improve specific treatment strategies.

Calcium sensitivity of residual force enhancement in rabbit skinned fibers.

Isometric force after active stretch of muscles is higher than the purely isometric force at the corresponding length. This property is termed residual force enhancement. Active force in skeletal muscle depends on calcium attachment characteristics to the regulatory proteins. Passive force has been shown to influence calcium attachment characteristics, specifically the sarcomere length dependence of calcium sensitivity. Since one of the mechanisms proposed to explain residual force enhancement is the increase in passive force that results from engagement of titin upon activation and stretch, our aim was to test if calcium sensitivity of residual force enhancement was different from that of its corresponding purely isometric contraction and if such a difference was related to the molecular spring titin. Force-pCa curves were established in rabbit psoas skinned fibers for reference and residual force-enhanced states at a sarcomere length of 3.0 μm 1) in a titin-intact condition, 2) after treatment with trypsin to partially eliminate titin, and 3) after treatment with trypsin and osmotic compression with dextran T-500 to decrease the lattice spacing in the absence of titin. The force-pCa curves of residual force enhancement were shifted to the left compared with their corresponding controls in titin-intact fibers, indicating increased calcium sensitivity. No difference in calcium sensitivity was observed between reference and residual force-enhanced contractions in trypsin-treated and osmotically compressed trypsin-treated fibers. Furthermore, calcium sensitivity after osmotic compression was lower than that observed for residual force enhancement in titin-intact skinned fibers. These results suggest that titin-based passive force regulates the increase in calcium sensitivity of residual force enhancement by a mechanism other than reduction of the myofilament lattice spacing.

Force-Sarcomere Length Relations in Patients with Thin Filament Myopathy Caused by Mutations in NEB, ACTA1, TPM2 and TPM3

Mutations in the nebulin gene (NEB), skeletal muscle alpha-actin1 gene (ACTA1), beta-tropomyosin 2 gene (TPM2) and alpha-tropomyosin 3 gene (TPM3) lead to thin filament myopathies, such as nemaline myopathy (NM), congenital fiber type disproportion (CFTD) and cap disease (CAP). A hallmark feature of these myopathies is muscle weakness. Here, we aimed to elucidate the effect of NEB, ACTA1, TPM2 and TPM3 mutations on thin filament length by determining the sarcomere length-dependence of force.Quadriceps biopsies from NM, CFTD, and CAP patients (n=18) with mutations in the NEB, ACTA1, TPM2 or TPM3 were compared to biopsies from controls (n=3). Using permeabilized muscle fibers, maximal active tension was determined at incremental sarcomere lengths (range 2.0-3.5 μm) to obtain the force-sarcomere length relationship.The maximal active tension (Fmax (in mN/mm2, mean±SD)) was significantly lower in biopsies from severe NEB (18±5), mild NEB (76±5), severe ACTA1 (54±13) and severe TPM3 (95±14) patients compared to biopsies of controls (164±17), whereas no significant changes in Fmax were observed in biopsies from mild ACTA1 (139±27), mild TPM2 (1201±8) and mild TPM3 (156±13) patients. The classification of severity is based on the age of onset.No shift in the force-sarcomere length relationship was observed in mild ACTA1, TPM3 and TPM2 patients. Interestingly, in contrast to patients with ACTA1, TPM2 and TPM3 mutations, fiber preparations from both mildly and severly affected NEB patients showed a leftward shift of the force-sarcomere length relationship (a leftward shift of the force-sarcomere length relationship indicates shorter thin filaments).Our data suggest that mutations in NEB result in the most pronounced changes in thin filament length. Insights in the mechanisms underlying weakness in patients with thin filament mutations are necessary to improve specific treatment strategies.

Does Calcium Sensitivity Increase after Active Stretch in Skinned Muscle Fibres?

Introduction Isometric force after active stretch of muscles is higher than the purely isometric force at the corresponding lengths. This property is termed residual force enhancement (RFE). Active force in skeletal muscle depends on calcium attachment characteristics to troponin. Passive force has been shown to influence calcium attachment characteristics in cardiac muscle, specifically the sarcomere length-dependence of Ca-sensitivity. Since one of the mechanisms proposed to explain RFE has been the increase in passive force that results from the engagement of titin upon activation and stretch, our aim was to test whether Ca-sensitivity was changed after active stretch and whether this change was related to titin.Methods Tension-pCa curves were established in three groups of fibres for reference and active stretch contractions at a sarcomere length of 3.0μm. Group 1 (n=13) was formed of skinned fibres without any chemical treatment. Fibres in Group 2 (n=12) were treated with trypsin before establishing the tension-pCa curves. In Group 3, fibres (n=9) were osmotically compressed by dextran T-500 after treatment with trypsin.Results and discussion After active stretch, Ca-sensitivity was increased in Group 1 fibres. When titin was eliminated using trypsin, this increased Ca-sensitivity was abolished. This means that titin is probably responsible for the increase in Ca-sensitivity. The mechanism by which titin may modulate Ca-sensitivity is by reducing the myofilament lattice spacing by generating a radial force that compresses the myofilaments. However, when, in the absence of titin, fibres were compressed, they did not show any difference in Ca-sensitivity between reference and active stretch contractions. These results suggest that Ca-sensitivity increases after active stretch compared to isometric contractions and that titin must be present in order for this increase to occur.

Depressed Frank–Starling mechanism in the left ventricular muscle of the knock-in mouse model of dilated cardiomyopathy with troponin T deletion mutation ΔK210

It has been reported that the Frank–Starling mechanism is coordinately regulated in cardiac muscle via thin filament “on–off” equilibrium and titin-based lattice spacing changes. In the present study, we tested the hypothesis that the deletion mutation ΔK210 in the cardiac troponin T gene shifts the equilibrium toward the “off” state and accordingly attenuate the sarcomere length (SL) dependence of active force production, via reduced cross-bridge formation. Confocal imaging in isolated hearts revealed that the cardiomyocytes were enlarged, especially in the longitudinal direction, in ΔK210 hearts, with striation patterns similar to those in wild type (WT) hearts, suggesting that the number of sarcomeres is increased in cardiomyocytes but the sarcomere length remains unaltered. For analysis of the SL dependence of active force, skinned muscle preparations were obtained from the left ventricle of WT and knock-in (ΔK210) mice. An increase in SL from 1.90 to 2.20μm shifted the mid-point (pCa50) of the force-pCa curve leftward by ~0.21pCa units in WT preparations. In ΔK210 muscles, Ca2+ sensitivity was lower by ~0.37pCa units, and the SL-dependent shift of pCa50, i.e., ΔpCa50, was less pronounced (~0.11pCa units), with and without protein kinase A treatment. The rate of active force redevelopment was lower in ΔK210 preparations than in WT preparations, showing blunted thin filament cooperative activation. An increase in thin filament cooperative activation upon an increase in the fraction of strongly bound cross-bridges by MgADP increased ΔpCa50 to ~0.21pCa units. The depressed Frank–Starling mechanism in ΔK210 hearts is the result of a reduction in thin filament cooperative activation.

Fast Skeletal Muscle Troponin Activation Increases Force of Mouse Fast Skeletal Muscle and Ameliorates Weakness Due to Nebulin-Deficiency

The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension–pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations <1 µM), CK-2066260 increased tension of NEB KO fibers to beyond that of WT fibers. Crossbridge cycling kinetics were studied by measuring ktr (rate constant of force redevelopment following a rapid shortening/restretch). CK-2066260 greatly increased ktr at submaximal activation levels in both WT and NEB KO fiber bundles. We also studied the sarcomere length (SL) dependence of the CK-2066260 effect (SL 2.1 µm and 2.6 µm) and found that in the NEB KO fibers, CK-2066260 had a larger effect on calcium sensitivity at the long SL. We conclude that fast skeletal muscle troponin activation increases force at submaximal activation in both wildtype and NEB KO fiber bundles and, importantly, that this troponin activation is a potential therapeutic mechanism for increasing force in NM and other skeletal muscle diseases with loss of muscle strength.

Sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy

To investigate whether sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy (FSHD). Sarcomeric function was evaluated by contractile studies on demembranated single muscle fibers obtained from quadriceps muscle biopsies of 4 patients with FSHD and 4 healthy controls. The sarcomere length dependency of force was determined together with measurements of thin filament length using immunofluorescence confocal scanning laser microscopy. X-ray diffraction techniques were used to study myofilament lattice spacing. FSHD muscle fibers produced only 70% of active force compared to healthy controls, a reduction which was exclusive to type II muscle fibers. Changes in force were not due to changes in thin filament length or sarcomere length. Passive force was increased 5- to 12-fold in both fiber types, with increased calcium sensitivity of force generation and decreased myofilament lattice spacing, indicating compensation by the sarcomeric protein titin. We have demonstrated a reduction in sarcomeric force in type II FSHD muscle fibers, and suggest compensatory mechanisms through titin stiffening. Based on these findings, we propose that sarcomeric dysfunction plays a critical role in the development of muscle weakness in FSHD.

Titin Mediates Sarcomere Length Dependence of Myocyte Power

According to the Frank-Starling relationship, ventricular pressure increases with end-diastolic volume. This is controlled, in large part, by sarcomere length (SL) dependence of cardiac myofibrillar force, loaded shortening, and power. Consistent with this, both myofibrillar force and power fall at shorter SL, however, when Ca2+ activated force and presumably thin filament activation levels are matched between short and long SL (by increasing the activator [Ca2+]) short SL actually yields faster loaded shortening and greater peak normalized power output (PNPO). A potential mechanism for faster loaded shortening at short SL is that as SL decreases titin becomes less taut which reduces the impedance of cross-bridges, a process that may be mediated by titin's interactions with the thick filament. We propose a more slackened titin yields greater myosin head radial and azimuthal mobility and these flexible cross-bridges are more likely to maintain thin filament activation, which would allow more force-generating cross-bridges to work against a fixed load resulting in faster loaded shortening. We tested this idea by measuring SL dependence of power at matched forces in rat skinned cardiac myocytes containing either N2B titin or a longer, more compliant N2BA titin. We predicted less overshoot in power by short SL in N2BA titin myocytes. Consistent with this, peak power did not overshoot but was actually less at short versus long SL at matched thin filament activation levels in N2BA-containing myocytes (Wt: ΔPNPO = +0.0121 ± 0.0115 (n=5), N2BA titin: ΔPNPO = −0.0566 ± 0.0493 (n=5)). These findings are consistent with SL per se modulating the mechanical properties of cross-bridges with this modulation being mediated by titin. This myofibrillar mechanism may help sustain ventricular power during periods of low preloads, and perhaps a breakdown of this mechanism is involved in impaired function of failing hearts.

PKA Effects on Cardiac Troponin and Myofibril Relaxation

Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for beta-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. We recently reported that PKA phosphorylation of cTnI alone, or in combination with cMyBP-C, is sufficient to suppress cardiac contractility and the sarcomere length dependence of contraction. Troponin C (cTnC) interaction with cTnI (C-I) is a critical step in contractile activation that may be modulated by cTnI phosphorylation. Here we tested the hypothesis that altering C-I interactions by PKA, or by phosphomimetic mutations in cTnI, directly affects myofilament relaxation. Using a fluorescent probe coupled to cTnC (C35S IANBD), the Ca2+ binding affinity to troponin complex (cTn) and C-I interaction affinity were monitored. Ca2+ binding to cTn (pCa50) was significantly decreased by PKA, when both whole cTn or cTnI alone were phosphorylated (pCa50 = 0.3 and 0.1 units, respectively). PKA phosphorylation of cTnI also weakened C-I interaction in the presence or absence of Ca2+, with a greater effect for the former. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild type (WT), nonphosphorylatable (S23/24A cTnI) or phosphomimetic (S23/24D cTnI) recombinant cTn. Mechanics were monitored at maximum and submaximal Ca2+. PKA treatment of WT or S23/24D cTnI exchange resulted in a significantly increased slow phase relaxation rate (kREL,slow) and decreased duration of slow phase relaxation (tREL,slow). These effects were largest at submaximal Ca2+. In contrast, exchange with S23/24A cTnI resulted in no effects on relaxation kinetics. Together these data suggest that weakened C-I interaction affinity from PKA phosphorylation results in increased myofilament relaxation kinetics. These effects may enhance early phase diastolic relaxation during beta-adrenergic stimulation. Supported by AHA7400069 (VR), AHA2090056 (DW), HL65497 (MR).