The Myosin Duty Ratio Tunes the Calcium Sensitivity and Cooperative Activation of the Thin Filament

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

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The Role of Thin Filament Calcium Sensitivity in Modulating Relaxation Time of Soleus and EDL Skeletal Muscle

Biophysical Journal ◽

10.1016/j.bpj.2019.11.804 ◽

2020 ◽

Vol 118 (3) ◽

pp. 122a

Author(s):

Connor Tyree ◽

Kyra Peczkowski ◽

Paul M. Janssen ◽

Jill Rafael-Fortney ◽

Jonathan P. Davis

Keyword(s):

Skeletal Muscle ◽

Relaxation Time ◽

Thin Filament ◽

Calcium Sensitivity

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The effect of altered temperature on Ca2(+)-sensitive force in permeabilized myocardium and skeletal muscle. Evidence for force dependence of thin filament activation.

The Journal of General Physiology ◽

10.1085/jgp.96.6.1221 ◽

1990 ◽

Vol 96 (6) ◽

pp. 1221-1245 ◽

Cited By ~ 69

Author(s):

N K Sweitzer ◽

R L Moss

Keyword(s):

Skeletal Muscle ◽

Cardiac Muscle ◽

Thin Filament ◽

Calcium Sensitivity ◽

Differential Sensitivity ◽

Fiber Types ◽

Tension Development ◽

Maximum Tension ◽

Filament Activation ◽

Muscle Types

The effect of changes in temperature on the calcium sensitivity of tension development was examined in permeabilized cellular preparations of rat ventricle and rabbit psoas muscle. Maximum force and Ca2+ sensitivity of force development increased with temperature in both muscle types. Cardiac muscle was more sensitive to changes in temperature than skeletal muscle in the range 10-15 degrees C. It was postulated that the level of thin filament activation may be decreased by cooling. To investigate this possibility, troponin C (TnC) was partially extracted from both muscle types, thus decreasing the level of thin filament activation independent of temperature and, at least in skeletal muscle fibers, decreasing cooperative activation of the thin filament as well. TnC extraction from cardiac muscle reduced the calcium sensitivity of tension less than did extraction of TnC from skeletal muscle. In skeletal muscle the midpoint shift of the tension-pCa curve with altered temperature was greater after TnC extraction than in control fibers. Calcium sensitivity of tension development was proportional to the maximum tension generated in cardiac or skeletal muscle under all conditions studied. Based on these results, we conclude that (a) maximum tension-generating capability and calcium sensitivity of tension development are related, perhaps causally, in fast skeletal and cardiac muscles, and (b) thin filament activation is less cooperative in cardiac muscle than in skeletal muscle, which explains the differential sensitivity of the two fiber types to temperature and TnC extraction. Reducing thin filament cooperativity in skeletal muscle by TnC extraction results in a response to temperature similar to that of control cardiac cells. This study provides evidence that force levels in striated muscle influence the calcium binding affinity of TnC.

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Elevated levels of phosphate increase reconstituted thin filament velocity and calcium sensitivity at low pH in an in vitro motility assay

The FASEB Journal ◽

10.1096/fasebj.27.1_supplement.1202.24 ◽

2013 ◽

Vol 27 (S1) ◽

Author(s):

Edward P Debold ◽

Thomas J Longyear ◽

Matthew A Turner ◽

Jonathan P Davis ◽

Joseph J Lopez

Keyword(s):

Thin Filament ◽

Calcium Sensitivity ◽

Low Ph ◽

Motility Assay ◽

In Vitro Motility Assay ◽

In Vitro Motility

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A Troponin T Variant Linked with Pediatric Dilated Cardiomyopathy Reduces the Coupling of Thin Filament Activation to Myosin and Calcium Binding

Molecular Biology of the Cell ◽

10.1091/mbc.e21-02-0082 ◽

2021 ◽

pp. mbc.E21-02-0082

Author(s):

Samantha K. Barrick ◽

Lina Greenberg ◽

Michael J. Greenberg

Keyword(s):

Dilated Cardiomyopathy ◽

Calcium Binding ◽

Thin Filament ◽

Troponin T ◽

Calcium Sensitivity ◽

Cellular Level ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Fluorescence Measurements

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well-understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an in vitro motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.

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cMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium

The Journal of General Physiology ◽

10.1085/jgp.202012782 ◽

2021 ◽

Vol 153 (3) ◽

Cited By ~ 1

Author(s):

Jasmine Giles ◽

Daniel P. Fitzsimons ◽

Jitandrakumar R. Patel ◽

Chloe Knudtsen ◽

Zander Neuville ◽

...

Keyword(s):

High Velocity ◽

Thin Filament ◽

Cardiac Myosin ◽

Low Velocity ◽

Shortening Velocity ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding ◽

Low Levels ◽

Cooperative Activation

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

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Abstract 448: Strain Rate Dependent Myosin Head Position Changes During Relaxation

Circulation Research ◽

10.1161/res.127.suppl_1.448 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Weikang Ma ◽

Melissa J Bukowski ◽

Alexandra R Matus ◽

Thomas C Irving ◽

Charles S Chung

Keyword(s):

Strain Rate ◽

Thin Filament ◽

Myosin Head ◽

Thick Filament ◽

Calcium Sensitivity ◽

Head Position ◽

Mechanical Control ◽

Myocardial Relaxation ◽

Rate Dependent ◽

Head Positioning

Impaired cardiac relaxation is present in nearly all cases of heart failure and possibly in up to 25% of the asymptomatic population. Myocardial relaxation is known to be biochemically modified by the calcium reuptake rate, thin filament calcium sensitivity, and crossbridge kinetics. Mechanical regulation of relaxation was thought to be regulated via afterload, but we have recently shown that a lengthening strain was sufficient to modify relaxation. Further, the relaxation rate is actually dependent on the strain rate, a relationship that we termed Mechanical Control of Relaxation. Computational modeling suggests that myosin detachment is a key mechanism underlying Mechanical Control of Regulation, but to date, no experimental evidence for this was available. The objective of this study was to determine if myosin head position changed in response to lengthening strains during relaxation. Intact cardiac trabeculae were mounted within the beamline of the Biophysical Collaborative Access Team (BioCAT) beamline at the Advanced Photon Source at Argonne National Laboratories. The trabeculae were paced and load-clamps were performed during time-resolved imaging of the equatorial axis, which primarily reflects myosin head positioning. Activation (pacing) caused the myosin head localization to shift from the thick filament to near the thin filament (increased I 1,1 /I 1,0 ratio). During stretch, there was a transient decline of the I 1,1 /I 1,0 ratio which recovered until relaxation was complete, when the ratio again reduced indicating myosin returned to the thick filament. These preliminary data suggest that Mechanical Control of Relaxation is caused by perturbations in myosin, but the late-diastolic kinetics suggests that the strain-rate dependent detachment does not lead to immediate deactivation of myosin heads. Modifications of myosin ATPase properties may reveal more specific regulatory targets, which may provide new insight and targets for treating impaired myocardial relaxation.

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Regulatory mechanism of length-dependent activation in skinned porcine ventricular muscle: role of thin filament cooperative activation in the Frank-Starling relation

The Journal of General Physiology ◽

10.1085/jgp.201010502 ◽

2010 ◽

Vol 136 (4) ◽

pp. 469-482 ◽

Cited By ~ 22

Author(s):

Takako Terui ◽

Yuta Shimamoto ◽

Mitsunori Yamane ◽

Fuyu Kobirumaki ◽

Iwao Ohtsuki ◽

...

Keyword(s):

Thin Filament ◽

Linear Regression Analysis ◽

Left Ventricular ◽

Active Force ◽

Force Measurements ◽

Systematic Understanding ◽

Cardiac Sarcomeres ◽

Length Dependent Activation ◽

Cooperative Activation

Cardiac sarcomeres produce greater active force in response to stretch, forming the basis of the Frank-Starling mechanism of the heart. The purpose of this study was to provide the systematic understanding of length-dependent activation by investigating experimentally and mathematically how the thin filament “on–off” switching mechanism is involved in its regulation. Porcine left ventricular muscles were skinned, and force measurements were performed at short (1.9 µm) and long (2.3 µm) sarcomere lengths. We found that 3 mM MgADP increased Ca2+ sensitivity of force and the rate of rise of active force, consistent with the increase in thin filament cooperative activation. MgADP attenuated length-dependent activation with and without thin filament reconstitution with the fast skeletal troponin complex (sTn). Conversely, 20 mM of inorganic phosphate (Pi) decreased Ca2+ sensitivity of force and the rate of rise of active force, consistent with the decrease in thin filament cooperative activation. Pi enhanced length-dependent activation with and without sTn reconstitution. Linear regression analysis revealed that the magnitude of length-dependent activation was inversely correlated with the rate of rise of active force. These results were quantitatively simulated by a model that incorporates the Ca2+-dependent on–off switching of the thin filament state and interfilament lattice spacing modulation. Our model analysis revealed that the cooperativity of the thin filament on–off switching, but not the Ca2+-binding ability, determines the magnitude of the Frank-Starling effect. These findings demonstrate that the Frank-Starling relation is strongly influenced by thin filament cooperative activation.

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