scholarly journals The structure of the native cardiac thin filament at systolic Ca2+ levels

2021 ◽  
Vol 118 (13) ◽  
pp. e2024288118
Author(s):  
Cristina M. Risi ◽  
Ian Pepper ◽  
Betty Belknap ◽  
Maicon Landim-Vieira ◽  
Howard D. White ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
Binding Sites ◽  
Short Range ◽  
Bound States ◽  
Thin Filament ◽  
Narrow Range ◽  
The Other ◽  
Thin Filaments ◽  
Troponin Complex ◽  
Myosin Binding

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

Troponin-Tropomyosin Control of Thin Filament Activity Revealed by Electron Microscopy and 3-D Reconstruction.

2000 ◽  
Vol 6 (S2) ◽  
pp. 88-89
Author(s):  
W. Lehman ◽  
V. Hatch ◽  
M. Rosol ◽  
V. Korman ◽  
R. Horowitz ◽  
...  
Keyword(s):  
Thin Filament ◽  
Wild Type ◽  
Thin Filaments ◽  
Mutant Proteins ◽  
3 Dimensional ◽  
Actomyosin Atpase ◽  
Helical Reconstruction ◽  
Troponin Complex ◽  
Cardiac Muscles ◽  
Myosin Binding

Muscle contraction and the actomyosin ATPase that drives the contractile process are switched on and off by changes in sarcoplasmic free Ca2+ -concentration. In skeletal and cardiac muscles, on-off switching is mediated by the actinassociated protein tropomyosin and by the troponin complex. While the details of this mechanism are still subject to debate, it is well-accepted that tropomyosin strands move to sterically block and unblock myosin binding sites on actin, thereby controlling actomyosin ATPase and consequently contraction. It is also well known that the Ca2+- dependency of the movement of tropomyosin on actin is governed by troponin.As a means of studying tropomyosin movement and the influence of troponin, we have used cryo-EM, negative staining and 3-dimensional helical reconstruction to define the positions of tropomyosin and troponin on thin filaments. We examined various preparations of native isolated filaments and filaments reconstituted with wild-type and mutant proteins.

scholarly journals Single molecule imaging reveals the concerted release of myosin from regulated thin filaments

2018 ◽  
Author(s):  
A. V. Inchingolo ◽  
M. Mihailescu ◽  
D. Hongsheng ◽  
N. M. Kad
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Calcium Binding ◽  
Thin Filament ◽  
Striated Muscle ◽  
Monte Carlo Model ◽  
Local Concentration ◽  
Single Molecule Imaging ◽  
Thin Filaments ◽  
Myosin Binding

AbstractRegulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which in turn shifts the position of tropomyosin on actin to expose myosin binding sites. The binding of the first myosin holds tropomyosin in a position such that more myosin binding sites on actin are available, resulting in cooperative activation. Troponin and tropomyosin also act to turn off the thin filament; however, this is antagonized by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation we use the RTF tightrope assay, in which single RTFs are suspended between pedestals above a microscope coverslip surface. Single molecule imaging of GFP tagged myosin-S1 (S1-GFP) is used to follow the activation of RTF tightropes. In sub-maximal activation conditions, S1-GFP molecules bind forming metastable clusters, from which release and rebinding of S1-GFP leads to prolonged activation in these regions. Because the RTFs are not fully active we are able to directly observe deactivation in real time. Using a Reversible Jump Markov Chain Monte Carlo model we are able to dynamically assess the fate of active regions. This analysis reveals that myosin binding occurs in a stochastic stepwise fashion; however, an unexpectedly large probability of multiple simultaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated, active process.

scholarly journals Single-molecule imaging reveals the concerted release of myosin from regulated thin filaments

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Quentin M Smith ◽  
Alessio V Inchingolo ◽  
Madalina-Daniela Mihailescu ◽  
Hongsheng Dai ◽  
Neil M Kad
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Calcium Binding ◽  
Thin Filament ◽  
Fluorescent Protein ◽  
Striated Muscle ◽  
Local Concentration ◽  
Single Molecule Imaging ◽  
Thin Filaments ◽  
Myosin Binding

Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.

Effect of Extracellular CA on the Phosphorylation of Myosin Binding Protein C. In Cardiac Muscle and the Phosphorylation on Force Generation

2000 ◽  
Vol 6 (S2) ◽  
pp. 74-75
Author(s):  
S. Winegrad ◽  
I. Kulikavskaya ◽  
G. McClellan
Keyword(s):  
Cardiac Muscle ◽  
Binding Sites ◽  
Protein C ◽  
Contractile Activity ◽  
Binding Protein ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding ◽  
Physiological Reactions ◽  
Binding Subunit

The affinity of the binding sites on troponin C (TNC) for Ca is sensitive to the state of phosporylation of troponin 1 (TN1). Two and possibly three other myofilament proteins can be phosphorylated by physiological reactions: the mosin binding protein C (MyBP-C), the regulatory light chain of myosin (RLC), and the tropomyosin binding subunit of troponin (TNT). In MyBP-C, the phosphorylation sites are specific for cardiac muscle, an occurrence that suggests a function unique to cardiac muscle. Phosphorylation of MyBP-C and RLC can be produced by Ca regulated kinases present in the myofibrils. Phosphorylation of RLC produces an increase in Ca sensitivity of contraction at submaximal concentrations of Ca without alteration of the maximum Ca activated force (Fmax). Absence of contractile activity decreases phosphorylation of RLC. One to two hours of quiescence in normal extracellular Ca reduces phophorylation substantially, and the level rises slowly when contractile activity is resumed.

scholarly journals High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding

2019 ◽  
Vol 116 (33) ◽  
pp. 16384-16393 ◽  
Author(s):  
Oleg S. Matusovsky ◽  
Alf Mansson ◽  
Malin Persson ◽  
Yu-Shu Cheng ◽  
Dilson E. Rassier
Keyword(s):  
Conformational Changes ◽  
High Speed ◽  
Supported Lipid Bilayer ◽  
Thin Filaments ◽  
Force Microscopy ◽  
Atomic Force ◽  
Activated State ◽  
Troponin Complex ◽  
Myosin Binding ◽  
Activation Conditions

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin–troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Regulation of Contraction in Striated Muscle

2000 ◽  
Vol 80 (2) ◽  
pp. 853-924 ◽  
Author(s):  
A. M. Gordon ◽  
E. Homsher ◽  
M. Regnier
Keyword(s):  
Binding Sites ◽  
Thin Filament ◽  
Striated Muscle ◽  
Force Development ◽  
Cross Bridge ◽  
Hydrolysis Products ◽  
Strong Binding ◽  
Regulation Of Contraction ◽  
Myosin Binding ◽  
Cross Bridges

Ca2+ regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A7TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca2+binding sites on TnC, conformational changes resulting from Ca2+ binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca2+ binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1–2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca2+-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the “open” probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A7TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca2+ binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca2+ regulates the strong binding of M·ADP·Pi to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca2+ activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A7TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca2+]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca2+ binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. These results and conclusions suggest possible explanations for differences between skeletal and cardiac muscle regulation and delineate the paths future research may take toward a better understanding of striated muscle regulation.

scholarly journals CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mert Colpan ◽  
Jessika Iwanski ◽  
Carol C. Gregorio
Keyword(s):  
Muscle Contraction ◽  
Cardiac Muscle ◽  
Adenylyl Cyclase ◽  
Thin Filament ◽  
Essential Role ◽  
Striated Muscle ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Thin Filaments ◽  
Pointed End

AbstractThe precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2’s function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2’s multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.

scholarly journals Comparing models with one versus multiple myosin-binding sites per actin target zone: The power of simplicity

2019 ◽  
Vol 151 (4) ◽  
pp. 578-592 ◽  
Author(s):  
Alf Månsson
Keyword(s):  
Binding Sites ◽  
Computational Cost ◽  
Spatially Explicit ◽  
Target Zone ◽  
Thin Filaments ◽  
Contact Points ◽  
Model Predictions ◽  
Myosin Binding ◽  
Myosin Motors ◽  
Strain Dependent

Mechanokinetic statistical models describe the mechanisms of muscle contraction on the basis of the average behavior of a large ensemble of actin–myosin motors. Such models often assume that myosin II motor domains bind to regularly spaced, discrete target zones along the actin-based thin filaments and develop force in a series of strain-dependent transitions under the turnover of ATP. The simplest models assume that there is just one myosin-binding site per target zone and a uniform spatial distribution of the myosin motor domains in relation to each site. However, most of the recently developed models assume three myosin-binding sites per target zone, and some models include a spatially explicit 3-D treatment of the myofilament lattice and thereby of the geometry of the actin–myosin contact points. Here, I show that the predictions for steady-state contractile behavior of muscle are very similar whether one or three myosin-binding sites per target zone is assumed, provided that the model responses are appropriately scaled to the number of sites. Comparison of the model predictions for isometrically contracting mammalian muscle cells suggests that each target zone contains three or more myosin-binding sites. Finally, I discuss the strengths and weaknesses of one-site spatially inexplicit models in relation to three-site models, including those that take into account the detailed 3-D geometry of the myofilament lattice. The results of this study suggest that single-site models, with reduced computational cost compared with multisite models, are useful for several purposes, e.g., facilitated molecular mechanistic insights.

scholarly journals The mechanism of thin filament regulation: Models in conflict?

2019 ◽  
Vol 151 (11) ◽  
pp. 1265-1271 ◽  
Author(s):  
Michael A. Geeves ◽  
Sherwin S. Lehrer ◽  
William Lehman
Keyword(s):  
Rate Constant ◽  
Binding Sites ◽  
Thin Filament ◽  
State Model ◽  
Muscle Contractility ◽  
Thin Filaments ◽  
Thin Filament Regulation ◽  
Three State Model ◽  
State Models ◽  
Two State Model

In a recent JGP article, Heeley et al. (2019. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812198) reopened the debate about two- versus three-state models of thin filament regulation. The authors review their work, which measures the rate constant of Pi release from myosin.ADP.Pi activated by actin or thin filaments under a variety of conditions. They conclude that their data can be described by a two-state model and raise doubts about the generally accepted three-state model as originally formulated by McKillop and Geeves (1993. Biophys. J. https://doi.org/10.1016/S0006-3495(93)81110-X). However, in the following article, we follow Plato’s dictum that “twice and thrice over, as they say, good it is to repeat and review what is good.” We have therefore reviewed the evidence for the three- and two-state models and present our view that the evidence is overwhelmingly in favor of three structural states of the thin filament, which regulate access of myosin to its binding sites on actin and, hence, muscle contractility.

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