Actomyosin Binding Research Articles

Effects of different electrostatic field intensities assisted controlled freezing point storage on water holding capacity of fresh meat during the early postmortem period

In this study, the effect of different intensity electrostatic fields on the water holding capacity (WHC) of fresh meat during the early postmortem period in controlled freezing point storage (CFPS) were investigated. Significantly lower cooking loss were found in low voltage electrostatic field (LVEF) and high voltage electrostatic field (HVEF) compared to the control group (CK) (p < 0.05). The myofibril fragmentation index and microstructure results suggested that the sample under HVEF treatment remained relatively intact. It has been revealed that the changes in actomyosin properties under electrostatic field treatment groups were due to the combination and dissociation of actomyosin binding into myofilament concentration, which consequently affects the muscle WHC. The study further demonstrated that the electrostatic field, especially HVEF, might increase the WHC of fresh meat by affecting the distribution of water molecules and physiochemical properties of actomyosin during the early postmortem period.

Kinetics of interaction of myosin-nucleotide complex and actin.

In the actomyosin cycle myosin hydrolyses ATP and binds actin, initially weakly and then strongly. The timing of myosin ATP hydrolysis and binding to actin remains elusive. We performed double-mixing transient experiments to characterize the kinetics of actomyosin interaction shortly after ATP binding to myosin. In the experiment we first rapidly pre-mixed rabbit skeletal myosin S1 and stoichiometric ATP and after a controlled delay rapidly added unregulated F-actin to the mixture. The first mixing produces mainly complexes of myosin.ATP and myosin.ADP.Pi, we expected to have small quantity of unreacted myosin and complex of myosin.ADP in the mixture. In the second mixing myosin binds actin. We detected transient fluorescence of pyrene labeled actin and changes in the light scattering after the second mixing. It is accepted that the change of fluorescence of pyrene labeled actin reflects strong actomyosin binding due to the conformational change of actin induced by bound myosin. One can suggest that light scattering reflects the formation of weakly bound actomyosin, since light scattering is sensitive to the size of the protein complex, and not the strength of binding. In our experiments we varied protein concentrations and the time of the delay between the first and the second mixing, which is the myosin-nucleotide incubation time. We fitted obtained transients to the solution of the system of differential equations, describing the evolution of this composite protein system. We discuss models of actomyosin interaction that correspond to the observed transient kinetics and the possible cooperativity of myosin interaction with unregulated actin.

Cardiac myosin velocity and force are dramatically improved with an alternative triphosphate substrate.

Cardiac myosin is the molecular motor that drives the pumping action of the heart; therefore, there is strong interest in gaining control over its function in an effort to develop more effective treatments for various forms of heart disease. We sought to determine if the force and motion generating capacity of β-cardiac myosin could be altered in vitro by replacing ATP with different isomers of an alternative energy source, azobenzene triphosphate (AzoTP). Using the ortho-isomer of AzoTP, cardiac myosin moved actin filaments 62% faster than ATP in a motility assay (p<0.001). Subsequent experiments suggested that the increased velocity may be the result of an accelerated rate of ortho-AzoDP-release from myosin. Cardiac myosin also generated 10% more force when using ortho-AzoTP compared to ATP in a laser trap assay (p<0.001), which was likely due to an increased rate of attachment to actin because the frequency of single actomyosin binding events observed in a three-bead laser trap assay was double that observed for ortho-AzoTP vs. ATP (p<0.001). In contrast to ortho-AzoTP, with the meta isomer of AzoTP cardiac myosin translocated actin filaments 90% slower than with ATP (p<0.001). This finding is likely do to a slowed rate of detachment from actin, based on preliminary single molecule findings. With the para-isomer of AzoTP no movement was detected in the motility assay, suggesting it may not bind to the nucleotide-binding site. Thus, these findings demonstrate that the function of beta-cardiac myosin can be manipulated using structural isomers of an alternative energy substrate (AzoTP). These findings therefore may provide important new insights into how to alter the function of cardiac myosin in vivo, which could help in the development of novel therapies to different forms of heart disease.

Influence of 2,3-Butanedione-Monoxime on the Interaction of Myosin with Actin in Healthy and in Congenital Myopathy

Congenital myopathies are a heterogeneous group of human skeletal muscle disorders characterized by muscle hypotonia and weakness. Myopathies have a wide range of clinical phenotypes, which makes it extremely difficult to develop approaches to their treatment. There are several pharmacological agents in clinical use or under clinical investigation for the treatment of cardiomyopathies whose mechanism of action can be used to treat congenital myopathies as well. One such agent is 2,3-butanedione-monoxime (BDM), a noncompetitive inhibitor of myosin ATPase activity used to suppress acute myocardial injury. The molecular mechanisms of inhibition of myosin by BDM in skeletal muscle have not been studied, therefore the aim of this work was to estimate the effect of BDM on the interaction of myosin with actin in the modeling of several ATPase stages in skeletal muscle fiber, in order to assess the prospects for the use of BDM for the treatment of congenital myopathies. We found that BDM enhances the rigidity of myosin binding to actin when modeling weak binding forms of these muscle proteins, which can slow down the transition of actomyosin from the AM ∙ ADP ∙ Pi to the AM ∙ ADP state and is one of the reasons for the decrease in myosin ATPase activity in the presence of BDM. When modeling successive stages of the ATPase cycle using ADP, AMPPNP, ATPγS, and ATP, the myosin heads gradually switch to a state of weak interaction with actin. In the presence of the regulatory proteins tropomyosin and troponin in the muscle fiber, BDM does not affect the formation of a weak form of actomyosin binding, but increases the number of myosin heads essential for force generation. BDM can be used to increase the efficiency of myosin conformational rearrangements in the presence of tropomyosin with the R90P mutation associated with congenital myopathy, since this reagent increases the number of myosin heads in the muscle fiber capable of effective conformational rearrangements in the ATPase cycle and partially inhibits the pathological effects of the mutation.

Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation.

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven-stranded beta sheet within the myosin head. Beta isoform-specific salt bridge D208:K450, restricting the flexibility of loop 1, was previously discovered in molecular dynamics simulations. Earlier it was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulate ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. To examine the hypothesis we expressed the wild type and mutant of the myosin head construct (1-843 amino acid residues) in differentiated C2C12 cells, and the kinetics of ATP-induced actomyosin dissociation and ADP release were characterized using stopped-flow spectrofluorometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP-induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. The flexibility of loop 1 does not affect the rate of ADP release from human cardiac actomyosin.

Myosin'S Powerstroke Occurs with Phosphate Still in the Active Site

A fundamental property of myosin is its ability to harness the power of ATP hydrolysis to generate force. A key unanswered question is the timing of the force-generating powerstroke relative to phosphate (Pi) -release from its active site. We examined the ability of single-headed myosin Va (MV) construct to bind to actin and generate a powerstroke in a single molecule laser trap assay in the presence of elevated Pi. We examined both WT MV and the impact of a mutation (S217A) in the conserved switch I region of the active site which slows actin-activated Pi-release. Upon binding to actin WT myosin rapidly (>500/s) generated a 7±0.5 nm powerstroke. In the presence of 30mM Pi, no decrease in the size (7±0.4 nm) or delay in the rate of the development of the powerstroke (>500/s) was observed, however the average duration of strong actomyosin binding (ton) was reduced in the presence of Pi (240±20 vs 113± ms, p<0.05). The reduced ton is consistent with Pi rebinding to the active site and accelerating detachment from actin. Therefore, this suggests myosin generates the powerstroke with Pi still in the active site. S217A generated a powerstroke with a similar rate (>500/s) and size (9±0.9 nm) to the WT, but ton was shorter, but not significantly different, than WT (143±20 ms, p=0.22 and 62±5 ms, p<0.05 for S217A in the absence and presence of Pi, respectively). Since the S217A mutant slows actin-activated Pi-release from the active site our results fit best to a model in which the powerstroke occurs with Pi still in the active site. Together these findings suggest that actin-binding, and not the release of Pi from the active site, trigger the conformational changes that lead to the powerstroke, and thus force-generation by myosin.

Electrostatic interactions in the SH1-SH2 helix of human cardiac myosin modulate the time of strong actomyosin binding.

Two single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the SH1-SH2 helix of the myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the SH1-SH2 helix modulate the affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1-843 amino acid residues) were expressed in differentiated C2C12 cells, and the duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. The mutant R694N showed a faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus indicating that electrostatic interactions within the SH1-SH2 helix region of human cardiac myosin modulate ADP release and thus, the duration of the strongly bound state of actomyosin.

CaATP prolongs strong actomyosin binding and promotes futile myosin stroke.

Calcium plays an essential role in muscle contraction, regulating actomyosin interaction by binding troponin of thin filaments. There are several buffers for calcium in muscle, and those buffers play a crucial role in the formation of the transient calcium wave in sarcomere upon muscle activation. One such calcium buffer in muscle is ATP. ATP is a fuel molecule, and the important role of MgATP in muscle is to bind myosin and supply energy for the power stroke. Myosin is not a specific ATPase, and CaATP also supports myosin ATPase activity. The concentration of CaATP in sarcomeres reaches 1% of all ATP available. Since 294 myosin molecules form a thick filament, naïve estimation gives three heads per filament with CaATP bound, instead of MgATP. We found that CaATP dissociates actomyosin slower than MgATP, thus increasing the time of the strong actomyosin binding. The rate of the basal CaATPase is faster than that of MgATPase, myosin readily produces futile stroke with CaATP. When calcium is upregulated, as in malignant hyperthermia, kinetics of myosin and actomyosin interaction with CaATP suggest that myosin CaATPase activity may contribute to observed muscle rigidity and enhanced muscle thermogenesis.

Acidosis decreases the Ca2+ sensitivity of thin filaments by preventing the first actomyosin interaction.

Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because it depresses the calcium (Ca2+) sensitivity of the myofilaments. However, the molecular mechanism behind this depression in Ca2+ sensitivity is unknown, providing a significant challenge to a complete understanding of the fatigue process. To elucidate this mechanism, we directly determined the effect of acidosis on the ability of a single myosin molecule to bind to a regulated actin filament in a laser trap assay. Decreasing pH from 7.4 to 6.5 significantly (P < 0.05) reduced the frequency of single actomyosin-binding events at submaximal (pCa 8-pCa 6) but not at maximal Ca2+ concentration (pCa 5-pCa 4). To delineate whether this was due to a direct effect on myosin versus an indirect effect on the regulatory proteins troponin (Tn) and tropomyosin (Tm), binding frequency was also quantified in the absence of Tn and Tm. This revealed that acidosis did not significantly alter the frequency of actomyosin binding events in the absence of regulatory proteins (1.4 ± 0.15 vs. 1.4 ± 0.15 events/s for pH 7.4 and 6.5, respectively). Acidosis also did not significantly affect the size of myosin's powerstroke or the duration of binding events in the presence of regulatory proteins, at every [Ca2+]. These data suggest acidosis impedes activation of the thin filament by competitively inhibiting Ca2+ binding to TnC. This slows the rate at which myosin initially attaches to actin; therefore, less cross bridges will be bound and generating force at any given submaximal [Ca2+]. These data provide a molecular explanation for the acidosis-induced decrease in force observed at the submaximal Ca2+ concentrations that might contribute to the loss of force during muscle fatigue.

Mechanisms Through Which Agents Of Muscle Fatigue, Acidosis And Phosphate, Inhibit Muscle Myosin Function.

PURPOSE: During muscle fatigue from intense activity, elevated concentrations of hydrogen ions (acidosis) and inorganic phosphate (Pi) inhibit muscle’s ability to generate force and motion. However, it is not clear exactly how these metabolic by-products reduce the force and enzymatic function of muscle’s molecular motor, myosin. METHODS: To determine these mechanisms we directly measured the effect of these fatigue agents on the force generating capacity of isolated myosin in a laser trap assay and on its ability to hydrolyze ATP in an ATPase assay. RESULTS: Acidosis (pH 7.4 vs. 6.5) in a mini-ensemble laser trap assay reduced myosin’s average force production by 20% (p < 0.05) due to a slowed rate of actomyosin binding. This conclusion was supported by the observation that acidosis slowed myosin’s ability to hydrolyze ATP by roughly 90% (p < 0.05) in a solution assay. By contrast elevated levels of Pi (0 vs. 10-15mM), in the presence of low pH (6.5), caused a similar reduction in force. However, this was likely due to an accelerated rate of myosin’s detachment from actin, because myosin’s ATPase rate also recovered back toward the control value (pH 7.4, no Pi) when Pi was added. CONCLUSION: Thus, these data provide unique insight into the molecular mechanisms that underlie the loss of muscle function during fatigue. In our current work we are using these findings to explore methods to mitigate these effects in vitro in a first step toward attenuating fatigue in diseases such as chronic heart failure.

High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing

Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.

Cardiac contractility, motor function, and cross-bridge kinetics in N47K-RLC mutant mice.

We have investigated the physiology and mechanical profiles of skinned papillary muscle fibers from transgenic mice expressing the N47K mutation in the myosin regulatory light chain (RLC), shown to cause hypertrophic cardiomyopathy in humans. The results were compared with wild-type (WT) mice, both expressing the human ventricular RLC. Rate constants of a cross-bridge (XB) cycle were deduced from tension transients induced by sinusoidal length changes during maximal Ca2+ activation, and were studied as a function of MgATP, MgADP, and Pi concentrations. N47K mutant showed slower XB cycles but higher actin-activated ATPase activity compared with WT. Consequently, N47K exhibited larger tension than WT. K0 (ADP association constant) and K4 (equilibrium constant of force generation) were larger in N47K, and K1 (ATP association constant) was slightly larger in N47K vs. WT, demonstrating stronger nucleotide binding and force generation abilities of the mutant, but no changes in rigor acto-myosin binding were observed. Tension per XB was similar among groups, but N47K exhibited more XB distribution in the attached state. Larger values of tension and higher ATPase in N47K suggested that more cross-bridges participated in tension production in the mutant myocardium compared with WT. In vivo analysis of heart function, performed in ~ 12.5-month-old mice by echocardiography and invasive hemodynamics, demonstrated a significant decrease in dP/dtmax -end-diastolic volume relationship, indicating a depression of ventricular contractility in N47K mice. Our findings suggest that the N47K mutation exerts its action through direct alterations of myosin motor function that ultimately result in pathological hypertrophic remodeling in N47K hearts.

Pseudo-Phosphorylation Mediated Rescue of R58Q-Linked Familial Hypertrophic Cardiomyopathy Phenotype

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myofibrillar disarray and often caused by mutations in genes encoding for major sarcomeric proteins. This study is focused on Arginine to Glutamine substitution (R58Q) in the ventricular myosin regulatory light chain (RLC) resulting in a malignant hypertrophic phenotype. We previously showed a mutation-induced decrease in the endogenous level of cardiac RLC phosphorylation in R58Q mice, resulting in compromised communication between the functional regions of the RLC and affecting the interaction of myosin and actin and cardiac muscle contraction. In this report we explored the therapeutic potential of pseudo-phosphorylation, in which Aspartic Acid is replaced for phosphorylatable Serine-15 (S15D) in the R58Q background. In vitro actin-activated myosin ATPase activity was assessed using S15D-R58Q and R58Q vs. WT reconstituted porcine cardiac myosin. R58Q largely decreased maximal ATPase activity, Vmax (∼30% lower) compared to WT. S15D-R58Q rescued the maximal ATPase activity to the WT level. The Michaelis–Menten constant (in μM) for R58Q (Km=4.04±0.45) was significantly increased compared to WT (Km=1.68±0.19), suggesting that a higher concentration of actin is needed to activate cross-bridges. S15D-R58Q-RLC showed a small but significant increase in Km (2.56±0.32), compared with WT. Steady-state acto-myosin binding was studied using fluorescently labelled actin to quantify the dissociation constants (Kd) and stoichiometry of binding (n) between myosin and actin in the absence of nucleotide. Binding of WT-RLC to actin was strong (Kd value =5.5 nM, n=0.89). A large decrease in binding (∼67-fold increase in Kd) was observed for R58Q-RLC. Kd of S15D-R58Q-RLC was only slightly increased (∼3-fold) compared with WT indicating a rescue in binding affinity by pseudo-phosphorylated RLC mutant. Our findings on S15D-R58Q support the idea that myosin phosphorylation may have important translational applications for the treatment of RLC induced hypertrophic cardiomyopathy. Supported by NIH-HL123255 (DSC).

Using Single Myosin and Small Myosin Ensembles to Examine Thin Filament Activation in a Laser Trap Assay

Thin filament activation in muscle requires both Ca++ binding to troponin and myosin strongly binding to actin. Despite extensive study, the relative importance of each of these processes to overall thin filament activation remains unclear. To determine the role of Ca++ we examined the effect of increasing Ca++ concentrations on the frequency of single molecule binding events in a single molecule laser trap assay using reconstituted thin filaments. The frequency of actomyosin binding events increased from 0.03 at low Ca++ (pCa7), to 0.30 at pCa6 to 0.44 events/sec at pCa5. Increasing the myosin concentration so that the regulated filament interacted with a mini-ensemble of myosin (∼6 heads) in the laser trap assay was performed to gain insight into the cooperative nature of actomyosin binding. Binding events were broadly distributed at the highest Ca++ (pCa5), generating forces from 0.2 to 6.75pN, but averaged 1.38±0.05pN. As Ca++ was decreased the high force events were lost, reducing the average force to 0.76pN at pCa6, 0.61 at pCa7 and 0.2pN at pCa9. In addition the total number of actomyosin interactions decreased from 3.7/sec at pCa5 to just 0.05/sec at pCa9. Finally, we measured filament velocity in a motility assay as a function of Ca++ and ATP to distinguish rigor activation vs. Ca++ activation in a large ensemble of myosin. At pCa9 filaments were activated at ATP levels <200 μM while at intermediate Ca++ levels (pCa6.5) filaments achieved only half of the maximum velocity observed at saturating Ca++ (pCa5). Preliminary modeling efforts suggest that these observations are consistent with a model where Ca++ regulates myosin's weak to strong binding transition but once bound strongly an actomyosin interaction activates the filament locally, facilitating neighboring molecules to bind.

Different actions of salt and pyrophosphate on protein extraction from myofibrils reveal the mechanism controlling myosin dissociation.

Myosin is the major functional protein in muscle foods for water retention, protein binding/gelation and fat holding/emulsification. To maximize its functionality, myosin needs to be released from thick filaments. Understanding of the mechanism controlling myosin extraction will help improve quality traits of meat products. The data obtained show that actomyosin binding is the rate-limiting constraint for myosin release in rigor condition. Magnesium pyrophosphate (MgPPi) increased myosin extraction by weakening actomyosin interaction and maximized myosin extraction at 0.4 mol L(-1) NaCl, which was not attained at 1.0 mol L(-1) NaCl in the absence of PPi. Interaction between myosin rod domains is another critical constraint for myosin extraction, which is, rather than PPi, salt dependent. Further, our data suggest that MyBP-C (myosin binding protein C) and M-line might not be of significance in the process of NaCl-induced myosin extraction, though further study was needed. Our study provides new insight into the mechanism that controls myosin extraction from intact sarcomere, which could be applied to maximize myosin function and to improve meat quality in practice.

Observing the pCa-Force Relationship with a 3-Bead Laser Trap Assay

Muscular force is highly dependent on the amount of free Ca++ in the myoplasm because actomyosin binding is carefully regulated by troponin (Tn) and tropomyosin (Tm). To gain molecular insight into the Ca++ dependence of force, we directly observed the effect of Ca++ on the force-generating capacity of a mini-ensemble of myosin (∼6 heads) interacting with a single reconstituted thin filament (actin+Tn+Tm) in a three bead laser trap assay at 100uM ATP. Maximum force decreased in a Ca++-dependent manner (6.75, 4.75, 3.25, and 1.85pN at pCa 5, 6, 6.5 and 7 respectively). Furthermore, the average forces were significantly different (p 1.5pN where only 0.1% of the events achieved this force level at pCa7. In addition, the total percent of time the myosin molecules were strongly bound to the thin filament were 41, 21, 16, and 14% at pCa 5, 6, 6.5 and 7 respectively. Overall, these data provide unique insight into the molecular events that underlie the Ca++-dependence of force generation in a regulated system.

Macromolecular Crowding Increases Cross-Bridge Performance via Reduction of ADP Affinity to Acto-Myosin

We used methods of transient kinetics to determine if crowding agent Ficoll70, added to the reaction solution, changes the rate-limiting step of acto-myosin cycle, to explain the two-fold increase of acto-myosin ATPase activity in the presence of Ficoll70. D.discoideum myosin II S1 and rabbit skeletal actin were used as a model system. We determined kinetic rates of ATP binding to myosin, myosin recovery stroke, acto-myosin binding and dissociation, with and without ADP. Our experiments showed that virtually every kinetic step of the cross-bridge cycle was affected by Ficoll. Addition of Ficoll70 slows the rates of ATP binding and actin binding to myosin, as well as the rate of acto-myosin dissociation. Affinity of ADP to acto-myosin was reduced two-fold in the presence of Ficoll70. ADP release from acto-myosin is the rate limiting step of D.discoideum myosin II ATPase cycle, therefore reduced ADP affinity is the reason of increased acto-myosin ATPase activity in the presence of Ficoll70. Addition of Ficoll70 does not change the rate limiting step of D.discoideum myosin II ATPase cycle.

Allosteric effects of cardiac troponin TNT1 mutations on actomyosin binding: A novel pathogenic mechanism for hypertrophic cardiomyopathy

The majority of hypertrophic cardiomyopathy mutations in (cTnT) occur within the alpha-helical tropomyosin binding TNT1 domain. A highly charged region at the C-terminal end of TNT1 unwinds to create a flexible “hinge”. While this region has not been structurally resolved, it likely acts as an extended linker between the two cTnT functional domains. Mutations in this region cause phenotypically diverse and often severe forms of HCM. Mechanistic insight, however, has been limited by the lack of structural information. To overcome this limitation, we evaluated the effects of cTnT 160–163 mutations using regulated in vitro motility (R-IVM) assays and transgenic mouse models. R-IVM revealed that cTnT mutations Δ160E, E163R and E163K disrupted weak electrostatic actomyosin binding. Reducing the ionic strength or decreasing Brownian motion rescued function. This is the first observation of HCM-linked mutations in cTnT disrupting weak interactions between the thin filament and myosin. To evaluate the in vivo effects of altering weak actomyosin binding we generated transgenic mice expressing Δ160E and E163R mutant cTnT and observed severe cardiac remodeling and profound myofilament disarray. The functional changes observed in vitro may contribute to the structural impairment seen in vivo by destabilizing myofilament structure and acting as a constant pathophysiologic stress.

Dilatation of the Constricted Human Airway by Tidal Expansion of Lung Parenchyma

In the normal lung, breathing and deep inspirations potently antagonize bronchoconstriction, but in the asthmatic lung this salutary effect is substantially attenuated or even reversed. To explain these findings, the prevailing hypothesis focuses on contracting airway smooth muscle and posits a nonlinear dynamic interaction between actomyosin binding and the tethering forces imposed by tidally expanding lung parenchyma. This hypothesis has never been tested directly in bronchial smooth muscle embedded within intraparenchymal airways. Our objective here is to fill that gap. We designed a novel system to image contracting intraparenchymal human airways situated within near-normal lung architecture and subjected to dynamic parenchymal expansion that simulates breathing. Reversal of bronchoconstriction depended on the degree to which breathing actually stretched the airway, which in turn depended negatively on severity of constriction and positively on the depth of breathing. Such behavior implies positive feedbacks that engender airway instability. These findings help to explain heterogeneity of airflow obstruction as well as why, in people with asthma, deep inspirations are less effective in reversing bronchoconstriction.

The Two-Dimensional Kinetics of Binding and Unbinding are Both Regulated by Myosin's Actin-Binding Loop

Binding of myosin to actin is a multi-step, ionic strength-sensitive process for which the kinetic properties of bond formation and rupture have been studied in solution. However, the solution (three dimensional) environment is a poor model of the pseudo-two dimensional geometry in which myofilament proteins interact in vivo. We therefore investigated the rate of actomyosin bond formation and rupture in a two-dimensional setting using dynamic force spectroscopy at physiological and reduced ionic strength. We previously showed that catch bond rupture is slowed at physiologic ionic strength (145 mM KCl) when compared to low ionic strength (25 mM), and that bond lifetime is maximal at the isometric force generated by a single myosin molecule. In the current study we confirmed this result and sought to determine whether this difference is mirrored in the rate of actomyosin bond formation. A laser trap was used to measure the time to bond formation (tb) between actin filaments and nucleotide-free heavy meromyosin (HMM) over a range of compressive loads. Two dimensional on-rates were determined from tb and were increasingly force-dependent with decreasing ionic strength. We examined the effect of ionic strength on the initial long-range ionic interaction constituting the first step of actomyosin binding by targeted tryptic digestion of HMM’s actin-binding loop. Our data at physiologic ionic strength suggest that the actin-binding loop contributes to the decreased 2D rate and load-dependence of bond formation. This is matched by reduced rates of actomyosin unbinding. These data provide useful parameters for modeling studies and suggest that the actin-binding loop of myosin contributes to both the binding and unbinding of myosin from actin.