Cardiomyopathy-associated mutations in tropomyosin differently affect actin–myosin interaction at single-molecule and ensemble levels

Galina V. Kopylova, Daniil V. Shchepkin, Salavat R. Nabiev, Alexander M. Matyushenko, Natalia A. Koubassova, Dmitrii I. Levitsky, Sergey Y. Bershitsky

In the heart, mutations in the TPM1 gene encoding the α-isoform of tropomyosin lead, in particular, to the development of hypertrophic and dilated cardiomyopathies. We compared the effects of hypertrophic, D175N and E180G, and dilated, E40K and E54K, cardiomyopathy mutations in TPM1 gene on the properties of single actin–myosin interactions and the characteristics of the calcium regulation in an ensemble of myosin molecules immobilised on a glass surface and interacting with regulated thin filaments. Previously, we showed that at saturating Ca2+ concentration the presence of Tpm on the actin filament increases the duration of the interaction. Here, we found that the studied Tpm mutations differently affected the duration: the D175N mutation reduced it compared to WT Tpm, while the E180G mutation increased it. Both dilated mutations made the duration of the interaction even shorter than with F-actin. The duration of the attached state of myosin to the thin filament in the optical trap did not correlate to the sliding velocity of thin filaments and its calcium sensitivity in the in vitro motility assay. We suppose that at the level of the molecular ensemble, the cooperative mechanisms prevail in the manifestation of the effects of cardiomyopathy-associated mutations in Tpm.

DOI

Acidosis affects muscle contraction by slowing the rates myosin attaches to and detaches from actin

Katelyn Jarvis, Mike Woodward, Edward P. Debold, Sam Walcott

The loss of muscle force and power during fatigue from intense contractile activity is associated with, and likely caused by, elevated levels of phosphate ( [Math Processing Error]) and hydrogen ions (decreased pH). To understand how these deficits in muscle performance occur at the molecular level, we used direct measurements of mini-ensembles of myosin generating force in the laser trap assay at pH 7.4 and 6.5. The data are consistent with a mechanochemical model in which a decrease in pH reduces myosin’s detachment from actin (by slowing ADP release), increases non-productive myosin binding (by detached myosin rebinding without a powerstroke), and reduces myosin’s attachment to actin (by slowing the weak-to-strong binding transition). Additional support of this mechanism is found by incorporating it into a branched pathway model for the effects of [Math Processing Error] on myosin’s interaction with actin. Including pH-dependence in one additional parameter (acceleration of [Math Processing Error]-induced detachment), the model reproduces experimental measurements at high and low pH, and variable [Math Processing Error], from the single molecule to large ensemble levels. Furthermore, when scaled up, the model predicts force-velocity relationships that are consistent with muscle fiber measurements. The model suggests that reducing pH has two opposing effects, a decrease in attachment favoring a decrease in muscle force and a decrease in detachment favoring an increase in muscle force. Depending on experimental details, the addition of [Math Processing Error] can strengthen one or the other effect, resulting in either synergistic or antagonistic effects. This detailed molecular description suggests a molecular basis for contractile failure during muscle fatigue.

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Cooperativity of myosin interaction with thin filaments is enhanced by stabilizing substitutions in tropomyosin

Daniil V. Shchepkin, Salavat R. Nabiev, Galina V. Kopylova, Alexander M. Matyushenko, Dmitrii I. Levitsky, Sergey Y. Bershitsky, Andrey K. Tsaturyan

Muscle contraction is powered by myosin interaction with actin-based thin filaments containing Ca2+-regulatory proteins, tropomyosin and troponin. Coiled-coil tropomyosin molecules form a long helical strand that winds around actin filament and either shields actin from myosin binding or opens it. Non-canonical residues G126 and D137 in the central part of tropomyosin destabilize its coiled-coil structure. Their substitutions for canonical ones, G126R and D137L, increase structural stability and the velocity of sliding of reconstructed thin filaments along myosin coated surface. The effect of these stabilizing mutations on force of the actin–myosin interaction is unknown. It also remains unclear whether the stabilization affects single actin–myosin interactions or it modifies the cooperativity of the binding of myosin molecules to actin. We used an optical trap to measure the effects of the stabilization on step size, unitary force and duration of the interactions at low and high load and compared the results with those obtained in an in vitro motility assay. We found that significant prolongation of lifetime of the actin–myosin complex under high load observed at high extent of tropomyosin stabilization, i.e. with double mutant, G126R/D137L, correlates with higher force in the motility assay. Also, the higher the extent of stabilization of tropomyosin, the fewer myosin molecules are needed to propel the thin filaments. The data suggest that the effects of the stabilizing mutations in tropomyosin on the myosin interaction with regulated thin filaments are mainly realized via cooperative mechanisms by increasing the size of cooperative unit.

DOI

Loop 2 of myosin is a force-dependent inhibitor of the rigor bond

Amy M. Clobes, William H. Guilford

Myosin’s actin-binding loop (loop 2) carries a charge opposite to that of its binding site on actin and is thought to play an important role in ionic interactions between the two molecules during the initial binding step. However, no subsequent role has been identified for loop 2 in actin-myosin binding. We used an optical trap to measure bond formation and bond rupture between actin and rigor heavy meromyosin when loaded perpendicular to the filament axis. We studied HMM with intact or proteolytically cleaved loop 2 at low and physiologic ionic strength. Here we show that the presence of intact loop 2 allows actomyosin bonds to form quickly and that they do so in a short-lived bound state. Increasing tensile load causes the transition to a long-lived state—the distinguishing behavior of a catch bond. When loop 2 was cleaved catch bond behavior was abrogated leaving only a long-lived state. These data suggest that in addition to its role in locating binding sites on actin, loop 2 is also a force-dependent inhibitor of the long-lived actomyosin complex. This may be important for reducing the duty ratio and increasing the shortening velocity of actomyosin at low forces.

DOI

Mechanical and kinetic properties of β-cardiac/slow skeletal muscle myosin

Bernhard Brenner, Nils Hahn, Eva Hanke, Faramarz Matinmehr, Tim Scholz, Walter Steffen, Theresia KraftWe aimed to establish reference parameters to identify functional effects of familial hypertrophic cardiomyopathy-related point mutations in the β-cardiac/slow skeletal muscle myosin heavy chain (β-cardiac/MyHC-1). We determined mechanical and kinetic parameters of the β-cardiac/MyHC-1 using human soleus muscle fibers that express the same myosin heavy chain (MyHC-1) as ventricular myocardium (β-cardiac). The observed parameters are compared to previously reported data for rabbit psoas muscle fibers. We found all of the examined kinetic parameters to be slower in soleus fibers than in rabbit psoas muscle. Somewhat surprisingly, however, we also found that the stiffness of the β-cardiac/MyHC-1 head domain is more than 3-fold lower than the stiffness of the fast isoform of psoas fibers. Furthermore, and different from rabbit psoas muscle, in human soleus fibers both the occupancy of force-generating cross-bridge states as well as the elastic extension of force-generating heads increase with temperature. Thus, a myosin head in the force generating states makes an increasing contribution to force with temperature. We support some of our fiber data by data from in vitro motility and optical trapping assays. Initial findings with FHC-related point mutations in the converter imply that the differences in stiffness of the head domain between the slow and fast isoform may well be due to particular differences in the amino acid sequence of the converter. We show that the slower kinetics may be linked to a larger flexibility of the β-cardiac/MyHC-1 isoform compared to fast MyHC isoforms.

DOI

Mechanical and kinetic properties of β-cardiac/slow skeletal muscle myosin

Bernhard Brenner, Nils Hahn, Eva Hanke, Faramarz Matinmehr, Tim Scholz, Walter Steffen and Theresia Kraft

We aimed to establish reference parameters to identify functional effects of familial hypertrophic cardiomyopathy-related point mutations in the β-cardiac/slow skeletal muscle myosin heavy chain (β-cardiac/MyHC-1). We determined mechanical and kinetic parameters of the β-cardiac/MyHC-1 using human soleus muscle fibers that express the same myosin heavy chain (MyHC-1) as ventricular myocardium (β-cardiac). The observed parameters are compared to previously reported data for rabbit psoas muscle fibers. We found all of the examined kinetic parameters to be slower in soleus fibers than in rabbit psoas muscle. Somewhat surprisingly, however, we also found that the stiffness of the β-cardiac/MyHC-1 head domain is more than 3-fold lower than the stiffness of the fast isoform of psoas fibers. Furthermore, and different from rabbit psoas muscle, in human soleus fibers both the occupancy of force-generating cross-bridge states as well as the elastic extension of force-generating heads increase with temperature. Thus, a myosin head in the force generating states makes an increasing contribution to force with temperature. We support some of our fiber data by data from in vitro motility and optical trapping assays. Initial findings with FHC-related point mutations in the converter imply that the differences in stiffness of the head domain between the slow and fast isoform may well be due to particular differences in the amino acid sequence of the converter. We show that the slower kinetics may be linked to a larger flexibility of the β-cardiac/MyHC-1 isoform compared to fast MyHC isoforms.

DOI