Lysine acetylation of F-actin decreases tropomyosin-based inhibition of actomyosin activity

Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys326 and Lys328, are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca2+ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys326 and Lys328 are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, nonspecific actin acetylation did not disrupt characteristic F-actin–Tpm binding. However, it significantly reduced Tpm-mediated inhibition of myosin attachment, as reflected by increased F-actin–Tpm motility that persisted in the presence of Tn and submaximal Ca2+. Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys326 and Lys328, also resulted in less inhibited F-actin–Tpm, implying that modifying only these residues influences Tpm's location and, potentially, thin filament regulation. To unequivocally determine the residue-specific consequences of acetylation on Tn–Tpm–based regulation of actomyosin activity, we assessed the effects of K326Q and K328Q acetyl (Ac)-mimetic actin on Ca2+-dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca2+ sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys328, modulates muscle contraction via disrupting inhibitory Tpm positioning.

The mechanism of thin filament regulation: Models in conflict?

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.

Investigation into the mechanism of thin filament regulation by transient kinetics and equilibrium binding: Is there a conflict?

Striated muscle contraction occurs when myosin undergoes a lever-type structural change. This process (the power stroke) requires ATP and is governed by the thin filament, a complex of actin, tropomyosin, and troponin. The authors have used a fast-mixing instrument to investigate the mechanism of regulation. Such (pre–steady-state kinetic) experiments allow biochemical intermediates in a working actomyosin cycle to be monitored. The regulatory focal point is demonstrated to be the step that involves the departure of inorganic phosphate (i.e., AM-ADP-Pi → AM-ADP). This part of the cycle, which lies on the main kinetic pathway and coincides with the drive stroke, is maximally accelerated ∼100-fold by the combined association of ligands (Ca[II] and rigor myosin heads) with the thin filament. However, the observed ligand dependencies of the rates of Pi dissociation that are reported herein are at variance with predictions of models derived from experiments where ATP hydrolysis is not taking place (and myosin exists in a nonphysiological form). It is concluded that the principal influence of the thin filament is in setting the rate of Pi dissociation and that physiological levels of regulation are dependent upon the liganded state of the thin filament as well as the conformation of myosin.

Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin’s thin-filament pointed-end assembly but does not affect tropomodulin’s assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin’s affinity for F-actin and affects leiomodin’s function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin’s function.