A role for actin flexibility in thin filament-mediated contractile regulation and myopathy

Abstract

Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformationdependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.

TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction

“…The cardiac tube diameters, which are directly proportional to cellular length, were measured during diastole and again after each treatment. As shown previously, WT dorsal vessel diastole involves less than full 100% relaxation in this preparation (15,17,51). This is evident from the effect of intracellular chelation and complete dissociation of Ca 2+ via EGTA/EGTA,AM, which increased the diameter by 2.6% compared to diastole, and from myosin inhibition via blebbistatin that caused another 3.7% increase (Fig.…”

“…No matter its primacy, this switch is considered insufficient for contractile activation; actions of myosin cross-bridges are also required. The switch is also insufficient for inhibition, which requires other features of Tn, Tpm, and actin (9,(12)(13)(14)(15)(16)(17). In particular, the N-terminal half of TnT, otherwise known as TNT1, may play a role in relaxation, to a degree that is an open subject of investigation.…”

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

“…Drosophila serves as a vehicle for transgenic actin production that can be purified in ample amounts needed for in vitro studies (22,49,54). The Act88F IFM actin isoform shares 92% identity with human ACTA skeletal actin, and previous in vitro studies have confirmed that the two behave indistinguishably in vitro (22,55).…”

“…Tissue-purified porcine cardiac Tpm was provided by Dr. Jeff Moore (University of Massachusetts-Lowell) and bovine cardiac isoforms of Tpm and Tn were provided by Dr. Larry Tobacman (University of Illinois at Chicago). Pools containing ectopically-expressed Act57B WT , Act57B K326Q , or Act57B K328Q actin were purified from Drosophila IFMs according to previously published protocols (22,49,54).…”

The remodelling of actin composition as a hallmark of cancer

“…4 ) [137] . Dysregulated actin dynamics can be seen when an alteration to the filament occurs since this disrupts the electrostatic contacts between the F-actin and TM [138] . Altering TM-binding would affect the binding of ABPs such as cofilin as well as the loss of inhibition of actin filament branching and nucleation at the leading edge by the Arp2/3 complex.…”