Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions

Abstract

J. Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions. Am J Physiol Heart Circ Physiol 308: H1248 -H1257, 2015. First published March 13, 2015; doi:10.1152/ajpheart.00834.2014 is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ϳ20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contractionrelaxation cycle may contribute to altered contractility and the development of FHC. left ventricle; mutation of regulatory light chain; polarization of fluorescence INTERACTIONS OF MYOSIN WITH actin and associated proteins (sarcomeric proteins) are responsible for muscle contraction (19,24,48). These interactions include the mode of attachment of actin to myosin during different physiological states and the kinetics of conformational changes of myosin when interacting with actin. Familial hypertrophic cardiomyopathy (FHC) has been recognized to be caused by mutations in all major sarcomeric proteins of the heart, including the myosin regulatory light chain (RLC) (1). Compared with the myosin heavy chain, mutations in the RLC are rare, but often associated with malignant outcomes (18,27,41). It is therefore important to find out whether these FHC-linked mutations result in alterations of the mode of attachment of myosin to actin, and/or whether they lead to alterations of kinetics of acto-myosin interactions. These changes may ultimately contribute to the development of FHC. In this report we have focused on the K104E (lysine to glutamic acid) substitution in the myosin RLC expressed in the heart of transgenic (Tg) mice (25). The mutation was identified in a Danish population by Andersen et al. (2) and was associated with left ventricu...

Impact of regulatory light chain mutation K104E on the ATPase and motor properties of cardiac myosin

“…6 B ; Huang et al, 2014 ). Previous work in this model also found an increase in the rate of cross-bridge binding to thin filaments and the rate of execution of the power stroke in contracting myofibrils from ex vivo left ventricles of Tg K104E mice ( Duggal et al, 2015 ). Purified myosin from this tissue also revealed a slightly slower second-order ATP-binding constant for Tg K104E compared with Tg WT myosin ( Duggal et al, 2015 ).…”

“…Previous work in this model also found an increase in the rate of cross-bridge binding to thin filaments and the rate of execution of the power stroke in contracting myofibrils from ex vivo left ventricles of Tg K104E mice ( Duggal et al, 2015 ). Purified myosin from this tissue also revealed a slightly slower second-order ATP-binding constant for Tg K104E compared with Tg WT myosin ( Duggal et al, 2015 ). Collectively, these findings in full-length α-cardiac myosin are contrary to those we observed in M2β-S1, supporting the notion that the effects of the mutation on intrinsic motor properties may be dependent on the length or isoform of the myosin backbone.…”

No Difference in Myosin Kinetics and Spatial Distribution of the Lever Arm in the Left and Right Ventricles of Human Hearts

“…Following labeling of recombinant peptide with SeTau-647 Maleimide, 5 nM of this protein was exchanged with the native LC of ventricular myosin in the exchange solution containing 15 mM KCl, 5 mM EDTA, 5 mM DTT, 10 mM KH 2 PO 4 , 5 mM ATP, 1 mM TFP, and 10 mM imidazole, pH 7 (Duggal et al, 2015 ; Huang et al, 2015 ). The reaction was allowed to occur at 30°C for 10 min.…”

Hereditary heart disease: pathophysiology, clinical presentation, and animal models of HCM, RCM, and DCM associated with mutations in cardiac myosin light chains

“…The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. Stopped-flow experiments revealed a significantly faster dissociation rate observed in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATPbinding rate for the K104E compared with WT myosin 21 . Barth et al reported a new skeletal muscle fiber type-I myopathy in three unrelated Dutch families with progressive cardiomyopathy and early infant deaths due to cardiac failure 11 , and the genetic cause was only recently identified by Wetermen et al to be a homozygous mutation in the last acceptor splice site of the myosin regulatory light chain 2 gene which results in use of a cryptic splice site upstream of the last exon causing a frameshift and replacement of the last 32 codons by 20 different codons 129 (Fig.…”