Myosin light chain phosphorylation to the rescue

Granzier, Henk; Tombe, Pieter P. de

doi:10.1073/pnas.1511455112

Cited by 9 publications

(12 citation statements)

References 23 publications

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“…These in vitro results gave basis to testing the S15D phosphimimic-mediated rescue of HCM phenotype in vivo [12]. We showed that S15D pseudophosphorylation of D166V-RLC mutant was sufficient to prevent the development of HCM in mice [12,28]. Future studies will explore and evaluate the therapeutic potential of pseudophosphorylation of R58Q-RLC in vivo to rescue its HCM phenotype.…”

Section: Discussionmentioning

confidence: 85%

Phosphomimetic‐mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain

et al. 2018

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Myosin regulatory light chain (RLC) phosphorylation is important for cardiac muscle mechanics/function as well as for the Ca2+-troponin/tropomyosin regulation of muscle contraction. This study focuses on the arginine to glutamine (R58Q) substitution in the human ventricular RLC (MYL2 gene), linked to malignant hypertrophic cardiomyopathy in humans and causing severe functional abnormalities in transgenic R58Q mice, including inhibition of cardiac RLC phosphorylation. Using a phosphomimic recombinant RLC variant where the phosphorylation site Ser-15 was substituted with an aspartic acid (S15D) and placed in the background of R58Q, we aimed to assess whether we could rescue/mitigate R58Q-induced structural/functional abnormalities in vitro. We show rescue of several R58Q-exerted adverse phenotypes in S15D-R58Q-reconstituted porcine cardiac muscle preparations. A low level of maximal isometric force observed for R58Q- versus WT-reconstituted fibers was restored by S15D-R58Q. Significant beneficial effects were also observed on the Vmax of actin-activated myosin ATPase activity in S15D-R58Q versus R58Q-reconstituted myosin, along with its binding to fluorescently-labeled actin. We also report that R58Q promotes the OFF state of myosin, both in reconstituted porcine fibers and in transgenic mouse papillary muscles, thereby stabilizing the super-relaxed state (SRX) of myosin, characterized by a very low ATP turnover rate. Experiments in S15D-R58Q-reconstituted porcine fibers showed a mild destabilization of the SRX state, suggesting an S15D-mediated shift in disordered-relaxed (DRX)↔SRX equilibrium towards the DRX state of myosin. Our study shows that S15D-phosphomimic can be used as a potential rescue strategy to abrogate/alleviate the RLC mutation-induced phenotypes and is a likely candidate for therapeutic intervention in HCM patients.

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Section: Discussionmentioning

confidence: 85%

Phosphomimetic‐mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain

et al. 2018

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“…In this study, we aimed to test a new method to reverse the effects of HCM, caused by the D166V mutation, using a highly effective strategy of AAV-mediated gene delivery. The proof-of-principle was recently published [19,36], where we showed that the functional, structural and histological phenotypes observed in HCM-D166V mice could be prevented by cardiac-specific expression of phosphomimic S15D-D166V. Importantly, experiments in S15D-D166V mice demonstrated that the depressed RLC phosphorylation in D166V myocardium was not a product of reduced cMLCK expression, and that the hypophosphorylation of myosin RLC in D166V hearts was most likely a result of mutationelicited steric constraints preventing cMLCK to effectively phosphorylate RLC at Ser15 [19,22].…”

Section: Discussionmentioning

confidence: 99%

Therapeutic potential of AAV9-S15D-RLC gene delivery in humanized MYL2 mouse model of HCM

et al. 2019

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Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on Aspartic acid-to-Valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in nonphosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-Associated Virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with Serine-to-Aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV-vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLC-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation.

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“…Therefore, combining in silico MD simulations and pseudophosphorylation is a powerful technique for understanding basic structure–function relationship and also the mechanistic basis of disease-causing mutations of RLC. Pseudophosphorylation not only improves systolic function but also avoids adverse effects resulting from the D166V RLC mutation (Granzier and de Tombe 2015). In such pseudophosphorylation, the phospho-mimic negative charge is constitutive, which means that in vivo, the heart cannot control the charged-state of RLC and dynamically respond to altered demands.…”

Section: In Vivo Modificationsmentioning

confidence: 99%

“…The drawback of this S15D mutation approach is that it will not easily be adapted to patients. A possible alternative is the manipulation of the activities of either cMLCK (increasing it) or cMLCP (lowering it) (Granzier and de Tombe 2015). The reason is that these enzymes are highly specific toward cRLC, and therefore the technique may be feasible without adversely affecting effects on other proteins (Kamm and Stull 2011).…”

Section: In Vivo Modificationsmentioning

confidence: 99%

Phosphorylation of the regulatory light chain of myosin in striated muscle: methodological perspectives

Chakravorty

Song

et al. 2016

Eur Biophys J

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Phosphorylation of the regulatory light chain (RLC) of myosin modulates cellular functions such as muscle contraction, mitosis, and cytokinesis. Phosphorylation defects are implicated in a number of diseases. Here we focus on striated muscle where changes in RLC phosphorylation relate to diseases such as hypertrophic cardiomyopathy and muscular dystrophy, or age-related changes. RLC phosphorylation in smooth muscle and non-muscle cells are covered briefly where relevant. There is much scientific interest in controlling the phosphorylation levels of RLC in vivo and in vitro in order to understand its physiological function in striated muscles. A summary of available and emerging in vivo and in vitro methods is presented. The physiological role of RLC phosphorylation and novel pathways are discussed to highlight the differences between muscle types and to gain insights into disease processes.

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Myosin light chain phosphorylation to the rescue

Cited by 9 publications

References 23 publications

Phosphomimetic‐mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain

Phosphomimetic‐mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain

Therapeutic potential of AAV9-S15D-RLC gene delivery in humanized MYL2 mouse model of HCM

Phosphorylation of the regulatory light chain of myosin in striated muscle: methodological perspectives

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