Essential light chain S195 phosphorylation is required for cardiac adaptation under physical stress

Scheid, Lisa-Mareike; Mosqueira, Matías; Hein, Selina; Kossack, Mandy; Juergensen, Lonny; Mueller, Marianne; Meder, Benjamin; Fink, Rainer H. A.; Katus, Hugo A.; Hassel, David

doi:10.1093/cvr/cvw066

Cited by 28 publications

(32 citation statements)

References 47 publications

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“…Finally, phosphorylation in myosin essential light chain has also been studied. Scheid et al (2016) investigated the role of the highly conserved Ser-195 phosphorylation site of the ELC using heterozygous adult zebrafish lazy susan (laz m647 ) in regulating contractile function in normal physiology and disease. After physical stress, heart function of laz heterozygous zebrafish severely deteriorated, causing heart failure and sudden death.…”

Section: Therapeutic Potential Of Myosin Rlc Pseudo-phosphorylationmentioning

confidence: 99%

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

Yadav

Szczesna‐Cordary

2017

Biophys Rev

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Many genetic mutations in sarcomeric proteins, including the cardiac myosin regulatory light chain (RLC) encoded by the MYL2 gene, have been implicated in familial cardiomyopathies. Yet, the molecular mechanisms by which these mutant proteins regulate cardiac muscle mechanics in health and disease remain poorly understood. Evidence has been accumulating that RLC phosphorylation has an influential role in striated muscle contraction and, in addition to the conventional modulation via Ca 2+ binding to troponin C, it can regulate cardiac muscle function. In this review, we focus on RLC mutations that have been reported to cause cardiomyopathy phenotypes via compromised RLC phosphorylation and elaborate on pseudo-phosphorylation rescue mechanisms. This new methodology has been discussed as an emerging exploratory tool to understand the role of phosphorylation as well as a genetic modality to prevent/rescue cardiomyopathy phenotypes. Finally, we summarize structural effects postphosphorylation, a phenomenon that leads to an ordered shift in the myosin S1 and RLC conformational equilibrium between two distinct states.

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Section: Therapeutic Potential Of Myosin Rlc Pseudo-phosphorylationmentioning

confidence: 99%

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

Yadav

Szczesna‐Cordary

2017

Biophys Rev

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“…Dissection procedures were published previously (Gupta and Mullins, 2010; Iorga et al, 2011; Singleman and Holtzman, 2011; Arnaout et al, 2014; Scheid et al, 2016). Nevertheless, they will be described in some detail to provide information for zebrafish based IVMAs within this protocol.…”

Section: Materials and Proceduresmentioning

confidence: 99%

“…The presence not only of the myosin heavy and light chains, but also of the regulatory proteins tropomyosin and troponin in the zebrafish cardiac extracts (Scheid et al, 2016) enables the analysis of calcium regulatory effects on filament sliding.…”

Section: Introductionmentioning

confidence: 99%

“…The usefulness of these protocols was shown in the identification of stress-induced phosphorylation at the ventricular essential myosin light chain (ELC) in zebrafish (Scheid et al, 2016). In this context, the proposed method using zebrafish ventricular and skeletal muscle proteins in the IVMA was used to understand the consequence of the causative mutation at the molecular functional level (Scheid et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

“…In this context, the proposed method using zebrafish ventricular and skeletal muscle proteins in the IVMA was used to understand the consequence of the causative mutation at the molecular functional level (Scheid et al, 2016). Taken together, this new protocol is optimized to study cardiac myosin and regulatory protein function as well as skeletal muscle actin from adult zebrafish at the molecular level in vitro .…”

Section: Introductionmentioning

confidence: 99%

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Extraction Protocols for Individual Zebrafish's Ventricle Myosin and Skeletal Muscle Actin for In vitro Motility Assays

et al. 2017

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The in vitro motility assay (IVMA) is a technique that enables the measurement of the interaction between actin and myosin providing a relatively simple model to understand the mechanical muscle function. For actin-myosin IVMA, myosin is immobilized in a measurement chamber, where it converts chemical energy provided by ATP hydrolysis into mechanical energy. The result is the movement of fluorescently labeled actin filaments that can be recorded microscopically and analyzed quantitatively. Resulting sliding speeds and patterns help to characterize the underlying actin-myosin interaction that can be affected by different factors such as mutations or active compounds. Additionally, modulatory actions of the regulatory proteins tropomyosin and troponin in the presence of calcium on actin-myosin interaction can be studied with the IVMA. Zebrafish is considered a suitable model organism for cardiovascular and skeletal muscle research. In this context, straightforward protocols for the isolation and use of zebrafish muscle proteins in the IVMA would provide a useful tool in molecular studies. Currently, there are no protocols available for the mentioned purpose. Therefore, we developed fast and easy protocols for characterization of zebrafish proteins in the IVMA. Our protocols enable the interested researcher to (i) isolate actin from zebrafish skeletal muscle and (ii) extract functionally intact myosin from cardiac and skeletal muscle of individual adult zebrafish. Zebrafish tail muscle actin is isolated after acetone powder preparation, polymerized, and labeled with Rhodamine-Phalloidin. Myosin from ventricles of adult zebrafish is extracted directly into IVMA flow-cells. The same extraction protocol is applicable for comparably small tissue pieces as from zebrafish tail, mouse and frog muscle. After addition of the fluorescently labeled F-actin from zebrafish—or other origin—and ATP, sliding movement can be visualized using a fluorescence microscope and an intensified CCD camera. Taken together, we introduce a method for functional analysis in zebrafish cardiac and skeletal muscle research to study mutations at the molecular level of thick or thin filament proteins. Additionally, preliminary data indicate the usefulness of the presented method to perform the IVMA with myosin extracted from muscles of other animal models.

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Interrogating Cardiovascular Genetics in Zebrafish

Liu

Renz

Hassel

2019

Genetic Causes of Cardiac Disease

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Essential light chain S195 phosphorylation is required for cardiac adaptation under physical stress

Abstract: Using adult heterozygous zebrafish, we show that ELC S195 phosphorylation is pivotal for adaptation of cardiac function to augmented physical stress and we provide novel mechanistic insights into the pathogenesis of ELC-linked cardiomyopathy.

Cited by 28 publications

References 47 publications

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

Extraction Protocols for Individual Zebrafish's Ventricle Myosin and Skeletal Muscle Actin for In vitro Motility Assays

Interrogating Cardiovascular Genetics in Zebrafish

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