Getting the skinny on thick filament regulation in cardiac muscle biology and disease

doi:10.1016/j.tcm.2013.07.004

Trends in Cardiovascular Medicine

Volume 24, Issue 4, May 2014, Pages 133-141

https://doi.org/10.1016/j.tcm.2013.07.004 Get rights and content

Abstract

Thin (actin) filament accessory proteins are thought to be the regulatory force for muscle contraction in cardiac muscle; however, compelling new evidence suggests that thick (myosin) filament regulatory proteins are emerging as having independent and important roles in regulating cardiac muscle contraction. Key to these new findings is a growing body of evidence that point to an influential and, more recently, direct role for ventricular myosin light chain-2 (MLC2v) phosphorylation in regulating cardiac muscle contraction, function, and disease. This includes the discovery and characterization of a cardiac-specific myosin light chain kinase capable of phosphorylating MLC2v as well as a myosin phosphatase that dephosphorylates MLC2v in the heart, which provides added mechanistic insights on MLC2v regulation within cardiac muscle. Here, we review evidence for an emerging and critical role for MLC2v phosphorylation in regulating cardiac myosin cycling kinetics, function, and disease, based on recent studies performed in genetic mouse models and humans. We further provide new perspectives on future avenues for targeting these pathways as therapies in alleviating cardiac disease.

Section snippets

Structural components of the cardiac motor

The major components within the cardiac thick filament that control muscle contraction include cardiac myosin and its regulatory proteins: (i) essential light chain (ELC), (ii) myosin regulatory light chain-2 (ventricular myosin light chain-2 (MLC2v) in cardiac muscle), and (iii) myosin-binding protein C (MyBP-C). Substructure analyses of myosin revealed that it is a hexameric protein consisting of a pair of myosin heavy chains and two pairs of light chains (two ELC and two MLC2) (Warrick and

MLC2v phosphorylation: at the heart of cross-bridge cycling kinetics and cardiac myofilament function

Compelling new evidence using integrated, computational, and genetic mouse models provided important mechanistic insights into a direct, independent, and indispensable role for MLC2v phosphorylation in regulating calcium-dependent cardiac muscle contraction and function (Sheikh et al., 2012). Most importantly, we were able to show the fluidity of these mechanisms to drive biological findings in cardiac muscle at increasing levels of complexity starting from the cross-bridge, to the myofilament,

Reconstructing a role for MLC2v phosphorylation in adult cardiac function and disease

MLC2v plays an essential role in early embryonic cardiac development and function (Chen et al., 1998); however, it was not until recent studies that its regulatory effects following phosphorylation were shown to play an essential role in adult heart physiology and function. Specifically, a growing number of genetic mouse models alongside human studies have paved the way toward identifying an essential role for MLC2v phosphorylation in adult cardiac torsion, function, and disease in vivo (Table 1

MLC2v phosphorylation as a potential therapeutic target for alleviating cardiac disease

A role for MLC2v phosphorylation in the adaptive response to stress-induced cardiac hypertrophy and disease was also identified when both young non-phosphorylatable MLC2v knock-in mutant mice, utilized at a stage when no structural defects or disease was observed, as well as cardiac MLCK null mice displayed an exacerbated response to cardiac dysfunction following pressure overload (Sheikh et al., 2012, Warren et al., 2012). Intriguingly, we showed that young mutant hearts displayed a

Conclusions and future directions

New findings from genetic mouse models strongly suggest critical roles for MLC2v phosphorylation in cardiac myosin cycling kinetics, contraction, torsion, function, and disease (Table 2). These studies have provided important relevance to human studies that highlighted a role for MLC2v phosphorylation in the pathogenesis of human cardiac disease (Table 1). Key to these findings is the identification of cardiac MLCK and myosin phosphatase, which provided additional proof toward an essential role

Acknowledgments

We thank Dr. Valeria Mezzano (UCSD, La Jolla, CA) for technical assistance with the figure. R.C.L. is a recipient of an American Heart Association Postdoctoral fellowship. F.S. and J.C are supported by grants from the National Institutes of Health.

References (51)

A.N. Chang et al.
Cardiac myosin is a substrate for Zipper-interacting protein kinase (ZIPK)
Journal of Biological Chemistry
(2010)
J. Chen et al.
Selective requirement of myosin light chain 2v in embryonic heart function
Journal of Biological Chemistry
(1998)
J.S. Davis et al.
The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation
Cell
(2001)
F.A. Dias et al.
The effect of myosin regulatory light chain phosphorylation on the frequency-dependent regulation of cardiac function
Journal of Molecular and Cellular Cardiology
(2006)
P. Ding et al.
Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo
Journal of Biological Chemistry
(2010)
Z. Grabarek
Structural basis for diversity of the EF-hand calcium-binding proteins
Journal of Molecular Biology
(2006)
C. Hidalgo et al.
Effect of diastolic pressure on MLC2v phosphorylation in the rat left ventricle
Archives of Biochemistry and Biophysics
(2006)
J. Huang et al.
Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy
Journal of Biological Chemistry
(2008)
M. Ikebe et al.
Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin
Journal of Biological Chemistry
(1986)
K.E. Kamm et al.
Signaling to myosin regulatory light chain in sarcomeres
Journal of Biological Chemistry
(2011)

R.J. Levine et al.

Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments

Biophysical Journal

(1996)

R. Okamoto et al.

Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart

Cellular Signalling

(2006)

J. Ratti et al.

Structure and interactions of myosin-binding protein C domain C0: cardiac-specific regulation of myosin at its neck?

Journal of Biological Chemistry

(2011)

A. Sanbe et al.

Abnormal cardiac structure and function in mice expressing non-phosphorylatable cardiac regulatory myosin light chain 2

Journal of Biological Chemistry

(1999)

S.B. Scruggs et al.

Ablation of ventricular myosin regulatory light chain phosphorylation in mice causes cardiac dysfunction in situ and affects neighboring myofilament protein phosphorylation

Journal of Biological Chemistry

(2009)

S.B. Scruggs et al.

A novel, in-solution separation of endogenous cardiac sarcomeric proteins and identification of distinct charged variants of regulatory light chain

Molecular and Cellular Proteomics

(2010)

D. Szczesna et al.

Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca²⁺ binding, and phosphorylation

Journal of Biological Chemistry

(2001)

C. Toepfer et al.

Myosin regulatory light chain (RLC) phosphorylation change as a modulator of cardiac muscle contraction in disease

Journal of Biological Chemistry

(2013)

M.P. Walsh et al.

Purification and characterization of bovine cardiac calmodulin-dependent myosin light chain kinase

Journal of Biological Chemistry

(1979)

T.P. Abraham et al.

Diastolic dysfunction in familial hypertrophic cardiomyopathy transgenic model mice

Cardiovascular Research

(2009)

H.A. AL Khayat et al.

Atomic model of the human cardiac muscle myosin filament

Proceedings of the National Academy of Sciences of the United States of America

(2013)

J.H. Brown et al.

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

Proceedings of the National Academy of Sciences of the United States of America

(2011)

J.Y. Chan et al.

Identification of cardiac-specific myosin light chain kinase

Circulation Research

(2008)

B.A. Colson et al.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

The Journal of Physiology

(2010)

J. England et al.

Heavy and light roles: myosin in the morphogenesis of the heart

Cellular and Molecular Life Sciences

(2013)

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Review articleGetting the skinny on thick filament regulation in cardiac muscle biology and disease

Abstract

Section snippets

Structural components of the cardiac motor

MLC2v phosphorylation: at the heart of cross-bridge cycling kinetics and cardiac myofilament function

Reconstructing a role for MLC2v phosphorylation in adult cardiac function and disease

MLC2v phosphorylation as a potential therapeutic target for alleviating cardiac disease

Conclusions and future directions

Acknowledgments

Journal of Biological Chemistry

Journal of Biological Chemistry

Cell

Journal of Molecular and Cellular Cardiology

Journal of Biological Chemistry

Journal of Molecular Biology

Archives of Biochemistry and Biophysics

Journal of Biological Chemistry

Journal of Biological Chemistry

Journal of Biological Chemistry

Biophysical Journal

Cellular Signalling

Journal of Biological Chemistry

Journal of Biological Chemistry

Journal of Biological Chemistry

Molecular and Cellular Proteomics

Journal of Biological Chemistry

Journal of Biological Chemistry

Journal of Biological Chemistry

Diastolic dysfunction in familial hypertrophic cardiomyopathy transgenic model mice

Cardiovascular Research

Atomic model of the human cardiac muscle myosin filament

Proceedings of the National Academy of Sciences of the United States of America

Visualizing key hinges and a potential major source of compliance in the lever arm of myosin

Proceedings of the National Academy of Sciences of the United States of America

Identification of cardiac-specific myosin light chain kinase

Circulation Research

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

The Journal of Physiology

Heavy and light roles: myosin in the morphogenesis of the heart

Cellular and Molecular Life Sciences

Review article
Getting the skinny on thick filament regulation in cardiac muscle biology and disease