Distinct sequences and post-translational modifications in cardiac atrial and ventricular myosin light chains revealed by top-down mass spectrometry

https://doi.org/10.1016/j.yjmcc.2017.04.002Get rights and content

Highlights

  • Identified N-terminal acetylation and methylation in RLCa and ELCa, respectively

  • Definitively localized phosphorylation site in mono-phosphorylated RLCa to Ser22

  • Identified and corrected errors in database sequences for swine RLCa and ELCa

  • Confirmed Nα-methylation as the N-terminal modification in RLCv and ELCv

  • Confirmed sequences of human atrial and ventricular myosin light chains

Abstract

Myosin is the principal component of the thick filaments that, through interactions with the actin thin filaments, mediates force production during muscle contraction. Myosin is a hexamer, consisting of two heavy chains, each associated with an essential (ELC) and a regulatory (RLC) light chain, which bind the lever-arm of the heavy chain and play important modulatory roles in striated muscle contraction. Nevertheless, a comprehensive assessment of the sequences of the ELC and RLC isoforms, as well as their post-translational modifications, in the heart remains lacking. Herein, utilizing top-down high-resolution mass spectrometry (MS), we have comprehensively characterized the sequences and N-terminal modifications of the atrial and ventricular isoforms of the myosin light chains from human and swine hearts, as well as the sites of phosphorylation in the swine proteins. In addition to the correction of disparities in the database sequences of the swine proteins, we show for the first time that, whereas the ventricular isoforms of the ELC and RLC are methylated at their N-termini, which is consistent with previous studies, the atrial isoforms of the ELC and RLC from both human and swine are Nα-methylated and Nα-acetylated, respectively. Furthermore, top-down MS with electron capture dissociation enabled localization of the sites of phosphorylation in swine RLC isoforms from the ventricles and atria to Ser14 and Ser22, respectively. Collectively, these results provide new insights into the sequences and modifications of myosin light chain isoforms in the human and swine hearts, which will pave the way for a better understanding of their functional roles in cardiac physiology and pathophysiology.

Introduction

The thick filaments of muscles, including cardiac, skeletal, and smooth muscles, are predominantly composed of myosin that, through interactions with actin (the principal component of thin filaments), mediates force production during muscle contraction [1], [2]. Myosin is a hexamer consisting of two heavy chains (MHCs), each of which is associated with an essential (or alkali) light chain (ELC) and a regulatory (or phosphorylatable) light chain (RLC) [3]. In striated muscles both the ELC and RLC, which bind and stabilize the lever-arm of the MHC [4], [5], play critical roles in the modulation of contractile function [6], [7], [8], [9], [10], [11]. In support of this, mutations in both the ELC and RLC are associated with the development of cardiac and skeletal muscle myopathies [12]. Additionally, phosphorylation of the RLC by the Ca2 +/calmodulin-dependent myosin light chain kinase (MLCK) represents a critical mechanism regulating contractility, especially in the heart [6], [11], [13], [14], [15], [16]. In particular, recent studies have convincingly demonstrated that loss of RLC phosphorylation leads to pathological cardiac hypertrophy and heart failure [16], [17], [18], [19]. These findings have generated substantial interest in the roles played by myosin light chains and their modifications in cardiac physiology and pathophysiology; thus, a comprehensive assessment of myosin light chain isoforms and their post-translational modifications (PTMs) in the heart is apropos.

A number of different myosin light chain isoforms have been identified to date, all of which belong to the EF-hand protein super-family of Ca2 +-binding proteins [20]. Four of these genes are expressed in the heart, with expression patterns that vary by chamber, developmentally, and in response to pathological stimuli [7], [10], [15]. The MYL3 and MYL4 genes encode the ventricular (ELCv) and atrial (ELCa) isoforms of the ELC, while the ventricular (RLCv) and atrial (RLCa) isoforms of the RLC are encoded by the MYL2 and MYL7 genes, respectively. Although expression of ELCv is primarily restricted to the ventricles of the heart, ELCa is expressed in both the atria and ventricles during normal embryonic development [7], [10]. In adulthood, however, expression of the ELCa is restricted to the atria, although re-expression in the ventricles occurs in response to pressure overload and heart failure [7], [10], [21]. On the other hand, RLCv expression is restricted to the ventricles both in the developing and adult heart [15]. Conversely, RLCa, like ELCa, is expressed throughout the heart early in development, and becomes restricted to the atria later in development [15].

Top-down mass spectrometry (MS) has gained considerable popularity as the premier approach for comprehensively characterizing proteins [22], [23], [24]. Unlike in conventional bottom-up MS, in which proteins are digested and the resulting peptides are analyzed by MS, intact proteins are analyzed in top-down MS, providing a global or “bird's eye” view of all protein species, including those containing sequence variations (due to mutations/polymorphisms or alternative splicing) and/or PTMs [22], [23], [24], [25], [26], [27], [28], [29], [30]. Following intact protein analysis, specific protein species of interest can be isolated and fragmented using a variety of tandem MS (MS/MS) techniques, including, but not limited to, electron capture dissociation (ECD) and collision induced dissociation (CID), to obtain sequence information and localize PTMs [22], [23], [24], [25], [26], [27], [28], [29], [30]. In particular, top-down MS with ECD represents a powerful method for the comprehensive characterization of proteins; especially those containing labile PTMs such as phosphorylation, which are frequently lost when proteins are fragmented using energetic dissociation methods such as CID [25]. Additionally, the use of high-resolution mass spectrometers in top-down MS studies offers unparalleled mass accuracy, which not only increases confidence in protein identification, but also in the identification of protein PTMs [31]. Herein, utilizing top-down high-resolution MS, we have characterized the sequences and N-terminal modifications of cardiac myosin light chain isoforms from human and swine, as well as the sites of phosphorylation in the swine proteins, towards a better understanding of the functional roles of these proteins in cardiac physiology and pathophysiology. Interestingly, we found that, whereas the ventricular ELC and RLC are Nα-tri-methylated, the atrial ELC is methylated at its N-terminus while the atrial RLC in both swine and human is Nα-acetylated; making the atrial RLC unique among cardiac myosin light chain isoforms. Importantly, we have also precisely localized the sites of phosphorylation in swine RLC isoforms from the ventricles and atria to Ser14 and Ser22, respectively. Although prior studies have reported atrial RLC phosphorylation, this represents the first study to definitively localize a site of phosphorylation in this isoform.

Section snippets

Methods

Detailed methods are found in Supporting information. To comprehensively characterize the sequences and PTMs of swine myosin light chain isoforms, myofilament-enriched extracts were prepared from the atrial and ventricular myocardium of 1–3 healthy adult Yorkshire domestic swine (Sus scrofa) (approximately 3 months of age) using the two-step extraction procedure described by Van Eyk et al. [32], [33] with modifications. Subsequently, protein extracts prepared from swine atrial or ventricular

Inhibition of phosphatase activity towards the RLC

To quantify RLC phosphorylation we employed quantitative online top-down LC-MS. This method provided robust and highly-reproducible measurement of RLC phosphorylation in cardiac tissue extracts prepared using the two-step extraction procedure described by Van Eyk et al. [32], [33] with modifications (Fig. S2, Supplemental results). By varying the concentration of phosphatase inhibitors used in the HEPES-based extraction buffer [32], [33], we determined that supplementation with 600 mM NaF (in

Characterization of cardiac myosin light chain isoform sequences

In this study, we utilized top-down high-resolution MS/MS to characterize the sequences of cardiac myosin light chain isoforms in the human and swine hearts (Fig. 2, Fig. 4, Fig. 5; Figs. S5, S9, S13, S14, S19) towards a better understanding of the functional roles of these proteins in cardiac physiology and pathophysiology.

In addition to verifying the sequence of human RLCa, we found that swine RLCa contained a 26 amino acid stretch not present in the UniProtKB/Swiss-Prot database sequence for

Disclosures

None.

Acknowledgments

The authors would like to thank Timothy A. Hacker for swine heart tissue samples and Rachel Heuer for critical reading of this manuscript. Financial support was kindly provided by NIH F31 HL128086 (to Z.G.), and NIH R01 HL109810 and R01 HL096971 (to Y.G.). YG would also like to acknowledge NIH R01 GM117058 and S10 OD018475.

References (69)

  • C. Toepfer et al.

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

    J. Biol. Chem.

    (2013)
  • P.J. Silver et al.

    Frequency-dependent myosin light chain phosphorylation in isolated myocardium

    J. Mol. Cell. Cardiol.

    (1986)
  • A. Sanbe et al.

    Abnormal cardiac structure and function in mice expressing nonphosphorylatable cardiac regulatory myosin light chain 2

    J. Biol. Chem.

    (1999)
  • C. Hidalgo et al.

    Effect of diastolic pressure on MLC2v phosphorylation in the rat left ventricle

    Arch. Biochem. Biophys.

    (2006)
  • T. Kampourakis et al.

    Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle

    J. Mol. Cell. Cardiol.

    (2015)
  • P. Haynes et al.

    Transmural heterogeneity of cellular level power output is reduced in human heart failure

    J. Mol. Cell. Cardiol.

    (2014)
  • J.S. Davis et al.

    The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation

    Cell

    (2001)
  • 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

    Mol. Cell. Proteomics

    (2010)
  • P. Ding et al.

    Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

    J. Biol. Chem.

    (2010)
  • A.N. Chang et al.

    Cardiac myosin is a substrate for zipper-interacting protein kinase (ZIPK)

    J. Biol. Chem.

    (2010)
  • H.E. Huxley

    The mechanism of muscular contraction

    Science

    (1969)
  • A.F. Huxley et al.

    Proposed mechanism of force generation in striated muscle

    Nature

    (1971)
  • I. Rayment et al.

    Three-dimensional structure of myosin subfragment-1: a molecular motor

    Science

    (1993)
  • I. Rayment et al.

    Structure of the actin-myosin complex and its implications for muscle contraction

    Science

    (1993)
  • H.L. Sweeney et al.

    Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function

    Am. J. Phys.

    (1993)
  • I. Morano

    Tuning the human heart molecular motors by myosin light chains

    J. Mol. Med. (Berl)

    (1999)
  • P.A. Hofmann et al.

    Effects of partial extraction of light chain 2 on the Ca2 + sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers

    J. Gen. Physiol.

    (1990)
  • S. Lowey et al.

    Role of skeletal and smooth muscle myosin light chains

    Biophys. J.

    (1995)
  • O.M. Hernandez et al.

    Myosin essential light chain in health and disease

    Am. J. Physiol. Heart Circ. Physiol.

    (2007)
  • D. Szczesna

    Regulatory light chains of striated muscle myosin. Structure, function and malfunction

    Curr. Drug Targets Cardiovasc. Haematol. Disord.

    (2003)
  • K. Poetter et al.

    Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle

    Nat. Genet.

    (1996)
  • M.C. Olsson et al.

    Basal myosin light chain phosphorylation is a determinant of Ca2 + sensitivity of force and activation dependence of the kinetics of myocardial force development

    Am. J. Physiol. Heart Circ. Physiol.

    (2004)
  • F. Sheikh et al.

    Mouse and computational models link Mlc2v dephosphorylation to altered myosin kinetics in early cardiac disease

    J. Clin. Invest.

    (2012)
  • S.A. Warren et al.

    Myosin light chain phosphorylation is critical for adaptation to cardiac stress

    Circulation

    (2012)
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