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