A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain

Abstract

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reversephase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/ MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method.

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

“…Thus, our studies offer insights and tools (computational and mouse models) to expand our understanding of how variations in myosin cycling kinetics across the heart wall contribute to decreased subendocardial workload and DCM. This is important, since we highlight a direct and early influence of loss of Mlc2v phosphorylation in heart disease and failure in vivo (Figure 2), suggesting a direct contribution of dysregulation of MLC2v phosphorylation in human cardiomyopathy and failure (6,8,(10)(11)(12)(13)(14). Recent studies suggest that the MLCK family of "cytoskeletal protein kinases," which target Mlc2v phosphorylation, might also have a broader role in mechanosignaling (43) and thus future studies focused on determining whether strain modulation of cardiac Mlck activity is a potential contributing factor to the mechanisms and outcomes highlighted by our models are warranted.…”

“…Understanding the molecular actions of MLC2v phosphorylation in the regulation of actin-myosin interaction kinetics in cardiac muscle is of key importance, since emerging evidence in humans suggests a critical role for MLC2v in human health. Several studies have shown that dysregulation of MLC2v via dephosphorylation is associated with human cardiomyopathies and heart failure (6,8,(10)(11)(12)(13)(14); however, mechanistic insights into its underlying role in heart disease still remain elusive.…”

“…Growing evidence suggests that this simple model cannot explain all aspects of striated muscle regulation and that myosin regulatory proteins, such as MLC2v, may have an influential role in regulating activation of cardiac muscle contraction via posttranslational modifications such as phosphorylation (6)(7)(8)(9)(10)(11)(12)(13)(14). In the 1980s, in vitro studies performed by Stull and colleagues noted a temporal association between the extent of Mlc phosphorylation and potentiation of isometric twitch force in skeletal muscle (5), suggesting that Ca 2+ binding to actin-bound regulatory proteins (troponin C) may not be the only process regulating striated muscle contraction.…”

p21-activated kinase improves cardiac contractility during ischemia-reperfusion concomitant with changes in troponin-T and myosin light chain 2 phosphorylation

“…3F). Our laboratory has shown that ablation of MLC2 phosphorylation decreases ventricular power, lengthens the duration of ventricular ejection, and may also modify other sarcomeric proteins (e.g., cardiac troponins) as substrates for kinases and phosphatases (21)(22)(23). The other differences that may be of significance are that Tm phosphorylation fell significantly with I/R in WT but not in Pak-KO hearts, and TnT phosphorylation increased with I/R in WT but not in Pak-KO.…”

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

“…However, human ventricular RLC may only be singly phosphorylated (Ser-15) (Fig. 1) or singly deamidated (Asn-14 to Asp-14) (Scruggs et al 2010;Scruggs and Solaro 2011). These sites are mostly phosphorylated by a cardiac-specific MLCK (cMLCK), encoded by MYLK3, which, in addition to its role in Ca 2+ sensitization of myofilaments (Sweeney and Stull 1986), may serve as an adaptive mechanism to rapidly facilitate sarcomere organization in cardiac myocytes in response to hypertrophic stimuli associated with increased intracellular Ca 2+ (Aoki et al 2000).…”