Sarah B. Scruggs

Ablation of Ventricular Myosin Regulatory Light Chain Phosphorylation in Mice Causes Cardiac Dysfunction in Situ and Affects Neighboring Myofilament Protein Phosphorylation

There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P-). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P-) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P-) displayed a significantly reduced response to β1-adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P-) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following β1-adrenergic stimulation, a mechanism prevented in TG-RLC(P-), thus providing a biochemical difference in β1-adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca2+ tension and Ca2+-ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca2+-activated tension and tension cost in TG-RLC(P-) fibers, with no change in Ca2+ sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to β1-adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.

The significance of regulatory light chain phosphorylation in cardiac physiology.

It has been over 35 years since the first identification of phosphorylation of myosin light chains in skeletal and cardiac muscle. Yet only in the past few years has the role of these phosphorylations in cardiac dynamics been more fully understood. Advances in this understanding have come about with further evidence on the control mechanisms regulating the level of phosphorylation by kinases and phosphatases. Moreover, studies clarifiying the role of light chain phosphorylation in short and long term control of cardiac contractility and as a factor in cardiac remodeling have improved our knowledge. Especially important in these advances has been the use of gain and loss of function approaches, which have not only testedthe role of kinases and phosphatases, but also the effects of loss of RLC phosphorylation sites. Major conclusions from these studies indicate that (i) two negatively-charged post-translational modifications occupy the ventricular RLC N-terminus, with mouse RLC being doubly phosphorylated (Ser 14/15), and human RLC being singly phosphorylated (Ser 15) and singly deamidated(Asn14/16 to Asp); (ii)a distinct cardiac myosin light kinase (cMLCK) and a unique myosin phosphatase targeting peptide (MYPT2) control phosphoryl group transfer;and (iii) ablation of RLC phosphorylationdecreases ventricular power, lengthens the duration of ventricular ejection, and may also modify other sarcomeric proteins (e.g., troponin I) as substrates for kinases and/or phosphatases. A long term effect of low levels of RLC phosphorylation in mouse models also involves remodeling of the heart with hypertrophy, depressed contractility, and sarcomeric disarray. Data demonstrating altered levels of RLC phosphorylation in comparisons of samples from normal and stressed human hearts indicate the significance of these findings in translational medicine.

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

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 reverse-phase 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.

An MRM-based Workflow for Quantifying Cardiac Mitochondrial Protein Phosphorylation in Murine and Human Tissue

The regulation of mitochondrial function is essential for cardiomyocyte adaptation to cellular stress. While it has long been understood that phosphorylation regulates flux through metabolic pathways, novel phosphorylation sites are continually being discovered in all functionally distinct areas of the mitochondrial proteome. Extracting biologically meaningful information from these phosphorylation sites requires an adaptable, sensitive, specific and robust method for their quantification. Here we report a multiple reaction monitoring-based mass spectrometric workflow for quantifying site-specific phosphorylation of mitochondrial proteins. Specifically, chromatographic and mass spectrometric conditions for 68 transitions derived from 23 murine and human phosphopeptides, and their corresponding unmodified peptides, were optimized. These methods enabled the quantification of endogenous phosphopeptides from the outer mitochondrial membrane protein VDAC, and the inner membrane proteins ANT and ETC complexes I, III and V. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of mitochondrial protein phosphorylation in cardiac physiology and pathophysiology. This article is part of a Special Issue entitled: Translational Proteomics.

Post-translational modification of cardiac proteasomes: functional delineation enabled by proteomics

Proteasomes are ubiquitously expressed multicatalytic complexes that serve as key regulators of protein homeostasis. There are several lines of evidence indicating that proteasomes exist in heterogeneous subpopulations in cardiac muscle, differentiated, in part, by post-translational modifications (PTMs). PTMs regulate numerous facets of proteasome function, including catalytic activities, complex assembly, interactions with associating partners, subcellular localization, substrate preference, and complex turnover. Classical technologies used to identify PTMs on proteasomes have lacked the ability to determine site specificity, quantify stoichiometry, and perform large-scale, multi-PTM analysis. Recent advancements in proteomic technologies have largely overcome these limitations. We present here a discussion on the importance of PTMs in modulating proteasome function in cardiac physiology and pathophysiology, followed by the presentation of a state-of-the-art proteomic workflow for identifying and quantifying PTMs of cardiac proteasomes.

Harnessing the Heart of Big Data

The exponential increase in Big Data generation combined with limited capitalization on the wealth of information embedded within Big Data has prompted us to revisit our scientific discovery paradigms. A successful transition into this digital era of medicine holds great promise for advancing fundamental knowledge in biology, innovating human health, and driving personalized medicine; however, this will require a drastic shift of research culture in how we conceptualize science and use data. An e-transformation will require global adoption and synergism among computational science, biomedical research, and clinical domains. A scarce number of scientific investigations have innovated clinical diagnosis, prognosis, and therapeutics, despite decades of research and enormities of National Institutes of Health (NIH)–funded research dollars.1,2 This situation requires a global reassessment of whether linear thought processes and reductionistic approaches alone can describe biological processes in a way that translates to valuable information on human systems. Information gleaned from population science using large Big Data data sets has perpetuated a shift in the paradigm of how we define and investigate health and disease in the individual patient.3 We are recognizing the profound value in unorthodox data types and in the integration of diverse data to describe individuals to sufficient depths for discerning clinical outcomes. Biomedicine, along with other fields, has been awakened and awed by the digital wave of major corporations such as Google and Amazon, who have revolutionized the Internet roadmap through developing and refining sophisticated data analytics platforms to accurately describe individual human behavior.4 The reality in biomedical science is that there are zettabytes of high-quality data sitting idly on servers and in cloud infrastructures, and an abundance of biomedical knowledge lies hidden within, yet only a small fraction of this wealth has been harvested. There is an immediate need for data science to …

Regulation of Acetylation Restores Proteolytic Function of Diseased Myocardium in Mouse and Human

Proteasome complexes play essential roles in maintaining cellular protein homeostasis and serve fundamental roles in cardiac function under normal and pathological conditions. A functional detriment in proteasomal activities has been recognized as a major contributor to the progression of cardiovascular diseases. Therefore, approaches to restore proteolytic function within the setting of the diseased myocardium would be of great clinical significance. In this study, we discovered that the cardiac proteasomal activity could be regulated by acetylation. Histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamic acid and sodium valproate) enhanced the acetylation of 20S proteasome subunits in the myocardium and led to an elevation of proteolytic capacity. This regulatory paradigm was present in both healthy and acutely ischemia/reperfusion (I/R) injured murine hearts, and HDAC inhibition in vitro restored proteolytic capacities to baseline sham levels in injured hearts. This mechanism of regulation was also viable in failing human myocardium. With 20S proteasomal complexes purified from murine myocardium treated with HDAC inhibitors in vivo, we confirmed that acetylation of 20S subunits directly, at least in part, presents a molecular explanation for the improvement in function. Furthermore, using high-resolution LC-MS/MS, we unraveled the first cardiac 20S acetylome, which identified the acetylation of nine N-termini and seven internal lysine residues. Acetylation on four lysine residues and four N-termini on cardiac proteasomes were novel discoveries of this study. In addition, the acetylation of five lysine residues was inducible via HDAC inhibition, which correlated with the enhancement of 20S proteasomal activity. Taken as a whole, our investigation unveiled a novel mechanism of proteasomal function regulation in vivo and established a new strategy for the potential rescue of compromised proteolytic function in the failing heart using HDAC inhibitors.

Site-specific quantitative analysis of cardiac mitochondrial protein phosphorylation

We report the development of a multiple-reaction monitoring (MRM) strategy specifically tailored to the detection and quantification of mitochondrial protein phosphorylation. We recently derived 68 MRM transitions specific to protein modifications in the respiratory chain, voltage-dependent anion channel, and adenine nucleotide translocase. Here, we have now expanded the total number of MRM transitions to 176 to cover proteins from the tricarboxylic acid cycle, pyruvate dehydrogenase complex, and branched-chain alpha-keto acid dehydrogenase complex. We utilized the transition set to analyze endogenous protein phosphorylation in human heart, mouse heart, and mouse liver. The data demonstrate the potential utility of the MRM workflow for studying the functional details of mitochondrial phosphorylation signaling. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Proteome Dynamics and Proteome Function of Cardiac 19S Proteasomes

Myocardial proteasomes are comprised of 20S core particles and 19S regulatory particles, which together carry out targeted degradation of cardiac proteins. The 19S complex is unique among the regulators of proteasomes in that it affects both the capacity and specificity of protein degradation. However, a comprehensive molecular characterization of cardiac 19S complexes is lacking. In this investigation, we tailored a multidimensional chromatography-based purification strategy to isolate structurally intact and functionally viable 19S complexes from murine hearts. Two distinct subpopulations of 19S complexes were isolated based upon (1) potency of activating 20S proteolytic activity, and (2) molecular composition using a combination of immuno-detection, two-dimensional-differential gel electrophoresis, and MS-based approaches. Heat shock protein 90 (Hsp90) was identified to be characteristic to 19S subpopulation I. The physical interaction of Hsp90 with 19S complexes was demonstrated via multiple approaches. Inhibition of Hsp90 activity using geldanamycin or BIIB021 potentiated the ability of subpopulation I to activate 20S proteasomes in the murine heart, thus demonstrating functional specificity of Hsp90 in subpopulation I. This investigation has advanced our understanding of the molecular heterogeneity of cardiac proteasomes by identifying molecularly and functionally distinct cardiac 19S complexes. The preferential association of Hsp90 with 19S subpopulation I unveils novel targets for designing proteasome-based therapeutic interventions for combating cardiac disease.

New insights into the functional significance of the acidic region of the unique N-terminal extension of cardiac troponin I.

Previous structural studies indicated a special functional role for an acidic region composed of residues 1-10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC-MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-ε (PKCε) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and ktr, but no change in half-maximally activating Ca(2+). In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca(2+)-sensitivity with no change in maximum tension or ktr. We further tested the impact of acidic domain modifications on myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well as cooperativity of activation of myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24). This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.