Pingbo Zhang

Multiple Reaction Monitoring to Identify Site-Specific Troponin I Phosphorylated Residues in the Failing Human Heart

Background— Human cardiac troponin I is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples. Methods and Results— On the basis of in silico prediction and liquid chromatography/mass spectrometry data, 14 phosphorylation sites on cardiac troponin I, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplantation patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared with samples obtained from nonfailing donor hearts (n=10 per group). Thirty mass spectrometry–based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding nonphosphorylated site. The results show that in heart failure there is a decrease in the extent of phosphorylation of the known protein kinase A sites (S22, S23) and other newly discovered phosphorylation sites located in the N-terminal extension of cardiac troponin I (S4, S5, Y25), an increase in phosphorylation of the protein kinase C sites (S41, S43, T142), and an increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cardiac troponin I (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at 3 novel sites was found to decline toward control after resynchronization therapy. Conclusions— Selective, functionally significant phosphorylation alterations occurred on individual residues of cardiac troponin I in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.

Multiplex assays for biomarker research and clinical application: translational science coming of age.

Over the last decade, translational science has come into the focus of academic medicine, and significant intellectual and financial efforts have been made to initiate a multitude of bench‐to‐bedside projects. The quest for suitable biomarkers that will significantly change clinical practice has become one of the biggest challenges in translational medicine. Quantitative measurement of proteins is a critical step in biomarker discovery. Assessing a large number of potential protein biomarkers in a statistically significant number of samples and controls still constitutes a major technical hurdle. Multiplexed analysis offers significant advantages regarding time, reagent cost, sample requirements and the amount of data that can be generated. The two contemporary approaches in multiplexed and quantitative biomarker validation, antibody‐based immunoassays and MS‐based multiple (or selected) reaction monitoring, are based on different assay principles and instrument requirements. Both approaches have their own advantages and disadvantages and therefore have complementary roles in the multi‐staged biomarker verification and validation process. In this review, we discuss quantitative immunoassay and multiple reaction monitoring/selected reaction monitoring assay principles and development. We also discuss choosing an appropriate platform, judging the performance of assays, obtaining reliable, quantitative results for translational research and clinical applications in the biomarker field.

PKCα-specific phosphorylation of the troponin complex in human myocardium: a functional and proteomics analysis.

Aims Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved. Methods and Results Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca2+] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m2) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m2). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca2+-sensitivity of force (pCa50u200a=u200a5.59±0.02) compared to cTn (DD) (pCa50u200a=u200a5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143. Conclusion PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca2+-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca2+-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser359 Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity

ABSTRACT The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr313-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδs Tyr313-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδs enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser359. We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser359. In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser359 phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr313 docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.

Troponin I alterations detected by multiple-reaction monitoring: how might this impact the study of heart failure?

Cardiovascular diseases such as heart failure (HF) represent the leading cause of death and morbidity in the western world. Cardiac troponin I (cTnI) is a key regulator of contraction and relaxation in the heart, and its function is well known to be modulated by phosphorylation. While there is some evidence that phosphorylation of cTnI is altered during HF, there has never been a comprehensive analysis to identify or quantify all the phosphorylation sites of cTnI in HF. In a recently published study, the authors developed multiple-reaction monitoring (MRM) assays, a targeted mass spectrometry (MS)-based quantitative method, to assess the extent of cTnI phosphorylation in control human hearts and those in HF [1]. Due to the unique characteristics of MRM, the authors were able to determine the absolute quantity of each phosphorylated residue. The data showed that human cTnI is phosphorylated at more sites than previously reported, with 14 modifiable amino acid residues, including six novel sites (S4, S5, Y25, T50, T180 and S198). Comparing HF with control hearts, almost all the sites had altered phosphorylation in disease. Overall, these findings provide novel insights into sitespecific phosphorylation of cTnI in human myocardial samples, and also suggest correlation of phosphorylation of cTnI with functional status and potential utility of site-specific phosphorylation of cTnI as a biomarker of HF status. Basics of multiple-reaction monitoring Until recently, the verification of clinical biomarkers has essentially depended on the development of affinity/antibody-based assays such as ELISA. Unfortunately, the development of assays based on specific antibodies has a number of drawbacks: nonspecific antibody binding, cross reactivity and epitope masking, as well as the cost and time of the developing reagents and the complexities of development of quality controlled clinical tests. It is even more challenging to develop an ELISA to quantify specific post-translational modifications of particular amino acid residues. By contrast, MRM is an antibody-free MS-based approach, and is highly quantitative and reproducible. The MRM assays are most often carried out using a triple quadripole mass spectrometer (e.g., Qtrap nanoflow LC-MS/MS, AB Sciex) to simultaneously target multiple peptides of one or more proteins. When targeting a specific modifiable amino acid residue in a constrained MRM assay, the modified peptides or unmodified counterpart peptides that are unique to a given protein are selected in the MS instrument and quantified. Absolute quantification can be obtained if a known quantity of labeled synthetic peptide (e.g., N) is used to develop a standardized curve. MRM assays can provide sensitive identification, quantification and validation of protein phosphorylation associated with Troponin I alterations detected by multiple-reaction monitoring: how might this impact the study of heart failure?

Heart Failure–Related Hyperphosphorylation in the Cardiac Troponin I C Terminus Has Divergent Effects on Cardiac Function In Vivo

Background: In human heart failure, Ser199 (equivalent to Ser200 in mouse) of cTnI (cardiac troponin I) is significantly hyperphosphorylated, and in vitro studies suggest that it enhances myofilament calcium sensitivity and alters calpain-mediated cTnI proteolysis. However, how its hyperphosphorylation affects cardiac function in vivo remains unknown. Methods and Results: To address the question, 2 transgenic mouse models were generated: a phospho-mimetic cTnIS200D and a phospho-silenced cTnIS200A, each driven by the cardiomyocyte-specific &agr;-myosin heavy chain promoter. Cardiac structure assessed by echocardiography and histology was normal in both transgenic models compared with littermate controls (n=5). Baseline in vivo hemodynamics and isolated muscle studies showed that cTnIS200D significantly prolonged relaxation and lowered left ventricular peak filling rate, whereas ejection fraction and force development were normal (n=5). However, with increased heart rate or &bgr;-adrenergic stimulation, cTnIS200D mice had less enhanced ejection fraction or force development versus controls, whereas relaxation improved similarly to controls (n=5). By contrast, cTnIS200A was functionally normal both at baseline and under the physiological stresses. To test whether either mutation impacted cardiac response to ischemic stress, isolated hearts were subjected to ischemia/reperfusion. cTnIS200D were protected, recovering 88±8% of contractile function versus 35±15% in littermate controls and 28±8% in cTnIS200A (n=5). This was associated with less cTnI proteolysis in cTnIS200D hearts. Conclusions: Hyperphosphorylation of this serine in cTnI C terminus impacts heart function by depressing diastolic function at baseline and limiting systolic reserve under physiological stresses. However, paradoxically, it preserves heart function after ischemia/reperfusion injury, potentially by decreasing proteolysis of cTnI.

Cardiovascular proteomics: Assessment of protein post-translational modifications

Proteomics is the study of proteins and it employs methods that characterize their diversity. It is based on detection, identification, and quantification of proteins, isoforms, and post-translational modifications (PTMs). Proteomics is used to study how the proteome is altered in response to changes in the environment, changes that allow the cell to adapt to acute and ongoing chronic stimuli. The stimuli almost always result in PTMs of specific proteins and a subset of these changes can result in rapid and dynamic changes to the cells. In the heart, the most commonly PTM studied is phosphorylation, yet, there is increasing recognition of the role of S-nitrosation, acetylation, and O-GlcNAcylation. With technology development, proteomics has become a powerful identification tool for discovery of new markers for prognosis, diagnosis, and therapy. This chapter outlines the basic analytical protein biochemical techniques used in proteomics with subsequent examples of biological application to analysis of cardiac subproteomes and PTMs.