Phosphorylation Of cTnT Research Articles

Phosphorylation and Fragmentation of the Cardiac Troponin T: Mechanisms, Role in Pathophysiology and Laboratory Diagnosis

Cardiac troponin T (cTnT), a protein essential for calcium-regulated, myofibrillar ATPase activity, is extremely sensitive to the action of a significant number of intra- and extracellular enzymes, the action of which causes post-translational modifications (PTMs) of amino acid structure and functioning cTnT. PTMs of cTnT may play important roles in the regulation of cardiac contractility. The vast majority of cTnT modifications involve the phosphorylation by a variety of Ser/Thr kinases, including PKC. At the same time, the activity of cTnT phosphorylation can change under physiological conditions and in some CVDs, including HF, AMI, and arrhythmias. Along with cTnT phosphorylation, cTnT fragmentation occurs, the activity of which can also change. This article discusses the mechanisms of cTnT phosphorylation and fragmentation, discusses the important role of these processes in the pathophysiology and laboratory diagnosis of some CVD, and notes promising directions for further research.

Modulations of CA2+ Sensitizers and Phosphorylation of cTnT in Dynamic Equilibrium of cTnC N-Domain

An increasing body of evidence suggests cTnC exists in dynamic conformational equilibrium and that Ca2+ binding regulates this equilibrium. However, little direct evidence has been offered to support these claims. The present study aims to directly monitor dynamic conformational equilibrium of cTnC and investigate how the dynamic equilibrium is modulated by the presence of cardiac troponin I (cTnI), cardiac troponin T (cTnT), Ca2+ sensitizers, and a Ca2+ desensitizing phosphomimic of cTnT (cTnT(T204E). To achieve the objective, a dye homodimerization approach is developed and implemented which allows for determination of the dynamic equilibrium between open and closed conformations in cTnC's hydrophobic cleft. Modulation of this equilibrium by other troponin subunits, Ca2+-sensitizers, and phosphomimic of cTnT(T204E) is characterized. Isolated cTnC contained a small open conformation population in the absence of Ca2+, which increased significantly upon addition of saturating levels of Ca2+. This suggests the Ca2+ induced activation of thin filament arises from an increase in the probability of hydrophobic cleft opening. Inclusion of cTnI increased the population of open cTnC while inclusion of cTnT had the opposite effect. Samples containing Ca2+-desensitizing cTnT(T204E) showed a slight but insignificant decrease in open conformation probability compared to samples with cTnT(wt), while Ca2+-sensitizer treated samples generally increased open conformation probability. These findings show that an equilibrium between open and closed conformations of cTnC's hydrophobic cleft are play a significant role in tuning the Ca2+ sensitivity of the heart.

FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation

FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca2+ and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca2+ and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca2+ sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca2+-sensitivity. The rate of Ca2+-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca2+-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca2+-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca2+-sensitivity and likely play an important role in cardiac regulation.

Structural and Kinetic Studies using FRET: Impact of Pseudo-Pkc Phosphorylation of Cardiac Troponin T on Calcium-Activated Thin Filament Regulation

Regulation of myocardial contractility is influenced by numerous signaling agents. One of the key methods for modification of cardiomyocyte function is phosphorylation of cardiac troponin (cTn), the Ca2+-dependent regulatory switch that controls thin filament activation, by isoforms of protein kinase C (PKC). PKC has been shown to phosphorylate the tropomyosin binding subunit of cTn (cTnT) at Thr-195, Thr-199, Thr-204 and Thr-285 (rat cTnT). The effect of these phosphorylations on cTn structure and dynamics remains largely unknown. To ascertain the site specific effects of PKC phosphorylation of cTnT on thin filament regulation, pseudo-phosphorylation of cTnT was combined with Forster resonance energy transfer (FRET) to monitor the transition between the Ca2+ and Mg2+ states of cTn. Single-cysteine cTnI mutants were labeled with IAEDENS at either Cys-151 or Cys-167, and a single-cysteine cTnC mutant was labeled at Cys-89 with DDPM. Different combinations of mutant cTnT and fluorophore labeled cTnI and cTnC were then incorporated into reconstituted thin filaments, which were subjected to calcium titrations, time resolved FRET structural measurements, and stopped flow kinetic measurements. Titrations showed a consistent pseudo-phosphorylation dependent increase in pCa50 values, and kinetics experiments showed decreased rates of Ca2+ dissociation in pseudo-phosphorylated mutants. Additionally, Mg2+ distances between Cys-167 of cTnI and Cys-89 of cTnC were reduced in the presence of pseudo-phosphorylation. Thus our results suggested that pseudo-phosphorylation of cTnT leads to sensitization of the Ca2+-dependent conformational transitions involved in cTn regulatory switching. However, a prior in situ study demonstrated a pseudo-phosphorylation dependent decrease in EC50 (JBC vol. 278, pp. 35135-44). These results may suggest PKC mediated phosphorylation of cTnT may represent a post-translational modification where cTn regulatory switching is sensitized to Ca2+, but also partially uncoupled from Tm movement.

Structure of the NH2-terminal variable region of cardiac troponin T determines its sensitivity to restrictive cleavage in pathophysiological adaptation

We previously reported that the NH2-terminal variable region of cardiac troponin T (cTnT) is removed by restrictive μ-calpain cleavage in myocardial ischemia–reperfusion [24]. Selective removal of the NH2-terminal variable region of cTnT had a compensatory effect on myocardial contractility [25]. Here we further studied this posttranslational modification under pathophysiological conditions. Thrombin perfusion of isolated mouse hearts and cardiomyocytes induced the production of NH2-terminal truncated cTnT (cTnT-ND), suggesting a role of calcium overloading. Ouabain treatment of primary cultures of mouse cardiomyocytes in hypokalemic media, another calcium overloading condition, also produced cTnT-ND. Exploring the molecular mechanisms, we found that cTnT phosphorylation was primarily in the NH2-terminal region and the level of cTnT phosphorylation did not change under the calcium overloading conditions. However, alternatively spliced cTnT variants differing in the NH2-terminal primary structure produced significantly different levels of cTnT-ND in vivo in transgenic mouse hearts. The results suggest that stress conditions involving calcium overloading may convey an increased sensitivity of cTnT to the restrictive μ-calpain proteolysis, in which structure of the NH2-terminal variable region may play a determining role.

Cell therapy enhances function of remote non-infarcted myocardium

Cell transplantation improves cardiac function after myocardial infarction; however, the underlying mechanisms are not well-understood. Therefore, the goals of this study were to determine if neonatal rat cardiomyocytes transplanted into adult rat hearts 1 week after infarction would, after 8–10 weeks: 1) improve global myocardial function, 2) contract in a Ca2+ dependent manner, 3) influence mechanical properties of remote uninjured myocardium and 4) alter passive mechanical properties of infarct regions. The cardiomyocytes formed small grafts of ultrastructurally maturing myocardium that enhanced fractional shortening compared to non-treated infarcted hearts. Chemically demembranated tissue strips of cardiomyocyte grafts produced force when activated by Ca2+, whereas scar tissue did not. Furthermore, the Ca2+ sensitivity of force was greater in cardiomyocyte grafts compared to control myocardium. Surprisingly, cardiomyocytes grafts isolated in the infarct zone increased Ca2+ sensitivity of remote uninjured myocardium to levels greater than either remote myocardium from non-treated infarcted hearts or sham-operated controls. Enhanced calcium sensitivity was associated with decreased phosphorylation of cTnT, tropomyosin and MLC2, but not changes in myosin or troponin isoforms. Passive compliance of grafts resembled normal myocardium, while infarct tissue distant from grafts had compliance typical of scar. Thus, cardiomyocyte grafts are contractile, improve local tissue compliance and enhance calcium sensitivity of remote myocardium. Because the volume of remote myocardium greatly exceeds that of the grafts, this enhanced calcium sensitivity may be a major contributor to global improvements in ventricular function after cell transplantation.

Protein kinase C α and ε phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium

Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCα- and PKCε-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca2+] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCα and PKCε equally reduced Ca2+ sensitivity in failing cardiomyocytes (ΔpCa50 = 0.08 ± 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca2+ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCα resulted in a modest significant reduction in passive force. Effects of PKCα were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCα. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca2+ sensitivity. Subsequently, PKCα treatment of donor cardiomyocytes reduced Ca2+ sensitivity (ΔpCa50 = 0.08 ± 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCα- and PKCε-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca2+ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium.

Impact of Cardiac Troponin T N-Terminal Deletion and Phosphorylation on Myofilament Function

Cardiac troponin T (cTnT) is a phosphoprotein that modulates cardiac muscle contraction through its extensive and diverse interactions with neighboring thin filament proteins. Its N-terminal half is the "glue" that anchors the troponin complex to tropomyosin-actin. Until now, studies aimed at investigating the role of the N-terminal tail region have not considered the effects of phosphorylation. To understand better the regulatory role of the N-terminal tail region of phosphorylated cTnT, we investigated the functional effects of N-terminal deletion (amino acids 1-91) and phosphorylation on Ca(2+) dependence of myofilament isometric force production, isometric ATPase rate, and thin filament sliding speed. Chemomechanical profiles were assessed in detergent permeabilized fiber preparations where the native troponin (cTn) was exchanged with recombinant cTn engineered to contain modified cTnT (truncated, phosphorylated) in the presence of wild-type cTnI and cTnC. Removal of the cTnT N-terminal amino acids 1-91 (cTnT-del) enhances myofilament responsiveness to nonsaturating Ca(2+) levels (the physiological range in cardiac myocytes). However, at saturating Ca(2+) levels, there is a reduction in isometric tension and ATPase rate. On one hand, phosphorylation of cTnT-del attenuates the sensitizing effect induced by truncation of the N-terminal tail, "resetting" myofilament Ca(2+) responsiveness back to control levels. On the other hand, it impairs isometric tension development and ATPase rate. Interestingly, phosphorylation of cTnT (cTnT-P) differentially regulates tension cost (an index of cross-bridge cycling rate): increased by cTn-del-P and decreased by intact cTn-wt-P. Like the isometric fiber data, sliding speed of thin filaments regulated by cTn-del is more sensitive to Ca(2+) compared with cTn-wt. Phosphorylation of cTnT (whether cTnT-del or -wt) depresses sliding speed and is associated with Ca(2+) desensitization of thin filament sliding speed.

Raf-1: a novel cardiac troponin T kinase

Phosphorylation of cardiac troponin is a key mechanism involved in regulation of contractile function. In vitro kinase assays revealed that lysates prepared from resting cardiomyocytes contain cardiac troponin I (cTnI) and cTnT kinase activity. cTnI phosphorylation is inhibited by pharmacologic inhibitors of PKA, PKC, Rho kinase and PKC effectors such as RSK and PKD; these kinase inhibitors do not inhibit phosphorylation of cTnT. Rather, cTnT phosphorylation is decreased by the Raf inhibitor GW5074. In vitro kinase assays show that recombinant Raf phosphorylates cTnT, and that Raf-dependent cTnT phosphorylation is abrogated by a T206E substitution; Raf does not phosphorylate cTnI. These studies identify Raf-dependent cTnT-Thr(206) phosphorylation as a novel mechanism that would link growth factor-dependent signaling pathways to dynamic changes in cardiac contractile function.

Abstract 1573: Phosphorylation of Cardiac Troponin I by Mammalian Sterile20-like Kinase Mst1

Mammalian sterile 20-like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of human heart cDNA library with a dominant negative Mst1 (K59R) as bait, cardiac troponin I (cTnI) was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiac myocytes, in which cTnI interacted with full length Mst1, but not with its N-terminal kinase fragment. In vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1 in its free form or in reconstituted troponin complex. In contrast, cardiac TnT was phosphorylated by Mst1 only when incorporated in the troponin complex. Mass spectrometric analysis indicated that Mst1 phosphorylates cTnI at Thr 31 , Thr 51 , Thr 129 , and Thr 143 . Substitution of Thr 31 with Ala substantially reduced Mst1-mediated cTnI phosphorylation by approximately 90%, while replacement of either Thr 51 orThr 129 , or Thr 143 with Ala reduced Mst1-catalyzed cTnI phosphorylation by approximately 60%, suggesting that Thr 31 is a preferential phosphorylation site for Mst1. Protein epitope analysis and binding assays showed that Mst1 mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. Our results suggest that Mst1 is a novel mediator of cTnI and/or cTnT phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions. This research has received full or partial funding support from the American Heart Association, AHA National Center.

ASK1 Associates with Troponin T and Induces Troponin T Phosphorylation and Contractile Dysfunction in Cardiomyocytes

There is increasing support for the idea that excessive production of proinflammatory mediators such as tumor necrosis factor (TNF) and reactive oxygen species (ROS) contribute to the pathogenesis of cardiac dysfunction. However, the mechanisms by which cytokine/ROS production mediates cardiac dysfunction have not been established. Given that apoptosis signal-regulating kinase 1 (ASK1) is highly expressed in cardiac muscle and that ASK1 is an important mediator in the signaling pathways induced by tumor necrosis factor, interleukin-1, and ROS, we used the yeast two-hybrid system with ASK1 as bait to identify ASK1 substrates from a human heart cDNA library. The cDNA encoding the cardiac troponin T (cTnT) was isolated. ASK1 specifically interacted with cTnT, but not cTnI, in vitro and in vivo via the C-terminal ASK1 domain. ASK1 specifically phosphorylated cTnT in vitro and in vivo. Mutations in cTnT (T194/S198) at an ASK1-phosphorylation consensus sequence significantly reduced phosphorylation by ASK1. ROS-induced ASK1 activation, cTnT phosphorylation, and contractile dysfunction in cardiomyocytes showed similar kinetics. Moreover, overexpression of constitutively active ASK1 induces cTnT phosphorylation and inhibits shortening and calcium transient in adult cardiomyocytes. We conclude that ASK1 plays an important role in regulation of cardiac contractile function by phosphorylating cTnT and may participate in cytokine/ROS-induced pathogenesis of cardiomyopathy and heart failure.

Transgenic incorporation of skeletal TnT into cardiac myofilaments blunts PKC-mediated depression of force.

Protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) and troponin T (cTnT) has been shown to diminish maximum activation of myofilaments. The functional role of cTnI phosphorylation has been investigated. However, the impact of cTnT phosphorylation on myofilament force is not well studied. We tested the effect of endogenous PKC activation on steady-state tension development and Ca(2+) sensitivity in skinned fiber bundles from transgenic (TG) mouse hearts expressing fast skeletal TnT (fsTnT), which naturally lacks the PKC sites present in cTnT. The 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment induced a 29% (46.1 +/- 2.5 vs. 33.4 +/- 2.6 mN/mm(2)) reduction in maximum tension in the nontransgenic (NTG) preparations (n = 7) and was inhibited with chelerythrine. However, TPA did not induce a change in the maximum tension in the TG preparations (n = 11). TPA induced a small but significant (P < 0.02) increase in Ca(2+) sensitivity (untreated pCa(50) = 5.63 +/- 0.01 vs. treated pCa(50) = 5.72 +/- 0.01) only in TG preparations. In TG preparations, (32)P incorporation was not evident in TnT and was also significantly diminished in cTnI, compared with NTG. Our data indicate that incorporation of fsTnT into the cardiac myofilament lattice blunts PKC-mediated depression of maximum tension. These data also suggest that cTnT may play an important role in amplifying the myofilament depression induced by PKC-mediated phosphorylation of cTnI.