Posttranslational modifications of cardiac troponin T: An overview

YJMCC-07603; No. of pages: 10; 4C: Journal of Molecular and Cellular Cardiology xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc

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Review article

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Posttranslational modifications of cardiac troponin T: An overview

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Alexander S. Streng a, Douwe de Boer a, Jolanda van der Velden b, Marja P. van Dieijen-Visser a, Will K.W.H. Wodzig a,⁎ Department of Clinical Chemistry, Maastricht University Medical Centre, Maastricht, The Netherlands Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands

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Cardiac troponin (cTn) is an important sarcomeric protein complex situated on the thin filament and is involved in the regulation of cardiac muscle contraction. This regulation is primarily controlled by Ca2+ binding to troponin C and in addition fine-tuned by the posttranslational modification of cTnI and cTnT. The vast majority of cTnT modifications involve the phosphorylation by protein kinase C (PKC) or other kinases and the N-terminal cleavage by caspase and calpain. In vitro studies employing reconstituted detergent-skinned fiber bundles and cell culture generally show a detrimental effect of cTnT phosphorylation on muscle contraction, which is backed by some in vivo studies finding increased cTnT phosphorylation in heart failure, but contradicted by others. In addition, N-terminal cleavage of cTnT is thought to be another factor influencing cardiac contraction. Time-dependent degradation of cTnT has been observed in human serum upon myocardial infarction. These molecular changes might influence the immunoreactivity of cTnT in the clinical immunoassay and have consequences for the clinical interpretations of these measurements. No consensus has yet been reached on the occurrence and extent of these observations and their underlying processes are subject of intense scientific debate. This review will focus on discussing these modifications, their implications on physiology and disease and summarizes the complex interplays of different enzymes on the molecular forms of cTnT and their associated effects. © 2013 Published by Elsevier Ltd.

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Article history: Received 21 May 2013 Received in revised form 18 June 2013 Accepted 8 July 2013 Available online xxxx

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Keywords: Cardiac troponin T Cardiac physiology Phosphorylation Fragmentation

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Introduction . . . . . . . . . . . . . Troponin T structure and nomenclature . cTnT phosphorylation . . . . . . . . . 3.1. PKC mediated phosphorylation . 3.2. Phosphorylation by other kinases 3.3. cTnT phosphorylation in disease . 4. cTnT fragmentation . . . . . . . . . . 5. Summary and conclusions . . . . . . . Disclosure statement . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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Abbreviations: AMI, acute myocardial infarction; ASK1, apoptosis signal-regulating kinase 1; CaMK II, Ca2 +/calmodulin-dependent protein kinase II; cTnT/I/C, cardiac troponin T, I, C; ESRD, end-stage renal disease; GFC, gel filtration chromatography; HF, heart failure; LV, left ventricle; MS, mass spectrometry; Pi, inorganic phosphate; PAK1/3, p21 activated kinase 1, 3; PKA/C/D, protein kinase A, C, D; PTM, posttranslational modification; PP1, PP2A, protein phosphatase 1, 2A; ROCK-II, rho-A-dependent protein kinase; RV, right ventricle; Tm, tropomyosin. ⁎ Corresponding author at: Department of Clinical Chemistry, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. Tel.: +31 43 387 66 94; fax: +31 43 3874692. E-mail address: [email protected] (W.K.W.H. Wodzig).

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1. Introduction

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The cardiac troponin (cTn) complex is a regulatory protein complex situated on the thin filament in cardiac muscle cells and consists of three distinct subunits. Cardiac troponin C (cTnC) binds Ca2+, cardiac troponin I (cTnI) inhibits the actomyosin crossbridge formation in diastole and cardiac troponin T (cTnT) provides for the binding of troponin to tropomyosin (Tm). Following an action potential, a sudden release of Ca2+-ions from the sarcoplasmic reticulum, triggered by the influx via the L-type Ca2+-channel (Ca2+-induced Ca2+-release), increases

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0022-2828/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.yjmcc.2013.07.004

Please cite this article as: Streng AS, et al, Posttranslational modifications of cardiac troponin T: An overview, J Mol Cell Cardiol (2013), http:// dx.doi.org/10.1016/j.yjmcc.2013.07.004

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(PTM) of several cardiac contractile proteins. The most prominent of these is protein phosphorylation under the influence of certain enzymes, called kinases. Proteins can be phosphorylated on the amino acids serine (Ser), threonine (Thr) and tyrosine (Tyr). The exact molecular mechanisms regarding these phosphorylations and other PTMs and the effect they have on cardiac contractility are still not fully understood. The different modification sites, target proteins and enzymes, all working in concert, complicate our understanding of the system. New phosphorylation sites and kinases are still being discovered on a regular basis [15,16] and differences in PTMs of contractile proteins have been shown to be present in heart failure (HF) and after acute myocardial infarction (AMI) [17–20]. It is unknown if these modifications are the results of the pathology itself or an adaptive mechanism to the disease in an attempt to maintain cardiac output. Much of the focus of cardiac myofilament protein phosphorylation has been on cTnI, where serine residues 23 and 24 (Ser-23/Ser-24) are phosphorylated by protein kinase A (PKA) and D (PKD) after βadrenergic receptor stimulation, resulting in an increased crossbridge cycling rate and reduced Ca2+-sensitization [21–23]. This so-called “fight-or-flight-response” contributes to the enhancement of systolic function and relaxation under conditions of heightened cardiac demand. While PKA represents the predominant kinase that regulates myofilament function in the healthy heart, several other kinases that target cTnI are up-regulated during cardiac disease. One of the most studied kinase involved in myofilament dysfunction is protein kinase C (PKC), which typically targets residues Ser-43, Ser-45 and Thr-145, although a recent study has identified multiple other sites on human cTnI, which may be targets of PKC [24]. After an AMI, cTnI phosphorylation of Ser-23/Ser-24 is decreased in mice, while at the same time phosphorylation of Ser-43 is increased, leaving the overall cTnI phosphorylation level constant [25]. This is consistent with impaired β-adrenergic stimulation leading to a decrease of PKA activation and a further reduction of cardiac output as a result of altered phosphorylation. In patients with HF, overstimulation of this β-adrenergic pathway has led to its desensitization and PKA-mediated phosphorylation is decreased [26]. Multiple

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its concentration in the cytosol from sub-micromolar to micromolar concentrations. At this level, Ca2+ binds to the regulatory domain of cTnC, resulting in a cascade of structural changes within the troponin complex. This causes the movement of Tm, exposing the myosin binding sites on the actin molecules and thus allowing for crossbridge formation and muscle contraction (Fig. 1). See references [1–5] for reviews. Generally, the more Ca2+ that is able to bind to the thin filaments, the higher the force of contraction. An increase in cardiac contractility may also lead to a higher force of contraction and allows for a larger volume of blood to be ejected during a single power stroke [6]. The myofilaments have a variable sensitivity to Ca2+, which influences Ca2+ binding in addition to the variable Ca2+ concentrations. A higher Ca2+-sensitivity of the myofilaments leads to an earlier contraction in systole and a delayed relaxation in diastole. In the laboratory, Ca2+-sensitivity is measured during steady state, whereas in vivo, the effects of Ca2+-sensitivity are less straightforward because the beating heart never truly is in steady state but is dynamically shortening, developing tension, relaxing and lengthening [7]. The energy for this heart beat is yielded by adenosine triphosphate (ATP) which is hydrolyzed by the enzyme ATPase at the myosin head. The enzymatic activity of ATPase best corresponds to the unloaded shortening velocity and correlates with the crossbridge cycling rate and the energy consumption of the myocyte [8–10]. In addition, a positive correlation was shown between the ATPase activity and the average heart rate of different species [9]. Other influencing variables are tension cost and the Hill coefficient, describing the economy and cooperativity of contraction, respectively. Tension cost is obtained by combining ATPase activity with the tension in the myofilaments (which is directly proportional to the resulting force) and so describes the economy – or energy cost – of the contraction [10]. The Hill coefficient is a measure of cooperativity. A higher cooperativity indicates a quicker response to an increasing Ca2+-concentration [11]. All these variables can be tuned in various ways to match the cardiac output to the cardiac demand of the body [12–14]. This tuning is regulated, among other things, by the posttranslational modification

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Fig. 1. Schematic of the sarcomere showing the actin (thin) and myosin (thick) filaments, A. Protein structure of the thin filament during muscle relaxation, B, and during muscle contraction, C. TnT, troponin T; TnI, troponin I; TnC, troponin C.

Please cite this article as: Streng AS, et al, Posttranslational modifications of cardiac troponin T: An overview, J Mol Cell Cardiol (2013), http:// dx.doi.org/10.1016/j.yjmcc.2013.07.004

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2. Troponin T structure and nomenclature

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Three genes are encoding for TnT in the human genome; each of which are expressed in different muscle types. These genes are TNNT1 (coding for slow skeletal troponin T; ssTnT), TNNT2 (coding for cardiac troponin T; cTnT) and TNNT3 (coding for fast skeletal troponin T; fsTnT). Early experiments by Anderson et al. showed that there are four cTnT isoforms expressed in the human heart, which were numbered in the order of decreasing molecular size [32,33]. Fig. 2 shows

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an alignment of the complete amino acid sequence of these isoforms; the human canonical isoform (cTnT1) and isoform 8 (cTnT2), which are mainly expressed in the fetal heart; isoform 6 (cTnT3), the dominant isoform in healthy adult heart; and isoform 7 (cTnT4), also expressed in the fetal heart, but found to be re-expressed in the adult failing heart [32,34], and the canonical forms of rat, mouse and bovine cTnT according to the UniProtKB/Swiss-Prot database (http://www.uniprot. org/uniprot/) [35,36], The numbering does not match because more isoforms were later discovered and again numbered in the order of size, separate from the naming convention as proposed by Anderson. As can be concluded from Fig. 2, cTnT is highly conserved between species, except for the “hypervariable” N-terminus (residues 1–79), which varies between species and isoforms. Indicated are the two cTnT–Tm binding sites (residues 89–127 and 215–240) [37] and the cTnT–cTnI binding site, also known as the “IT-arm” (residues 234–284) [38,39]. Also shown are the discovered cTnT phosphorylation sites and the cleavage sites of μ-calpain and caspase-3 discussed in this paper. The

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reviews have been published about the phosphorylation of cTnI [27–31], which has been intensively studied. Less attention has been given to the phosphorylation of cTnT, which is also thought to influence cardiac contraction. The focus of this paper will be to extensively discuss the advances made towards cTnT phosphorylation and fragmentation and their effects on the physiology and pathophysiology of cardiac muscle contraction.

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Fig. 2. Alignment of human, mouse, rat and bovine cTnT isoforms. Alignment of the proteins was performed using the UniProtKB/Swiss-Prot database (http://www.uniprot.org/align/) [35,36]. Human isoforms shown are the canonical (fetal) isoform (cTnT-1), a second fetal isoform (cTnT-2), the healthy adult cTnT isoform, (cTnT-3) and a third fetal isoform, found to be re-expressed in the adult failing heart (cTnT-4). Mouse, rat and cow cTnT all represent their respective canonical forms. * (asterisk) indicates positions which have a single, fully conserved residue. : (colon) indicates conservation between groups of strongly similar properties. . (period) indicates conservation between groups of weakly similar properties. A blank indicates no conservation of the residue. Highlighted sections of the protein indicate various binding sites, phosphorylation sites and proteolytic cleavage sites, as indicated.

Please cite this article as: Streng AS, et al, Posttranslational modifications of cardiac troponin T: An overview, J Mol Cell Cardiol (2013), http:// dx.doi.org/10.1016/j.yjmcc.2013.07.004

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3. cTnT phosphorylation

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3.1. PKC mediated phosphorylation

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The earliest reports of cTnT phosphorylation date back to the 1980s, where the groups of Gusev [42] and Villar-Palasi [43] identified Ser-2 as a phosphorylation site in cardiac muscle. At that time, the kinases responsible were thought to be a certain “cTnT kinase” and phosphorylase kinase [43,44]. Further research towards this specific phosphorylation site has been halted shortly thereafter in favor of PKC mediated phosphorylation sites. The association of cTnT with PKC has been shown by Kuo et al. who reported that PKC catalyzes at least 2 mol of phosphate (Pi)/mol of cTnT and that the phosphorylation of cTnT is inhibited by the presence of Tm – also a substrate of PKC – to which it is firmly bound [45,46]. In a follow-up study, bovine cTnT was phosphorylated in vitro by incubation with PKC for different time periods. The increase in phosphorylation at different sites on cTnT after prolonged incubation was shown by autoradiography of tryptic peptide maps. Subsequent Edman sequencing determined the preferred site of cTnT phosphorylation at Thr-294 (human canonical sequence), and identified Thr-204, Thr-213 [47], and Ser-208 [48,49] as additional phosphorylation sites. Table 1 summarizes the advances made towards the understanding of cTnT phosphorylation. In general, PKC phosphorylation of cTnT reduces the binding affinity of cTnT for Tm. This lowers troponin's ability to expose the blocked myosin-binding sites and thus results in a decreased Ca2+-dependent actomyosin ATPase activity [50,51]. In an attempt to determine the site-specific effects of PKC-mediated cTnT phosphorylation, Sumandea et al. created different recombinant cTnT proteins with glutamic acid or alanine residues mutated at the four PKC phosphorylation sites [52]. These residues mimic (Glu) and prohibit (Ala) phosphorylation at those sites and were combined in various ways to search for functional effects. Using this technique, Thr-213 was identified as a “functionally critical site” as a glutamic acid modification of this site alone resulted in a significant decrease of maximum tension, actomyosin ATPase activity, myofilament Ca2+-sensitivity and cooperativity. This finding was confirmed when Thr-213 was subsequently phosphorylated by PKC-α [52]. The effects of the other PKC sites (Thr-203, Ser-208 and Thr-294) are less unequivocal. Glutamic acid mutation of these three sites had no effect when Thr-213 was mutated to alanine [52,53]. This may suggest that these sites play no functional role, but the authors did not actually phosphorylate those residues. Interestingly, a previously performed study in transgenic mouse hearts did show a functional role of one or more of these residues. Fast skeletal TnT, which lacks residues Thr-204, Ser-208 and Thr-294, was expressed in mouse hearts. After induced PKC phosphorylation, the maximum tension in these transgenic fibers was higher than in wild type and a small but significant increase, rather than a decrease, in Ca2+-sensitivity was observed [54]. This indicates that the absence of residues Thr-204, Ser-208 and Thr294 blunts the depression of force and the decrease in Ca2+-sensitivity. Thus, Thr-213 may not be the sole functional phosphorylation site in cTnT as the other three residues could also have a detrimental effect on cardiac contractility.

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3.2. Phosphorylation by other kinases

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Other kinases than PKC have been implicated in cTnT phosphorylation as well. Free bovine cTnT was phosphorylated in vitro by Ca2 +/ calmodulin-dependent protein kinase II (CaMK II), but its effect disappeared in the reconstituted cTn–Tm-complex [65,66]. Apoptosis signal-regulating kinase 1 (ASK1) is implicated in intracellular signaling cascades triggered by stress stimuli like proinflammatory cytokines and reactive oxygen species [67]. Under the influence of these stimuli, ASK1 was shown to specifically phosphorylate cTnT at Thr-204 and Ser-208 in vitro and in adult rat cardiomyocytes, which resulted in reduced contractility after treatment with hydrogen peroxide [68]. A completely new phosphorylation site was discovered by Vahebi et al. in their experiments towards rho-A-dependent protein kinase (ROCK-II). In vitro phosphorylation of mouse muscle fibers by ROCK-II

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Up to eleven different PKC isoforms have been identified, which can be classified as conventional (α, β, βII, γ), novel (δ, ε, η, θ) and atypical (ζ, λ and ι) [55]. These isoforms each act as different kinases in themselves and induce different physical effects upon phosphorylation of cTnT. In vitro phosphorylation of purified bovine troponin by recombinant PKC isoforms showed that PKC-α and PKC-δ phosphorylated cTnI more than cTnT. PKC-ζ phosphorylated cTnT on an unknown site, but had almost no interaction with cTnI [49]. Interestingly, several years later, Wu and Solaro found the exact opposite with an affinity pulldown-assay using cTnT and cTnI from primary rat ventricular cardiomyocytes. According to their immunoblots, PKC-α and PKC-δ interact solely with cTnT while PKC-ζ strongly and solely interacts with cTnI [56]. PKC-ε interacts with cTnI as well as cTnT in both studies [49,56] and is thought to be the most abundant PKC isoform in adult ventricular myocytes [57,58]. The apparent conflicting effect of PKC-ζ is an interesting observation which might be prescribed to interactions of PKC-ζ with dephosphorylation pathways. PKC-ζ has been shown to interact with p21 activated kinase (PAK)1 [56,59], a kinase which phosphorylates cTnI [60], but is also known for its activation of protein phosphatase (PP)2A [61] which has been implicated in threonine dephosphorylation of cTnI and cTnT [61,62]. This supports the findings of Wu and Solaro who were surprised to observe a decrease, rather than an increase, in troponin phosphorylation after expression of PKC-ζ within the cardiomyocytes [56]. A likely explanation for the differences in the described studies [49,56] are the different phosphorylation techniques. In vitro phosphorylation relies on incubation in buffers containing kinases and other components, but likely lack dephosphorylation components. In situ studies are performed by indirect inducement of phosphorylation within viable cardiomyocytes. This does allow for unexpected results like dephosphorylation upon up-regulation of a kinase. Adding support to this hypothesis is a study where an increase in both cTnT and cTnI phosphorylation is observed after inhibition of PKC-ζ. This reduces PP2A activity and as such reduces cTn dephosphorylation, leading to an increase in phosphorylation. Interestingly, this results in an improvement of cardiac contractility, but the authors did not investigate whether cTnI or cTnT, and if so; which sites, are responsible [63]. Dephosphorylation can work as a compensatory mechanism to reverse the reduced contractility as a result of troponin phosphorylation. PP1 dephosphorylation of cTnT returned reduced ATPase activity and Ca2+-sensitivity, caused by PKC phosphorylation, back to control values in rat cardiomyocytes [64]. PP1 does not have a specific target site to dephosphorylate cTnT, however, Thr-213 is the least sensitive to PP1, while being specifically targeted by PP2A [56,64], suggesting different roles for the two phosphatases. A map of the involved pathways for cTnT phosphorylation and dephosphorylation is given in Fig. 3. This illustrates the underlying mechanisms and interplays between various kinases, phosphatases, messenger proteins and intracellular second messengers.

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different isoforms and the structure of troponin T have been reviewed in [39–41]. The annotation of targeted residues in the studies reviewed in this paper varies due to the use of different animal models (species), different isoforms (namely isoforms 1, 3, and 4 according to Anderson) and the in- or exclusion of the initiating methionine. These differences are mainly caused by the variable nature of the N-terminus of cTnT and complicate the comparison of different studies. For clearness and consistency, we decided to use the annotation of the canonical human isoform of cTnT (Fig. 2, cTnT-1; http://www.uniprot.org/uniprot/P45379), including the initiating methionine, regardless of the specific isoforms/ species used in the separate papers, throughout this review.

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Please cite this article as: Streng AS, et al, Posttranslational modifications of cardiac troponin T: An overview, J Mol Cell Cardiol (2013), http:// dx.doi.org/10.1016/j.yjmcc.2013.07.004

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A.S. Streng et al. / Journal of Molecular and Cellular Cardiology xxx (2013) xxx–xxx t1:1 t1:2

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Table 1 Summary of the known cTnT phosphorylation sites, their targeting kinases and their physiological effect.

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Reference

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Kinase

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Analytical

Effect

Additional findings

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Ser-2

“cTnT kinase”

In vitro dog

SDS-PAGE, autoradiography

Not investigated

None

Ser-2 Ser-2

“cTnT kinase” Unknown

In vitro bovine (Healthy) in vivo human/mouse

Gel filtration, chromatography Not investigated Top–down MS Not investigated

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Villar-Palasi, 1981 [43] Gusev, 1983 [42] Sancho Solis, 2008 [74]/Zhang, 2009 [75] Jaquet, 1995 [65]

Thr-204

CaMK II

In vitro bovine

HPLC

Not investigated

t1:8

Noland, 1989 [47]

PKC

In vitro bovine

Not investigated

t1:9

Jideama, 1996 [49]

Peptide mapping, Edmansequencing, autoradiography Skinned muscle fiber measurements, peptide mapping, autoradiography

Thr-204, Ser208, Thr-294

PKC

t1:12

Sumandea, 2009 [70]

Ser-208, Thr213, Ser-285, Thr-294

PKC-α

In vitro mouse

t1:13

Sumandea, 2003/ 2004 [52,53]

Thr-213

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Pfleiderer, 2009 [71] Vahebi, 2005 [69]

Skinned muscle fiber measurements

Autoradiography SDS-PAGE, MS, skinned muscle fiber measurements

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PKC-α, Mouse mutants PKC-βII, PKC-ε mimicking phosphorylation, in vitro mouse Thr-213 Raf-1 In vitro rat Ser-285, Thr-294 ROCK-II In vitro mouse

Inhibition of cardiac contractility Reduces maximal tension

ASK1 is activated by ROS. ASK1 does not phosphorylate cTnI Effect was blunted in TG mice expressing fsTnT in the heart

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Montgomery, 2001 [54]

Langendorff perfusion, autoradiography Skinned muscle fiber measurements, SDS-PAGE, WB, autoradiography MS, skinned muscle fiber measurements, motility assay, phosphospecific immunoblots

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In vitro human, ex vivo/in situ rat In vitro mouse

ASK1

Reduces ATPase activity and Ca2+-sensitivityc Increased Ca2+-sensitivityb

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He, 2003 [68]

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PKC-α, PKC-δ, In vitro bovine PKC-ε, PKC-ζb

cTnT contains 0.8 mol Pi/mol cTnT pSer-2 is the only in vivo phosphorylation site in healthy heart CaMK II does not phosphorylate the reconstituted cTn–Tm complex None

Reduces maximal tension, ATPase activity, Ca2+sensitivity and myofilament sliding speed. Reduces tension cost

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Thr-204, Thr213, Thr-294 Thr-204, Ser208, Thr-213, Thr-294, unknown site† Thr-204, Ser-208

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Reduces maximal tension, ATPase activity, and Ca2+sensitivity

Not investigated. Reduces maximal tension, reduces ATPase activity, reduces Ca2+-sensitivity

N-terminus deletion decreases cTnT–Tm binding affinity, decreases maximal tension and maximal ATPase activity Phosphorylation increases tension cost after N-terminus deletion Thr-213 is a “functionally critical” phosphorylation site

Raf-1 does not phosphorylate cTnI None

cTnT indicates cardiac troponin T; CaMK II, Ca2+/calmodulin-dependent protein kinase; PKC, protein kinase C; ASK, apoptosis signal-regulating kinase; ROCK, rho-A-dependent protein kinase; TG, transgenic; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; MS, mass spectrometry; HPLC, high pressure liquid chromatography; NMR, nuclear magnetic resonance; WB, western blotting; ROS, reactive oxygen species; Tm, tropomyosin; cTnI, cardiac troponin I; and fsTnT, fast skeletal troponin T. a Amino acid numbering is based on the human canonical cTnT isoform (cTnT1). b PKC-ζ phosphorylated cTnT at an unknown site and caused an increase in Ca2+-sensitivity without influencing ATPase activity. c Result of PKC-α and PKC-ε mediated phosphorylation of cTnT.

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resulted in a decrease of maximum myofilament tension and MgATPase activity (both by 15%) and a decrease in Ca2+-sensitivity [69]. Analysis with mass spectrometry (MS) identified the cTnT phosphorylation sites of ROCK-II at Ser-285 and Thr-294. Myosin light chain and cTnI phosphorylation, while present, did not contribute to the observed functional effects [69]. Later, PKC-α was also implicated in the phosphorylation of this site [70]. More recently, Raf-1 kinase has been implicated in the phosphorylation of Thr-213 [71], the functionally critical cTnT phosphorylation site as proposed by Sumandea and colleagues [52]. PAK3 has also been linked to cTnT phosphorylation, although no sites could be identified and the observed increased Ca2+-sensitivity was attributed to cTnI phosphorylation [60].

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3.3. cTnT phosphorylation in disease

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Recently, scientists have debated the physiological relevance of many of these identified phosphorylation sites of cTnT and other myofilament proteins [72,73]. The above reviewed studies all used purified or recombinant troponin, which was in vitro phosphorylated by several selected kinases and subsequently reconstituted. Often, only the troponin complex itself was reconstituted, instead of the entire filament. In one study, CaMK II phosphorylation of Thr-204 was present in free cTnT and the cTn-complex, but absent in the reconstituted cTn–Tmcomplex [65,66]. A possible explanation for this is that the kinase affinity for that site is decreased due to steric hindrance of Tm, which binds to cTnT close to that location [39].

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t1:16 t1:17 t1:18 t1:19 t1:20 t1:21

In vivo studies simply have not been able to reproduce the results shown in vitro. MS studies of healthy rat, mouse and human heart tissue unambiguously show that cTnT is either monophosphorylated on Ser-2 or unphosphorylated [74,75]. The significance of this site has not yet been elucidated, but it could be possible that it plays a functional role in healthy cardiomyocytes. In addition, it needs mentioning that it is possible to miss phosphorylation sites using top–down MS and as such not seeing one does not rule out its existence. Considering that the measured in vitro effects of cTnT phosphorylation generally indicate a reduced myofilament contractility (see Table 1) and that PKC is upregulated in response to hypertrophic signaling and HF [76–78], one could hypothesize that cTnT phosphorylation is increased in pathological states (summarized in Table 2). However, in two separate studies of postmortem non-failing donor hearts and endstage failing explanted hearts (immediately snap-frozen in liquid nitrogen), no difference in average phosphorylation of cTnT was observed [79,80]. Interestingly, Messer et al. determined that in failing as well as non-failing human hearts the average phosphorylation level was 3.1 mol Pi/mol cTnT; indicating that at least two other residues need to be phosphorylated besides Ser-2 [80]. In a recent study where congestive heart failure was induced in rats, increased phosphorylation was found at two residues (Thr-213 and Ser-285, both targets of PKCα), using phosphospecific antibodies [81]. In that study however, Belin et al. found that phosphorylation of cTnT at those sites was increased by 50% in both right and left ventricle failing heart tissue as compared to control [81], which is in contradiction with earlier studies in failing human tissue [79,80]. In addition, they found significant differences in

Please cite this article as: Streng AS, et al, Posttranslational modifications of cardiac troponin T: An overview, J Mol Cell Cardiol (2013), http:// dx.doi.org/10.1016/j.yjmcc.2013.07.004

317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343

A.S. Streng et al. / Journal of Molecular and Cellular Cardiology xxx (2013) xxx–xxx

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6

344 345

E

D

Fig. 3. Different pathways of cTnT phosphorylation and dephosphorylation. An arrow indicates stimulation, an arrow with a minus sign (–) indicates dephosphorylation. Circled molecules are messenger proteins and receptor ligands, yellow indicates second messengers, G-proteins and other signaling molecules, green indicates kinases and red molecules indicate phosphatases. cTnT is depicted schematically at the bottom of the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

t2:1 t2:2

Table 2 Summary of all available literature to date studying cTnT phosphorylation in pathological states.

C

E

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348 349

R

346 347

t2:3

Reference

Disease

t2:4

Kameyama, 1998 [82]/ Noguchi, 2003 [83] Van der Velden, 2003 [79] Messer, 2007 [80]

LVH ➔ cardiac failure LV of Dahl saltsensitive rats End stage Human LV heart failure End stage Unspecified human heart failure heart tissue Induced LVH Rat LV and RV or CHF

Belin, 2011 [81]

t2:8

Walker, 2010 [25]

t2:9 t2:10

Van der Velden, 2011 [87] Avner, 2012 [86]

t2:11

Dubois, 2011 [88]

t2:12

Eiras, 2006 [89]

t2:13 t2:14 t2:15 t2:16

O

C

Q3t2:7

Change in cTnT phosphorylation

Measured effect

Additional findings

Reduction of bulk cTnT phosphorylation

Decrease in Ca2+-sensitivity

PKC is likely involved in the transition to failure cTnI and MLC-2 phosphorylation is reduced 3.1 mol Pi/mol cTnT

a

No identified change No identified change

2+

Increase in Ca -sensitivity, no change in maximal tension Increase in Ca2+-sensitivity, reduced sliding speed Twofold increase in PKCa 45% decrease in maximal tension in RV and LV, decrease expression in RV and a fourfold in Ca2+-sensitivity in LV increase in LV [116]

MI of the LAD coronary artery MI of the LCx coronary artery MI of the LAD coronary artery MI of left coronary artery (rats) MI ➔ LVR (humans)

Mouse LV

Increase of bulk cTnT phosphorylation, 87% increase of pThr-213b in RV, 24% in LV after CHF No change in pSer-285b No identified changea

Pig LV

No identified changea

Mouse LV

No identified change

Rat LV and plasma Human plasma

Atrial fibrillation

Human RA

Decrease of pSer-208b in rat LV and Not investigated Circulating p-cTnT could be a new prognostic biomarker for LVR plasma following infarction b Decrease of pSer-208 in human plasma indicative of LVR Increase in cTnT mono-phosphorylation. Decrease in maximal activated force, reduction of crossbridge kinetics

N

t2:6

Investigated tissue/material

U

t2:5

An earlier in vivo study employing Dahl salt sensitive rats found a decrease of cTnT phosphorylation in failing hearts [82,83]. However, they did find that inhibiting PKC activity by blocking endothelin receptors halted the development of failure, suggesting a PKC-mediated mechanism. The authors argued that a reduced bulk cTnT phosphorylation could mask the increase of phosphorylation on a specific, functional, site. Consistent with the findings of Belin, they also found a decrease of Ca2+-sensitivity in the failing hearts [83].

T

350 351

phosphorylation and contractility between the right (RV) and left (LV) ventricle (LV showed higher total cTnT phosphorylation, RV showed a higher phosphorylation of Thr-213, and Ca2+-sensitivity was higher in the RV than the LV). Also, the physiological effects measured in this study (a decrease in Ca2+-sensitivity and a 45% decrease in maximum myofilament force [81]) did not correspond to the effects seen in human heart tissue (an increase in Ca2+-sensitivity and myofilament sliding speed and an unchanged maximum myofilament force [79,80]).

Not investigated

Redistribution of level of cTnI phosphorylation

Increased Ca2+-sensitivity, decrease of maximal tension Increased Ca2+-sensitivity

LVH indicates left ventricular hypertrophy; CHF, congestive heart failure; MI, myocardial infarction; LAD, left anterior descending; LCx, left circumflex; LVR, left ventricular remodeling; LV, left ventricle; RV, right ventricle; RA, right atrium; cTnT, cardiac troponin T; cTnI, cardiac troponin I; PKC, protein kinase C; MLC-2, myosin light chain 2; and p-, phosphorylated. a No cTnT phosphorylation data was shown. b Amino acid numbering is based on the human canonical cTnT isoform (cTnT1).

Please cite this article as: Streng AS, et al, Posttranslational modifications of cardiac troponin T: An overview, J Mol Cell Cardiol (2013), http:// dx.doi.org/10.1016/j.yjmcc.2013.07.004

352 353 354 355 356 357 358 359

A.S. Streng et al. / Journal of Molecular and Cellular Cardiology xxx (2013) xxx–xxx

381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396

401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422

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379 380

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5. Summary and conclusions

483

F

375 376

R

373 374

N C O

371 372

U

369 370

O

Cardiomyocyte apoptosis following AMI and HF can cause proteolytic degradation of cTns by caspase-3 and μ-calpain (also known as calpain-I) [20,91,92]. Di Lisa et al. found that μ-calpain, which is active at low, micromolar, Ca2+-concentrations, is more effective at degrading troponin than m-calpain, which is active at millimolar Ca2+-concentrations [92]. Also interesting is that troponin phosphorylated by PKC is more sensitive to cleavage by calpain than unphosphorylated troponin [92]. Calpain is a protease which nonspecifically degrades free cTnT [93]. However, when cTnT is in complex with cTnI and cTnC, it selectively cleaves the N-terminal, variable region between residues 78–79 in mouse cardiomyocytes in vitro, altering binding affinities of cTnT with cTnI and Tm [93,94]. The human sequence has a serine instead of a leucine at residue 79, see Fig. 2, but it is unknown if (and if so; how) that influences the cleavage. It was recently determined that deletion of the highly acidic part of the N-terminus (residues 1–52 in human cTnT) removes 23 negative charges (25 when Ser-2 is phosphorylated) which highly affects the binding affinity of cTnT for Tm and reduces maximal activated tension and Ca2+-sensitivity. In contrast, deletion of residues 53–78 has no effect on cTn–Tm binding, decreases cooperativity and increases Ca2+-sensitivity [94]. In addition to calpain, caspase-3 cleaves cTnT between Asp-98 and Asp-99, but only when it is in complex with cTnI and TnC and not as free cTnT [91]. Sumandea et al. found that caspase cleavage of cTnT reduces maximum myofilament tension by 24%, reduces ATPase activity by 23% and increases Ca2+-sensitivity [70]. These

367 368

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366

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4. cTnT fragmentation

364 365

observations suggest a specific functional mechanism of degradation rather than random destructive breakdown of structural proteins. Necrosis of cardiac tissue as seen after myocardial infarction is accompanied by the release of different proteolytic enzymes from the lysosomes. For cTnI it is generally accepted that it is highly susceptible to proteolysis. The occurrence of immunoreactive cTnI fragments in human serum after myocardial infarction has been confirmed by multiple groups and is undisputed [20,95,96]. Degradation and changes in the molecular forms of cTnI have consequences for the immunoreactivity of the antibodies used in the various clinical assays [97,98]. This results in varying outcomes when measuring the same serum sample with different cTnI assays employing different antibodies, complicating the clinical interpretation of those measurements. Further research on cTn degradation and the effect it has on clinical measurements is required. With respect to cTnT degradation upon myocardial infarction, the literature is not unequivocal. cTnT fragmentation in AMI patient serum was shown by different groups using gel filtration chromatography (GFC) [98] and immunoblots [20,99,100]. Extensive fragmentation of cTnT was also found in serum of patients suffering from end stage renal disease (ESRD) [101,102]. However, both claims were disputed in studies using GFC and it was argued that cTnT in ESRD and AMI patient serum is present predominantly in the intact and complexed form [103,104]. No consensus was reached in this discussion [105,106], but a recent study by Cardinaels et al. clearly shows the existence of cTnT fragments in the serum of AMI patients, identified using antibodies from the clinical cTnT-assay by Roche diagnostics [107]. Cardinaels confirms the data by Michielsen et al. who first showed that the degradation of cTnT in serum after AMI is progressive. Intact cTnT rapidly disappears from serum after the ischemic event and progressively smaller cTnT fragments appear in time [100]. It is plausible that in ESRD, these fragments accumulate due to reduced clearance, possibly resulting in an overestimation of cTnT concentrations [101,108]. As indicated for cTnI, degradation of cTnT in human serum can also have consequences for the immunoreactivity of the antibodies used in the cTnT immunoassay and may influence the clinical cTnT measurements. Developing immunoassays specifically aimed at detecting intact, fragmented, or phosphorylated cTnT can be of help to investigate the pathophysiology of troponin degradation and might result in clinical applications. If cTnT is being fragmented, the question remains if it solely takes place in the bloodstream after release or if a portion of it is being degraded inside the cardiomyocyte as part of some regulatory mechanism. Hard evidence regarding in situ cTn degradation is absent, but some studies provide interesting clues that may suggest the formation of cTn fragments in viable cardiomyocytes. For example, two studies showed cTnT fragments in healthy human heart tissue homogenates, implicating its degradation in situ [109,110]. In addition, two in vitro studies, using vastly different approaches, also support this hypothesis. Using metabolic inhibition of primary rat cardiomyocytes, cTnT and cTnI were shown to be released from the cells both intact and fragmented [111]. Lastly, a pilot study using ischemic mouse HL-1 cardiomyocytes reported a decrease in intracellular cTn concentration combined with the formation of cTn fragments before the onset of cell death [112]. However, when cTnT was spiked in cTnT negative serum and incubated at 37 °C for prolonged periods of time, a clear degradation of cTnT was observed [113]. These preliminary observations may lead to the hypothesis that troponin is selectively cleaved inside the cardiomyocyte (possibly underlying the transition of left ventricular hypertrophy to failure [114]) and is subsequently progressively degraded upon its release in the circulation.

D

398

362 363

T

397

The discrepancy between these studies can be explained by the use of different species (human and rat). Van der Velden et al. showed that there are large discrepancies between the different models [84] and in an informative discussion; Marston and de Tombe debated the advantages and disadvantages of animal models versus the use of human tissue [85]. In that “point/counterpoint”-article, it is argued that animal models cannot give a more accurate representation of the human heart than the human heart itself. On the other hand, human control samples are disputed which are often post-mortem donor hearts undergoing autolysis and exposed to high levels of catecholamine and inotropic support agents following death. To illustrate the difference between rodent and human tissue, the authors show that Ca2+-sensitivity is increased in human failing hearts and decreased in the heart tissue of small rodents following induced heart failure [85], which is completely in line with the studies described above [79–83]. Several studies have been performed on contractile protein phosphorylation after AMI in mice and pigs [25,86,87]. No difference has been shown in total cTnT phosphorylation after MI. However, sitespecific analysis using phosphospecific antibodies or MS was not performed in those studies. In contrast, a recent study did show a difference in cTnT phosphorylation in rats after induced MI [88]. In that study, Dubois et al. used a polyclonal antibody specific towards the phosphorylated Ser-208 residue and sensitive for both human and rat cTnT and saw a decrease in Ser-208 phosphorylation in rat LV tissue and in rat plasma 2 months after infarction. In human plasma, a decrease of Ser208 phosphorylation measured immediately after AMI proved to be a predictor for left ventricular remodeling one year after the event [88]. This study suggests a prognostic application in the clinic for phosphorylated cTnT. An increase in cTnT phosphorylation is also reported in patients with atrial fibrillation [89]. This increased phosphorylation was accompanied with a decrease of maximal activated force and a reduction in crossbridge kinetics, while Ca2+-sensitivity was unchanged, in comparison to patients with sinus rhythm [89]. There was no difference in phosphorylation of myosin light chain 2 or cTnI while the activity of PP1 and PP2A was increased [89,90]. This may suggest a specific mechanism of phosphorylation, targeting functional sites and counteracting the effects of phosphatases.

E

360 361

7

425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482

Cardiac troponin is an important regulatory protein complex that is 484 essential in maintaining cardiac contractility and dynamically changes 485 and adapts to variations in cardiac demand. In this review, we have 486

Please cite this article as: Streng AS, et al, Posttranslational modifications of cardiac troponin T: An overview, J Mol Cell Cardiol (2013), http:// dx.doi.org/10.1016/j.yjmcc.2013.07.004

506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528

C

532

None.

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looked specifically at changes in the tropomyosin-binding subunit of troponin: cTnT. As summarized in Table 1, in vitro studies showed that the phosphorylation of cTnT reduces ATPase activity, reduces maximal myofilament tension and reduces Ca2+-sensitivity. However, in vivo, the only site shown to be phosphorylated in healthy tissue is the N-terminal Ser-2. There is no consensus on the nature of cTnT phosphorylation during pathologic states as the few studies that were performed to investigate this show conflicting results. The animal species used are thought to influence the outcome, as well as differences in measurement strategies like total phosphorylation versus sitespecific phosphorylation. Nevertheless, some interesting observations have been summarized in Table 2 and may warrant further investigation. Especially the reports of Belin and Dubois show promising results, illustrating an increased PKC expression during heart failure and subsequent increases in PKC-mediated cTnT phosphorylation featuring the adverse effects discussed earlier. Also prominent are the observed differences between the right and left ventricle and the finding that cTnT phosphorylation status in the heart correlates with the phosphorylation state of circulating cTnT in plasma following an AMI, which may be a predictor for the development of left ventricular remodeling. Due to their cardiac specificity, cTnI and cTnT are actively being used as biomarkers for cardiac injury like AMI, myocardial ischemia and heart failure. Better understanding of the way cTnT changes on a molecular level can be important in interpreting cTnT levels in the clinic and may lead to new measurement techniques offering additional information about the disease state of the patient. Dubois et al. already showed that cTnT phosphorylation can be used as a prognostic marker for left ventricular remodeling after AMI. Following the same line of thought, fragmentation patterns of circulating cTnT may be used to differentiate between different cardiac diseases. Lin et al. proved this concept for cTnI and introduced a qualitative cTnI assay employing a plethora of antibodies able to determine cleavage products, phosphorylation sites and complex formation [115]. Despite the fact that posttranslational modifications of cardiac contractile proteins have been thoroughly researched over the past few decades, the role of cTnT modifications in health and disease is still unclear. More research in this area is needed to fully understand the complex interplay between the disease state, cTnT modifications and the adverse effects within the cardiomyocyte. We feel that the focus of future research should lie on site-specific cTnT phosphorylations and their effects on cardiac contractility in different pathologic states. A better understanding of the mechanism and function of cTnT phosphorylation and degradation may lead to the development of new diagnostic and prognostic tools as well as novel therapeutic interventions.

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Please cite this article as: Streng AS, et al, Posttranslational modifications of cardiac troponin T: An overview, J Mol Cell Cardiol (2013), http:// dx.doi.org/10.1016/j.yjmcc.2013.07.004

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