Physiological and pathological status of troponin in heart

Physiological Action of Troponins I, T, and C

Troponin comprises three subunits, troponin I, troponin T, and troponin C, which work in concert with tropomyosin and filamentous actin (thin filament) in a highly cooperative manner to control and regulate striated muscle (skeletal and cardiac) contraction. Contraction occurs through the calcium-dependent interaction of the thin and thick filament (primarily composed ofmyosin) (16-20). In diastole, at reduced cellular calcium concentration, there is a low probability of interaction between the thick and thin filament and, as such, force production is low and the muscle is relaxed. With systole, calcium floods the cell, inducing a complex series of conformational changes within the troponin complex, ultimately promoting the interaction between actin and myosin (thin and thick filament) and resulting in force.

The troponin subunits each play a unique functional role (16-20). cTnT is named for its ability to anchor troponin onto the actin-tropomyosin thin filament through a number of Ca2+-dependent and Ca2+-independent interactions. cTnC is the Ca2+ sensor. The binding of an additional calcium to troponin C during systole alters its conformation and interaction with cTnI, initiating the Ca2+-dependent signal to the rest ofthe thin filament. cTnI acts as a switch, turning muscle contraction on and off. Contraction occurs when cTnC binds the regulatory calcium, allowing the Ca2+-regulatory region of cTnI to bind cTnC. This initial interaction pulls over the cTnI inhibitory region from its sites on actin-tropo-myosin toward cTnC, promoting a cascade of conformational changes throughout the thick and thin filaments that allows actin and myosin to interact (Fig. 1).

Fig. 1. Schematic representation of human cTnl. (A) Representation of linear amino acid sequence of human cTnl (residues 1-209). Important biological regions are shown: the troponin I (Tnl) inhibitory region ( ED ); the troponin I calcium-regulatory region ( □ ); and a region to the C-terminus that binds actin-tropomyosin (■), which anchors the troponin I molecule in place. Phosphorylation sites for protein kinase A (PKA), PKC, and p21-activated kinase (PAK) are shown as P. The zigzag points to the C-terminal cleavage site that occurs with global ischemia in isolated rat hearts. (B) Schematic of troponin I (modified from ref. 18) showing various regions just described including secondary cleavage sites shown to occur in isolated rat hearts with more severe ischemia.

Fig. 1. Schematic representation of human cTnl. (A) Representation of linear amino acid sequence of human cTnl (residues 1-209). Important biological regions are shown: the troponin I (Tnl) inhibitory region ( ED ); the troponin I calcium-regulatory region ( □ ); and a region to the C-terminus that binds actin-tropomyosin (■), which anchors the troponin I molecule in place. Phosphorylation sites for protein kinase A (PKA), PKC, and p21-activated kinase (PAK) are shown as P. The zigzag points to the C-terminal cleavage site that occurs with global ischemia in isolated rat hearts. (B) Schematic of troponin I (modified from ref. 18) showing various regions just described including secondary cleavage sites shown to occur in isolated rat hearts with more severe ischemia.

Phosphorylation and Proteolysis of Troponin

The action of each troponin subunit is regulated through extensive interactions with each other and actin-tropomyosin. Likely to ensure that the complex remains associated under most conditions, some of these protein-protein interactions are extremely strong. Others are less tight and are primarily involved in the regulation of the troponin and thin filament response to changes in the cellular calcium concentration. Like most important cellular regulators, cTnI is under tight regulatory control, primarily through signaling pathways that ultimately lead to phosphorylation of specific serine and threonine residues. Although many different kinases (PKC and PAK) have been shown to be able to phosphor-ylate cTnI (and cTnT) in vitro, only PKA phosphorylation has been shown to occur in vivo (20) (Fig. 1). PKA is stimulated in response to physiological ^-adrenergic stimulation, which also results in the phosphorylation of numerous other important calcium-response proteins such as phospholamban and the ryanodine receptor (21). The extent of cTnI and cTnT phosphorylation is affected by two factors: the individual activity of the kinase and phosphatase and the accessibility of the potential phosphoamino acid. The latter is primarily dependent on the conformation of the protein and, hence, the cellular calcium concentration as well as the presence and local influence of any other posttrans-lational modification.

Degradation

Phosphorylation

Phosphorylation

Degradation

Degradation

Phosphorylation

Phosphorylation

Degradation

- C-terminus (ending at 192)

- N-terminus (starting at 63)

- N-terminus (starting at 73)

- PKA (reduced levels)

- protein kinase C

- p21 activated kinase

- PKA (increased levels)

Fig. 2. Potential different posttranslational modified forms to troponin I that may occur under physiological and pathological conditions.

Further diversity can arise when cTnI and/or cTnT undergoes specific and selective pathological proteolysis with ischemia (19,20,22). The proteolysis in human heart was initially speculated from a series of experiments on isolated rat hearts that underwent global ischemia (17,18,20,22). There is an initial C-terminal proteolysis of cTnI followed by two N-terminal proteolysis cleavages that subsequently occur with increasing degree (severity) of ischemia. Although not present in larger animals (dogs and pigs), it is clear that proteolysis of cTnI does occur in human hearts (more detail in a later section). These modifications of the troponins could give rise to a dizzying number of phosphorylated and degraded species of cTnI and cTnT. Confirmation of this diversity is currently lacking, primarily owing to the lack oftools capable of assessing and monitoring the phosphorylation and degradation status of each amino acid residue in a robust and high-throughput manner. Even so, this combination of physiological and pathological posttranslational modifications, each of which gives important information about the function of the contractile apparatus, has the potential to become the next level of diagnostics (Fig. 2).

Several intrinsic characteristics of cTnI govern, at least in part, the form of this molecule that is present in the serum. As mentioned, the interactions between cTnI and the other troponin subunits in the myocyte can be extremely tight and require harsh denaturing conditions (8 M urea and 1M salt) to dissociate into dimers or monomers. These strong interactions make it difficult for the intact molecules to be released from one another. As well, cTnI and cTnT are essentially insoluble as monomers and aggregate at neutral pH. Both molecules have enhanced solubility when bound to each other or to cTnC. Furthermore, because of its high pi (9.5), resulting from its large number ofpositively charged amino acid residues, cTnI is "sticky" and will bind to negatively charged proteins (e.g., troponin C). cTnT, although having a more neutral pi (~5.0), is also "sticky," owing to its charge distribution, which is clustered. The N-terminus of cTnT is predominately acidic with many

Aggregation of Troponin Subunits

Fig. 3. Schematic of cTnl complex isolation from tissue and serum of patient who underwent coronary artery bypass graft (CABG). Either tissue homogenate or serum was incubated with an anti-cTnl antibody covalently coupled to beads. The beads were extensively washed to remove any unbound and nonspecifically bound protein. This can be seen in the Western blot (lane 1, sample loaded [serum]; lanes 2-6, wash steps), where there is minimal cTnl eluted. The majority of the cTnl remained bound to the antibody-bead. An aliquot of the bead (cTnl complex-antibody-bead) was boiled and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotted for the three troponin subunits. In tissue, all three subunits were observed with equal quantity. In serum, primarily cTnl and cTnC were observed, indicating that cTnl is either a monomer or bound to cTnC in serum. Protein identification was confirmed by digesting the remaining aliquot of the bead (cTnl complex-antibody-bead) and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In tissue, all three troponin subunits were identified, whereas peptide fragments of only cTnl and cTnC were observed in sample isolated from serum (see ref. 25 for more details).

Fig. 3. Schematic of cTnl complex isolation from tissue and serum of patient who underwent coronary artery bypass graft (CABG). Either tissue homogenate or serum was incubated with an anti-cTnl antibody covalently coupled to beads. The beads were extensively washed to remove any unbound and nonspecifically bound protein. This can be seen in the Western blot (lane 1, sample loaded [serum]; lanes 2-6, wash steps), where there is minimal cTnl eluted. The majority of the cTnl remained bound to the antibody-bead. An aliquot of the bead (cTnl complex-antibody-bead) was boiled and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotted for the three troponin subunits. In tissue, all three subunits were observed with equal quantity. In serum, primarily cTnl and cTnC were observed, indicating that cTnl is either a monomer or bound to cTnC in serum. Protein identification was confirmed by digesting the remaining aliquot of the bead (cTnl complex-antibody-bead) and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In tissue, all three troponin subunits were identified, whereas peptide fragments of only cTnl and cTnC were observed in sample isolated from serum (see ref. 25 for more details).

negatively charged amino acid residues whereas the C-terminal portion of the molecule is basic and contains mainly positively charged amino acids.

These properties indicate, especially for cTnl, that these molecules will most likely not be found as monomers in the serum but, rather, bound to one or both of the other cardiac troponin subunits or to other serum protein(s). Data from the literature support this assumption and have demonstrated indirectly that cTnl in the serum is most likely com-plexed predominately with cTnC (23,24). Using an anti-troponin I antibody, our laboratory (25) captured cTnI-containing complexes from the serum of patients with AMI and proved directly that cTnI is bound to cTnC in serum (Fig. 3). However, it is not always the case that these two subunits are bound. It is important to recognize that the phosphorylation and degradation status of cTnI can dramatically alter the affinity of cTnI for cTnC.

Fig. 4. Representative Western blots of cTnl and cTnT from human myocardium or serum. (A) One-dimensional SDS-PAGE (12%) Western blot of cTnl of biopsy obtained from patient undergoing CABG taken from left ventricle in area remote to ischemic region before surgery and after cross-clamp removal. (B) One-dimensional SDS-PAGE (12%) Western blot of cTnl serum obtained from three different patients with AMI diagnosed using clinical parameters and elevated serum troponin I based on clinical chemistry assay. (C,D) One-dimensional SDS-PAGE (12%) and two-dimensional gel electrophoresis (pi 4-7; 12% SDS-PAGE) Westernblot of cTnT from left ventricle tissue obtained from transplanted heart rejected by recipient (C) or biopsy obtained from patient who underwent CABG (D).

Fig. 4. Representative Western blots of cTnl and cTnT from human myocardium or serum. (A) One-dimensional SDS-PAGE (12%) Western blot of cTnl of biopsy obtained from patient undergoing CABG taken from left ventricle in area remote to ischemic region before surgery and after cross-clamp removal. (B) One-dimensional SDS-PAGE (12%) Western blot of cTnl serum obtained from three different patients with AMI diagnosed using clinical parameters and elevated serum troponin I based on clinical chemistry assay. (C,D) One-dimensional SDS-PAGE (12%) and two-dimensional gel electrophoresis (pi 4-7; 12% SDS-PAGE) Westernblot of cTnT from left ventricle tissue obtained from transplanted heart rejected by recipient (C) or biopsy obtained from patient who underwent CABG (D).

For example, truncation of the N-terminus (which can occur with ischemia [secondary site in Fig. 1]) will remove both PKA phosphorylation sites and a major cTnl-cTnC binding site, greatly reducing the affinity between the modified cTnI and cTnC. In this case, the circulating modified form of cTnl most likely will be bound to other positively charged proteins present in the serum at higher concentration than cTnC. As such, the actual composition ofthe cTnl complex in serum will be dictated in part by it posttranslational status in the tissue (degraded and/or phosphorylated) (Fig. 4). The same applies to cTnT.

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