Potential for different fragments for different pathophysiological processes

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"Cytosolic" vs "Structural" Pools

Evidence for Existence of Two Cellular Pools

Based on the bimodal release kinetics of cTnl, it is assumed that there are at least two pools oftroponin (26,27). They have been labeled cytosolic and structural, but the studies that have been conducted have usually made that distinction predicated on the ability to displace the troponin based on the buffer necessary rather than immunohistochemistry. Displacement with gentle buffers has been suggested to be associated with a "cytosolic pool" and those associated with harsher biochemical procedures with the so-called structural pool. Although conceptually difficult to envision either cTnl or cTnT (owing to their poor solubility and "stickiness") floating freely around in the cytosol, it is possible that low levels of troponin are present in the cytosol or loosely bound to the myofilament during their incorporation (or removal) from the thin filament. Little is known about the process involved in renewal and regeneration of the thin filament, which is fundamental to understand the origin of these two physically different pools.

Analyses performed during the development of the troponin assays suggest that for cTnl, roughly 3.7% of the total troponin found in the heart is in this cytosolic or perhaps better termed early releasable pool (26), and roughly 5% for cTnT (27). Although a relatively modest percentage of the total concentrations of these proteins, it is in fact a very large amount. In comparative terms, the early releasable pool of cTnl and cTnT is very similar to the amount of CK-MB found in myoctyes. It is this pool that is thought to cause the initial increases observed in patients with AMI. Subsequent release and the subsequent elevations of the cardiac troponins, which can persist for 10-14 d or even longer, are thought to be owing to the degradation of the structurally bound pool as the area of injury is remodeled. Initially, the area becomes infiltrated by inflammatory cells that utilize lysosomal enzymes to degrade the injured material, both cells and matrix and then monocytes and fibroblasts begin to repair the area with the generation of a collagen matrix, that eventually leads to fibrosis (28). Exciting data have suggested that in some circumstances new cardiomyocytes may be interdigitated with this process (29). These two pools are thought to be important in the kinetics of troponin release because of the reasonably prompt increase in the cardiac troponin in the clinical setting and the long persistence in the circulation after an acute insult. It is known from studies in which purified cardiac troponin has been injected in animals that the half-life of single chains and/or complexes in plasma is relatively short, in the range of 2-4 h (30). Thus, persistent elevations more likely reflect the continuing degradation, rather than delayed clearance. Even in patients with renal dysfunction, clearance curves do not appear to be altered (31).

Evidence for Release Limited to Cytosolic Pool

Cytosolic pools may be of some importance from the point ofview ofboth pathophysiology and distinguishing reversible from irreversible myocyte injury. It is thought that perhaps the cytosolic pool could be released with injurious stimuli that are not apt to cause irreversible cellular injury. The ability to show such release experimentally and confirm that the cells are injured but have not succumbed is problematic. A good experimental example comes from monitoring skeletal troponin I release from the respiratory muscles of rats breathing against an inspiratory resistive load for which careful detection of skeletal troponin I (slow and fast skeletal troponin I monitored independently) was related to the time course of changes in arterial blood gases, respiratory drive (phrenic activity), and pressure generation (Fig. 5) (32). Modified forms of fast skeletal troponin I were detected in serum midway through loading coinciding with muscle fatigue and long before hypercapnic ventilatory failure (when it is assumed that cellular necrosis occurs) (32).

For the heart, there are several clinical situations in which transient elevations in cardiac troponin are observed with very rapid clearance that would suggest that irreversible

Fig. 5. Release of fast skeletal troponin I (FsTnI) during inspiratory resistive loading. A Western blot of serum obtained from a rat undergoing inspiratory resistive loading shows increased detection of specifically FsTnI during muscle fatigue prior to organ failure. The various Western blots represent different time points in the events leading up to inspiratory resistive loading-induced pump failure with cellular necrosis most likely occurring. (For more details see ref. 32.)

Fig. 5. Release of fast skeletal troponin I (FsTnI) during inspiratory resistive loading. A Western blot of serum obtained from a rat undergoing inspiratory resistive loading shows increased detection of specifically FsTnI during muscle fatigue prior to organ failure. The various Western blots represent different time points in the events leading up to inspiratory resistive loading-induced pump failure with cellular necrosis most likely occurring. (For more details see ref. 32.)

injury is not present. The two most commonly cited are exercise (33,34) and pulmonary embolism (PE) (35,36). It is now clear that at the extremes of exercise, termed vital exhaustion, when animals are pushed to extreme physical activity, troponin degradation in myocardium and release into plasma occurs (37). In addition, relatively minor transient increases in troponin levels have been observed in humans after extreme exercise (33,34). What is particularly interesting is that the elevations are not persistent but quite transient, usually observed during the first day, and are usually very minor in magnitude. The question then has been raised, could such elevations represent transient release from the cyto-solic pool without implying that exercise causes irreversible myocyte injury? Such speculation would fit with other studies that have failed to show an adverse effect from the minor elevations and a bias, from many studies that exercise may even be good for cardiac structure and function. Perhaps a better example of this phenomenon is PE (35,36). PE causes an acute increase in pulmonary resistance and pulmonary pressures, which increases the wall stress in the right ventricle. If the right ventricle is hypertrophied, it can tolerate higher pulmonary resistances and generate higher pressures than if the ventricle has not previously been conditioned in some way that results in a hypertrophic response. However, in experimental models, it is difficult to cause irreversible damage to the right ventricle (38,39). This is, in large part, because it is generally thin and, therefore, can receive nutrients from the cavity, and it has a gradient for collateral blood flow because ofthe lower pressure in the right ventricle compared with the left ventricle and the right ventricle manifests a dual blood supply as well. Thus, the increases in troponin seen in acute PE that evolve rapidly and are gone by 40 h have been used to suggest that such transient elevations may be a reflection of reversible injury. If so, this suggests that a cytosolic pool that releases the troponin and perhaps the structural pool, which would only likely be degraded in response to cell death, may be preserved. Such speculation would fit with the experimental and clinical data that suggest it is difficult to injure irreversibly the right ventricle (38,39).

Potential Diagnostic Application

To the extent that the hypothesis that the release of troponin in the clinical and experimental examples just discussed is limited to the cytosolic pool is correct, this paradigm would imply that if one could characterize the fragments within the cytosolic pool and distinguish them from the fragments in the structural pool, one might begin to at least answer the question of reversible vs irreversible damage. For example, it does appear that the troponin within the cell is in the form offree chains and it is known that larger fragments, complexes, are released with severe ischemic insults (23-25).

It is conceivable but unproven that the nature of those fragments could depend on the nature ofthe injurious stimulus. As mentioned previously, modification oftroponin fragments is believed to occur in tissue. Thus, the heterogeneity of fragments demonstrated may be related in part to residence time in the myocardium (40,41). It is likely that the duration in the tissue is a function, at least in part, of blood flow, with larger cellular proteins being washed out more slowly than membrane-bound constituents (40,41).

potential of fragment analysis Clinical and Experimental Examples

The opportunity to use fragment analysis to determine reversible vs irreversible injury is but a small part of the potential of this exciting approach. If the conversion and release of these forms is not rapidly followed by degradation in the plasma by proteases that further process the fragments, it is conceivable that one could observe different fragments with different diseases (Fig. 4). There are several bases from which to anticipate that these differential patterns may exist. First, as articulated in detail in the preceding section, there is the notion of a "cytosolic pool" containing mostly free proteins (polypeptide chains) in contrast to the structural pool. Second, there is the additional clinical example of renal failure. It is well known that cTnT is more frequently elevated than cTnI (42) even when assays thought to have similar clinical sensitivities and other circumstances are used. The reasons for this discrepancy are unclear. However, investigators have raised the possibility that the fragments that occur in renal failure may be different (43). In keeping with the fact that the antibody recognition sites for troponin T are quite close together, fragments of cTnT have been documented to include very small ones (Fig. 4). By contrast, the antibody recognition sites for cTnI, though not terribly distant, are said to be much farther apart. It has been proposed that processing in patients with renal failure could eliminate some of the epitopes for the detection of cTnI and could even open additional epitopes for the detection of cTnT. Fragment analysis may permit exploration of this important issue that carries significant clinical implications. Third, experimental models suggest several additional settings in which specific modifications of troponin may occur. For example, Feng et al. (44) showed that acute increases in preload lead to proteolysis of cTnI and release most likely via a calpeptin-mediated mechanism. This mechanism might also be relevant both to a dialysis population in whom intravascular volumes go up and down frequently, and to a heart failure population in whom rapid changes in preload could lead to the release oftroponin. Feng et al.'s (44) data suggest that the cells eventually die as the result ofapoptosis. This finding raises the possibility that troponin fragments may be released in other situations in which apoptosis occurs (45).

N-terminal degradation of cTnl can occur both with ischemic-induced damage (46) and with simulated microgravity (47) and is thought to occur during normal myocyte turnover (48) (Fig. 2). However, the actual N-terminal degradation products appear to differ based on the physiological (residues 1-26, 1-27, or 1-30 being proteolyzed [47]) and pathological conditions (1-63 and 1-73 being proteolyzed) (46) (Fig. 4). Furthermore, in a low flow ischemic model in dogs, it was cTnT, not cTnl, that underwent degradation (49), indicating that monitoring the fragmentation (or absence of degradation) of both molecules could lead to additional clinical insight.

Clearly, understanding differences in the type of fragments that are derived may lead to improved diagnosis ofthe underlying pathological process, particularly in settings in which it is challenging to discriminate conditions on clinical grounds. For example, because the most common cause ofheart failure is ischemic heart disease, it is difficult to know whether or not troponin elevations in patients presenting with heart failure represent underlying coronary heart disease, supply/demand ischemia, or the effects of heart failure independent of either of those disease entities. Because increased concentrations of troponin are present in patients with idiopathic cardiomyopathy, it is clear that coronary artery disease is not necessary for the release oftroponin to occur (50). It is conceivable that the processing of fragments is different owing to variation in the injurious stimulus, and if so, identification of the proper fragment might lead to a better diagnosis. This approach will be highly sensitive to the location in which fragment processing occurs. If all processing occurs within myocardium and no processing occurs in plasma, the detection of specific fragment patterns will be far more simple. On the other hand, if serum proteases alter the fragments after release from myocytes, it may become much more complicated to determine which patterns exist owing to the underlying pathophysiology vs subsequent processing in the blood.

Improvement in Assays for Troponin

Finally, it may well be that the sensitivity of the present methods for the measurement of troponin may be improved by reliably detecting complexes when they are present, and being able to denature them so that more epitopes are available for detection with conventional antibodies. The antibodies used in commercial assays vary tremendously. Some are capable of detecting single chains extremely well, whereas others tend to detect complexes (51). These issues of antibody specificity have posed problems, with two assays as notable examples. An early cTnT assay had a lack of a totally specific capture antibody oftroponin T (52), which led to false positive results. Another assay had an antibody to the carboxy-terminal end where cleavage occurred in plasma, rendering the protein undetectable (53). Thus, one way to begin to move toward increased sensitivity, harmonization of testing, and obviation of interferences may be to denature the complexes.

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