Matrixmetalloproteinases and tissue inhibitors of metalloproteinases: Immunhistochemical markers in the diagnosis of lethal myocardial infarctions?
Introduction
Diagnosing a lethal myocardial infarction is a common task in forensic routine work, since such cases often occur sudden and unexpected. Because survival times of myocardial infarctions can be very short, macroscopically visible alterations of the myocardial tissue may not be present. Therefore, immunohistochemical examinations have been established in forensic medicine. An ideal marker for the detection of myocardial infarctions should appear early after the onset of ischemia and should be specific for this pathological event. In the past, evaluation of possible new markers has been difficult since they have mainly been tested on human myocardial tissue drawn during autopsies. In such cases, there typically is a lack of certain information, especially the exact survival time after the infarction or a possible reperfusion of the ischemic myocardium. Because of this, defining the precise chronological occurrence of new markers has been challenging. We have been able to adapt an animal model, the isolated Langendorff heart, so that it solves this problem and allows the investigation of possible new markers of myocardial infarctions especially with a view to the time of their appearance [15]. Furthermore, the method allows the generation of infarctions with and without reperfusion, as well as the preparation of different kinds of “control hearts”. By combining experiments with the Langendorff-model and the examination of human tissue samples, an extensive evaluation of possible markers for myocardial infarctions is possible.
In the present study we tested two matrix metalloproteinases (MMP), MMP-2 and MMP-9, as well as one tissue inhibitor of metalloproteinases (TIMP), TIMP-1, for their use as immunohistochemical markers of acute myocardial infarctions. MMPs and TIMPs can be found in many tissues and organs. MMPs are important for the degradation of peptides, mainly of the extracellular matrix (eM), and therefore take part in many physiological and pathophysiological processes. Their function in the heart has been subject of extensive research and is especially well known for MMP-2 [5], [12]. This enzyme is not only involved in the development of the heart and vessels, it is also activated under pathological conditions like infarctions or chronic heart failure. The activation can be triggered by various stimuli like oxidative stress, ischemia/reperfusion injury or inflammatory cytokines. As a consequence, MMP-2 activity can not only be found in the eM but also in the cardiomyocytes. Intracellular MMP-2 is responsible for the degradation of several proteins, amongst them troponin. In the eM, MMPs take part in developmental processes, as described above, and the remodelling due to injuries and diseases.
With a view to ischemia and infarctions not only MMP-2, but also other MMPs and TIMPs have been studied [3], [6], [7], [9], [10], [16], [18], [20], [21]. Degradation of collagen fibres starts only a short time after the onset of ischemia and is in a large part attributed to an increased activity of MMPs already present in muscle tissue, though animal models have also detected an increased expression of the enzymes during the post-ischemic time course. Herzog et al. detected an increased activity of MMP-2 approximately one hour after an infarction and an increased activity of MMP-9 approximately two hours after an infarction in a rat model with infarctions without reperfusion [9]. The alterations, however, were not as distinct as they were one day after the infarction. The study of Cleutjens et al. presents results of a rat model with myocardial infarctions without reperfusion that show increased amounts of TIMP-1 mRNA in infarcted myocardial tissue about six hours after the onset of ischemia [3]. Romanic et al. detected a markedly increased expression of the active form of MMP-9 and a slightly increased expression of the active form of MMP-2 within one day after infarction in a rabbit model with myocardial infarctions without reperfusion [18]. Furthermore, their study revealed a decreased expression of the active form of TIMP-1 within the first four days after an infarction.
In clinical studies it has been discussed that the concentration of MMPs and TIMPS in a patient’s serum might be usable as a prognostic marker after an infarction [13], [19]. The inhibition of MMPs, for example by the application of TIMPs or doxycycline, is also being suggested as a therapy after myocardial infarction, since it inhibits extensive cardiac remodelling which is known for increasing the risk of fatal (short or long term) complications [2], [8].
These experimental and clinical findings make MMPs and TIMPs also interesting for forensic use. Since no studies regarding the immunohistochemical detection MMPs/TIMPs after myocardial ischemia in post mortem drawn tissue have been published to date, our project aimed on answering the following questions:
- 1.
Can MMP-2, MMP-9 and TIMP-1 be detected immunohistochemically in myocardial tissue after early ischemia/infarction?
- 2.
If yes, how early do MMP-2, MMP-9 and TIMP-1 appear/increase in the detectable amount after the onset of ischemia.
- 3.
Does the detectable amount of MMP-2, MMP-9 and TIMP-1 depend on a reperfusion of the ischemic myocardium?
- 4.
Can alterations of the detectable amount of MMP-2, MMP-9 and TIMP-1 only be seen after myocardial infarctions or can they also be found under other pathophysiological conditions?
Section snippets
Material and methods
Animal experiments were performed in compliance with the German legislation on protection of animals, as well as the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The protocol for the Langendorff-system was approved by the local Animal Ethics Committee (project no. O 27/11).
The examination of human myocardium drawn during autopsies was approved by the ethical committee of the Medical Faculty of the
General staining pattern
Positive staining for MMP-2 was found mainly in the eM. Only few cases of the human study group showed increased amounts of MMP-2 in the cardiomyocytes. This fact was taken into account in the staging system. Positive staining for MMP-9 and TIMP-1 was found exclusively in the intracellular space.
Study hearts rat
As an unexpected secondary finding, the markers could also be detected in myocardium surrounding mechanical lesions of the tissue caused by the ligation and the ECG-electrodes. Although this was not our
Discussion
In clinical research, new markers have been discovered for the diagnosis or the prognosis of myocardial infarctions. Those markers are also interesting for forensic case work. In two previous studies we were able to demonstrate that dityrosine, a protein product of oxidative stress, can be used as a marker for lethal myocardial infarctions with only a short survival time [14], [15].
In the present study we combined experiments performed with the isolated Langendorff heart and examinations of
Summary
Our experiments with the Langendorff-model and post mortem drawn, human tissue samples showed that immunohistochemical staining of myocardial tissue after an infarction/ischemia indeed presents changes in the detectable amounts of MMP-2, MMP-9 and TIMP-1. However, in human cases these changes were mainly seen after a longer survival time, questioning the use of the markers for the detection of acutely lethal infarctions. They might rather be used as a second attempt when other, established
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declarations of interest
None.
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