Minocycline Treatment Upon Reperfusion Inhibits Myocardial Ischemia/Reperfusion Induced

Minocycline treatment upon reperfusion inhibits myocardial ischemia/reperfusion induced apoptosis

H.J. de Haas

R.H.J.A. Slart, MD, PhD Department of Nuclear Medicine and Molecular Imaging

R.A. Tio, MD, PhD Department of Cardiology

H.H. Boersma, PharmD, PHD, Hospital Pharmacy

Cardiovascular Imaging Group Groningen

January 11th – may 31st 2010

Abstract

OBJECTIVES

This study aims to determine whether minocycline reduces myocardial Ischemia and reperfusion injury (IRI)-induced apoptosis when administered upon reperfusion, using in vivo 99mTc-Annexin A5 imaging.

BACKGROUND

Although reperfusion is central in the treatment of Acute Myocardial Infarction, reperfusion also causes damage to the heart by triggering apoptosis. Minocycline (Mc) is an antibiotic with anti-inflammatory and antiapoptotic properties. It is used clinically for treatment of acne and urethritis. In animal models of cerebral Ischemia and reperfusion (I/R) and in patients with ischemic stroke, Mc treatment upon reperfusion reduces apoptosis directly through inhibition of apoptotic signaling pathways and indirectly through inhibition of inflammation. In myocardial I/R, minocycline treatment before ischemia reduces apoptosis, infarct size and improves functional recovery through comparable mechanisms. It is not known whether minocycline treatment upon reperfusion reduces myocardial IRI.

METHODS

Mice were exposed to myocardial ischemia reperfusion. Mc was administered to a subgroup of animals upon reperfusion (n=7), another subgroup (n=6) remained untreated. As a control, another group of mice underwent a sham procedure (n=5). After the procedure, the radiotracers99mTc-Annexin A5 and201Tl were administered. Subsequently, in vivo dual-tracer99mTc-Annexin A5 and201Tl SPECT imaging was performed to measure myocardial apoptosis. After imaging, the mice were sacrificed and hearts were harvested. Then myocardial apoptosis was evaluated in vitro by deoxyuridine triphosphatenick-end labeling (TUNEL) staining.

RESULTS

SPECT imaging showed a 40% lower 99mTc-Annexin A5 uptake in Mc-treated mice vs non-treated mice (1.31±0.32%ID/g vs. 2.15±0.52%ID/g). Moreover, Annexin uptake in Mc treated mice was not significantly higher than in sham-operated mice (0.92 ± 0.35 %ID/g). Qualitative analysis of TUNEL staining confirmed these findings.

CONCLUSIONS

We demonstrate that Mc treatment upon reperfusion reduces myocardial IRI-induced apoptosis. Therefore, Mc is a promising therapeutic agent for the treatment of myocardial IRI in reperfused AMI.

Table of contents

Introduction 4

Pathology IRI: ischemic phase 4

Pathology IRI: Reperfusion phase 5

Methods of detecting Programmed Cell death 6

Minocyline (Mc) as an anti-IRI therapeutical 7

Goal of this study 8

Materials and methods 9

Materials 9

Labeling protocol of 99mTc-Annexin A5 9

Study design 9

IRI procedure 10

In vivo 99mTc-Annexin A5SPECT imaging 11

Reconstruction of SPECT images 11

Analysis of 99mTc Annexin A5spect images. 11

Immunohistologic analysis of apoptosis 12

Statistical analysis 13

Results 14

99mTc Annexin A5 imaging 14

Immunohistochemical analysis of apoptosis 14

Immunohistochemical analysis of apoptosis 15

Discussion 16

Advantages and Limitations 16

Recommendations for further research. 16

Conclusion 20

Appendix A 21

Introduction

Acute myocardial infarction (AMI) is one of the leading causes of mortality and morbidity worldwide(1). Even optimal therapy can not prevent the occurrence of acute death in 10% of AMI patients and the development of heart failure in yet another 25%(1).

AMI is caused by the acute and complete occlusion of one or more of the coronary arteries, which supply the heart muscle with blood. The occlusion causes ischemia (cessation of the blood flow) to the tissue distal to it, which within 30 minutes causes cell death and thereby loss of tissue. This is called irreversible ischemic injury, which is one of the most important causes of morbidity and mortality(1;2). Clearly, the goal of all treatments for AMI is to reduce the amount of irreversible ischemic damage, reduce the mortality and improve (heart functional outcomes). To accomplish this, it is essential to restore the perfusion to the ischemic area (reperfusion) as soon as possible(3). In the past decades, great improvements have been made in the development of so-called reperfusion therapies. The first step was the invention of pharmaceutical thrombolytic therapies, which in a significant number of patients successfully dissolved the occlusion and restored perfusion. In the 1970’s, reperfusion therapy really took off with the invention and subsequent optimization of the percutaneous coronary intervention (PCI). With this technique, a balloon-bearing catheter is navigated through the femoral artery through the abdominal aorta to the occlusion in the coronary artery. Then, the catheter is moved into the occlusion, which subsequently is removed by inflation of the balloon. After this, a small wire mesh tube called a stent is placed at the original position of the occlusion to reduce the chances of restenosis(4)In the 40 years since the invention of the technique, refinements such as the development of stents, which prevent reocclusion have resulted in high success rates. Nowadays, successful reperfusion is accomplished in 75% of cases(3). However, the death and morbidity rates remain high, which indicates that there are still some hurdles to take before the treatment of AMI can be claimed to be optimal. A great opportunity for improving the results of the intervention lies in the logistical aspects. A large study showed that in the US, the median time between arrival in the hospital with signs of AMI and start of PCI is 180 minutes. Also it was found that in only 4.9% of patients the intervention started within 90 minutes, which is recommended by the guidelines(3). However, even successful PCI within the recommended timeframe cannot completely prevent damage to occur. Obviously it is not possible to start PCI before 30 minutes after onset of ischemia, and therefore it is not possible to prevent all ischemic injury. However, there is a second reason which is more counterintuitive and offers a target for additional therapies. Although the net effects of reperfusion therapy are highly beneficial, reperfusion itself also causes damage to the previously ischemic myocardium. This process is called ischemia reperfusion injury (IRI) (1;2;5). IRI is a complex group of effects, which together result in cell death of potentially viable cardiomyocytes. Results of animal studies indicate that IRI is responsible for up to 50% of the infarct size (1). Therefore large scientific efforts have gone into developing anti-IRI. Clearly, the main goal of anti-IRI therapies is to reduce programmed cell death, and thereby save functional tissue and improve the outcomes. To develop anti-IRI therapies, knowledge of the pathology of IRI and programmed cell death is necessary.

Pathology IRI: ischemic phase

The ischemic period damages the cardiomyocytes severely. This predisposes to the development of IRI, and if reperfusion is not accomplished on time, eventually leads to necrosis(2). The pathology of ischemic injury has been excellently reviewed elsewhere (2;6;7). Here, the ischemic changes are briefly summarized.
Within seconds after the onset of ischemia, the oxygen stores of the cardiomyocytes (heart muscle cells) and the surrounding capillaries are exhausted. This causes a metabolic shift from oxidative phosphoryllation to anaerobic glycolysis (AG) as the main source of the cellular energy molecule ATP. However, the ATP yield of AG is much lower than that of its oxidative counterpart. Therefore, most energy-consuming processes slow down significantly or cease to occur. The most important of these are the contractions of the cardiomyocytes and the activity of electrolyte transporters. This directly and indirectly results in severe alterations of the cellular molecular contents. The amount of K+ and Mg+ decrease, while accumulation of radical oxygen species (ROS), metabolic breakdown products such as lactate and ions such as Ca2+, Na+, Cl-, and H+ occurs. On balance, the alterations cause an increase in the osmotic pressure, which causes cellular swelling. Also, some of these molecules are directly toxic. The most important of these are Ca2+ and ROS. Ca2+ activates lipases, which together with the ROS damage cellular membrane and cause more influx of water and subsequent cellular swelling. Furthermore, Ca2+ activates proteases which damage organelles and the cytoskeleton and thereby make the cells more vulnerable to swelling. In addition, Ca2+ and ROS damage the mitochondria and thereby predisposes the cells to IRI.
Many of the aforementioned alterations amplify themselves or others. Collectively, they eventually result in massive cellular swelling, until disruption of the cellular membrane occurs, and the cell dies. This causes spillage of the toxic contents of the cells. These damage other cells and prime the tissue for an inflammatory response upon reperfusion. This uncontrolled mechanism of cell death is called necrosis.

Only timely reperfusion can reverse this process and prevent necrosis. Reperfusion saves the cells by causing washout of the accumulated harmful molecules and replenishing cellular stores of oxygen and nutrients. This results in repair of cellular damage and restoration of physiologic cellular metabolism and function.

Pathology IRI: Reperfusion phase

Besides its profound beneficial effects, reperfusion also causes damage (IRI) to the heart, by causing cell death to a subset of severely damaged, but potentially viable cardiomyocytes in the ischemic area(1;2). Evidence is mounting that IRI as opposed to non-reperfused ischemia, predominantly leads to apoptosis(8;9), the most well known form of programmed cell death (PCD). Apoptosis is an energy dependent process which is characterized by organized degradation of organelles, fragmentation of DNA and cellular shrinkage(9). Apoptotic cells, as opposed to necrotic cells are removed from the tissue by phagocytes, before they spill their contents(9;10). Apoptotic cells are recognized by phagocytes, because they present the phospholipid phospatidylserine (PS) on their membranes (10). Although apoptosis is a less destructive form of cell death than necrosis, apoptotic loss of cardiomyocytes causes loss of heart function(9). Therefore it is an important target for anti-IRI therapies and cardioprotective therapies in general (9;11-15).

The mechanisms by which reperfusion triggers apoptosis in these cells, is complex and still under debate, but it has been determined that ROS and CA2+ and neutrophils are the key players(2;16;17). The oxygen which is transported to the cells causes recommencement of ATP production by the mitochondria which are still functioning(18). However, when oxygen is reintroduced to damaged mitochondria, this leads to a burst of ROS. Another burst in ROS is caused by the invasion of neutrophils to the tissue, which were attracted by proinflammatory factors released during ischemia and reperfusion(17;19). The ROS cause damage to the membrane, which leads to influx of even more Ca2+ to the cells(2). Then the ROS, together with the Ca2+ cause the mitochondria to release several pro-apoptotic proteins, such as cytochrome C and Smac/DIABLO (9;20). These proteins activate a cascade of proteolytic enzymes called caspases, whih in the presence of ATP execute the organized degradation of the cell and the DNA (9;21). Also, the presentation of PS on the membranes of apoptotic cells is downstream effect of the caspases(22).

All in all, the inflammatory response and the apoptotic cascade seem as promising targets for the reduction of reperfusion injury (11;23-25).

Methods of detecting Programmed Cell death

Due to the important role of apoptosis in not only IRI but also in many other physiologic and pathologic processes, such as embryonic development, response to therapy in oncologic tumors, and transplant rejection, large efforts have gone into developing techniques to measure the amount of apoptosis, both in vitro and in vivo. Appropriate targets for these methods are I) DNA breaks and II) PS expression, which both are hall marks of apoptotic cells. Two widely used methods for apoptosis detection which are based on aforementioned hallmarks of apoptosis include:

I) TUNEL staining: Double stranded DNA breaks are the target for the golden standard of in vitro apoptosis detection: the TUNEL staining(26). This staining can be performed on cell as well as on tissue sections. Kits for this staining are commercially available from various manufacturers. Roughly, the active components of these kits are are terminal deoxynucleotidyl transferase (TDT), and dUTP. TdT is an enzyme that recognizes the DNA breaks, and subsequently catalyzes the binding of dUTP’s to the broken strands. The dUTP’s are labeled (usually fluorescently) so that apoptotic cells, whose nuclei contain dUTP’s, can be detected by microscopy.

II) Annexin A5: PS expression on the cellular membrane, the second hallmark of apoptosis is also receiving more and more interest. This interest was boosted by the discovery that the human protein Annexin A5 binds to PS with high affinity and specificity. Annexin A5 is a member of the Annexin A5family, a group of 160 human proteins which share a Ca2+ dependent affinity for phopholipids. In 1994, the first Annexin A5based in vitro apoptosis assay was developed(27). Since then, Annexin A5has been labeled with many labeling molecules to allow for apoptosis detection in cell cultures and tissue sections. Also, Annexin A5can be used as a probe (a target-binding) molecule for nuclear imaging. For nuclear imaging, probes are labeled with radioisotopes, which emit gamma-radiation. This radiation can be detected by nuclear imaging camera’s single photon emission computed tomography (SPECT) and positron emission tomography (PET) depending on the isotope. Computers then are used to transform (reconstruct) the camera data to 3D images. In these images it is shown where in the body the radiolabeled probe accumulated. The tracer accumulates on the place where the target molecule is most strongly expressed. In the case of Annexin A5 this is PS on apoptotic cells.
To allow for nuclear imaging, Annexin A5 has been labeled with a variety of radioisotopes using various labeling protocols (see appendix A). The SPECT radioisotope 99mTc has been shown to be by far the most promising one(28). The most feasible way to bind Annexin A5 to 99mTc is by conjugating Annexing with 6-Hydrazinopyridine-3-carboxylic acid (HYNIC), to which 99mTc can be bound in the nuclear imaging laboratory in a few relatively easy steps(28). In vivo 99mTc-Annexin A5SPECT imaging has been successfully used to show apoptosis in a large collection of animal models, namely experimental models of cardiac allograft rejection , tumor response to cytostatic therapy, of myocardial ischemia/reperfusion (I/R)were used(20;28;29).
Also, feasibility of apoptosis imaging and measuring with 99mTc-Annexin A5it has been shown in patients with vulnerable atherosclerotic plaques, various oncologic tumours, and reperfused myocardial infarction. In the studies where this was examined, the Annexin A5uptake strongly corresponded to the amount of apoptosis as shown by immunohistochemistry(20;28).

Minocyline (Mc) as an anti-IRI therapeutical

Since the discovery of IRI, many studies focused on developing anti IRI therapies. Although animal studies have shown cardioprotective effects of a wide variation of therapeutics such as a cocktail of glucose-insulin-potassium (30), EPO(31), antioxidants (32;33) and others, the results of clinical studies regarding these methods have yielded negative or inconclusive results (1;34). This indicates the need for developing novel anti-IRI therapies(1;34).