In recent years the mitochondria have been recognized as regulators of cell death via both apoptosis and necrosis in addition for cell survival. Cellular dysfunctions induced by intra- or extracellular come together on the mitochondria and induce a sudden increase in permeability of the inner mitochondrial membrane, causing the opening of the mitochondrial permeability transition pore to open in the inner mitochondrial membrane with subsequent loss of ionic homeostasis, matrix swelling and outer membrane rapture. The detailed molecular mechanisms underlying the mPTP induced cellular dysfunction during cardio pathology such as in ischaemia or reperfusion injury remain to be explicated. However, a growing body of evidence does support the concept of pharmacological inhibition of the mPTP as an effective and promising strategy for the protection of the heart ischaemia and myocardial reperfusion injury. This review summarizes and discusses 1) the properties of the mPTP: structure and function, 2) the role of mPTP in ischaemia and myocardial reperfusion injury, and 3) the inhibition of mPTP opening as cardioprotection.
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National Heart, Lung, and Blood Institute, USA
Coronary Heart disease
Heart diseases are the main causes of in the UK, which accounts for about 35% of deaths each year (198, 000 people each year). Coronary heart disease (CHD) being the leading cause of death in the United Kingdom around one third in men and one 6 in women die from this disease every year. Coronary heart disease causes around 94, 000 deaths each year and most commonly in the age group of 65-74 year old individuals (1).
Fig. 1. A view of the heart with coronary heart disease, where the coronary artery is being blocked by fatty substances.
Fig. 2. A view of reperfusion treatments which are used in clinical environments. Trying to reduce the severity of damage to the heart muscle tissue during myocardial ischaemia.
CHD can be described as what happens when the blood supply to the heart is being blocked or narrowing of blood vessels by a build-up of fatty substances in the coronary arteries (Fig. 1), this is known as Atherosclerosis. If the arteries are completely blocked, consequently there is no blood supply to the heart, and can cause a Myocardial infarction, more commonly known as a heart attack. This can however be treated by reperfusion, which is
re-oxygenation of heart muscle tissue, trying to reduce the severity of damage to the muscle tissue, which can occur during myocardial ischaemia. Some types of reperfusion treatments (Fig. 2) which are used clinically are thrombolytic therapy, percutaneous coronary intervention (PCI), more commonly known as angioplasty, and Coronary bypass graft surgery.
Surprisingly, the process of myocardial reperfusion can itself induce cardiomyocyte death. This phenomenon is called myocardial reperfusion injury (2, 3).
Lethal Myocardial-Reperfusion Injury
Reperfusion is an absolute requirement for successfully decrease the infarct size of an ischemic heart; it both reduces mortality and morbidity. On the other hand, it has been proven that both reversible and irreversible signs of injury of the cardiac myocyte (6). Myocardial reperfusion injury indicates the injurious metabolic, functional, or structural consequences of restoring coronary blood flow that might be reduced, avoided, or reversed by modifying the conditions of reperfusion. Lethal myocardial reperfusion injury refers to the death of myocytes that were still alive at the beginning of reperfusion (2).
The main mediators which lead to lethal myocardial reperfusion injury are several sudden biochemical and metabolic changes which occur during the period of myocardial ischaemia to the ischemic myocardium during myocardial reperfusion. These changes involves mitochondrial reenergization, the production of reactive oxygen species (ROS), intracellular [Ca2+] overload, restoration of physiological pH, and inflammation of the blood vessel, all of these changes interact with each other to mediate cardiomyocyte death through the opening of the mitochondrial permeability transition pore (mPTP) and the stimulation of cardiomyocyte contracture. This opening of the mitochondrial permeability pore (mPTP) occurs by uncoupling oxidative phosphorylation and inducing mitochondrial swelling and rapture. Making the mPTP open is the most important mediator for myocardial reperfusion injury. This can be seen in the figure (Fig. 3) below, which shows the pathogenesis of lethal myocardial ischaemia reperfusion injury (4, 5).
Fig. 3. Here the pathogenesis of myocardial ischaemia reperfusion injury is described. With an emphasis on the mitochondrial permeability transition pore (mPTP).
The mPTP as a mediator of Ischaemia-Reperfusion Injury
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Crompton (et al.1987) (3) were the first to propose that mitochondrial permeability transition pore may play a role in cardiac reperfusion injury. It has been proven that mPTP opening occurs on reperfusion of the ischemic heart, and there are large indications on that mPTP is an important cause of ischaemia/reperfusion injury (4, 5). For the heart to recover from and myocardial infarct or reperfusion injury, the mitochondria has to return to full functionality, if this doesn't occur it would lead to necrotic cell death-which means the muscle cells are fully damaged and there is no way for it to return to its normal function. However, before this stage there are many factors which play a big role and that contribute to this injury which were described very briefly in the earlier segment. During ischaemia, cytosolic Ca2+ becomes elevated, while the mitochondrions are still polarized, Ca2+ is driven out of the matrix through the uniporter. Hence, when the mitochondria depolarizes, the accumulated matrix Ca2+ flows down the electrochemical gradient back (either through the uniporter, Na-Ca exchange or mPTP) into the cytoplasm, contributing to the myocytes to contract rigorously. This has been shown in a study by Hausenloy (et al. 2004) of an adult rat cardiomyocyte being loaded with tetramethylrhodamine methyl ester (TMRM) so that it can be shown in fluorescent imagining, this is shown in figure 4, below (7).
Hausenloy et al. 2004The mitochondrial permeability transition pore is non-selective channel in the inner mitochondrial membrane. Opening of the channel collapses the mitochondrial membrane potential, uncouples oxidative phosphorylation resulting in ATP depletion and cell death (6). During myocardial ischaemia the mitochondrial PT pore remains closed, only to be open during the first few minutes of reperfusion in response to overload of mitochondrial calcium (Ca2+) and phosphate, oxidative stress, ATP depletion and physiological pH correction. Therefore, the mitochondrial PTP is crucial in determining of myocardial lethal reperfusion injury (7, 8).
Fig. 4. An adult rat cardiomyocyte has been induced with oxidative stress over time. Image A shows the cardiomyocyte before oxidative stress. Image B the cardiomyocyte being induced with oxidative stress and the arrows are showing the mitochondrions. Image C we can see a "wave" of depolarization where the fluorescent has been intensified can we see the mitochondria PT pore opening. Image D the entire cardiomyocyte has undergone mitochondrial depolarization. Image E shows the collapse of mitochondrial membrane potential, ATP depletion and the cell undergoes rough contractions. This can describe what happens during ischaemia myocardial reperfusion injury.
Fig. 5. The model structure, the mechanism of the mPTP opening and impermeable state of mPTP is illustrated here in this image.
Halestrap et al. 2002Furthermore, by describing the structure of the mitochondrial PTP we might perceive a better understanding of its function. There have been many speculations on the molecular composition of the mPTP. However, they don't know the exact components of the mitochondrial permeability transition pore. All that is known is that it is made of protein channel in the inner membrane (IM) of the mitochondria, which was thought to be adenine nucleotide translocator (ANT) and the only known component is Cyclophilin-D (CyP-D) in the matrix. mPTPs are Ca2+, red-ox, voltage, and pH sensitive, such that the mPTP opening is increased by matrix [Ca2+], ROS (reactive oxygen species), membrane depolarization, and high pH. However, Ca2+ and ROS are the most important inducers of mPTP opening. Formation of disulfide bonds between critical thiol groups on the protein in the IM allowing CyP-D to bind and promote the opening of the mPTP. Other modifiers promoting mPTP opening including inorganic phosphate (Pi), which enhances matrix Ca2+ uptake. The most potent inhibitor of mPTP opening in isolated mitochondria is the cyclophilin binding protein Cyclosporin A (CsA), which attaches to the CyP-D, and in that way prevents interaction with other PTP components. Sangliferhrin A (SfA) is also a potent inhibitor, which binds to a different site on CyP-D.
Fig. 5. The model structure, the mechanism of the mPTP opening and impermeable state of mPTP is illustrated here in this image.
Halestrap et al. 2002
The mechanism and structure can be seen in the figure below (Fig. 5) (8, 9).
As said earlier, the mitochondrial permeability transition pore remains closed during ischaemia and only opens during reperfusion. This has been proven by some studies made by Halestrap et al. 2004 which is demonstrated using the "Hot-DOG" technique, measuring
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the mitochondrial entrapment of a radioactive marker, which has been loaded into the cytosol of the heart. In this technique Lagendorff-perfused heart are firstly loaded with [Â³H]2-deoxyglucose (Â³H-DOG), which builds up in the cytosol as Â³H-DOG-6-phosphate (Â³H-DOG-6-P) but it can only enter the mitochondria when the mitochondrial PT pore is open. The extent to which the Â³H-DOG-6-P enters the mitochondria can be determined in the presence of EGTA, which reseals the pores and so entraps the Â³H-DOG-6-P within the mitochondria. Measurement of the H content of the mitochondria gives a quantitative value for the extent of pore opening, provided that suitable controls and corrections are performed to account for variations in
Halestrap et al. 2004
Halestrap et al. 2004
Fig. 6. Shows how the Â³H-DOG-6-P in mitochondria is used as an indicator of pore opening. Corrections are made variations in cell loading with DOG and mitochondrial recovery.
Fig. 7. Time dependence of MPTP opening during reperfusion. The opening of the MPTP was detected using the DOG preloading technique. Hearts were subjected to 30 min of global ischaemia before reperfusion for
the time shown.the initial loading of the heart with 3H-DOG and the recovery of intact mitochondria. The latter is determined using citrate synthase and this measurement might also be a useful refinement to the published NAD+ technique, shown below in Fig. 6 (8, 9). As in Fig. 7 we can see using the H-DOG entrapment technique we have been able to confirm that the pores remain closed during the 30 minutes of ischaemia however, opens on reperfusion (reoxygenation) with the time course that reflects the pH to return to its standard values in the pre-ischemic phase. To investigate the contributions of pathological conditions, such as ischaemia/reperfusion to pore opening, the heart can be loaded with Â³H-DOG either before ischaemia (DOG-preloading) or during reperfusion after ischaemia (DOG-postloading). DOG-preloading allows the extent of pore opening that occurs during the initial phase of ischaemia/reperfusion but it does not take into account whether or not some of the pores close again. Hence, DOG-postloading only detects those mitochondria that retain open pores. As a result, by comparing these two results provides an estimate of those mitochondria whose pores have opened and closed again as reperfusion continues. Nevertheless, the Â³H-DOG technique is not without its own limitations, it cannot detect mitochondrial pore opening of cells that have undergone extensive necrosis due to the rapture of the plasma membrane (11). In these cells the mitochondrial inner membrane will also rapture and all of the Â³H-DOG entrapped in both the cytosol and mitochondrial will be lost during isolation procedure and will therefore not be detected. By this technique we can determine that the mitochondrial permeability transition pore is a critical determinant of lethal reperfusion injury, and such as it is an important new target for cardioprotection.
The mPTP as a target for cardioprotection
If mitochondrial PTP opening represents a critical step in ischaemia/ reperfusion injury or other cardio pathologies, the interventions that prevent mPTP opening should be beneficial.
It would be. There is increasing evidence that almost any procedure that reduces reperfusion injury is associated with either the decrease in mPTP opening, or an increase in subsequent pore closure. This might be viewed directly through either direct inhibition on the pore with agents such as CsA and SfA, or through an indirect inhibition associated with a decrease in factors responsible for mPTP opening, by modulating calcium overload, oxidative stress, ATP depletion or intracellular pH (12).
Targeting CyP-D with Cyclosporin A (CsA) and Sanglifehrin A (SfA) to inhibit the mPTP
Fig.8. A table of the size of the infarct when the heart has been perfused with SfA or CsA within the first 15 minutes of reperfusion comparing with one that was given SfA or CsA after (delayed) 15 min of reperfusion.Nazareth et al. (13) were the first to demonstrate protection with CsA using isolated cardiomyocyte of anoxia and reoxygenation, and this has later been confirmed by others (14, 15). Furthermore, it has been proven that CsA is a potent inhibitor by other procedures such as, Langendorff- perfused hearts subjected to global ischaemia/ reperfusion (16,17), as well as in animals subjected to reperfusion (18). However, there is a problem with the use of CsA, it is that it can induce additional effects through inhibition of the Ca2+-regulated protein phosphate, calcineurin. To overcome this, CsA analogues ([MeAla6]CsA, 4-metyl-val-CsA), a similar compound in structure however differs a bit in its chemical compound, which are without the effect on calcineurin can be used (16, 17, 19), and as can Sanglifehrin A (SfA)(20). However, CsA and SfA provides a fairly small inhibition of the primary mPTP opening during reperfuison after prolonged ischaemia. This is probably due to high concentrations of Ca2+ and ROS during the first few minutes of reperfusion (17, 19) since it is known that CsA does not inhibit the mPTP when there is a high calcium overload and oxidative stress (21). There have been recent study on the effect of cyclosporing on reperfusion injury in acute myocardial infarction by Piot et al. In this study they used cyclosporine A at the time of Percutaneous coronary intervention (PCI, or angioplasty) would limit the size of the inract during myocardial infarction. They had randomly choosen 58 patients diagnosed
Piot et al. 2008with acute ST-elevation myocardial infarction to reviece either an intravenous bolus ot CsA or normal saline (control group) immidetley before undergoing PCI suergery (22). The infarct size was then assed from all the pateints by measuring the release of creatine kiase and troponin I. The results proved that cyclosporine A did significantly reduce the infarct size compared to the control group which can
be seen in the figure 9.
Fig. 9. The assessment of Infarct Size by biomarker measurement.
Serum creatine kinase was measured frequently for 3 days after coronary reperfusion. Curves for the control and cyclosporine groups are shown in Panel A, Cyclosporine administration (Adm.) resulted in a significant reduction in infarct size approximately 40% as measured by creatine kinase release. The serum troponin I measurements were taken just as frequently for 3 days after the coronary reperfusion. The curves for both the control group and the cyclosporine group are shown in Panel B. CsA Adm. did not show any significant reduction in infarct size as measured by troponin I release. However, this was onlya very smallpilot trial, and this have to be confirmed by a larger clinical trial before this will be used clinically (22).
Inhibition of mPTP by reducing intracellular Ca2+ overload.
A potent inhibitor of mPTP opening is low pH, and a key inducer for mPTP opening is overload of calcium, subsequently we can say that my maintaining a low pH and decrease the Ca2+ overload will provide protection again mPTP opening, cardioprotection. Several groups have demonstrated that maintaining acidic extracellular pH during reoxygenation after a period of anoxia can protect muscle cells from total damage (23). In contrast, low physiological pH during the ischemic phase is very harmful for the myocytes, probably because Na+ /H+ exchange is stimulated, loading the heart with Na+ and CaÂ²+, since the Na+/H+ exchange allows the Ca to flow into to the matrix leading to mPTP opening. However, inhibitors of the Na+/H+ exchanger such as amiloride and cariporide are meant to protect the hearts from reperfusion injury by reducing the accumulation on the Na+ and CaÂ²+ influx during the ischemic phase (24). As mentioned earlier keeping a low physiological pH to the normal physiological levels would, either these two of these would inhibit mPTP opening.
Inhibiting mPTP opening with free radical scavengers including pyruvate and propofol.
Reducing oxidative stress through the use of radical scavengers is known to offer some protection against reperfusion injury (25, 26). However, there may be indirect effects on the mPTP opening in view of the fact that oxidative stress is known to inhibit plasma membrane ion pumps , leading to calcium overload (). Consequently protection by free radical scavengers could affect the opening of mPTP through both indirect and direct effects. There have been studies on effects of two free radical scavengers on mPTP opening, pyruvate (27) and the anaesthetic propofol (28), in both cases it has been proven that they have a cardio protective effect which are associated with reduced opening of the mPTP.
Pyruvate's ability to protect the heart against ischaemia / reperfusion injury, or anoxia /reoxygenation injury has been known for a few years (29, 30). Furthermore, pyruvate stimulates the heart during reperfusion because, unlikely to glycogen or glucose, it does not require ATP for activation before it metabolises. Halestrap et al. has confirmed that when the heart has been induced with 10 mM pyruvate before ischaemia and been maintained during reperfusion it has significantly improved the recovery of the heart, and that it is associated with a great reduction of mPTP opening during the initial time of the reperfusion phase. In addition to this, it has also shown that when pyruvate has been loaded during reperfusion, almost all the "open" mPTPs close (29). Therefore it was proven that once the mPTP are open they can close up again and let the heart recover fully during the reperfusion phase. Furthermore, these studies even showed that pyruvate has an effect on other metabolic changes which occurs during ischaemia/ reperfusion, by causing a larger build up of intracellular lactic acid during ischaemia and slow the return of the intracellular physiological pH to its normal values, inhibiting the opening of mPTP (29). By this we can clearly see that pyruvate can be seen as yet another mechanism which can protect the heart against reperfusion injury.
Another free radical scavenger might be the anaesthetic propofol, which is used clinically, during cardiac surgery and in postoperative sedation (28, 31). It may also inhibit plasma membrane calcium channel (28). However, when the concentrations are a bit higher than when used clinically, propofol may inhibit mPTP opening, it has been demonstrated that propofol protects Langendorff- perfused hearts against reperfusion injury and damage to the heart caused by oxidative stress (32, 33)..
This has been demonstrated by Halestrap et al. by using the Â³H-DOG- technique to confirm that this kind of protection does inhibit the mPTP opening (28). However, studies have not shown any significant effect of propofol on inhibition of mPTP opening. When propofol is added to the isolated heart mitochondria, at the same concentration at which it is induced with is clinical anaesthesia, there were no observations of the inhibition of mPTP opening. However, studies have not shown any significant effect of propofol on inhibition of mPTP opening (28), which may suggest that the protective effect of propofol may be questionable. They even demonstrated that there is a cardio protective effect of propofol on the functional recovery of the working heart of a rat by doing a cold cardioplegic ischemic arrest, which may be seen as much closer view of a situation experienced in open heart surgery (28).
Both pyruvate and propofol provide examples of reagents as protection of the heart from reperfusion injury associated with the reduction of mPTP opening in vivo. Furthermore, these studies by Halestrap et al. and others have shown that these reagents may be useful as cardio protection, but more likely to be solutions used in cardio surgery.
Protection by ischemic Preconditioning does involve inhibition of mPTP opening.
One of the most effective procedures for cardio protection is ischemic preconditioning (IPC), when the heart is subjected to concise (3- 5 min) ischemic periods with intervening recovery periods before a prolonged period of ischaemia is initiated. However, the proper mechanism of the protection by IPC is still under discussion, several mediators such as receptor-dependent and receptor-independent triggers, and intracellular mediators have been suggested that they play a crucial role in IPC(34- 36). In view of the fact that mPTP opening is a critical factor in reperfusion injury, it may perhaps be expected that IPC leads to inhibition of PTP opening, and has now been proven by publication around 4000 studies about IPC. The effects of ischemic- preconditioning (37) are first correlated to the inhibition of mPTP opening in isolated mitochondria and myocyte models. The results from these studies were later confirmed by direct measurements of mPTP opening in isolated hearts subjected to Ischemic preconditioning with subsequent global ischaemia or reperfusion (11). The H-DOG technique demonstrated as well that IPC did reduce the PTP opening during reperfusion and thereby reduces the chances for the pores opening.
Fig.10. Mitochondrial PTP opening measured by [3H]-2-deoxyglucose (DOG)-preloading method in isolated and Langendorff-perfused rat hearts. Hearts were perfused with or without 0.2 Î¼M cyclosporine A (CsA) or sanglifehrin A (SfA),or subjected to ischemic preconditioning (IPC) by two cycles of 5 min global ischemia interspersed with 5 min reperfusion
prior to 30 min global ischemia and 30 min reperfusion.
Javadov et al. 2007What is interesting here is that, under these conditions ischemic preconditioning has a greater cardio protective effect compared to the direct inhibition of Cyclosporin A or Sanglifehrin A, which were mentioned earlier, this can be seen in figure10.
Modified from Hausenloy et al. 2004
Fig. 11. A Diagram showing the by inhibiting mPTP opening with either CsA or SfA during preconditioning phase abolished protection associated with IPC. There have been further demonstrations on this by Hausenloy et al. where they investigated if there would be any significant difference when IPC is used with CsA or SfA when its treated, showed a shocking result of that it did not give any kind of cardio protective function whatsoever, instead when used together it abolished any kind of protection that IPC would provide to the heart figure 11 (below).
As a conclusion, we can state that by opening the mPTP changes the mitochondrion from just being an organelle that provides ATP to maintain heart function into a device of "destruction", inducing cell death. The swelling that occurs when the pore opens can lead to outer membrane rupture, leading to cell death, either apoptosis or necrosis. Mitochondrial PTP opening has been demonstrated under pathological conditions including cardiac ischemia/ reperfusion when mitochondria are overloaded with Ca2+ in parallel with ROS generation and ATP depletion. Existing data support the central hypothesis that PTP plays a crucial role in determining life or death. There has been many studies which have been proven to have cardio protective effect by inhibiting mPTP opening. These studies involve pharmacological interventions which inhibits the mPTP opening, by making the pores remain closed this is used either directly (CsA and SfA) or indirectly (propofol, pyruvate, and preconditioning methods), to provide protection of the tissue during reperfusion injury or ischaemia. However, this has clear implications for improving the outcome of open heart surgery or thrombosis. In cardiac surgery pharmacological agents can be administrated, it actually has already been in use(see Ref. 22), prior to ischaemia and reperfusion. Similarly, following coronary thrombosis the blocked arteries must be cleared with clot busting enzymes or PCI (angioplasty) and the period of prospect for a successful outcome might be prolonged by the prior studies or suitable inhibitors of the mPTP. In view of the growing body of evidence of PTP opening as an endpoint of cell damage, clarifying the precise mechanisms of pore opening and its consequences will be necessary for further development of specific inhibitors to be used as therapeutic tools in the clinical setting.