The Caspase System and Muscle Degradation Tim Parr [1]Our last speaker of the day is Dr. Tim Parr. Dr. Parr graduated in 1987 from the University of Southampton with a degree in physiology, biochemistry, and nutrition, and he tells me these are all clinically associated degrees. I m laughing here because I m reading his handwriting. Okay. We ll let him. Well, forget all that since I ve been working in caspase for here s your award. Oh thank you very much. Get my award out. Right, to introduce myself, I m from the UK, I flew in last night, so I m still suffering from jet lag. I am going to talk about a scenario that we ve been recently working on, but for over 15 years, I ve been working on calpains with a group which contained Peter Buttery and Ron Bardsley. Peter Buttery has been working on protein turnover, protein nutrition for oh a very long time. The person at the front of the list obviously, I m at the top left because I m speaking the person at the front of the list is Caroline Kemp, whose work I m going to describe today and who s also in the audience and will have a poster tomorrow, hopefully, if we can find it because it s somewhere between here and Chicago, and she ll be standing next to it and will be able to give you a lot more details about her work if you can t find me. So what I m going to talk about today is the caspase system. I m going to [2]first tell you where I m from, so this is the Sutton Bonington Campus sorry, the picture hasn t come out very well but this was the UK, the red arrow is Nottingham. This is Errol Flynn in tights, Robin Hood. Nottingham is where Robin Hood comes from. We don t walk around in tights in Nottingham, so don t go to Nottingham under that impression, or else you ll be in trouble. [3]So what I m going to talk about, I m going to give an introduction to caspases. Forgive me, some of you in the audience are already familiar with the area, but I think because it s a relatively new area in the field of meat quality, I think it s worth going through what caspases are and what they do and then go on to some of the roles in muscle function. And then I m going to show you some very, I d say preliminary data, Caroline would disagree with me, but it is preliminary data, really novel data that we ve had a look at caspase system in pigs and look at the activity associated with postmortem development in tenderness, and then I m going to go for a conclusion at the end. [4]So this doesn t come out very well, but I ll go through it. Cell death. Cell death can be by necrotic death or it can be by apoptosis. There are two distinct pathways. Necrotic death is extremely disruptive. Essentially, you end up with a cell that swells, you end up with some proteolysis, but ultimately, you end up with rapid disruption and a cell that lyses and essentially in vivo in a response to all the contents which have leached out. Apoptosis, on the other hand, is a ordered process. It s associated with a process which ends up with a compartmentalization of the contents of the cell into little packets which can be dealt with by the normal physiological mechanisms inside the body. So with this process, you end up with no inflammation. This is an ordered process affecting the DNA, the DNA get fragmented, and the cell structure gets chronically orientated. Apoptosis is the process. Programmed cell death is a specifically ordered process which occurs during distinct stages of development. Apoptosis occurs all the time. Essentially, for this process to occur, we have proteolytic enzymes and we have the caspases. [5]So the caspases are a large group of proteolytic enzymes. There have been 14 identified so far. They are proenzymes. They have to be activated to become fully active within a cell, and the pro caspase consists of a single peptide chain which contains various domains within it. It contains a leader domain and these two subunits here which form the functional proteolytic enzyme, and during activation, stimuli, which I ll come to in a minute, this gets activated, it forms a tetramer of two individual units which exposes an active site, which can then carry out proteolysis. The pro domain is usually lost, but it s not necessarily lost, so you can have different molecular weight forms which exist inside the cell in the active state. Page 1
[6]They can be subdivided into different groups, so you ve got initiator caspases, which are involved in activating the process of apoptosis, the executioner caspases, and then the cytokine dependent pathways. I m going to principally focus on these today in association with muscle protein turnover. Then associated with these particular proteolytic enzymes are specific inhibitors with various names with incredibly long definitions to these names, so I m not going to go into them, but essentially they re defined as inhibitors of apoptosis or IAPs. Then you ve got regulators and activators. Some of these activators have not been fully characterized, but the best characterized ones are of the Bcl 2 family, which can be divided into Bcl like proteins which are antiapoptotic and Bax like proteins which are proapoptotic and then associated proteins which were involved in the activation of caspases. So it s a complex system of hierarchy, a checks and balances system which is under stringent control in terms of activation. [7]So to go through some of the principles of activation, and there are several pathways involved. So caspase activation cascades, so this is the plasma membrane, we have a death receptor on the membrane, a death ligand and there are various death ligands associated with this process, which is associated with initiated caspases, so in this receptor pathway, its procaspase 8 becomes activated to caspase 8, then activates pro caspase 3. Caspase 3 in the activated state is the actual proteolytic enzymes which are involved in the process of degrading selective substrates within a cell. So this is called the extrinsic pathway and it s ligand/receptor mediated, predominantly. [8]Then this is my attempt to mitochondria. Caroline says it looks like a bit of small intestine, but I think it looks like a very good piece of mitochondria. So this is a mitochondria, so a mitochondria. My artistic skills aren t very good, but I think it does look like a piece of mitochondria. This process is the intrinsic pathway, and it essentially starts at the level of the mitochondria. Associated with the mitochondria, you have these proteins: Bax and Bac, Bcl xl and Bcl 2. Bcl 2, to remind you, is antiapoptotic; Bax proteins are proapoptotic. Under certain stimuli, usually associated with particular types of stress, these proteins are activated by quite a complex process, but I haven t got the time to go through it in here, but essentially these actors pause which allow for the release of cytochrome c from mitochondria. This stimulates the activation of procaspase 9 through the apoptosome, which is a complex of several proteins which actually requires ATP and cytochrome c in order to activate caspase 9. This goes on to activate caspase 3 or caspase 7 with the execution of caspases. Caspase 3 activated is involved in cell death. This is the intrinsic pathway or mitochondrial mediated pathway. [9]And then we have the ER. My ER was as good as my mitochondria, but you can get the general impression. This is the ER. This process in terms of involving the ER in a caspase activation cascade involves the activation of caspase 12. This is now, I think, relatively conclusively involves milli calpain and it s associated with the release of calcium from the ER. Procaspase 12 is activated in association with milli calpain to give you caspase 12, which again can activate these executioner type caspases, which give you cell death. [10]Associated with these processes are the inhibitors. These inhibitors tend to act at this point down here. On the points where these individual caspases have been activated, they re modifying the response. [11]In terms of muscle cells, muscle cells, obviously as you all know, are multinucleated cells. They re distinctly different from these mononuclei cells like liver and kidney where apoptosis is a regular occurrence. The fact that you re dealing with multinuclei in a cell probably gives apoptosis a distinct role within muscle cells, and this is under a matter of debate, but if you look at the literature, the type of apoptosis that takes place in these multinuclei cells is probably going to be slightly different from these mononuclei cells in terms of the DNA degradation component, selective DNA of selective nuclei. So in mature skeletal muscle, there is not strong evidence for a death receptor mediated response. There are, in fact, high levels of Bax and Bcl 2, which suggest a mitochondrial mediated pathway may be involved, and there s also the potential involvement of ER mediated Page 2
responses of the calcium involvement either through caspase 12 and milli calpain, but calcium mediated effects also happen through mitochondrial mediated pathways. [12]So caspases in muscle involvement, their upregulation is associated with conditions of atrophy, dystrophies, burn injury, and there s considerable literature suggesting they re associated with sarcopenia, the wasting of muscle in old age. During muscle cell differentiation and culture processes which inhibit caspase 3, you tend to get reduced myotube formation and associated muscle specific proteins. Those are particularly the transcription factors associated with muscle differentiation. There s also evidence, particularly from a group in Israel, that there may be an interaction between caspases and calpastatin associated with this differentiation event. [13]So the caspase substrates are, like calpain substrates, specific. In fact, they have a slightly higher degree of specificity. In fact, they cut at aspartic acid residues. They also cut at glutamic acid residues so the specificity is becoming slightly less stringent, but in essence, you get an ordered degradation, a specific targeting of specific proteins, and this is predominantly by these executioner caspases, caspase 3, caspase 6, and caspase 7, so a specific range of substrates cleaved and many of these substrates are also cleaved by calpains. [14]So sorry, this probably doesn t come out very well for you to read at the back, but essentially at the top here, we have caspase 3 and calpain and a list of substrates which were listed by Kevin Wang in 2000 which are cleaved by both of these proteolytic enzymes. To go down the list, you ve got cytoskeletal proteins like spectrin, you ve also got things like actin on the list, you ve also got focal adhesion kinase, calpastatin, protein kinase C, and also some of the proteins, Bcl 2 and Bax, which are involved in the control of apoptosis itself. [15]Caspases in recent times have been strongly associated with ischemic damage, so in brain ischemia, we have dysregulation of normal apoptosis, so we have activation of apoptotic like processes. This is usually associated with calcium overloaded, we ve got activation of caspases, cleavage of key substrates, and one of the predominant things you see is spectrin cleavage. Usually, this ischemic insult is not only associated with brain, it s associated with other tissues, but it s accompanied by changes in calpain activity. The two things usually go hand in hand, potentially associated with ischemic events. [16]So this has led to a hypothesis which is derived from the fact that in living organisms, a lack of nutrients and oxygen leads to cell death and apoptosis, and this is associated with ischemic induced death. So a colleague in the meat science field, Ahmed Ouali who works at INRA in France, came forward with a model that at slaughter, animals were essentially bled, the cells were derived of nutrients and oxygen, and in his terminology, he contends that muscles have no alternative but to engage towards suicide cell death, apoptosis, caspase activation, postmortem proteolysis. There is no hard evidence from this group that this process takes place. [17]So at the time when Caroline started her Ph.D., we had an hypothesis that we set about trying to investigate, that conditions postmortem lead to caspase activation in skeletal muscle, and these proteases contribute to early postmortem proteolysis, extending the ischemic model. Ischemia is similar but not the same as death at slaughter. Ischemia is associated usually with a reperfusion event, and that is usually associated with activation of a lot of apoptotic events, so that our objectives have been to look at whether fundamentally, are caspases expressed in skeletal muscle, a very fundamental work. Are caspases active postmortem, and do caspases cleave myofibril proteins? Are they having an effect on the structure which is associated with tenderness? [18]So in the first study that Caroline carried out, we did a very basic study. Really, we tookfour muscle groups and we determined the levels of myosin heavy chain as an indication of muscle type, so we took trapezius, psoas, longissimus dorsi, and semitendinosus, and we really wanted to investigate whether there was a relationship Page 3
between muscle type and caspase expression, just a very simple experiment. But the experiment was really there to look at the use of these anti caspase antibodies because there were a lot of caspase antibodies available on the market. They are predominantly for human and rats, we were using them in pigs, and also whether we can actually detect any caspase activity in the muscles concerned. [19]So this is a bit busy, but the general impression is this is slow myosin, two muscle types, trapezius, psoas. These are the faster types, LD, semitendinosus, and then different caspase isoforms determined by Western blotting, so caspase 3, the two distinct isoforms, pro and active, different isoforms detected by different antibodies. You can see from that just looking at that globally, there is no association with a particular muscle type, but we could detect these different caspase isoforms. [20]Also, probably more importantly, we could detect caspase activity across these muscle groups, so trapezius, psoas, LD, semitendinosus, and in the LD muscle, when we used a caspase specific inhibitor, we got complete inhibition of caspase 3/7 activity. Assay that we used detects caspase 3 and 7, not just caspase 3. [21]So the conclusions from this, very basic conclusions, there was caspase activity and protein could be detected using commercially available antibodies and activity assays. Caspase activity and the level of proteins was found to differ between muscles, but there wasn t any relationship to slow myosin heavy chain, and the caspase system protein levels and activity weren t associated with a specific muscle type. [22]So our next experiment was really looking at whether these caspases were active postmortem. If they were, we weren t going to go any further. So in porcine longissimus muscle, we basically sampled at various time points. We took these time points based on the studies associated with calpains to see if there was activation early in this process because that s where we would expect this activation initiation event to take place. We looked at samples and analyzed them for caspase activities, caspase 3 and caspase 7 and caspase 9, and in situ, we tried to assess caspase substrates to see if there was any actual caspase activity in situ using these specific substrates. So this is poly ADP ribose polymerase and alpha spectrin. These are substrates which are used in terms of their proteolytic cleavage to give an indication of the apoptosis is taking place, and also we looked at shear force associated with these LD samples. [23]So this is caspase 3/7 activity. We can t distinguish between 7 or 3 so it s grouped together, and we ve got this decline in activity over a eight day period, and the general effect was that the later time points were significantly different from [24]the earlier time points, and this was mirrored by caspase 9 activities, so these assays are either florescence based assays or luminance based assays, again a decline, most rapid decline early in postmortem declining over the eight day period, so that we could detect activity early in the postmortem period after death. [25]With this shear force, and lo and behold, with our samples, we managed to get quite a big range, so we correlated these, so this is shear force against the change in caspase activity ratios. So this is shear force at eight days and there was a significant correlation in the very small group that we looked at. I must emphasize, very preliminary data in terms of this relationship, but there was a suggestion that there was a relationship between both caspase 9 and caspase 3/7 activity. This indication is a ratio between zero hour and 32 hours, giving an indication of the change in caspase activity because these assays, caspase 9 assay and caspase 3/7 assay, activate everything which is there, so you were measuring the total caspase activity. [26]Sorry, this doesn t come out very well. The blot on the bottom hasn t come out from this projector very well, so I ll probably have to describe it to you, but we looked at power. This is poly ADP ribose polymerase. This is one of the main Page 4
caspase specific substrates found in vivo and is used virtually by most of the people who are interested in apoptosis in human beings as an indicator that apoptosis is taking place. So this antibody detects a specific substrate, which is generated by caspase mediated cleavage of 116 kilodalton power. So during caspase mediated cleavage, you get a specific 89 kilodalton product which is detected by a 89 kilodalton specific antibody, and we were able to detect early in this postmortem period the appearance, though this doesn t come out very well but you can get the general idea, that we could detect this 89 kilodalton product being produced during the postmortem period which is produced by caspase mediated activity. [27]The other substrate that we looked at in terms of in taking samples and looking at it in situ was the production of a product which is generated from spectrin, so alpha spectrin can be cleaved by calpain and it can be cleaved by caspase. During calpain cleavage, you generate 150 kilodalton and 145 kilodalton product. During caspase cleavage, you end up with 150 kilodalton product and 120 kilodalton product, and this was defined by Kevin Wang in 2000. [28]There are caspase specific spectrin product antibodies which would detect these ends and using these again this has not come out very well we could detect a 120 kilodalton product, but one of the main observations of this was that the 150 kilodalton product predominated, suggesting there is a lot of calpain cleavage over caspase cleavage. But when we looked at the 120 kilodalton product at two hours you can t visibly see it on here because it doesn t come out very well, and I apologize for that at two hours, it was correlated to shear force. Again, preliminary data. It just so happened we had a spread in shear force associated with these animals. [29]So the predominant observation was this, was we could actually detect caspase activity postmortem and its early stages of postmortem where we can detect it, where it s most active. We can detect caspase activity in situ. This is supported by the presence of protein fragments that are specifically generated by caspase mediated proteolysis, and these are markers of apoptosis in vivo, so there is some caspase mediated activity there, we can detect it using the assay, and we have found some associations between early caspase activity and shear force at eight days. [30]What we tried to answer then was whether caspase 3 is actually capable of degrading myofibrillar proteins, the proteins that you see being degraded during postmortem conditioning phases. So what we did is we expressed human caspase 3, recombinant caspase 3 and this was expressed in E. coli, affinity purified and its activity assessed using inhibitors and the standard assay. So we were able to determine the activity of this recombinant caspase, we isolated myofibrils from porcine LD and incubated with recombinant caspase 3, and we analyzed the proteins by SDS PAGE and Western blotting and MALDI TOF mass spec. And I m just holding my breath to see if you can actually see anything on the next slide. [31]Yes, you can, just. So this is different units of recombinant caspase incubated for 37 degrees for 24 hours. Really crude experiment just to see if we can get caspase mediated cleavage off myofibril proteins. [32]What we found is a decrease in this band, which we tentatively identified as desmin and a decrease in this band, which we tentatively identified as troponin I. [33]Using MALDI TOF, we isolated bands from similar gels and it was identified by MALDI TOF mass spec. as desmin and troponin I in these regions, so as we increased the amount of caspase 3, we get a disappearance of this band and disappearance of this band down here. [34]We ve got other bands that increase intensity with increasing amounts of recombinant caspase 3, predominantly associated with 32 kilodaltons, 28 kilodaltons, and down at the 18 kilodalton regions. [35]And we identified those bands as actin, troponin T, and myosin light chain. We seem to remember here this is predominantly the actin band, there is actually small Page 5
amounts of actin being generated, but the troponin T band, we ve got a distinct increase in the band here. So there is cleavage of myofibril proteins seen in myofibrils incubated with increasing units of caspase 3. [36]What we did next was to see if there was an effect of co incubation with either calpastatin or EDTA, so we have recombinant caspase with EDTA. We see a very similar band cleavage pattern as we saw on the last slide. Recombinant caspase 3 with calpastatin again we get a similar band cleavage pattern as we saw in the last slide and if we have recombinant caspase 3 inhibitor, we don t see these bands, predominantly these bands here being produced, so we can get degradation inhibition using caspase inhibited. It s not inhibited by EDTA or calpastatin, things that you would expect to inhibit calpains. [37]And at four degrees, if we incubate myofibril proteins with four degrees with 10 units of recombinant caspase 3 at ph 5.8, conditions which are similar to what are found in muscles during the postmortem conditioning phase, we again get the cleavage of these bands, desmin and predominantly the appearance of this band down here at 38 kilodaltons and one at 32 kilodaltons. Again, the figure hasn t come out very well, but you can see the 28 kilodalton band over this eight day period at four degrees. [38]So in conclusion, recombinant caspase is capable of degrading myofibril proteins. It s not massive, but it probably may contribute. It was active and caused proteolysis at four degrees at ph 5.8. This is the buffer which is a specific buffer which keeps its ph constant, which are conditions similar to postmortem muscle, and incubations for up to eight days showed an increased degradation of various identified proteins, desmin, troponin I and troponin T, proteins which we know are cleaved postmortem. [39]So overall conclusions, caspases can be detected in skeletal muscle postmortem. Different muscles exhibit different levels of caspases and also levels of inhibitors, and the interesting thing is that a lot of these inhibitors were first identified in skeletal muscle, so skeletal muscle contains rather a lot of these apoptotic inhibitors. Caspase activity changes during the postmortem conditioning period over the early period of postmortem. Caspase activity is present in situ postmortem, and human recombinant caspase 3 degrades specific myofibril proteins. That is, porcine myofibril proteins. [40]There is evidence that caspases potentially are involved in postmortem degradation and some of our data just support that idea. There is increasing evidence that there is a calpain and caspase interruption, particularly during ischemic conditions. And before I came to this conference, I just did a quick search of the Web of Science using calpain and caspase as keywords, and you can see there s a large increase in the number of papers which have this calpain caspase association, out of a total of 147 calpain hits for 2006. Whether it s through an interaction with calpains or it s caspases themselves having a direct effect on meat quality (i.e., myofibril degradation) remains to be resolved, but very strong interest within the clinical field in terms of this interaction, the interaction between calpains and caspases. It is associated with an ischemic event. This is where most of these publications are coming from. Caspases do cleave substrates which are associated with the cytoskeleton, substrates which are potentially involved in the development of tenderness, suggesting that they may interact with caspases in this process. [41]But for meat quality and having worked on calpains for 12, 15 years, the evidence to date indicates that the calpains system has a significant role in meat quality. It s a major factor in generating meat tenderness, but as with Ahmed Ouali, that I tend to agree that there are potentially other systems likely contributing to this process. They may be involved directly in this process through myofibril degradation, or they may be involved in an associated process in terms of partly controlling calpain activity, i.e., acting upstream. [42]And with that, I ll take any questions. Page 6
[42]Questions and answers. Page 7