The Caspase System: a Potential Role in Muscle Proteolysis and Meat Quality?

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1 CURRENT TOPICS IN MUSCLE GROWTH & DEVELOPMENT The Caspase System: a Potential Role in Muscle Proteolysis and Meat Quality? Tim Parr, Ron Bardsley, Peter Buttery & Caroline Kemp Introduction Meat quality is the generic term used to describe the properties and perceptions of meat which are generally considered to be colour, flavour, texture, tenderness and juiciness (Maltin et al., 2003), with tenderness considered as one of the most important traits (Miller et al., 200). Meat tenderisation occurs primarily from the weakening of the myofibrillar structure in muscle fibres via the actions of proteolytic enzymes (Sentandreu et al., 200). There are several proteolytic systems that could have a potential role in postmortem proteolysis and meat tenderisation, including the cathepsins, the multicatalytic protease (or proteasome) and calpains. The contribution of cathepsins to meat tenderisation is no longer considered to be a strong candidate as they are contained within the lysosomes and appear not to have access to the myofibrillar structure at critical stages of the post-mortem proteolysis period (Hopkins and Taylor, 2004). There is also little association between cathepsin activity and the variation in tenderness in meat (Whipple et al., 990). Although the proteasome is found at relatively high levels in skeletal muscle and is well established as having a role in muscle function, it is thought that it does not contribute to meat tenderisation as it is unable to cleave myofibrillar proteins to generate a similar pattern to that seen in situ during the conditioning period (Taylor et al., 995). Calpains are probably the most extensively studied protease family with regard to meat science and it is widely accepted that calpain-mediated proteolysis does play a major role in the process of meat tenderisation (Sentandreu et al., 200). Although there is considerable evidence to support the role of the calpain system in the postmortem conditioning period and the development of meat tenderness, it has been suggested that the calpain system cannot be solely responsible Tim Parr University of Nottingham Nutritional Sciences, Sutton Bonington Campus College Road, Loughborough, Leicestershire, LE2 5RD, UK. tim.parr@nottingham.ac.uk Proceedings of the 60 th American Meat Science Association Reciprocal Meat Conference (pp. 5-56) June 7-20, 2007, Brookings, South Dakota for all the biochemical and structural changes that take place, and therefore other proteolytic systems must be involved (Jiang, 998). Recently, several reviewers have reexamined the role of proteolytic systems in postmortem proteolysis and meat tenderisation and have proposed that the caspase family of proteases could be activated postmortem and influence the rate of tenderisation (Sentandreu et al., 200; Herrera-Mendez et al., 2006; Ouali et al., 2006). The Caspase Protease System Caspase Structure and Function Caspases are a family of intracellular cysteine aspartatespecific proteases (Alnermi et al., 996). To date 4 members of the caspase family have been identified and phylogenetic analysis indicates that there are two subfamilies, namely those involved in the inflammatory response and those involved in apoptosis (Earnshaw et al., 999). Apoptosis or programmed cell death is the organised dismantling of the cell, characterised by cell shrinking, DNA fragmentation, membrane blebbing and the formation of apoptotic bodies without inducing an inflammatory response (Wyllie et al., 980). This contrasts with necrosis which usually involves an inflammatory response. The caspases involved in apoptosis can be further subdivided into initiator caspases such as caspases 8, 9, 0 and 2 and effector caspases such as 3, 6 and 7, depending on their position in the cell death pathway (Earnshaw et al., 999). Initiator caspases are activated in response to pro-apoptotic signals and in turn activate the downstream effector caspases that are involved in the direct targeting and cleavage of specific proteins resulting in cell disassembly (Fuentes-Prior and Salvesen, 2003). Caspase Activation Pathways There are three main caspase activation pathways; the extrinsic pathway, the intrinsic pathway and the endoplasmic reticulum (ER)-mediated pathway (Figure ). The extrinsic pathway, also referred to as the death receptor pathway, is triggered by cell surface receptors. The initiator caspases, caspases 8 and 0, are activated via this type of receptormediated signalling (Boatright and Salvesen, 2003). The intrinsic pathway involves caspase 9 and is activated in response to environmental stress such as hypoxia and 60 th Annual Reciprocal Meat Conference 5

2 Fas FLIP Pro-caspase 8 FADD Pro-caspase 9 Bcl-2 Mitochon- dria Pro-caspase 2 Caspase 8 Apoptosome Cytochrome c Initiator caspases (8, 9, 0, 2) Caspase 2 Caspase 9 ER Pro-caspase 3 Caspase 3 IAP Smac/DIABLO Effector caspases (3, 6, 7) Cell Death Figure. Schematic diagram of the intrinsic, extrinsic and ER-mediated apoptosis pathways showing the caspases in- volved in each pathway. FADD Fas associated death domain, IAP- inhibitor of apoptosis, Smac - secondary mito- chondrial activator of caspases, DIABLO - direct IAP-binding protein with low pi (adapted from Holcik, 2002). ischemia, acting via mitochondrial damage and release of cytochrome C (Earnshaw et al., 999). The ER-mediated pathway is activated via stress directly upon the ER, such as 2+ a disruption in Ca homeostasis, which in turn activates initiator caspase 2. Caspase Regulation Inadvertent activation of caspases can have devastating effects and is therefore strictly regulated to protect from inappropriate caspase-directed proteolysis. There are a number of mechanisms that are involved in the regulation of caspase activation, including those acting via the Bcl-2 family, IAPs (Inhibitors of Apoptosis Proteins), ARC (Apoptosis Repressor with CARD (caspase recruitment domain)) or FLIP (FLICE (Fas associated death domain-like IL-β-converting enzyme)-inhibitory protein) (Earnshaw et al., 999). Interac- outlined in Figure tions between these inhibitory proteins and caspases are. Caspase Substrates Caspases function in a mainly executive role, switching off protective pathways and turning on downstream activities, which in turn lead to cellular destruction (Earnshaw et al., 999). To date more than 280 targets of caspasemediated proteolysis have been identified (Fischer et al., 2003). Proteins targeted and cleaved by caspases include cytoskeletal proteins, nuclear structural proteins, cytokines, membrane receptors, proteins involved in DNA synthesis and repair, RNA synthesis and also transcription factors (Schuh and Schulze-Osthoff, 998). Interactions between the Calpain and Caspase Systems There is increasing evidence for interactions between the calpain and caspase protease systems, predominantly focus- ing on the protein substrates that are targeted. Indeed, some authors have claimed that certain calpain isoforms and caspase family members exhibit virtually identical substrate specificity (Wang, 2000). Furthermore, Nakagawa and Yuan (2000) demonstrated that a disruption in Ca 2+ homeostasis as a result of ischemic injury, induced calpain-mediated activation of caspase 2 and that the anti-apoptotic protein Bcl-XL was cleaved by m-calpain transforming it into a proapoptotic protein. The endogenous calpain inhibitor calpastatin is also cleaved by caspases, 3 and 7, generating distinct degradation patterns (Wang et al., 998). Therefore if caspases are indeed active in the muscle postmortem they may influence meat quality either directly or by proteolysis of calpastatin. If the latter, this in turn could result in the activation of calpain which are proven to be involved in meat tenderisation and thus reduced toughness. The potential for interaction between the calpain and caspase systems was highlighted further in a study by Neumar et al. (2003), who demonstrated that calpain inhibition though over- 3 activity and expression of calpastatin promoted caspase apoptosis. Caspases and Skeletal Muscle Caspase expression has shown to be up-regulated in a number of skeletal muscle diseases and conditions including sarcopenia (Leeuwenburgh, 2003), Duchenne muscular dystrophy (Martini, 200) and hypoxia/ischemia (Adams et al., 200). Quite apart from their probable involvement in skeletal muscle atrophy, caspases have also been shown to play key roles in skeletal muscle development, with caspase expression being essential for normal muscle differentiation during myogenesis (Fernando et al., 2000). 52 American Meat Science Association

3 Table. Changes in caspase 3/7 and 9 activities and protein levels of poly (ADP-ribose) polymerase (PARP 89kDa) and alpha II spectrin breakdown degradation product 20 kda (SBDP20), the caspase-mediated proteolysis break- down products, over time in porcine longissimus dorsi. Time h n Caspase 3/7 activity fluorescence/ µg protein Caspase 9 activity luminescence/ µ g protein PARP 89 kda units / mg protein SBDP20 units / mg protein ND ND ND.08 s.e.d P value P<0.00 P<0.00 P<0.00 P<0.00 ND = not detectable arbitrary densitometry units Caspases and Postmortem Proteolysis In meat animals the process of exsanguination occurs during slaughter, depriving all cells and tissues of nutrients and oxygen (Herrera-Mendez et al., 2006). After death essentially normal metabolism continues in muscle for a short time, but inevitably muscle cells will soon start to die. It has been suggested that apoptosis rather than necrosis is the most likely process by which cell death occurs (Sentandreu et al., 2002). A very reasonable hypothesis is that the process of slaughter and subsequent exsanguination initiates apoptotic pathways which results in the activation of caspases, which could in turn contribute to early postmortem proteolysis and meat tenderisation. Muscle fibre type and the Caspase System The fibre type composition of different skeletal muscles in livestock is known to influence the biochemical events involved in tenderisation (Maltin et al., 2003). In our recent study, several components of the caspase system were analysed across longissimus dorsi (LD), trapezius (TZ), psoas (PS) and semitendinosus (ST) muscle samples in Large White pigs. This work has shown that caspases and the skeletal muscle-specific caspase inhibitor ARC can be detected in all these muscle types and that levels of expression and activity vary significantly; for example, there is 3 fold more caspase 2 in ST in comparison to LD muscle (Kemp et al., 2006a). This suggests that different muscles will vary in their capacity to carry out caspase-mediated proteolysis. However our research found no association between the pattern of caspase activity and the fibre type profile of particular muscle types based on myosin heavy chain analysis (Kemp et al., 2006a). Caspase Activity across the Postmortem Conditioning Period The study described above indicated that caspases were present in a range of muscle types and that caspase activity could be found at and around the point of slaughter, indi- cating that apoptotic processes are in place and potentially primed to make a contribution to postmortem proteolysis (Earnshaw et al., 999). In a subsequent preliminary study with ten animals, we examined the levels of caspase activity in porcine LD muscle during the meat conditioning period up to eight days post-mortem (Kemp et al., 2006b). The activities of effector caspases 3 and 7 (combined activity assay) and the initiator caspase 9 were found to decrease over the postmortem conditioning period (Table ). Caspases 3/7 and 9 activities were highest in the early stages of the postmortem conditioning period, with less than 6% of at death activity remaining after 92 h. Amongst the individual animals, the post-mortem changes in caspase activity were quite variable and the ratio of activities at 0 and 32 h was taken as an indication of the residual caspase activity in an individual animal during the early postmortem period. Thus a high value of the ratio could indicate that most of the caspase had been activated and used prior to autolytic removal during the previous 32h conditioning. There was a negative relationship between shear force and the 0:32 h ratio of caspase 9 (r = -0.68, P = 0.044) (Figure 2A) and caspase 3/7 activities (r = -0.62, P = 0.053) (Figure 2B). Caspase substrates alpha II spectrin and poly (ADP-ribose) polymerase (PARP) were analysed in muscle samples taken across the conditioning period and their specific caspase-mediated degradation products were detected by Western blot analysis (Table ). The caspase-specific cleavage products of PARP and alpha II spectrin are both known indicators of apoptosis and the changes observed in their protein levels corresponded with those detected in caspase activities. In addition there was also a negative relationship between shear force and the level of the caspase generated alpha II spectrin 20 kda degradation product (r = -0.75, P = 0.02) (Figure 2C). These preliminary findings indicate that changes in caspase activity and caspase-mediated cleavage do in fact take place in muscle during the conditioning pe- 60 th Annual Reciprocal Meat Conference 53

4 Shear Force kg Shear Force kg Shear Force kg Casp 9 0:32 h Activity Ratio Casp 3/7 0:32 h Activity Ratio SBDP20 express/ mg protein Figure 2. Relationships observed between porcine longissimus dorsi 8 day shear force and A) caspase 9 0:32 h activity ratio; B) caspase 3/7 0:32 h activity ratio, where the ratio of activities at 0 and 32 h being taken as an approximation of the change in caspase activity during the early postmortem period and C) protein levels of the specific caspase 3 spectrin 20 kda degradation product at 2 h post mortem, n = 0. riod and that this appeared to be associated with the development of more tender meat. Recombinant Caspase 3 Proteolysis of Myofibrillar Proteins To gain an indication whether caspases were capable of degrading myofibrillar proteins we carried out in vitro experiments examining the effect of co-incubation of recombinant caspase 3 on purified myofibrils. A full length human recombinant caspase 3 (kind gift from Henning R. Stennicke, Nova Nordisk, Bargværd, Denmark) was expressed in E.coli and purified to homogeneity. Myofibrils were extracted from porcine LD muscle according to the procedure of Goll et al. (974) and incubated with recombinant caspase 3 (rc3) in a buffer designed to simulate conditions found in muscle postmortem, according to Winger and Pope (98). The purified myofibrils were incubated with either increasing concentrations of rc3 or for different duration. The resulting digests were examined by SDSpolyacrylamide gel electrophoresis. Although there was a visible change in some bands, indicating degradation of myofibrillar proteins, the major proteins myosin heavy chain and actin appeared to be mainly intact. However there was a visible decrease in the band intensity of proteins identified as desmin and troponin I (based on apparent molecular weight) which correlated with increasing concentrations of rc3 (Figure 3A). In addition the appearance of bands at approximately 32, 28 and 8 kda was observed, which were presumed to be the result of caspase 3- mediated protein degradation. Using MALDI-TOF mass spectrometry these bands were identified as being derived from actin, troponin T and myosin light chain respectively. These degradation patterns were not observed in myofibrils incubated in the presence also of the caspase 3 specific inhibitor Ac-DEVD-CHO (Figure 3B). However coincubation with either the endogenous calpain inhibitor calpastatin or the Ca 2+ chelator EDTA in the presence of rc3 still showed an increase in band intensities at 32, 28 and 8 kda (Figure 3B), suggesting that conditions where calpain is inhibited still permitted recombinant caspase 3 activity. Myofibrils were subsequently incubated with rc3 at 4 C for up to 8 d to simulate the postmortem conditions in vitro. Incubation of isolated myofibrils with rc3 over this time period resulted in the visible degradation of a number of protein bands identified as desmin and troponin I, with troponin I disappearing completely after 8 d of incubation (Figure 3C). The 28 kda troponin T degradation product, which has been shown to positively associate with meat tenderness (Huff-Lonergan et al., 996), increased, as did the 8 kda myosin light chain degradation product. In comparison these degradation patterns were not observed in myofibrillar preparations incubated for 8 d at 4 C in the absence of rc3 (Figure 3C), indicating that rc3 was responsible for the degradation observed. These incubation studies have shown that rc3 is capable of causing proteolysis of key myofibrillar proteins under conditions that are similar to those found in the muscle in the postmortem conditioning period. 54 American Meat Science Association

Conclusion The ultimate tenderness of meat is to a large extent dependent on the degree of alteration and weakening of myofibrillar structures and has been largely attributed to endogenous

5 Conclusion The ultimate tenderness of meat is to a large extent dependent on the degree of alteration and weakening of myofibrillar structures and has been largely attributed to endogenous proteolytic enzymes (Sentandreu et al., 2002). Our research into the potential role of caspases in the postmortem conditioning period and meat tenderness has shown that caspases are expressed in a number of different muscle types and their activity can be detected across the postmortem conditioning period, on a similar time scale as µ- calpain. Additionally caspase-mediated myofibrillar degradation patterns detected in vitro are similar to those observed in situ. These preliminary investigations have indicated that the caspase proteases appear to fulfil some of the criteria that were stipulated by Koohmaraie (988) as a requirement for a protease system to influence meat quality. Firstly caspases are endogenous to skeletal muscle fibres and secondly it appears that they have the ability to degrade myofibrillar proteins. Our investigations are seeking to build on our observations to investigate whether caspases do contribute to the process of meat tenderisation in situ and whether variability within this system or in its response to stimuli at slaughter could be contributory to the factors that are known to influence meat quality. References Adams V; Gielen S; Hambrecht R & Schuler G. (200). Apoptosis in skeletal muscle. Front Biosci 6, D-D. Alnemri ES; Livingston DJ; Nicholson DW; Salvesen G; Thornberry NA,Wong WW & Yuan J. (996). Human ICE/CED-3 protease nomenclature. Cell 87, 7. Boatright KM & Salvesen GS. (2003). Mechanisms of caspase activation. Curr Opin Cell Biol 5, Earnshaw WC; Martins LM & Kaufmann SH. (999). Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68, Fernando P; Kelly JF; Balazsi K; Slack RS & Megeney LA. (2002). Caspase 3 activity is required for skeletal muscle differentiation. Proc Natl Acad Sci U S A 99, Fischer U; Janicke RU & Schulze-Osthoff K. (2003). Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ 0, Fuentes-Prior P & Salvesen GS. (2004). The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem J 384, Goll DE; Young RB & Stromer MH. (974). Separation of subcellular organelles by differential and density gradient centrifugation. In Proc 27th Ann Recip Meat Conf, pp Herrera-Mendez CH; Becila S; Boudjellal A & Ouali A. (2006). Meat ageing: Reconsideration of the current concept. Trends in Food Science & Technology 7, Holcik M. (2002). The IAP proteins. Trends Genet 8, 537. Hopkins DL & Taylor RG. (2004). Post-mortem muscle proteolysis and meat tenderisation. In Muscle Development of Livestock Animals, ed. te Pas M, Everts M & Haagsman H, pp CAB International, Cambridge, MA, USA. Figure 3. The effects of incubating porcine myofibrils with recombinant caspase 3 (rc3). A) The effect of increasing concentrations of rc3 at 37 o C for 24 h. B) The effect of co-incubation: lane 0 units rc3 + 5 mm EDTA, lane 2 no rc3 + 5 mm EDTA, lane 3 0 units rc µl semi-purified calpastatin, lane 4 0 units rc3 + Ac-DEVD-CHO (0. µg/µl). C) The effect of incubating myofibrils for 8 d at 4 o C with or without 0 units rc3. In both figures the major degradation products generated by caspase-mediated proteolysis are indicated by their molecular weights. Abbreviations: M, myosin heavy chain; α, α-actinin; D, desmin; A, actin; T, troponin-i. Huff-Lonergan E; Mitsuhashi T; Beekman DD; Parrish FC, Jr.; Olson DG & Robson RM. (996). Proteolysis of specific muscle structural proteins by mu-calpain at low ph and temperature is similar to degradation in postmortem bovine muscle. J Anim Sci 74, Jiang ST. (998). Contribution of muscle proteinases to meat tenderization. Proc Natl Sci Counc Repub China B 22, th Annual Reciprocal Meat Conference 55

6 Kemp CM; Parr T; Bardsley RG & Buttery PJ. (2006a). Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles. Meat Science 73, Kemp CM; Bardsley RG & Parr T. (2006b). Changes in caspase activity during the postmortem conditioning period and its relationship to shear force in porcine longissimus muscle. J Anim Sci 84, Koohmaraie M. (988). The role of endoproteases in meat tenderness. In Proceedings of the 4st Annual Reciprocal Meat Conference, pp Laramie, WY, USA. Leeuwenburgh C. (2003). Role of apoptosis in sarcopenia. J Gerontol A Biol Sci Med Sci 58, Maltin C, Balcerzak D, Tilley R & Delday M. (2003). Determinants of meat quality: tenderness. Proc Nutr Soc 62, Martini F. (200). Fundamentals of Anatomy and Physiology, vol. 5th Edition. Prentice Hall, NJ, USA. Miller MF; Carr MA; Ramsey CB; Crockett KL & Hoover LC. (200). Consumer thresholds for establishing the value of beef tenderness. J Anim Sci 79, Nakagawa T & Yuan J. (2000). Cross-talk between two cysteine protease families. Activation of caspase-2 by calpain in apoptosis. J Cell Biol 50, Neumar RW; Xu YA; Gada H; Guttmann RP & Siman R. (2003). Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis. J Biol Chem 278, Ouali A; Herrera-Mendez CH; Coulis G; Becila S; Boudjellal A; Aubry L & Sentandreu MA. (2006). Revisiting the conversion of muscle into meat and the underlying mechanisms. Meat Science 74, Sentandreu MA; Coulis G & Ouali A. (2002). Role of muscle endopeptidases and their inhibitors in meat tenderness. Trends in Food Science & Technology 3, Stroh C & Schulze-Osthoff K. (998). Death by a thousand cuts: an ever increasing list of caspase substrates. Cell Death Differ 5, Taylor RG; Tassy C; Briand M; Robert N; Briand Y & Ouali A. (995). Proteolytic Activity of Proteasome on Myofibrillar Structures. Molecular Biology Reports 2, Wang KKW; Posmantur R; Nadimpalli R; Nath R; Mohan P; Nixon RA; Talanian RV; Keegan M; Herzog L & Allen H. (998). Caspasemediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch Biochem Biophys 356, Wang KKW, (2000). Calpain and caspase: can you tell the difference? Trends in Neuroscience 23, Whipple G; Koohmaraie M; Dikeman ME; Crouse JD; Hunt MC & Klemm RD. (990). Evaluation of attributes that affect longissimus muscle tenderness in Bos taurus and Bos indicus cattle. J Anim Sci 68, Winger RJ & Pope CG. (98). Osmotic properties of post-rigor beef muscle. Meat Science 5, Wyllie AH; Kerr JF & Currie AR. (980). Cell death: the significance of apoptosis. Int Rev Cytol 68, American Meat Science Association

The Caspase System: a potential role in muscle proteolysis and meat quality? Tim Parr

The Caspase System: a potential role in muscle proteolysis and meat quality? Tim Parr Caroline Kemp, Ron Bardsley,, Peter Buttery Division of Nutritional Sciences, School of Biosciences, University of