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

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1 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 Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK

2 Sutton Bonington Campus University of Nottingham

3 Introduction Introduction Caspases Role in muscle function Experimental data in pigs Caspase activity postmortem Conclusion

4 Cell death Necrosis vs apoptosis Apoptosis Modified from [Van Cruchten, 2002]. Ordered process affecting nuclei and cell structure Proteolytic enzymes caspases

5 Caspase family Caspases Cys proteases 14 different caspases pro caspase pro ~p20 ~p10 pro ~p20 ~p10 Activated caspase ~p20 ~p10 ~p10 tetramer ~p20 Shi, 2002.

6 Caspase family: a functional hierarchy Initiator Caspases 2, 8, 9, 10, 12 Executioner Caspases 3, 6, 7 Cytokine dependent pathways Caspases 1, 4, 5, 11, 13, 14 Inhibitors, Inhibitor of APoptosis (IAP) XIAP, FLIP, ARC Regulators and Activators Bcl 2, anti apoptotic; Bax pro apoptotic Apoptotic protease activating factor 1 (Apaf1)

7 Caspase Activation Casade Death ligand Death receptor Pro caspase 8 Extrinsic pathway Ligand/receptor mediated Caspase 8 Pro caspase 3 Cell death Caspase 3

8 Caspase Activation Casade Death ligand Death receptor Bcl X L Bcl 2 Pro caspase 8 CytoC Bax Bak Pro caspase 9 Apoptosome (Apaf 1) Caspase 8 Pro caspase 3 Caspase9 Intrinsic pathway mitochondriamediated Cell death Caspase 3

9 Caspase Activation Casade Death ligand Death receptor Bcl X L Bcl 2 Pro caspase 8 CytoC Bax Bak Pro caspase 9 Apoptosome (Apaf 1) ER Caspase9 Caspase 8 Pro caspase 3 Pro caspase 12 Ca 2+ Caspase 12 m calpain Cell death Caspase 3

10 Caspase Activation Casade Death ligand Death receptor Bcl X L Bcl 2 Pro caspase 8 CytoC Bax Bak Pro caspase 9 IAP Caspase9 Apoptosome (Apaf 1) ER Caspase 8 Pro caspase 3 Pro caspase 12 Ca 2+ IAP Caspase 12 m calpain Cell death Caspase 3

11 Muscle function Distinction between apoptosis in Mononuclei cells..liver, kidney etc Muscle multinuclei cells In mature skeletal muscle Not strong evidence for the Death receptor mediated response High levels of Bax and Bcl 2 suggests mitochondrial mediated pathway may be involved Also potential involvement of ER mediated response Ca 2+ involved caspase 12, m calpain

12 Caspases in muscle function Up regulation of caspases in muscle atrophy conditions Muscular dystrophies Congenital myopathy Burn injury Sarcopenia Muscle cell differentiation in cell culture Caspase 3 inhibition or homologous deletion leads to reduced myotube formation and muscle specific proteins

13 Caspase substrates Specific cleavage at specific positions Cut at Aspartic acid residues Ordered degradation Predominantly by Caspases 3, 6 or 7 Specific range of substrates cleaved Many of these substrates are also cleaved by calpains

14 Caspase 3 and calpain substrates Wang 2000

15 Ischaemic caspase activation Brain ischaemia Dysregulation of normal apoptosis Associated with Ca 2+ overload Activation of caspases Cleavage of key substrates Spectrin cleavage is a typical outcome Other ischaemic insults are associated with caspase activation Accompanied with changes in calpain activity

16 Role of caspase in meat development? In living organisms lack of nutrients and O 2 leads to cell death (apoptosis) Association with ischaemic induced death A proposed model by Ahmed Ouali et al (2002, 2006) At slaughter Animals are bled Cells are deprived of nutrients and O 2 Muscles engage towards suicide Caspase activation Postmortem proteolysis

17 Role of caspase in meat development? Hypothesis: Conditions postmortem lead to caspase activation in skeletal muscle and these proteases contribute to early postmortem proteolysis As proteolysis impacts on tenderness caspases are potential factors involved in generating tenderness variability. Objectives Are caspases expressed in skeletal muscle? Are caspases active postmortem? Do caspases cleave myofibril proteins?

18 Caspase Isoforms in Porcine Skeletal Muscles Determine levels of Myosin Heavy Chain (MHC) levels as an indication of muscle type in pigs (n=4) Trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) Investigate whether there was a relationship between muscle type and caspase expression Use of anti caspase Ab (across species) Caspase activity analysis

19 Caspase system variation across muscles 1.2 TZ PS LD ST relative level of protein a a b b a a a a a b b c a b b b a a b c a a b a 0 slow MHC caspase 3 32kDa caspase 3 20kDa caspase 8 18kDa caspase 12 50kDa ARC Within an isoform values with different superscript are significantly different p<0.01

20 Variation in Caspase 3/7 activity between muscles 2500 Fluor /mg wet wt tissue b b c d 0 a LD + DEVD CHO TZ PS LD ST Values with different subscripts are significantly different, P<0.01

21 Conclusions Caspase activity and protein could be detected using commercially available Abs and activity assay Caspase activity and level of protein was found to differ between different muscles No clear relationship between smhc and caspase system protein levels and activity

22 Postmortem caspase activity Porcine longissimus dorsi (LD) Large White gilts 81.2±1.98kg (n=10) Sampling at 0, 2, 4, 8, 16, 32 and 192 h after slaughter Samples analysed for caspase activities Caspase 3/7 and caspase 9 In situ assessment of caspase substrates Poly (ADP ribose) polymerase Alpha spectrin LD chops assessed for shear force

23 Postmortem Caspase 3/7 Activity Fluor/ug protein Time (h) 32 and 192 h activity significantly lower than all other time points, P<0.001

24 Postmortem Caspase 9 Activity Lum /ug protein Time (h) 0, 2 and 4 h activity significantly higher than at 8, 32 and 192 h, P<0.001

25 Shear Force v Caspase Activity 7 6 Shear force range 3.07 to 5.99kg Caspase 9 Caspase 3/7 Shear Force kg :32 h Caspase Activity Ratio Relationship between SF and change in caspase 9 activity, r = 0.68, P=0.044 change in caspase 3/7 activity r = 0.62, P=0.053

26 Caspase activity in situ Poly (ADP ribose) polymerase (PARP); a main caspase specific substrates found in vivo PARP DNA bind Cat + Caspase DNA bind 24kDa Cat 89kDa specific Ab 116kDa 89kDa 89kDa cleavage product detected postmortem hour 89kDa

27 Caspase activity in situ αii spectrin (214kDa) Calpain cleavage 150kDa 145kDa Caspase cleavage 150kDa (SBDP150) Caspase specific cleavage fragment 120kDa (SBDP120) Wang 2000

28 Caspase activity in situ αii spectrin (214kDa) Caspase specific SBDP120 and non specific SBDP150 detected PM Calpain cleavage 150kDa SBDP150 SBDP kDa Caspase cleavage SBDP120 protein levels peak at 2 h PM Caspase specific cleavage fragment 150kDa (SBDP150) 120kDa (SBDP120) Wang 2000 Shear Force (kg) r = 0.75, P< hr SBDP120 (abs units) /mg protein

29 Conclusions Caspase activity was detected postmortem In the early stages of the postmortem conditioning period caspases are most active Caspase activity in situ is supported by the presence of protein fragments that are specifically generated by caspase mediated proteolysis Markers for apoptosis in vivo Associations found between caspase activity early postmortem and shear force (8d).

30 Caspase activity and myofibril proteins Is effector caspase 3 capable of degrading myofibrillar proteins? Recombinant human caspase 3 (rc3) was expressed in E.coli, affinity purified and activity assessed. One unit of rc3 activity was defined as the amount of enzyme that cleaved 2.94 mm substrate/min/µg protein Myofibrils were isolated from porcine LD muscle and incubated with rc3 Proteins were analysed by SDS PAGE, Western blotting and MALDI TOF mass spec.

31 Effect of rc3 Concentration Units rc3 Myosin 105kDa ( actinin) Incubated at 37 o C for 24 hours 55kDa (Desmin) Actin 32 kda 28 kda 22kDa (Troponin I) 18 kda 17 kda 11 kda

32 Effect of rc3 Concentration Decreasing band intensity Units rc3 Myosin 105kDa ( actinin) 55kDa (Desmin) Actin 32 kda 28 kda 22kDa (Troponin I) 18 kda 17 kda 11 kda

33 Effect of rc3 Concentration MALDI TOF MS identified Units rc3 Myosin 105kDa ( actinin) Desmin 55kDa (Desmin) Actin 32 kda 28 kda Troponin I 22kDa (Troponin I) 18 kda 17 kda 11 kda

34 Effect of rc3 Concentration Increasing band intensity Units rc3 Myosin 105kDa ( actinin) 55kDa (Desmin) Actin 32 kda 28 kda 22kDa (Troponin I) 18 kda 17 kda 11 kda

Effect of rc3 Concentration on Myofibril Proteins MALDI MS identified 0 1 2 5 10 20 Units rc3 Myosin 105kDa ( actinin)

35 Effect of rc3 Concentration on Myofibril Proteins MALDI MS identified Units rc3 Myosin 105kDa ( actinin) Desmin 55kDa (Desmin) Actin Actin Troponin T Troponin I Myosin LC 32 kda 28 kda 22kDa (Troponin I) 18 kda 17 kda 11 kda

Effect of Calpastatin and EDTA on rc3 rc3 + + + EDTA + + Calpastatin + Caspase 3 inhib + Incubation at 37 o C for 24 hours with rc3 (10 units)

36 Effect of Calpastatin and EDTA on rc3 rc EDTA + + Calpastatin + Caspase 3 inhib + Incubation at 37 o C for 24 hours with rc3 (10 units) Degradation inhibited by Caspase inhibitors Not inhibited by EDTA calpastatin Myosin actinin Desmin Actin 32 kda 28 kda Troponin I 18 kda 17 kda 11 kda

Effect of rc3 on Myofibril Proteins at 4 o C ve 0 1 2 5 8 day M D A W blot for Desmin 0 1 2 5 8 day 55 kda 32

37 Effect of rc3 on Myofibril Proteins at 4 o C ve day M D A W blot for Desmin day 55 kda 32 kda 28 kda T 18 kda 17 kda 11 kda 10 units rc3 Incubation at 4 o C at ph 5.8 Over 8 day period degradation observed

38 Conclusions rc3 is capable of degrading myofibril proteins rc3 was active and caused proteolysis at 4 C and ph 5.8 conditions similar to postmortem muscle Incubating for up to 8 d showed increased degradation of various identified proteins desmin, troponin I, troponin T.

39 Overall Conclusions Caspases can be detected in skeletal muscle postmortem Different muscle exhibit different levels of caspases and also an inhibitor Caspase activity changes during the postmortem conditioning period Caspase activity is present in situ postmortem Caspase 3 degrades specific myofibril proteins.

40 Caspase/Calpain interactions There is increasing evidence that there is a calpain and caspase interaction particularly in ischaemic conditions publication frequency Year Keyword: calpain and caspase Web of Science Calpain 2006 = 447 Whether it is through an interaction with calpains or it is caspases themselves having an effect on meat quality remains to be resolved.

41 Meat quality The evidence to date indicates the Calpain system has a significant role in meat quality. a major factor in generating variability in tenderness..other systems are likely to contribute

42 Sutton Bonington Campus University of Nottingham

CC2.04_t.txt The Caspase System and Muscle Degradation Tim Parr

CC2.04_t.txt The Caspase System and Muscle Degradation Tim Parr 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,