Meat Science 92 (2012) 252–259

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Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Review

Advances in apoptotic mediated proteolysis in meat tenderisation

Caroline M. Kemp a,⁎, Tim Parr b
a
Central Community College, Grand Island, NE 68802, USA
b
Division of Nutritional Sciences, School of Biosciences, Sutton Bonington Campus, The University of Nottingham, Leicestershire, LE12 5RD, UK

a r t i c l e i n f o a b s t r a c t

Keywords: Meat tenderness is considered to be one of the most important attributes of meat quality; however it is also
Caspases one of the most variable. Ultimate meat tenderness is influenced by the amount of intramuscular connective
Apoptosis tissue, the length of the sarcomere and also the proteolytic potential of the muscle. Post-mortem proteolysis
Post-mortem proteolysis by endogenous proteases causes the weakening of myofibril structures and associated proteins, which results
Meat tenderisation in tenderisation. The caspase proteolytic system was first identified to be a potential contributor to post-
mortem proteolysis and tenderisation in 2002. Since then research has both supported and challenged this
hypothesis. The purpose of this review is to examine the experimental evidence available for caspases'
involvement in post-mortem proteolysis, and to highlight cross-talk between this proteolytic system and the
calpain system, a known contributor to meat tenderisation.
© 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
2. Apoptotic pathways and caspase activation and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
3. Apoptosis in muscle post-mortem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
4. Caspase-mediated post-mortem proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
5. Interactions between the caspase and calpain proteolytic systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

1. Introduction mortem changes in myofibrils in vitro. To date four main proteolytic

Meat quality is a generic term used to describe properties and
perceptions of meat and generally comprised colour, flavour, texture,
tenderness, and juiciness (Maltin, Balcerzak, Tilley, & Delday, 2003).
The mechanisms responsible for the development of these meat
qualities are interdependent, highlighting the complexity of the
conversion of muscle into meat (Ouali et al., 2006). Proteolysis of key
myofibrillar proteins by endogenous enzymes within the muscle fibre
is the largest contributing factor to meat tenderness, a trait highly
valued by consumers. In 1988, Koohmaraie defined the criteria for a
proteases involvement in post-mortem proteolysis and therefore
meat tenderness: it must be endogenous to the skeletal muscle, it
must have access to myofibrils, and it must be able to mimic post-
systems have received the attention of meat scientists, calpains,
cathepsins, proteasomes and caspases. The calpain proteolytic system
is probably the most extensively researched protease family with
regard to meat science and it is widely accepted that calpain activity,
specifically μ-calpain does contribute to meat tenderisation
(Koohmaraie & Geesink, 2006). However, which of these remaining
proteases are involved and the extent their contribution has on meat
quality is still debated. This review focuses on the role of the caspase
proteolytic system and apoptosis on muscle structure and biochem-
istry, with regard to meat tenderness, examining experimental
evidence that both reject and support their involvement.
2. Apoptotic pathways and caspase activation and regulation

Apoptosis and caspases were first implied to be involved in post-

⁎ Corresponding author. Tel.: + 1 308 398 7384; fax: + 1 308 398 7399. mortem proteolysis and meat tenderisation by Sentandreu, Coulis,
E-mail address: carolinekemp@cccneb.edu (C.M. Kemp). and Ouali (2002) who hypothesised that after slaughter skeletal

0309-1740/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2012.03.013
 C.M. Kemp, T. Parr / Meat Science 92 (2012) 252–259
muscle will engage in cell death, with apoptosis rather than necrosis
being the most likely candidate. Apoptosis is classified as highly
organised programmed cell death. It is characterised by undergoing
the processes of disassociation from neighbouring cells via cell
shrinkage, mitochondrial depolarisation, chromatin condensation,
DNA fragmentation, membrane blebbing and the formation of
apoptotic bodies (Wyllie, Kerr, & Currie, 1980). One of the major
physiognomies of apoptosis is the maintenance of the plasma
membrane during the process, thus preventing cellular component
discharge and damage to adjacent cells (Green, 2011). This process of
apoptosis is orchestrated by the family of cysteine aspartate-specific
proteases; caspases. Caspases were first implicated in apoptosis with
the identification that the human protease responsible for the
activation of interleukin-1β-converting enzyme (ICE) is homologous
to a product of the Caenorhabditis elegans death gene 3 (CED-3),
which is required for cell death in the nematode (Yuan, Shaham,
Ledoux, Ellis, & Horvitz, 1993). Caspases' involvement in apoptosis
was established further by the observations that caspase inhibitors
prevented apoptosis, caspases are responsible for the majority of
proteolytic cleavage that leads to cell death and that deficiencies in
caspase expression caused defects in apoptosis (Chang & Yang, 2000).
Fourteen members of the caspases protease family have been
identified and are expressed in a number of different tissues and cell
types (Nakagawa & Yuan, 2000); however some seem to be species
specific, with caspase 13 appearing to be expressed in bovine only
(Koenig, Eckhart, & Tschachler, 2001) and caspase 11 only found in
the rat and the mouse (Fuentes-Prior & Salvesen, 2004). Phylogenetic
analysis of caspases indicates that there are two main subfamilies;
caspases 1, 4, 5, 11 and 13 appear to be predominantly involved in the
inflammatory response system, whilst caspases 3, 6, 7, 8, 9, 10 and 12
are involved apoptosis. These apoptotic caspases can be further
subdivided into initiator caspases such as 8, 9, 10 and 12, and effector
caspases such as 3, 6 and 7, depending on their position in the cell
death pathway (Earnshaw, Martins, & Kaufmann, 1999). All caspases
are synthesised in the cytosol of cells as inactive zymogens. Whilst
initiator caspases are monomeric in their inactive conformation,
executioner caspases are dimeric (Denault & Salvesen, 2008).
Activation of apoptosis by caspases constitutes a minimal two-
step signalling pathway. The apical (initiator) caspases are activated
Extrinsic Pathway
Fas
FADD
FLIP
Pro-caspase 8/
Pro-caspase 10
ER-
Pro-caspase 12
medi
Caspase 8/
ated
Caspase 10
Path
way
Caspase 12
ER Pro-caspase 3
Caspase 3
Cell Death
Fig. 1. Schematic diagram of the intrinsic, extrinsic and ER-mediated apoptosis pathways and the caspases associated with each one. FADD—Fas associated death domain, IAP—
inhibitor of apoptosis, Smac—secondary mitochondrial activator of caspases, DIABLO—direct IAP-binding protein with low pI, FLIP—FLICE inhibitory protein, ARC—apoptosis
repressor with caspase recruitment domain.
Adapted from Holcik (2002).
253
in response to apoptotic stimuli, once activated; these caspases
directly activate the executioner (effector) caspases downstream by
limited proteolytic cleavage (Earnshaw et al., 1999). In caspase
mediated apoptosis there are three main pathways of activation
(Fig. 1): the first pathway, involving caspase 9, is termed the intrinsic
pathway and originates from within the cell in response to
environmental stress such as hypoxia and ischemia. This cellular
stress causes physiological changes to the cell including mitochon-
drial outer membrane permeability, a reduction in the inner
mitochondrial membrane's transmembrane potential and cyto-
chrome c release, leading to the formation of the apoptosome, a
complex essential for caspase 9 activation (Boatright & Salvesen,
2003). The second pathway, known as the extrinsic pathway, is the
pathway through which caspases 8 and 10 are activated. This
pathway is sometimes referred to as the death receptor pathway,
and involves the assembly of a cell membrane receptor–adaptor–
caspase complex. The assembly of this complex and the apoptosome
formed in the intrinsic pathway transforms a molecular signal into
proteolytic activity (Denault & Salvesen, 2008). Caspase 12 is a
unique initiator caspase, as its activation does not require the
assembly of a molecular platform like caspases 8, 9 and 10. Caspase
12 was initially thought to be an inflammatory response caspase due
to its high homology with caspases 1, 4, 5 and 11. However further
research has demonstrated that it is involved in cell death, initiating
the apoptotic cascade via stress directly upon the endoplasmic
reticulum (ER) (Nakagawa et al., 2000).
Executioner caspases are activated by the initiator caspases
through cleavage of the linker unit separating the large and small
subunits of their catalytic domain. This simple proteolysis of
executioner caspases is sufficient to gain maximal activity (Denault
& Salvesen, 2008). Caspases 3 and 7 are probably the most
extensively researched caspases; their high homology and substrate
and inhibitor specificity makes distinguishing between them virtually
impossible. However, caspases 3 and 7 do have differences in their
prodomains, which are thought to be involved in their subcellular
targeting (Fuentes-Prior & Salvesen, 2004). Caspase 6 is also classified
as an effector caspase due to its short prodomain, however its target
substrates are completely different to those of caspases 3 and 7
suggesting that the functions of caspases 3, 7 and 6 do not overlap
Intrinsic Pathway
Bcl-2
Pro-caspase 9 Mitochondria
Initiator caspases
Apoptosome Cytochrome c
Caspase 9
IAP Effector caspases
Smac/DIABLO
254 C.M. Kemp, T. Parr / Meat Science 92 (2012) 252–259
(Boatright & Salvesen, 2003). Caspases are among the most specific
proteases, requiring an aspartic acid residue at the C-terminal side of
the cleavage point of the substrate (Earnshaw et al., 1999). To date at
least 1000 cellular substrates have been identified as targets of these
executioner caspases (Green, 2011), including the myofibrillar pro-
teins actin, myosin, desmin, α-actinin, and troponin-T (Kemp & Parr,
2008).
Inadvertent activation of caspases can have devastating effects
and is therefore strictly regulated to prevent inappropriate activity
and apoptosis. There are a number of mechanisms involved in the
regulation of caspase activation, with the mode of regulation
dependent upon the nature of the initial stimulus (Earnshaw et al.,
1999). The Bcl-2 family can be divided into anti- and pro-apoptotic
members. Pro-apoptotic members are associated with both the
intrinsic and extrinsic pathways, interacting with one another,
caspases and also other proteins associated with these pathways
such as cytochrome c to promote and amplify the cell death signalling
pathway. Whereas the anti-apoptotic members of the Bcl-2 family
appear to promote cell survival by sequestering the actions of the
pro-death members (Fuentes-Prior & Salvesen, 2004). FLIPs (FLICE
(Fas associated death domain-like IL-1β-converting enzyme)-
inhibitory protein) are specifically associated with the extrinsic
pathway preventing the recruitment of caspases 8 and 10 to the cell
membrane, acting as a competitive inhibitor (Chang & Yang, 2000).
Inhibitors of apoptosis (IAPs) are one of the most important control
points of apoptosis. IAPs bind directly to specific caspases, via their
BIR (baculoviral IAP repeat) domains functionally sequestering them
(Fuentes-Prior & Salvesen, 2004). The caspase inhibitor apoptosis
repressor with caspase recruitment domain (ARC and is almost
exclusively expressed in myogenic cells and therefore may be an
important regulator of apoptosis in the skeletal muscle, Koseki,
Inohara, Chen, & Núñez, 1998). Protein levels of ARC have been
shown to vary between different porcine skeletal muscles post-
mortem, suggesting different inhibitive capacities in the different
muscles sampled (Kemp, Parr, Bardsley, & Buttery, 2006). Unfortu-
nately, there is currently little knowledge of caspase regulation in
skeletal muscle post-mortem; thus further investigation of expres-
sion of these inhibitors post-mortem would be valuable. The
intricacies of these pathways and caspases' activations and regula-
tions are well beyond the scope of this review and readers are
directed to the relevant reviews (Boatright & Salvesen, 2003; Denault
& Salvesen, 2008; Earnshaw et al., 1999).
3. Apoptosis in muscle post-mortem
The hypothesis that muscle engages in a form of cell death post-
mortem was further elucidated in 2006 by Herrera-Mendez et al. and
Ouali et al. who hypothesised that the hypoxic/ischemic conditions
that are induced through the process of slaughter and exsanguination
could activate caspases and apoptosis in the skeletal muscle post-
mortem, in a similar way to that observed during neuronal or cardiac
ischemia. In this section we will review the current evidence
supporting apoptosis as the mode of cell death in muscle post-
mortem.
Heat shock proteins (Hsp) are a family of protective proteins,
primarily functioning to prevent degradation and structural damage
of proteins from apoptotic processes in cells (Beere, 2005), with Hsp
27 and 70 identified to directly inhibit both the intrinsic and extrinsic
apoptotic pathways (Gotoh, Terada, Oyadomari, & Mori, 2004; Paul et
al., 2002). In longissimus thoracis (LT) muscle from Charolais bulls, the
gene DNAJA1 was identified to be down-regulated in muscle samples
with high meat quality and its expression inversely related to
tenderness (Bernard et al., 2007). DNAJA1 encodes Hsp40, a co-
chaperone of Hsp70, and this complex can directly inhibit apoptosis
through prevention of translocation of the pro-apoptotic protein Bax
to the mitochondrial membrane, which triggers the release of
cytochrome c, and initiates the activation of the intrinsic pathway
and caspase 9 (Gotoh et al., 2004). Protein levels of Hsp70, as
determined by 2D gel electrophoresis and mass spectrometry
analysis, were also found to be decreased in LT muscle from
Norwegian Red bulls at 48 h post-mortem, as well as two spots
corresponding to Hsp27 (Bjarnadottir, Hollung, Faergestad, &
Veiseth-Kent, 2010). Additionally, this group determined a lower
abundance of Hsp70 in electrically stimulated muscles; a known
tenderising technique, in comparison to non-stimulated muscles,
indicating that electrical stimulation could indirectly accelerate
apoptosis and post-mortem tenderisation via its actions on Hsp70
(Bjarnadóttir, Hollung, Høy, & Veiseth-Kent, 2011). Bernard et al.
(2007) also identified Hsp27 to be down-regulated in tender muscle
samples. Hsp27 is involved in the regulation and stabilisation of
myofibrillar proteins, and protection of actin filaments (Paul et al.,
2002). Thus, its down-regulation could indicate increased actin
degradation, which is a key indicator of apoptosis and a caspase
substrate. Furthermore, the gene encoding for caspase 3 was one of
the genes identified to be up-regulated in muscle samples with high
meat quality. However, this up-regulation of the caspase 3 gene was
only detected in the 15 month age group not the 19 month (Bernard
et al., 2007). Collectively, these data suggest that the down-regulation
of the anti-apoptotic properties of DNAJA1, Hsp70 and Hsp27 could
facilitate cell death and caspase activity during the post-mortem
conditioning period and subsequently increase meat tenderisation.
Additionally, muscle analysis on tough versus tender LT muscle
samples from Charolais bulls by 2D electrophoresis identified a
significant increase in proteolytic products in the tender group.
Further analysis of these proteolytic revealed that six of these
products originated from the inner and outer membranes of
mitochondria. Detection of these products occurred very early during
the post-mortem conditioning period and thus could not have been
derived from meat ageing. These data indicate an increase in
mitochondrial membrane disruption in the tender group, which
occurs early in the intrinsic apoptotic pathway and is subsequently
involved in caspase activation (Laville et al., 2009).
Whilst these studies indicate apoptosis as a potential cell death
mechanism in muscle cells post-mortem, they do not provide direct
evidence. Key characteristics of apoptosis include cell shrinkage,
mitochondria alteration, DNA fragmentation, chromatin condensa-
tion and degradation of specific cytoskeletal proteins including actin,
a known caspase substrate (Green, 2011). Recently, Becila et al.
(2010) examined three different hallmarks of apoptosis during the
first 72 h following exsanguination. Phosphatidylserine phospholipids
externalisation was detected in the gastrocnemius and plantaris
muscle samples from Wistar rats as early as 1 h post-mortem.
Translocation of phosphatidylserine phospholipids signals a cell's
dying status to neighbouring cells; however it occurs irrespective of
the cell death pathway. Thus, to elucidate the process of cell death cell
shrinkage and actin degradation were determined. Cell shrinkage is
associated with an increase in the extracellular space, whilst cell
swelling, a key characteristic of necrosis, causes a reduction in the
extracellular space (Green, 2011). The extracellular space between
individual muscle fibres increased significantly between 1 and 24 h
post-mortem, with the extent of shrinkage estimated to be between
30 and 33% at 24 h (Fig. 2). Furthermore, actin degradation was
detected in muscle samples, with expression of an actin derived
fragment at 32 kDa increasing sharply immediately after death and
peaking at 3–4 h post-mortem. The observation of this 32 kDa actin
fragment is in agreement with proteome analysis in porcine muscle
samples post-mortem (Lametsch, Roepstorff, & Bendixen, 2002;
Lametsch, Roepstorff, Moller, & Bendixen, 2004). Whilst the amount
of actin degradation products detected in comparison to the amount
of intact actin in skeletal muscle myofibrils is relatively small, it
has been suggested that even limited proteolysis of actin could alter
the actin/myosin rigour bond and minor degradation may have an
 C.M. Kemp, T. Parr / Meat Science 92 (2012) 252–259
Fig. 2. Cell shrinkage in rat gastrocnemius muscle excised at (a) 1 h and (b) 24 h post-
mortem. Inset rat LD muscle excised ante-mortem under anaesthesia, where fibres are
in close contact (Becila et al., 2010).
influence on meat tenderness (Huff-Lonergan, Zhang, & Lonergan,
2010; Lametsch et al., 2004).
In a separate study, morphological and biochemical cell changes
associated with apoptosis were assessed in longissimus dorsi (LD),
psoas and semitendinosus (ST) skeletal muscles from crossbred bulls
across the post-mortem storage period (Cao et al., 2010). Condensa-
tion and margination of nuclear chromatin were detected between
30 min and 1 d, formation of apoptotic bodies by d 4, and DNA
fragmentation peaked at d 7 post-mortem. Whilst these apoptotic
physiognomies were identified in all three muscles sampled, more
chromatin condensation and apoptotic bodies were detected in the
LD; in turn this could be indicative of more apoptotic-mediated
proteolysis (Cao et al., 2010). In the LD, the proteolytic capacity is the
major determinant of tenderness (Koohmaraie, Kent, Shackelford,
Veiseth, & Wheeler, 2002), which could provide an explanation of the
differences observed between the LD, ST and psoas muscles. However,
at d 7 more DNA fragmentation was observed in the psoas muscle,
and this difference could be a reflection of fibre type composition of
the muscle, as the psoas muscle contains more slow oxidative fibres
(Kemp, Parr et al., 2006), which have a greater volume of
mitochondria and, thus, maybe more susceptible to mitochondrial-
associated apoptosis (Cao et al., 2010). In these two separate studies,
Cao et al. (2010) and Becila et al. (2010) provided evidence that cell
death in post-mortem muscle occurs via apoptosis not necrosis.
Furthermore, these data indicate that initiation of apoptosis occurs
immediately after death, and therefore the conversion of muscle into
meat starts by the onset of the apoptotic process a few minutes post-
mortem.
255
4. Caspase-mediated post-mortem proteolysis
The caspase proteolytic system was first directly examined in
skeletal muscle post-mortem by Kemp, Parr et al. (2006), who
identified diverse protein expression and activity of various compo-
nents of the caspase system in different porcine muscles. More
specifically, differences were observed between initiator caspases;
whilst high levels of caspase 9 activity were detected in muscle
samples, very low and no expression of the active isoforms of
caspases 8 and 12, respectively, were detected. These findings were
further concurred by Pulford et al. (2009) who identified significantly
higher caspase 9 activity than caspase 8 activity in beef muscle
samples. Furthermore, caspase 9 activity across the post-mortem
conditioning period has been found to positively correlate to activity
of effector caspases 3/7 (Kemp, Bardsley, & Parr, 2006; Kemp, King,
Shackelford, Wheeler, & Koohmaraie, 2009). Collectively these data
indicate that the predominant apoptotic pathway active in skeletal
muscle post-mortem is the intrinsic pathway, involving caspase 9.
Thus, supporting the hypothesis of Ouali et al. (2006), the process of
death and exsanguination would induce hypoxia and ischemia within
muscle cells, which in turn would activate the intrinsic apoptotic
pathway.
Caspase activity has been demonstrated to change across the post-
mortem conditioning period, with the highest activities always
detected in the early phase (Kemp, Bardsley et al., 2006; Kemp et
al., 2009; Pulford et al., 2009). This change in caspases 3/7 and
caspase 9 activity during the early phase of conditioning has been
identified to have a negative relationship with shear force in porcine
LD muscle; thus, the more caspase activated, the greater the change
and the lower the shear force value, and therefore the higher the level
of meat tenderness (Kemp, Bardsley et al., 2006). In this study a
negative relationship was also detected between shear force and
expression of the caspase generated alpha II spectrin 120 kDa
degradation product (SBDP120). The appearance of this SBDP120
has also been detected in chicken pectoralis superficialis muscle
samples treated with apoptotic inducers (Chen et al., 2011). Caspase-
mediated cleavage of full length 214 kDa alpha II spectrin generates
signature 150 and 120 kDa degradation products, and detection of
these peptides is considered an excellent marker for caspase
mediated apoptosis (Nath et al., 2000). The degradation of alpha II
spectrin during apoptosis could significantly compromise the mem-
brane permeability and also cytoskeletal integrity (Wang, 2000), a
process that is associated with meat tenderisation (Taylor, Geesink,
Thompson, Koohmaraie, & Goll, 1995).
An association between caspase activity and meat quality was
also detected in callipyge sheep (Kemp et al., 2009). Callipyge sheep
are characterised by heavy muscling in the pelvic limb and loin
region, but not the thoracic region, and decreased meat tenderness
which is associated with elevated calpastatin content (Koohmaraie,
Shackelford, Wheeler, Lonergan, & Doumit, 1995). Kemp et al.
(2009) detected higher caspase 3/7 activity and also caspase 9
activity in muscles which are affected by the phenotype, in normal
lambs than callipyge lambs, across the post-mortem storage period.
Collectively these findings indicate that during the conditioning
period caspases are both active and capable of targeting and
cleaving their specific substrates, adding to the evidence that
apoptosis is the mode of cell death in skeletal muscle post-
mortem and caspases have a role in meat tenderisation. However,
in a similar study to that conducted in porcine LD muscle,
Underwood, Means, and Du (2008) examined changes in caspase
3 activity and protein expression in beef muscle samples during
post-mortem ageing. In this study only the inactive isoform of
caspase 3 not the active isoform was detected, and no association
between caspase 3 activity determined immediately after slaughter
and muscles that developed into beef with extreme shear force
values was found.
256 C.M. Kemp, T. Parr / Meat Science 92 (2012) 252–259
One of the criteria specified by Koohmaraie (1988) for a protease
involvement in post-mortem proteolysis and meat tenderisation is
that it must be able to mimic in situ post-mortem changes in
myofibrils in vitro. Recently there have been a number of studies
examining this benchmark using myofibrils purified from sheep, pigs,
chicken and cattle muscle. In porcine LD myofibrils co-incubated with
recombinant caspase 3; Kemp and Parr (2008) demonstrated
caspases ability to replicate myofibril degradation patterns in situ.
MALDI-TOF mass spectrometry identified degradation of myofibril
proteins including desmin and troponin I, and proteolytic products at
32, 28 and 18 kDa, found to arise from the degradation of actin,
troponin T and myosin light chain, respectively. These degradation
patterns were not observed in myofibrils incubated with the caspase
specific inhibitor DEVD-CHO.
The degree of alteration and weakening of myofibrillar structures
directly affects ultimate meat tenderness, with modification of
several key proteins including myosin, actin, desmin and troponin,
during the ageing period. Myosin and actin are the two most
abundant proteins in the skeletal muscle myofibrils. Traditionally,
these proteins were considered not to undergo major changes during
the conditioning period; however, increased sensitivity in proteomics
has led to the identification that they are susceptible to proteolysis by
endogenous proteases (Lametsch et al., 2002, 2004). Desmin is an
intermediate filament protein localised at the periphery of the
myofibrillar Z-disc (Taylor et al., 1995). Degradation of desmin
during post-mortem ageing has been reported in numerous studies
and its proteolysis appears to correlate with tenderisation (for review
see Huff-Lonergan et al., 2010). Troponins T and I are both degraded
in muscle post-mortem (Ho, Stromer, & Robson, 1996; Huff-Lonergan
et al., 1996), a positive association between troponin T breakdown
and tenderness has been identified (Huff-Lonergan et al., 1996), and
the appearance of troponin T degradation products is considered an
excellent indicator of proteolysis (Ho et al., 1996). Degradation of the
large structural proteins titin and nebulin is also thought to
contribute to tenderisation, by increasing the fragility of myofibrils
in the I-band, and decreasing the stability of the actin filaments (Huff-
Lonergan et al., 2010; Taylor et al., 1995). The extent and rate of
proteolysis of these key myofibrillar proteins during ageing and
subsequent impact on tenderisation are outside the range of this
article and readers are directed to the review by Huff-Lonergan et al.
(2010).
In a similar study to that performed in porcine LD myofibrils,
Huang, Huang, Xu, and Zhou (2008) reported limited proteolysis in
myofibrils isolated from chicken pectoralis superficialis muscle
incubated with DEVD-CHO. Degradation of desmin and of the large
structural proteins nebulin and titin was severely attenuated in the
a) Normal LD
b) Callipgye LD
Fig. 3. Representative Western blots loaded with 21 μg myofibrillar protein samples probed for desmin. a) Normal animal LD. b) Callipyge animal. Muscle samples were incubated
with oxidative stressors fenretinide or H2O2, recombinant caspases 3 (rC3), the caspases inhibitor DEVD-CHO, or in treatment buffer alone (control) for 0, 1, 2, 7, and 21 d at 4 °C.
The major immune-reactive band for desmin is indicated (Kemp & Wheeler, 2011).
DEVD-CHO treatment, as was troponin T until 7 d post-mortem,
which corresponded with the appearance of proteolytic products at
32 and 28 kDa. The caspase-specific inhibitor did not affect μ- or m-
calpain activity, indicating that the limited proteolysis observed in the
DEVD-CHO treated myofibrils must be attributed to its direct
inhibition of caspases. In a continuation of this study, this research
group subsequently analysed the effects of apoptotic inducers on
caspase 3 activity and myofibrillar disruption during post-mortem
ageing in chickens (Chen et al., 2011). All three apoptotic inducing
treatments examined in this study resulted in a significant increase in
caspase 3 activity, and the appearance of a 30 kDa proteolytic product
assumed to originate from troponin T degradation and the generation
of the caspase specific alpha II spectrin 120 kDa degradation product
(SBDP120). Inhibition and activation of the caspase proteolytic
system have also been demonstrated in myofibrils isolated from LD
and supraspinatus muscles from normal and callipyge sheep (Kemp &
Wheeler, 2011). Overall, results showed treatment with apoptotic
inducers hydrogen peroxide and fenretinide reduced protein expres-
sion of the inactive latent isoform of caspase 9, indicating activation of
the intrinsic apoptotic pathway. Furthermore, these apoptotic in-
ducers and also recombinant caspase 3 increased proteolysis of
myofibrillar proteins including desmin and troponin T, whilst the
caspase specific inhibitor DEVD-CHO inhibited degradation (Fig. 3).
Oxidative stress has also been reported to increase both calpain and
caspase-mediated degradation of myofibrillar proteins, myosin heavy
chain, α-actinin, actin and troponin I in rat myofibrils, with increasing
levels of stress causing a stepwise escalation of proteolysis (Smuder,
Kavazis, Hudson, Nelson, & Powers, 2010). Cumulatively, these data
show that manipulation of the caspase system through either
induction or inhibition of activity can directly affect degradation of
myofibrillar proteins.
However, whilst these studies have provided some substantial
evidence for caspases involvement in being able to replicate myofibril
degradation patterns in situ, in beef conflicting data has been
reported. Huang, Huang, Zhou, Xu, and Xue (2011) demonstrated
significant degradation of titin and nebulin and an increase in
proteolytic fragments at 32 and 28 kDa; assumed to arise from actin
and troponin T breakdown, in beef LT myofibrils incubated with
caspase 3. In contrast, no such degradation patterns were observed in
caspase 3 treated ST myofibrils in comparison to the control or μ-
calpain treated samples (Mohrhauser, Underwood, & Weaver, 2011).
These observations led the authors to conclude that μ-calpain, not
caspase 3, is responsible for the degradation of myofibrillar proteins
during beef ageing. One can only speculate that the differences
observed in all of these in vitro myofibril studies could be attributed
to species differences, muscle type, breeds or even the pre- and post-
 C.M. Kemp, T. Parr / Meat Science 92 (2012) 252–259
slaughter environment. In vivo different muscles respond to the same
stimuli to give differing patterns of muscle wasting, for example in
sarcopenia there is wasting of specific muscle fibre types, with loss of
type II fibres and retention of Type I, suggesting intrinsic differences
in wasting potential (Evans, 2010). All of the above mentioned factors
are known to affect post-mortem proteolysis and meat tenderisation.
Thus, these studies only further highlight our need for continued
research in this area to increase our understanding of the conversion
of muscle into meat process, and the role of the caspase proteolytic
system.
Predominantly, research into caspases involvement in post-
mortem proteolysis and tenderisation has focused on the effector
caspases 3 and 7 and little attention has been paid to effector caspase
6. Recently, post-mortem proteolytic patterns in bovine LT myofibrils
incubated with either caspase 3 or caspase 6 were examined (Huang
et al., 2011). Caspases 3 and 6 were both identified to cause significant
degradation of the large structural proteins titin and nebulin; however
degradation patterns of other myofibrillar proteins differed. Caspase 6
induced greater degradation of the intermediate filament protein
desmin, in comparison to caspase 3, whilst the appearance of
proteolytic fragments at 32 and 28 kDa, previously identified to
originate from actin and troponin T degradation respectively (Kemp &
Parr, 2008), was significantly higher in the caspase 3 incubated
samples. Thus, whilst caspases 3 and 6 can both produce myofibril
degradation, their patterns are distinct from one another, highlighting
both the differences and also the similarities between these caspases
in their target substrate specificity. In a continuation from this study,
Huang, Huang, Ma, Xu, and Zhou (2012) examined the effects of the
caspase 6 specific inhibitor VEID-CHO on myofibrillar protein
degradation. Marginal retardation of proteolysis of proteins nebulin,
desmin and troponin T was detected in VEID-CHO incubated samples
in comparison to control, suggesting that caspase 6 has minimal role, if
any, in post-mortem degradation and meat tenderisation.
Meat tenderness is not just influenced by proteolysis, but
temperature, pH, sarcomere length, and connective tissue/collagen
content of the muscles can all affect meat quality and directly or
indirectly impact a muscles' proteolytic potential. Ultimate pH (pHu)
has been widely used as an indicator of potential meat tenderness
(Young, West, Hart, & van Otterdijk, 2004). LT beef muscle samples
exhibiting a low pHu had consistently greater levels of caspase 3/7
activity than those with intermediate or high pHu, suggesting that a
more vigorous rate of apoptosis was initiated in the low pHu muscles
(Pulford et al., 2009). Cell models have demonstrated that acidic
conditions are favoured for maximum induction of apoptosis
(Nilsson, Johansson, Johansson, Kågedal, & Ollinger, 2006), thus a
low pHu in muscle samples would be conducive to an optimal
environment for apoptosis and caspases activation. Additionally,
caspase 3/7 activity was significantly higher at 1 and 3 h post-
mortem in muscle samples destined to have low pHu, suggesting
caspases' 3/7 potential as an early indicator and predictor of high
meat quality and tenderness (Pulford et al., 2009). In contrast, μ-
calpain activity in low pH conditions (pH 6) is reduced in comparison
to neutral conditions (pH 7.5) (Camou, Marchello, Thompson, Mares,
& Goll, 2007; Carlin, Huff-Lonergan, Rowe, & Lonergan, 2006; Geesink
& Koohmaraie, 2000; Maddock, Huff-Lonergan, Rowe, & Lonergan,
2005), with some studies identifying 6.5 as an optimal pH for μ-
calpain activity (Carlin et al., 2006; Geesink & Koohmaraie, 2000;
Maddock et al., 2005). Furthermore, the rate of pH decline post-
mortem is known to significantly affect μ-calpain activity. A rapid pH
decline can lead to μ-calpain inactivation (Maddock et al., 2005;
Pomponio, Ertbjerg, Karlsson, Costa, & Lametsch, 2010; Veeramuthu
& Sams, 1999), whereas an intermediate pH decline allows for more
optimal conditions for μ-calpain activity (Maddock et al., 2005).
However, the effect of ultimate pH and rate of pH decline on caspase
activity has not been thoroughly examined in muscle post-mortem
and is an area that warrants further investigation.
257
5. Interactions between the caspase and calpain proteolytic
systems
The calpain proteolytic system is known to be a major contributor
to post-mortem proteolysis and meat tenderisation, with μ-calpain
considered to be the primary protease. Nonetheless, silencing the μ-
calpain gene does not completely attenuate post-mortem proteolysis
in skeletal muscle (Geesink, Kuchay, Chishti, & Koohmaraie, 2006)
and thus other proteases must be considered. There has been
increasing evidence of interactions between the calpain and caspase
protease systems; this has predominantly focused on the substrates
that are targeted including the proteins actin, actinin, myosin,
spectrin, vimentin, and troponin I (Smuder et al., 2010; Wang,
2000). However, the similarities between the calpain and caspase
protease systems are not just limited to the substrates that they
cleave. Caspase 12 can be activated by calpains in response to a
disruption in the Ca 2 + homeostasis in the ER. The amount of Ca 2 +
required for this cleavage was found to be at millimolar not
micromolar levels, suggesting that m-calpain is responsible for
caspase 12 activation. Milli-calpain can also cleave the anti-
apoptotic protein Bcl-XL, which associates with the mitochondrial
membrane, into a pro-apoptotic molecule, and thus could potentially
contribute to the induction of the intrinsic apoptotic pathway
(Nakagawa & Yuan, 2000). Calpains can also act as negative
regulators of apoptosis, cleaving caspases 3, 7, 8 and 9 at distinctive
sites to generate inactive isoforms (Chua, Guo, & Li, 2000). Thus the
cross-talk between these two families appears to be multifaceted.
Calpastatin is a known caspase substrate, with cleavage by
caspases 3 and 7 producing distinct proteolytic fragments (Wang et
al., 1998). Upon examination of caspase 3/7 and calpastatin activity
across the post-mortem conditioning period a negative relationship
was identified between peak caspase 3/7 activity at 8 h and
calpastatin activity at 0 and 2 d in the LD muscle from normal
lambs, but not callipyge lambs (Kemp et al., 2009). Caspase-mediated
calpastatin degradation is thought to compromise plasma membrane
and cytoskeletal integrity, resulting in an increase in calcium levels
and calpain activation (Wang, 2000). Caspases could therefore
modulate post-mortem proteolysis and meat tenderisation via its
actions on calpastatin. Interpretation of this negative relationship was
that whilst caspase 3/7 activity may contribute to the decrease in
calpastatin in normal lambs, in callipyge animals the calpastatin
content is overwhelming, and caspase activity is simply not sufficient
enough to overcome it. Consequently, in the callipyge phenotype
calpastatin activity is the over-riding factor in post-mortem
proteolysis.
Furthermore, inhibition of calpains through either up-regulation
of calpastatin or via the actions of the calcium chelator EGTA resulted
in up-regulated caspase activity and also accelerated apoptosis
(McGinnis, Gnegy, Park, Mukerjee, & Wang, 1999; Neumar, Xu,
Gada, Guttmann, & Siman, 2003). In porcine LD myofibrils co-
incubated with recombinant caspase 3 and either calpastatin or
EDTA an increase in caspase mediated proteolysis of key myofibrillar
proteins myosin heavy chain, actin and troponin T was observed
(Kemp & Parr, 2008). The exact mechanism behind this up-regulation
remains unclear, although one could speculate that this increase in
caspase mediated proteolysis is a compensatory mechanism. How-
ever, whilst treatment with apoptosis inducers fenretinide and
hydrogen peroxide, and also recombinant caspase 3 can increase
proteolysis in callipyge animals, which exhibit elevated levels of
calpastatin, in comparison to control treatment, their effects were not
substantial enough to overcome the limited proteolysis that these
animals exhibit (Fig. 3b; Kemp & Wheeler, 2011). The results from
these studies accentuate the complexity of post-mortem proteolysis
and meat tenderisation, and suggest that further research is required
to understand the mechanisms behind it and the interactions
between different proteolytic systems.
258 C.M. Kemp, T. Parr / Meat Science 92 (2012) 252–259
1600
WT
1400
KO
Fluor/mg protein
1200
1000
800
600
* Shifts in energy status and myofibrillar stability. Journal of Agricultural and Food
400
200
0 low-voltage electrical stimulation. Meat Science, 89, 143–149.
Age (weeks)
Fig. 4. Caspase 3/7 activity levels in skeletal muscle of wild type (WT) and μ-calpain
knockout mice (KO) at 3, 5, 10, 20 and 30 weeks of age. Activity expressed as arbitrary
units of fluorescence/mg protein ± SEM. Significant effect of age and genotype
(P b 0.0001, P b 0.05, respectively). Genotypes × age interaction observed at 5 weeks
(*P b 0.05). (Kemp et al., in press).
More recently, an interaction was observed between these two
proteolytic systems in μ-calpain knockout mice (Kemp, Oliver,
Wheeler, Chishti, & Koohmaraie, submitted for publication). This
study examined the role of the calpain system in skeletal muscle
using genetically altered mice that do not express the μ-calpain gene to
further elucidate the importance of μ-calpain in protein turnover and
its subsequent effects on skeletal muscle growth and development.
Proteolytic systems are an essential component of muscle protein
turnover, with evidence suggesting that the calpain protease family
plays a vital role (Goll, Neti, Mares, & Thompson, 2008). Results from
this study detected significantly higher caspase 3/7 activity in knockout
mice in comparison to wild type, and this increased activity was
particularly apparent in the younger mice when the animals were
rapidly growing (Fig. 4). This data suggests that caspases were up-
regulated to compensate for the lack of μ-calpain and to ensure normal
skeletal muscle growth and development, as no differences in skeletal
muscle mass were detected. Thus, highlighting further the complexities
of the interactions between these two protease systems.
6. Conclusion
The rate and extent of meat tenderisation are known to be highly
variable, and are influenced by numerous factors which are
interdependent upon one another. One of these factors is the content
and activity of endogenous proteases within the skeletal muscle. The
calpain system, in particular μ-calpain, has been identified to be a
major contributor to post-mortem proteolysis, and its specific
inhibitor calpastatin greatly influences meat tenderness. However, it
has been proposed that post-mortem proteolysis and meat tender-
isation are a multi-enzymatic process including calpains, cathepsins,
proteasomes and also the caspase system. Although evidence has
identified apoptosis, not necrosis, as the primary mode of cell death in
skeletal muscle post-mortem, there still remains some ambiguity in
the extent to which the caspase system contributes to post-mortem
proteolysis and meat tenderisation. Whilst some studies have
provided support for a role for the caspase proteolytic system in the
conversion of muscle into meat, others have dismissed it. Conse-
quently we must continue our efforts in research to further our
understanding of the caspase system post-mortem, the interactions
between this and other proteolytic systems, and the overall complex
muscle to meat conversion process.
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