Callipyge lambs
Small Heat Shock Proteins May Be Related to Toughness in Loins of Callipyge Lambs Yuan H. Brad
1 Kim ,
Moriah
• Small heat shock proteins (sHSPs) are a family of chaperone proteins that have a protecYve role in cell death (known as anY-‐apoptoYc acYvity)2. • It has been recently suggested that their chaperone and/or anY-‐ apoptoYc funcYons could be adversely related to the postmortem meat tenderizaYon process by hindering proteolyYc enzymes from breaking down structural myofibrillar proteins during aging process2. • While several proteomic studies found that higher sHSP acYvity (or expression) were observed in tougher meat compared tender counterparts, the role of sHSP in toughness of callipyge lambs has never been invesEgated. • We hypothesized that meat toughness of callipyge lambs could be associated with their up-‐regulaEon/elevated acEvity of sHSP, which in turn retards meat tenderizaEon process, as illustrated in the schemaYc drawing below:
• Less troponin T degradaYon was observed in callipyge lamb samples than in others across postmortem aging (Fig. 10 & 11; P < 0.05).
• Warner-‐Bratzler shear force and cook loss were measured on each of the lamb chop samples. • The experimental design was a randomized complete block design, and data were analyzed by using mixed procedure of SAS. Means were separated (F-‐ test, P < 0.05) by significant differences.
Results
0.3 0.25 0.2
CC
0.15
CN
0.1
NC
0.05
NN
0
0
3 6 Postmortem days
9
Figure 11. Troponin T degradaYon product over different aging Ymes.
1.8
• Loins from Callipyge (CN) lambs showed higher shear force (Fig. 4) and higher cook loss values (Fig. 5) than loins from other genotypes (P < 0.05).
Figure 12. RepresentaYve Western blots for HSP27 and aB-‐crystallin (9 days postmortem)
8
Cook Loss
7
30
6 5 4 3 2 1 0
25 20 15 10 5 0
CC
CN NC Genotypes
NN
CC
CN
NC
NN
Genotypes
Figure 4. Shear force values across four genotypes
Figure 5. Cook loss across four genotypes
• Higher levels of intact µ-‐calpain (80-‐kDa subunit) were observed across aging Ymes in CN samples as compared with that of other genotypes (P < 0.05), indicaYng less acYvity of µ-‐calpain (Fig.6). Callipyge lambs showed higher calpastaYn than their normal counterparts (Fig. 7). 0.7 0.6
Figure 7 (right). Intact µ-‐calpain (80-‐kDa subunit) indicates lesser autolysis and therefore less acYvity of loins from CN.
Objective
0.35
1.6
Figure 6 (above). RepresentaYve Western blots for µ-‐ calpain (9 days postmortem) and calpastaYn (15 min postmortem)
Figure 3. Proposed schemaEc mechanisms illustraEng the involvement of sHSP in the meat tenderizaEon process of Callipyge lambs
Figure 10. RepresentaYve Western blot for degradaYon products of troponin-‐T (9 days postmortem).
• More intact desmin was observed in callipyge lamb samples than in others across postmortem aging (Fig. 8 & 9).
The objecEve of this study was to determine an involvement of sHSP in tenderness development of loins from callipyge lambs during postmortem aging.
0.5 0.4
CC
0.3
CN
0.2
NC
0.1
NN
0
0
3 6 Postmortem days
1.8 1.7 1.6
CC
1.4
CN
1.3 1.2
NC
1.1
NN
1 0.9 0.8
0
3
6
9
Postmortem days
Figure 8 (above). RepresentaYve Western blot for intact desmin (9 days postmortem).
Figure 9. Intact desmin of loins from four lamb genotypes over a period of aging.
1.4
CC 1.2
CN NC
1
NN
0.8 0.6 0
3
6
9
Postmortem days Figure 13. Intact HSP27 loins from four lamb genotypes over a period of aging.
Conclusions • A higher expression of HSP27 was found in the CN genotype, which coincided with higher levels of intact desmin and troponin T, less μ-‐ calpain autolysis, and eventually higher shear force values as compared to other genotypes. • This observaEon leads us to postulate that the up-‐regulaEon of HSP27 could be related to meat toughness in callipyge lamb meat by delaying muscle protein degradaEon during aging, possibly through its anE-‐apoptoEc funcEon. • Further studies for the elucidaEon of the specific mechanisms (chaperoning and anE-‐apoptosis) and possible differences in sHSP behaviors across different muscle fiber types would be highly warranted.
9
1.5
• Higher levels of intact HSP27 were found in CN samples than in other genotypes across aging Ymes (Fig. 12 & 13; P < 0.05). • aB-‐crystallin was found to have a significant aging effect but not genotype effect.
Intact HSP27
Figure 2. RepresentaYve photos of loins from normal (LeE) and Callipyge (right) sheep from the current study – The loin from Callipyge shows bigger loin eye size than the loin from normal sheep.
• 4 lambs from 4 genotypes (CC, CN, NC, and NN) were slaughtered (a total of 16 lambs). Muscle samples from Longissimus dorsi (LD) were collected immediately post-‐harvest (15 min), as well as aEer 3, 6, and 9 days of postmortem aging. Table 1. Four genotypes analyzed in this study. • Western blots were performed to Genotype Phenotype observe desmin, troponin T, µ-‐ Paternal+/Maternal+ (NN) Normal calpain, calpastaYn, HSP27 and aB-‐ +/MaternalC (NC) Paternal Normal crystallin. Band intensity from blots C/MaternalC (CC) Paternal Normal w a s q u a n Y fi e d u s i n g U V P PaternalC/Maternal+ (CN) Callipyge Visionworks Analysis SoEware.
Cook Loss (%)
Figure 1. Normal (2, 4) vs Callipyge (1,3) Sheep A photo courtesy of Dr. Samuel Jackson,
Callipyge
Results, Continued
Intact µ-‐calpain
Normal
Chris
1 Bidwell
Methods
Shear Force (kg)
• Callipyge lambs are well known for their muscular hypertrophy of the loin and rump and their efficient feed-‐to-‐gain conversion. Their meat is leaner, but noYceably tougher and less palatable than meat from normal counterparts1. • The primary cause for callipyge meat toughness is an up-‐regulaYon of calpastaYn, which blocks the proteolyYc acYvity of µ-‐calpain and decreases the tenderness potenYal that could be achieved in aging1.
Jolena
2 Waddell ,
Troponin T degradaEon product
Background
1 Penick ,
Intact desmin
Traci
1 Cramer ,
References • 1Delgado, E.F., Geesink, G.H., Marchello, J. A., Goll, D.E., Koohmarie, M. (2001) The calpain systems in three muscles of normal and callipyge sheep. J. Anim. Sci. 79, 398-‐412 • 2 Ouali, A. et al. (2013). Biomarkers of meat tenderness: Present knowledge and perspecYves in regards to our current understanding of the mechanisms involved. Meat Science, 95(4), 854-‐870. • Image of callipyge vs. normal sheep courtesy of Jackson, S.P. (1993-‐05). InvesYgaYon of the producYvity and carcass composiYon of sheep with a muscular hypertrophy gene. (Doctoral dissertaYon). Retrieved from Texas Tech University Libraries. h_p://hdl.handle.net/2346/18187
1 Kim ,
Moriah
• Small heat shock proteins (sHSPs) are a family of chaperone proteins that have a protecYve role in cell death (known as anY-‐apoptoYc acYvity)2. • It has been recently suggested that their chaperone and/or anY-‐ apoptoYc funcYons could be adversely related to the postmortem meat tenderizaYon process by hindering proteolyYc enzymes from breaking down structural myofibrillar proteins during aging process2. • While several proteomic studies found that higher sHSP acYvity (or expression) were observed in tougher meat compared tender counterparts, the role of sHSP in toughness of callipyge lambs has never been invesEgated. • We hypothesized that meat toughness of callipyge lambs could be associated with their up-‐regulaEon/elevated acEvity of sHSP, which in turn retards meat tenderizaEon process, as illustrated in the schemaYc drawing below:
• Less troponin T degradaYon was observed in callipyge lamb samples than in others across postmortem aging (Fig. 10 & 11; P < 0.05).
• Warner-‐Bratzler shear force and cook loss were measured on each of the lamb chop samples. • The experimental design was a randomized complete block design, and data were analyzed by using mixed procedure of SAS. Means were separated (F-‐ test, P < 0.05) by significant differences.
Results
0.3 0.25 0.2
CC
0.15
CN
0.1
NC
0.05
NN
0
0
3 6 Postmortem days
9
Figure 11. Troponin T degradaYon product over different aging Ymes.
1.8
• Loins from Callipyge (CN) lambs showed higher shear force (Fig. 4) and higher cook loss values (Fig. 5) than loins from other genotypes (P < 0.05).
Figure 12. RepresentaYve Western blots for HSP27 and aB-‐crystallin (9 days postmortem)
8
Cook Loss
7
30
6 5 4 3 2 1 0
25 20 15 10 5 0
CC
CN NC Genotypes
NN
CC
CN
NC
NN
Genotypes
Figure 4. Shear force values across four genotypes
Figure 5. Cook loss across four genotypes
• Higher levels of intact µ-‐calpain (80-‐kDa subunit) were observed across aging Ymes in CN samples as compared with that of other genotypes (P < 0.05), indicaYng less acYvity of µ-‐calpain (Fig.6). Callipyge lambs showed higher calpastaYn than their normal counterparts (Fig. 7). 0.7 0.6
Figure 7 (right). Intact µ-‐calpain (80-‐kDa subunit) indicates lesser autolysis and therefore less acYvity of loins from CN.
Objective
0.35
1.6
Figure 6 (above). RepresentaYve Western blots for µ-‐ calpain (9 days postmortem) and calpastaYn (15 min postmortem)
Figure 3. Proposed schemaEc mechanisms illustraEng the involvement of sHSP in the meat tenderizaEon process of Callipyge lambs
Figure 10. RepresentaYve Western blot for degradaYon products of troponin-‐T (9 days postmortem).
• More intact desmin was observed in callipyge lamb samples than in others across postmortem aging (Fig. 8 & 9).
The objecEve of this study was to determine an involvement of sHSP in tenderness development of loins from callipyge lambs during postmortem aging.
0.5 0.4
CC
0.3
CN
0.2
NC
0.1
NN
0
0
3 6 Postmortem days
1.8 1.7 1.6
CC
1.4
CN
1.3 1.2
NC
1.1
NN
1 0.9 0.8
0
3
6
9
Postmortem days
Figure 8 (above). RepresentaYve Western blot for intact desmin (9 days postmortem).
Figure 9. Intact desmin of loins from four lamb genotypes over a period of aging.
1.4
CC 1.2
CN NC
1
NN
0.8 0.6 0
3
6
9
Postmortem days Figure 13. Intact HSP27 loins from four lamb genotypes over a period of aging.
Conclusions • A higher expression of HSP27 was found in the CN genotype, which coincided with higher levels of intact desmin and troponin T, less μ-‐ calpain autolysis, and eventually higher shear force values as compared to other genotypes. • This observaEon leads us to postulate that the up-‐regulaEon of HSP27 could be related to meat toughness in callipyge lamb meat by delaying muscle protein degradaEon during aging, possibly through its anE-‐apoptoEc funcEon. • Further studies for the elucidaEon of the specific mechanisms (chaperoning and anE-‐apoptosis) and possible differences in sHSP behaviors across different muscle fiber types would be highly warranted.
9
1.5
• Higher levels of intact HSP27 were found in CN samples than in other genotypes across aging Ymes (Fig. 12 & 13; P < 0.05). • aB-‐crystallin was found to have a significant aging effect but not genotype effect.
Intact HSP27
Figure 2. RepresentaYve photos of loins from normal (LeE) and Callipyge (right) sheep from the current study – The loin from Callipyge shows bigger loin eye size than the loin from normal sheep.
• 4 lambs from 4 genotypes (CC, CN, NC, and NN) were slaughtered (a total of 16 lambs). Muscle samples from Longissimus dorsi (LD) were collected immediately post-‐harvest (15 min), as well as aEer 3, 6, and 9 days of postmortem aging. Table 1. Four genotypes analyzed in this study. • Western blots were performed to Genotype Phenotype observe desmin, troponin T, µ-‐ Paternal+/Maternal+ (NN) Normal calpain, calpastaYn, HSP27 and aB-‐ +/MaternalC (NC) Paternal Normal crystallin. Band intensity from blots C/MaternalC (CC) Paternal Normal w a s q u a n Y fi e d u s i n g U V P PaternalC/Maternal+ (CN) Callipyge Visionworks Analysis SoEware.
Cook Loss (%)
Figure 1. Normal (2, 4) vs Callipyge (1,3) Sheep A photo courtesy of Dr. Samuel Jackson,
Callipyge
Results, Continued
Intact µ-‐calpain
Normal
Chris
1 Bidwell
Methods
Shear Force (kg)
• Callipyge lambs are well known for their muscular hypertrophy of the loin and rump and their efficient feed-‐to-‐gain conversion. Their meat is leaner, but noYceably tougher and less palatable than meat from normal counterparts1. • The primary cause for callipyge meat toughness is an up-‐regulaYon of calpastaYn, which blocks the proteolyYc acYvity of µ-‐calpain and decreases the tenderness potenYal that could be achieved in aging1.
Jolena
2 Waddell ,
Troponin T degradaEon product
Background
1 Penick ,
Intact desmin
Traci
1 Cramer ,
References • 1Delgado, E.F., Geesink, G.H., Marchello, J. A., Goll, D.E., Koohmarie, M. (2001) The calpain systems in three muscles of normal and callipyge sheep. J. Anim. Sci. 79, 398-‐412 • 2 Ouali, A. et al. (2013). Biomarkers of meat tenderness: Present knowledge and perspecYves in regards to our current understanding of the mechanisms involved. Meat Science, 95(4), 854-‐870. • Image of callipyge vs. normal sheep courtesy of Jackson, S.P. (1993-‐05). InvesYgaYon of the producYvity and carcass composiYon of sheep with a muscular hypertrophy gene. (Doctoral dissertaYon). Retrieved from Texas Tech University Libraries. h_p://hdl.handle.net/2346/18187
