Manipulating connective tissue turnover to improve meat quality
Peter Purslow Dept. Food Science Ontario Agricultural College University of Guelph, Guelph, Ontario, Canada [email protected] Anatomy of intramuscular connective tissue (IMCT)
Artwork courtesy Dr L-T Lim
Purslow (2010) J. Bodyw. Mov. Ther. 14: 411-417 NaOH extraction of muscle reveals collagenous structures
Perimysial thickness ~ 10-20x endomysial thickness
Purslow & Trotter (1994) J Musc. Res. Cell Motil. 15: 299-304. Perimysial collagen varies most between 14 bovine muscles
Muscle Perimysial Endomysial collagen collagen (%DM) (% of DM) Extensor carpi radialis 4.76 1.20 Infraspinatus 4.30 0.58 Sternocephalicus 3.37 0.76 Supraspinatus 2.38 0.66 Rhomboideus 2.09 0.89 Splenius 2.12 0.56 Subscapularis 1.96 0.65 Pectoralis profundus 1.62 0.89 Triceps brachii cap. long. 1.80 0.46 Complexus 1.44 0.71 Gluteus medius 1.23 0.64 Gastrocnemius 1.15 0.63 Obliquus intern. abdom. 0.54 0.55 Serratus ventralis 0.43 0.47
Values are means of measurements from 6 Friesian bovine animals
Amount of perimysial collagen varies by 10:1 whereas endomysium varies only by 3:1 Purslow (1999). Proc. 45th ICoMST (Yokohama) 210-219.
Intramuscular connective tissue (IMCT)
• Why is IMCT important to meat tenderness?
• Can we degrade IMCT post-mortem?
• Can the amount of IMCT be changed?
• Can the quality of IMCT be changed?
Intramuscular connective tissue (IMCT)
• Why is IMCT important to meat tenderness?
-Despite usually being only 2-10% of proteins in meat, collagenous component is a major contributor to toughness
- Variations in collagen content between cuts is a major determinant of expected eating quality, and hence retail value
- Variations in collagen maturity impact toughness of meat from older animals USDA Weekly Retail Beef Feature Activity 13 May 2011 Advertised Prices for Beef to Consumers at Major Retail Supermarket Outlets ending during the period of 05/13 thru 05/19 (prices in dollars per pound)
Beef Cut Wtd. Av. Retail price Filet Mignon 12.39 per lb Boneless NY strip steak 8.05 per lb Sirloin steak 5.24 per lb Rump steak 4.99 per lb Flat brisket 3.76 per lb
These beef cuts show a price differential of 329% - Not based on nutritional value, but on expected eating quality
www.ams.usda.gov/mnreports/lswbfrtl.pdf Acessed 19 May 2011 Ranking of Beef Muscles for Tenderness By Chris R. Calkins, Ph.D. and Gary Sullivan, University of Nebraska
Brisket Fillet NY strip Rump
www.beefresearch.org/.../BeefResearch/Ranking%20of%20Beef%20Muscles%20for%20Tenderness.pdf Acessed 19 May 2011 Relationship between toughness and collagen content of 18 beef muscles
Total and soluble collagen content accounted for 45 % of the overall variation in toughness after cooking at 60C for 20 min and 34% after cooking at 75°C for 1 h 75C for 1 hr.
Dransfield, E (1972) Intramuscular Composition and Texture of Beef Muscles. J. Sci. Food Agric. 28: 833-842 Ageing causes some IMCT degradation in raw meat....
Fig. 2 from Nishimura, T (2010) Animal Science Journal 81, 21–27
Ageing reduces strength of perimysium from raw meat, but no reduction is seen in meat cooked above 60°C
Lewis et al (1991) Meat Sci. 30: 1-12. A series of muscle physiology and functional biomechanics studies indicates:
Variations in the amounts and spatial distribution of perimysial IMCT appear related to muscle function in vivo
- so small prospects of manipulating IMCT content and retaining a healthy animal Conclusions from previous studies
• Intramuscular connective tissue has a strong contribution to cooked meat toughness
• Postmortem storage does degrade IMCT – but this is not significant after cooking
• Variability in IMCT expression between muscles is notable and can closely be related to in vivo functional roles
• “Background toughness” concept; IMCT component of toughness not considered to be easily changed Ranking of Beef Muscles for Tenderness By Chris R. Calkins, Ph.D. and Gary Sullivan, University of Nebraska
“ Background effect
The term background effect relates to connective tissue located throughout a muscle. This connective tissue retains considerable strength throughout extended periods of cooler aging. Thus, even when the actomyosin effect is very low, background toughness will be caused by this connective tissue.
Two aspects of connective tissue come into play relative to tenderness. First is the amount. The more connective tissue (comprised primarily of the protein collagen) the less tender the meat. Typically, muscles of locomotion (those found in the thoracic and pelvic limbs of animals) have more connective tissue and are less tender.
The second feature of connective tissue is its heat-induced solubility….. It is important to note that older animals have more cross-links within collagen than younger animals, meaning the collagen of older animals is less soluble when heated. Therefore, older animals provide meat that is less tender. “
www.beefresearch.org/.../BeefResearch/Ranking%20of%20Beef%20Muscles%20for%20Tenderness.pdf Acessed 19 May 2011 Alternative for manipulation?
Long residence time of collagen More cross-linked with age
Tougher meat
Immature cross-links are functionally directed during synthesis
Mature cross-links: if NOT functionally required
- could reduce amounts by increasing turnover of IMCT? Degradation Synthesis MATURE IMMATURE Collagen Collagen
Mature collagen High in heat stable cross-links
Immature collagen Low in heat stable cross-links Turnover of IMCT
Principal matrix-degrading enzymes: MMPs (matrix metallproteinases) (Inhibitors – TIMPs) MMP family – 28 enzymes with preferred substrates MMP 1, 2, 3, 7,9, 10,11, 12, 13, 15, 16 TIMP family – 4 inhibitors Found to be expressed in skeletal TIMP 1, 2, 3, muscle
Nutrition and mechanical stimuli both known to affect turnover of IM-ECM
Adaptation of muscle and muscle hypertrophy is known to involve increased expression of a range of MMPs Normal activation of MMPs in vivo is by proteolysis. Latent (pro-) form of e.g. MMP-2 has MW= 72k: cleavage of pro- domain yields smaller active enzyme ~65k . Denaturation/renaturation of pro-form during zymography activates 72 k MW pro-form, as does activator AMPA
Figures from: Zhang et al (2009) Vascular 17:147-157 Our current cell culture models
of pathways affecting IMCT turnover
Physical stress Challenges Oxidative stress Hormonal signals…
Muscle cells ( myocytes)
Cell models Fibroblasts (cell line specific responses)
MMP activity TIMP levels Outcomes Cell signaling Collagen synthesis..... Who and what controls turnover of IMCT?
Which cells maintain and turnover which parts of IMCT? - myoctes, satellite cells, fibroblasts ?
What is the role of factors such as: • nutrition (including vitamins) • mechanical stress • hormones • growth factors • oxidative stress in expression of MMPs, TIMPs and CT components?
- can we use any of these to manipulate IMCT turnover? Rationale: endomysial and perimysial networks have to be expanded to allow for muscle fibre hypertrophy Dietary control of muscle growth rate influences collagen turnover/solubility
Kristensen et al (2002) J. Anim Sci 80: 2862-71.
O Pigs + Dietary WBSF % soluble MFI Treatment 4day /N collagen 1day
AA AA 45.3 34.5 86.6
RA RA 43.6 35.2 798.1 AR Live WgtLive AR 51.3 36.9 93.3 RR RR 49.7 39.2* 75.5
LD muscle studied 0 90 165d
Therkildsen et al. 2002 (Animal Science 75: 367-377 ) In each period also report significant increases in soluble A=ad lib R=60% of adlib. collagen with restricted feeding (i.e. slower growth). Level of animal nutrition and collagen turnover: Av. Daily Gain vs. Heat soluble collagen
ALFALFA GRASS CONCENTRATE LD: R= 0.449 (P<0.05) LD: R= -0.388 (P<0.05) LD: R= 0.494 (P<0.05) ST: R= 0.387 (NS) ST: R= 0.231 (NS) ST: R= 0.186 (NS)
LD ST LD ST 45 45 LD ST 45 40 40 40 35 35 35 30 30 30 25 25 25 20 20 20 15 15 15 10 5 10 10 0 5 5 % heat soluble collagen soluble % heat 0.5 1.5 2.5 b0 0 c a 0.5 1 1.5 2 2.5 0.5 1.5 2.5 3.5 Av. Daily Gain, kg/d
Collagen solubility in LD with increasing ADG shows different trend on grass v. alfalfa & grain – diet component effect? Trends suggest that diets affected the collagen stability of the two muscles in very different ways
Archile-Contreras et al (2010). Meat Sci. 86:491-497 Functionally different muscles have different IMCT content, composition
Is capacity for IMCT turnover intrinsically different between different muscles?
If collagen synthesis and (some) degradation is controlled by fibroblasts -
Are fibroblasts from different muscles phenotypically different? Fibroblasts from three different beef muscles: 25 a
• Have different proliferative capacity (a) 2 20 - b • Express MMP-2 differently 15 c 10
pg of MMP of pg 5 and Latent (b) activity of MMP-2 of fibroblasts (at passage 1 after isolation) 0 LD ST SMD
180
2 (b) Factors affecting IMCT turnover may have - 170 different effects in different muscles. 160 a MMP - a Responses of fibroblasts may be tailored 150 a to a specific muscle environment. 140
130 pg of Pro of pg 120 Archile-Contreras et al .J. Anim. Sci. LD ST SMD 88: 4006-4015
Environmental conditions
Social stress Housing
Fasting Feeding strategy
Physiological conditions Exercise
Altered collagen metabolism in muscle? Oxidative stress activates pro-MMP-2
Kandasamy et al (2010) Cardiovasc Res 85: 413-423
“Oxidative stress (ONOO−) and cellular glutathione (GSH) react with the critical cysteine residue in the highly conserved PRCGVPD domain,...... resulting in an active, full-length enzyme” Environmental conditions
Social stress Housing
Fasting Feeding strategy
Physiological conditions Exercise
Altered collagen metabolism? Net effect of reactive oxidative species (ROS) is to decrease collagen synthesis by muscle fibroblasts
Archile-Contreras AC, Purslow PP. (2011) Oxidative stress may affect meat quality by interfering with collagen turnover by muscle fibroblasts. Food Research International, 44: 582–588 Environmental conditions
Social stress Housing
Fasting Feeding strategy
Physiological conditions Exercise
Altered collagen metabolism
Can vitamins (E,C) counteract effects of oxidative stress on collagen turnover ?
Study effects of ROS + vitamins on cell cultures of muscle fibroblasts 1800
LD ST 1600 a a
1400
control a a 1200
1000
800
600 b b 400 b d
200 c c Percent change from the from change Percent 0 0.1 mU/mL 100 µM Vit E 50 µM Vit C 50 µM Vit C & E 50 µM Vit C & 100 X/XO 0.1 mU/mL X/XO 0.1 mU/mL X/XO 0.1 mU/mL X/XO µM Vit E + 0.1 mU/mL X/XO
X/XO : superoxide generating system based on xanthine/ xanthine oxidase 50 LD ST
40 control a 30 b c 20 e
10 e e e d d
0 Percent change from the from change Percent -10 f 0.1 mU/mL 100 µM Vit E 50 µM Vit C 50 µM Vit C & E 50 µM Vit C & X/XO 0.1 mU/mL X/XO0.1 mU/mL X/XO0.1 mU/mL X/XO 100 µM Vit E + 0.1 mU/mL X/XO
In the presence of ROS, vitamins E and C increase both MMP activity and collagen synthesis by muscle fibroblasts.
Vitamins E and C may increase the repair and remodelling/turnover of IMCT
Effects in all cases are muscle - specific
Archile-Contreras et al (2011) Journal of Agricultural and Food Chemistry 59: 608-614
Location of collagenase/gelatinase activity in skeletal muscle
Which cells maintain and turnover which parts of IMCT in muscle? - myoctes ? - fibroblasts of endomysium, perimysium, epimysium?
Use gelatinase in-situ zymography (ISZ) to study spatial variations of enzyme activity within muscle tissue
Cha & Purslow (2010) Comp. Biochem. Physiol. A: .156, 518-522 Rat soleus muscle – in-situ gelatinase zymography
Gelatinase activity is at borders of muscle fibres and fascicles
Activity strongly increased with MMP activator, and blocked by MMP inhibitor
Muscle cells variably express pro-MMPs (activated by AMPA) – related to size/fibre type?
G – normal gelatinase ISZ GM – GM1429 inhibitor specific to MMPs E – EDTA A- AMPA activator
Location of MMP activity
• Collagenase/gelatinase activity primarily located at muscle fibre/fascicle borders • Muscle cells in tissue sections are seen to express high levels of pro-MMPs intracellularly • Expression of MMPs from muscle cells possibly more important than expression from fibroblasts in remodelling IMCT • Variations in expression of MMPs may be muscle fibre-type specific Effect of stress hormone on MMP expression: Epinephrine (Adrenaline)
• Epinephrine is a general agonist of - & β- adrenergic receptors • Epinephrine increases calpastatin in muscle, by a signalling pathway associated with c-AMP dependent kinase (PKA)
• β -adrenergic chronic agonists (e.g. clenbuterol, ractopamine, cimaterol, salbutamol) mimic this effect, = growth promoters
Beta-agonist Muscle fibroblasts (NOR-10) ractopamine
Culture medium expression of MMP-2 and TIMP-1 expressed as percentage of the non-treated control in skeletal muscle fibroblasts (NOR-10) and myoblasts (C2C12) in response to ractopamine treatment. Serum-free conditioned media were supplemented with or without 2 or 10 uM myoblasts ractopamine and cultured for (C2C12) additional 6 or 24 hours.
Increase in both MMP-2 activity and TIMP-1 significant in myoblasts only Epinephrine & Ractopamine
In addition to effects on myofibrillar protein synthesis and metabolism in muscle cells:
• Epinephrine increases MMP-2 expression and activity • Ractopamine also increase MMP-2 activity – but increases TIMP-1 (inhibitor) expression much more
Possible that ractopamine may impact collagenous as well as myofibrillar components of meat toughness
In general, greater effects on activity secreted by myoblasts than on muscle-derived fibroblasts at physiologically-relevant levels.
Cha & Purslow, submitted Conclusions
Collagen stability in growing animals may be affected by nutrition levels, vitamins
Muscle location Fibroblasts from different
muscles show differences in MMP expression/activity
Muscle cells more receptive to stimuli affecting MMP activity than fibroblasts
MMP expression may vary Cardiac vs. between muscle fibre types skeletal Intramuscular connective tissue (IMCT) – an immutable “background”?
• Is IMCT important to meat tenderness?
• Can we degrade IMCT post-mortem?
• Can the amount of IMCT be changed?
• Can the quality of IMCT be changed?
Degradation Synthesis MATURE IMMATURE Implications for meat quality Collagen Collagen
Our series of studies shows that there are multiple factors which can affect the balance between expression and activity of IMCT-degrading enzymes and synthesis of new collagen (with less heat-stable crosslinks) by both muscle cells and fibroblasts.
The potential effects of any in vivo treatments based on these factors on meat tenderness can be assessed in future studies
However, muscle fibre type differences and fibroblast phenotype differences between muscles may provide extra complexity; a treatment applied to the whole animal may affect different muscles differently Archile-Contreras AC, Mandell IB, Purslow PP. (2010) Cha, MC, Purslow PP (2010) Matrix metalloproteinases are less Disparity of dietary effects on collagen characteristics and essential for the in-situ gelatinolytic activity in heart muscle than in toughness between two beef muscles. Meat Science, skeletal muscle. Comparative Biochemistry and Physiology - Part A: 86,491-497 Molecular & Integrative Physiology 156, 518-522
Archile-Contreras AC, Mandell IB, Purslow PP. (2010) Phenotypic differences in matrix metalloproteinase 2 Cha MC, Purslow PP (2010) The activities of MMP-9 and total activity between fibroblasts from three bovine muscles. gelatinase respond differently to substrate coating and cyclic J. Anim. Sci. 88: 4006-4015. mechanical stretching in fibroblasts and myoblasts. Cell Biology International, 34(6), 587-91 Archile-Contreras AC, Purslow PP. (2011) Oxidative stress may affect meat quality by interfering with collagen turnover by muscle fibroblasts. Archile-Contreras A, Cha M, Mandell I, Miller S, Purslow PP (2011) Food Research International 44: 582-588. Vitamins E and C may increase collagen turnover by intramuscular fibroblasts; potential for improved meat quality. Journal of Agricultural and Food Chemistry 59: 608-614
Funding: Discovery Grant (NSERC) & OMAFRA