Manipulating connective tissue turnover to improve 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 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 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 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 60C for 20 min and 34% after cooking at 75°C for 1 h 75C 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. , 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