Dr Tim Parr Dr John Brameld Muscle Fibre Type and Meat Quality

Muscle Fibre Type and Meat Quality
Dr Tim Parr
Dr John Brameld
Division of Nutritional Sciences
School of Biosciences
School Biosciences
Food production and consumption research
Outline
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•
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Introduction to muscle fibre development
Fibre types and typing
Fibre type and meat Quality
Muscle growth
– Total Fibre Number
– Cross Sectional Area
• Potential relationship to meat Q
Skeletal muscle cells (Myo-blast, -tube, fibre)
Skeletal muscle cells in vitro
100µm
100µm
Proliferating
(undifferentiated)
myoblasts
100µm
Differentiated
myotubes
Section of skeletal muscle
- fibres are multinuclear
Muscle specific precursor
cells are derived from the somite
Myoblasts are formed in the myotome and dermamyotome
regions of the somite (mesoderm origin)
Wnt signalling & myogenesis
• Wnt’s are secreted signalling proteins (paracrine
or autocrine)
• Exposure to Wnt’s (& some other factors)
induces determination of muscle-cell lineage
• Disruption of Wnt signalling results in
transdifferentiation of myoblasts to adipocytes
• Transdifferentiation process could be utilised to
enhance Intra Muscular Fat (IMF) deposition
– Enhancing meat quality
What determines a muscle cell from other
cell types ?
• Muscle specific transcription factors (DNA-binding
proteins)
– Family of very similar TFs termed Muscle or Myogenic
Regulatory Factors (MRFs)
– All are bHLH type transciption factors
– Included are MyoD, myf5, Myogenin and MRF4
– Induced by Wnt’s (& other factors)
Induced expression of MRF
genes (e.g. MyoD) results in conversion to
muscle cell type
What do the MRFs do ?
• Switch on all the other genes that make a muscle cell a
muscle cell (particularly myogenin)
– Creatine phosphokinase (CPK) - enzyme marker of
differentiation, muscle specific form
– desmin
– α-actin
• Proteins that reflect Fibre type
– Myosin heavy chain (determining fibre type)
– troponin
– tropomyosin
Regulation of myogenesis
Regulation of myogenesis
Determination/
Commitment
Regulation of myogenesis
Proliferation/
cell division
Determination/
Commitment
Regulation of myogenesis
Growth factors
+/Proliferation/
cell division
Determination/
Commitment
Regulation of myogenesis
Growth factors
+/Proliferation/
cell division
Determination/
Commitment
Withdraw from cell cycle, Align,
Fuse & Terminally Differentiate
Regulation of myogenesis
Growth factors
+/Proliferation/
cell division
+/-
+/Single multinuclear
cell
Determination/
Commitment
Withdraw from cell cycle, Align,
Fuse & Terminally Differentiate
Growth Factor effects on muscle cell proliferation and
differentiation
Growth Factor effects on muscle cell proliferation and
differentiation
IGF’s and myogenesis
• Insulin-like growth factors appear to be unique in
that they can act as mitogens and differentiation
factors
– concentration-dependent
• IGF’s shown to increase myogenin expression
(cultured cells)
• Knockout of IGF-II (transfected with antisense) in a
spontaneously differentiating muscle cell line
prevents differentiation (autocrine IGF-II effect)
Regulation of Muscle fibres
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•
•
•
•
Numbers of fibres
Types of fibres
Satellite cells
Postnatal growth
Protein metabolism
Muscle fibres
• Basic muscle cell
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–
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–
Highly specialised, terminally differentiated
10-100 µm diameter
mm – cm long
Contains all typical cell organelles (mitochondria, ER,
ribosomes, etc)
• Major difference to other cells
– Multinuclear cells
– Contain myofibrils (contractile proteins)
– Specialised ER – sarcoplasmic reticulum
Skeletal muscle
• A particular muscle is made up of 1000’s of fibres
– Collected together to form bundles
– Surrounded by connective & adipose tissue
Skeletal muscle
• A particular muscle is made up of 1000’s of fibres
– Collected together to form bundles
– Surrounded by connective & adipose tissue
– The quantity of connective tissue (collagen) negatively
influences quality
– Quantity of adipose tissue influence quality
• Marbling: In pork2.5-3% intramuscular fat suggested to
improve flavour, juiciness and tenderness (Devol et al
1988)
• Higher content = negative consumer perception
Skeletal muscle anatomy
Skeletal muscle fibres
Muscle fibre type
• Classic histochemistry staining
• Type I slow oxidative
• Type IIA fast oxidative glycoyltic
• Type IIB fast glycoyltic
Maltin et al 2001
– This has been subsequently split into IIX and IIB
Muscle fibre Types- defined at different levels
• Physiological: defined at whole muscle then single
fibre by the speed of contraction
• Biochemical contractile characteristic: defined by the
acto-myosin ATPase
- reflects speed of contraction
• Metabolic: defined by the metabolic enzymes that
regenerate ATP, glycolytic through to oxidative
- metabolism to support contraction
• Molecular: expression of genes especially the Myosin
Heavy Chain (MyHC) genes.
Examination and defining fibres
• Acto-myosin ATPase staining
– Low pH the ATPase of slow fibres are active
– At high pH ATPase of fast fibres are active
pH 4.7
Choi et al 2009
pH10.4
– Allows staining of three types of fibre Type I, IIA and IIB
• Reflects contractile speed
• IIA and IIB difficult to define
Examination and defining fibres
• Metabolic based stains
– Slow type fibres have high oxidative metabolism (aerobic)
• Oxidative metabolism enzymes and electron transport chain
– Fast type fibres have high glycolytic metabolism (anaerobic)
• Glycolysis capacity
pH9.2
Maltin et al 2001
– Staining of Type I, IIA and IIB
• Based on metabolic component
Examination and defining fibres
• Molecular analysis
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Based on the expression of the four adult MyHC isoform genes
Type I, IIA, IIX and IIB; associated with fibres slow to fast
However large mammals don’t express MyHCIIB (pigs do)
Immunocytochemistry methods and in situ hybridisation on fibres
pH 4.7
MHC IIX
riboprobe
– Muscle fibres can co-express MyHC forms (esp fast)
Histochemistry vs molecular analysis
The molecular characterisation (average fibre
type characteristics in muscle) Ab approach
Western blot for MyHC
Anti-slow MyHC
Sazili et al 2005
Anti-fast MyHC
Relationship between W-blot MyHC Antibody and
Histochemistry
Sazili et al 2005
Examination and defining fibres
• Gene structure of MyHC isoforms
– Cluster of the thre fast MyHCs on chromosome 19 (cattle)
– The gene for MyHC Type I (MYH6) is on a separate
chromosome 10 (cattle) in tandem with cardiac myosin.
Muscle fibre types
MyHC (pigs)
MyHC
(large mammals)
Not a absolute scale
I
I
IIA
IIA
IIX
IIX
IIB
Muscle fibre types
I
IIA
IIB
Classical labelling of fibre type
Not a absolute scale
Metabolic characteristics of fibres
Lefaucher 2010
Simple relationships of fibre type composition to
meat quality ?
• Oxidative fibres
– Mitochondria, more phospholipids and myoglobulin
• Glycolytic fibres
– More glycogen, high Ca-ATPase activity, high glycolysis
• Implications for cold shorting and rigor
• Expectation that glycolytic muscle will have rapid
decline in pH due to high capacity for lactate
generation.
– High glycolytic potential
– STRESS and level of glycogen influences
Simple relationships of fibre type
composition to meat quality ?
• Fast Glycolytic fibres (Type II, X and B)
– More glycogen, high Ca-ATPase activity, high glycolysis
• Increased fast twitch glycolytic fibres in pigs
– Increases rate and extent of pH in meat
– Paleness
– Decreases water holding capacity
• But this relationship does not always hold, although it
is generally correct
Simple relationships of fibre type composition to
meat quality ?
• In Pigs Psoas major (mixed muscle type), faster pH
decline than slow Semispinalis and fast Longissimus
Lefaucher 2010
At slaughter circa pH 7
Simple relationships of fibre type
composition to meat quality ?
• Slow oxidative fibres (Type I, IIA (?))
– Mitochondria, more phospholipids and myoglobulin
• Increased slow twitch oxidative type I
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decreases rate and extent of pH in meat
Decrease paleness
improve water holding capacity
Phospholipids improve flavour
• Type I fibres susceptible to rapid glycogen breakdown
and Dark Firm Dry meat
Simple relationships of fibre type composition to
meat quality ?
• Across species the majority of studies have indicated
+ve relationship between SO fibres and tenderness plus
juiciness but there is not a clear general relationship
longissimus lumborum
Vastus lateralis
Muscles from same animal different relationships
Maltin et al 2003
Simple relationships of fibre type composition to
meat quality – proteolysis activity?
• Calpain:calpastatin (Ouali &Talmant 1990)
– Higher in Fast glycolytic muscles
– Low in Slow oxidation muscles
Sazili et al 2005
High slow MyHC = high calpastatin
Marbling - Intra-Muscular Fat (IMF)
Picture marbling
• IMF associated with
– juiciness, flavour and tenderness (indirectly)
– found within intramuscular adipocytes
– Deposited later in growth
• High muscularity with high glycolytic activity leads to
low IMF development
– Oxidative (red) muscle tends to have more IMF
• BUT no strict association with fibre type
– Semitendinosus Red (48% Type I) lower IMF than
white (20% Type I) (Hocquette et al 2010)
Summary
• Fibre type
– Defined by contraction, metabolism and MyHC expression
• Fibre type reflects metabolic activity
• Meat quality
– Size of fibres
– Glycolysis potential, effects on pH
• Slow oxidative fibres generally good for meat quality
• Some relationships between fibre type and meat quality
but there are numerous interacting factors
– Muscle type, species, breed, genotype, nutrition,
environment, slaughter conditions
and pm processing
Muscle mass is related to……..
• Total Number of
fibres TNF
– fixed a birth
• Cross Sectional
Area CSA
– alters with growth
– stimuli
• Length
These factors also potentially influence
meat quality
Development of Total Number of Fibres
• Primary Fibres
– First ones formed (early embryo)
– Normally become slow oxidative (SO) fibres (type I).
Also called red or β fibres.
• Secondary Fibres
– Form around the primary fibres
– Normally become fast oxidative glycolytic (FOG, type
IIA) or fast glycolytic (FG, type IIB) fibres. Also called
α-red or α-white fibres.
Primary and secondary fibre formation
Fast fibres (2°)
Slow fibre (1°)
ATPase staining for fast fibres
1° muscle fibres
2° muscle fibres
tend to form
tend to form
type I (slow) fibres
type II (fast) fibres
Proliferation and differentiation of
myoblasts mainly takes place in utero
Timings for appearance of fibres
Primary
Secondary
Tertiary
Length of
Gestation
Reference
Poultry
3-7 df
8-16 df
-
21 days
Rat
14-16
df
30 df
17-19 df
-
22 days
Bandman & Rosser
(2000)
Wilson et al (1988)
30-35 df
-
68 days
Dwyer et al (1995)
35 df
55 df
0-15 dpn
114 days
Sheep
32 df
38 df
62-76 df
145 days
Lefaucher et al
(1995)
Wilson et al (1992)
Bovine
60 df
90 df
110 df
278-283 days
Human
56 df
90 df
110-120
df
280 days
Guinea
pig
Pig
Gagniere et al
(1999)
Draeger et al (1997)
Genetic effects on myogenesis and
relationship to meat quality
The greater TNF the greater the capacity for growth
double muscled
Genetic effects on myogenesis
• Double muscling in cattle (e.g. Belgian Blue)
– Due to increased number of muscle fibres at birth
– Appears to be due to increased activity of and time
exposed to mitogens coupled with delayed
differentiation
– Mutation in myostatin gene (also called GDF-8, a
member of the TGFβ family) resulting in production of
inactive protein
– TGFβ inhibits myoblast proliferation (and differentiation)
→ removed an inhibitor.
Exogenous Hormone effects on
myogenesis
• In pigs it has been demonstrated that exogenous
growth hormone (GH) given at a particular stage
of pregnancy increases the number of muscle
fibres in resulting offspring
• May relate to IGFs
– GH ↑ IGF-I expression/production in many tissues
Total fibre number (x1000)
Effect of GH in Pregnancy on muscle Fibre Number
of Progeny at Birth
• GH administered at early
gestation (10-24 days)
increased muscle fibre
number
• Similarly, doubling maternal
dietary intake at early
gestation (25-50 days) also
increased muscle fibre
number (Dwyer et al, 1994)
400
350
300
250
200
150
100
50
0
Cont
GH (10- GH (50- GH (8024)
64)
94)
Rehfeldt et al (1993)
Effects on meat quality
• Increasing TNF
• Myostatin variants (Belgian Blue)
– Increased TNF
– Increased hypertrophy of glycolytic fibres
• Good meat quality
• Attributed to lower total collagen with higher solublity
(Ngapo et al 2002)
Muscle mass is related to……..
• In farm species fibre
number is set at or
close to birth
• Post-natally extra fibres
not formed only
increase in size (length
& CSA)
- Hypertrophy
Selection for growth
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•
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•
Compare domesticated vs wild animals
Selection for growth rate and leaness
Shift to more glycolytic and less oxidative fibres
Increase CSA of fibres
High muscularity is associated
with large type IIB fibres
• Associated with mass
JSR Geneconverter
JSR Ltd UK
Stages of muscle growth
• Muscle fibre number is set at or close to birth. This means
that:
– Extra fibres cannot be formed post-natally, they can only
increase in size (length & width)
– However, there are satellite cells which are able to
proliferate and fuse (differentiate) with existing fibres
(repair mechanism and also growth)
• Stimulated with many Growth factors
• Muscle cell DNA content ↑ during post-natal growth
• But, ↑ in size mainly due to ↑ in protein content
What are Satellite cells ?
• Small mononuclear cells residing between the
sarcolemma & basement membrane
• Evenly distributed across muscle fibre surface (↑ density at
neuromuscular junctions)
• Represent 2-10% of total muscle nuclei in adult muscle
(more in young animals – decrease with age)
• Potential functions of satellite in transdifferentiation
• Muscle cells myoblasts to adipocytes
• Benefits for IMF???
Skeletal muscle anatomy
Satellite cells associated with muscle
fibres
Satellite cells
Muscle fibre hypertrophy
• ↑ width due to ↑ no. of myofibrils in the fibres
• ↑ length due to addition and/or lengthening of
sarcomeres
– Synthesis of myofibrillar proteins
• ↑ no. of nuclei (↑ DNA) gives fibres a greater
capacity to synthesise proteins
• Increase in protein content and increase CSA
Hypertrophy – protein accretion
AMINO
ACIDS
Synthesis
Degradation
PROTEIN
Accretion = synthesis – degradation
Growth both are influenced
Regulation
• Protein synthesis and degradation are controlled
separately (and are each vast areas of current
research)
• Factors involved in their regulation include nutrients,
hormones, physiological state (e.g. infection) and
functional demand (exercise/ work)
Factors affecting muscle protein synthesis and breakdown
Growth factor effects (selected example)
•
•
•
•
Beta-adrenergic agonists
Strong growth promoters increase hypertrophy
Switch to fast type muscle
Changes in expression of Myosin Heavy chain to faster
forms movement toward Type IIX and IIB(?)
• Decrease fat
• Tendency to DFD (decrease glycogen)
• Associated with decreased meat quality (shear force )
– the maximum increase being 146% !!!
Beta agonist trial – sheep
• Animals treated for 6 days with
– Dietary beta-agonist
• (cimaterol) at 10 ppm
– Growth hormone (long acting single dose)
• bST @ 150 mg/40kg BW
• Treatment at 60 days and 120 days of age
• Examination of muscles
– Histochemistry and MyHC mRNA levels
Change in diameters
Effects of beta-agonist and GH treatment on ST fibre diameters in 60d
old male lambs
50
45
40
Diameter (µm)
35
30
Beta agonist
Control
GH
25
20
15
10
5
0
SO
FOG
FG
Fibre type
ALL
Change in diameters
Effects of beta-agonist and GH treatment on ST fibre diameters in
120d old male lambs
50
*
*
45
*
40
Diameter (µm)
35
30
Beta agonist
Control
GH
25
20
15
10
5
0
SO
FOG
FG
Fibre type
ALL
Change in % fibre type
Effects of beta-agonist and GH treatment on ST % fibre types in 120d
old male lambs
60
*
50
Fibre type %
40
Beta agonist
Control
GH
*
30
20
10
0
SO
FOG
Fibre type
FG
Change in % of MyHC isoforms
assessed by qRT-PCR
Switch to faster fibre type
• But is it the fibre type per se or associated changes
• With beta agonist there is a change in proteolysis
systems
– Decrease protein degradation
– Inhibits post-mortem proteolysis capacity
• Increases in calpastatin
– Endogenous inhibitor calpain
– Micro-calpain is responsible for tenderness
Calpastatin changes with beta agonists
- cattle fed beta-agonists 28days
Calpastatin
mRNA *
1000
900
800
activity
700
600
500
400
Muscle
weight
**
300
Calpastatin
*
200
µ-
100
*
0
C
Parr et al. (1992)
β
C
β
m*
C
β
C
β
C
β
Summary growth fibre type and meat Q
• Increase CSA detrimental
• Hypertrophy of fast oxido-glycolytic fibre are detrimental
– Switch to fast fibre
– Intrinsic nature to be low quality
• BUT is it change in CSA per se?
– Other associated factors
– Changes Ca metabolism (halothane pigs)
– Changes in glycogen (RN carrier pigs)
Summary
• Fibre type
– Defined by contraction, metabolism and MyHC expression
• Slow oxidative fibres generally good for meat quality
• Fast glycolytic fibres generally band for meat quality
• Some relationships between fibre type and meat quality but
there are numerous interacting factors
– Muscle type, species, breed, genotype, nutrition, environment,
slaughter conditions and pm processing
• Factors influencing growth affect fibre type (TNF and CSA)
• Assocation of fibre type to meat quality may be due to a change
in the muscle which gives a change in fibre type