Muscle Fibre Types and Regulation Notes

Muscle Fibre Types and Regulation

The Force-Velocity Relationship

Illustrates the relationship between force/power and velocity in muscle contraction.
Key parameters:
- $F_{max}$ : Maximum force.
- $P_{max}$ : Maximum power.
- $V_{max}$ : Maximum velocity.
A.V. Hill mentioned in relation to this relationship.

Lecture Aims

Identify the basis upon which skeletal muscle fibres are classified.
Examine the physiological properties of different fibre types.
Examine how plastic our muscle fibres are and the mechanisms underlying how they might change

Red or White?

Muscle fibres are classified based on myoglobin content:
- Slow-twitch fibres: Red, high myoglobin content.
- Fast-twitch fibres: White, low myoglobin content.
Example: Turkey breast (white) vs. Turkey leg (red) (Ranvier 1873).

Mechanical Response to a Single Electrical Impulse

Characterized by:
- TPT: Time to peak tension.
- Pt: Peak tension.
- ½RT: Half relaxation time.
Example values: 10N tension, 10ms duration.
EMD: Electro-mechanical delay.

Classification of Fibre Types - Twitch Speed Determinants

Activation: Rate of $Ca^{2+}$ release from the sarcoplasmic reticulum (SR).
Cross-bridge kinetics: Myosin ATPase activity/myosin isoform composition.

Tetanus

Mechanical response to multiple stimuli in slow and fast muscles.
Unfused tetanus.
Fused tetanus.

Frequency Response Relationship

Force production relative to stimulation frequency.
Slow fibres reach 100% force at lower frequencies compared to fast fibres.
Slow fibres: 50% force at 20Hz, 100% force at 50Hz.
Fast fibres: 50% force at 50Hz, 100% force at 100Hz.

Motor Unit Classification (Burke et al. 1973)

Slow-fatigue resistant (SR).
Fast-fatigue resistant (FFR).
Fast-fatiguable (FF).

Classification Based on Speed, Substrate, and Enzyme Characteristics (Peter et al. 1972)

Slow-twitch:
- High oxidative enzyme activity (HAD, SDH, CS).
- Low glycolytic enzyme activity (LDH, PFK, PHOS).
Fast-twitch oxidative:
- Medium oxidative and glycolytic enzyme activity.
Fast-twitch glycolytic:
- Low oxidative enzyme activity.
- High glycolytic enzyme activity.

Early Classification of Muscle Fibres

Property	SLOW (ST)	FAST (FT(a))	FASTEST (FT(b))
Twitch Properties	ST	FFR	FF
Colour (Myoglobin)	Red	White	White
Twitch & Metabolism	SO	FO	FG
*ST: Slow Twitch
*FFR: Fast Fatigue Resistant
*FF: Fast Fatigable
*SO: Slow oxidative
*FO: Fast oxidative
*FG: Fast glycolytic

Myosin ATPase

ATP hydrolysis:
- $ATP arrow ADP + P_i + Energy$
M.ATP represents the myosin-ATP complex.

Fibre Types Characterized Using ATPase Histochemistry

Type I fibres.
Type IIa fibres.
Type IIb fibres (IIx in humans).
Pre-incubation at different acid and alkali pHs allows identification of different fibre types

The Myosin Cross-Bridge (Rayment 1993)

Heavy chains.
Light chains (Essential Light Chain - ELC, Regulatory Light Chain - RLC).
Actin binding site.
ATP pocket.
Neck region.
Motor domain.
50kD, 25kD, 20kD sizes

Myosin Heavy Chain (MHC) Isoforms

MHC-I (Slow).
MHC-IIA (Fast).
MHC-IIX (Fast) (formerly IIb, sometimes IId).
SDS-PAGE used to resolve MHC isoforms.
Rodents have IIA, IIX, and IIB isoforms.

Does IIB Isoform Exist in Humans?

Yes, but in specialized muscles (e.g., extra-ocular and laryngeal muscles - Andersen et al. 2002).

Co-expression of MHC Isoforms

Hybrid fibres can express:
- MHC-I/MHC-IIA.
- MHC-IIA/MHC-IIX.
Observed in single fibres (Andersen et al. 2004).

Distribution of MHC Isoforms

Vastus Lateralis muscle example (Andersen et al. 1994).
Healthy Young:
- MHC-I
- MHC-IIA
- MHC-IIX
- MHC-I/IIA.
- MHC-IIA/IIX.

Match Between mRNA and Protein (Andersen & Schiaffino 1997)

In situ hybridization.
ATPase Histochemistry.
Immunohistochemistry.
Monoclonal antibody.
Fibre 31: MHC-I mRNA matches MHC-I protein.
Fibre 32: MHC-IIA mRNA matches MHC-IIA protein.
Fibre 33: MHC-IIX mRNA matches MHC-IIX protein.

Other Important Contractile Proteins

Exist as fast and slow isoforms.

Major Myosin Heavy Chain (MHC) and Light (MLC) Isoforms in Mammalian Skeletal Muscle (Schiaffino & Reggiani 1996)

	Developing Muscles	Fast Muscles	Slow Muscles
MHC	Emb-MHC, Neo-MHC	MHC-IIA, MHC-IIB/IIX(d)	MHC-I (beta slow)
MLC (alkali)	MLC1emb, MLC3f	MLC1f, MLC3f	MLC1s
MLC (regulatory)	MLC2f	MLC2f	MLC2s
*Gene family

Myosin Light Chain Isoforms

SDS Page - different composition of gel compared to MHC – smaller proteins!

Multiplicity of Protein Isoform Expression

Highlighted in striated muscles (Schiaffino & Reggiani 1998).

Classification of Muscle Fibres (Revisited)

Property	SLOW (ST)	FAST (FT(a))	FASTEST (FT(b)*)
Twitch Properties	ST	FFR	FF
Twitch & Fatigue	ST	FFR	FF
Colour (Myoglobin)	Red	White	White
Twitch & Metabolism	SO	FO	FG
ATPase (Histochemistry)	Type I	Type IIa	Type IIb*
Myosin Heavy Chain (SDS-PAGE)	MHC-I	MHC-IIA	MHC-IIX
*IIb is IIx in humans

Lecture Aims (Revisited)

To identify the basis upon which skeletal muscle fibres are classified.
To examine the physiological properties of different fibre types.
To examine how “plastic” our muscle fibres are and the mechanisms underlying how they might change.

Muscles with Different Functions

Have different fibre compositions (Harridge et al. 1996).
Soleus: High MHC-I.
Vastus Lateralis: Mixed MHC-I, MHC-IIA, MHC-IIX.
Triceps Brachii: Higher in MHC-IIA/IIX.

Human Single Fibre Function In Vitro

Chemically skinned fibre segment.
Force transducer and motor attached.
pCa = 4.5
SDS-PAGE used to indentify MHC

Human Muscle Function In Vitro

Single fibres are chemically skinned using detergent (glycerol / Triton X100).
Fibres are electrically dead, without a membrane potential.
Activated in vitro using solutions containing $Ca^{2+}$ , ATP, and factors like PCr.
Experiments at 12-15°C.
Isometric force, force during shortening measured to create force-velocity curve.
Vmax calculated from extrapolation of F-V curve.
Power calculated.
$V_0$ (unloaded maximum shortening velocity) calculated from slack test manoeuvres.

Isometric Force in Human Skinned Fibres

Measured at max $Ca^{2+}$ activation (pCa 4.5), 12°C, and sarcomere length 2.5µm.
Fibre diameter measured for CSA calculation.
Note: Skinned fibres may swell, overestimating CSA by ~20%.

Force & Power - Velocity Relationships

Of different human fibre types (Bottinelli et al. 1996).
Data normalized for differences in fibre CSA, length, and volume.
Experiments at 12°C.
IIx, IIa, I

Large Range of Vmax Values

Observed in fibres containing the same MHC isoform.
*IIX fibres may still be called IIb here!

Myosin Light Chain Composition

Do differences in myosin light chain composition explain the variability in $V_0$ in fibres containing the same MHC isoform?

In Vitro Motility Assay

Speeds of fluorescently labelled actin filaments moved by myosin motors are calculated.
Allows the study of myosin behavior outside the constraints of the sarcomere.
Myosin (MHC-IIa) extracted from human single fibres moving fluoresced actin filaments.

Actin Sliding Filaments

actin sliding filaments ( $V_f$ ) on myosin isoforms before (PRE) and after (POST) training in young (YO) subjects
*YO MHC1 MHC2A
*Canepari et al. (2005) J Appl Physiol

Differences in Behaviour Between Species

Table 1. Maximum shortening velocity (V) determined with the slack test protocol in rabbit and in mouse single muscle fibres
*Think scaling!

Force Velocity Relationship and Power

Same power, but different optimal velocities.
Arrangement of sarcomeres (series and parallel) determines muscle function.
Series: Velocity.
Parallel: Force.

What Gives a Muscle Good Endurance?

$O_2$ delivery.
- Capillary supply.
$O_2$ utilization.
- Mitochondria.

Mitochondria

Power stations of cells; sites of oxidative phosphorylation.
Contain enzymes for:
- Fat metabolism (β-oxidation).
- Krebs cycle.
- Electron transport.

Activities of Glycolytic and Oxidative Enzymes

(μmoles. min-1 g wet weight-1)
Type I, Type IIa, Type IIx
Glycolytic:
- Phosphorylase.
- Phosphofructokinase.
Oxidative:
- Succinate Dehydrogenase.
- Citrate Synthase.

Capillaries and Lipid Content

Higher capillaries around type 1 fibres - ↑ perfusion
Higher lipid content in type 1 fibres

Metabolic Differences between Fibre Types

Type I fibres are for “endurance”:
- High mitochondrial density.
- High capillary density.
- High myoglobin content.
- High triglyceride content.
- High oxidative enzyme activity.

Differences Between Fibre Types (Relative to Slow)

Property	Slow (MHC-I)	Fast (MHC-IIA)	Super Fast (MHC-IIX)
$V_{max}$	-	↑	↑↑
Power	↑	↑↑
$V_{opt}$	-	↑	↑↑
Force	↑?	↑?
Rate of Force Rise	-	↑	↑↑
Relaxation		↑	↑↑

Differences Between Fibre Types (Relative to Slow, Continued)

Property	Fast (MHC-IIA)	Super Fast (MHC-IIX)
Fatigue Resistance		
Economy		
Mitochondria		
Cap. Density		

Summary I

Types and physiological function.
Adult human muscle may contain one (or more) of three MHC isoforms I, IIa & IIx (formally known as IIb).
Single fibre studies show that MHC expression is the prime determinant of $V_{max}$ .
In terms of power generation fibres follow the order: IIx → IIa → I.

Muscle Fibre Type Distribution in Athletes

Elite sprint and endurance athletes differ in their muscle fibre type distribution (Costill et al. 1976).
100 m sprinters and Distance runners.

Twin Studies

Nature or nurture? (Komi et al. 1977).
MZ twins of both sexes had almost identical muscle fibre composition.
The heritability estimate was 99.5% for males and 92.8% and for females.

Fibre Type Transformation and Training

Sprint / power training: MHC-IIX ↔ MHC-IIA ↔ MHC-I

Effects of Sprint / Power Training on MHC Expression

Andersen et al. 1994 general sprint training.
Adams et al. 1995 weight training.
Harridge et al. 1998 3s sprint cycling.

Rapid Down Regulation of MHC-IIX mRNA

*Rapid switch-off of the human myosin heavy chain IIX gene after heavy load muscle contractions is sustained for at least four days. Andersen JL, Gruschy-Knudsen T (2018)

Transformation of Vastus Lateralis

To a fast phenotype with spinal cord injury (Andersen et al. 1998).

Training Induced Changes

One of the earliest training studies showed no effect of endurance training on main fibre type composition (but a IIb(x) to IIa shift) (Andersen & Henriksson (1977).

Cross-Reinnervation Model

Demonstrating a slowing of contractile characteristics of fast muscle (FDL) when re-innervated by a motor nerve normally supplying a slow muscle (soleus) in the cat

Chronic Low-Frequency Electrical Stimulation

Induces a fast-to-low MHC transformation in animal muscle (Pette, D 1998).

No Switching of the Molecular Motor

(Myosin Heavy Chain Isoform expression) following prolonged sprint training. Harridge et al. (2002).

Increase in Number of Fibres Containing MHC-I mRNA

*In spinal cord injured tibialis anterior muscle following low- frequency electrical stimulation training. Vissing et al. (2005)

Compare and Contrast

*In situ hybridization measuring mRNA.

Mis-match Between mRNA & Protein

Evidence of fibre transformation and its direction.

Transforming I → IIA?

*Fibre 34 is a hybrid fibre containing both MHC-I and IIa isoforms at the protein level, but only MHC-IIA mRNA suggesting it is transforming from I - IIA

Calcineurin Hypothesis

*Blockade of the calcineurin pathway with cyclosporin A increased the type II fibre percentage in rat soleus muscle. Chin et al. (1998)

Possible Mechanism of Fibre Switching

Blockade of calcineurin with cyclosporin A led to an increase in the percentage of fast fibres.

Nuclear Translocation of NFATc1-GFP

*Is induced in fast muscle fibres by stimulation with a tonic low-frequency pattern Tothova et al.2006

Signalling Pathways Controlling Muscle Fibre-Type Specification.

*Slow-to-fast or fast- to-slow fibre-type switching in mammalian skeletal muscle.

Summary II

IIa and IIx isoforms appear to interchange readily with a change in activity.
Fast-to-slow switching of MHC isoforms is possible but, in human skeletal muscle there is little evidence from longitudinal studies and certainly is not easily achieved with voluntary exercise.
Fatigue resistance can improve in the absence of a switch in fibre type (as defined by the molecular motor).
The mechanism of MHC switching seems to relate (primarily) to a $Ca^{2+}$ activated calcineurin signalling process.

Training by Low-Frequency Stimulation

*Of tibialis anterior in spinal cord- injured men. Harridge et al. (2002)

Improvement in Fatigue Resistance

*Through training. Harridge et al. (2002)

One Legged Training

*Increased capillary density, mitochondrial content and peak oxygen uptake achieved when cycling with the specific leg that was measured. Saltin et al. (1976)

Endurance Training Increases Oxidative Enzyme Activity

**Levels in muscle Maughan, Gleeson & Greenhaff (1997)

PGC-1α

Is “a” master regulator of mitochondrial biogenesis and also regulates other “slow” genes
Lin et al. 2002

What Stimulates the Master Regulator PGC-1α?

AMPK (energy stress).
CaMK (Particularly CaMK II, activated by ↑ $Ca^{2+}$ during contraction).
p38MAPK.
SIRT 1 (Caused by the rise in lactate and NAD+).
CREB (cAMP Response Element Binding protein) (rise in circulating catecholamines through the β-adrenergic receptor).

Endurance Training Increases Capillary Density

*Saltin et al. (1976)

Increased Capillary Density Through Angiogenesis

*Changes in capillary density surrounding different fibre types before and after 12 weeks of endurance training. (From Ingjer 1979)

Functional Consequence of More Capillaries

*Training increases capillary number and increases transit time Richardson et al. 1999

What Stimulates Angiogenesis?

*Exercise up regulates angiogenic growth factors

What are the Signals Provided by Exercise Which Upregulate VEGF?

*See Hoier B and Hellsten Y. (2014) for good review

Summary III

Exercise induces an increase in mitochondrial enzyme activity and number - mitochondrial biogenesis.
PGC-1α is the master regulator of mitochondrial biogenesis.
More mitochondria allow greater flux through “aerobic” system – facilitates O2 utilisation.
Training induces the growth of new capillaries - angiogenesis.
This increases mean transit time - facilitates O2 delivery.
VEGF is a master regulator of angiogenesis.

Related Adaptations in Muscle to Endurance Training

*Overall, the effect of training (alongside mitochondrial biogenesis and angiogenesis) is for fats to referentially utilised to spare glycogen. *Essentially, metabolic health and sensitivity to insulin is ensured by having a high energy turnover – i.e. exercise !

Muscle Fibre Types and Regulation Notes