Regulation of Muscle Mass1st November 2010Learning Objectives:

Describe the relation between muscle mass and mortality in chronic disease.Outline the developmental steps from myoblast to myofibre.Describe the transcriptional control of gene expression in skeletal muscle using the protein skeletalmuscle α-actin as an example.Describe the mechanisms responsible for skeletal muscle hypertrophy including the role of IGF-1.Describe the mechanisms controlling skeletal muscle atrophy by ubiquitin ligases.Describe the relationship between the hypertrophic and atrophic signaling pathways referred toabove.

Slides 1-7

Chachexia is the loss of muscle mass (systemic wasting of muscle) and occurs in many chronicdiseases including cancer, AIDS, heart disease and chronic obstructive pulmonary disease(COPD).Muscle mass is an important regulator of survival/mortality in chronic illnesses, and the loss ofmuscle mass is more closely associated with death than many standard prognostic indicators.

E.g. COPD is a lung disease in which smoking results in the damage to the lung tissue andrestricts the amount that people can breathe.In this disease, FEV1 (expressed as a % of the normal value) is a classic marker of diseaseprogression and severity), so normally FEV1 is expected to be a good predictor of how wellpeople survive.In limiting cases it is predictive (i.e. if something else doesn’t get you, the inability to breathesurely will).However, this relationship affects only a few people and something else is important indetermining survival in many others.One better predictor of mortality is muscle mass, in this case, measured by quadricepsstrength. Not only are muscle mass and strength important predictors of how long we willlive for, the ability to use muscle (i.e. move) is also related to the quality of patient lifemeasured using a questionnaire. So the amount and usability of our muscle is important.

Ageing and loss of muscle mass:

There is an increased risk of 1 year mortality at a BMI of less than 22 at the age of 65.In people over 50, the unintended loss of 10% body mass in a year increases the likelihood ofmortality by 60%.

The loss of muscle mass is a co-morbidity for many chronic diseasesThe effects of cachexia on the survival of patients with COPD:

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cachectic

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cachectic

Quadriceps weakness is also associated with an increased mortality – and therefore can also beused as an effective predictor of mortality.

Slides 8-15 – How does a cell become a muscle cell?

Muscle fibres

To understand how muscle mass is regulated, it is useful to understand where skeletal musclecells come from and the basics about how their differentiation is controlled.Adult skeletal muscle cells are multinucleate fibres, with the nuclei all around the edge of the cellbut this is not the way they start out.

If we look at the skeletal muscle in an embryo we can see that the nuclei are down the middleof the fibres but we still have one cell with lots of nuclei.

But like all other cells, skeletal muscle cells start life as individual cells with a single nucleus.There are two ways we could go from here to a multinucleated cell, and they are:

To divide the nucleus multiple times without dividing the cell, as happens in theformation of megakaryocytes (platelet production)To fuse cells together – which is how the skeletal muscle syncytium is formed(syncytium is one cell with many nuclei)

Skeletal muscle

Nuclear division without cell division ?

Fusion of mononuclear cells ?

So we start with several skeletal muscle myoblasts, and under the appropriate conditionsthese will stop proliferating, start to express markers of the differentiated skeletal musclephenotype and fuse together to form a myotube.

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This process is followed by the maturation of these myotubes into myofibres giving us ourskeletal muscle.

These steps in differentiation are principally controlled by a set of 4 transcription factors, whichare proteins of the basic-helix-loop-helix family. There are 4 of these transcription factors intotal:

MyoD

Which was identified using a differential screen for genes that were up-regulated whena particular line of fibroblasts was treated with aza-cytidine (which causes de-methylation of DNA, and allows these cells to convert into other cell types,particularly myocytes).

Transfection of MyoD into a number of different cells converts them into myoblaststhat can subsequently fuse into myotubes.

Myf5, MRF-4 and myogenin

These have been shown (using gene targeting) to regulate specific parts of thedifferentiation of skeletal muscles.

Skeletal muscleMyoD/Myf-5

Myogenin

MRF-4

myoblast

myotube

myofiberMyoD and Myf-5 play redundant roles in the differentiation of myoblasts into myocytes(myocytes refer to the fully developed muscle cell found in muscles)Myoblasts then require myogenin to drive the formation of myotubes.Finally, MRF-4 is required for the maturation of these myotubes into myocytes. Once expression of these transcription factors is activated, there is a positive feedback system whichmaintains and enhances their expression. Fusion to a myofibre is not the end of the story – as there are many different types of myofibres:

Some have fast contraction kinetics, contain few mitochondria and so rely on anaerobicmetabolism to provide energy, and are useful for fast responses and for strength.Others have slower contraction kinetics, contain more mitochondria and are useful forendurance activities.Different muscles have different functions and activities, so express different amounts of eachfibre type.

The fraction of each fibre type we have is important in determining how good we are at a particular

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The fraction of each fibre type we have is important in determining how good we are at a particularactivity.In a patient population, the fraction of Type I (Slow Fibres) is very important as it dictates how farthey can move (and therefore their quality of life too).The relationship between activity and fibre type is not one way, as the more someone moves in aday, the more MHC1 they tend to express in their quadriceps).

Further specification

Type IIXType IIB Type IIA Type I

oxidativeglycolytic

Power Endurance

Fast slow

Skeletal muscle fibre type

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How much do you move in a day (min) How much do you move in a day (min)

Activity modulates myosin type

Amount of MHCI

Amount of MHCIIa

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Which myosin predominates dictates endurance

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Amount of MHCI

Amount of MHCIIa

Which myosin predominates dictates endurance

Slide 16 – How is muscle phenotype controlled?Obviously we can control muscle mass by regulating the amount of message made and the amount ofprotein made from each message.

DNA

RNA

Protein

Transcription

Translation

Replication

How is muscle phenotype controlled?

Slide 17-20 – Regulating the amount of messageTranscription is controlled by differential activation of the promoter region of specific target genesusing distinct transcription factors. In determining how a cell becomes a skeletal muscle cell, we need to consider the factors that controlgene expression in skeletal muscle cells. We have already considered the 4 myogenic basic helix loop helix proteins, but there are someadditional regulators of skeletal muscle-specific gene expression. For skeletal muscles, we need to consider what factors should be built into the system to allowexpression of the gene in skeletal muscle cells, and what factors are likely to increase or reducethe expression of such a gene.

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the expression of such a gene. The easiest place to start is to look at the control of the contractile protein and consider when thegene needs to be expressed and what it needs to respond to.

The first thing to consider is that we want the gene expressed in skeletal muscle once theyhave become skeletal muscle cells. The easiest way to do this is to use the same transcriptionfactors that are involved in causing the cells to become skeletal muscle cells in the first place. It is also important to consider that these proteins are contractile proteins, and they need tobe able to respond to changes in demand. These proteins make up a significant portion of themuscle cell and making them therefore consumes a significant portion of the cell’s energy.We only want to have enough and not too much. The organism also needs to be able to growand respond to changes in demand, so we need a system that will respond to that.

How do we achieve this control of protein amount?

There are two things we could regulate:1) The amount of protein2) OR the amount of mRNA production at the level of transcription

To do that, we increase/decrease the binding of proteins to the DNA predominantly in thepromoter region of the gene we are interested in.

The regulation of skeletal muscle contractile protein expression can be illustrated using skeletal alpha-actin as an example:

To determine that the protein is expressed in the right tissue (i.e. skeletal muscle only), we can usetissue-specific transcription factors that bind to the promoter regions of the gene. In skeletal muscle, the bHLH (basic-helix-loop-helix) transcription factors (that have already beendescribed) bind to a recognition sequence in the appropriate promoter and here in the skeletalactin promoter, we have the appropriate binding sites for the myoD class of factors.

As we don’t want some of the proteins made when the cells are still proliferating, we can switchoff gene expression, by inhibiting these myogenic transcription factors with similar proteins likeId and twist.

Skeletal muscle actin

I’m a contractile protein gene

I need to be expressed in a skeletal muscle cell

I don’t want to be expressed during cell

proliferation

MyoD, myogenin, MRF4, Myf5

(myogenic transcription factors)

SRF

SRF, MEF-2, NF-AT

TATA

I Need to respond to changing work load

Id, TwistSwitch of gene expression while the cells are still proliferating – so inhibit myogenic TF during this process.

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Skeletal muscle actin

I’m a contractile protein gene

I need to be expressed in a skeletal muscle cell

I don’t want to be expressed during cell

proliferation

MyoD, myogenin, MRF4, Myf5

(myogenic transcription factors)

SRF

SRF, MEF-2, NF-AT

TATA

I Need to respond to changing work load

Id, TwistSwitch of gene expression while the cells are still proliferating – so inhibit myogenic TF during this process.

We also have binding sites for a range of different transcription factors that respond toenvironmental factors related to the demand for the protein.

So there are binding sites for:Serum Response Factor (SRF)Nuclear factor of activated T-Cells (NF-AT)Myocyte enhancer factor 2 (MEF2)

The amount and activity of these factors is important in determining which fibre typepredominates.

The mechanisms that control transcription factor activity can be quite complex but allow the cell torespond to different environmental ques. For example:

Increasing contract occurs alongside increased average Ca2+ concentration in the cell, asCa2+ is required for contraction to occur.This increase in Ca2+ triggers an increase in the activity of two systems:

The CaMKIV and calcineurin system Calcinuerin targets the transcription factor NF-AT present in the cytoplasm, andcauses it to translocate into the nucleus, where it targets the transcription factorMEF-2.

CaMKIV targets MEF-2 directly.

In both cases, there is an increase in activity leading to an increase in the expressionof the target gene (in this case, the skeletal muscle alpha-actin).

The regulation of MEF-2 is not direct – it involves a second protein: histone deacetylases(HDAC4 and 5) which inhibit the expression of genes when they are in the nucleus, byincreasing the positive charge on the histone residues (which causes the DNA to becomemore compact and less accessible).HDAC4 and 5 bind to MEF-2 when they are in the nucleus and inhibit the expression ofgenes to which MEF-2 is bound.

CaMKIV phosphorylates these HDACs, and causes them to exit the nucleus andbind to the 14-3-3 proteins in the cytoplasm. MEF-2 can then bind other factors thatacetylate the histones, and open up the chromatin and increase gene expression.

Type IIXType IIB Type IIA Type I

Skeletal muscle fibre type

SRF SRFSRF SRF

NFATC1, 2, 3,4NFATC2, 3, 4NFATC2, 3, 4NFATC4

PGC-1a

MyoD MyoD myogenin

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Slides 21-24 – Regulating the Amount of Protein

There are two sides to regulating the amount of protein:Firstly, the amount of protein synthesisSecondly, regulating the amount of protein breakdownIt is the net turnover that is important

Once you are an adult, these two processes are in reasonable equilibrium, so that we don’t keepgrowing and the amount of muscle we have stays relatively constant, but this is a dynamic process sothat if you increase the amount of exercise you do, the amount of protein synthesis ANDdegradation increase, but synthesis increases more than degradation, so that we make more muscle.In chronic disease, the amount of breakdown decreases, but so does the amount of synthesis, andthe breakdown occurs to a greater degree, resulting in muscle loss.

Fractional changes in muscle protein homeostasis

Normal exercise Chronic disease

Immobility/ acute disease

synthesis

Breakdown

The regulation of protein levels involves both activity and hormonal control. For example, growthhormone, testosterone and IGF-1 all act to promote muscle growth, while glucocorticoids andmyostatin inhibit muscle protein synthesis. Both IGF-1 and myostatin will be focused in more detail – both of these proteins will also affectmRNA, but their effects on synthesis and protein degradation will be considered in greater detail.

Regulation of protein synthesisIncreased anabolic hormones or activity activate protein synthesis

Increased catabolic hormones or reduced activity activate protein

breakdown

Muscle protein synthesis

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Regulation of protein synthesisIncreased anabolic hormones or activity activate protein synthesis

Increased catabolic hormones or reduced activity activate protein

breakdown

Muscle protein synthesis

IGF-1

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myostatin

Slides 25-28 – Regulation of Protein SynthesisSummary of the factors involved in protein synthesis machinery:

Proteins are made by translating the mRNA code into a polypeptide chain, one amino acid at a time.We can consider two phases of this process:

One is the initiation phase where a ribosome binds to the 5’ end of an mRNA moleculeThe second is the elongation phase where the ribosome moves along the mRNA adding moreamino acids as it goes.

We are mostly interested in the initiation phase, because the rate of protein synthesis can be moreeffectively increased by increasing the rate of initiation than by increasing the rate of elongation.(This doesn’t mean that elongation isn’t also a target).

IGF-1 is a small peptide hormone that is part of the insulin family.

The predominant source of circulation IGF-1 is the liver, where it is made in response togrowth hormone.However, some tissues, including skeletal muscle can produce IGF-1 locally, and in musclechanges in IGF-1, expression occur in response to stretch and load on the muscle. IGF-1 signals by binding to the IGF-1 Receptor, a tyrosine kinase that exists in themembrane as a preformed homodimer. Binding of IGF-1 to this receptor causes aconformational change that activates the tyrosine kinase portion of the protein leading to thephosphorylation of IRS-1.One of the immediate targets of IGF-1 signalling is P13Kinase, which in turn phosphorylatesand activates protein Kinase B (also known as Akt). Here we have the activation of aclassic protein kinase cascade, which allows for the amplification of a signal as one kinase canphosphorylate more than one target at each step in the chain.In skeletal muscle, there are two Akt isoforms, that we need to consider:

Akt1Akt2

These proteins appear to be coupled to different components because knockout of Akt1 inmice leads to growth retardation, while knockout of Akt2 leads to Type 2 Diabetes.It appears in part from this, but also from direct assays of IGF-1 and insulin function, thatAkt1 is a target of the IGF-1, whereas Akt2 is a target of the insulin receptor. We will now consider two targets of Akt that are involved in increasing protein synthesis aspart of the hypertrophy signaling cascade:

1) glycogen synthase kinase (GSK3β)

GSK3β inhibits the activity of one of the initiation factors elf-2B, so inhibitsprotein synthesis by blocking the loading of Met-tRNA into the ribosome.Inactivation of GSK3β by Akt releases this block, leading to increased RNAsynthesis.

2) mTOR complex (Mammalian target of Rapamycin)This, on the other hand, phosphorylates S6 kinase, leading to increasedphosphorylation of the ribosomal protein s6.

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One further target of this system is the elF4E binding protein, which sequesterselF4E. Phosphorylation of elF4E binding protein reduces its binding of elF4Eallowing elF4E to participate in the initiation of protein synthesis.

eIF4-E

7mG

eIF4E-BP

mTORP

eIF4E-BP•

Slides 32-43: Myostatin and the Regulation of Protein Breakdown

The opposite of muscle protein synthesis and hypertrophy is muscle breakdown and atrophy.It is important to note that although we largely consider this a pathological process, it is a normal andimportant system, which allows us to use the large amount of stored energy in skeletal muscle protein,during times of starvation. Myostatin was discovered by identifying the gene that was mutated in hypermuscular breeds ofanimals. These breeds include the Belgian blue cows and bully whippets. The animals don’t produceany of the protein myostatin- an inhibitor of muscle growth. Not only is myostatin an inhibitor ofmuscle growth but it also affects fibre type and these animals have a lot of fast fibre but very littleslow fibre. The cows are therefore not as good for meat production as the quality is not high (tootough) and the dogs may run fast but are susceptible to cramp. However, in a normal person, changes in myostatin do coincide with muscle wasting and a loss ofstrength; as shown in the graph below where weak COPD patients have more myostatin than strongones, and the amount of myostatin they make is inversely correlated with the amount of activity theyperform.

Myostatin in COPD patients

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Normalised Myostatin mRNA (AU)

6 Minute Walk (meters)

One effect of increased myostatin is the increased expression of two proteins called MuRF1 andatrogin. Not only are these proteins increased by myostatin, but they are also markedly upregulatedin a number of different atrophic conditions.

Atrophy

Inhibition of protein synthesis

Protein breakdown

Inactivity Reduced energy statusInflammation

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Atrophy

Inhibition of protein synthesis

Protein breakdown

Inactivity Reduced energy statusInflammation

MuRF1 and atrogin are both ubiquitin ligases, as such, their job is to add ubiquitin moieties toproteins. Ubiquitin is a small protein that functions in the cell, primarily as label that indicates that aprotein is to be degraded by a proteosomal system. So an increase in the expression of MuRF1 andatrogin suggests that we will see increased protein degradation.

Ubiquitin dependent degradation

How are the expression of these proteins controlled, apart from in response to myostatin?

FOXO1 and FOXO3 are a pair of transcription factors that are important in the regulation of theexpression of the genes for MuRF1 and atrogin.The transcription factors FOXO1 and FOXO3 and their activity are regulated by proteinphosphorylation and nuclear localization. The active form of each protein is not phosphorylatedand is present in the nucleus, whilst the inactive form is phosphorylated and becomes localized in thecytoplasm.

How else is MuRF controlled ?

MuRF1Foxo

FoxoP

Akt PI-3K

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How else is MuRF controlled ?

MuRF1Foxo

FoxoP

Akt PI-3K

The regulation of the activity of FOXO transcription factors is therefore also an important aspect ofthe regulation of atrophy, and it involves 2 key components:

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the regulation of atrophy, and it involves 2 key components:

Firstly, Akt, which phosphorylates and inhibits FOXOSecondly, AMP Kinase, which activates FOXO

AMP kinase is a protein that is central to the ‘energy sensing system’. It is activated by AMP – amolecule that is present at low concentrations, but is in equilibrium with ADP. The equilibrium allowsthe cell to measure its energy status in the following way:

A muscle contraction leads to the conversion of ATP into ADP and most (but not all) of theADP is turned back into ATP by the creatine kinase reaction.Therefore some ADP remains.The resulting small increase in ADP after a period of contractile activity is measurable byNMR spectroscopy of contracting muscle. Thus a sign of heavy ATP use is the increase in ADP, which results in the conversion of 2molecules of ADP into 1 ATP and 1 AMP. This does not have a big effect on ATPconcentrations but increases the levels of AMP markedly as the amount of AMP in the cell isvery low.

Energy status

ATP ADP + Pi

2ADP ATP + AMPAdenylate kinase

Intracellular concentrations of AMP are very low You should remember that it is not the absolute concentration change that is important intriggering a response but the relative change so the percentage reduction in ATP is small butthe percentage increase in AMP is very large. Hence when energy levels are low ADPincreases and AMP increases by a much larger margin. This increase in AMP is then picked up by AMPK, which becomes activated. Activation ofAMPK can be both allosteric (an enzyme combining with a regulatory molecule) andthrough post translational modification (phosphorylation).

The stimulation of FOXO activity by AMPK may seem surprising as it is byphosphorylation and we have just said that phosphorylation of FOXOs leads to inhibitionthrough nuclear exclusion. However AMPK phosphorylates a different part of the proteinand this phosphorylation leads to activation of the transcription promoting activity ofFOXO.

AMPK has two other affects that are related to energy metabolism in muscle cells.

Firstly it inhibits protein synthesis by inactivating the mTOR complex. Given the expense

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Firstly it inhibits protein synthesis by inactivating the mTOR complex. Given the expenseof making protein this is a sensible short term move and can be seen to be closely associatedwith protein breakdown. The second effect of AMPK activation is an increase in mitochondriogenesis. This has theeffect of increasing the energy available from each glucose molecule, as the energy yieldfrom each glucose is higher when it is fully oxidised by the Krebs cycle rather than byglycolysis. Mitochondriogenesis is obviously a much longer term effect and may be restrictedto a subset of fibres.

How do these signalling pathways interact?These pathways are interrelated. Indeed as you have seen there are at least 2 nodal points mTOR and Foxo with activation of Aktincreasing the activity of mTOR and protein synthesis and inhibiting the activity of Foxo. Conversely AMPK increases the activity of Foxo but suppresses the activity of mTOR.

FoxO a nodal point for the regulation of protein turnover

Growth inhibitorsTGF-b, myostatin

Myostatin is now a target for therapeutic intervention and there are a number of ways that peopleare trying to combat muscle wasting by inhibiting the ability of myostatin to bind to its receptor orthe signalling of the receptor complex.

Methods to inhibit myostatin signalling

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Follistatin

Myostatin propeptideMyostatin antibody

Soluble Act IIRB ECD

Act IIRB

Alk 4/5SB-431542 SB-505124

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Imidazo-pyridines

SMAD2/3P

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Methods to inhibit myostatin signalling

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Follistatin

Myostatin propeptideMyostatin antibody

Soluble Act IIRB ECD

Act IIRB

Alk 4/5SB-431542 SB-505124

A-83-01

Imidazo-pyridines

SMAD2/3P

Muscle Diseases1st November 2010Learning Objectives:

Introduce the major categories of muscle diseases:Muscular dystrophiesCongenital myopathiesMetabolic & mitochondrial myopathiesInflammatory myopathiesNeurogenic changesSecondary changes in systemic pathological conditions

Outline the relevant clinical, pathological and molecular changes of the most common muscledystrophies Outline the relevant clinical, pathological and molecular changes of the most common congenitalmyopathies

Provide the basic knowledge on metabolic and mitochondrial myopathies

Describe the major features of inflammatory myopathies

Briefly describe the changes in skeletal muscle secondary to systemic pathological conditions.

Classification of Muscle Diseases

Rapidly growing fieldOver 300 diseases knownClassification based on phenotype, genetics, metabolic/functional tests, pathology

DivisionsPrimary (Muscle is the primary organ of disease)Secondary (Muscle is affected as part of a systemic disease)

Clinical Assessment of the Patient:

HistoryFamily history (consanguineous parents, other siblings etc.)Age of onsetMotor milestones (sitting, crawling, walking)Presenting symptoms: Type, severity of symptomsPain (intensity, at rest or exertional)CrapsMedications (statins, steroids)

ExaminationMotor milestones (sitting, crawling, walking)Distribution and severity of weaknessMuscle bulk (wasting, hypertrophy)Laxity, contractures, stiffnessGaitReflexesSkin changes (rush, hyperkeratosis)CK, EMG, NCV, MRI-Scans

Values of CK (Creatine Phosphokinase) is usually 25-200IU/L – an increase indicatesmuscle damage (very important)Lactate (when mitochondrial myopathy is suspected)Inflammatory markers (ANCA, ANA etc) in inflammatory myopathiesEMG – diagnostic in myasthaenia, helpful in distinguishing neurogenic and myopathicdiseasesMRI Scans – Identifies selective muscle involvements

Types of muscle disease:

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Types of muscle disease:

• Neurogenic• Myasthaenia

• Dystrophic• Myopathic

Neurogenic:- Problems at the neurone i.e. extrinsic to the muscle (i.e. secondary to the muscle –usually due to disease of peripheral nerves or anterior horn cells)Myasthenia:- Problems at the neuromuscular junction

Myopathic:- Problems/pathology in the muscle itself (i.e. primary or intrinsic to the muscle)Dystrophy:- Any of several hereditary diseases of muscular system characterized by weakness andwasting of skeletal muscles

Congenital MyopathiesTypically non-progressive.Static weakness present from birth or infancy.Most patients achieve the ability to walk independently.Typically the serum CK are normal or only mildly elevated, indicating low degree of muscledamageCharacterised by generalized muscle thinning and muscle weakness

Severity depends on the degree of disruption of the excitation/contraction coupling or the severity ofdysfunction of sarcomeric proteins – not so much on the ‘class of disease’Mild Congenital Myopathies:

Proximal Muscle Weakness:Difficulties in walking fastDifficulties in runningDifficulties in getting up from the floor

Axial WeaknessScoliosis commonRespiratory muscle weakness common even in ambulant patients

Nemaline Myopathies:This type of congenital disease is caused by a different gene/protein defect but give a similarpathology, but different clinical phenotype.

NebulinAlpha Actin (ACTA1)Beta Tropomyosin (TPM2)Alpha Tropomyosin (TPM3)Troponin T1 (TNNT1)

Muscular Dystrophies:General Aspects:

Congenital (birth or first 6 months of life)Childhood onset / Adult Onset

Duchenne (only childhood) and BeckerLimb girdle muscular dystrophiesOther

Often progressive weaknessVariable severity: from congenital to adult onset depending on the subtype (many subtypesare recognized)Weakness is usually proximal (Rarely distal)

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Weakness is usually proximal (Rarely distal)Respiratory muscle weaknessLimitation of joint movements often accompanies the weaknessMuscular atrophy often with associated pseudohypertrophy in the same patient.

Muscular Dystrophies: Class Proteins involved…

Transarcolemma (Duchenne, sarcoglycanopathies, others)EnzymesNuclear envelopeExtracellular matrix (Collagen VI)SarcomereOthers (Cytoskeleton, ER proteins of unknown function)

Dystrophies (Pathology)Wide variation in fibre sizesNecrosisFibrosis, fatHypercontracted fibresSplit and whorled fibresInternal nucleiBasophilic fibres

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Duchenne Muscular Dystrophy1 in 3500 male births (gene Xp21 – 79 exons – 427kDa Protein)Symptomatic <1 year – 5 yearsLoss of independent walking 6-12 yearsDeath usually around mid-teens to early twenties30% significant learning difficultiesTrials testing gene transfer

Investigations:CK: 50-100x normalDeletion in gene detectable in 70%Muscle biopsy:

Histology DystrophicImmunocytochemistry shows absent dystrophin

Motor difficultiesLate walking in 50% >18 monthsToe walkingUnable to run or jumpFallsStairs and climbing hardGower’s ManoeuvreProgressive joint contractures scoliosisWeakness of the Respiratory muscles

“Typical Becker”Mean age of onset of symptoms is around 11 yearsCalf pains, slower than peersVariable progressionLoss of walking ability (only in 18%) – Mean age at loss of walking is around 37 yearsScoliosis extremely uncommon (an abnormal lateral curve to the vertebral column)Dilated cardiomyopathy, however, very common

Dysferlinopathy – Dystrophies may occur late in life

Proximal involvementVery high Creatine KinaseOnset is usually around 2nd to 3rd decadeSlow progressiveChromosome 2p13.355 ExonsMissense, deletions, insertionsUbiquitous proteins of 230kDa unknown function – repair of membranes

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Ubiquitous proteins of 230kDa unknown function – repair of membranesDifferent phenotypesSome mutations associated with inflammation – mistaken as polymyositis

Energy in Muscle FibresATP/ADP is the immediate source of energyOxidative phosphorylationAnaerobic glycolysis (use of glucose stored as glycogen lipids)Creatine Kinase ReactionAdenylate Kinase Reaction Associated Pathologies:

Mitochondrian myopathiesGlucose metabolismLipid storage diseases

Mitochondrial Myopathies:Inheritance for the motherOnset at any ageVariable severityMultisystemic (Brain, muscle, heart, sensory system)Affects mitochondrial or nuclear DNAImpairment of respiratory chain enzymes

DiagnosisHigh LactateBiopsy (not always)Test of respiratory chain enzymesDNA Analysis

Typically involving multiple systems:Mitochondria Encephalopathy Lactate Acidosis Stroke

Variable age of onsetSeizure, dementia, myopathyExercise intolerance, hearing loss, short statureA3243G in more than 95% of cases

GlycogenosisLysosomal (Acid Maltase deficiency – digests glycogen to glucose)Non-lysosomal (enzymes involved in production or catalysis of glycogen and enzymes inglycolysis), Also deficit of glycogen synthase 1 – No glycogenOnset at any ageExercise intoleranceCardiac involvementRhabdomyolysis

Myophosphorylase deficiency or McArdle’s DiseaseGlycogen Phosporylase

Removes glycose residues from Alpha-(1,4)-linkages within glycogen moleculesProduct of reaction: Glucose-1-phospate

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Product of reaction: Glucose-1-phospateLeads to:

Exercise IntoleranceAge of Inset 10-50 yearsSecond wind phenomenon

Pathology:PYGM gene – 11q13Autosomal recessive80 known types of mutationCommon stop codon mutation in exon 1 (nonsense ARG49)No protein expression or unstable proteinMainly impairment of aerobic glycogenolysis.

Disorders of Lipid MetabolismFatty acid are released from fat and used in muscle after long exercise when glycogen is exhausted toproduce AcetylCoA to feed the Krebs Cycle and produce ATPFeatures depend on the enzyme affected:Onset at any ageVariable weaknessIntolerance to exerciseCardiac involvementRhabdomyolysis

Inflammatory MyopathiesPathology caused by an inflammatory reaction against muscle fibresCommon in adultsUsually immune-mediatedSlow or acute onsetVariable distribution of weaknessUsually high CKUsually steroid responsive DermatomyositisPolymyositis (rare)Inclusion body myositisOverlap syndrome (in course of Lupus, Rheumatoid Arthritis)Anti-Synthetase AntibodiesUnusual forms (pipestem; COX-Negative polymyositis)

DermatomyositisChildren and adultsAcute onsetSkin and muscle involvementLife threatening in childrenHigh CK (up to 20,000 IU/L)Steroid ResponsiveCD4/CD20 in perimysium – upregulation of MHCI; C5b-9 in capillaries

Neonatal myosin

T lymphocytes

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Neonatal myosin

T lymphocytes

Sporadic Inclusion Body MyositisMost common inflammatory myopathy after 50 yearsAsymmetrical, distal weaknessNormal CKProgressiveSteroid unresponsiveRimmed vacuoles, endomysial CD8 lymphocytes – MHCI upregulation – no C5b-9Unknown pathogenesis

Myopathies Secondary to Other ConditionsInfectionsCancer (Paraneoplastic myopathies)Endocrine-related myopathies (i.e. thyroid diseases)Toxic myopathies (drug induced)Systemic diseases (sarcoidosis, diabetes)Excessive exercise or disuse

Increasingly common secondary myopathies

Ageing – By far the most commonest ‘disease’ of muscleSarcopaenia: Less muscle, more fat

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Skeletal Muscle Contraction Notes1st November 2010Learning Objectives:

Outline  the  sequence  of  steps  in  the  “chain  of  command”  leading  to  skeletal  muscle  contraction  and  be  ableto  explain  its  significance  in  terms  of  normal  and  disease  states.

Motor units:Define, sketch and label and explain how the motor unit functions. Describe motor unit size, distribution within a muscle, and fibre type.Describe changes in motor units that result from of denervation and re-innervation.

Fibre types:Describe the properties (physiological, structural, and metabolic) of the 3 main types in human

skeletal muscle. List and briefly describe the methods that can be used to distinguish them

Muscle plasticity, changes in performanceDescribe briefly the changes in strength and endurance that results from training and from disuse. Describe satellite cells and how they are involved in skeletal muscle repair.Describe changes in muscle that occur during aging.Define the following:Atrophy: decrease in size of muscle fibres.Eccentric exercise: muscle fibres are active while lengthening. Occurs in normal activities, for

example biceps brachii does it when a heavy load is being slowly lowered by extending the elbow joint.Hyperplasia: increase in number of cells. Probably doesn’t happen in human skeletal muscle.Hypertrophy: enlargement of existing muscle fibres.

Satellite cells: small cells in skeletal muscle. One nucleus and little cytoplasm (in contrast the musclefibres) and located around the outside of muscle fibres. Dormant usually, but can proliferate and migrateto a damaged area of muscle fibres. Can differentiate and produce contractile proteins.

Definition:A motor unit is one motor neurone and all the muscle fibres it innervates (has synapses with,NMJs)

MotoneuronMotor neurone

Muscle fibres

NMJ

Functional Anatomy:Cell bodies of motor neurons are in the anterior horn of the spinal cordAxons of motor neurons lie in the ventral rootBranched terminals of motor axons are within muscle

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Chain of commands for voluntary skeletal muscle contraction:

Brain

Spinal cord

Peripheral nerve

Neuromuscular Junction

Muscle fibre membrane

Transverse tubular system

Ca release

Actin - myosin activation

Cross-bridge formation

Chain of Command for voluntary skeletal muscle contraction

Motor unit location

FORCEPOWER

Brain

Spinal cord

Peripheral nerve

Neuromuscular Junction

Muscle fibre membrane

Transverse tubular system

Ca release

Actin - myosin activation

Cross-bridge formation

Chain of Command for voluntary skeletal muscle contraction

Motor unit location

FORCEPOWER

(Lateral)Small distal muscle, fewer motor units, smaller motor units

Large proximal muscle, more motor units, larger motor units

But there are some exceptions (eye muscles and facial muscles – small muscles with many, small motor units)Motor Unit Architecture: Arrangement within the muscle

Each section shows the location of muscle fibres belonging to one motor unit.Muscle fibres are localisedBUT they are not segregated from muscle fibres belonging to other motor units.

Muscle Fibre TypesSlow (Type I)

Slow, Oxidative (SO)Slow Twitch and Shortening speedHigh Fatigue ResistanceHigh myoglobin content, Rich capillary supply and many mitochondriaHigh levels of oxidative enzymes

Fast, Fatigue Resistant (Type IIA)Fast, Oxidative, Glycolytic (FOG)Fast Twitch and Shortening speedHigh Fatigue ResistanceHigh myoglobin content, Rich capillary supply and many mitochondriaMedium/High levels of oxidative enzymes

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Medium/High levels of oxidative enzymes

Fast, Fatigable (Type IIB)Fast, Glycolytic (FG)Fast Twitch and Shortening speedLow Fatigue resistanceLow myoglobin content, poor capillary supply and few mitochondriaLow glycogen levels

Motor Unit Type

I IIA IIB (S, SO) (FR,FOG) (FF, FG)

Property

Twitch speed slow fast fastMax shortening speed slow fast fastFatigue resistance high high low

Red colour dark dark paleMyoglobin content high high lowCapillary supply rich rich poorMitochondria many many fewOxidative enzymes high med/high lowGlycogen low high low

Alkaline ATPase low high highAcid ATPase high low moderate

Myosin isoformAntibody staining orelectrophoresis I IIA IIB

Histological

Protein

Contractile profile

Identification

Metabolic profile

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Motor Unit Type

I IIA IIB (S, SO) (FR,FOG) (FF, FG)

Property

Twitch speed slow fast fastMax shortening speed slow fast fastFatigue resistance high high low

Red colour dark dark paleMyoglobin content high high lowCapillary supply rich rich poorMitochondria many many fewOxidative enzymes high med/high lowGlycogen low high low

Alkaline ATPase low high highAcid ATPase high low moderate

Myosin isoformAntibody staining orelectrophoresis I IIA IIB

Histological

Protein

Contractile profile

Identification

Metabolic profile

Duration of force in an isometric twitch

Is much longer lasting for S fibres

FF fibre type

FR fibre type

S fibre type

Is about the same for FF and FR fibres

Maximum Shortening Speed:This is proportional to the maximum speed that thick and thin filaments can slide over each otherIt is set by the maximum rate of the myosin-actin crossbridge cycle. Myosin isoform is different in slowthan fast muscle fibres, while actin is consistent (i.e. the same).Important for function because shortening speed giving maximum power is about 1/3 maximumshortening speed.So fast fibres produce high power at higher shortening speed than slow fibres.

Dependence of Power on Speed of Shortening

0

100

200

300

400

0 2 4 6velocity (fibre lengths/s)

Pow

er (m

W/g

mus

cle)

fast fibresslow fibres

Repeated Isometric Tetani:Fatigue Resistance = Capacity to sustain forceSlow fibres are very fatigue resistantFR fibres are fatigue resistant. Force stays reasonably high.FF fibres are easily fatigued. Force declines rapidly.

S fibresIsometric Force

2 min5 min

FR fibres

1st

FF fibres

1min30 s

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S fibresIsometric Force

2 min5 min

FR fibres

1st

FF fibres

1min30 s

N.B. All the muscles in a motor unit are the SAME type (i.e. S, FR or FF)The central nervous system activates specific motor units with the contractile properties that match the task tobe performed.

MotoneuronType I

Type IIBType IIA

Muscle Plasticity: How and why muscle changes

Training – for strength, for enduranceDisuseInjury & RepairAgeing

Strength Training – increases force produced during voluntary contractionType of training – high force, few repetitionsPart, but not all, is due to muscle hypertrophy (increase in muscle fibre size)

Hypertrophy (Whole Muscle) – Hypertrophied muscles has the same number of fibres. The diameter of eachfibre has increased (see next page diagram)

before

after

Each dot represents a fibre

Hypertrophy (One Muscle Fibre) – Hypertrophied muscle fibre has more contractile filaments, and thereforemore crossbridges producing more force

before

after

Each dot represents a contractile filament (myosin & actin)

Strength Training – increases force produced during voluntary contraction.

Type of training – high forces, few repetitionsPart, but not all, is due to muscle hypertrophy (increase in muscle fibre size)N.B. NO Hyperplasia occurs, i.e. the number of fibres in a muscle appears to be geneticallydetermined, and is NOT increased by strength training.

Quadriceps contraction: knee extension

Biceps femoris contraction: knee flexion

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Quadriceps contraction: knee extension

Biceps femoris contraction: knee flexion

Training Example

Task: isometric knee extension

fixed

During training experiment:Observe: increasing strength of knee extension and a decrease in the activity of an opposing muscle.

During training experiment:Observe: increasing strength of knee extension and a decrease in the activity of an opposing muscle.

Quadriceps contraction: knee extension

Biceps femoris contraction: knee flexion

At start of training

Quadriceps contraction: knee extension

Biceps femoris relaxed

After training

Conclusion: the improvement in the strength of knee extension is partly due to increased strength of kneeextensor muscle AND partly due to a NEURAL MECHANISM (task specific learning, or skill): learn tokeep opposing muscle relaxed.Endurance Training – Increases duration of performance, improves fatigue resistanceTypes of training – many repetitionse.g. Response of human adductor pollicis to endurance training in 8 human subjects:

Metabolic Adaptations:Increased capillary densityIncreased mitochondrial content and oxidative enzyme activityBut these adaptations decline quickly after stopping training

Non-Metabolic AdaptationsA change myosin isoform may occur

DisuseDisuse à leads to weakness and fatigue

Absence of load-bearing is the most potent cause e.g. bed restAnti-gravity muscles are the most affected (e.g. quadriceps)Mechanisms – Reduced recruitment of motor units and atrophy

Atropy – whole muscle

before

after

Atropied muscle has same number of fibres as before.

Diameter of each fibre has decreased.

Each dot represents a fibre

However, this is REVERSIBLE – effects are the opposite of training effects

Injury and RepairCommon causes:

DiseaseExternal TraumaInternal TraumaEccentric Exercise

Denervation and reinnervation – results in clustering of fibres of the same type (rather than mosaicpattern)’Muscle fibre damage and repair:

Calcium (Ca2+) enters through the damaged membraneMacrophages soon enterThe macrophages remove the debrisMeanwhile, the infarct also spreads along the muscle fibre leading to the death of the fibreSatellite cells become activated, which signal the myoblast activationMyoblasts form myotube, which make actin and myosinRepair complete

N.B. Damage and Repair oof muscle fibres and the role of satellite cells

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1.2.3.4.5.6.7.

N.B. Damage and Repair oof muscle fibres and the role of satellite cells

Satellite cell activation

Ca2+ enters

Macrophages remove debris

Macrophages enter

Infarct spreads

Myoblasts

DEATH OF FIBRE

Myotube

REPAIR COMPLETE

Membrane damagedDamage and Repair of muscle fibres – Role of Satellite Cells

Satellite cell activation

Ca2+ enters

Macrophages remove debris

Macrophages enter

Infarct spreads

Myoblasts

DEATH OF FIBRE

Myotube: make actin & myosin

REPAIR COMPLETE

Membrane damagedDamage and Repair of muscle fibres – Role of Satellite Cells

Satellite cell activation

Ca2+ enters

Macrophages remove debris

Macrophages enter

Infarct spreads

Myoblasts

DEATH OF FIBRE

Myotube

REPAIR COMPLETE

Membrane damagedDamage and Repair of muscle fibres – Role of Satellite Cells

Satellite cell activation

Ca2+ enters

Macrophages remove debris

Macrophages enter

Infarct spreads

Myoblasts

DEATH OF FIBRE

Myotube

REPAIR COMPLETE

Membrane damagedDamage and Repair of muscle fibres – Role of Satellite Cells

AgeingDecline in performance with age occurs even in highly trained subjectsMuch of this strength loss is due to a decrease in muscle mass

Maximal voluntary torques of dorsiflexor and plantarflexor muscles of

male and female subjects vs. age

Up to age 52, no age effect

After 52, steady decline

Mean number of motor units for under 60’s

Lower limit of the range of number of motor units for under 60’s

Motor neuron loss also contributes to functional changes during aging

207 healthy subjects 7months to 97 years

Number of motor units vs. age

Motor units in extensor digitorum brevis muscle

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Mean number of motor units for under 60’s

Lower limit of the range of number of motor units for under 60’s

Motor neuron loss also contributes to functional changes during aging

207 healthy subjects 7months to 97 years

Number of motor units vs. age

Motor units in extensor digitorum brevis muscle

Definitions:Atrophy: decrease in size of muscle fibres.Eccentric exercise: muscle fibres are active while lengthening. Occurs in normal activities, for

example biceps brachii does it when a heavy load is being slowly lowered by extending the elbow joint.Hyperplasia: increase in number of cells. Probably doesn’t happen in human skeletal muscle.Hypertrophy: enlargement of existing muscle fibres.

Satellite cells: small cells in skeletal muscle. One nucleus and little cytoplasm (in contrast the musclefibres) and located around the outside of muscle fibres. Dormant usually, but can proliferate and migrateto a damaged area of muscle fibres. Can differentiate and produce contractile proteins.