Muscle energetics in exercising horses

Dominique-Marie Votion1,2,*,†, Rachel Navet3,†,Veronique Anne Lacombe4, Francis Sluse3, Birgitta Essen-Gustavsson5, Kenneth William Hinchcliff6, Jose-LuisL. Rivero7, Didier Serteyn1 and Stephanie Valberg8

1Equine Teaching Hospital, University of Liege, Liege, Belgium2Equine European Centre of Mont-Le-Soie, University of Liege, Liege, Belgium3Laboratory of Bioenergetics, University of Liege, Liege, Belgium4College of Pharmacy, The Ohio State University, Columbus, OH, USA5Department of Clinical Sciences, Swedish University of Agricultural Sciences,Uppsala, Sweden6Faculty of Veterinary Science, University of Melbourne, Werribee, Victoria, Australia7Muscle Biology Laboratory, Department of Comparative Anatomy and PathologicalAnatomy, Faculty of Veterinary Science, University of Cordoba, Cordoba, Spain8Department of Veterinary Population Medicine, University of Minnesota, St Paul, MN,USA

* Corresponding author: dominique.votion@ulg.ac.be

Submitted 28 February 2007: Accepted 17 September 2007 Review

AbstractAn optimally functional musculoskeletal system is crucial for athletic performance and even minor perturbations canlimit athletic ability. The introduction of the muscle biopsy technique in the 1970s created a window of opportunityto examine the form and function of equine skeletal muscle. Muscle histochemical and biochemical analyses haveallowed characterization of the properties of equine muscle fibres and their influence on, and adaptation to, physicalexertion. Analyses of exercise responses during standardized treadmill exercise and field studies have illustrated therole of cellular energetics in determining athletic suitability for specific disciplines, mechanisms of fatigue, adaptationsto training and the affect of diet on metabolic responses. This article provides a review of the tools available to studymuscle energetics in the horse, discusses the muscular metabolic pathways and summarizes the energetics of exercise.

Keywords: muscle biopsy; exercise; glycogen; glucose; lipid; muscle fibre

Introduction

The tremendous range of athletic capacity of equine

athletes can be attributed to both years of genetic selec-

tion for prowess for a particular form of exercise as well

as the remarkable plasticity of muscle, which readilyadapts to physical training. The introduction of the

muscle biopsy technique and the expanding array of

histochemical, biochemical and molecular applications

developed over the last three decades have improved

our understanding of muscle structure, function,

adaptation to training and limitations of performance.

In this review,modernmethods to assess the relationship

between the physical characteristics of skeletal muscle

and the biochemical response and adaptation to exercise

will be reviewed; the complexity of muscle energetics

in equine athletes will also be briefly summarized.

Supplemental detailed reviews of muscle biochemistryand physiology can be found elsewhere1–3.

Techniques to measure metabolic andcontractile properties of muscle

Thefirst part of this paper discusses the techniques avail-

able for assessment of substrate metabolism, especially

in relation to the contractile properties of the muscle

fibres. These techniques allow a quantitative and†Both authors have equally contributed to the review.

Equine and Comparative Exercise Physiology 4(3/4); 105–118 DOI: 10.1017/S1478061507853667

qCambridge University Press 2008

qualitative assessment of the effects of exercise, training

and dietarymanipulation on substratemetabolism at the

level of the muscle and the whole body.

Muscle biopsy techniqueThe percutaneous needle muscle biopsy technique

was introduced to equine research by Lindholm and

Piehl4 as well as by Snow and Guy5. Since that time,

it has proved to be an invaluable tool in defining the

histological, histochemical and biochemical properties

of equine skeletal muscle. Standardization of the site ofthe muscle biopsy is imperative because equine skel-

etal muscles have a heterogeneous distribution of

muscle fibre types within the muscle6. Deeper regions

within locomotory muscles have contractile and meta-

bolic characteristics similar to those of postural

muscles7–9. In addition, fibre types vary among differ-

ent muscles in the same horses as well as across

horses and breeds10–17. The selection of the specificmuscle to biopsy therefore is of critical importance

and will depend on its propulsive or postural role.

Many studies of equine athletes utilize the gluteus

medius muscle or the semitendinosus muscle because

of its importance in locomotion and demonstrated

metabolic adaptations to exercise and training18–26.

Other investigators have also used the triceps brachii,

along with the masseter26 (as a non-exercise muscle

control sample site).

When sampling site and depth are consistent and

potentially involve several sites21,23,24,27, repeatable

results are obtained28. Muscle biopsy has provided

a wealth of information regarding the histochemical,

biochemical and metabolic properties of various

muscles within the same horse9,29,30, between horses11

and breeds15,21 as well as responses to exercise25,31,32.A detailed review of the technique for performing percu-

taneous needle muscle biopsy and sample preparation

can be obtained elsewhere3–5,21,22,33.

Contractile properties of muscle fibresThe contractile machinery provides the fibre’s ability

to shorten and lengthen through a highly organized

structure. Contractile speed varies according to both

the myosin heavy-(MyHC) and light-chain isoforms

expressed at the protein level in a given muscle fibre.

The contractile strength of a fibre is directly related tothe cross-sectional area of that fibre.

Histochemical studies of contractility

Fibre typing of equine skeletal muscle first relied uponhistochemical analysis, which revealed the acid and

alkali stabilities of the myofibrillar adenosine tripho-

sphatase (ATPase) activity in each fibre type4,5. Slow-

twitch fibres (type I fibres) have low specific mATPase

activity and often have smaller cross-sectional areas,

whereas fast-twitch fibres (type II fibres) have high

activity and often the largest cross-sectional areas.

Slow- and fast-twitch fibres can be further subdivided

into four types based on the sensitivity of the myosin

ATPase enzyme to acid or alkaline preincubation.

Classification then included types I, IIA, IIB and inter-

mediated or IIC fibres29,34,35. The speed of contraction

was found to be fastest in type IIB fibres, intermediate

in type IIA and slowest in type I fibres36. The type IICfibres, which were mainly found in foals29, would indi-

cate fibres’ transformation.

Immunohistochemical studies of myosin heavy

chains

The development of monoclonal antibodies for specific

MyHC isoformsprovidedamoreaccuratemeans todiscern

specific fibre types in equine skeletal muscle. Immuno-

histochemical staining identified several MyHC isoforms

in equine muscle8,37 that are encoded for by separate

genes38. These included three MyHC isoforms that definefive fibre types: pure I, IIA and IIX fibres and the hybrids

(i.e. coexistence of two MyHC isoforms) I þ IIA and IIA

þ IIX fibres39 (Fig. 1). In situ hybridization with RNA

probes specific for each MyHC isoform shows that

the majority of fibres express identical mRNA and

protein isoform,whereas hybrid fibres present amismatch

between coexpression at the protein rather than the

mRNA level40,41. Type IIB fibres identified previously bythe myofibrillar ATPase histochemical technique were

shown to represent type IIX fibres, whereas the true IIB

MyHC isoform commonly found in rodents was not

identified in equinemuscle39. Further, no cDNAs encoding

the IIB gene have been identified in horses40,42. Thus,

although requiringamorecomplexanalysis, immunohisto-

chemistry provides more specific and accurate inform-

ation regarding MyHC isoform(s) in equine muscle thantraditional histochemical analysis8,25,43.

Metabolic properties of muscle fibresThe metabolic properties of equine muscle can be

evaluated through histochemical and tinctorial stains,

as well as by biochemical and molecular assays.

Histochemical assessment of enzyme activities

Tinctorial and histochemical stains of enzyme activities

reveal distinctive metabolic capacities in various fibre

types. Modified Gomori Trichrome stains or stains

coupled to the activity of mitochondrial enzymes such

as a nicotinamide adenine dinucleotide reductase

(NADH) or succinate dehydrogenase (SDH) readily

demonstrate the oxidative capacity of skeletal musclefibres. Amylase–periodic acid Schiff’s stains may also

be useful in examining oxidative capacity as the

number of capillaries surrounding each muscle fibre

can be assessed with this stain. However, oxidative

capacity and capillaries are not univocally correlated.

The glycolytic capacity of muscle fibres can be evaluated

Votion et al.106

using stains for phosphorylase, phosphofructokinase

(PFK) or glycerol-3-phosphate dehydrogenase (GPDH)

enzyme activity. The phosphorylase stain has thedisadvantage of being dependent on in situ glycogen,

whereas stains coupled to PFK enzyme activity are

hampered by the lability of PFK. The GPDH stain was

developed to assess the capacity of cytosolic GPDH to

reduce nicotinamide adenine dinucleotide (NADþ) to

NADH. However, it is unclearwhether the GPDH assayed

truly represents glycolytic capacity or whether it also or

alternatively measures oxidative capacity via flavineadenine dinucleotide (FAD)-dependent GPDH in mito-

chondria44. Nevertheless, the activity of this enzyme is

highly correlated with that of other enzymes directly

involved in glycolysis (discussed in Quiroz-Rothe and

Rivero45). The oxidative capacity of the fast-twitch

fibres has, in earlier studies, been evaluated using either

SDH or NADH stains, and fibres were then classified as

FT (low oxidative) or FTH (high oxidative) in additionto ST fibres4,46.

Histological stains for energy substrates

The major sources of energy for muscle contraction

are intramuscular glycogen and triglycerides, as well

as blood-borne glucose and fatty acids. Periodic acidSchiff’s stains readily demonstrate the presence of gly-

cogen in myofibres and Oil red O stains highlight the

presence of lipid droplets47. Sequential staining of

muscle sections for glycogen content and contractile

properties have been used to assess the pattern of

recruitment of muscle fibres19,46–53.

Immunofluorescent and immunohistochemical

stains

Detailed identification of structures within myofibrescan be accomplished using monoclonal antibodies

coupled to fluorescent tags. The location of insulin-

sensitive glucose transporters GLUT-4 within intra-

cellular storage pools and in their active position

within the sarcolemma in equine muscle has been

characterized by this method54,55. Furthermore, local-

ization of isoforms of the Ca2þ-ATPase (SERCA)

within equine skeletal muscle has been accomplishedby SERCA immunohistochemistry45,56.

Biochemical assays

Amorequantitativemeans to assessmetabolic capacity of

skeletal muscle is to measure the activity of enzymes or

substrates in whole muscle homogenates or on pools offibres or single fibres of identified type. Frequently used

markers of oxidative capacity include assays of citrate

synthase (CS) or SDH activity within the Krebs cycle or

3-OH-acyl-CoA dehydrogenase (HAD) in free fatty acid

oxidation. Glycolytic capacity is often assessed by deter-

mining lactate dehydrogenase (LDH) activity or PFK

activity. The LDHusually indicates the capacity for lactate

production. The activity of hexokinase (HK) is used toevaluate the capacity for phosphorylation of glucose.

Assessment of the concentrations of triglycerides, glyco-

gen, glucose-6-phosphate, pyruvate, lactate and adenine

nucleotides provides further information regarding

the metabolic state of muscle at the time of biopsy.

Metabolite analyses on whole muscle must be evaluated

FIG. 1 Serial frozen sections of M. gluteus medius from a representative horse stained for immunocytochemistry and enzyme histochem-istry. Sections were stained with monoclonal antibodies against specific myosin heavy chain (MyHC) isoforms (upper part, from left toright: A–D): BA-D5 (A, anti-MyHCI), SC-71 (B, anti-MyHCIIA), BF-35 (C, anti-MyHCs I and IIA) and S5-8H2 (D, anti-MyHCs I and IIX).Other sections (lower part, from left to right: E–G) were stained for quantitative histochemistry of succinate dehydrogenase (E) andglycerol-3-phosphate dehydrogenase (F). (G) a-Amylase-periodic acid Schiff for visualizing capillaries. The four MyHC-based musclefibres are labelled in all serial sections

Muscle energetics in exercising horses 107

with caution as this represents only a mean value for the

metabolic responses in different fibre types57.

Gene transcription

A rapidly developing application for equine muscle biop-

sies is the useof real-timeRT-PCRwhichprovides ameans

to evaluate gene transcription within muscle under vary-

ingmetabolic stimuli. Itwas recently used to assessGLUT-

4 gene transcription and glycogen branching enzyme

gene transcription in equine muscle58,59.

Structure and function of muscle fibre typesCo-localization of contractile and metabolic staining

within individual fibres shows that, in untrained horses,

muscle fibre types often have characteristic metabolic

properties. In general, type I fibres have the highest oxi-

dative capacity and lipid stores aswell as the lowest glyco-

lytic capacity and glycogen stores. In contrast, type IIX

fibres have the lowest oxidative capacity and lipid

stores and the highest glycolytic capacity and glycogenstores in theuntrained state. Type IIAfibres are intermedi-

ate in these capacities. Some overlap exists, however,

among muscle fibre types resulting in a continuum

rather than exclusive metabolic and contractile proper-

ties (see Table 1)44,45,60,61.

At rest, type I motor units are primarily recruited for

posture. Their smaller size, high lipid stores, high oxi-

dative capacity and high capillary density make themideally suited to resist fatigue through oxidative

metabolism. At exercise, motor units are recruited in

the rank order I ! IIA ! IIAX ! IIX depending on the

intensity and duration of exercise. The large cross-

sectional area of type IIX fibres, as well as their high

glycogen stores and glycolytic capacity, makes them ide-

ally suited for high-intensity maximal aerobic and anaero-

bic exercises. However, high-force type IIX fibres areless resistant to fatigue than type I fibres (Table 1).

Assessment of whole-body substrate utilizationand energy partitioning during exerciseAlthough themeasurementof tissue samples frommuscle

biopsy described above provides useful information

about metabolic and contractile profiles of muscle

fibres, these local and systemic measures are ‘static’

measures that do not allow quantitative assessment of

the actual rates of substrate use62. Metabolic studies in

humans andmore recently in horses utilize stable isotope

tracer methodology in combination with indirect calori-

metry for calculation of whole-body substrate oxidation

rates63–66. Using a constant rate infusion of a stable

isotopically labelled tracer of glucose, rates of glucoseproduction (mainly hepatic glucose production) and uti-

lization (mainly muscle glucose uptake) can be estimated

during submaximal exercise62,63. Thus, in contrast with

measurement of blood glucose concentrations, this tech-

nique allows one to monitor the dynamics of glucose

turnover during exercise or after feeding67. Furthermore,

indirect calorimetry can be used to calculate whole-body

oxidation rates, derived frommeasurement of the horse’soxygenconsumption, carbondioxideproduction and res-

piratory exchange ratio (RER). Themain assumptions are

that measure of gas exchange at the level of the lung

(measured by RER) accurately reflects the actual meta-

bolic gas exchange at the cellular level (RQ) and that

proteincontribution isnegligible. Fromconcurrent calcu-

lations ofwhole-body rates of carbohydrate oxidation and

the rate of plasma glucose disappearance, it is also poss-ible to estimate the contribution by blood glucose and

muscleglycogen (plus lactate) to total carbohydrate utiliz-

ation64. The main assumption is that rate of glucose dis-

appearance is equal to the actual oxidation rate of

glucose derived from the blood, although it is possible

that some glucose is used for glycogen resynthesis

during low-to-moderate-intensity exercise64. The main

limitation is that this state-of-the-art technique canonly measure substrate utilization during submaximal

exercise. A further disadvantage is the cost associated

with the isotope and the instrumentation required for

measurement of isotopic enrichment62. Detail review of

these techniques can be found elsewhere62.

Muscular metabolic pathways

The second part of this paper discusses the muscular

metabolic pathways. The ATP hydrolysis supports the

cross-bridge cycle as well as the activity of ion pumps

Table 1 Fibre-type features of equine skeletal muscle

Variables Properties of muscle fibre types

Specific ATP activity From low to high: I , IIA , IIAX , IIXSpeed for shortening From slow to fast: I , IIA , IIAX , IIXResistance to fatigue From high to low: I . IIA . IIAX . IIXPower output From low to high: I , IIA , IIAX , IIXMetabolic propertiesGlycogen content From low to high: I , IIA , IIAX , IIXOxidative capacity From high to low: I $ IIA . IIAX . IIXGlycolytic capacity From low to high: I , IIA , IIAX , IIX

Morphologic characteristicsFibre size (cross-sectional area) From small to large: I # IIA , IIAX , IIXCapillary bed From high to low: I $ IIA . IIAX . IIX

Votion et al.108

and channels participating in excitation–contraction

coupling, in particular, the sarcoplasmic reticulum

calcium ATPase pump. Since the local ATP reserves

can only sustain contraction for a few seconds and

ATP needs to be produced at the site of its utilization,

oxidative phosphorylation from glucose and lipid

substrates is the main pathway for ATP synthesis under

aerobic conditions.Under most physiological conditions, glucose

entrance across plasma membranes into the muscle

cell is the rate-limiting step in glucose utilization68. Glu-

cose transport occurs primarily by facilitated diffusion

that uses a family of structurally related proteins

(GLUT-1 to GLUT-12) as glucose carriers. For instance,

GLUT-4 is the major isoform in the skeletal muscle68.

Whereas GLUT-1 and GLUT-5 isoforms are mainly assoc-iatedwith the cell surface and are not insulin stimulated,

the translocation of the GLUT-4 protein from an intra-

cellular (non-active) pool to the plasma membrane

(active site) is largely regulated by insulin- and contrac-

tion-dependent processes69.

Glycolysis begins with glucose-6-phosphate obtained

either from blood glucose and phosphorylation by HK

or from mobilized stored intracellular glycogen andproceeds through a series of steps to produce pyru-

vate (Fig. 2). The rate-limiting step in glycolysis is the

conversion of fructose-6-phosphate to fructose-1,

6-diphosphate by the enzyme PFK, whose activity

is regulated by the ATP/ADP (ADP:adenosine dipho-

sphate) ratio. When this ratio decreases the activity

of PFK increases, thus resulting in a greater production

of pyruvate. Under anaerobic conditions, pyruvate inthe cytoplasm is converted to lactate by LDH. Anaero-

bic glycolysis is efficient in terms of kinetics but not in

terms of ATP synthesis yield per glucose. With aerobic

metabolism, pyruvate is transported into the mito-

chondrion and undergoes oxidative decarboxylation

to form acetyl-coenzyme A (acetyl-CoA). Oxidative

metabolism is highly efficient in terms of energy

yield but not in terms of kinetics.Fatty acids from circulating very-low-density lipopro-

teins or from stored muscle triglycerides are a prime

substrate for aerobic metabolism. Sarcoplasmic short-

and medium-chain fatty acids (fewer than ten atoms

of carbon) can freely enter the mitochondrial matrix,

where they formacyl-coenzymeA (acyl-CoA). In contrast,

long-chain fatty acids are esterified first as acyl-CoA

and then as acylcarnitine by carnitine palmitoyltransfer-ase I and II (CPT I and CPT II) before they cross

the mitochondrial membranes. b-Oxidation starts with

acyl-CoA oxidation catalysed by the HAD, culminating

in the formation of acetyl-CoA (which enters the Krebs

cycle–also named the tricarboxylic acid cycle or citric

acid cycle) and the production of shortened acyl-CoA

(two C fragments are removed byb-oxidation sequence),

with concurrent reduction of one FAD and one NADþ.

Metabolites resulting from aerobic glycolysis and

fatty acid b-oxidation enter the Krebs cycle as acetyl-

CoA. In this mitochondrial process, several oxidation

steps are involved that result in the formation of

oxaloacetate, which may be used to repeat the Krebs

cycle. The oxidants utilized are NADþ and FAD. For

each acetyl-CoA that undergoes the whole process,

three NADH þ Hþ, one FADH2 and one GTP are gener-ated. The reduced coenzymes will be reoxidized by

the electron transport chain (see further) to provide

ATP. Fibres that contain a lot of mitochondria have

higher oxidative capacity than fibres poorly furnished

with that organelle.

Pathways of oxidative energy conservation

The reducingpower of various substrates is converted into

phosphate potential through the process of respiratory

chain and ATP synthase: oxidoreduction energy

of reduction substrates converges into two reduced

coenzymes, NADH þ Hþ and FADH2, which deliver theirelectrons to the electron transport chain. During the elect-

ron transport from coenzymes to oxygen, a proton elect-

rochemical gradient is built by proton pumps. The ATP

synthesis relies on the consumption of this proton gra-

dient. The respiratory chain is made up of four complexes

(complex I–IV) named as follows: I, NADH–ubiquinone

oxidoreductase; II, succinate–ubiquinone oxidoreduct-

ase; III, ubiquinol–cytochrome c oxidoreductase and IV,cytochrome c oxidase.

Energetics of exercise

This last part reviews the energetics of exercise. The rela-

tive contribution of aerobic and anaerobic pathways for

energy production, as well as the source of energy,

depends on the type, intensity and duration of theexercise. Training and nutrition are also important deter-

minants of the pathway used for energy production.

Muscle responses to exercisesAll the fibres of a singlemotor neuron (i.e. that respond toits action potential) belong, in general, to the same fibre

type and are dispersed throughout the muscle mass (for

details, see Rivero and Piercy3). Glycogen inmuscle is uti-

lized during most types of exercise and glycogen utili-

zation increases with increasing work intensity, being

most pronounced during the first work bouts during

interval work19,70,71. During low-intensity submaximal

exercise, muscle triglycerides, blood glucose and freefatty acids (released from adipose tissue and/or liver

stores) are the main sources of energy for type I and IIA

fibres72. With prolonged low-intensity exercise, the

uptake of free fatty acids by muscle increases substan-

tially, gradually becoming the major source of energy47.

During extremely prolonged low-intensity exercise

Muscle energetics in exercising horses 109

FIG. 2 Interconnection between the contractile apparatus and pathways of energy metabolism. Action potential causes the sarcoplasmic reticulum to release large quantities of calcium ions(Ca2þ) that enable muscle contraction. When muscle stimulation ceases, the sarcoplasmic reticulum Ca2þ-ATPase (SERCA) pumps back Ca2þ into the sarcoplasmic reticulum. There areseveral pathways for adenosine triphosphate (ATP) synthesis, which may simplistically be subdivided into anaerobic and aerobic pathways. The anaerobic pathway takes place in the cytosoland includes the coupling of phosphocreatine (PC) to adenosine diphosphate (ADP) by the creatine kinase (CK), deamination of adenosine nucleotides (with formation of adenosine mono-phosphate (AMP) and inosine monophosphate (IMP)) and glycolysis. Activity of the PFK is considered as an indicator of the capacity for anaerobic metabolism. Assay of GPDH is consideredas a measure of the glycolytic capacity of the muscle cell. Blood glucose across the sarcolemma may be increased in response to exercise owing to glucose transporter type 4 (GLUT-4),which is moved to the cell surface from intracellular sites. The activity of hexokinase (HK) is used to evaluate the capacity for phosphorylation of glucose, which is the first step of glycolysisfrom blood glucose. The aerobic pathway involves several mitochondrial processes: free fatty acid b-oxidation, the Krebs cycle and the electron transport chain. Activity of the 3-OH-acyl-CoAdehydrogenase (HAD) is considered as a measure of lipid oxidation capacity, activity of the citrate synthase (CS) as an indicator of Krebs cycle activity and activity of the succinate dehydro-genase (SDH) as a measure of oxidative capacity of the muscle cell. The pyruvate resulting from the cytosolic glycolysis may enter the Krebs cycle or, in the absence of oxygen, be convertedto lactate by the lactate dehydrogenase (LDH). Activity of LDH is considered as a measure of the capacity for anaerobic metabolism. Reduced forms of nicotinamide adenine dinucleotide(NADH þ Hþ) and of flavine adenine dinucleotide (FADH2) that are produced during glycolysis and the Krebs cycle are used by the electron transport chain to regenerate ATP

Votio

net

al.

110

further motor units are recruited, including IIAX, and

finally the type IIXmotor units48,72. Catabolismof protein

and amino acid metabolism may also contribute to pro-

vision of energy during this type of tough endurance

exercise73.

In horses performing high-intensity maximal exer-

cise, type IIX motor units are rapidly recruited in

addition to type I, IIA and IIAX fibres, anaerobic glyco-lysis is the main source of energy and large amounts of

lactate are produced leading to acidosis71,74,75. Muscle

can temporarily generate ATP using breakdown of

phosphocreatine and through the muscle adenylate

kinase pathway57,74–77. Cleavage of phosphocreatine

enables transfer of the phosphate group to ADP in

order to reconstitute ATP. This source of energy is

rapidly depleted, since the amount of phosphocreatinestored is very small. With the adenylate kinase path-

way, one ATP and one adenosine monophosphate

(AMP) are generated using two ADP molecules. The

AMP molecule can then be deaminated to ionosine

monophosphate (IMP) within the purine nucleotide

cycle, with concurrent production of ammonia. High

lactate, ammonia and IMP levels and low ATP levels

are seen after intense exercise in the muscle ofStandardbreds and especially in type II fibres57,78,79.

To sustain muscle contraction beyond the first few

minutes of exercise, horses rely heavily on glycogen,

which is the obligate substrate to sustain anaerobic

ATP production70. The importance of muscle glycogen

reserves as an anaerobic energetic fuel to prevent

fatigue during high-speed exercise has been high-

lighted in studies that manipulated muscle glycogenstores after strenuous exercise80,81. For instance, sub-

stantial depletion of muscle glycogen stores in horses

before exercise is associated with decreased anaerobic

capacity, during a subsequent high-speed exercise

test80,81. Replenishment of muscle glycogen stores by

glucose infusion after glycogen-depleting exercise

restores anaerobic capacity, evident as restoration of

the maximum accumulated oxygen deficit and runtime to fatigue during high-speed exercise81. Similarly,

a 41% decrease in muscle glycogen concentration

impaired the capacity for work in horses dragging a

sled, suggesting a reduction in anaerobic capacity82.

During intense exercise, excessive production of

lactic acid occurs in association with fatigue and is

believed to alter metabolism of the muscle cells83

and induce sarcoplasmic reticulum dysfunction84,85.For example, low pH inhibits the enzyme PFK, thus

decreasing efficiency of the anaerobic glycolysis.

Furthermore, the glycolytic enzymes are in close proxi-

mity to the sarcoplasmic reticulum Ca transport

system86. Thus, impaired glycolytic flux and selective

depletion of muscle glycogen associated with sarco-

plasmic reticulum may impair excitation–contraction

coupling and calcium flux, and thus may contribute

to fatigue87,88. On the other hand, products of AMP

deamination stimulate glycolytic enzymes. Fatigue is

also thought to be related to the accumulation of Pi,

ADP, AMP and further degradation products77,89,90 con-

comitant with depletion of glycogen stores80,81

observed in the type I and IIA oxidative fibres51,77.

Depletion of sources of energy in these fibres will

induce progressive recruitment of type IIX fibres andfurther lactate production. Thus, the metabolic

response to such exercise is highly influenced by the

muscle fibre composition and by the oxidative and gly-

colytic capacities of the muscle fibres. Those proper-

ties are most probably responsible for the different

resistance to fatigue observed between horses91.

Capillary supply of muscles, and especially of type

IIB fibres, is of importance for aerobic capacity andexercise tolerance in Standardbred trotters92.

In prolonged low-to-moderate-intensity (submaxi-

mal) exercise, lipids are the predominant source of

energy. However, glycogen depletion (observed in

type I and IIA oxidative fibres49,93) also likely contrib-

utes to fatigue71 because acetyl-CoA produced by free

fatty acid oxidation needs oxaloacetate (produced

from pyruvate) to enter the Krebs cycle and proceedto sufficient ATP synthesis through oxidative phos-

phorylation (see Fig. 2). Evidence of the role of

muscle glycogen as a major energy substrate during

submaximal exercise was clearly demonstrated by

combined use of isotopic tracer and indirect calorime-

try methods. Intramuscular carbohydrates (primarily

glycogen) account for c. 50% of the total energy expend-

iture during submaximal exercise performed at 30%of maximum oxygen uptake during the early phase of

the exercise (0–30min). However, as the exercise dur-

ation increases, glycogen utilization counts for only

20% of the total energy expenditure, with a concomitant

increase in lipid utilization at 60–90min of exercise63.

Conversely, with increased work intensity (from 30 to

60% of maximum O2 uptake), there is an increase in

the rates of both muscle glucose uptake and muscle gly-cogen breakdown, with concomitant decrease in lipid

oxidation (Fig. 3). Indeed, the increase in energy expen-

diture associated with increased workload was

met almost entirely by an increase in muscle glycogen

utilization, which was c. 60% of the total energy expen-

diture63 (see Fig. 3). Administration of carbohydrate

before or during exercise, either in the form of glucose

(2 g kg21, PO or 34.9mmol kg min21 IV) or a grainmeal (corn, 51.4 kJ of digestible energy kg21 of diet),

enhanced glucose use and decreased lipid oxidation

during subsequent submaximal exercise, without alter-

ing the contribution of muscle glycogen utilization to

total energy expenditure63,65 (see Fig. 4). These studies

clearly demonstrate the influence of nutrition on

substrate utilization and energy partitioning during

subsequent exercise. However, it is worth noting

Muscle energetics in exercising horses 111

that feeding of forage 2–3 h before exercise has a mini-

mal effect on substrate utilization during exercise

(Fig. 4).

Because glycogen is a limiting fuel for contracting

muscle for both high- and low-to-moderate-intensity

exercise, the capacity to maximize muscle glycogen

replenishment after exercise is an important factor

for optimizing performance in horses competingon successive days or on several occasions during the

same day94. However, complete resynthesis of muscle

glycogen pool requires 48–72 h after exercise93,95–97,

after conventional or high-carbohydrate diet. Intraven-

ous glucose administration hastened glycogen stores

replenishment after strenuous exercise81 but, in the

equine muscle glycogen depleted by exercise, starch-

rich meals failed to enhance GLUT-4 gene expression,GLUT-4 protein content and muscle concentrations

within the first 24 h after exercise59,96. A 30% increase

in muscle glycogen concentrations in horses fed

starch-rich meals was observed only 72 h after exercise

compared with horses fed conventional diet96. Mechan-

isms underlying the slow glycogen replenishment

after exercise in horses are not well known, but

may include limited ability of the small intestine todigest starch, slower activity of the muscle glycogen

synthase enzyme and lower recruitment of GLUT-4

at the plasma membrane compared with values

reported in humans and rodents96,98. For instance,

glycogen synthase activity increased twofold after

FIG. 3 Contribution of energy from different substrate sourceswith work intensity and time. With increasing work intensitythere is an increase in muscle glycogen utilization, with aconcomitant reduction in the rate of lipid oxidation in equineskeletal muscle. Contribution of energy from different substratesources during the 20- to 30-min (at 30% of maximum O2

uptake) and 35- to 45-min (at 60% of maximum O2 uptake)periods of exercise in six horses. Modified from Geor et al.63

and used with permission from the Journal of AppliedPhysiology.

FIG. 4 Relative caloric contributions from oxidation of muscle glycogen, lipid and blood glucose during exercise. Administration of carbo-hydrates (either in the form of oral glucose or starch-rich meals) before exercise increases plasma glucose utilization and decreases lipidutilization, without altering the use of muscle glycogen during the 30- to 60-min period of a submaximal exercise (at 50–55% maximumO2 uptake). Panel A. Relative caloric contributions from oxidation of muscle glycogen, lipid and blood glucose in six horses during exer-cise after oral administration of water or glucose (2 g kg21). ** Values significantly different for both glucose and fat utilization in glucosecompared with water trial, P , 0.05. Modified from Geor et al.64 and used with permission from the Journal of Applied Physiology.Panel B. Relative caloric contributions from oxidation of muscle glycogen, lipid and blood glucose during exercise after withholdingfeed, feeding hay or feeding grain 90min before exercise. * Values significantly different for fat utilization in grain compared with hay trial,P , 0.05. Significant increase in the caloric contribution from oxidation of glucose in the grain trial compared with the fast and feedingtrials was noticed during the 5- to 30-min period of exercise. Modified from Jose-Cunilleras et al.65 and used with permission from theJournal of Applied Physiology.

Votion et al.112

glycogen-depleting exercise in horses, whereas a five- to

ten-fold increase was reported in humans under similar

condition96. Furthermore, in contrast with other

species, GLUT-4 protein content is unchanged or only

mildly increased immediately after exercise96,99, and

IV glucose infusion after exercise enhanced muscle gly-

cogen synthesis but attenuated the increase in GLUT-4

protein content99. Because of the slow rate of glycogensynthesis (up to 72 h), the interval between exercise

bouts may be inadequate for complete or partial restor-

ation of muscle glycogen stores, which may contribute

to a decline in performance during subsequent exercise

in the horse94. The effect of pre-exercise and, more

recently, post-exercise feeding on muscle glycogen

substrate and exercise performance have been well

studied, but are beyond the scope of this review.

Contractile and metabolic profile in relation to

performance

Specific athletic abilities are greatly influenced by gen-

etic factors, and significant variations in muscle fibre

composition are observed among breeds and types of

horses known to have a predisposition for specific dis-ciplines11,15. For example, Arabian horses, known to

have high endurance capacities, have in their locomo-

tory muscles a greater percentage of type I fibres than

Thoroughbreds horses, known to be sprinters14. In

addition, muscle fibre proportion correlates with per-

formances49,100. Among endurance horses, the better

performers have higher percentages of type I and IIA

fibres, larger type I and IIA fibres, higher activitiesof oxidative enzymes, higher lipid oxidation capacity

and lower percentages of type IIX fibres27,101. On

the contrary, performances requiring short duration,

high-intensity exercise are correlated with high percent-

ages of type II fibres100,102–104. Standardbred trotters

with higher racing performance have a higher type

IIA:IIB ratio and more high-oxidative type IIB fibres

than horses with a lower racing performance105,106.The proportion of slow- to fast-contracting fibres is

thought to be somewhat inherited17,100,103 and to be

influenced by age15,25,107 and gender15,100,108; how-

ever, it may be modified by training programmes109,110

(see below).

Muscle responses to trainingIt can be predicted that a range of fibre type distributions

are required for success at elite levels within each disci-

pline. Linked to plasticity of muscle energetics, specifictraining has the ability significantly to change the fibre-

type composition, metabolic properties, fibre size and/

or capillarization within skeletal muscle20,21,110,111.

For example, endurance-trained horses show some

enlargement of type I and IIA fibres (hypertrophy) and

an increased number of capillaries surrounding type I

fibres in their locomotory muscles. However, these

changes are not consistently observed amongst different

breeds21. In Standardbred trotters, a period of intensive

training will rapidly increase the oxidative capacity and

the capillary density in muscle112. Long-term draught

training programmes in Andalusian horses modify

MyHC composition, with an increase in high-oxidative

fibres and a decrease in fast-glycolytic fibres113. InStandardbred trotters, there is an increase in the type

IIA:IIB ratio and in oxidative capacity in skeletal muscle

after a draught-loaded interval-training programme30.

Regular training in Standardbred trotters from 18

months of age until 3–4 years of age resulted in significant

changes inmuscle composition,witha shift among type II

fibres towards fast oxidative IIA fibres and an increase in

oxidative capacity114. Well-trained racing Standardbredtrotters have a high IIA:IIB fibre ratio and a high oxidative

capacity105. Changes in muscle fibre oxidative capacity

were also observed in 2- to 3-year-old trained Thorough-

breds35,115–118. In regularly well-trained Thoroughbreds,

additional high-intensity training programmes have the

potential to increase anaerobic capacity119,120, which

might be of particular interest in ensuring the final accele-

ration necessary to compete satisfactorily in a race.However, high-intensive training might increase the risk

of lameness120 and might contribute to overtraining.

Muscle concentration of glycogen stores may also be

influenced by training, since it has been shown that

low-intensity121 (in Haflinger ponies) or high-intensity

training programmes increase muscle glycogen

stores119,122. The GLUT-4 gene expression increases in

the hours following glycogen-depleting exercise59,which might contribute to refurnishing the glycogen

stores used during exertion. Glucose transport capacity

from blood to muscle is increased in trained muscle

owing to an increase of GLUT-4123. Furthermore, training

favours development of fibres’, capillary network which

results in better muscle perfusion and greater blood

nutrient availability.

Thus, it appears that training increases the overallcapacity of the muscle to respond to specific demands.

Fibre-type transitions would occur in a ranked and

sequential mode40, such as type IIX ! IIAX ! IIA ! I,

according to the energetic requirement enforced by

training (i.e. type IIA fast oxidative fibres for

sprinters and type I slow oxidative fibres for endur-

ance performers).

Despite apparent healthiness and maintenance oftraining, some Standardbred horses show diminished

performances. These may be red cell hypervolaemic

horses which, compared with normovolaemic horses,

have a higher oxidative capacity in type II fibres, a

lower capillary supply43,124,125 and a lower percentage

of the type IIX isoform43. In these poor performers, it

is assumed that a change in MyHC isoforms from IIX to

IIA occurs. This might explain why these horses were

Muscle energetics in exercising horses 113

unable to complete a sprint (which requires fully

recruitment of type IIX fibres).

It is important to realize that not only training but

also dietary compositions may influence glycogen sto-

rage and metabolic response to exercise52,126,127. Age

and gender are also important factors to consider, as

these influence fibre-type composition and, in both

Standardbreds and Thoroughbreds, stallions have ahigher type IIA:IIB ratio compared with mares114,128.

Perspectives

Metabolic adaptations to every type of physical exer-

cise are the result of very wide-ranging responses of

muscle cells (among others) to intracellular signals

that are provided by metabolic pathway activities,and are the consequences of cellular stress (in its

widest meaning). The simplest description of these

so-called retrograde responses could be: changes in

metabolic by-product concentrations and up-or-down

regulation of transcription of DNA coding for specific

enzymes (proteins) that control the flux of metabolic

pathways, i.e. the sources of these by-products. If

modulation of transcription is followed by changes intranslation into proteins, then up-or-down regulation

of enzyme concentrations (activities) occurs that can

modify the rate at which the by-products are pro-

cessed. A new steady state is therefore achieved.

While genomes are rather constant entities, their pro-

tein complements, the so-called proteomes, differ from

cell to cell. Furthermore, depending on the cell life

cycle and environmental factors, protein expressionpatterns are constantly adapted in terms of splicing

isoforms, expression levels or post-translational modifi-

cations following their biogenesis. Accordingly, these

complex and dynamic protein networks cannot be

fully characterized by gene expression analysis alone,

making proteomics (i.e. the study of proteomes) the

most unavoidable field in protein science. Proteomics

encompasses a two-step analysis of proteomes including(1) a large-scale protein profiling followed by (2) protein

identification by mass spectrometry and bioinformatic.

Protein expression proteomics (also called compara-

tive proteomics), which describe proteome variations

induced under various conditions, could be the

‘golden way’ of studying cellular adaptations to exer-

cise at the energy metabolism level, as well as at the

structural protein level sustaining mechanical work.

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