\
The Cell
Frank C. Mooren
Cellular Architecture
The cell represents the smallest unit of a living
organism and exists either as a free and migrating
cell, like blood cells, or as a cell embedded within a
tissue complex. Despite many contacts with the
extracellular matrix or neighboring cells, the single
cell functions independently and autonomously
within a certain range. It is surrounded by a cell
membrane that is responsible for perception of
hormonal information and for controlling the
exchange of ions, substrates, and other substances.
The cell interior is organized and structured by the
cytoskeleton and consists of the cytosol and various
membrane-bound organelles. Such a classes: integral and peripheral proteins. This
compartmentalization enables the cell to carry out
numerous functions and processes synchronously
for instance, energy generation and dissipation,
protein synthesis and protein degradation.
Membranes
Membranes coat the entire cell as well as
intracellular organelles. This structural homogeneity
of cellular membranes is a prerequisite for vesicular
exchange across the cell membrane and between
cellular organelles. All membranes consist of lipids
and proteins as their major components. The
relative amounts of proteins and lipids can vary
significantly between different cell types and
between different organelles. Likewise, the protein
content of neuronal myelin membranes is about
20% dry weight but Increases to about 80% in the
mitochondrial membrane. The membrane lipids
belong to different lipid classes such as glycerol-
phospholipids, phosphosphingolipids, glycosphin-
golipids, and sterols. The major membrane lipids
are phospholipids, which form a bilayer matrix with the
polar regions facing the hydrophil extracellular fluid
and the cytosol. The complex lipids like
phosphosphingolipids are involved in cellular signaling
processes or in identifying cells like gly- cosphingolipids
that carry blood group antigens in the erythrocyte
membrane. Cholesterol, another complex lipid, is by far
the most commonly found sterol in membranes. While
its content is high in the. plasma membrane, it is
nearly absent in the mitochondrial membrane. The
reason for this diversity of the chemically distinct lipids
in any given membrane is largely unknown.
Membrane proteins can be divided into two
classification denotes the degree of treatment
required to release the proteins from the membrane.
Peripheral proteins that usually associate with lipid
bilayer in a noncovalently bound form can be
released from the membrane by relatively gentle
methods like washing of the membranes in buffers
of different ionic strength and pH or in the presence
of divalent cation chelators such as EDTA. In
contrast, integral membrane proteins are partially
embedded in the bilayer and can be released from
the bilayer only through destruction of its
structural integrity using detergents or organic
solvents. However, this distinction between
peripheral and integral membrane proteins
emphasizes the relative strength of attachment but
does not clearly define the mode of attachment to
the bilayer. The attachment might depend on
electrostatic or hydrophobic interactions, or the
protein might be covalently bound to fatty acids or
phospholipids that serve as an anchor within the
lipid bilayer.
 4 Mooren
The Cell 5

Integral proteins, which span the entire
Table 1.1 Classification
membrane, of Actin-Associated
are called transmembrane proteins. TheyProteins
exocytosis, or phagocytosis, but also for cyto-
may have a single transmembrane segment or
multiple transmembrane segments and are
amphiphilic. The portions of the proteins that face
the polar solvents are enriched in amino acid
residues with polar and ionized side chains, whereas
the parts of the molecule that are in contact with
the lipid bilayer contain primarily nonpolar amino
acid residues.
The concept of the plasma membrane as a fluid
mosaic model describes the plasma membrane as a
highly flexible and mobile structure. For more than
three decades it has been known that all
biomembranes display a lateral and transversal
asymmetry for both constituents, proteins and
lipids, suggesting that the mobility of molecules is
still somewhat restricted (Bretscher 1972). The
transversal asymmetry of membrane proteins
depends on the way in which the protein is originally
inserted into the membrane. Generally the
translocation of proteins across the bilayer is
negligible. In contrast, the lateral mobility of proteins
is much higher, allowing the enhancement of
receptor protein density in specialized areas of the
cell or in contact zones to other cells or membranes.
Likewise, transversal lipid asymmetry can be
found in the plasma membrane. While the choline-
containing phospholipids, phosphatidylcholine and
sphingomyelin, are primarily located in the cells
outer leaflet, the aminophospholipids phos-
phatidylserine and phosphatidylethanolamine are
primarily exposed to the intracellular environment.
This property can be used for diagnostic purposes,
for example to determine apoptotic cells. In apoptotic
cells, the membrane phospholipid
phosphatidylserine is translocated from the inner to
the outer leaflet of the plasma membrane. The
dissipation of phospholipid asymmetry is facilitated
by calcium-dependent and Mg-ATP-inde- pendent
activation of scramblase. The asymmetry of plasma
membrane lipids under physiological conditions is
maintained through the activation of two active
transporters, the phospholipid translo- case
(floppase) and the aminophospholipid trans- locase
(flipase) (Diaz & Schroit 1996; Tang et al. 1996;
Bitbol & Devaux 1988). In addition, there is evidence
for a lateral inhomogeneity of the lipid composition
of the plasma membranes resulting in certain lipid
microdomains. Furthermore, the cytoskeleton plays
an important role in transversal and lateral
distributions of membrane components. This is true
not only for the membrane
vesicle movements, which involve endocytosis,
skeletal interactions with the membrane, which play
a role in determination of asymmetry and
stabilization of microdomains as well as plasma
membrane stability (Manno et al. 2002). '
Cytoskeleton
The cytoskeleton is responsible for the cells shape
and structure. The cytoskeleton is not a solid and
static framework. Instead, it is highly dynamic and is
involved in a variety of processes such as cellular
movement and locomotion, cell division,
phagocytosis, volume regulation, and cell signaling.
Furthermore, it is involved in the organization of
vesicular transport inside the cell and in directing
vesicular endocytotic and exocytotic processes.
The cytoskeleton consists of three major types of
filamentous proteins: actin filaments or
microfilaments, intermediate filaments, and
microtubules (Frixione 2000). Actin filaments are
small (about 8 nm thick) filaments of polymerized
actin, a 43- kDa globular protein. Polymerization is
dependent on ATP and cations, most likely
potassium and magnesium. There are a number of
actin-associ- ated proteins that are important for the
structure and function of the actin network (table
1.1); for example, spectrine can serve as a cross-
linker of actin filaments. Moreover, it connects the
actin cytoskeleton to several other membrane
proteins and ion transport systems. The latter
function has also been established for ankyrin,
another actin- associated protein. Interactions
between ankyrin and the sodium pump, sodium
channels as well as H*-K+ ATPases have been shown
(Denker & Barber 2002).
The actin cytoskeleton is primarily located in the
cell periphery. In polarized cells like epithelial cells,
the structure of the actin meshwork differs between
the basolateral and the apical part. Moreover, the
actin cytoskeleton builds up the core of cellular
extensions like microvilli.
The structure of the actin cytoskeleton is highly
dynamic and can rapidly change upon cellular
activation or stress. Therefore the actin filament
turnover is linked to intracellular signaling processes
adapting the cytoskeletal network to the
physiological demands, for example during cell
activation or motility (Mitchison & Cramer 1996).
Important effectors on the actin cytoskeleton are the
PI 3-kinase and small GTPases including Rho, Rac,
and CDC-42. Each of these has specific inter-

Actin-associated proteins Name Function

Actin monomer binding Profilin, cofilin, Bind monomeric G actin; regulate the
proteins thymosin availability of polymerizable actin; actin
disassembly
Actin filament capping Geisolin, villin, Regulate monomer addition at the filament
proteins leufactin end
Actin filament nucleating ARP2/3 Generate new filaments
proteins
Actin filament binding
proteins Cross-linking Spectrin, fimbrin Connect filaments to form a 3-dimensional
proteins Membrane Tafin, ankyrin, ezrin network Connect filaments to the plasma
anchors Motor proteins Myosin 1, II, V, VI membrane Drive vesicular transport
actions with particular actin-associated proteins
inducing the formation of filopodia, lamellipodia,
and stress fibers (Nunoi et al. 2001).
Microtubules are polymers made of tubulin, a well-
characterized heterodimeric protein. Microtubules
originate from the centrosome, an area in the cytoplasm
characterized by the presence of centriols, and require
cations and GTP for polymerization. The microtubule
network is located below the actin cytoskeleton
spanning the distance from the plasma membrane to
the nucleus. Because microtubules are polarized, their
parallel arrangement within the cell defines the polarity
of the ceil. The microtubule cytoskel- _. eton can rapidly
assemble into a variety of distinct configurations in
order to carry out different tasks. During mitosis,
microtubules build up the mitotic spindle responsible
for the chromosomes movement. During interphase,
microtubules are responsible for the arrangement of
cellular organelles and play a role in the movement of
vesicles between organelles and the plasma membrane
(Allan et al. 2002).
Similar to the situation with the actin
cytoskeleton, there exist also a number of
microtubule-associated proteins (MAPs) that serve
in stabilization and cross-linking of microtubules.
Furthermore, MAPs convey signaling molecules
such as kinases and phosphatases to the
microtubule cytoskeleton (Gundersen & Cook
1999). Motorproteins of the kinesin and dynein
complexes are involved in the directed movement of
vesicles. While dynein 1 (CD1) drives the cargo
toward the minus ends of microtubules, most
members of the kinesin superfamily drive the
vesicle movement in the opposite direction (Allan et
al. 2002; King 2000).
Finally, another group of cytoskeletal filaments,
the intermediate filaments, should be mentioned.
This group is more heterogeneous than those dis
cussed so far. Encoded by more than 50 known
genes, intermediate filaments consist of associated
tetramers of fibrous proteins and are divided into six
major classes (Fuchs & Weber 1994). One group
encompasses the ceratines, which can be found
predominantly in epithelial cells. Another group
contains vimentin, found predominantly in
mesenchymal cells, and desmin, expressed in all
muscle cells. Intermediate filaments can be found in
cell-cell contact zones and around the nuclear pores.
A major function of several intermediate filaments is
to stabilize cellular architecture against mechanical
forces (Coulombe et al. 2000).
Cell Organelles
The cytosol contains a number of membrane- bound
structures called cell organelles. Because of their
different sizes and densities, organelles can be
isolated after cellular homogenization by density
gradient centrifugation. Organelles fulfill different
functions in cellular metabolism and enable the cell
to separate the multiple biochemical reactions, for
example synthetic and degradation pathways, or
energy-producing and -consuming reactions (figure
1.1).
Nucleus
The cell nucleus is usually the largest cell organelle
and is composed of two membranes, forming the
nuclear envelope, which is tethered to the
endoplasmic reticulum (figure 1.1a). The nucleus
houses the genetic information of the cell encoded in
the sequence of DNA nucleotides. This information is
fundamental for cellular homeostasis leading to the
synthesis of proteins that determine structure and
function of the cell. Usually the DNA is associated
with proteins forming a fine network of threads
called chromatin. For cell division, the
 j

The Cell 7

DNA protein aggregates will be condensed, form* ing the ER handles a number of different functions such
the 46 chromosomes of a human cell. as
During the interphase the genetic information is
post-translational modification of proteins,
transcribed into RNA, which has to be translocated
from the nucleus to the cytosol for translation into support of the vesicular transport machinery,
assistance in generating tertiary protein i
an amino acid sequence at the ribosomes.
Additionally, RNA and proteins that build the structures,
ribosomes have to be transferred to the cytosol. This lipid biosynthesis,
macromolecular exchange between the nucleus and calcium storage, and
the cytoplasm takes place through nuclear pores
detoxification.
that result from the fusion of the two membranes of
One can speculate that the apparently
the nuclear envelope. These circular openings are
homogeneous tubular system of the ER consists of
approximately 800 angstroms in diameter and occur
several microdomains and subdomains characterized
at regular intervals along the surface of the nuclear
by specific biochemical markers. Recent
envelope. A supramolecular structure referred to as
investigations of the intracellular traffic between the
the nuclear core complex and of approximately 125
ER and Golgi apparatus describe these organelles as
MDa is an important controlling element for the
highly dynamic in structure, making a precise
import and export of macromolecules (Weis 2003).
definition and description difficult. Likewise,
Finally, the nucleolus can be easily localized within
subdomains such as the transitional elements or the
the nucleus. This filamentous region is responsible
so-called ER-Golgi intermediate compartment
for decoding ribosomal RNA and assembling RNA
(ERGIC) have been defined. It was found that any
and protein components of ribosomal subunits.
part of this secretory pathway has its unique set of
Ribosomes resident or marker proteins that define its structural
Decoding of the genomic information and translating and functional properties. These resident proteins
it into an amino acid sequence are performed - at the either do not enter transport vesicles or - are
ribosomes. Ribosomes are macromolecular _ retrieved from the Golgi apparatus, when they have
complexes consisting of more than 50 different escaped the ER. For this purpose resident proteins
ribosomal proteins and a number of ribosomal RNA possess amino acid sequences like the di- lysin motif
molecules. Typically each ribosome is composed of a targeting a protein to the ER (Teasdale & Jackson
large and a small subunit. The ribosomal subunits 1996).
are assembled in the nucleolus region of the nucleus. The Golgi Apparatus
Ribosomal proteins therefore have to be imported into
The Golgi apparatus consists of about six to eight
Infest
the nucleus to associate with RNA. The complete
membrane-bound flattened cisternae arranged in
subunits are'sbA^
then exported through the nuclear pores
parallel order. It is a dynamic organelle that receives,
; v
&^\^3pt?GSrfp. -

into the cytosol, where the subunits form the

post-translationaily modifies, and sorts proteins for
functional ribosome. The ribosomes either are located
delivery to various destinations (figure 1.1c).
free in the cytosol or are attached to the endoplasmic
Functionally, the two sides of the Golgi apparatus
reticulum to produce housekeeping or secretory
behave differently. At one side, the so-called cis-GoIgi
proteins, respectively. For protein synthesis, however,
network (CGN), newly synthesized material from the
a ^fe.
number of additional enzymes, like RNA
ER is imported. At the opposite side, the trans-Golgi i
polymerases, are required.
network (TGN), the material is exported after sorting
Endoplasmic Reticulum and packaging. Between these steps the post-
The endoplasmic reticulum (ER) is a membrane- translational modification of proteins takes place, for
bound organelle that consists of a network of example the linkage of carbohydrates to proteins to
cisternae and tubules stretching throughout the form glycoproteins.
e Morphology and function of the Golgi apparatus i
cytosol (figure 1.16). Morphologically a smooth and I
a rough endoplasmic reticulum can be are highly dependent on intact cytoskeletal
Figure 1.1 Electron micrographs of cellular organelles; (a) Nucleusstructures.
(nu), mitochondria (mi); (b) endoplasmic
Depolarization reticulum
of microtubules (er); (c)
results in
distinguished, the latter being characterized by the
Golgi apparatus (golgi); (d) lysosomes (ly). zymogen granula (zy);a (e) mitochondrium
fragmentation of in a Golgi
the cross-section of human
apparatus skeletal
(Thyberg &
attachment
muscle outer of ribosomes.
(om),This apparent structural space (im), matrix (ma), and myofibrils (fi); (f) subsarcolemmat
membrane cristae (cr), intermembrane
Moskalewski 1999). The different organization of 1
simplicity should not obscure the fact that
(ss) and intermyofibrillar (imf) mitochondria of a human muscle fiber. Micrographs a-d were taken from pancreatic acinar
cells, which are an epithelial ceil type. The bar in figures a-d indicated 1 pun.
Micrographs A-D by the courtesy of Dr. M.M. Lerch, Ernst-Moritz-Arndt Universitat Greifswaid; micrographs E-F from H. Hoppeier & M. Fluck, Med.
Sci. Sports Exerc. 35:95-104, 2003, with permission.
t

6
 s Mooren
..microtubule network in polarized epithelial cells and
in fibroblasts therefore results in different
localizations of the Golgi apparatus (McNiven &
Marlowe 1999). In addition, maintaining the proper
structure and function of the Golgi apparatus
depends on a number of mechanochemical enzymes
like dyneins, kinesins, and myosins, which are
involved in formation and movement of Golgi-
derived vesicles (Allan et al. 2002).
Mitochondria
Mitochondria are oval-shaped organelles measuring
1 to 2 (j,m in length and 0.1 to 0.5 jxm in diameter.
Mitochondria are separated from the cytosol by an
outer membrane while their interior is further
compartmentalized by a membrane system. The
inner membrane divides the mitochondrias interior
into the intermembrane space and the matrix
(figure l.le). The inner membrane is folded several
times, forming sheets or tubules that have been
named christae mitochondrialis. Novel insights into
the mitochondrial structure owing to recent
technical advances revealed that mitochondrias
shape and structure are variable and depend on
source and conformational state. The christae
structure can vary from tubular to complex lamellar
structures. It has been shown that they are
connected to the intermembrane space by tubular
structures that might have important implications
on metabolite diffusion and reaction rates (Frey &
Manneila 2000). The two mitochondrial membranes
are different with respect to their ion permeability.
While the outer membrane is highly permeable for
molecules up to 5,000 kDa, the inner membrane
demonstrates low ion permeability comparable to
that of the plasma membranes. Therefore the
composition of the inner membrane space is
comparable to that of cytosol. While the
mitochondria matrix contains the enzymes of the
Krebs cycle, the inner membrane carries the
enzymes responsible for oxidative phosphorylation
like cytochrome C.
Mitochondria are the only organelles that contain
separate and semiautonomous genomic information
on the mitochondrial DNA. This DNA codes for
about 13 different proteins involved in oxidative
phosphorylation and for ribosomal and transfer
RNAs.
The role of mitochondria in cell homeostasis,
however, does not seem to be limited to energy
metabolism. There is evidence for a role of
mitochondria in intracellular signaling.
Mitochondrias role as a calcium storage and
buffering organelle is well known (Rutter et al.
1998). Recent obser
vations from several different cell types indicate a
certain spatial distribution of mitochondria within
the cytosol that results in the modulation of
intracellular calcium signals (Tinel et al. 1992).
Moreover, mitochondria are involved in the
initiation of programmed cell death (apoptosis).
Numerous close contacts between mitochondria,
the ER, and the cytoskeleton were observed,
emphasizing the dynamic interaction between
these structures (Rizzuto et al. 1998). Finally, there
is evidence for a spatial heterogeneity of the
mitochondria population within a cell. In the
skeletal muscle cell, mitochondria from the
subsarcolemmal (SS) and the intermyofibrillar
(IMF) region can be distinguished that differ with
respect to their biochemical properties and their
adaptation^ response to exercise (see later).
Vesicular Structures
Within the cytosol, at least three different vesicular
structures can be defined: peroxisomes, lyso- somes,
and endosomes (figure l.ld). These membrane-bound
organelles account for about 3% of the total volume.
Peroxisomes contain a number of different enzymes
that are involved in lipid degradation and
detoxification. Lysosomes are the final site of
degradation and digestion of incorporated
extracellular raacromolecules and of obsolete
intracellular material. However, there is increasing
evidence that lysosomes and their precursor
structures may have additional functions such as
cytotoxicity and antigen processing/presentation.
Lysosomes are loaded with a number of degrading
enzymes like hydrolases and esterases and are
characterized by an acidic pH value (Hunziker &
Geuze 1996).
Endosomes represent a heterogeneous
population of intracellular vesicular structures.
Endo- somal compartments can be defined
according to various aspects such as function,
morphology, or composition. The definition of the
endosomal compartment is sometimes difficult
because there are overlaps with other
compartments, especially the lysosomal
compartment. The term early endosorne" reflects an
operational definition for this primary vesicle
docking site either for endocytotic vesicles from the
plasma membrane or as an acceptor compartment
for vesicles from the trans-Golgi network. While early
endosomes usually are located toward the cell
periphery, late endosomes can be found closer to the
nucleus (Gruenberg 2001). From the early
endosomes, material can be directed back to the
plasma membrane via the recycling endosorne, as it
occurs
10 Mooren
The Cell 9

toduring
the cytosol
receptor forrecycling.
degradation Otherby pathways
the proteasome..
are the is Finally, a complex
not present protein machinery
on lysosomal membraneshas been
(Clague
(Bonifacino
transfer of & Weissmann
material to the1998). Cargo transport
late endosomes, which described
1998). Finally,that itis has responsible
been observed for membrane
that the
from
may thefuseER with to iysosomes
the Golgi network
for final is performed and
degradation, via fusion.
material Studies
during the fromtrafficsynaptic
throughvesicle exocytosis
the endosomal
the ER-Golgi across
transcytosis intermediate
the cytosolcompartment.
from one During plasma in neurons have
compartment is moregiven and insight into a protein
more acidified. This iscom-
due
the cargo passage
membrane domainthrough to the the opposite
Goigi apparatus,domain plex
to theknown
activity as
of athe core complex,
K*-fT-ATPase. The pH which
of theseems
early
post-translational
(Katzmann et al.processes are performed early
2002). Morphologically, and operative
endosomalalsocompartment
in other cell istypes. about After6.0;
docking,
late
cargo is packed again and transported
endosomes tend to be tubular while late endosomes for secre- the first contact between the
endosomes and Iysosomes are characterized by vesicular and target
pH
tion to the
are more plasma(Gruenberg
spherical membrane.2001). Another pathway
It is becoming membrane,
values of about vesicle
5 andand eventarget
below. SNARE
This declinemolecules
in pH
directs
more and digestive enzymes
more apparent thatlikeevery hydrolases and
stage is defined interact
seems to withbeeach other. Synaptobrevin
important for dissociation is the of
esterases
by special toward
markers or thecoat lysosomal compartment
proteins. Vesicles coated relevant
incorporated vesicular
receptorSNARE complexes molecule,
as well while
as for thethe
via
withthe clathrin
endosomalrepresentcompartment. the Tobest- avoid defined
mem- target
shift ofSNARE consists
vesicular marker of proteins
syntoxin (Mukherjee
1 and SNAP-25. et al.
brane depletion pathway,
internalization of the ER and istoused,
which retrieve lost ER
for example, After
1997).the fusion event the core complex is released
proteins, a retrieval pathway
for internalization exists from the
of ligand-occupied Golgi
receptors. by the action of NSF (n-ethylmaleimide-sensitive
apparatus to the ER as well as from
Ligand-receptor coupling induces the exposure of the intermedi- Cytosolic
factor), whichTraffic
is recruited to the membrane by
ate compartment to the ER.
signaling motifs that are recognized by a complex of adaptor proteins called SNAPs (no relation to
In theand
adapter endocytotic route, the The
clathrin molecules. imported
rate of material
clathrin- Cytosolic traffic
SNAP-25). Despite refersthe toprogress
the multiple exchange of
in understanding
isdependent
directed endocytosis
first to the atendosomal
the plasmacompartment,
membrane is vesicles
the between and
mechanical the intracellular
structural basis organelles as well
of membrane
from which it by
also regulated canrabbe5, transported
a small GTPase. via different
Rab 5 is as with athe
fusion, plasmaofmembrane
number questions (figure
remain1.2). There
openfor
routes.
also a For marker example,
of the internalized
early endosome receptors can
or sorting exist several
example, how routes
specificityin order to establishfusion
of membrane the
be recycled to
endosome and the is
plasma membrane;
involved material early
in promoting can communication
arises (Rizo & between
Siidhof 1998). organelles and plasma
be sorted for
endosome finalRab
fusion. digestion
11 and in rabthe7 andlysosomes
9 are otheror membrane and to ensure that none of the organelles
can be transferred to the trans-Golgi
small GTPases and marker proteins for the recycling network; and Exercise and the Cell
will be depleted of membrane components. In the
finally material can be transported
and the late endosome, respectively. Another marker from one side synthetic route, proteins are first translocated into
ofofthetheplasma
late membrane
endosometo is the the
opposite side.
(ci)-mannose 6- the ERand
Acute where they undergo
chronic exercisefolding
boutsand arematuration.
important
Such vesicular
phosphate receptor transport
(M6PR). It is found highlyconcentrated ER quality control
dependent on physiological stimuliguarantees
known to that affectonly correctly
metabolism
aonnumber but The similarity in membrane as
of factors.
late endosomes folded
well proteins
as organare released Acute
structure. to theexercise
cell surface.
bouts,
structure of the plasma membrane and the intra- ' " especially Otherwise those
proteins are are
that retro-translocated
maximal or supramaxi-
cellular organelles is an important prerequisite mal, often result in structural damage to tissues
for their structural communication via vesicles. followed by a more or less long period of impaired
Budding, movement, and fusion of vesicles are function. Tissue repair leads to an adaptive
moreover dependent on an intact cytoskeletal response, usually making the tissue more resis-
network that arranges the different pathways and tant to subsequent stimuli. Regular and chronic
that, in combination with the cytoskeletal-associ- exercise at a moderate workload is followed by
ated proteins, is necessary for vesicle movement. an adaptive training response that results in an
To address vesicles to a final target, a number of enhanced functional capacity of the organism,
code and marker proteins are used. Various vesicle presenting clinically as an increased maximal
coat proteins like clathrin or COP I and II have been oxygen capacity and enhanced muscular power
described that have different functions in vesicular and fatigue resistance. Both local damage and
transport. Clathrin-c.oated vesicles, the first coated adaptive response have a cellular and molecular
vesicles to be observed (in 1964), are involved in the component, which is discussed in the following
internalization pathway (Roth & Porter 1964). chapters. The present chapter is an overview of
However, some non-clathrin-depen- dent endocytotic current knowledge about the effects of exercise on
events have been identified also. aspects of the cellular structure such as membrane
COP I and COP II vesicles seem to be involved in the composition, cytoskeletal network, and organelle
transport to and from the ER (Allan et al. 2002; function.
Teasdale & Jackson 1996). Moreover, a number of
marker proteins have been characterized, such as Exercise Effects on Plasma
small GTPases (rab 5,11, 7, 9) or special amino acid Membrane Integrity and
motifs like the d-lysine or d-arginine motifs that
characterize the different organelles and vesicles Composition
and drive them to the target (Clague 1998). These Both acute and chronic exercise affect the integ-
marker sequences exist not only for membrane rity and the composition of the plasma membrane.
Figure 1.2 Schematic diagram of the
proteins, but also for soluble proteins of the cytosolic traffic Depending
organization (for detailson
seethe intensity
text). ERGIC, and duration,
endoplasmic acute Golgi
reticulum-
intermediate compartment; EE, early endosome; LE, late endosome; RE, recycling endosome; Lys, lysosome; Q O,
organelles matrix.
organelle/vesicle targeting motifs; / cargo unmodified/modified.
 The Cell
exercise damages cellular membranes (Temiz et al.
2000; Overgaard et al. 2002). This effect is either
delayed or blunted in trained individuals, suggesting
an adaptational process during chronic exercise that
can at least in part counteract the harmful effects
associated with acute exercise (Senturk et al. 2001;
Vincent et al. 1999; Yalcin et al. 2000; Venditti & Di
Meo 1997).
In red blood cells, the exercise-induced plasma
membrane damage is indicated by an enhanced
osmotic fragility and an impaired deformability
(Senturk et al. 2001; Yalcin et al. 2000). After an
exercise test, structural alterations of plasma
membrane of leucocytes have been described, which
were associated with an increased cellular calcium
content (Caimi et al. 1997). Azenabor and Hoffman-
Goetz (2000) reported increased intracellular calcium
levels and calcium influx in mouse thymocytes after
exhaustive exercise. Recently, we could confirm these
data in human lymphocytes immediately after an
exhaustive exercise test at 80% V02max. These
alterations were time dependent, since these effects
were not observed at later time points after the test.
The increase in basal calcium was interpreted as an
enhanced calcium influx across the plasma
membrane due to exercise-associated structural
damage, because the calcium sequestration was
unaffected in these cells (Mooren et al. 2001).
A number of factors have been proposed to be
responsible for plasma membrane structural
changes observed during and after exercise.
Oxidative stress is one factor that can induce
structural and functional alterations. Since it is
difficult to measure free radicals because of the
short life span of these species, scientists focus on
determining free radical tissue damage such as lipid
peroxidation. Exercise-induced lipid peroxidation has
been demonstrated in a number of studies (Dillard et
al. 1978; Child et al. 1998; Cooper et al. 2002).
Moreover, a correlation between lipid oxidation and
exercise intensity was reported (Azenabor &
Hoffman-Goetz 2000). Markers of free radicals could
be diminished after application of antioxidants and
allopurinol, an inhibitor of xanthine oxidase (see
chapter 9) (Senturk et al. 2001; Vina et al. 2000).
Other factors that must be considered are
metabolic and mechanical stress. Increased plasma
lactate levels and, subsequently, an enhanced lactate
influx into red blood cells may also affect cell rigidity
(Szygula 1990). Red cell deformability decreases with
increasing lactate levels under in vitro conditions.
This effect might be mediated
11
by an osmotic shrinkage of lactate-loaded
erythrocytes that could be further aggravated by an
exercise-associated fluid shift (Brun 2002).
Finally, mechanical stress affects plasma
membranes. In endothelial cells, shear stress has
been shown to be beneficial because it results in
decreased adhesion of blood cells and therefore
prevents atherogenesis (Cooke 2003). On the other
hand, the mechanical stress in contractile tissue can
result in a decreased cellular integrity. Structural
damage is pronounced after eccentric exercise as
indicated by the release of intracellular enzymes and
macromolecules like creatine kinase and myosin
heavy chains into the blood (Overgaard et al. 2002).
Exercise-induced structural changes in the
plasma membrane can be delimited by training
(Yalcin et al. 2000). One reason might be the up-
regulation of defense mechanisms such as radical
scavenging and depleting mechanisms (for details see
chapter 9) (Vincent et al. 1999). Exercise-induced
cellular hypertrophy is associated with an
enlargement of the plasma membrane. Cell
capacitance measurements indicate that the cell
membrane surface area of cardiomyocytes increases
under endurance training-induced cardiac growth
(Mokelke et al. 1997). Furthermore it .could be
shown that regular exercise alters the lipid
composition of both plasma and mitochondrial
membrane (Dohm et al. 1975). In long distance
runners, an increased ratio of phosphatidylcholine to
phosphatidylethanolamine and a decreased ratio of
cholesterol to total phospholipids were observed
(Nakano et al. 2001). This finding was associated
with an enhanced deformability of the athletes
erythrocytes. After four weeks of treadmill training, a
change in the structural composition of rat skeletal
muscle membrane was observed with a decrease in
polyunsaturated fatty acids (Helge et al. 1999). These
data were partially confirmed in humans after six-
week exercise training of low intensity (Andersscn et
al. 1998). Furthermore, a muscle type-specific
phospholipid fatty acid pattern was observed. The
white quadriceps, a muscle dominated by fast-twitch
glycolytic fibers, demonstrated a significantly higher
degree of unsaturated fatty acids than soleus
muscle, which consists mainly of slow oxidative
fibers, or the red quadriceps muscle, which contains
both slow oxidative and oxidative glycolytic fibers.
This might be explained by the contribution of lipids
that the fiber type utilizes for fueling oxidative
phosphorylation, and emphasizes the impact of
metabolism on membrane composition.
1! | E-C coupling j
j' I dysfunction
12 | Mooren

Mechanical Stress and Cytoskeleton (figure 1.4; Friden & Lieber 2001). Whether these
events are solely responses to the mechanical
Mechanical stress is an important effector and
stimulus or the effect of activated intracellular
modulator of the cytoskeletons structure and
proteases remains unclear. Several studies have
organization. Cytoskeletal alterations depend on the
demonstrated an increase in basal intracellular
intensity of the stimulus. Submaximal stimuli
calcium concentrations in muscle fibers after
usually result in a reorganization of the structure,
exercise, which can activate calcium-dependent
while supramaximal stimuli often disrupt the
proteases such as calpain. Belcastro (1993) reported
cytoskeletal network followed by regenerating and
both an increased calcium affinity of calpain and an
remodeling processes. Examples of both responses
increased calpain activity in hindlimb muscles after
are given here while a distinction is made between
exercise. Interestingly, calpain preferentially affects
muscle and non-muscle cells.
cytoskeletal proteins while it interferes less with the
Exercise may damage fibers in the active
myofilaments. Another proposed pathway involves
muscles, especially when the exercise is relatively
calcium-dependent activation of phospholipase A2,
intense, is of long duration, and includes eccentric
known to damage cell membranes by cracking
contractions. Clinically this presents as muscular
membrane lipids (Armstrong 1990). While
discomfort and pain in the stressed muscles that
pharmacological inhibition of phospholipase A2 was
peak 24 to 48 h after exercise, known as delayed-
effective in attenuating the stimulus-induced
onset muscle soreness (DOMS; Friden & Lieber
membrane damage in skeletal muscle, it was not
2001). DOMS is directly related to the eccentric
responsible for myofibrillar disruption (Jackson et
component of exercise, a form of exercise
al. 1984; Duncan 1988).
characterized by forcible lengthening of a
The other hypothesis focuses on a primary sar-
contracting muscle. This type of exercise generates
colemmal damage. This should result in both
higher tension than any other form of contraction
disintegration of the lipid bilayer and disconnection
(Proske & Morgan 2001).
of the calcium release units (feet structure), which
Which is the primary event of muscle injury after
consists of membranous (voltage-dependent Ca2+
exercise is still controversial (figure 1.3). One
channel; dihydropyridine receptor, DHPR) and
hypothesis claims a primary intracellular event in
submembranous components (intracellular Ca2*
which overstretched and disrupted sarcomere's
channel; ryanodine receptor, Ryr) together with
represent the initial damage. Cytoskeletal
cytoskeletal elements. A disturbance of ion
disruption, especially of the desmin intermediate
homeostasis and of the excitation-contraction (EC)
filament network, has been shown to occur early
coupling follows (Warren et al. 2001). Supporting
after eccentric exercise. Subsequently,
evidence comes from studies in mouse muscle.
morphological studies show sarcomeres out of
Exercise-induced tension deficit was reversible after
register with one another and Z-disc streaming and
application of caffeine, known to
broadening

Mechanical
Loss of
load
function
(especially
(e.g force
eccentric
decline).
exercise)

Ryr/DHPR Ca-
re!ease unit (feet
structure)
 B Ca-dependent
1 protease activation

Figure 1.3 Postulated sequences of events leading from mechanical load to muscular loss of function.
Adapted from U. Proske & D.L. Morgan, 2001, J. Physiol. 537.2:333-345.
14 Mooien
The Cell 13

proteins, membrane lipids, and ion channels (Ali & biogenesis.

and should helpMoreover,
the muscle a to number
withstand of further
other
Schumacker 2002). Some ion channels have been physiological
stress. Other and pathophysiological
hypotheses focus onconditions have
an increased
identified that change their open probability upon been shown
resistance to enhance
against stress ofmitochondrial
the cytoskeleton biogenesis,
and the
membrane stretching, leading to a subsequent influx such
cell as electrical
membrane (Gibalastimulation,
et al. 1995).creatine depletion,
of calcium concomitant with an activation of hyperthyroidism,
Currently there isand
almostmitochondrial
no information respiratory
available
calcium-dependent intracellular signaling pathways deficiency.
about the effect of exercise on the cytoskeleton of
(Sachs & Sokabe 1990). Mitochondrial
non-muscle biogenesis
cells. However, oneiscan
a complex process
find analogies In
covering
other various
areas steps
such such
as hemodynamics events
as signaling and
Mechanical Stimuli and leading to research.
cardiovascular transcriptionA numberof nuclear and
of interesting
mitochondrialhavegenes, protein
made and lipid about
synthesis,
Membrane Traffic observations been recently the
proteinof import
effect into mitochondria,
shear stress on the endothelial and theircell
Mechanical stimuli including shear stress, osmotic
assembly into The
cytoskeleton. the enzyme
blood complexes (figure 1.5).
flow constantly exposes
pressures, and the like apply forces to the plasma
membrane resulting in alterations of membrane endothelial cells in Mechanisms
Initial Signaling vivo to hemodynamic forces that
tension, one intrinsic variable of plasma change with the onset of exercise and depend on the
Muscle contraction is activated by and associated
membranes. A rough approximation of plasma type and intensity of exercise. The shape and the
with numerous intracellular signaling events, one of
membrane tension is given by LaPlaces law. Other cytoskeletal organization of the endothelial cells
which is increases in cytosolic calcium. Calcium is
contributing factors are the attachment of depend on the Intensity of the shear stress applied.
known to be an important second messenger.
cytoskeleton and hydrostatic pressure across the In regions of the aortic tree exposed to low shear
Voltage-induced calcium influx releases calcium
membrane (Dai &Sheetz 1999). Usually cells stress, the cells are polygonal and have only a few
from the sarcoplasmic reticulum. Besides facilitating
respond to increases in membrane tension with an stress fibers. In contrast, in regions of high shear
actin/myosin cross-bridging, calcium serves as an
enhanced exocytosis rate, leading to an enhanced stress, the cells are more elongated and contain
activator of a number of kinases and phosphatases.
cell surface as indicated by capacitance numerous stress fibers. These findings could be
The differential activation of transcription factors by
measurements. This mechanism is used as a trigger confirmed by in vitro experiments using flow
distinct calcium signaling patterns seems to be
for secretory cells, for example the release of atrial chambers (Barakat 1999; Galbraith et al. 1998).
responsible for myosin isoform expression, thereby
natriuretic factor (ANF) and angiotensin II from Morphologically, enhanced shear stress results in
modulating the muscle phenotype (Chin et al. 1998;
stretched cardiac myocytes (Sadoshima & Izumo more stress fibers and a rearrangement of the
Wu et al. 2001; Allen & Leinwand 2002; Chin et al.
1997). In contrast, a decrease in membrane tension cytoskeleton. This includes thicker intercellular
2003) (see chapter 13). For a long time it was
is followed by an enhanced endocytosis rate junctions, more apical microfilaments, and more
unclear whether increases in cytosolic calcium
resulting
Figure 1.4inElectron
a recovery of membrane
micrograph tension.damaged
of eccentrically Several microtubule organizing centers (Galbraith et al,
would be propagated into intracellular organelles.
human skeletal
exceptions muscle.
to these rulesNotecanthebe lack
found,of registry
leading ofto 1998).
Using the recombinant calcium-sensitive photo-
neighboring
the myofibrils, that
assumption the Z-disc
mechanicalstreamingstimuli
and Several mechano-sensors have been identified in
dissipation, and the sarcomeric disruption. protein aequorin, modified by addition of a
predominantly affect the rate of membrane turnover the cell membrane. On the luminal side of
Micrograph courtesy of Dr. J. Friden, Department of Hand Surgery, mitochondrial target in the sequence, the calcium
(Apodaca endothelial cells, vascular endothelial growth factor
Sahlgrenska 2002). Exocytosis
University and endocytosis
Hospital, SE-413 45, Goteborg, seem to
Sweden. concentration within the mitochondrial matrix of
be coupled
Reprinted, processes
by permission, from J.leading
Friden. to a continuous receptor (a receptor tyrosine kinase) can serve as a
living cells could be determined (Rizzuto etal. 1992;
replacement of the cell surface. Membrane tension mechano-sensor. Similarly, integrins work on the
Rutter et al. 1998). This approach revealed that
therefore can be adjusted depending on which abluminal side (Fisher et al. 2001; Shyy &Chien
upon physiological stimulation, the cytosolic calcium
release the
process sarcoplasmic calcium pool and thereby
predominates. 2002). Dynamic interaction between mechano-sen-
increase was accompanied by an increase of the
bypassing
Cellular the voltage-dependent
mechano-sensors include calcium-induced
integrins, ion sitive integrins and extracellular matrix proteins
mitochondrial matrix calcium concentration, which
calcium release
channels, (Warren
and the et al. 1993;
cytoskeleton, Balnave
while & Allen
downstream results in the activation of many downstream
reached levels sufficiently high to activate matrix
1995).
signaling pathways involve intracellular messengers signaling molecules. This includes activation of
dehydrogenases. Another calcium-dependent
like While the exact
calcium, cyclicseries
AMP,of andinitialthe
events leading of
activation to serine/threonine and tyrosine kinases located in the
pathway was demonstrated by Wu et al. (2002) using
muscle damage remains open, there
phosphorylation/dephosphorylation are obvious
cascades. For a cell membrane and focal adhesions and induction of
a transgenic mouse approach. The authors could
signs detailed
more of impaired cellular the
description integrity
readerasisindicated
referred byto phosphorylation cascades of signaling molecules;
show that a downstream effector kinase of the
the release
some excellentof recent
intracellular
reviews molecules such as
(Hamill & Martinac examples are the Ras-MEKK-JNK signaling pathway
calcium signaling pathway, the calcium/calmodulin-
creatine
2001; kinase,
Davies 1995;lactate
Morris dehydrogenase
& Homann 2001). (LDH), or and members of the Rho small GTPase family such
dependent protein kinase, augmented mitochondrial
myosin heavy chains. Subsequently, an as RhoA, which is functionally linked to MAPK
DNA replication and mitochondrial biogenesis as
Mitochondrial
inflammatory response Biogenesis starts with deposits of signaling, cell migration, and the organization of
well as up-regulation of mitochondrial enzymes. This
fibronectin and the invasion of leucocytes, leading to theactin-based cytoskeleton (Shyy & Chien 2002).
effect is induced by expression of the peroxisome
Chronic stimulationand
tissue remodeling of skeletal
adaptation muscle
of theresults in
muscle Shear stress-induced formation of stress fibers and
proliferator-activated receptor y coactivator-1 (PGC-
structural
fibers. A second andboutfunctional adaptations
of eccentric exercise, of
repeated cell elongation and alignment could be inhibited by
1).
mitochondria. With respect to exercise,
within days or weeks, produces much less damage. endurance RhoN19, a dominant negative mutant of RhoA (Li et
There is evidence, however, that the calcium
training
The structuralis the basismost
of thispowerful
adaptational stimulus
processfor is al. 1996). Other endothelial cell shear stress
increase alone is not sufficient for induction
mitochondrial
proposed to be the formation of extra sarcomeres, mechano-sensors that have been proposed include
which should improve the muscles working range G-proteins, intercellular junction
16 Mooren
The Cell 15

induce mitochondrial biogenesis as well. This could increased in the triceps muscle about 18 h after the
be an indication that in addition to the role of exercise. Likewise, NRF-1 and NRF-2 were detected
depleted energy pools, the turnover rate of energetic between 12 and 18 h after exercise (Baar et al.
phosphates can induce mitochondrial biogenesis 2002). Additional transcription factors involved in
(Hood 2002). |t ATP I Activation of mitochondrial biogenesis are activator protein-1 (AP-
mRNA : Protein
The role turnover
of intracellular kinases
| kinases and and 1) and peroxisome proliferator-activated receptor a
expression /expression |
phosphatases for induction of mitochondrial
l phosphatases and y (PPARa/y). The latter factor appears to be
of genes and
biogenesis ist [Ca
far2+from clear. Although there is much responsible for the |transcriptional
| encoding assembly of
control
, Altered wof
] in |e.g., protein,
evidence that contractile
cytosol and factivity activates a number enzymes involved in mitochondrial
mitochondrial I multisubunit j betaphenotype;
oxidation
| kinase C,
of Acute
different kinases, for example protein kinase C
mitochondria; (figure|1.6) (Hoppeler & Fluck
proteins 2003).
respiratory metabolic
exercise | AMP kinase,
and ERK and MAP kinases (for details chapter 13), 4 I complexes adaptation to
I MAP kinases Regulation of20
[e.g., Tom Cellular mRNA Levels
their roles in mitochondrial
It Electron biogenesis have to be exercise
determined. transport Protein cytochrome
expressionc,l depends
e.g., on
COX, the amount of mRNA
available within
I COX III a cell, which
| importin turn depends on the
TranscriptionandofV0Nuclear2
and one hand on the transcriptional
l machineryactivity and on the
Mitochondrial Genes other hand on mRNA stability. For mitochondrial
Although there are only a few Minutes
I---------------
mitochondrial gene biosynthesis both processes seem to be important.
Seconds Minutes-hours Hours-days Days-weeks Weeks
products (two ribosomal proteins, 22 transfer RNAs,Time Recent experiments by Freyssenet et al. (1999)
and 13 polypeptides), they are nonetheless indicated that the postexercise levels of mRNA
important for establishing complete oxidative increased initially due to an increase mRNA stability
Figure 1.5 Established
capacity. sequence ofby
This is supported exercise-induced
the fact that events
the leading to mitochondrial
followed biogenesis.transcriptional
by an enhanced Mitochondrial biogenesis is a
activation.
complex process depending on aDNA sequence of signaling andinanabolic eventsresults
These with different time prior
confirmed constants (for details that
experiments see text).
had
number of mitochondrial copies changes
Reprinted, by permission, from D. Hood, 2001, "Plasticity in skeletal, cardiac, and smooth muscle: Invited review: Contractile activity- induced,"
response to endurance training, suggesting a role at shown an increase of cytochrome oxidase activity
Journal of Applied Physiology 90:1137-1157.
the pretranscriptional level (Murakami et al. 1994; that was higher than the increase in its mRNA
iwai et al. 2003). The presence of two genomes (Flood et al. 1989).
responsible for mitochondrial biosynthesis makes
- Mitochondrial Protein Synthesis
coordination
of mitochondrial between the Cellular
biogenesis. transcription calcium of increase
both factor (see later) (Lai & Booth 1990; Freyssenet et al.
Besides amino acid sequence coding regions,
genomes
subsequent necessary. In chronically
to application of a calcium stimulated rat
ionophore 1994; Bergeron et al. 2001). An important
regions coding for ribosomal RNA and tRNA can be
anterior
resulted tibialis muscle, Hood
in the expression of only et aal.
few(1989)nuclear could
genes downstream effector molecule that is activated by
found on the mitochondrial genome, suggesting an
demonstrate a similar time
encoding mitochondria! course in
proteins, mRNAothers
- while levels that
of decreases in ATP and phosphocreatine is the AMP-
autonomous translational regulation of protein
the cytochrome oxidase subunit HI, which is
would be critical to mitochondrial biogenesis were not activated protein kinase (AMPK). AMPK activity is
synthesis. This is supported by measurements in
mitochondrialiy
affected. Therefore encoded,otherand IV, whichseem
co-stimuli is nucle-
to be increased, during exercise in the skeletal muscle.
skeletal muscle that revealed different rates of
arly encoded,
necessary in relation tobiogenesis,
for mitochondrial cytochrome one oxidase
of which However, recent observations indicate that this
protein synthesis and different adaptations to
activity.
is the cellular pool of high-energy phosphate bonds. response depends on the muscle type. Endurance
exercise between the two populations of
During the last
Interestingly, decade et
Jouaville a number
al. (1999) of transcription
found that training caused an enhanced expression of a
mitochondria, IMF and S3 mitochondria. Under
factors have been
mitochondrial calciumdiscovered,
levels such correlate as nuclear
with an subunit of AMPK in rat red quadriceps muscle but
nonadaptive steady state conditions, rates of protein
respiratory
enhancement factor 1 and 2 (NRF-1,
in mitochondrial NRF-2), that
ATP concentration. not in soleus or white quadriceps (Durante et al.
synthesis were found to be higher in IMF than in SS
activate several nuclear genes encoding for 2002). Activation of AMPK by administration of 5-
ATP/AMP Ratio
mitochondrial and ATP
enzymes. Turnover
However, these transcription
mitochondria. Acute exercise resulted in a decrease
amino- imidazole-4-carboxamide ribofuranoside
Cellular areenergy status is reflected by the ATP/AMP of protein synthesis in both fractions, but with
factors also involved in the coordinated (A1CAR) increases expression of some mitochondrial
ratio and phosphocreatine content, different time constants. In contrast, chronic
transcription of the nuclear and and appears as
mitochondrial enzymes like citrate synthase and cytochrome C
a central stimulator of mitochondrial biogenesis. In exercise reduces protein synthesis only in the IMF
genomes, since NRF-1 sites exist in the promoter (Oj'uka et al. 2000; Winder et al. 2000). On the other
addition to the physiological stimuli such subpopulations, but without any impairment in the
region of the mitochondrial transcription factor as A hand, expression of fatty acid synthase and the
endurance training, numerousmitochondrial
recent experimental chronic contractile activity-induced cytochrome C
(TFAM), which stimulates DNA pyruvate kinase gene is inhibited (Foretz et al. 1998).
conditions leading to a disturbance in energy oxidase activity increase (Connor et al. 2000). There
transcription and replication. With the discovery of In addition to its role in mitochondrial biogenesis,
metabolism increased the mitochondrial content of is evidence from a number of studies that the
PGC-1 (PPARy-coactivator-1), an important enhanced AMPK activity was associated with
skeletal muscles. Depletion of seems cellular endurance training-induced increase in
coactivator of mitochondrial biogenesis to increases in GLUT4 protein content.
phosphocreatine and ATP pools by chronic feeding mitochondrial oxidase capacity is more pronounced
have been identified (Wu et al. 1999). PGC-1 Interrupting ATP production by pharmacological
of [3-guanidinopropionic acid was followed by an in SS than in IMF mitochondria (Krieger et al. 1980;
coactivates NRF-1 and is involved in the stimulation dissipation of the electrochemical gradient using
increase Hoppeler et al. 1973). Recent observations revealed
of GLUT4ofexpression.
mitochondrial oxidative
Recent studies capacity
suggest of fast-
that mitochondrial uncoupling agents is another way to
twitch skeletal muscles due to an enhanced activity a more differentiated regulation, since single
single bouts of exercise induced the expression of activate gene transcription important for
of mitochondrial enzyme activities in both subpopuiations seem to
these transcription enzymes, an up-regulation
factors (Pilegaard et al. 2003). of mitochondrial biogenesis. However, mitochondrial
cytochrome C mRNA,PGC-1 and activation of the nuclear respond independently to the training stimulus
After 3-h swimming, mRNA was detected in biogenesis can be activated without substantial
respiratory (Bizeau et al. 1998). Furthermore, high-intensity
red muscles factor-1 (NRF-1)
while PGC-1 transcription
protein was changes in cellular ATP level as indicated by the fact
interval training
that exercise of moderate intensity can
18 The Cell
Mooren
the-inner membrane (Koehler 2000). Again this
translocase consists of several subunits closely
assembled with mitochondrial HSP70 and the
nucleotide exchange factor MGRP E, which function
as an ATP-dependent translocation motor. TIM-22 is
also associated with a number of proteins (TIM-18,
TIM-54). With the help of a family of small proteins
in the mitochondrial intermembrane space, proteins
are guided to the TIM-22 complex. Insertion of the
precursor into the inner membrane is dependent on
membrane potential. Therefore dissipation of the ion
gradient by uncoupling agents reduces the protein
import into the inner mitochondrial membrane.
In chronically stimulated skeletal muscles, an
adaptation of members of the protein import
machinery (e.g., MSF, cytosolic and mitochondrial
HSP70, TOM 20) has been shown (Takahashi et al.
1998). Twenty-week treadmill training increased the
HSP60 expression in rat muscle (Samelman et al.
2000). A differential regulation of mitochondrial
protein import has been shown for the
mitochondrial membranes and for the mitochondrial
subpopulations. The rate of protein import is higher
in IMF mitochondria than in SS mitochondria
(Takahashi & Hood 1996).
Mitochondrial Phospholipid Synthesis
As indicated in the preceding discussion, the
structural basis of mitochondria is formed by a
double membrane system. Therefore expansion of
Figure 1.6 Signaling and transport pathways leading to mitochondrial biogenesis. TOM/TIM, translocase of the outer/ inner
the mitochondrial reticulum during mitochondrial
membrane; JNK, c-Jun NH2-terminal kinase; AMPK, AMP-activated protein kinase; PPAR, peroxisome proliferator- activated
biogenesis requires the enhanced synthesis of
kinase; p38, mitogen activated protein kinase; AP-1, activator protein
various phospholipids as components of the
in comparison to continuous endurance training
was shown to be more effective in stimulating fatty
acid oxidation in IMF than in SS mitochondria,
suggesting a differential effect of the training
regimes on the biogenesis of mitochondrial
subpopulations (Chilibeck et al. 1998, 2002).
Mitochondrial Protein Assembly
An optima! oxidative phosphorylation capacity
requires the structural assembly and interaction of
both nuclear- and mitochondrial-encoded proteins.
Therefore biogenesis of mitochondria depends on an
effective transfer system responsible for the import
of precursor proteins from the cytosol as well as for
the export of mitochondrially coded proteins. For
this purpose, mitochondria! membranes contain
several protein transiocases along with a number of
chaperones and processing enzymes for assistance
in the translocation process. The first step for
translocation of a protein
17
membrane system. Lipid synthesis is performed in
the ER, followed by transport and sorting of the
lipids to the outer and inner mitochondrial
membranes. In contrast, cardiolipin, which is
involved in the translocation process of the TIM-23
complex, is synthesized at the inner mitochondrial
membrane (Schiame & Haidar 1993).
Endurance training is followed by a significant
increase of mitochondrial lipid synthesis. The ratio
of protein to lipid plus protein content of
mitochondria, however, remains stable during
endurance training, suggesting a correlation
between protein and lipid synthesis (Davies et al.
1981). Measurements of Hood and colleagues,
however, indicated a different time constant of
protein and lipid synthesis, the latter being faster
(Hood et al. 1994; Takahashi &Hood 1993).
Conclusion
The development of novel techniques has enabled
fascinating insights into the cells structure and
function. Its description as a steady state, a term
generally used for biochemical reactions, seems
applicable for the structural components of a cell as
well. Under resting conditions, and even more under
phosphatase
activated conditions, dynamic, interactions and
s (INK; p38)
transitions between various cellular components
exist and allow rapid and extensive adaptations!
responses. The following chapters give an overview of
our 1;current
knowledge
NRF, nuclear respiratoryregarding
factor. how cells and
subcellular structures face and respond to the
exercise challenge.
into mitochondria is to attach a targeting sequence
to the amino terminus. Next, the protein can bind to
the translocation complex TOM (which stands for
translocase in the outer mitochondria! membrane),
which consis ts of several subunits responsible for
recognition, binding, and translocation of the protein
(figure 1.6; Lithgow 2000).
A number of cytosolic chaperones guide the
precursor proteins to the transiocases and prevent
their misfolding and aggregation. Examples of these
chaperones are cytosolic HSP70 and mitochondrial
import stimulating factor (MSF).
After passing the TOM complex, proteins targeted
for the mitochondrial inner membrane and for the
matrix have to pass another translocase system
named TIM (translocase of inner membrane). TIM
consists of two different complexes, the TIM-23
complex responsible for transport of proteins into
the matrix and the TIM-22 complex responsible for
mediating protein insertion into
 Chapter 2 fiSIB
Cellular Life Span
Laurie Hoffman-Goetz, Joe Quadriiatero, and Harshna Patel
Research supported by a grant from the
Natural Sciences and Engineering Research Council of Canada
Cell Cycle and Tissue Turnover
fn order for proper growth to occur and life to be
sustained, cell renewal and reproduction are
required (Singh et al. 2000). Tissue turnover and
homeostasis are dependent on the relationship
between cellular proliferation, differentiation, and
death and are essential for the generation and
maintenance of complex tissue architecture.
Alterations in the balance between the signals that
lead to cell death and cell renewal can lead to
undesired tissue atrophy or tissue growth (King &
Cidlowski 1998; Pucci et al. 2000). The cell cycle is
an integral factor controlling cellular proliferation
and tissue turnover. The cel! cycle is a highly ordered
and tightly regulated process that assesses both the
intracellular and extracellular environments,
including factors such as extracellular growth
signals, cell size, and DNA integrity, at multiple
checkpoints. Progression through the cell cycle
ultimately results in the replication and distribution
of genetic material from one ceil generation to the
next (Ho & Dowdy 2002; Israels & Israels 2001;
Schafer 1998).
Cell Proliferation
Extracellular signals (such as growth factors,
hormones, and mitogens) play an important role in
cell proliferation, development, differentiation, and
survival (Talapatra & Thompson 2001).
Growth factors accomplish this by interacting with
specific receptors. Steroid hormones, such as
testosterone, estrogen, and cortisol, influence
cellular growth and differentiation by penetrating
the plasma membrane and acting on nuclear
receptors, thereby exerting direct effects on genomic
regulatory mechanisms. Polypeptide growth factors,
such as epithelial growth factor (EGF), insulin-like
growth factor-1 (TGF-1), and interleu- kin-2 (IL-2),
cannot penetrate the cell membrane and instead act
on specific plasma membrane receptors. These cell
surface receptors include enzyme-linked receptors,
such as tyrosine kinase and serine/threonine kinase
receptors, receptors associated with cytoplasmic
kinases, and the seven transmembrane domain
receptors (Martinez Arias & Stewart 2002).
Steroid hormone receptors are located in the
cytoplasm of the cell or nucleus and include the
receptors for androgens, estrogens, glucocorticoids
and mineralocorticoids, thyroid hormone, retinoids,
and vitamin D. Ligand-receptor binding results in an
allosteric change that leads to heat shock protein
dissociation, receptor dimerization, the binding of
the receptor to specific DNA elements, and gene
transcriptional activities (Kumar & Thompson 1999;
Sheppard 2002; Weigel 1996). Tyrosine kinases are a
family of enzymes that phos- phorylate or transfer a
phosphate group from ATP to tyrosine residues on
specific cellular proteins, upon binding of ligand or
growth factor. Tyrosine
19
 20 Hoffman-Goetz, Quadrilatero, and Patel
Cellular Life Span 21

kinases can be divided into two categories, receptor
Table 2.1 Mammalian Cyclins, Associated cdks, Cell Cycle Stage of Involvement, and
(e.g., epithelial growth factor receptor, fibroblast can enter the cell cycle at early G,; similarly, G 1 cells
Function During
growth factor Cell insulin-like
receptor, Cycle Regulation
growth factor-1 deprived of external growth stimulation can exit into
receptor) and nonreceptor (e.g., src, fgr, ctk, abl, and
fak) tyrosine kinases. The activity of tyrosine-specific
protein kinases is known to be associated with
various oncogenes that alter cellular growth (Cross
& Dexter 1991; Goel et al. 2002; Haluska & Adjei
2001; Hubbard 1999; Hubbard &TiU 2000).
Growth factors stimulate the cell to enter the cell
cycle; however, absence or early withdrawal of
growth factors will result in the cells returning to a
quiescent state (Schafer 1998). Growth factors
prevent cell death through various signaling
pathways, including the Jak/STAT, PJ3K/Akt, and
Ras/ MAPK pathways (Talapatra & Thompson
2001). However, lack of growth factor availability
results in the loss of mitochondrial function and
leads to cell death (Talapatra & Thompson 2001).
Cell Cycle and Mitosis
The cell cycle is divided into the G0, G,, S, G2, and M
phases. The G0 phase is a quiescent nonproliferating
state. The G, and G2 are gap phases during which
required cellular components such as RNA, proteins,
and enzymes are synthesized and accumulated for
use in the subsequent stage. During the S or
synthesis phase DNA replication occurs, while M
phase or mitosis involves the division of nuclear and
cellular components (Zafonte et al. 2000). The G,, S,
and G2 phases can be grouped into the interphase
whereas the M phase can be further delineated as
six distinct subphases each with specific
morphological characteristics. These include
prophase, prometaphase, metaphase, anaphase,
telophase, and cytokinesis. During prophase,
nuclear chromatin begins to condense and
centrosomes separate to form the mitotic spindles.
During prometaphase, the nuclear envelope is
broken down, which allows the chromosomes to
associate with the spindle fibers. Metaphase is
characterized by the lining up of the chromosomes
at the equator of the cell and disappearance of the
nuclear membrane. During anaphase, the
centromeres split, allowing the sister chromatids to
separate and move toward opposite poles of the
cells. At telophase, the nucleolus reappears and
there is a formation of a new nuclear membrane.
The final result, known as cytokinesis, culminates
with the formation of two genetically identical
daughter cells (Boisover et al. 1997; Singh et al.
2000; Van De Graaff & Fox 1995).
..Upon appropriate growth stimulation, G0 cells
G0. Although growth factor stimulation is needed for
a cell to enter into G, and proliferate, this stimulus
is essential only in the first two-thirds of the G,
phase. This point between the early and late G 1
represents an irreversible commitment to undergo
one cell division and is termed the restriction point
(Ho & Dowdy 2002). This restriction point divides
the cell cycle into a growth factor-dependent phase
(early GJ) and growth factor-independent phase (late
G, to M). Therefore, following initial stimulation, cells
can complete the remainder of the growth cycle
through mitosis in the absence of further exposure
to growth factors or mitogens (Jones & Kazlauskas
2001; Lundberg & Weinberg 1999).
Normal cells do not progress from one phase to
the next unless the events of the preceding phase
have been correctly completed. Progression of cells
through the cell cycle is controlled at specific stages,
particularly at the G, to S and G2 to M transitions.
These transition stages are referred to as cell cycle
checkpoints (Poster et al. 2001; Sherr 1996).
Specifically, cyclins are the major regulators of the
cell cycle because of thejr association with cyclin-
dependent kinases (cdks). Cdks belong to a family of
serine and threonine protein kinases and are
dependent on the presence of cyclins for activation
(Pucci et al. 2000). Normally, cdks are inactive, but
once bound to their cyclin counterpart they form
active cyclin- cdk complexes. These complexes
phosphorylate critical serine and threonine sites on
the target cells, which ultimately stimulate gene
expression of transcription factors and critical cell
cycle components (Foster et al. 2001; Lundberg &
Weinberg 1999; Zafonte et al. 2000). A variety of
cyclin-cdk complexes are formed during distinct
phases of the cell cycle and are responsible for the
phosphorylation of a particular set of target proteins
(Lundberg & Weinberg 1999). Currently, 16 cyclins
(cyclin A, B,, B2, C, D,, D2, D3, E, F, G,, G2, H, I, K,
T,, and T2) as well as 9 cdks (cdk 1-9) have been
identified in mammalian cells. Table 2.1 summarizes
selected mammalian cyclins, their associated cdks,
cell cycle stage of involvement, and defined functions
(Johnson & Walker 1999; Kong et al. 2000; Leclerc
& Leopold 1996).
During the G, phase there is an increased
expression of cyclin D (D,, D2, D() as well as cdk4
and cdk6. Progression through late G, is coupled
with an increased expression of cyclin E and cdk2

Cyclins Associated cdk Cycle phases Function

A cdk1(cdc2), S Participates as a negative feedback loop for E2F
cdk2 regulation
B,.Ba cdkl C2toM Regulation of cyclin degradation during mitotic exit
C cdk8 G0tS Phosphoryiates RNA polymerase II
Phosphorylates Rb and Ri>-related proteins that
^2' ^3 cdk4, cdk6 G0toS regulate the activity of transcription factors (e.g., E2F
family)
E cdk2 G, to 5 Maintains Rb in a hyperphosphorylated state therefore
acting as a positive feedback loop for E2F
F ? G, to M Regulates cyclin B, localization to the nucleus
and is required for transition from G, to S phase.
Binding of cyclin A with cdk2 allows DNA synthesis
to occur during the S phase. Cyclin A associates
with cdkl later on in S phase, and increased
expression of cyclin A and B with cdkl propels the
cell to exit G2 and complete mitosis (Israels & Israels
2001). These cyclin-cdk complexes are regulated by
the varying expression of cyclins and cdk inhibitors.
Two families of cdk inhibitors are involved in cell
cycle control. The Cip/Kip family includes p21 and
p27, which function at various points of the cell
cycle. In particular, these cdk inhibitors target cdk2,
-4, and -6. The second family includes the inhibitors
of cyclin-dependent kinase 4 (INK4) genes, plG*
and pi 9^ (Israels & Israels 2001). Figure 2.1 shows
a schematic representation of the cell cycle and
various regulator components affecting progression
through the various stages.
Telomeres
Telomeres are DNA-protein structures found at the
ends of all linear eukaryotic chromosomes. This
cap protects the chromosome ends from
degradation that would normally occur during
double DNA strand breaks. Telomeres also function
to prevent illegitimate recombination of chromosome
ends, which would lead to unstable chromosome
forms. Therefore, telomeres are critical for the
proper function, integrity, and stability of the
chromosome and ultimately establish a stable linear
chromosome end. In addition, it has been proposed
that telomeres have a central role in determining the
number of times a cell can divide (Blackburn 1991;
Perrem & Reddel 2000).
Telomere DNA usually consists of double-
stranded sequences, with a single G-rich strand that
protrudes at the 3N end and forms a singlestrand
overhang (Price 1999). In mammalian cells, the
length of the double-strand repeat (TTAGGG/
CCCTAA)n ranges from a few to tens of kbp, whereas
the length of the single-strand overhang (TTAGGG)n
is usually no more than a few hundred nucleotides
(Coljins 2000). The number of telomeric repeats
differs from one organism to another. Telomeric
length also varies in different mammalian tissues as
well as from one chromosome to another
(Buchkovich 1996). Friedrich et al. (2000) found that
telomere length was significantly shorter in
leukocytes compared to skin (fibroblasts) and
synovial tissue (fibrocytes) of the same patient and
was consistent with the faster replicating properties
of leukocytes compared to the other tissues.
During DNA replication, a gradual shortening of
the telomere occurs. Because DNA polymerases
work only in the 5N to 3N direction and require
short oligonucleotides as primers, shortening of the
telomeric DNA is expected during each cell cycle
(Buchkovich 1996). Telomere terminal transferase or
telomerase is a telomeric-specific ribonucleoprotein
reverse transcriptase that is important in telomere
maintenance. Telomerase is composed of a
structural RNA and two proteins, telomeric repeat
binding factor 1 (TRF1) and TRF2. Telomerase
utilizes an integral RNA component as a template for
de novo synthesis and adds single- stranded
telomeric repeats to the chromosomes 3N end. Both
TNF1 and -2 have been hypothesized to aid in the
docking and holding of the enzyme to the telomeric
DNA (Collins 2000; Counter 1996; Perrem & Reddel
2000).
 22
24 Hoffman-Goetz,
Cellular Ouadrilatero.
Hoffman-Goetz, Quadrilatero,
Life Span and
andPatel
Patel 23

..in1.4
be stimulating
+ 0.1 kbendothelial
longer thancell migration.
those of memory However, cells tion
cells,ofwhich
the basement
interact to. membrane
produce by activated
proteases, TGF-P (3)
both processes can be inhibited
from the same donors and were independent of age. by angiogenesis endothelial
that furthercell induces
migration changes and inproliferation,
the pericytes andand (4)
inhibitors
These such suggest
findings as angiostatin
that cell anddivision
endostatin (Nie &
is ongoing maturation
myofibroblasts. of the neovasculature.
TGF-j3 is a homodymeric
Honn 2002). the life span and that memory cells
throughout polypeptide
Angiogenesis thatisisinitiated
strongly in response
expressedtoin hypoxic
sites or of
Endothelial
undergo more cells have the
extensive cellintrinsic
divisionsability
thanto naiveform ischemic
tissue morphogenesis
conditions that (Robertsresultet al.
in 1986).
the release of
tubelike
cells. structures
A later studyafter endothelial
by Weng cell proliferation
and colleagues (1997) cytokines
Angiogenesis
from variousis controlled
sourcesby(Griffioen
a balance & between
Molema
and migration
showed that meanthrough TRF lengths extracellular matrix.
were significantly 2000).
angiogenic
Relaxation
and angiostatic
of the blood factors.
vesselSomeis a prerequisite
angiogenic
Endothelial
greater cells elongate
in germinal centerand align to form compared
B-lymphocytes a sprout, for
factors
endothelial
act directly
cells toby enter
stimulating
the angiogenic
cells to cascade.
migrate,
andnaive
to the lumen
and memoryis formedcells. by a Differentiation
curvature within fromeacha This
proliferate,
is mediated
or form abyvessel, nitricwhereas
oxide others (NO), can an
endothelial
naive to a cell (Qian etcenter
germinal al. 1997). Individual
lymphocyte sprouts
results in endothelium-derived
indirectly activate host relaxingcellsfactor
(macrophages,
(EDRF), and mastby
elongate andlengthening
significant eventually join, of forming loops through
the telomere, while up-regulation
cells, lymphocytes) of fibroblast
to release growth endothelial
factor-2 (FGF-2)
growth
which blood begins
subsequent to flow. However,
differentiation to ain ordermemory to form B- factors.&The
(Ziche Morbidelli
direct-acting2000). factors
VEGFincludeis then acidic
ableand to
a stable vasculature
lymphocyte results and in prevent
significantceil deathtelomeric by stimulate
basic fibroblastic
vasodilation growth andfactors
affect(aFGF
the permeability
and bFGF),
apoptosis, endothelial
shortening that may cells must interact
be controlled by with mural
telomerase and
VEGF, vascular
and angiopoietin
tone via (Nie endothelial
& Honn 2002).NO production
Indirect-
cells such as pericytes in small vessels and smooth
activity. (Griffioen
acting factors& Molemainclude 2000;
tumor Ziche
necrosis
& Morbidelli
factor-a 2000).
(TNF-
muscle cells within large vessels (Griffieon & The
a), TGF-j3,
increaseandin platelet- microvascularderived permeability
endothelial cell to
Angiogenesis
Molema 2000). This can be achieved through the proteins
growth is factor/thymidine
a crucial step in angiogenesis.phosphorylase Leakage (PD-of
production of platelet-derived growth factor (PDGF), plasma
ECGF/TP). proteins
These factorsand the are briefly
formation describedof an in
Angiogenesis,
which acts as athe formation
mitogen of new capillary PDGF
and chemoattractant. blood extravascular
table 2.2. However, fibrin gel angiogenesis
are sufficient inhibitors
for endothelial
exist to
vessels
induces from preexisting
differentiation of thevasculature,
mural cells involves
into cell
counterbalance
growth (Folk-the man effects
1997). of the
VEGF angiogenic
can also factors.
induce
complex
pericytesinteractions
or smooth muscle among endothelial
via cell-to-cellcells, matrix
contact- the
Theseexpression
factors include
of proteases
thrombosponsdin,
and receptorsinterferon,that are
proteins,
dependentand soluble factors,
processes. Transforming leadinggrowth
to endothelial
factor-P crucial
tissue for inhibitors
tissue remodeling
of MMP and (TIMP),
prevention
angiostatin,
of cell
cell proliferation, migration,
(TGF- (3) is found on both mural cells and vessel formation. and death
endostatin,
in endothelialand cells platelet
(Farrara factor-4
2001). and are
Normal
endothelial tissue growth is characterized by dependence summarized
For endothelialin table cells
2.3. to enter into new areas of the
on new vessel formation for the supply of oxygen and body, they must detach from the basement
nutrients as well as removal of waste products (Ziche membrane via proteolysis. Matrix metalloprotein-
Table 2.2 Angiogenesis
& Morbidelli 2000). However, Growth
regardless Factors
of the cause, ases (MMP) are endopeptidases that are secreted,
new vessels always grow out of preexisting vessels. activated, and then degraded in the extracellular
Figure
The2.1blood
A schematicvessels representation
in the body of norma!
functionceil cycle
as a and selected regulatory elements involved in cellular progression.
matrix. MMP facilitate the invasion of endothelial
Following growth factor stimulation, normal cells progress through the stages of the cell cycle with the aid of various
transport compartment for the blood and play a cells through the basement membrane andavailability
entrance
regulatory factors. Cycle progression is positively influenced by growth factor (e.g., insulin-like growth factor-1)
major role in maintaining homeostasis in various into avascular
(prior to the restriction point), cydins (e.g., cyciin D), cyclin-dependent kinases (e.g.,tissuecdk4), andin oncogenes
response (e.g., to angiogenic
c-myc) and
ways. Thebyblood
is inhibited directly contacts
tumor suppressor genes (e.g., thep53)endothelial stimuli (e.g.,
as well as cdk inhibitors (Stelterp21).Stevenson 1999). MMP are secreted
cells on the vessels composed of pericytes, smooth by a number of cells including fibroblasts,
muscle cells, fibroblasts, basement membranes, and inflammatory cells, epithelial cells, and endothelial
an Progressive
extracellularshortening
matrix within the subendothelium
of chromosome ends is telomerase activity is low or absent in replicating
cells.
(Griffioen
intimately&coupledMolema with 2000). The endothelial
cellular division ofcells normalform cells, telomere lengths
As endothelial ceils leave of their
the original
chromosome will
sites, they
asomatic
monolayercells and and are actively
results in the involved in several
so-called end- gradually decrease
proliferate and can (Goynsinvade&Laveryboth2000).vascular and
regulatory
replication processes
problem. This in istheattributable
body including to the Immune
avascular tissues.response
Angiogenic is factors
dependent such as on VEGF the
coagulation
inability of the DNA polymerasesintegrity
and metabolism. Tissue to complete and . expression
and bFGF and can differentiation
directly stimulate of specific responsive
endothelial cell
homeostasis
replication ofare theregulated
3N end by duea to balance between cell
their requirement cells following
proliferation viaancGMP-mediated
antigenic challenge. Stimuli
activation of that
the
proliferation
for an upstream and cellRNAdeath.primer, Modulation
ultimatelyofresulting
angiogenic in result in the proliferation
mitogen-activated protein kinaseand expansion
(MAPK) family of normal(Nie
function
the shrinking is achieved through regulation
of the chromosome ends withof each the &lymphocytes
Honn 2002).should However, alsoinresult
order infor the induction
endothelial of
cells
survival
successive of endothelial
cell cycle. cellsThis
during vessel cell
affects repaircycle and telomerase
to activity. This
create a network, they mustwould migrate
serve to maintain
toward and
angiogenesis,
reproductive whereby capacity unnecessary,
(McEachern et newlyal. formed
2000; telomere
within length tissue.
the vascular and reproductive potential.
As the cells proliferate,
vessels undergo regression
Wynford-Thomas 1996). and apoptosis
Cessation of (Nor
cell Hathcock et is
plasminogen al. converted
(1998) found to that
plasmin unstimulated
by the
&Polverini
proliferation 1999).is indicative of cellular aging and mouse T-lymphocytes
plasminogen activators expressed
u-PA andlowt-PA. to virtually
Plasmin
The senescence.
cellular adult vasculature Normaliscells usually quiescent.
in culture possess In undetectable
degrades levels laminin,
fibronectin, of telomerase
and the proteinactivitycore butof
response to angiogenic stimuli,
a maximum number of cellular divisions called the such as vascular expressed significant
proteoglycans. Plasmin telomerase activity after
is considered the inmost
vivo
endothelial
Hayflick limit growth factor (VEGF)
(Perrem &Reddeland basic Telomeric
2000). fibroblast antigen stimulation.
important protease for Weng et al. (1995)
mobilization found from
of FGF-2 that
growth
length isfactor (bFGF),
predictive endothelial
of replication cells undergo
capacity in normala as donor age increased, from
the heparin sulfate proteoglycans in the extracellular24 to 75 years,
series
cells in ofvitrotightly
(Allsopp controlled
et al. 1992; events
Harley toet form
al. 1990), new terminal
matrix poolrestriction
(Griffioen & fragment
Molema (TRF) 2000). length
Aside from (an
capillary vessels. activity
and telomerase These steps include
is either (1) initiation
absent or at very of estimate of cell
stimulating telomere size) decreased
proliferation, VEGF and inbFGF
both play
naivea
the
low angiogenic
levels in many response, adult(2)somatic
dissolu cells (Chiu et al. and memory CD4+ lymphocytes. Furthermore, TRF
role
1996; Hiyama et al. 1995). Moreover, if lengths of naive CD4+ lymphocytes were found to

Factor Function Receptor

DIRECT ACTING
Basic fibroblast growth factor Endothelial mitogen, survival factor, FGFR-4
(bFGF/FGF-2) angiogenesis inducer; inducer of Flk-1
expression
Acidic fibroblast growth factor Endothelial mitogen and angiogenesis FGFR-4
(aFGF/FGF-1) inducer
Vascular endothelial growth Endothelial mitogen, survival factor, and Flk-1
factor/Vascular permeability factor permeability inducer Flt-1
(VEGF/VPF)
 Angiogenin
In vivo-acting angiogenesis inducer with Angiogenin
RNase activity receptor
Epidermal growth factor EGFR "
Weak endothelial mitogen, inducer of VEGF
expression
Tumor necrosis factor-a (TNF-a) In vivo-acting angiogenesis inducer; TNFR-55
endothelial mitogen (low concentrations) or
inhibitor (high concentrations); inducer of
VEGF expression
Platelet-derived growth factor (PDGF) Mitogen and motility factor for endothelial PDGFR
cells and fibroblasts; in vivo angiogenesis
inducer
Thymidine phosphorylase (TP)/Piatelet- Receptor
derived endothelial cell growth factor In vivo-acting angiogenesis factor unknown
(PD-ECGF)
Transforming growth factor-p (TGF-p) in vivo-acting angiogenesis Inducer or
TGF-BR I, II,
endothelial growth inhibitor; inducer of Ml
VEGF expression

INDIRECT ACTING
inhibitors Function
Plasminogen fragment; systemically acting
Cellular Life Spanangiogenesis inhibitor. 25
Angiostatin Inhibits endothelial ceil proliferation by unknown mechanism.
Endostatin Proteolytic fragment of collagen XVIII. Systemically acting angiogenesis
Table 2.3 Inhibitors of Angiogenesis
inhibitor. Inhibits endothelial cell proliferation and angiogenesis by
unknown mechanism.
Thrombospondin-1 Binds to CD36 on endothelial cells and inhibits angiogenesis by
(TSP-1) unknown mechanism.
Tissue inhibitors of Blocks breakdown of extracellular matrix and endothelial cell invasion
metalloproteinases by inhibiting MMPs.
(TIMPs)'
Platelet factor 4 (PF4) Inhibits angiogenesis by interfering with bFGFR.

Prolactin Inhibits angiogenesis by bFGF and VEGF-Induced proliferation to

endothelial cell (S phase arrest).
inhibits EGF and laminin-dependent endothelial cell motility and
EGF fragment angiogenesis.
Interferon-a (IFN-a) Inhibits angiogenesis by antimigratory and antimitotic effect on
endothelial cells; blocks bFGF production by parenchymal cells.
Interferon-p (IFN-p) Inhibits endothelial cell proliferation and migration.
interferon-7 (IFN-7) Inhibits endothelial cell proliferation and migration.
lnter!eukin-12 (IL-12) Inhibits angiogenesis by stimulating IFN-7-
26 Hoffman-Goetz, Quadrilatero, and Patel

Apoptosis -~ and execute the apoptotic program through cleavage

of several vital proteins (caspase 3, caspase 6,
Apoptosis is a highly regulated, evolutionarily
caspase 7). Three pathways result in caspase
conserved pathway that is essential for normal
activation and subsequent apoptosis. Figure 2.2 is a
embryonic development, regulation of the immune
simplified illustration of the pathways to apoptosis
system, hormone-dependent atrophy, chemically
and necrosis.
induced cell death, and tissue homeostasis (Cohen
In the first pathway, cell death is associated with
1997; Phaneuf & Leewenburgh 2001; Sjostrom &
mitochondria permeability changes. This is known
Bergh 2001). Apoptosis can also be induced by a
as the death-by-neglect pathway because it involves
number of deleterious stimuli or stressors including
deprivation of cells of necessary survival stimuli
DNA damage, heat, hormonal triggering, anticancer
(e.g., growth factors or co-stimulators), which then
drugs, and physical stress. Since apoptosis does not
leads to the rapid increase in the permeability of
result in the release of intracellular material into the
mitochondria membranes and the release of
extracellular space, it usually does not lead to an
cytochrome C (Budd 2001). Various factors,
inflammatory response. Apoptosis is an active
including irradiation, glucocorticoids (GC), nitrogen
process that requires participation of the dying cell
monoxide, reactive oxygen species (ROS), and certain
and changes in cellular biochemistry and
chemotherapeutic drugs, induce apoptosis by this
morphology, leading to DNA cleavage, nuclear
biochemical mechanism. Cytochrome C, localized on
condensation and fragmentation, and changes in
the outside of the inner mitochondrial membrane
membrane lipid distribution (Bidere & Senik 2001).
and intermembrane space (Hirsch et al. 1997),
Apoptotic cells are rapidly endocytosized by
functions as a cofactor with a protein known as
neighboring healthy cells or phagocytosized by
apoptosis activating factor-1 (Apaf-1) to stimulate
professional phagocytes that recognize a number of
caspase 9 and initiate the apoptosis pathway.
signals on the surface of condemned cells, such as
Cytochrome C is released into the cytosol, binds to
phosphatidyiserine residues that pass through the
Apaf-1, and forms a complex known as dATP. This
plasma membrane. Apoptosis results in the
complex then activates procaspase 9 to caspase 9
irreversible, internucleosomal fragmentation.of
and eventually results in the activation of caspase 3
genomic DNA and the fragmentation of the cell into
Angiogenesis and Telomeres (van Cruchten &Cell van Death
den Broeck 2002). This
membrane-bound apoptotic bodies (Los et al. 2001;
pathway seems to be controlled by a group of
Phaneuf & Leewenburgh
The relationship 2001). The
between angiogenesis and complex
telomere proteins that regulate
The processes of cell apoptosis.
death, cell These proteins (e.g.,
proliferation, and
sequential process of
length/telomerase apoptosis
activity has involves an effector
been investigated. Bcl-2,
the ceilBax, and are
cycle Bcl-x) are codedlinked
inexorably for by (Evan
the genes of
et al.
phase,
Franco aetpointal. of no return,
(2002) a degradation
indicated phase,
that telomerase- the Bcl-2
1995). Two(B-cell
terms lymphoma)
have been family
usedandto may function
describe the
and a clearance phase. In the effector
deficient mice (Terc have shorter telomere lengths phase,
to regulate
process of cellmitochondrial
death, necrosis and permeability via the
apoptosis. Necrosis,
endonucleases and cellular caspases
and impaired angiogenic potential, Mean TRF are activated
mitochondrial
a term introduced permeability
by Virchowtransition
in the 19th pore, with
century,
and mitochondrial
lengths were founddysregulation
to be shorter occurs (Salvesen
in endothelial &
cells consequent
refers to a release
passiveof process
cytochrome C intoby
induced thecatabolic,
cytosol.
Dixit 1999). At the point of no return,
from the iliac arteries (a site of higher hemodynamic cells are
Theor second
toxic, pathological pathway
eventsof(Grogan
apoptosis
et al.involves
2002).
destined
stress and to cell
die. turnover)
Reorganization of thetocytoskeleton
compared iliac veins. triggering
Recently, itbyhas binding of ligandsthat
been suggested to death-inducing
necrosis is not
and nuclear chromatin
Furthermore, telomeric breakdown
DNA loss was characterize
greater in thethe membrane
a mechanism receptors
of cell (Newton
death at& all Strasser
but is 2001).
rather This
the
degradation phase. Finally,
iliac artery compared to thethe removal
internal of apoptotic
thoracic artery pathway
collectionis ofknown as the receptor-ligand-
morphological changes occurring mediated in
cells byofmacrophages
(a site low hemodynamic and other
stresscells
andoccurs
normallyas part
free pathway of apoptosis.
cells postmortem (MajnoTwo& important
Joris 1995). ligands in this
Necrotic cell
of the clearance phase (Aigner 2002).
from atherosclerotic plaques), and was more pathway
death isareassociated
Fas ligandwith (FasL), which inflammatory
specific binds to the
The effector phase of apoptosis involves the
dramatic with increasing donor age (Chang & Harley Fas receptor,
changes and TNF-a
in dying (an inflammatory
and adjacent cells. In cytokine),
contrast,
activation of cytosolic enzymes known as caspases.
1995). Of clinical importance is the finding that which binds
apoptosis to the
refers TNFR1
to the set receptor. Fas events
of cell death and TNFR1with
Caspases are cysteine proteases that cleave the
human telomerase reverse transcriptase (hTERT) have corresponding
particular morphologicalcytoplasmic regions known
characteristics such as
carboxy terminal of aspartic acid (Asp) residues
mRNA is expressed by vascular endothelial cells of conserved
blebbing and death domain
nuclear (DD) responsible
fragmentation (Kerr 1971; for
(e.g., cysteine aspartases). Caspases are present in
astrocytic tumors but is absent in normal brain signaling
Kerr et al. transduction
1972). Wyllie and in apoptosis. Transduction
colleagues (1980) linked
an inactive form in the cytoplasm in virtually every
(Pallini et al. 2001); hTERT mRNA was related to the occurs through the
the observation binding of patterns
of stereotyped the Fas-associated
of induced
cell (Newton & Strasser 2001; Villa et al. 1997). To
histological grade of the tumor. Collectively, these death domain with procaspase
DNA fragmentation 8, the activation
in gel electrophoresis with the of
date, 14 caspases have been identified and
studies suggest that telomere length and telomerase caspase 8, andFurthermore,
term apoptosis. the eventual conversion
apoptosis describes of
implicated in cell death. Caspases cleave precursors
activity play a key role in angiogenic potential and procaspase
an active cell3 into
deathcaspase
process 3. that occurs under both
to produce mature cytokines (caspase 1, caspase
may have implications for tumor therapy and age- Not surprisingly,
normal physiological lymphocytes
and express Fas. FasL
pathophysiological
11), initiate the propagation of apoptotic death
associated vascular diseases, such as is mainly expressed
conditions. by T-lymphocytes
These processes after in further
are described
signals (caspase 8, caspase 9),
arteriosclerosis. detail in the sections that follow.

Bogota
28 Cellular Quadrilatero,
Hoffman-Goetz, Life Span and Patel 27

independent .p{,the receptor-ligand pathway and the (SODD), another cytoplasmic protein, may be
mitochondrial pathway. involved in limiting apoptosis signaling by death
The commitment to cell death is based on a receptors since it also has a death domain and
number of factors. For example, receptor clustering interacts with Fas (Musci et al. 1997).
results in caspase activation to generate the first Regardless of the pathway through which
proteolytic signal. Commitment to other pathways is apoptosis occurs (i.e., through death by neglect or
dependent on whether the extent of damage death by triggering of death receptors), certain
outweighs the ability of the cell to repair itself, which morphological changes occur in the dying cell
is in itself dependent on the ratio of proteins that (Hacker 2000). These morphological consequences of
initiate apoptosis (e.g., Fas, p53) to the proteins that apoptosis are (1) loss of attachment of the apoptotic
inhibit or block apoptosis (e.g., Bcl-2) (Phaneuf & cell to other cells and the extracellular matrix, (2)
Leewenburgh 2001). Low levels of damage beyond the occurrence of protrusions from the plasma
ability of the cell to repair itself result in postmitotic membrane (blebs), (3) condensation of the nucleus
arrest and cell survival. More severe damage leads to and fragmentation of genomic DNA, (4) dilation of the
the activation of apostat, induction of death signals, endoplasmic reticulum and release of ribosomes, and
and the downstream activation of executioner (5) disintegration of the cell into apoptotic bodies that
caspases. are membrane- bound vesicles varying in size and
As noted earlier, several factors trigger apoptosis, composition (Hacker 2000). Apoptotic bodies are
including growth factor deprivation, cytokines, engulfed by neighboring ceils, especially
members of the TNF family, TGF-3, GC, retinoids, macrophages. Under some circumstances apoptotic
cytotoxic chemicals, and many more. Apoptosis can cells that are not phagocytosized show features of
also be induced by cell injury resulting from toxicity, necrosis without inflammation (van Cruchten & van
ischemia and reperfusion injury, oxidative stress, den Broeck 2002).
inflammation, and irradiation (Fellstrom & Zezina
2001). The apoptotic pathway in lymphocytes can be Necrosis
prevented by various activating stimuli, including
specific antigen recognition;" provision of essential The term necrosis means the irreversible and
growth factors (such as IL-2), and engagement of co- dramatic changes that are visible by microscopy after
stimulators (such as CD28 on T-cells by B7 cell death (Majno & Joris 1995). Most pathology
molecules on antigen-presenting cells). In response to textbooks define necrosis based on whether the
apoptotic stimuli, inhibitory genes may be up- morphological changes are due to enzymatic
regulated, leading to the synthesis of apoptosis digestion of ceils (e.g., Iiquefactive necrosis) or due to
inhibitory proteins. representation
One of the best denaturation of proteins (e.g., coagulative necrosis).
Figure 2.2 Schematic of thecharacterized
pathways to apoptosis and necrosis, (a) Ligation of death receptors with
apoptosis inhibitory protein Necrosis is characterized by osmotic swelling,
cytokines, such as FasL and TNF-a,isleads
Bcl-2, which blocks
to activation of death receptors (e.g., Fas, TNFR1, TNFR2, TRAIL), activation of the
cytochrome
caspase cascade,C release (b) Stress to the
from mitochondria,
and apoptosis, binds Apaf-ROS,rupture
cell (e.g., GC, and of the plasma
growth membrane,
factor withdrawal) and
leads the release
to MPT formationof
1,
andand inhibitsC release
cytochrome activation of cytosol,
into the caspasewhich is in part &
9 (Newton cytosolic
regulated by thecontents
expressioninto theand
of Bd-2 extracellular environment.
Bax. The formation of the
dAPT complex
Strasser initiates
2001). the activation
Bcl-2 insertsof theintocaspase outer andNecrotic
the cascade cells retain
leads to apoptosis, the toshape
(c) Stress of the reticulum
the endoplasmic nucleus,
(ER) may lead tomembrane
mitochondrial cellular apoptosis via activation
and maintains the of caspase 12.although
integrity chromatin of stress
(d) Greater magnitudes condensation,
will bypass thelate DNA
apoptotic
pathway and lead to necrosis, (e) Sustained
of the mitochondria by allowing the export of H ions accumulations
+ of degradation,
intracellular and
calcium pyknosis
(Ca 2
*) can (nuclear
activate shrinkage
calpains, leadingand
to
necrosis, (f) Decreased intracellular ATP availability can lead to cell basophilia) can
death via necrosis. occur (Chautan et al. 1999;
through ion channels (Budd 2001). A family of
proteins that contain the Bci-2 homology domain 3 Schweichel & Merker 1973). Moreover, necrosis is
region (e.g.,by Bax, Bad,and Bim, characterized_y/_ by the relatively slow disintegration of
activation antigens theBid) counteracts
cytokine IL-2. When the caspase 12 transgenic knockout (KO) mice
actions of Bci-2. Apoptosis signaling by death ligands the cell in which the plasma membrane integrity
mature T-lymphocytes are repeatedly stimulated by (Nakagawa et al. 2000). The basis for this pathway
isantigens,
also regulated breaks down and the cellular contents are
Fas andinFasL a number of ways. In Cytotoxic
are co-expressed. humans is the finding that when mice are challenged with
and a T-lymphocytes
variety of animal enzymatically degraded and released (Hacker 2000).
(CD8*) have a species,
specialized secreted
form of Fas- and the ER-stressing agents, tunicamycin and thap-
membrane-anchored decoy receptors The end result of necrosis is pathological
Fas ligand-mediated apoptosis. In CDS* preventT-cells, sigargin, apoptosis occurs in fibroblasts from
apoptosis inflammation. Whereas apoptosis is considered
binding ofsignaling
Fas-FasL by neutralizing
leads to the releaseFasLofand TNF-
perforins heterozygous (caspase 12+/~) mice but not in
related immunologically silent, a hallmark of necrotic death
at the apoptosis-inducing ligandand (TRAIL)
the (Newton & :
target cell membrane release of caspase \2~ ~ animals. In contrast, when
Strasser is the involvement of inflammatory cells. The
granzyme2001).B intoMoreover,
the targetincytosol
T-lymphocytes,
and inductiondeliveryof homozygous knockouts were given Fas-specific
of FasL to (Martin
the cell&surface involvement of these inflammatory cells (especially
apoptosis Green is under
1995; vanstrict control.
Cruchten & antibodies or synthetic GC (which activate the
FasL is stored in lytic granules, and degranulation in macrophages and neutrophils) and the release of
van den Broeck 2002). receptor ligand pathway and the mitochondrial
response to pathway
target cellforrecognition chemoattractants to Simulate neutrophil emigration
A third apoptosis leads to thetermed
has been rapid pathway of apoptosis, respectively), no differences
delivery of FasL to the cell surface. Silencer of death distinguish necrosis from
the endoplasmic reticulum (ER)-mediated were found in apoptosis. Thus, it has been
domain
mechanism and has been identified by studies in suggested that an ER-mediated mechanism of
apoptosis must be
 30 CellularOuadrilatero,
Hoffman-Goetz, Life Span and Patel 29
31

apoptosis.
recent
It is apparentHowever,
approach to this
that identifydistinction
although apoptotic may
the mechanisms cells be using
more
and For example,
exercise
target-cells and various
represents
through calpain
the inhibitorsangiogenesis
physiological
mitochondrial have been
mechanism
apparent than
flurochrome-labeled real. When
Annexin apoptosis
the circumstances of cell death vary, the end result V, which occursbinds on a
to shown
within to protect
mature against apoptosis
differentiated
(cytochrome C and Apaf-1) as weli as through in lymphoid
tissue. cells
Exercise the
large scale,
phosphatidylserine for example
(PS) that
is always the same: the disappearance of cells. Theis during
externalized embryonic
on the (Sarin et al. 1995; Squier et al.
receptor-mediated ligand mechanism, resultingthe
enhances 0 2 transport 1994;
conductance Wang 2000
between ). in
development,
membrane
impact of cell largefornumbers
of death
damaged or apoptotic
tissues, of phagocytic
organs, cells (Vermes
systems, ceils
andet Whatactivation
factors trigger
microcirculation
caspase and cells to respond
(Cainmitochondria,
& Freathy 2001).andwith necro-
increases
TGF-Pj also,
appear,
al. 1995).although
organisms dependsitbecause
However, is unclear
upon theAnnexinwhether
biological V binds these
contextto are
PS
in sis compared
.the number
suppresses pRb to
and apoptosis? Although
size of mitochondria
expression (Fan et al.many of the
in skeletal
1996), and
professional
residues
which it within
occurs. phagocytes
damaged
In somecells, or
cases, simply
thiscell neighboring
technique
death is may a same
musclestimuli
experiments and trigger both
the activity
with cell
of death
pAb-deficient enzymes pathways,
mice controlling the
indicate
parenchymal
pick
normal up physiologicalcells that
both apoptotic take
and up thecells.
necrotic
consequence ofcellular debris
To provide
development more toxicmetabolism.
oxidative
widespread the stressor,
apoptosis in the
Skeletal more
a varietymuscle likely it is sues,
ofangiogenesis,
tis-... that
(apoptotic
specificity
and bodies)
differentiation (Majno
during& embryogenesis.
in distinguishing Joris 1995).from In
apoptotic necrotic
other necrosis
determined
including will
skeletalensue.
by an
muscleAdditionally,
increase which
in capillary
(Zachsenhaus et cell death
al. density,
1996).
cells,
cases, Unlike the apoptosis,
cell use occurs
death of necrotic as acellpathophysiological
propidium death
iodide does (PI) not is pathway (apoptosisratio,
capillary-to-fiber or necrosis)
or both, occurs reflectsfollowing
a vital
involve activation
recommended
response to injury sinceof
or caspases.
PI enters
toxic insult. However,
necrotic the cellsabsence
but is Detection
injury depends
adaptation of Apoptosis
to upon
exercisethe intensity
and allows of the forinjury
improved and
of caspase
excluded activity ones.
from apoptotic does PInot is a eliminate
DNA stain and the the level capacity
aerobic of intracellular ATP available
(02 transport, (Leist et and
conductance, al.
Apoptosis and Cell Cycle
involvement
can therefore of
be other
used enzyme
both for cascades
cell cycle involved
analysis andin There
1997; are
Nicoterra
extraction) many
of et techniques
al.
skeletal 1998).
muscles for
Other
(Amaralthe detection
cellular
et al. factors,
2001). of
proteolysis.
to differentiate Calpains
necrotic (suchfrom as apoptotic
calpain p)cells. have Table
been apoptosis
including
These changes in cells,
short or
occur each
prolonged
after withaerobic advantages
opening of the
endurance and
As
2.4 described
implicated
summarizes inearlier, the cell
cell approaches
death cycleused
(Wang consists
2000), of
tosince the G0
they
measure limitations.
mitochondrial The major
training characterized approaches
permeability
by low-resistance for the detection
transition and high- pore of
or rest phase,
inactivate in
apoptosis the
caspases G or protein synthesis
cells. t(Chua et al. 2000) and shift cells phase, the apoptosis
(Kowaitowski are via
et al. 2001),
repetition contractile morphological
the concentration
activity changes
(Amaral et al.of2001; by
pro-
Sfrom
or DNA an replication
apoptotic phenotypephase, the to G2 a phase extending
necrotic one. microscopy,
apoptotic
Gustafsson to Western
antiapoptotic
et al. 1999). blotting, gel electrophoresis
proteins
Angiogenesis (Denecker
is the result et al.to
of
from
Calpains the canend be of activated
DNA replication
physiologicallyto eventual
by stimuli cell identify
2001), characteristic
and the DNA
magnitude
prolonged imbalances between the metabolic patterns,
of histo-
oxidative chemical
stress
division,
that induce
Effect
and the of
M or
sustained
Exercise
mitosis phase.
elevation
onof The
Cell
Ca2+ control
in the of techniques,
(Lemasters
requirements ELISA,
1999), of and flow tissue
influence
the cytometric
the decision andanalysis. point of
perfusion
the Proliferation and Cell Death
cell
cytosol. cycle
The is through
endogenous the action
inhibitor,of cyclin proteins
calpastatin, deathMorphological
by
capabilitiesapoptosis
of the changes
or death
vasculature inby condensed
necrosis
(Gavin et (see
al. DNAfigureof
2000a).
(e.g.,
protects A, B,against
D, E) and by cdks.
caipain- Cyclin necrotic
mediated and cdksdeath. form apoptotic
2.2). cells
The decision
Therefore, can tobeoxygenation
decreased detected
commit by microscopy.
to apoptosis,
resulting necrosis,
from this For
complexes;
Exercise
Nonetheless, andthe
has by
thechanging
potential
distinction the to concentration
betweenmodulate and
caspase-cell example,
or cellularstaining
imbalance stasis
causes isofshown
cells tissues
the with hematoxylin
to becomeand
diagrammaticaily eosin
inhypoxic
figure
activity
proliferation
mediated of apoptosis
these
and complexes,
cell
and death cell division
through
calpain-mediated can be
cytokines,
necrosis orand
2.3.with acridinea orange
produces variety or Hoeschst can
of metabolites be used in
implicated to
initiated
hormones, or stopped,
growth
is far from absolute. or
factors, cells
and can
metabolicbe induced
pathways. to identify apoptotic cells but
vessel growth, including adenosine, ADP, lactic acid, requires careful
undergo
Some of these apoptosis.
factors have The been relationship between
well characterized interpretation and technical
nicotinamide derivatives, experience, since
and prostaglandins of the.
apoptosis
in exercise andmodels,
the cell cyclesuch isasbest VEGF illustrated
and NO by the in apoptotic and necrotic ceils
E series. The mechanisms underlying capillary may not be easily
p53 tumor
vascular suppressor
endothelial gene. This
responses gene others,
whereas encodes suchfor distinguished
proliferation remain (van Cruchtenunknown, & van butden it isBroeck
likely that 2002).a
the p53 protein,
as "expression of whose
cyclinsexpression
and telomeraseis increased in The molecular
number of biology
factors technique
are required of Western to blotting
initiate
follow-'
lymphocytes, ing DNAhave damage not (e.g., been following oxidative
systematically using antibodies
physiological angiogenesisagainst caspase to exercise.
in response substrates,
stress
investigated.or irradiation)
The following(Mathieu section briefly et al.describes apoptosis-related
1999). - However, it is suggested proteins, that cyclins,
these factors and act tumorin
Expression of the p53 protein induces
exercise effects on angiogenesis, proliferation, and the expression suppressor genes can be used
concert to increase vascularity to promote oxygento identify apoptosis in
of the in
death p21 gene, muscle
skeletal which blocksand immune cyclin-cdk cells.complexes ceil populations. Limitations of
delivery to the tissue cells by increasing the capii- this method have
and ultimately leads to Gj to S phase cell cycle arrest been reviewedsurface
lary-to-fiber by McCarthyarea interfaceand Evan and (1998).
increasing Gel
Angiogenesis
(Stew r
art & Pietenpoi 2001). This interaction is electrophoresis
maximal blood flow. to demonstrate a characteristic DNA
further strengthened by the findings that mutations ladderDuring patternexercise, of localinternu-
muscle cleosomaloxygen tension DNA
The which
formation are of newseencapillaries is an essential fragmentation detects
to pi6, often in tumors, result in a falls; and exercise, under apoptotic
conditions cells. of restricted DNA
lossadaptive
of activityresponse
by the of skeletal
p53 tumor muscle to repeated
suppressor gene. fragmentation
blood flow, further as reduces
demonstrated the oxygen in tension.
situ byA the
The loss in p53 activity cripples the cells ability to technique known as TUNEL (terminal
mobilize p21 for inducing senescence and to mobilize deoxynucleotidyl transferase mediated dUTP nick end
pro-apoptotic proteins such as Bax to induce labeling) has been used as the gold standard. The
Table 2.4 Commonly Used Methods to Detect the Occurrence of Apoptosis in Cells
apoptosis (Lundberg & Weinberg 1999). Elevated TUNEL method has been used to detect the early
levels of the p53 protein induce the expression of Bax stages of cell death that cannot be visualized by
proteins, which induce greater apoptosis (Kamesaki routine histologic staining with hematoxylin- eosin.
1998). DNA fragmentation also occurs in necrotic cells;
Other genes, including the retinoblastoma, Rb hence the specificity of this method is problematic
gene, regulate the cell cycle and influence apoptosis (Saikumar et al. 1999; van Cruchten & van den
(Evan et al. 1995; van Cruchten & van den Broeck Broeck 2002). Enzyme-linked immunosorbant assay
2002). The Rb gene and its protein products (ELISA) methods can be used to measure caspase
(pRb/pl05, pRb2/pl30 and pl07) have growth- activity in apoptosis. Although caspase activity is
suppressive properties in that they inactivate indicative of apoptosis, limitations are similar to
transcription factors and negatively control the cell those of Western blotting: measurement of apoptosis
cycle between the Gs and S phases (Tonini et al. in cell populations but not in individual cells. Flow
Figure pRb
2002). 2.3 Schematic
also functions representation of celt progression
as an antiapoptotic factor.to necrosis or apoptosis.
cytometry techniques Followingare triggering
a by extracellular (e.g.,
growth factors and antigens)
For example, TGF-pj induces apoptosis in and/or intracellular factors (e.g., pro- vs. antiapoptotic proteins), ceils commit to cycle
progression, to maintenance (stasis), or to a death pathway (involving necrosis or apoptosis). MTP = mitochondrial
permeability transition pore; ATP = adenosine triphosphate.

Method to detect
apoptosis Strengths Limitations
Morphological Technique well validated; uses Difficulty in identifying apoptotic
microscopy common histological dyes; most versus necrotic celis.
labs have access to microscopy
equipment.
TUNEL-gel Considered "gold standard" for Difficulty in identifying apoptotic
electrophoresis verification of DNA laddering. versus necrotic cells.
Caspases-ELISA Caspase(s) activity definitive Application to cel! populations and
indication of apoptosis. not single ceils; requires
micropiate reader which may be
expensive; wiii not detect caspase-
independent apoptosis.
Phosphatidylserine (PS) Useful for detecting early May pick up PS in necrotic cells;
externalization-flow apoptotic cells. requires expensive flow cytometry
cytometry equipment.
32 Hoffman-Goetz, Quadrilatero, and Patel
possible mechanism that mediates angiogenesis is
muscle hypoxia during exercise. Breen et al. (1996)
showed that VEGF mRNA is increased fourfold after
a single treadmill run in rat muscle and is increased
further when exercise is performed under
hypoxemia. It was also demonstrated that FGF-2
mRNA was increased with exercise under hypoxemia
and that bFGF and TGF-P were increased after 1 h of
exercise. Similar findings were also obtained in
human muscle biopsies taken after 40 to 60 min of
knee extensor exercise (Richardson et al. 1999).
Chronic nerve stimulation has also been shown to
increase VEGF mRNA levels in skeletal muscles after
one and three days (Breen et al. 1996; Hang et al.
1995). This increase in VEGF gene expression is also
correlated with increase in hypoxia-inducible factor
(H1F) mRNA in working muscles after 30 to 45 min
of knee extensions (Gustafson et al. 1999). However,
change in mRNA is not necessarily followed by an
increase in protein, but VEGF protein is increased
almost threefold after three days of chronic electrical
nerve stimulation (Annex et al. 1998). Asano and
colleagues (1998) also showed that serum levels of
VEGF were increased during high-altitude swim
training in humans. VEGF has been considered a
potent angiogenic factor since it is up-regulated after
both exercise and electrical stimulation. Thus,
increased VEGF protein levels should provide an
important stimulus for angiogenesis, especially
during the early phase of the training program. At
present there is only one study that shows both
increased VEGF and vessel density with training
(Amaral et al. 2001). Lloyd et al. (2002) did not detect
increased muscle capillarity during the early time
period of training when the VEGF mRNA changes
were most apparent. However, increase in capillarity
was observed beginning at day 12 of training. Since
angiogenesis has been observed as early as five days
after the onset of chronic muscle stimulation, it is
likely that the angiogenic process was already
ongoing at earlier time points, during the dominant
phase of the VEGF response. However, a sustained
exercise stimulus, occurring over a number of days,
may be necessary in order for the development of
angiogenesis to be fully manifested.
Furthermore, inhibition of VEGF with a
neutralizing antibody completely blocked
angiogenesis induced by exercise, further supporting
the relationship between exercise, VEGF, and
angiogenesis. The angiogenesis-promoting factor
action of VEGF is produced primarily through VEGF
binding to its two receptors, Flk-1 and Fit-1 (Gavin et
al. 2000a). Early gene expression of Flt-1 and not
Flk-1 in response to exercise suggests that differ- -
ences exist in their functions. Flk-1 is critical for
embryonic endothelial cell differentiation and
vasculogenesis. Flt-1 is important in the organization
of the developing vasculature. However, both
receptors may be needed in angiogenesis since Flt-1
and Flk-2 gene expression can be induced by hypoxia
(Sandner et al. 1997). Unfortunately, the exact
mechanisms of action of both receptors are not clear
yet and need to be further elucidated.
In addition, constitutive VEGF protein expression
was confirmed in muscle fibers, vascular smooth
muscle (VSM) of larger vessels, interstitial fibroblasts,
and other cell types (Brown et al. 2001). Expression
of VEGF in VSM of arterial vessels in exercise-
induced angiogenesis is hypothesized to be due to
adenosine availability, which helps produce MMP
from VSM. The MMP mobilize VEGF or encourage
migration of mesenchymal cells participating in the
arteriolization of capillaries. Endothelial cell-
stimulating angiogenic factor (ESAF) is linked with
capillary growth in stimulated muscles; this activates
the pro-forms of metalloproteinase gelatinase A,
collagenase, and streptolysin, which are implicated in
capillary growth (Hansen-Smith et al. 1998).
Hypoxia-inducible factor 1 (HIF-1) has been
shown to be required for the hypoxia-induced
increase in VEGF (Forsythe et al. 1996). Both HIF- la
and HIF-1 p mRNA levels increase in vivo along with
increases in VEGF, However, a cause-effect
relationship cannot be established, since a similar
stimulus or mechanism affects the transcription or
stabilization, or both, of VEGF and HIF-1 mRNA
(Gavin et al. 2000b).
Nitric oxide (NO) also appears to be an important
regulator of endothelial cell growth and angiogenesis.
It is known to be important in blood flow regulation
during exercise and is a cellular signal regulating
mitochondrial respiration (Gavin et al. 2000b). As
mentioned previously, hypoxic exercise produces a
greater increase in VEGF mRNA levels than does
exercise alone. However, NO has been shown to
inhibit VEGF up-regulation through inhibition of
HIF-1 in aortic smooth muscle cells (Liu et al. 1995).
Therefore, since NO is important for vasodilation, it is
expected that NO should increase VEGF levels rather
than cause a decrease. Nonetheless, NO may regulate
VEGF gene expression differentially depending on the
specific tissue. NO increases VEGF mRNA levels via
guanylate cyclase activity in human A-l 72
glioblastoma cells and human Hep G2 hepatocellular
carcinoma cells. NO stimulates soluble guanylate
 Cellular Life Span
cyclase, which converts GTP to the intracellular
second messenger cGMP (Pilz et al. 1995). Thus,
VEGF does not function alone in exercise-induced
angiogenesis in skeletal muscle. Capillary growth in
stimulated muscles can be suppressed by treatment
with inhibitors of either nitric oxide synthase (NOS)
or prostaglandin production (Brown et ai. 2001).
Inhibition of NOS attenuates the exercise- induced
increases in VEGF mRNA (Gavin et al. 2000b).
However, the evidence is still preliminary and further
investigation is needed to elucidate the effects of
exercise on VEGF and VEGF receptor expression.
Although the presence of VEGF appears to be
essential to initiate and facilitate angiogenesis, its
actions are not sufficient as the sole agent to
complete the process. The angiopoietins are essential
for normal vascular remodeling. Both angiopoietins
(Angl, Ang2) bind to the Tie-2 receptor and thus
compete with each other. Angl functions to ensure
vessel stability while Ang2 has the opposite effect
(Maisonpierre et al. 1997). Therefore, the Ang2: Angl
ratio is thought to determine whether the net effect
of the angiopoietins is to stabilize or destabilize the
vasculature. An increase in the Ang2:Angl ratio
(destabilization) is indicative of being proangiogenic.
For example, Ang2 is up-regulated at the leading
edge of new vessel growth (Maisonpierre et al. 1997).
Preliminary evidence indicates that exercise,
particularly ischemic exercise, leads to a shift in
Ang2:Angl mRNA expression ratio that would
support destabilization of the vasculature. Further,
studies in transgenic mouse myocardium show that
Ang2 has a synergistic effect with VEGF on capillary
growth, while Angl antagonizes VEGF action
(Visconti et al. 2002). In the model proposed by
Lloyd and colleagues (2002), the Ang2:Angl mRNA
ratio was elevated after a single day of exercise,
thereby suggesting that destabilization of existing
muscle vasculature is an early event in exercise-
induced angiogenesis. In accordance with VEGF
mRNA expression profiles, the up-regulation of
angiopoietin receptor (Tie-2) mRNA was delayed
relative to the up-regulation of ligand mRNA.
However, the initial increase in the Ang2:Angl mRNA
ratio coincided with the up-regulation of VEGF
mRNA (Lloyd et al. 2002). The wprk by Lloyd and
colleagues is the first to show that a coordinated
activation of VEGF and the angiopoietin system
occurs during physiological angiogenesis in skeletal
muscle. Thus, a balance among these three factors
appears to be necessary for normal vessel growth.
33
Although hypoxia is a known regulator of VEGF
mRNA, its role in the exercise response has been
questioned. Breen et al. (1996) showed that VEGF
mRNA was unregulated in aerobic and hypoxic
exercise conditions. This suggests that other signals
associated with muscle contraction are sufficient to
increase VEGF mRNA levels. These signals could be
produced in response to flow-dependent stimuli,
mechanical changes induced by load bearing within
the muscle during exercise, or both. Increased
shear stress during exercise may play an important
role in modulating angiogenic factor gene
expression. Shear stress increases NO levels, and
Noris et al. (1995) found that NO activates HIF-1, a
major regulator of VEGF expression, in the absence
of hypoxia. In addition, increased stretch has been
shown to increase VEGF mRNA expression in
skeletal muscle (Rivilis et al. 2002). Although shear
stress, hypoxia, and mechanical stimuli are all
potential stimulators of VEGF gene expression in
response to exercise, it is presently unknown which
of these stimuli actually account for the higher
concentrations of this important angiogenic growth
factor.
Differential effects of angiogenesis factors on
various tissue sites play an important role in exer-
ciserinduced angiogenesis. For example, the lungs -
respond to hypoxia with vasoconstriction of the
.vasculature whereas in skeletal muscle, hypoxia
causes vasodilation of the vasculature. In addition,
capillary growth following exercise takes several weeks
to appear, making it difficult to ascertain the effects of
angiogenic factors in the long term.
Proliferation in Skeletal Muscle
Satellite cells are undifferentiated mononuclear
precursor muscle cells that are located between the
sarcolemma and basal lamina of the fiber (Bodine-
Fowier 1994). Evidence suggests that muscular
activity can stimulate satellite cells to advance from
their initial quiescent state (Yan 2000). This has led
several authors to investigate the effect of various
exercise protocols on muscle satellite cell activity.
Smith et al. (2001) found that 30 min of decline
running (-16 grade, 15 m/min) significantly
increased soieus muscle satellite ceil proliferation
(1.0 0.2% of fibers) compared to that in
nonexercise control rats (0.4 0.2% of fibers)
following two but not one, four, or seven bouts of
the same task. McCormick and Thomas (1992)
found that progressive treadmill training for 10
weeks resulted in an increase in soieus satellite cell
mitotic activity from 0.52 0.13 nuclei/mm2
34 Hoffman-Goetz, Quadrilatero, and Patel
to. ,1.28 0.33 nuclei/mm2 in exercised rats.
Furthermore, Tamaki et al. (2000) found that a
single bout of heavy hindlimb weightlifting
significantly increased muscle mitotic activity in
young (14-20 weeks) but not in old (>120 weeks)
rats. It is currently unclear whether and how
exercise affects cell cycle-specific components.
Proliferation in Immune Cells
Following mitogenic and antigenic stimulation,
quiescent immune cells (Gg) progress through the
cell cycle and are governed by the same molecules
as other cell lineages. However, some cell cycle
components interact with lymphocytes in a unique
manner and therefore play a critical role in immune
function. For example, T-lymphocytes do not express
cyclin Dr Therefore proliferation through the G,
phase is dependent on cyclins D 2 and D3 as well as
cdk4 and cdk6. Furthermore, p27 is constitutively
present in quiescent T-lympho- cytes and down-
regulated in the Gj phase following stimulation
(Balomenos & Martinez-A 2000; Chitko-McKown &
Modiano 1997).
Exercise has been shown to modulate many
aspects of immune function, including cell
proliferation (Pedersen & Hoffman-Goetz 2000).
Several investigators have examined the role of
physical activity on lymphocyte proliferation. Here
we describe some representative findings rather
than providing a comprehensive and exhaustive
review of the area. Nieman et al. (1994) found that
45 min of high (80% V0 2max)-intensity treadmill
running was associated with a 21% decrease in
concanavalin A-stimulated lymphocyte proliferation
1 and 2 h postexercise compared to baseline values
following correction for changes in CD3*
lymphocytes. However, moderate (50% V02max)-
intensity running had no effect on lymphocyte
proliferation. Short-duration (20 min) submaximal
cycling at 50% peak work capacity significantly
increased lymphocyte proliferation in young (26 3
years) but not elderly (69 5 years) subjects (Mazzeo
et al. 1998). A recent study (Green et al. 2002)
showed that 60 min of running at 95% ventilatory
threshold significantly decreased
phytohemagglutinin-induced peripheral blood
mononuclear cells (PBMC) proliferation but had no
effect on NK-depleted PBMC or PBMC adjusted for
CD3+ percentage. Although studies that measure
lymphocyte proliferation, as determined by 3H-
thymidine incorporation (or other radionucleotides),
are suggestive of changes in the cell cycle with
exercise, further research is needed
to pinpoint specific regulatory components (e.g.,
cyclin-cdks).
Telomeres and Telomerase Activity
The proliferation of satellite cells following muscle
injury plays a critical role in muscle recovery (Smith
et al. 2001). In skeletal muscle degenerative
conditions such as muscular dystrophy, satellite cells
are forced to undergo repeated bouts of cellular
proliferation to repair continuous skeletal muscle
damage. This leads to satellite cell exhaustion due to
repeated cycles of regeneration and telomeric loss.
Indeed, muscular dystrophy is associated with a 14-
fold greater rate (187 bp/year vs. 13 bp/ year) of
telomeric loss than that in healthy controls (Pecary
et al. 2000; Di Donna et al. 2000). Exercise also
causes muscle to undergo repeated bouts of
regeneration that could potentially affect telomeric
length. Moreover, exercise affects lymphocyte and
satellite cell proliferation, and these relationships
have led some authors to suggest a possible link with
alterations in telomere length and telomerase activity
in these tissues with exercise.
Two recent studies have addressed the effect of
exercise on telomere length or telomerase activity.
Radak et al. (2001) found that 60 min of swimming
five times per week for eight weeks (moderate
exercise), or a gradual increase in swimming
duration by 30 min/week (from 60 min during the
first week to 270 min by the last week), did not alter
skeletal muscle telomerase activity in Wistar rats.
Furthermore, BDF1 mice implanted with the S-l 80
sarcoma and exposed to various exercise conditions
did not have any differences in liver telomerase
activity. Bruunsgaard et al. (1999) found that mean
TRF length in blood mononuclear cell (BMNC), CD4*,
and CD8+ lymphocytes was significantly shorter in
elderly (median age 78) compared to younger (median
age 23) subjects. Furthermore, TRF lengths were
significantly decreased in BMNC and CDS'
lymphocytes of younger subjects and CD4
lymphocytes of elderly subjects following maximal
bicycle exercise lasting 17 to 20 min. Further
research is warranted given the limited data on
exercise-induced alterations in telomere length and
telomerase activity.
Apoptosis in Heart
and Skeletal Muscle
Cardiomyocytes undergo apoptosis in response to
ischemia and reperfusion injury. Gottlieb et al.
(1994) were among the first to demonstrate
 Cellular Life Span
apoptosis in rabbit myocardium subjected to
metabolic inhibition and recovery. Others have
shown increased Fas mRNA undergoing hypoxia-
induced myocyte death in rats (Tanaka et al. 1994),
as well as ultrastructural changes (e.g., DNA
fragmentation, membrane blebbing) indicative of
apoptosis that were especially pronounced during
reperfusion (restoration of oxygen, serum, and
glucose following a designated period of deprivation)
compared with the initial ischemia (deprivation of
oxygen, serum, and glucose) (Umansky et al. 1995).
Expression of Bcl-2, Bax, and Fas proteins in rat
myocytes has been reported post-ischemia (Kajstura
et al. 1996). As noted earlier, Fas and Bax are potent
cell death triggers whereas Bcl-2 functions to
increase cell survival. While it is clear that ischemia
can lead to cardiac apoptosis and necrosis, what
accounts for apoptotic injury to myocytes during
reperfusion? Reperfusion of ischemic tissue is
associated with the generation of oxygen free radicals
and acute leukocyte activation (Kloner et al. 1989;
Sussman & Bulkley 1990). ROS are generated
through a variety of mechanisms, including the
metabolism of arachidonic acid and respiratory burst
activity from leukocytes (which generates superoxide
anion, singlet oxygen, hypochlorous acid, and other
reactive species). ROS can damage DNA by oxidation
of purine and pyrimidine bases, most especially
guanine (Loft & Poulsen 1996), leading to apoptosis
of the injured cell.
Ca2+ also plays a role in cell death during
reperfusion injury. Following reperfusion, a large
increase in intracellular Ca2+ occurs with resultant
mitochondria Ca2+ accumulation. These events lead
to decreased ATP production and MTP formation,
which can lead to cell death (Suleiman et al. 2001;
Xu et al. 2001; Wang et al. 2002). Narayan et al.
(2001) found that rat ventricular myocytes subjected
to simulated ischemia and reperfusion showed
increased apoptosis (Annexin V staining), but not
when exposed to simulated ischemia alone. Following
simulated reperfusion, free Ca2+ concentrations
within the mitochondria increased from 111 14 nM
during baseline to 214 22 nM and 382 22 nM in
Annexin-negative and Annexin-positive cells,
respectively. Furthermore, calcium preconditioning
of myocytes maintains mitochondria Ca2+
homeostasis, preventing MPT formation and
apoptosis (Xu et al. 2001). However, ischemia-
reperfusion injury is not the only trigger for non-
necrotic cell death of cardio- myocytes.
Overstretching results in apoptosis in rat myocytes
and non-myocytes, together with
35
increased expression of Fas (Cheng et al. 1995).
However, cardiac adaptation totreadmill training in
rats is not associated with increased apoptosis of
myocytes (Jin et al. 2000).
Cell death occurs in immature skeletal muscle in
response to a variety of injurious stimuli. Some have
suggested that whether the response is apoptosis or
necrosis depends upon the maturity of the muscle
cell. Carraro and Franceschi (1997) hypothesized
that the normal response to injury by immature
muscle cells is apoptosis whereas the typical
response in adult muscle fibers to the same
inducing stimulus is necrosis. Evidence that age and
development influence apoptosis or necrosis
pathways comes from studies in neonatal rat
skeletal muscle (Kaminska & Fidzianska 1996) and
from observations of human embryos at different
stages of development (Carraro & Franceschi 1997).
Apoptosis has been demonstrated under non-
pathological conditions that stress muscle cells,
such as eccentric exercise, or conditions of atrophy,
such as prolonged traction and limb unweighting. As
a rule, damage to skeletal muscle is greater with
repeated contractions in which the muscle
undergoes lengthening (eccentric exercise) than with
activity involving shortening (concentric) or isometric
contractions. Biral and colleagues (2000) used a
model of sustained eccentric exercise, with repeated
contractions while the muscle was extended. With
use of the TUNEL method to detect apoptosis in rat
soleus and extensor digitorum longus, the
disappearance of the dystrophin-based membrane
skeleton and an elevated expression of Bax and
caspase 3 in individual muscle fibers were
demonstrated. Thus, apoptosis occurs in normal,
mature skeletal muscle fibers in response to extreme
functional demands.
Is there any evidence for apoptosis of skeletal
muscle fibers with less extreme workload demands?
Although tentative, evidence of apoptosis in adult
C57BL/6 mouse tibialis anterior muscle following 12
h of voluntary wheel running has been reported,
with myofiber content of Bax, Fas, and ubiquitin
correlated with the appearance of apoptotic
myonuclei (Podhorska-Okolow et al. 1998). Apoptosis
results in the elimination of myonuclei or satellite
ceils, or both, from atrophying rat muscle fibers with
hindlimb unweighting (Allen et al. 1997).
Apoptosis of Immune Cells
An area of increasing research is the effect of
exercise on apoptosis of immune cells. Strenuous
exercise regulates several factors that alter
36 Hoffman-Goetz. Ouadrilatero, and Patel
lymphocyte apoptosis in a number of ways. Eor~.
example, increased GC secretion, growth factor
withdrawal, ROS generation, and increased plasma
TNF-a are some of the signals that induce apoptosis
in immune cells (Phaneuf & Leewenburgh 2001); and
some of these factors (GC, TNF-a) are notably elevated
after strenuous, prolonged, or muscle-damaging
exercise. Apoptotic loss could contribute to the well-
documented decrease in the number and function of
lymphocytes postexercise (Hoffman-Goetz
Quadrilatero 2003; Pedersen & Hoffman-Goetz 2000).
The effect of high-intensity exercise on the
induction of lymphocyte apoptosis has been
repeatedly demonstrated. One of the earliest studies
to address this was that of Concordet and Ferry
(1993), who found DNA fragmentation (using the
TUNEL assay) of thymus following two treadmill
runs to exhaustion (separated by a 24-h rest) in
rats. Thymocyte apoptosis could be blocked by
administration of a GC receptor antagonist, RU-486
(mifepristone). In vitro studies indicate that
corticosterone exposure (at levels observed after
exercise) is associated with increased expression of
Annexin V-positive thymocytes (Hoffman-Goetz
&Zajchowski 1999). Increased oxygen consumption
and hyperventilation with exhaustive exercise lead
to the generation of ROS; ROS contribute to
thymocyte cell death as measured by lipid peroxides
and the influx of Ca2* ion into thymocytes (Azenabor
& Hoffman-Goetz 1999, 2000). Antioxidant
supplementation inhibits exercise-induced
leukocyte DNA damage (Hartmann et al. 1995) and
thymocyte apoptosis (Lin et al. 1999). Increased cas-
pase 3 activity was demonstrated in developing
thymocytes (but not mature splenocytes) after a
single intensive bout of exercise in mice (Patel &
Hoffman-Goetz 2002), providing evidence that
apoptosis occurs in GC-sensitive lymphoid tissues
via caspase-dependent mechanisms. Lagranha et al.
(2004) found that a single bout of intense treadmill
running significantly increased Bax and Bcl-x s (pro-
apoptotic) gene expression while at the same time
decreasing Bcl-x, (anti-apoptotic) gene expression
and mitochondrial membrane potential in
neutrophils from mature rats.
What is the mechanism for the apoptosis of
thymocytes in rats or mice after strenuous exercise?
We hypothesize that intense exercise leads to
apoptosis through both receptor-ligand signaling
and mitochondrial pathways (shown
diagrammatically in figure 2.2). Which pathway
is activated depends on the whether the exercise
triggers a hormonal response involving ACTH and
cortisol/corticosterone release, a metabolic response
involving ROS generation, or generation of
inflammatory cytokines such as TNF-a. In an
exercise bout of longer duration (e.g., 60-120 min)
and moderate intensity (e.g., <75% of VOzmax)
associated with activation of the hypothalamic-
pituitary-adrenal (HPA) axis (Ronsen et al. 2001),
& corticosterone release will trigger the mitochondrial
pathway of apoptosis, activating procaspase 8 and,
ultimately, caspase 3. In an exercise bout of high
intensity (e.g., >75% of V02max) and shorter duration
(e.g., <30 min), respiratory oxidant stress will occur
(Raidai et al. 2000), also leading to induction of
mitochondrial damage and cytochrome C release and
triggering of the caspase pathways. The type of
exercise also modifies the apoptotic pathway that
ensues once the minimum intensity and duration
thresholds have been exceeded. For example,
exercise that involves muscle damage (e.g., downhill
running) leads to the release of inflammatory
cytokines (e.g., TNF-a) and the induction of the
receptor-ligand mechanism of cell death. In contrast,
intense exercise that occurs without muscle damage
could induce apoptosis in lymphocytes primarily
through the mitochondrial pathway (see figure 2.4).
According to this model, short-duration sub-
maximal treadmill exercise (Hoffman-Goetz et al.
1999) should not induce significant apoptosis in
thymocytes or other susceptible lymphocytes.
Similarly, high-volume exercise of very low intensity,
which occurs with wheel running, would also not
induce apoptosis in lymphocytes (Hoffman-Goetz &
Fietsch 2002). Thus, variation in findings of
apoptosis effects among exercise studies may be due
to exercise intensity and duration continuum;
exercise that fails to lead to either an elevation in GC
or the generation of ROS or the release of
inflammatory cytokines will not be associated with
apoptotic events in lymphocytes. Moreover,
differences in the sensitivity of lymphocytes to these
apoptotic-inducing factors will also influence the
extent of cell death. For example, the primary
lymphoid compartments (thymus and bone marrow)
contain many double positive (004X08 ") and double
negative (CD4XD8") lymphocytes compared with
secondary lymphoid compartments (spleen, nodes,
blood), which have primarily single positive cells
(CD4+ or CD8*)- Characterization of the sensitiv-
 Cellular Life Span 37

Low Low

a>
s w Inflammatory-Receptor
Metabolic-Mitochondria o' o O
cytochrome c pathway
s ligand pathway

Low s. High Low

Figure 2.4 Model of exercise-induced lymphocyte apoptosis. Model predicts general exercise conditions under which apoptosis
will occur. Reactive oxygen species (ROS) and glucocorticoids (GC), released in response to high-intensity or long- duration
exercise or both, trigger the mitochondrial-cytochrome C pathway of apoptosis in lymphocytes. Other hormonal triggers (e.g.,
catecholamines) may be involved. Tumor necrosis factor-ot (TNF-a), released during muscle damage or exercise associated
with inflammatory events, triggers the receptor-ligand pathway of apoptosis in lymphocytes. Below some intensity and
duration threshold, exercise-induced apoptosis of lymphocytes does not occur because of absence of physiological triggers.

ity of these different subpopulations to ROS, GC, Conclusion

and other apoptotic stimuli may help to explain the
differential apoptosis in lymphocyte compartments
after exercise.
There are limited data from human studies on
the effects of exercise on the induction of apoptosis
in lymphocytes. Treadmill exercise to exhaustion
(Mars et al. 1998; Niess et al. 1996), treadmill
exercise above the aerobic-anaerobic threshold
(Hartmann et al. 1994), and treadmill exercise (80%
of V02max) to exhaustion (~30 min) (Mooren et al.
2002) were associated with induction of apoptosis in
human lymphocytes. In exercise of lower intensity
(60% of V02max) for a short duration (~30 min), no
apoptosis was observed in human lymphocytes.
Steensberg et al. (2002) found an increase in the
percentage (but not absolute numbers) of human
blood lymphocytes expressing early markers of
apoptosis after exercise at 75% of V0 2max for 2.5 h.
Since an increase in both plasma cortisol and
plasma isoprostanes (a measure of oxidative stress)
was observed, it is likely that the mitochondrial
pathway was activated, leading to the percentage
increase in apoptotic lymphocytes. Further, Hsu et
al. (2002) found decreased mitochondrial membrane
potential and increased DNAfragmentation in
peripheral blood leukocytes from subjects who
performed consecutive bouts of aerobic exercise at
60% and 85% but not 35% VO,max.
Diverse factors (cytokines, growth factors, cell cycle
components) regulate cell proliferation. An equally
diverse and often bewildering group of intracellular
and extracellular factors are involved in the
signaling of cell apoptosis. How these factors
converge to trigger the physical processes of cellular
replication, cellular stasis, or cellular apoptosis has
been the object of intense scientific research in the
past 20 years. Better understanding of the
relationship between level of expression of
oncoproteins such as c-myc and p53, survival
proteins such as the products of Bcl-2, and
components of the cell cycle (cyclins-cdks) will be
important for understanding how apoptotic
resistance develops in cancer cells and how cellular
senescence occurs. It is clear that with appropriate
signals, cardiac and skeletal muscle cells, as well as
lymphocytes, undergo apoptosis. The decision to
commit to the cellular apoptosis program in
response to an exercise challenge will ultimately
reflect a combination of extracellular influences
(e.g., glucocorticoids, generation of ROS, presence of
inflammatory cytokines) and intracellular influences
(e.g., ATP availability, short or prolonged opening of
the mitochondrial permeability transition pore,
concentration of pro-apoptotic to antiapoptotic
proteins, activation of oncogenes) acting upon
muscle and immune cells.