Download Role of proto-oncogenes in the control of myocardial cell growth and

Clinical Science (1991) 80,405-411
405
Editorial Review
Role of proto-oncogenes in the control of myocardial cell
growth and function
MICHAEL D. GAMMAGE 1
AND
JAYNE A. FRANKLYN 2
Departments of 'Cardiovascular Medicine and 2Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, U'K,
INTRODUCTION
A search for abnormal genes associated with oncogenesis
has led to the identification of a group of highly conserved
normal genes, whose products contribute to the physiological regulation of cell proliferation and differentiation
and to the development of the organism [1]. There is
increasing evidence that the proteins that are encoded by
these normal cellular genes, termed proto-oncogenes,
comprise a regulatory cascade involved in the propogation of growth signals from the cell-surface membrane to
the nucleus [2]. Evidence for the role of proto-oncogenes
in signal transduction within the cell, and hence in the
regulation of growth, function and differentiation, has
arisen from recognition that specific genes encode a
variety of growth factors, surface membrane receptors,
membrane-associated protein kinases and GTP-binding
proteins, as well as cytoplasmic serine and threonine
protein kinases and nuclear transactivating proteins [3].
Normal proto-oncogenes may be modified to transforming oncogenes involved in the malignant proliferation
of cells by processes of mutation, amplification or translocation. Modified forms of proto-oncogenes, termed
viral or v-oncogenes, are found in the genome of oncogenic retroviruses and are responsible for the ability of
these viruses to transform cells and induce tumours.
While considerable insight into normal signal transduction processes has been gained by investigation of the
abnormal oncogene products associated with tumour
development, it is the role of their normal cellular
counterparts in cell physiology upon which the present
review will focus. Evidence for a role of specific protooncogene products in signal transduction in a variety of
cell types is first outlined; evidence for their specific role
Correspondence: Dr M. D. Gammage, Department of Cardiovascular Medicine, University of Birmingham, Queen Elizabeth
Hospital, Edgbaston, Birmingham B15 2TH, U.K.
in the growth and function of the myocardial cell is then
reviewed.
PROTO-ONCOGENES AND PEPTIDE GROWTH
FACTORSAND THEIR RECEPTORS
Recognition that the products of cellular proto-oncogenes
might exert a general role in the reguation of cell growth
arose from the fortuitous observation that proto-oncogene-encoded proteins included previously. recognized
growth factors and their receptors [4]. For example, the
protein product of c-sis is one chain of the heterodimeric
mitogen, platelet-derived growth factor [5]. In addition,
several proto-oncogenes encode homologues of acidic
and basic fibroblast growth factors (FGFs), while other
related genes, such as int-1, have been postulated to
encode as yet undefined peptide growth factors. Furthermore, several classes of growth factor receptor have been
identified which share structural identity with protooncogene-encoded proteins. The product of c-erb B has
been demonstrated to encode part of the epidermal
growth factor receptor, while c-frns encodes the receptor
for the haemopoetic growth factor, colony-stimulating
factor. Both receptor isoforms for platelet-derived growth
factor resemble the products of the kit and the c-frns
genes, while the product of c-mas possesses seven transmembrane domains and so resembles receptors for
adrenergic and muscarinic agents; c- mas itself has
recently been proposed to encode a neuronal angiotensin
II receptor [4].
PROTO-ONCOGENES
PROTEINS
AND
SIGNAL-COUPLING
Various membrane-associated protein kinases, such as the
product of the src gene, and cytoplasmic protein kinases,
such as the product of c-mos, have been implicated in
signal propogation resulting from growth factor binding at
the cell-surface membrane. The guanine-nucleotide-
406
M. D. Gammage and J. A. Franklyn
binding proteins (G-proteins) and their proto-oncogeneencoded homologues, the family of ras gene products, are
perhaps the best characterized class of receptor-activated
signal transducers. In the case of the p-adrenoceptor,
these G-proteins exchange bound GDP for GTP and
assume an active configuration, the activated protein then
stimulating adenylate cyclase with resultant increases in
intracellular cyclic AMP. Other members of the
G-protein family are coupled to other receptors and activate effectors such as ion channels and phospholipase C,
the latter generating diacylglycerol and inositol trisphosphate as intracellular messengers [6]. The critical role
of ras proteins in this process has been revealed by their
structural similarity to well-characterized G-proteins and
by their ability to bind and hydrolyse GTP. Conversely, it
has emerged that many oncogenic ras proteins have lost
this ability to hydrolyse GTP, and these ras oncogene
products act predictably as constitutive growth factor
signals involved in uncontrollable cell proliferation.
PROTO-ONCOGENES
AND
INTRANUCLEAR
MEDIATORS OF GROWTH FACTOR EFFECTS
There is increasing evidence that several classes of protooncogene-encoded proteins found within the nucleus
regulate cell growth and function more directly than the
proto-oncogene products that are related to growth
factors, their receptors or intracellular coupling proteins.
These nuclear proteins include receptors for the thyroid
hormone tri-iodothyronine (T3 ), which are the products of
a family of related c-erb A genes [7], and the c-jun gene
product, which is a further well-defined DNA-binding
transcription factor, AP-l. It has been suggested that the
jun protein, together with the nuclear products of the
c-myc and c-fos genes, may be essential to the ability of
growth factors to regulate gene transcription [41.
The products of the c-jun, c-jos and c-myc proto-oncogenes are often considered in conjunction in terms of their
transcriptional regulatory effects because all three are
rapidly induced by a variety of growth stimuli and are
found within the nucleus. While stimulation of expression
of these gene products may be necessary to elicit a growth
response, the specificity of their effect upon growth
remains unclear. Transient stimulation of the synthesis of
fos, myc and jun proteins is triggered by a number of
diverse trophic signals, including peptide growth factors,
phorbol esters and physiological stress [4, 8], as outlined
below with reference to the myocardial cell. Fos proteins,
like the products of the c-erb A or Tj-receptor gene
family, have been shown to bind directly to regulatory
sequences of DNA, but both binding and transactivation
by the fos protein are known to be dependent upon the
formation of a heterodimeric molecule with the product
of c-jun; the fos and jun proteins share a helical structure
that aligns five leucine residues required for the formation
of the dimer adjacent to a domain rich in basic amino
acids essential for binding to DNA [9]. The majority of
trophic stimuli that increase c-fos and c-jun expression
also increase the expression of c-myc, and, like the fos
protein, the myc protein binds to DNA (without the need
for heterodimer formation) and directly stimulates gene
transcription.
Having outlined the major proto-oncogene products
implicated at present in the regulation of cellular signal
transduction, the remaining part of this review will
examine the changes in gene expression associated with
myocardial cell growth and the evidence for the role of
some of the specific proto-oncogene products described
in determining these changes.
PATHOPHYSIOLOGICAL REGULATION OF MYOCARDIAL GROWTH AND GENE EXPRESSION
Although mitotic division of myocardial muscle cells
ceases soon after birth, adaptive growth of the heart
occurs in response to a variety of physiological and pathological states which increase cardiac work. The growth
results from myocardial cell hypertrophy in the absence
of cell proliferation [10]. Myocardial cell hypertrophy
may result from diverse haemodynamic and humoral
stimuli, the development of hypertrophy in turn having
important clinical consequences in man. Although hypertrophy represents an important adaptive process that
enables the heart to compensate for overloading, whether
due to abnormal haemodynamic demands as in aortic
stenosis or to the loss of functional myocardial tissue as
occurs after myocardial infarction, the cells of the hypertrophied heart are not normal. In its early stages, hypertrophy unloads the cells of the failing heart by adding new
sarcomeres, resulting in an energy-sparing effect, but
long-standing hypertrophy increases the cell volume
occupied by myofibrils and potentially exacerbates an
energy deficit [11]. The realization that such functional
disorders of the myocardium are intimately involved in
the poor prognosis associated with the failing heart, and
that in hypertension an increase in heart muscle mass is an
independent risk factor for cardiovascular morbidity and
mortality [11], has led to detailed investigation of the
molecular changes involved in the hypertrophic response
of the heart to various stimuli.
Alpert & Gordon [12] first demonstrated that myosin
ATPase activity is reduced in failing hearts. This change
reflects an altered pattern of synthesis of the myosin
heavy chains (MHCs), which, in turn, not only has the
capacity to determine the rate of energy liberation by
myosin, but also determines muscle-shortening velocity or
rate of energy consumption. The switch to 'slow' or
low ATPase myosin production found in the overloaded
heart reduces myocardial contractility but improves
mechanical efficiency and is energy-sparing. The appearance of abnormal isoforms of key myocardial proteins,
such as myosin, as part of the hypertrophic response to an
altered cardiac load is one of several examples of a change
in expression of different members of a multigene family
expressed in the heart [13].
The synthesis and functional consequences of the
expression of different members of the family of genes
that encode the MHCs have been studied extensively in
the rat myocardium. The expression of the VI MHC,
which is comprised of two a-MHC subunits, leads to high
Proto-oncogenes and the heart
myosin ATPase activity and rapid shortening velocity,
while the expression of a V3 MHC, comprising two pMHC molecules, results in a protein product with low
ATPase activity and a slow shortening velocity. In the rat
ventricle, it is clear that mechanical overload, such as that
induced by constriction of the aorta, is associated with a
switch to the preferential synthesis of the V3 isoform [13]
resultant upon increased transcription of the P-MHC
gene and reduced transcription of the a-MHC gene [14].
A change in MHC gene expression has also been
observed in the mechanically overloaded human heart
[15], although in man changes are confined to the atria,
since only a slow-myosin isoform is synthesized in the
ventricles in the normal adult state. In human atria, a
decrease in the proportion of fast (a) MHCs has been
reported to parallel increases in left atrial pressure [16]
and left atrial enlargement [15]. Overload has also been
reported to alter the expression of myosin light chains in
the human heart, with the appearance in the atria of a
ventricular isoform of the myosin light chain [17].
Expression of the P-MHC is a normal feature of the
foetal, rather than the adult, ventricular myocardium in
the rat, while the pattern of atrial myosin gene expression
in man found in association with atrial overload is also
normally confined to the foetus. Adult myocardial cells
thus respond to mechanical overload not only by increasing protein synthesis, but at the same time by switching to
preferential production of foetal isoforms of a variety of
functionally important proteins. The phenomenon of
'foetal' protein expression in the pressure-loaded ventricle
has been demonstrated not only for the MHCs and
myosin light chains, but also for creatine kinase, a-actin
and p-tropomyosin in the rat [18-20] and atrial natriuretic factor in both man and the rat [21]. It has been
postulated that this reversion to the expression of 'foetal'
protein isoforms reflects the fact that adult myocardial
cells are terminally differentiated, so that for such cells to
regain the capacity for rapid protein synthesis possessed
in the foetal state, the pattern of protein synthesis has to
revert to that seen earlier in ontogeny. Although the
functional consequences of changes in MHC gene expression have been clarified, the relevance of changes in the
pattern of expression of other multigene families remain
obscure.
It is clear that changes in haemodynamic load exerted
in vivo are not exclusive determinants of the pattern of
expression of the MHC genes or other families of related
proteins found in the myocardium. We and others have
demonstrated that the induction of hypothyroidism in the
rat is associated with an increase in pre-translational
expression of P-MHCs in the ventricle, associated with a
switch to synthesis of the 'foetal' V3 myosin isoform [22].
Other endocrine factors, including gonadal steroids and
glucocorticoids, have been shown to alter MHC gene
expression in a similar way [23]. Interestingly, ventricular
hypertrophy in the rat resulting from thyroid hormone
administration may also be associated with stimulation of
P-MHC gene expression [24], in apparent contrast to the
stimulatory effect of thyroid hormone deficiency on PMHC expression mentioned above.
407
It remains enigmatic how signals of mechanical cardiac
overload or endocrine signals can be transduced to an
increase in cardiac mass and can furthermore provoke
'foetal' gene expression. Several pieces of evidence
suggest that processes which stimulate hypertrophic
growth of cardiac myocytes are coupled to control of
cardiac mass through the signal transduction pathways of
some of the proto-oncogene-encoded proteins believed to
convey growth factor effects. This evidence, outlined
below, comprises reported influences of hypertrophic
stimuli on the expression of specific proto-oncogenes in
the myocardium, as well as findings from studies in vitro
directly implicating specific proto-oncogene products in
the control of myocardial cell growth and gene expression.
PEPTIDE GROWTH FACTORS, ADRENERGIC
AGENTS AND MYOCARDIAL CELL GROWTH AND
GENE EXPRESSION IN Vl1RO
The ability to culture ventricular myocytes in vitro has
facilitated the investigation of factors which have been
implicated in studies of intact animals as playing a part in
controlling myocardial gene expression, and which may
be critical to the propogation of mechanical stimuli of
hypertrophy. It has been shown using such models in vitro
that specific serum constituents, comprising peptide
growth factors, can regulate both growth and expression
of specific genes in cardiac myocytes. Withdrawal of
growth factors by maintenance of cells in serum-free
media has been reported to advance the differentiation of
cardiac muscle cells [25], while undefined serum components, distinct from T3 and adrenoceptor agonists,
stimulate myocyte growth and protein synthesis [26].
Treatment of cells in vitro with the peptide growth factor,
transforming growth factor-,8 (TGF-f3) has been shown to
alter both MHC and actin gene expression in a pattern
resembling that observed after pressure overload in vivo
[21]. Despite the presence of the thyroid hormone
thyroxine (T4 ), a-MHC expression was inhibited and that
of the ,8-MHC gene was induced in the absence of any
change in cell number. FOFs have also been implicated in
the direct control of myocardial gene expression, both
acidic and basic FOFs inducing reciprocal effects upon aand ,8-MHC gene transcription [21]. Identification of both
TOF-,8 and FOF mRNAs in the myocardium, providing
evidence for local synthesis [21], together with their
effects on myocardial gene expression and growth, provide
evidence for a possible paracrine or autocrine role of
growth factors in pressure-overloaded hypertrophy.
In addition, the a I-adrenergic agonist noradrenaline
has been shown to increase cell volume and cell protein
content in the absence of DNA synthesis when administered in vitro, again mimicking changes seen in hypertrophy [27]. Noradrenaline has itself been implicated in
the genesis of myocardial hypertrophy in the intact animal
[28], with some groups suggesting that this adrenergic
agent has a fundamental growth-controlling function.
The evidence outlined above, that the products of
proto-oncogenes are involved in the signal transduction
408
M. D. Gammage and J. A. Franklyn
pathways mediating the actions of these hypertrophic
agents such as peptide growth factors and adrenergic
receptor agonists, has led to attempts to define the
specific role of proto-oncogene-encoded proteins in
determining cardiac cell growth and gene expression.
PROTO-ONCOGENE EXPRESSION IN INTACT
ANIMAL MODELS OF CARDIAC HYPERTROPHY
Several recent studies have demonstrated that pre-translational expression of the c- myc gene is stimulated by an
increase in haemodynamic load in the intact rat [28].
Mulvagh et al. [18] reported that constriction of the
abdominal aorta, and hence an imposition of increased
load upon the left ventricle, resulted in a 25% increase in
left ventricular mass at 10 days, associated with the
induction of c-myc expression in both the left ventricle
and atrium. Sustained expression of c-myc was more
pronounced in the atria than in the ventricle and was
more evident in younger than in older animals, in accord
with the known age-dependent ability of the cardiac
chambers to respond to a haemodynamic load. Izumo et
al. [20] later described transient induction of both c-myc
and c-fos occurring within an hour of acute pressure overload and thus preceding the expression of 'foetal' isoforms
of contractile proteins and atrial natriuretic factor. We
have reported that constriction of the aorta results in a
marked increase in left ventricular mass at both 3 and 9
days after operation, hypertrophy being associated with
stimulation of f3-MHC gene expression. In addition, we
demonstrated stimulation of c-myc, c-fos and Harvey-ras
mRN As at 3 days, changes no longer found at 9 days [29].
Other studies have demonstrated a rapid and transient
increase in c-myc and c-fos mRNAs, associated with
a gradual and more sustained increase in H-ras
mRNA [30]. These changes have been shown to be
specific, in that levels of c-erb A, c-erb B, sis and myb
mRNAs are unaffected by pressure-overloaded hypertrophy. Additional evidence is nonetheless required to
establish that augmented expression of the protooncogenes described is essential for the genesis of a
hypertrophic response to increased load upon the heart.
Stimulation of proto-oncogene expression is not
confined to models of cardiac hypertrophy associated
with haemodynamic overload. Administration of the f3adrenergic agonist isoprenaline in vivo has been reported
to lead to a rapid increase in myocardial c-fos mRNA in
several rodent species [31], while in the rat, c-jos stimulation was also reported to result from treatment with the
a-adrenergic agonist phenylephrine, as well as with histamine and prostaglandin E i- Since the adrenergic agents
employed have been shown to exert a hypertrophic effect
on the myocardial cell both in vivo and in vitro, these
findings lend support to a role for the fos gene product in
mediating a hypertrophic response.
Proto-oncogene expression has been investigated in
two further intact animal models of ventricular hypertrophy, namely spontaneously hypertensive rats and
thyroid-hormone-induced hypertrophy. Increased expression of c-myc mRNA in the hearts of l O-week-old spon-
taneously hypertensive rats has been reported, both
hypertension and ventricular hypertrophy being present
by this age. In addition, augmented expression of c-myc in
the cultured aortic smooth muscle cells of the same
animals in response to serum treatment of serumdeprived cultures was described [32]. Other studies have
failed to confirm a difference in proto-oncogene expression between spontaneously hypertensive animals and
their normal controls [33]. Conflicting results have also
been reported for thyroid-hormone-induced hypertrophy
in terms of effects upon contractile protein gene expression [20, 24, 34], but our own studies have demonstrated
that stimulation of f3-MHC gene expression, as well as
stimulation of the c-myc and c-fos proto-oncogenes, is
confined to the right ventricle, despite a similar hypertrophic influence of T 3 treatment on both ventricles.
These findings are associated with an increase in right
ventricular systolic pressure with thyroid hormone treatment, in the absence of marked changes in left ventricular
systolic pressure or mean aortic pressure [35], and suggest
that proto-oncogene stimulation may not in fact be an
essential prerequisite for the development of ventricular
hypertrophy and furthermore reflects an increased
pressure load in the right ventricle in this model rather
than a direct influence of T 3 • This view is supported by a
study demonstrating that T4 administration is without
effect upon a non-working heterotopically transplanted
rat heart, despite inducing both hypertrophy and changes
in myosin isoenzyme expression in the beating heart in
situ [34]. There is nonetheless strong evidence for direct
regulatory actions of thyroid hormones on expression of
the MHC genes, actions mediated by binding of ligandassociated c-erb A or T 3 receptor proteins to specific
regulatory sequences of the a- and f3-MHC genes [36].
Coronary flow has also been demonstrated to be a
determinant of c-fos and c-myc gene expression in the isolated beating heart [37].
While the studies of myocardial hypertrophy in intact
animal models outlined provide circumstantial evidence
implicating the products of a variety of specific protooncogenes in the transduction of hypertrophic signals and
hence in the genesis of myocardial hypertrophy, further
support for their role has arisen from the investigation of
proto-oncogene regulation and function in vitro.
STUDIES OF PROTO-ONCOGENE EXPRESSION IN
VJ1RO
Primary culture of neonatal cardiac myocytes was first
employed as a tool for the investigation of protooncogene regulation by Starksen et al. [27], who demonstrated that direct stimulation of cells by a [-adrenergic
agents, phorbol esters and serum induced an increase in
cell size in non-dividing cardiac myocytes. They also
demonstrated that noradrenaline induced a rapid increase
in c-myc gene expression and that serum and the phorbol
ester, phorbol 12-myristate 13-acetate, both exerted a
similar effect. These studies provided good evidence for a
direct effect of a hypertrophic stimulus on c-myc gene
expression, but since stimulation of the synthesis of myc
Proto-oncogenes and the heart
protein was transient and preceded an increase in cell size
it is clear that c-myc expression may be necessary, but not
sufficient, for the development of cell hypertrophy.
Studies attempting to define the intracellular function
of c-myc gene products in other cell types in vitro have
shown that they are likely to play important roles not only
in the control of hypertrophic growth but also in the
control of cell differentiation. It has been demonstrated
that down-regulation of c-myc is an early response to
interventions that inhibit cell proliferation and promote
cell differentiation. It has also been reported that antibodies to myc proteins or anti-sense oligonucleotides
(that bind native c-myc mRNA and prevent synthesis of
myc protein) specifically block DNA replication, in
accord with a proposed critical role of myc protein in cell
division [38, 39]. While these findings are not directly
relevant to the non-dividing myocyte, it is clear that
myoblasts infected with the viral myc gene fail to differentiate, a finding in accord with the association of augmented expression of c-myc with expression of 'foetal'
protein genes in several models of cardiac myocyte hypertrophy and with the parallel decline in c-myc and 'foetal'
protein gene expression evident in cardiac muscle during
embryonic development [40]. Further direct evidence for
a growth-mediating effect of the c-myc gene product has
arisen recently from a study of transgenic mice manipulated to exhibit constitutive c-myc expression in the heart.
Increased myocardial c-myc expression was associated
with cardiac enlargement by the time of birth [41].
While the role of the c-myc gene product in the control
of cell growth and differentiation has thus been partly
defined in cells other than those in the myocardium, it is
likely that other related factors play central roles in the
control of these processes. Several such factors including
myo Dl, myogenin and myfgene products, which d~splay
structural identity with myc proteins, have been implicated in the control of differentiation of skeletal muscle
cells [42]. These related gene products are believed to
fold into helix-loop-helix structures involved in dimerization, while a second region shows some sequence identity
with the DNA-binding domains of the c-fos and c-jun
gene products [43]. There is thus speculation that cardiacspecific equivalents of the skeletal muscle myo D 1,
myogenin and myf proteins which are critically important
to control of myocardial cell differentiation will be
defined.
Induction of c-myc has been shown to be preceded by
c-fos induction in a variety of cell systems,. and like my~,
fos protein synthesis is stimulated by dlve~se trop~c
signals. Furthermore, like the myc protem, studies
employing anti-sense oligonucleotides have de~o~strated
that synthesis of fos is essential for DNA replication and
that myoblasts infected with a viral c-fos gene are unable
to differentiate [44].
Gene-transfer and anti-sense-oligonucleotide-blocking
experiments examining ras gene product functions have
produced similar findings to studies of myc and fos
proteins [45]. In skeletal muscle lines, activated variants of
ras genes prevent the appearance of the proteins
associated with myogenic differentiation, including
409
muscle creatine kinase, sarcomeric actin, acetylcholine
receptors and voltage-gated sodium and calcium channels
[46, 47]. A major difference between expression of the
normal ras genes and expression of c-myc and c-fos,
which points to a different role in cell function, is that
there is little variation in the level of ras expression in the
heart in the embryonic, neonatal and adult state [40]; thus
there is little association with changes in the pattern of
expression of myocardial multigene families.
CONCLUSIONS
The precise mechanisms that modulate cardiac gene
expression in adaptation to hypertrophic stimuli remain
elusive. The nature of the hypertrophic signals themselves, aside from mechanical overload, are also elusive,
with various investigators emphasizing potential contributions of mechanical stress, increased oxygen consumption, coronary artery perfusion pressure and humor~
factors, including peptide growth factors, adrenergic
agents and angiotensin II [11, 21, 48, 49]. Whatever the
nature of the initiating or sustaining hypertrophic signal,
molecular changes in the proteins synthesized by the
myocardium play a major part in the adaptation of the
hypertrophied heart to chronic overload and may
influence the long-term prognosis in patients with congestive heart failure [11].
Although the roles of proto-oncogene products in
linking the hypertrophic signal to the reprogramming of
myocardial gene expression remain to be elucidated and
are at present largely speculative, rapid progress in this
field promises new insights into the pathogenesis of the
cardiomyopathy of overload. It is clear that there is a wide
variation in the individual hypertrophic response seen in
man. In many hypertensive patients left ventricular hypertrophy is well tolerated and adaptive in m~taining
cardiac output against the abnormal haemodynarmc load;
however, it is established that an increase in heart muscle
mass is an important risk factor for cardiovascular
morbidity and mortality above the risk posed by hypertension per se [50, 51]. Indeed the presence of cardiac
hypertrophy may be a more powerful predictor of
prognosis than the level of blood pressure or age ~52].
Improved understanding of the molecular mechamsms
that determine the development of cardiac hypertrophy
may therefore have profound therapeutic implications if
such understanding points the way to means of modulating the hypertrophic process in the myocardium.
ACKNOWLEDGMENTS
We are grateful to the British Heart Foundation, the Wellcome Trust and the Central Birmingham Health
Authority for financial support.
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