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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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