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Signaling Pathways in Cardiac Myocyte Hypertrophy
J Mol Cell Cardiol 29, 2873–2892 (1997)
Growth Factors and Cardiac Hypertrophy: Review Article
Signaling Pathways in Cardiac Myocyte Hypertrophy Martin A. Hefti, Beatrice A. Harder, Hans M. Eppenberger1 and Marcus C. Schaub Institute of Pharmacology, University of Zurich, CH-8057 Zurich; and 1Institute of Cell Biology, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland M. A. H, B. A. H, H. M. E M. C. S. Signaling Pathways in Cardiac Myocyte Hypertrophy. Journal of Molecular and Cellular Cardiology (1997) 29, 2873–2892. When a heart responds to increased workload it does so by hypertrophy. This is characterized by an increase in cell size in the absence of cell division, and is accompanied by distinct qualitative and quantitative changes in gene expression. The use of cardiomyocytes in cell culture has identified, besides mechanical loading, a range of substances, such as cytokines, growth factors, catecholamines, vasoactive peptides and hormones, involved in mediating cardiac myocyte hypertrophy, and has enabled the molecular dissection of the pathways involved in signal transduction. Many different pathways are activated in response to different hypertrophic stimuli, and a growing number of crosslinks are being characterized between these pathways. Recent evidence suggests a central role for Ras in transmitting signals from G-protein coupled receptors, from growth factor receptors and from cytokine receptors not only down the Raf-MEK-ERK pathway to the nucleus, but also to various other cytosolic effectors. The evaluation of distinct morphological phenotypes, together with biochemical data on gene regulation, suggests that interactions between different signaling pathways take place. Each stimulus provokes a typical cellular phenotype and different stimuli may act alone or in concert in a synergistic, antagonistic or permissive manner. Consequently, hypertrophy of cultured cardiomyocytes cannot simply be characterized as the reversal to the fetal gene expression program. Thus, hypertrophic growth of the heart may similarly be the result of a complex combinatorial action of various stimuli, which may also lead to different morphological and biochemical phenotypes with distinct physiological properties. 1997 Academic Press Limited K W: Cell culture; Cardiomyocytes; Cytoskeleton; Hypertrophy; Growth factors; Signal transduction.
Aim of this Review The use of cardiac cells in culture is a recent development which allows to study hypertrophy in an in vitro model at the cellular level. The purpose of this review is to draw attention to the variety in signaling pathways and cellular responses triggered by various stimuli which are able to induce cardiac hypertrophy. In addition, we point out some of the recently recognized evidence for cross-talk between these pathways and between different signals and their cellular responses. The individual hypertrophic stimuli identified by using cultured cardiomyocyte cell models, are likely to be balanced in vivo, even during compensatory hypertrophy. The disturbance of any
single factor, i.e. increase or decrease, may however tip the balance for the worse. Precise knowledge of the cellular responses to individual hypertrophic factors is a prerequisite, since established as well as new therapeutic strategies in cardiology involve either enhancement or counteraction of their effects. These strategies may include (i) systemic application of growth factors, cytokines or thyroid hormones, (ii) blocking the receptors of these hypertrophic factors, or (iii) interference with their production by the antisense oligodeoxynucleotide approach. We have described the relationship between the phenotype of hypertrophic cardiomyocytes in culture and that occurring in different forms of in vivo cardiac hypertrophy in more detail elsewhere (Schaub et al., 1997).
Please address all correspondence to: Dr Marcus C. Schaub, Institute of Pharmacology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
0022–2828/97/112873+20 $25.00/0
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1997 Academic Press Limited
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Table 1 Abbreviations, names and explanations Cells ARC, adult rat cardiomyocytes NRC, neonatal rat cardiomyocytes Proteins involved in hypertrophy a-cd actin, alpha cardiac actin a-sk actin, alpha skeletal muscle actin a-sm actin, alpha smooth muscle actin ACE, angiotensin converting enzyme ANF, atrial natriuretic factor MHC, myosin heavy chain MLC, myosin light chain SERCA2, Ca-pump of sarcoplasmic reticulum Growth factors and related proteins EGF, epidermal growth factor aFGF, acidic fibroblast growth factor bFGF, basic fibroblast growth factor FGFR, FGF receptor IGF, insulin-like growth factor IGFBP, IGF binding protein IGFR, IGF receptor PDGF, platelet-derived growth factor TGF, transforming growth factor Hormones, vasoactive peptides and catecholamines AT-II, angiotensin-II ET-1, endothelin-1 ISO, isoproterenol (isoprenaline) PE, phenylephrine T3, 3,5,3’-triiodo--thyronine TBP, T3 binding protein TR, T3 receptor Components mentioned in G-protein signaling AC, adenylate cyclase cAMP, cyclic adenosine monophosphate DG, 1,2-diacylglycerol G-protein, heterotrimeric GTP-binding protein GPR, G-protein-coupled receptor IP3, inositol-1,4,5-trisphosphate PA, phosphatidic acid PI, phosphatidylinositol PIP2, phosphatidylinositol-4,5-bisphosphate PIP3, phosphatidylinositol-3,4,5-trisphosphate PKA, protein kinase-A PKC, protein kinase-C PLC, phospholipase-C RA, retinoic acid Cytokines CT-1, cardiotrophin-1 IL-1b, Interleukin-1b
In view of space limitation, reviews which contain references to primary information are often cited. Table 1 presents a list of names and abbreviations used in this review.
Cardiac Myocytes in Culture Hypertrophy is an adaptational process of heart muscle cells to hemodynamic overload from various causes. Adult cardiomyocytes are terminally differentiated cells which have lost the ability to divide.
Proteins mentioned in cytokine signaling CR, cytokine receptor JAK, Janus kinase STAT, signal transducer and activator of transcription Proteins mentioned in growth factor signaling Cdc42, homologous to yeast cell division cycle gene 42 ERK, extracellular signal regulated kinase; ERK1=p44; ERK2=p42 GAP, GTPase activating protein GDS, guanine nucleotide dissociation factor, a GEF GEF, guanine nucleotide exchange factor Grb2, growth factor receptor bound protein-2 IRS, insulin receptor substrate JNK, c-Jun N-terminal kinase MAP, microtubule associated protein MAPK, mitogen activated protein kinase MAPKAPK, MAPK activated protein kinase MAPKK, MAPK kinase MAPKKK, MAPKK kinase MEK, mitogen activated ERK activating kinase MEKK, MEK kinase Nck, adaptor protein containing SH2/SH3-domains NRTK, non-receptor tyrosine kinase PI3K, phosphoinositide 3′-kinase PLD, phospholipase-D Rac, a member of the Ras superfamily Raf, a MAPKKK Ral, Ras-related protein Ras, a monomeric GTPase (p21) RGL, RAL-GDS-like protein Rho, Ras homologous RK, reactivating kinase (p38) RKK, RK kinase RSK, ribosomal S6 kinase RSTK, receptor serine/threonine kinase RTK, receptor tyrosine kinase SAPK, stress-activated protein kinase (JNK) SEK, SAPK kinase SH domain, Src homology domain Shc, SH2-domain containing adaptor protein Shp2 (Syp), SH domain containing protein tyrosine phosphatase Smad, vertebrate homologues of Drosophila Mad protein (Mothers against dpp) Sos, mammalian homologues of Son-of-sevenless (a Drosophila gene product), a GEF Src, derived from the Rous sarcoma virus oncoprotein
The increase in heart muscle is thus achieved by an increase in myocyte size rather than number (Anversa et al., 1986). However, hypertrophy does not simply mean more of the same, but involves specific qualitative alterations in gene expression as well as in cell phenotype (Swinghedauw, 1990; van Bilsen and Chien, 1993). Experiments in vivo are unable to distinguish between the relative contributions of the different factors leading to specific biochemical and morphological changes observed in cardiac hypertrophy. Long-term responses cannot be studied in
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isolated heart preparations, because these are not viable for more than a few hours. Cell to cell interactions between identical and different cell types modulate the hypertrophic reaction. The myocytes comprise 70–80% of the heart mass, but represent only 20–30% of total cardiac cells (Long, 1996), whereas fibroblasts, neurons and endothelial cells account for the majority in numbers. Subsequently, it is difficult to attribute certain properties of this tissue to any one cell type. Recent progress in cellular cardiology stems mainly from the development of experimental procedures for primary cell cultures (Jacobson and Piper, 1986). Adult ventricular cardiomyocytes in culture undergo drastic morphological remodeling from the elongated in vivo structure to a spheroidal flat shape with numerous extensions (Jacobson, 1977; Claycomb and Palazzo, 1980; Eppenberger et al., 1988; Nag et al., 1996). Cells hypertrophy to two–three times the original volume (Messerli et al., 1993), resume spontaneous rhythmic beating, seek contact with one another and, eventually, form a coherent 2-dimensional layer (Eppenberger et al., 1994). The initial breakdown and reassembly of myofibrils and regaining of contractile function, together with the accompanying biochemical changes, essentially, recapitulates the events associated with hypertrophy in vivo (van Bilsen and Chien, 1993; Schaub et al., 1997). This pattern of remodeling has also been described for chick embryonal (Lin et al., 1989; Handel et al., 1991; Rhee et al., 1994) as well as for neonatal rat cardiomyocytes (Atherton et al., 1986). Cardiomyocytes in primary culture thus present a suitable in vitro model for detailed analysis of the hypertrophic reaction at the cellular level with regard to individual stimuli and to their signaling pathways. No permanent cardiac cell lines exhibiting these characteristics in culture have been obtained yet. Myocyte cell lines which express some cardiac specific genes, however, have been derived from embryonal cardiac cell clones by retroviral transformation, by culture condition manipulation or from tumors in adult atrial tissue (Doetschmann et al., 1993; Borisov and Claycomb, 1995; Eisenberg and Bader, 1996; Engelmann et al., 1996; Habara-Ohkubo, 1996). These cells retain their proliferative capacity and exhibit an embryonal phenotype with only limited differentiation. Cardiomyocytes from immature animals (embryonic, fetal, neonatal) are easier to culture and have been most widely used. Adult ventricular cardiomyocytes, however, differ from immature cells. They are larger, have a more developed transverse tubular system, grow more slowly in culture
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and have a restricted potential for differentiation (Claycomb, 1983). Adult cardiomyocytes in culture may therefore present a more accurate model for myocardial hypertrophy in vivo. The main argument for using adult cardiomyocytes lies in the fact that they are derived from a terminally differentiated heart tissue and that it is this type of cell which also in vivo is confronted with hypertrophic stimuli, such as overload or other pathological events. On the other hand, the adult ventricular myocyte, in contrast to immature cells, has been refractory to conventional procedures for gene transfer, such as electroporation or calcium phosphate precipitation. Kirshenbaum et al., (1993) reported, however, a transfection success of 90% for adult cardiomyocytes with an adenoviral vector system.
Molecular Characterization of Cellular Hypertrophy Despite the differences between immature and adult rat cardiomyocytes, in principle, both cell types react with a characteristic succession of changes in gene expression (Glennon et al., 1995). Some of these changes are taken as markers of hypertrophy. These markers include the so-called early response genes (e.g. egr-1, hsp70, c-fos, c-jun, c-myc) which are activated within 30 min of exposure to a hypertrophic stimulus. Expression of these genes in response to various growth stimuli has been associated in other cell types with cell cycle regulation and induction of proliferation (Riabowol et al., 1988). In the heart, this transient response seems to represent a general pattern of growth induction in terminally differentiated cells that have lost the ability to undergo DNA replication (Izumo et al., 1988; Komuro et al., 1988; van Bilsen and Chien, 1993). Subsequently, re-expression (or recapitulation) of “fetal genes” such as b-MHC, ask actin and ANF may occur after 6–12 h. An upregulation of constitutively expressed contractile proteins, such as ventricular MLC-2 (Lee et al., 1988) and a-cd actin (Long et al., 1989), may follow after 12–24 h in culture. In cultured ventricular ARC, significant accumulation of the reexpressed proteins, e.g. b-MHC (Nag and Cheng, 1986; Eppenberger et al., 1988), a-sm actin (Eppenberger-Eberhardt et al., 1990) and ANF (Eppenberger-Eberhardt et al., 1993, 1997) can only be demonstrated after several days in culture. Using cultured cardiomyocytes, a number of neurohumoral factors and cell mediators which
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induce hypertrophic reactions have been identified (summarized in Fig. 1). These include a-adrenergic agonists, ET-1, AT-II and various polypeptide growth factors. In addition, cytokines (IL-1b, CT-1) and T3 have recently been recognized as hypertrophic stimuli in cardiomyocyte cell cultures (Palmer et al., 1995; Gosteli-Peter et al., 1996; Wollert et al., 1996). The hypertrophic effects of pressure/volume overload in vivo may depend on mechanical stretch. Stretching of cardiac myocytes cultured on deformable surfaces has indeed been shown to induce the cellular features of hypertrophy, as well as to activate multiple signal transduction pathways (Watson, 1996).
Intracellular Signaling Pathways One general aim, not only in cellular cardiology, is to attribute distinct phenotypic features to defined stimuli and signaling pathways. In contrast to the fast electrochemical signal propagation in nervous and skeletal muscle tissue, the regulation of longer lasting intracellular reactions comprises biochemical and structural alterations, and many operate via reversible covalent protein modifications, such as phosphorylation. Phosphorylation is brought about by two types of protein kinases, i.e. transmembrane receptor kinases (RTK and RSTK in Fig. 1) and a large number of various intracellular kinases. The latter include the non-receptor tyrosine kinases (NRTKs) of the Src and Jak families, which may serve as intermediates in transducing membrane receptor signals to the serine/threonine kinases (PKAs, PKCs, MAP kinases) operating further downstream in the pathways (Figs 1 and 2). Protein phosphorylation can be reversed by two families of protein phosphatases which specifically dephosphorylate phosphoserine/threonine or phosphotyrosine residues. A discussion of the phosphatases is beyond the scope of this review (see Hunter, 1995; Streuli, 1996). A cascade of successive transduction steps allows signal enhancement and diversification at branching points and thus permits combinatorial interactions between multiple pathways (Fig. 1). The pathways discussed in this section have been elucidated using different cell systems, and many have been shown to be operative in cardiac cells too (Sugden and Bogoyevitch, 1995). Cell membrane receptors and intracellular signaling proteins are highly conserved between mammalian species and the triggering events for cellular hypertrophy in
humans are likely to resemble closely those in the various animal models used (Glennon et al., 1995).
Growth Factors and their Signaling Pathways The classic peptide growth factors can be divided into five families: EGFs, FGFs, IGFs, PDGFs and TGFs. Each family comprises multiple members, where individual growth factor properties, besides induction of cell growth and proliferation, are manifold and range from being highly specific to being more general. FGF, TGFb, IGF and PDGF have been shown to cause cardiomyocyte hypertrophy in in vitro systems (Long, 1996). In the heart, PDGF is produced by non-myocytes. FGF and TGFb can be produced by cardiac myocytes and non-myocytes (Speir et al., 1992; Brand and Schneider, 1995) and therefore, these two factors may act in a paracrine or autocrine manner in the heart. IGF is produced predominantly in the liver and circulates in substantial amounts in the blood, where it may be bound to a number of IGF binding proteins (IGFBPs, six members of a homologous protein family ranging in size from 24–45 kDa) (Blakesley et al., 1996). The work on cardiac hypertrophy has concentrated mainly on the effects of FGF, TGFb and IGF and these studies have been done with cultured NRC and ARC. FGF The FGF gene family comprises nine members which share 40–50% sequence homology (Basilico and Moscatelli, 1992). The two prototypes are aFGF (FGF-1, 140 amino acids, IEP of 5.6) and bFGF (FGF-2, 154 amino acids, IEP of 9.6). They are highly conserved among various mammalians and chicken (90% sequence homology). Neither contains a classic signal sequence to direct its secretion through membranes (Friesel and Maciag, 1995). Most of the other FGFs contain such a functional signal sequence and their genes were originally identified as oncogenes. It is assumed that for secretion the prototype FGFs form disulfide bonded homodimers and exocytosis may be involved (Bastagli et al., 1995). In addition, an intracrine mode of action has been proposed where bFGF may be active after nuclear translocation without necessarily exiting the cell (Logan, 1990). aFGF and bFGF also occur as isoforms with higher molecular weights (22 instead of 17 kDa), resulting from alternative initiation of translation. A specific sequence of Lys-Lys-Pro-Lys near the N-terminus
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Hypertrophy in Cultured Cardiomyocytes Mechanical stress
Cardiac hypertrophic stimuli
[Cytokines] IL-1β CT-1
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STAT
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PKC
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TR Transcription
Figure 1 Cardiac hypertrophic stimuli initiate multiple signal transduction pathways. A whole range of substances and subsequently activated intracellular pathways are involved in mediating the hypertrophic phenotype in cultured cardiomyocytes. Evidence discussed in the text suggests a central role for the Ras-Raf-MEK-ERK pathway in transmitting these stimuli to the nucleus. Arrows indicate the major routes of signal transduction and illustrate some of the interactions between different pathways. Open arrowheads denote pathways to cytosolic substrates. Binding of TGFb to the primary receptor (RSTK with open box) enables this type-II receptor to bind and phosphorylate the type-I receptor (RSTK with black box), which then propagates the signal. A direct pathway linking mechanical stress to gene expression is thought to be operative (dashed arrow). For abbreviations see Table 1.
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IGF-IR
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Figure 2 Growth factor signaling through receptor tyrosine kinases. Various proteins containing SH2 domains are able to interact with activated growth factor receptors such as FGFR or with IRS to initiate multiple downstream responses. Each of these proteins binds to a different phosphorylated site on the activated receptor, recognizing surrounding features of the polypeptide chain in addition to the phosphotyrosine residue. One downstream event is the activation of Ras by Sos. The relay system downstream from Ras involves three distinct cascades of protein phosphorylation, known as the MAP kinases cascades. As discussed in the text, recent work has shown that many additional effectors can interact with Ras to initiate distinct responses, some of which are depicted here. It is likely that for a given stimulus only a subset of all possible interactions shown here actually take place. The specific effect of a given stimulus may be the result of a complex combinatorial activation of individual pathways depending on spatial and temporal abundance of relevant proteins in the cell in question. For abbreviations see Table 1.
targets these larger peptides to the cell nucleus. Their function within the nucleus is not known. The high molecular weight forms predominate in neonatal rat hearts and the low molecular weight forms in the adult heart (Liu et al., 1993). Their balance seems to depend on thyroid hormone levels, the higher the T3 level the lower the high molecular weight FGF isoforms. Overexpression of both low and high molecular weight forms displays similar effects in cultured embryonic chicken cardiomyocytes (Pasumarthi et al., 1994). All FGFs associate with the glycosaminoglycan heparin and with heparan sulfate proteoglycan of the outer cell surface. The association of FGFs with the extracellular matrix
provides potential storage sites from which they may be released upon injury and activation of matrix degrading enzymes. aFGF and bFGF have identical effects but aFGF is 30–100 times less potent. aFGF and bFGF are both found in the heart in myocytes and in non-myocytes. Their content is higher in atria than in ventricles suggesting a possible correlation with a higher regenerative capacity of the atria (Kardami and Fandrich, 1989). IGF-I IGF-I and IGF-II share structural and biological function homology with pro-insulin and are cleaved to release active monomeric proteins with 70 and
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67 amino acids respectively (Blakesley et al., 1996). The smaller IGF-II is more prominent during fetal development and IGF-I persists during adult life. Most of the functions of both are mediated via IGFI receptors. Complex formation of IGFs with the IGFBPs is required for transport through membranes and may control the interaction of the IGFs with receptors. The IGF-I receptor is structurally similar to the insulin receptor (White and Kahn, 1994). Both are synthesized as single chains that become glycosylated and cleaved, producing a- and b-subunits. The mature receptor is a disulfide-bonded heterotetramer with two 125 kDa a-chains oriented extracellularly, and two 95 kDa b-chains extending through the membrane and containing tyrosine kinase activity. Although pituitary secretion of growth hormone stimulates the production of IGF-I primarily in the liver (endocrine mode), most tissues are able to synthesize IGF-I locally (autocrine/paracrine mode).
TGFb TGFb belongs to a superfamily of over 20 regulatory proteins consisting of factors distantly related (30– 40% sequence homology) including the activins and bone morphogenetic proteins (BMPs). TGFb now designated TGFb1, shares 70–80% sequence homology with its other family members TGFb2, TGFb3 (1, 2 and 3 are mammalian forms), TGFb4 (avian) and TGFb5 (Xenopus). TGFb1 is synthesized as a 390 amino acid precursor protein, which is cleaved to yield an N-terminal remnant and a Cterminal monomeric TGFb segment. The mature TGFb consists of a disulfide bonded dimer (25 kDa) which is secreted as a latent complex non-covalently associated with its processed “latency peptide” (75 kDa) and an additional binding protein (135 kDa) (Sporn and Roberts, 1992; Kingsley, 1994; Long, 1996). Cultured NRC and ARC release TGFb from day 1 on into the culture medium ¨ (Roberts et al., 1992; Schluter et al., 1995). The three mammalian forms of TGFb bind to three types of receptors found on most mammalian cell types. The type-III receptors (200–400 kDa proteoglycan) have no known signaling function (Brand and Schneider, 1995). Type-I and type-II receptors are distantly related Ser/Thr kinases (RSTKs). Ligand binding by the primary receptor (type-II) enables it to bind the type-I receptor. In the resulting heteromeric complex, the type-II receptor phosphorylates the type-I receptor, which then propagates the signal (Fig. 1). Smad proteins have recently been identified as the first family of putative
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´ TGF-b signal transducers (Massague et al., 1997). Upon phosphorylation by activated receptors, Smads form complexes, move into the nucleus, associate with DNA binding proteins and activate gene transcription.
Receptor tyrosine kinase signaling The receptors of most growth factors, with the exception of TGF-b and IGF-II, are transmembrane tyrosine kinases (RTKs) (Ullrich and Schlessinger, 1990). Binding of growth factors such as EGF, PDGF and FGF leads to receptor dimerization which in turn enables the two cytoplasmic domains to crossphosphorylate each other on multiple tyrosine residues (Heldin, 1995). Dimerization is a general mechanism for activating enzyme-linked receptors with a single transmembrane domain. In each case the autophosphorylated tyrosines serve as highaffinity binding sites for a number of intracellular signaling proteins (Fig. 2). Although these proteins have varied structures and functions, they usually share two highly conserved non-catalytic modular binding domains, termed SH2 and SH3 (for Src homology), since they were first found in a tyrosine kinase encoded by the Rous sarcoma virus oncogene src. Each of these proteins binds to a different phosphorylated site on the activated receptor, recognizing surrounding features of the polypeptide chain in addition to the phosphotyrosine (Cohen et al., 1995). The interaction between SH2 domains and the phosphorylated receptors allows the recruitment of cytosolic substrates to the plasma membrane. The IGF-I receptor acts in a slightly different way. This receptor is a tetramer where ligand binding induces an allosteric interaction of the two halves and phosphorylates its catalytic domains. This activates them to phosphorylate separate proteins called IRS on multiple tyrosines (Waters and Pessin, 1996). The phosphotyrosines on IRS then serve as high-affinity binding sites for the docking and activation of intracellular signaling proteins containing SH2 domains, many of which can also bind to activated receptor tyrosine kinases directly (Fig. 2). Once bound, these proteins can be further regulated by phosphorylation or conformational changes. Different RTKs probably bind different combinations of these signaling proteins and therefore initiate different responses. PLC-c binds to receptors of PDGF, EGF and FGF via its SH2 domains. Subsequent phosphorylation stimulates the catalytic activity of PLC-c to hydrolize PIP2, which produces the second messengers IP3
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and DG (Lee and Rhee, 1995). In addition, activated PLC-c1 may associate with actin of the cytoskeleton by its SH3 domain. PIP2 itself has also been shown to interact with a number of actin-binding proteins such as a-actinin, profilin or vinculin. It seems likely, therefore, that PLC-c isoenzymes represent one link from growth factor signaling to the organization of the cytoskeleton. PI3K binds to activated RTKs via SH2 domains in its regulatory p85 subunit. Its catalytic p110 subunit is able to phosphorylate several phosphoinositides at their 3position and, thus, PI3K has been implicated in many intracellular pathways (Carpenter and Cantley, 1996). In addition to PI3K and PLC-c, many other SH2-domain containing proteins bind to RTKs, such as Src (a ubiquitous NRTK), Nck (an adaptor protein containing one SH2 and three SH3 domains), Shp2 (a phosphotyrosine phosphatase also known as Syp) as well as the two adaptor proteins Shc and Grb2 (Pawson, 1995) (Fig. 2).
Ras in Signal Transduction Ras proteins were first discovered as the hyperactive product of mutant ras genes which promote cancer by disrupting the normal controls on cell proliferation and differentiation. Like the other monomeric GTPases and the trimeric G-proteins, Ras proteins function as switches, cycling between two conformational states, active when GTP is bound and inactive when GDP is bound. In cells containing a ras oncogene, the Ras oncoprotein is locked in the GTP-bound state as a result of its defective GTPase and is likely to generate a constitutive signal even in the absence of growth factor stimulation. Two classes of signaling proteins regulate the activity of Ras and its relatives by influencing the transition between the active and inactive states (Boguski and McCormick, 1993). GTPase activating proteins (GAPs) increase the rate of hydrolysis of bound GTP by Ras, thereby inactivating it (Fig. 2). These negative regulators are counteracted by guanine nucleotide exchange factors (GEFs) which promote the exchange of bound GDP for GTP. In mammalian cells, Ras is activated for example by Sos, a GEF named after its Drosophila counterpart “Son-of-sevenless”. Sos is associated with Grb2 via the latter’s two SH3domains. Similarly, phosphorylated Shp2 can serve as a docking site for Grb2 as has been shown for the PDGF receptor (Sun and Tonks, 1994). Phosphorylated Shc has been implicated in the Ras signaling pathway by virtue of its association with the Grb2 adaptor molecule. Interestingly, Shc might
act as a go-between that relays messages from Gprotein coupled receptors to Ras (see below).
Ras effectors Multiple signaling pathways are activated by Ras (Fig. 2). Molecules that interact with GTP-bound Ras and transmit the signal, are referred to as Ras effectors (Marshall, 1996). Each of these effectors may be activated by Ras-GTP via a unique interaction on a particular site on Ras. PI3K seems to be one of these Ras effectors, binding in this case, however, is mediated by the p110 subunit. PKCf and RGL (a dissociation factor similar to Ral-GDS) have also been shown to interact with activated Ras. Nucleotide-exchange factors for Ral-GTPases, such as Ral-GDS, are the newest members of putative Ras effectors (Feig et al., 1996). Ral, in turn, may link growth factor signaling to lipid second messengers via PLD and to two members of the Rho family of small GTPases, namely cdc42 and Rac-1. PLD catalyses the hydrolysis of phosphatidylcholine to PA and choline. PA may be subsequently metabolized to yield DG, an activator of PKC. The two Rho family GTPases cdc42 and Rac-1 are involved in the regulation of the actin cytoskeleton (Symons, 1996) and they have also been shown to stimulate two distinct MAP kinases, the JNK/SAPK and the p38 MAP kinase (Feig et al., 1996). Microinjection experiments in Swiss 3T3 fibroblasts have revealed that cdc42 can activate Rac, which in turn activates Rho (Nobes and Hall, 1995) and recent mutational analysis has revealed that the ability of Rac and cdc42 to activate MAP kinase cascades and to induce cytoskeletal rearrangements is mediated by distinct, independent downstream signals (Lamarche et al., 1996).
MAP Kinases The relay system that transmits signals from Ras to the nucleus involves three distinct cascades of protein phosphorylation, each with three levels of chemically defined reactions: (i) MAPKKKs phosphorylate MAPKKs on serine or threonine residues; (ii) MAPKKs are dual-specificity protein kinases that phosphorylate the MAPKs on a threonine and a tyrosine residue in a Thr-X-Tyr motif; and (iii) once activated, the MAPKs phosphorylate serine or threonine residues in various nuclear and extranuclear substrates (Fig. 2) (Seger and Krebs, 1995; Denhardt, 1996). The three mammalian MAPK families are defined by being phosphorylated in a
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distinct motif. These three motifs are Thr-Glu-Tyr (T-E-Y) for ERKs, Thr-Phe-Tyr (T-P-Y) for the JNK/ SAPKs and Thr-Gly-Tyr (T-G-Y) for p38/RK. The first MAPKs to be identified were p44 (ERK1) and p42 (ERK2). ERK1 and ERK2 are proline-directed kinases, i.e. they generally phosphorylate Ser/Thr residues to the left of proline. Many important substrates for MAPKs are nuclear transcription factors that are thought to be phosphorylated after MAPK translocation to the nucleus (Treisman, 1996). Thus, there appears to be two reasonably discrete pathways under the influence of Ras: (i) the Raf-MEK-ERK pathway activated by growth factors, mitogens and tumor promoters and (ii) the MEKK-SEK-JNK/SAPK pathway that is mainly activated by various stressors and cytokines (Fig. 1). The pathways leading to activation of p38/RK, the third family of MAPKs, are not well understood yet (Denhardt, 1996). Extranuclear substrates for ERKs comprise the MAPs such as MAP-1, MAP-2, MAP-4 and tauprotein (Seger and Krebs, 1995). Their phosphorylation by ERKs is part of the regulation of microtubular rearrangements and cellular morphology. It has been shown that rabbit cardiac titin can be phosphorylated by the proline-directed ERK1 (Sebestyen et al., 1995). Additional substrates for ERKs are downstream serine/threonine kinases termed MAPK-activated protein kinases (MAPKAPK), such as p90 (RSK). ERKs may also phosphorylate upstream components in the signaling cascade that lead to their activation, thus demonstrating feedback regulation. Numerous studies have addressed the role of Ras, Ras effectors and the molecules of the MAPK cascades in cardiac signal transduction. It has been suggested, that in ventricular muscle, Ras activity may regulate “the basic transcriptional machinery” (Abdellatif et al., 1994). Introduction of a constitutively active Ras molecule into NRCs induced both genetic (expression of the c-Fos and ANF genes) and morphological effects (changes in the organization of the contractile apparatus) associated with hypertrophy (Thorburn et al., 1993). Furthermore, expression of a dominant inhibitory Ras mutant could prevent activation of ANF gene expression by PE. However, in a different study, coexpression of an inhibitory Ras mutant, together with a constitutively active Gq a-subunit, did not abolish the latters stimulation of ANF gene expression (La Morte et al., 1994). It has also been shown that Raf-1 and the downstream ERKs are necessary and sufficient for gene expression changes but not for cytoskeletal actin changes associated with PE-induced cardiac myocyte hypertrophy
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(Thorburn et al., 1994). However, activation of Ras is sufficient for both these changes and this may now be explained in view of the multiple pathways leading to extranuclear responses which can be activated by Ras. Adding to complexity, constitutively active MEK1 and ERK2 have been shown to stimulate ANF, b-MHC and a-sk- actin expression, whereas dominant negative mutants of these two molecules inhibited PE-induced ANF expression to a certain extent (Gillespie-Brown et al., 1995). Similarly, antisense oligodeoxynucleotides against the initiation of translation of the p42 and p44 MAPK isoform mRNAs inhibited development of the morphological features of hypertrophy (sarcomerogenesis, and increased cell size) in NRC exposed to PE. PE-induced activation of the ANF promoter and induction of ANF mRNA were also attenuated (Glennon et al., 1996). In a contrasting study, dominant-interfering mutants of p42 and p44 MAPK as well as the use of PD 098059, an inhibitor of MEK, failed to block PE induced ANF expression (Post et al., 1996). Apart from technical differences and the sometimes difficult interpretation of results obtained with mutant molecules and more or less specific inhibitors with respect to the in vivo situation, these findings may also reflect pathway redundancy, i.e. that multiple or different pathways may be potentially activated in response to a hypertrophic stimulus. Many interactions, e.g. concerning the precise interactions of various GTPases of the Ras superfamily are still unclear or have yet to be identified, but are likely to depend on the spatial and temporal abundance of the relevant regulatory proteins, such as the GAPs and GEFs, but also on the specific cell in question. Interestingly, RhoA has been suggested to function in a pathway, separate from, but complementary to Ras and is involved in Gq a-signaling in cardiomyocytes (Sah et al., 1996). Very recently, it has been suggested, that an effector-like function of Ras-GAP may predominate in the cardiac background, since abolishing GAP binding to Ras interfered with Rasdependent expression in transfected NRCs (Abdellatif and Schneider, 1997). Thus, effector-like functions of Ras-GAP, may be at least in part responsible for the global increase of RNA and protein synthesis in cardiac hypertrophy.
Signaling Through G-protein Coupled Receptors Catecholamines and vasoactive peptides (AT-II and ET-1) are all reported to induce anabolic responses
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with cardiac myocyte growth. The specific receptors for all of these mediators belong to the G-protein linked superfamily of homologous heptahelical transmembrane proteins. G-proteins, in turn, are a large family of heterotrimeric GTP-binding regulatory proteins such as Gq, Gi or Gs and are each composed of an a-, b- and c-subunit (Gudermann et al., 1996). G-protein function has historically been attributed to the GTP-bound a-subunit which is released from the bc-dimer upon receptor stimulation. However, recent work has suggested that the bc-subunits may play an equally prominent role in effector regulation. Numerous studies have shown that for instance ET-1 stimulates the PIP2 signaling pathway producing the two messengers DG and IP3 through a phospholipase-Cb catalysed hydrolysis of PIP2 (Fig. 1) (Sugden and Bogoyevitch, 1996). IP3 induces release of Ca2+ from intracellular Ca-stores. DG remains in the plain of the membrane for translocation and activation of members of the PKC family (Jaken, 1996; Spiegel et al., 1996). This family consists of cPKCs (classical, requiring DG and Ca2+), nPKCs (novel, requiring only DG) and aPKCs (atypical, activation mechanism unknown, probably not depending on DG). The tumor-promoting phorbol esters can activate the phospholipid-dependent cPKCs and nPKCs by substituting for DG. Active PKC has a number of nuclear and cytosolic substrates (Nishizuka, 1995). The a-isoform of PKC, for example has been shown to directly activate Raf-1 (Kolch et al., 1993), and thus provides a link to the signaling pathway that is activated by growth factor receptors. PKC-mediated activation of the MEKK-SEK-JNK/SAPK cascade has also been described as a consequence of GPR stimulation (Denhardt, 1996). In addition, signaling from GPR can be directly crosslinked to Ras (Fig. 1). ATII induced Ras activation in NRC involves a pathway linking a member (Fyn) of the Src family to Shc and Grb2-Sos (Sadoshima and Izumo, 1996). Recently, the bc-subunit of G-proteins have been suggested to be involved in a pathway from GPRs to Ras. This has been shown in the established cell lines like COS-7 (fibroblast-like simian kidney cells) and PC12 (phaeochromocytoma cells), where the bc-subunits of Gq, Gs and Gi are involved in this transmission (Crespo et al., 1995; Dikic et al., 1996; Lutrell et al., 1996). Taken together, these findings may offer an explanation for previous studies that reported Ras involvement for example, in the a1adrenergic receptor agonist PE-induced hypertrophic response of cardiac myocytes (Thorburn et al., 1993). The a-subunits of the G-proteins are known to be responsible for activation (Gq) of PLCb
as well as for stimulation (Gs) or inhibition (Gi) of adenylate cyclase, which is one of the main signal routes leading to PKA. PKA activity results directly in nuclear as well as cytosolic events. Some of these effects may be linked to changes in cytosolic levels of Ca2+ (Fig. 1). An interesting inhibitory effect has been demonstrated, in that PKA can interfere with the Ras-Raf-cascade by inhibiting the latter (Sugden and Bogoyevitch, 1995; Denhardt, 1996). Additional crosslinks are known to connect GPR signals to early events of the growth factor (RTK) and cytokine receptor (CR) induced pathways (Fig. 1). In the Rat-1 fibroblast cell line, the EGF receptor becomes rapidly tyrosine phosphorylated upon stimulation with ET-1 (Daub et al., 1996). In these cells, this ligand-independent mechanism of RTK activation is crucial for ET-1 induced activation of ERKs. In vascular smooth muscle cells, AT-II leads to activation of IGF-I receptor by tyrosine phosphorylation, probably via an intermediate NRTK (Delafontaine et al., 1996). It has also been shown that AT-II can modulate cytokine responses directly by activating the JAK-STAT cascade in cultured cardiac fibroblasts (Brecher, 1996).
Endothelin-1 The endothelium-derived vasoconstrictor ET-1 is a peptide of 21 amino acids. Two additional homologues (ET-2 and ET-3) were later identified. After cleavage from a large precursor peptide, ET-1 is released from vascular endothelium and other cells including cardiomyocytes (Suzuki et al., 1993). The ET peptides bind to three receptor subtypes (ET-A, ET-B and ET-C). The ET-A receptor probably mediates the vasoconstrictor effects and the ET-B receptor may induce vasodilation by causing release of NO and/or prostacyclin from endothelial cells. The ET-A receptor is the predominant species in rat hearts and is coupled to both the Gq and Gi species of G-proteins (Hilal-Dandan et al., 1994). In ventricular NRC, ET-1 induces a characteristic pattern of gene expression: (i) immediate early genes (c-fos, c-jun, egr-1); (ii) the early genes ANF, bMHC and a-sk actin, and later (iii) the genes for ventricular MLC-2 and a-cd actin (Sugden and Bogoyevitch, 1996). These transcriptional changes may involve the Gq-Ras-Raf-ERK pathway (Fig. 1). However, ET-1 also affects gene expression and causes hypertrophy via the Ras-MEKK1-SEK-JNK pathway (Bogoyevitch et al., 1996). Beside the effects on the transcriptional level, ET-1 also stimulates protein synthesis on the translational level. The mechanism of a positive inotropic response of
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the heart muscle to ET-1 is not yet clear. It has also been reported that ET-1 causes cellular injury in cardiac myocytes in vivo and, recently, it was shown that the survival rate of rats with heart failure is improved by long-term treatment with the ET-A receptor blocker BQ-123 (Sakai et al., 1996).
Angiotensin-II AT-II is the main effector molecule of the reninangiotensin system which is responsible for pressure and volume homeostasis (Bernstein and Berk, 1993). Renin which is synthesized by the kidney and secreted into the blood, hydrolyses the decapeptide angiotensin-I (AT-I) from the N-terminus of angiotensinogen. AT-I is converted to the octapeptide AT-II by the dipeptidyl carboxypeptidase ACE. In addition, a chymotrypsin-like protease (chymase) has been identified which is though to represent the main pathway for conversion of AT-I to AT-II in the human heart (Urata et al., 1990). ATII is a potent vasoconstrictor, increases aldosterone secretion, enhances central sympathetic outflow, catecholamine release from adrenal medulla and promotes release of vasopressin. Because of its hypertrophic effect on cardiomyocytes, it is also considered to be a growth factor. Most of these biological effects are mediated through the AT-1 receptor, although several other receptor subtypes have been identified (Timmermans et al., 1993). AT-II is able to induce hypertrophy of rat cardiomyocytes and hyperplasia in cardiac non-myocytes, both actions being mediated by the AT-I receptor (Sadoshima and Izumo, 1993). Electron microscopy reveals that immunoreactive AT-II is preferentially localized in what appears to be secretory granules in ventricular myocytes. AT-II may then be secreted from cardiomyocytes and act in an autocrine fashion by binding to the AT-I receptor which in turn associates with Gq and Gi-proteins (Sadoshima and Izumo, 1996). Important effects of AT-II have been demonstrated using cardiac fibroblasts and vascular smooth muscle cells where AT-II interacts with the IGF-I system (Delafontaine et al., 1996). For one, it stimulates cardiac IGF-I expression. However, due to a change in the balance between the various IGFBPs, the level of circulating IGF-I is lowered. This may account for the low metabolic activity which leads to weight loss in the AT-II infused animal. In cardiac fibroblasts, AT-II induced activation of the JAK-STAT-pathway and the MAP kinases, leads to changes in transcription and to cell proliferation.
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Catecholamines The transcriptional alterations typically associated with the hypertrophic reaction which is induced via the Gq-Ras-Raf-ERK or via the PKC signaling pathway in ventricular NRC is usually attributed to a1adrenergic stimulation, e.g. by epinephrine (Simpson et al., 1991). Catecholamine-activated signaling pathways in NRC and their phenotypic features resemble those induced by either AT-II or ET-1. This phenotype induced by a-adrenergic or ET-I stimulation can be inhibited by application of retinoic acid (RA) to NRC (Zhou et al., 1995). NRC express functional retinoic acid receptors which belong to the nuclear receptors of the steroid hormone receptor superfamily. Upon stimulation, these receptors may interfere with the transcription of those genes, which are stimulated by the GPR mediated signals. The a1GPR may couple to different G-proteins, among which are Gq and a 74 kDa Gh species (Terzic et al., 1993). Both b1- and b2-receptors usually couple to Gs when activated. It has recently been demonstrated in NRC that both a- and b-adrenergic agonists are able to activate MEK and ERK, albeit by different signaling pathways (Bogoyevitch et al., 1996). Accordingly, an increase in mRNA for a-sk actin is observed on either a- or b-adrenergic stimulation in NRC (Martin et al., 1996). However, b-adrenergic stimulation of NRC enhances accumulation of a-MHC (Rupp et al., 1991), while b-MHC mRNA and protein accumulation is activated by a-adrenergic stimulation (Waspe et al., 1990). The elevation of intracellular Ca2+ rather than cAMP appears important for the activation of ERK by b-adrenergic agonists, where a direct Gs-mediated coupling of the -type Ca-channel could be operative (Yatani and Brown, 1989; Bogoyevitch et al., 1996). Dual effects of b-adrenergic stimulation on ERK in COS-7 cells are due to a bcsubunit mediated activation of Ras and an inhibitory effect induced by the a-subunit via PKA on Raf further downstream (Crespo et al., 1995). The existence of these two opposing mechanisms (Fig. 1) may explain some of the differences observed between aand b-stimulation in heart cells.
Mechanical Stress Deformation of the cardiomyocyte as it occurs in vivo during contraction, or in vitro under artificial conditions, presents a mechanical stress for the cellular structures. This mechanical loading may present the most important physiological stimulus for cardiac hypertrophy. Cardiomyocytes kept in
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long-term culture spontaneously contract rhythmically, but can also be subjected to passive stretch. Mechanical loading induces a host of responses associated with cellular hypertrophy and with structural remodeling (Watson, 1996). Acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents (Hu and Sachs, 1996). These currents seem to arise from stretch-activated ion channels. Different ion-specific and non-specific mechanosensitive channel types have been described (Sackin, 1995). Stretch-activation is a membrane or membrane-cytoskeleton phenomenon and does not seem to involve soluble cytosolic messengers. However, such ion fluxes may result in lowering or increasing the cytosolic free Ca2+ levels which, in turn, may affect gene expression in the cell nucleus directly (dashed arrow in Fig. 1), or may modulate the signaling by other stimuli. Indeed, stretch was found to induce transient activation of PKC followed by Raf-1 and MEKK (both on the level of MAPKKK) and the successive components of the MAPK signaling cascade in a sequential time order over several minutes in isolated neonatal rat cardiomyocytes (Yamazaki et al., 1995a). Activation of PKC and MAP kinases can also be elicited by ATII and lead to hypertrophy; and AT-II has been reported to be secreted from cardiomyocytes under mechanical stress (Sadoshima et al., 1993). On the one hand, the stretch induced activation of Raf-1 and MAP kinases can only be suppressed partially by an AT-1 receptor inhibitor, and, on the other hand, also the stretch induced activation of MAPK can only partially be attenuated by inhibition of PKC (Yamazaki et al. 1993). In contrast, AT-II evoked activation of Raf-1 and MAP kinases can be blocked completely by the PKC inhibitor staurosporine (Yamazaki et al., 1995a). In adult rat and feline cardiomyocytes in culture, AT-II does not seem to be required for the anabolic processes (increased protein synthesis and expression of Na–Ca-exchanger mRNA) induced by mechanical load (Kent and McDermott, 1996). Taken together, externally applied stretch may activate multiple signaling pathways in ventricular NRC. One component of the stretch-induced effects operates by the release of AT-II, that acts in an autocrine and/or paracrine mechanism. However, AT-II was shown to be only partly responsible for the stretch-induced hypertrophic response involving activation of the MEK-ERK route (Yamazaki et al., 1995b). Thus, an alternative signaling pathway must be mediated by stretch which could involve mechano-sensitive membrane structures resulting in an increase of intracellular Ca2+ as a consequence of membrane deformation.
Cytokines and their Signaling Pathways Cytokines comprise signaling molecules that act as local mediators in cell–cell communication, such as colony-stimulating factors, interferons and interleukins. IL-1b, a 17 kDa protein initially termed endogenous pyrogen, is primarily known as mediator of inflammation and to play a key role in immune responses. Using the NRC culture model, IL-1b has recently been reported to induce a novel form of cardiac myocyte hypertrophy that is characterized by an increase in protein content, but an absence of the fetal program of a-sk actin or bMHC gene expression (Patten et al., 1996). This contrasts with the hypertrophy induced by PE, vasoactive peptides and other growth factors (see Table 2). In fact, IL-1b inhibits PE-induced a-sk actin and b-MHC gene expression. Transient transfection studies with a-sk actin and b-MHC promoter constructs indicate that the repression may occur at the level of transcription via activation of the negative transcription factor YY1. In contrast, an upregulation by IL-1b of mRNA levels for b-MHC and ANF was reported in NRC, concomitant with a down regulation of three Ca-regulatory genes (SERCA2, Ca-release channel and voltage dependent Ca-channel) (Thaik et al., 1995). For IL1b little is known concerning the immediate postreceptor events, although activation of the JNK/ SAPK and p38 MAP kinase cascade has been shown (Saklatvala et al., 1996). IL-1b seems to act in paracrine fashion since it is expressed in cardiac fibroblasts, but not in cardiomyocytes (Long, 1996). Cardiotrophin-1, a 21.5 kDa protein, is a novel member of the interleukin-6 family of cytokines and was isolated by expression cloning based on its ability to induce an increase in cell size in NRC (Pennica et al., 1995). CT-1 activates another form of cardiac myocyte hypertrophy that is different from the phenotype seen after a-adrenergic stimulation with PE. CT-1 induces a distinct set of immediate early genes as well as the ANF gene, but not the a-sk actin gene, and leads to a change in overall cell shape (Wollert et al., 1996). The overall cell shape is significantly elongated in culture with sarcomere assembly in series rather than in parallel. This is reminiscent of the morphological changes observed in hearts under volume as opposed to pressure overload (Gerdes et al., 1988). CT-1 is expressed in cardiomyocytes and acts in an autocrine way. CT-1 has been shown to signal via the gp130-LIFRb (leukaemia inhibitory factor receptor) dependent pathways (Wollert et al., 1996) and continuous activation of gp130 was shown to cause myocardial hypertrophy in vivo (Hirota et al., 1995).
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Hypertrophy in Cultured Cardiomyocytes Table 2 Effects of growth factors and other hypertrophic stimuli on mRNA and/or protein accumulation of markers of hypertrophy. Data for pressure overload (PO) and in part for T3 come from adult rat hearts in vivo. All other data was obtained from cultured neonatal or adult rat cardiomyocytes∗ (see text for details).
a-MHC b-MHC a-skeletal muscle actin a-smooth muscle actin SERCA2 ANF References
PO†
bFGF
TGFb
IGF-I
T3
IL-1b
ET-1
PE
↓ ↑ ↑ ↑ ↓ ↑ 1–6
↓ ↑ ↑ ↑ ↓ ↑ 7–9
↓ ↑ ↑ ↑ ↓ ↑ 10
n§ ↑ ↑ ↓ n ↓ 7–8 and 11–13
↑ ↓ ↑ ↑ ↑ ↑ 7 and 14–21
↓ ↑/↔ ↔ n ↓ ↑ 22–23
↑ ↑ ↑ n n ↑ 24–25
↓/↔ ↑ ↑ n ↓ ↑ 22 and 26–28
∗ Results are taken from refs: (1) Black et al., 1991; (2) Calderone et al., 1995; (3) Izumo et al., 1988; (4) Komuro et al., 1989; (5) Lompre et al., 1991; (6) Schwartz et al., 1986; (7) Gosteli-Peter et al., 1996; (8) Harder et al., 1996; (9) Parker et al., 1990b; (10) Brand and Schneider, 1995; (11) Donath et al., 1994; (12) Florini and Ewton, 1992; (13) Ito et al., 1993; (14) Dubus et al., 1993; (15) Kessler-Icekson, 1988; (16) Ladenson et al., 1988; (17) Lompre et al., 1984; (18) Nag and Cheng, 1986; (19) Rohrer and Dillmann, 1988; (20) Rohrer et al., 1991; (21) Winegrad et al., 1990; (22) Patten et al., 1996; (23) Thaik et al., 1995; (24) Sugden and Bogoyevitch, 1996; (25) Wang et al., 1992; (26) Bishopric et al., 1987; (27) Knowlton et al., 1991; (28) Waspe et al., 1990. † PO, pressure overload in rat heart in vivo induced by aortic constriction. ‡ PE, phenylephrine (a1-adrenergic stimulation). § n, not known at present.
The general features of cytokine signaling briefly discussed here are true also for the IL-6 family, of which CT-1 is a member. Cytokine receptors are not tyrosine kinases themselves, but upon ligand binding interact with associated proteins, the Janus kinases (JAKs), that phosphorylate tyrosine residues on both themselves and the receptors. Thereby, they provide docking sites for various downstream effector proteins: adaptor molecules such as Shc, various NRTKs and STATs (Ihle, 1995; Briscoe et al., 1996; Waters and Pessin, 1996). Thus, the CRs are able to signal via several downstream routes (Fig. 1). In addition, multiple JAK and STAT isoforms provide different specific interactions with particular CRs. The mode of activation for the STAT transcription factors is unique in that they are activated by tyrosine phosphorylation directly at the membrane level. Activated STATs form dimers, translocate to the nucleus and bind response elements to induce transcription.
Thyroid Hormone Signaling Cardiovascular disorders are frequently observed in hyperthyroidism and hypothyroidism. Cardiac performance can be directly correlated to the thyroid hormone serum level (Feldman et al., 1986). Heart rate, contractility, ejection fraction and coronary blood flow are all increased in hyperthyroidism, while peripheral vascular resistance is reduced concomitantly. These changes are accompanied by cardiac hypertrophy and may eventually result in heart failure. On the other hand,
thyroid hormone deficit is associated with decreased contractility and cardiac dilation. Both changes can be reversed by redressing the unbalanced thyroid hormone status (Ladenson et al., 1992). The active thyroid hormone is 3,5,3′-triiodo--thyronine (T3) which arises by deiodination of -thyroxine (T4) in the periphery. About 0.3% of serum T3 is in a free form which can be taken up by cells by an energy dependent process. Although the cell nucleus is the primary site of T3 action, it is still unclear how it is actually transported into the nucleus. Different cytosolic T3 binding proteins (TBP) have been identified which seem to direct translocation of T3 either to mitochondria or to the nucleus in vitro (Ichikawa and Hashizume, 1995). The nuclear T3 receptor (TR) is an acidic non-histone protein. It belongs to the superfamily of hormone responsive nuclear transcription factors, which are similar in structure and mechanism of action, including the receptors for steroid hormones, vitamin-D and RA (Lazar, 1993). Two TR genes exist on two different chromosomes giving rise to TRa (46 kDa) and TRb (55 kDa). At least two alternative mRNA splice products exist for each gene (TRa1, TRa2, TRb1, TRb2). The C-terminal portion is important for ligand binding and for interactions between receptors. The DNA-binding domain of TR extends over 69 amino acids (52–120 in TRa, 106–174 in TRb) and includes two zinc finger motifs. TRa2 does not bind T3. This variant is expressed in most tissues and may inhibit binding of activated TRs to DNA. Transcriptionally active forms include monomers, homodimers and heterodimers with other nuclear
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protein partners, such as the retinoid-X receptor (whose ligand is RA). The T3–TR complexes interact with specific sequences in DNA regulatory regions and modify gene expression. T3 causes both increases and decreases in gene expression and may also influence the stability of mRNA (Brent, 1994). The expression patterns of TR mRNA varies in a developmental and tissue-specific way and are regulated by T3. Cardiac muscle contains TRa1, TRa2 and TRb1. T3 upregulates TRb1 and downregulates TRa1 and TRa2 mRNA. T3 regulates a number of genes specific for cardiac muscle proteins which are directly involved in contractile function and intracellular Ca-handling. The positive chronotropic and inotropic effects of T3 on rat hearts in vivo have been shown to be mediated by catecholamines (Zimmer et al., 1995). This may be explained by the finding that T3 specifically stimulates expression of the b1-adrenergic receptor gene in cultured NRC (Bahouth, 1991). Thus, T3 may be considered to be permissive for the increased cardiac catecholamine responsiveness. On the other hand, the induction of cardiac hypertrophy by T3 (i.e. increased RNA/ DNA ratio and increased ratio of left ventricular weight over body weight) is not prevented by aand b-adrenoceptor blockade and, therefore, seems to represent a direct effect of T3 (Zimmer et al., 1995). This would conform with the notion that T3 regulates several proteins of the contractile apparatus as well as additional markers of hypertrophy such as ANF (see Table 2). In fact, the promoter regions of the genes of a-MHC (Tsika et al., 1990), a-sk actin (Collie and Muscat, 1992) and SERCA2 (Rohrer et al., 1991) have all been shown to contain T3 responsive elements. In contrast to most of the other hypertrophic stimuli, T3 upregulates a-MHC and downregulates b-MHC (Lompre et al., 1984). In addition, T3 specifically upregulates the mRNA of three subunits (a2, a3 and b) of the sarcolemmal Na-K-ATPase in cultured NRC (Orlowski and Lingrel, 1990). It has recently been shown, that T3 represses PKCa and PKCe expression in the neonatal and PKCe in the adult rat heart (Rybin and Steinberg, 1996). Thus, the developmental change in PKC isoform expression may be due to the surge of thyroid hormone levels shortly after birth (Walker et al., 1980).
Individual Hypertrophic Stimuli, Gene Expression and Cross-talk The biochemical profiles of some typical genetic markers of hypertrophy in response to pressure
overload in adult rat hearts in vivo and in response to several individual hypertrophic stimuli on cultured cardiomyocytes are summarized in Table 2. The data is presented as up- or downregulation of mRNA or of the corresponding proteins. Some data for T3 stems from experiments with rats in vivo, while all other data comes from cultured NRC or ARC. Pressure overload (PO) in vivo invariably leads to an increase in heart mass. A significant increase in cell size has been reported for ARC treated with bFGF (Harder et al., 1996) and for NRC treated with either IGF-I (Ito et al., 1993), IL-1b (Palmer et al., 1995), ET-1 (Ito et al., 1991) or a1-adrenergic stimulation (Simpson, 1985). Cell size of ARC treated with IGF-I is not increased in comparison to control cells (Harder et al., 1996). This may represent a difference in response capacity between NRC and ARC. The response profiles for the genetic markers listed in Table 2 are identical for treatment with bFGF, TGFb and also for pressure overload in vivo. All results for TGFb come from NRC in culture (Brand and Schneider, 1995), while results for bFGF come in part from NRC (Parker et al., 1990b) and in part from ARC (Gosteli-Peter et al., 1996, Harder et al., 1996). Treatment with bFGF upregulates mRNA and protein levels of ANF and a-sm actin in both NRC and ARC. A reciprocal regulation of a-sk actin mRNA was reported for aFGF (downregulation) and bFGF (upregulation) in normal NRC as well as in NRC transfected with a promoterdriven reporter gene (Parker et al., 1990a, 1990b). In these two studies, aFGF was employed at the same concentration as bFGF (1.4 n), which is optimal for the latter. However, it is known that aFGF is almost 100 times less potent than bFGF (Gospodarowicz et al., 1987). The data for only four of the listed genetic markers is available for IGF-I. In contrast to the just mentioned stimuli, IGF-I downregulates a-sm actin and ANF in ARC (Donath et al., 1994; Gosteli-Peter et al., 1996; Harder et al., 1996). For a-sm actin, downregulation of both mRNA and protein has been shown. The upregulation of b-MHC (Florini and Ewton, 1992) and a-sk actin in NRC is accompanied by an upregulation of additional proteins such as troponin-I and ventricular MLC-2 (Ito et al., 1993). This upregulation of several sarcomeric proteins seems to correspond with the observed increase in cell size in NRC. For T3 treatment, data for all proteins in Table 2 except a-sm actin have been obtained from rat hearts in vivo: MHC (Lompre et al., 1984), a-sk actin (Winegrad et al., 1990), ANF (Ladenson et al., 1988), and SERCA2 (Rohrer and Dillmann,
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1988). Upregulation of SERCA2 was also shown in cultured NRC (Rohrer et al., 1991). The changes concerning the MHC isoforms in vivo are also observed in NRC (Kessler-Icekson, 1988) and in ARC (Nag and Cheng, 1986; Dubus et al., 1993; GosteliPeter et al., 1996). Upregulation of both mRNA and protein for a-sm actin has been shown in ARC (Gosteli-Peter et al., 1996). The data with IL-1b, ET-1 and PE all comes from NRC (for refs. see Table 2). For b-MHC, either upregulation (Thaik et al., 1995) or no change (Patten et al., 1996) was observed under treatment with IL1b. For a-MHC either downregulation (Patten et al., 1996) or no change (Waspe et al., 1990) was seen with PE. Taken together, each hypertrophic stimulus elicits its own characteristic profile on the molecular level of gene regulation. bFGF and TGFb present an exception, in that they induce the set of selected molecular markers in vitro in the same way as PO does in vivo. Several experimental findings imply that crosstalk between different signaling pathways activated by individual stimuli may be effective. We have shown, recently, that T3 is permissive for the action of bFGF and IGF-I in that these two growth factors require around 1 n T3 in the medium in order to differentially regulate the a-sm actin gene in ARC. With saturating T3 concentrations, bFGF potentiates the effects of T3 alone (Gosteli-Peter et al., 1996; Schaub et al., 1997). Another example concerns TGFb, which on its own does not induce any overall protein synthesis or cell growth in either NRC or ARC (Parker et al., ¨ 1990a; Schluter et al., 1995). In ARC isoproterenol (ISO) alone also fails to induce protein synthesis and only the combination of both TGFb and ISO ¨ leads to cell hypertrophy (Schluter et al., 1995). TGFb is thus considered to represent the causal factor that induces hypertrophic responsiveness to b-adrenoceptor stimulation in ARC. In NRC, ISO has been shown also to stimulate cardiac nonmyocytes to produce TGFb3 which, in turn, provokes hypertrophy in the myocytes (Long et al., 1993). Therefore, this is an example of cross-talk between two signals that involve at least two different types of cells. The a1-adrenergic agonist PE did not operate in this manner. It is likely that in the intact heart tissue even more complex interactions between different cell types take place (Long, 1996). Using the in vitro model of cultured NRC, RA has recently been reported to suppress two hypertrophic markers induced by PE (Zhou et al., 1995). RA inhibits the expression of an ANF-luciferase gene construct under the stimulation of either PE or ET1 and prevents PE induced increase in cell size.
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These results suggest the possibility that a balance may exist in vivo by which cardiac hypertrophy can be counteracted by RA. Another example of antagonistic interaction concerns the IL-1b mediated inhibition of PE induced expression of mRNA for a-sk actin and b-MHC (Patten et al., 1996). In NRC, it has also recently been observed that co-treatment with IL-1b reversed the T3 effects on both MHC isogenes (Patten and Long, 1996). Whereas a-MHC mRNA expression was reduced in the presence of IL-1b and T3, b-MHC mRNA was re-expressed with IL-1b co-treatment. This reversal of T3 effects is suggested to occur at the transcriptional level (C.S. Long, 1997, personal communication). Taken together, the examples presented here illustrate the fact that different hypertrophic stimuli may act in concert to induce a variety of morphological and biochemical phenotypes.
Conclusions Using cell culture models, a whole range of stimuli have been identified as mediators of cardiomyocyte hypertrophy. This in vitro approach has also enabled the molecular dissection of some of the pathways involved in the intracellular transduction of these signals. We have pointed out some recently discovered points of cross-talk between different signaling pathways and the subsequent activation of a characteristic set of genes. So, lateral cross-talk between different signal pathways may occur upon stimulation by more than one factor at a time. These stimuli may then act alone, or in concert in a synergistic, antagonistic or permissive manner. Thus, there is no such thing as a general type of “hypertrophic reaction” or a reversion to the socalled “fetal gene expression program” during growth stimulation. The induction of such a variety of morphological and biochemical phenotypes raises, of course, the question about its physiological significance. The hypertrophic growth of the heart may similarly be the result of a complex combinatorial action of various stimuli, which may also lead to different morphological and biochemical phenotypes with distinct physiological properties.
Acknowledgement The work reported here has been supported by a grant from the Swiss National Science Foundation (31–40694.94) and from the Swiss Heart Foundation (1995) to M.C. Schaub.
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Growth Factors and Cardiac Hypertrophy: Review Article
Signaling Pathways in Cardiac Myocyte Hypertrophy Martin A. Hefti, Beatrice A. Harder, Hans M. Eppenberger1 and Marcus C. Schaub Institute of Pharmacology, University of Zurich, CH-8057 Zurich; and 1Institute of Cell Biology, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland M. A. H, B. A. H, H. M. E M. C. S. Signaling Pathways in Cardiac Myocyte Hypertrophy. Journal of Molecular and Cellular Cardiology (1997) 29, 2873–2892. When a heart responds to increased workload it does so by hypertrophy. This is characterized by an increase in cell size in the absence of cell division, and is accompanied by distinct qualitative and quantitative changes in gene expression. The use of cardiomyocytes in cell culture has identified, besides mechanical loading, a range of substances, such as cytokines, growth factors, catecholamines, vasoactive peptides and hormones, involved in mediating cardiac myocyte hypertrophy, and has enabled the molecular dissection of the pathways involved in signal transduction. Many different pathways are activated in response to different hypertrophic stimuli, and a growing number of crosslinks are being characterized between these pathways. Recent evidence suggests a central role for Ras in transmitting signals from G-protein coupled receptors, from growth factor receptors and from cytokine receptors not only down the Raf-MEK-ERK pathway to the nucleus, but also to various other cytosolic effectors. The evaluation of distinct morphological phenotypes, together with biochemical data on gene regulation, suggests that interactions between different signaling pathways take place. Each stimulus provokes a typical cellular phenotype and different stimuli may act alone or in concert in a synergistic, antagonistic or permissive manner. Consequently, hypertrophy of cultured cardiomyocytes cannot simply be characterized as the reversal to the fetal gene expression program. Thus, hypertrophic growth of the heart may similarly be the result of a complex combinatorial action of various stimuli, which may also lead to different morphological and biochemical phenotypes with distinct physiological properties. 1997 Academic Press Limited K W: Cell culture; Cardiomyocytes; Cytoskeleton; Hypertrophy; Growth factors; Signal transduction.
Aim of this Review The use of cardiac cells in culture is a recent development which allows to study hypertrophy in an in vitro model at the cellular level. The purpose of this review is to draw attention to the variety in signaling pathways and cellular responses triggered by various stimuli which are able to induce cardiac hypertrophy. In addition, we point out some of the recently recognized evidence for cross-talk between these pathways and between different signals and their cellular responses. The individual hypertrophic stimuli identified by using cultured cardiomyocyte cell models, are likely to be balanced in vivo, even during compensatory hypertrophy. The disturbance of any
single factor, i.e. increase or decrease, may however tip the balance for the worse. Precise knowledge of the cellular responses to individual hypertrophic factors is a prerequisite, since established as well as new therapeutic strategies in cardiology involve either enhancement or counteraction of their effects. These strategies may include (i) systemic application of growth factors, cytokines or thyroid hormones, (ii) blocking the receptors of these hypertrophic factors, or (iii) interference with their production by the antisense oligodeoxynucleotide approach. We have described the relationship between the phenotype of hypertrophic cardiomyocytes in culture and that occurring in different forms of in vivo cardiac hypertrophy in more detail elsewhere (Schaub et al., 1997).
Please address all correspondence to: Dr Marcus C. Schaub, Institute of Pharmacology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
0022–2828/97/112873+20 $25.00/0
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Table 1 Abbreviations, names and explanations Cells ARC, adult rat cardiomyocytes NRC, neonatal rat cardiomyocytes Proteins involved in hypertrophy a-cd actin, alpha cardiac actin a-sk actin, alpha skeletal muscle actin a-sm actin, alpha smooth muscle actin ACE, angiotensin converting enzyme ANF, atrial natriuretic factor MHC, myosin heavy chain MLC, myosin light chain SERCA2, Ca-pump of sarcoplasmic reticulum Growth factors and related proteins EGF, epidermal growth factor aFGF, acidic fibroblast growth factor bFGF, basic fibroblast growth factor FGFR, FGF receptor IGF, insulin-like growth factor IGFBP, IGF binding protein IGFR, IGF receptor PDGF, platelet-derived growth factor TGF, transforming growth factor Hormones, vasoactive peptides and catecholamines AT-II, angiotensin-II ET-1, endothelin-1 ISO, isoproterenol (isoprenaline) PE, phenylephrine T3, 3,5,3’-triiodo--thyronine TBP, T3 binding protein TR, T3 receptor Components mentioned in G-protein signaling AC, adenylate cyclase cAMP, cyclic adenosine monophosphate DG, 1,2-diacylglycerol G-protein, heterotrimeric GTP-binding protein GPR, G-protein-coupled receptor IP3, inositol-1,4,5-trisphosphate PA, phosphatidic acid PI, phosphatidylinositol PIP2, phosphatidylinositol-4,5-bisphosphate PIP3, phosphatidylinositol-3,4,5-trisphosphate PKA, protein kinase-A PKC, protein kinase-C PLC, phospholipase-C RA, retinoic acid Cytokines CT-1, cardiotrophin-1 IL-1b, Interleukin-1b
In view of space limitation, reviews which contain references to primary information are often cited. Table 1 presents a list of names and abbreviations used in this review.
Cardiac Myocytes in Culture Hypertrophy is an adaptational process of heart muscle cells to hemodynamic overload from various causes. Adult cardiomyocytes are terminally differentiated cells which have lost the ability to divide.
Proteins mentioned in cytokine signaling CR, cytokine receptor JAK, Janus kinase STAT, signal transducer and activator of transcription Proteins mentioned in growth factor signaling Cdc42, homologous to yeast cell division cycle gene 42 ERK, extracellular signal regulated kinase; ERK1=p44; ERK2=p42 GAP, GTPase activating protein GDS, guanine nucleotide dissociation factor, a GEF GEF, guanine nucleotide exchange factor Grb2, growth factor receptor bound protein-2 IRS, insulin receptor substrate JNK, c-Jun N-terminal kinase MAP, microtubule associated protein MAPK, mitogen activated protein kinase MAPKAPK, MAPK activated protein kinase MAPKK, MAPK kinase MAPKKK, MAPKK kinase MEK, mitogen activated ERK activating kinase MEKK, MEK kinase Nck, adaptor protein containing SH2/SH3-domains NRTK, non-receptor tyrosine kinase PI3K, phosphoinositide 3′-kinase PLD, phospholipase-D Rac, a member of the Ras superfamily Raf, a MAPKKK Ral, Ras-related protein Ras, a monomeric GTPase (p21) RGL, RAL-GDS-like protein Rho, Ras homologous RK, reactivating kinase (p38) RKK, RK kinase RSK, ribosomal S6 kinase RSTK, receptor serine/threonine kinase RTK, receptor tyrosine kinase SAPK, stress-activated protein kinase (JNK) SEK, SAPK kinase SH domain, Src homology domain Shc, SH2-domain containing adaptor protein Shp2 (Syp), SH domain containing protein tyrosine phosphatase Smad, vertebrate homologues of Drosophila Mad protein (Mothers against dpp) Sos, mammalian homologues of Son-of-sevenless (a Drosophila gene product), a GEF Src, derived from the Rous sarcoma virus oncoprotein
The increase in heart muscle is thus achieved by an increase in myocyte size rather than number (Anversa et al., 1986). However, hypertrophy does not simply mean more of the same, but involves specific qualitative alterations in gene expression as well as in cell phenotype (Swinghedauw, 1990; van Bilsen and Chien, 1993). Experiments in vivo are unable to distinguish between the relative contributions of the different factors leading to specific biochemical and morphological changes observed in cardiac hypertrophy. Long-term responses cannot be studied in
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isolated heart preparations, because these are not viable for more than a few hours. Cell to cell interactions between identical and different cell types modulate the hypertrophic reaction. The myocytes comprise 70–80% of the heart mass, but represent only 20–30% of total cardiac cells (Long, 1996), whereas fibroblasts, neurons and endothelial cells account for the majority in numbers. Subsequently, it is difficult to attribute certain properties of this tissue to any one cell type. Recent progress in cellular cardiology stems mainly from the development of experimental procedures for primary cell cultures (Jacobson and Piper, 1986). Adult ventricular cardiomyocytes in culture undergo drastic morphological remodeling from the elongated in vivo structure to a spheroidal flat shape with numerous extensions (Jacobson, 1977; Claycomb and Palazzo, 1980; Eppenberger et al., 1988; Nag et al., 1996). Cells hypertrophy to two–three times the original volume (Messerli et al., 1993), resume spontaneous rhythmic beating, seek contact with one another and, eventually, form a coherent 2-dimensional layer (Eppenberger et al., 1994). The initial breakdown and reassembly of myofibrils and regaining of contractile function, together with the accompanying biochemical changes, essentially, recapitulates the events associated with hypertrophy in vivo (van Bilsen and Chien, 1993; Schaub et al., 1997). This pattern of remodeling has also been described for chick embryonal (Lin et al., 1989; Handel et al., 1991; Rhee et al., 1994) as well as for neonatal rat cardiomyocytes (Atherton et al., 1986). Cardiomyocytes in primary culture thus present a suitable in vitro model for detailed analysis of the hypertrophic reaction at the cellular level with regard to individual stimuli and to their signaling pathways. No permanent cardiac cell lines exhibiting these characteristics in culture have been obtained yet. Myocyte cell lines which express some cardiac specific genes, however, have been derived from embryonal cardiac cell clones by retroviral transformation, by culture condition manipulation or from tumors in adult atrial tissue (Doetschmann et al., 1993; Borisov and Claycomb, 1995; Eisenberg and Bader, 1996; Engelmann et al., 1996; Habara-Ohkubo, 1996). These cells retain their proliferative capacity and exhibit an embryonal phenotype with only limited differentiation. Cardiomyocytes from immature animals (embryonic, fetal, neonatal) are easier to culture and have been most widely used. Adult ventricular cardiomyocytes, however, differ from immature cells. They are larger, have a more developed transverse tubular system, grow more slowly in culture
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and have a restricted potential for differentiation (Claycomb, 1983). Adult cardiomyocytes in culture may therefore present a more accurate model for myocardial hypertrophy in vivo. The main argument for using adult cardiomyocytes lies in the fact that they are derived from a terminally differentiated heart tissue and that it is this type of cell which also in vivo is confronted with hypertrophic stimuli, such as overload or other pathological events. On the other hand, the adult ventricular myocyte, in contrast to immature cells, has been refractory to conventional procedures for gene transfer, such as electroporation or calcium phosphate precipitation. Kirshenbaum et al., (1993) reported, however, a transfection success of 90% for adult cardiomyocytes with an adenoviral vector system.
Molecular Characterization of Cellular Hypertrophy Despite the differences between immature and adult rat cardiomyocytes, in principle, both cell types react with a characteristic succession of changes in gene expression (Glennon et al., 1995). Some of these changes are taken as markers of hypertrophy. These markers include the so-called early response genes (e.g. egr-1, hsp70, c-fos, c-jun, c-myc) which are activated within 30 min of exposure to a hypertrophic stimulus. Expression of these genes in response to various growth stimuli has been associated in other cell types with cell cycle regulation and induction of proliferation (Riabowol et al., 1988). In the heart, this transient response seems to represent a general pattern of growth induction in terminally differentiated cells that have lost the ability to undergo DNA replication (Izumo et al., 1988; Komuro et al., 1988; van Bilsen and Chien, 1993). Subsequently, re-expression (or recapitulation) of “fetal genes” such as b-MHC, ask actin and ANF may occur after 6–12 h. An upregulation of constitutively expressed contractile proteins, such as ventricular MLC-2 (Lee et al., 1988) and a-cd actin (Long et al., 1989), may follow after 12–24 h in culture. In cultured ventricular ARC, significant accumulation of the reexpressed proteins, e.g. b-MHC (Nag and Cheng, 1986; Eppenberger et al., 1988), a-sm actin (Eppenberger-Eberhardt et al., 1990) and ANF (Eppenberger-Eberhardt et al., 1993, 1997) can only be demonstrated after several days in culture. Using cultured cardiomyocytes, a number of neurohumoral factors and cell mediators which
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induce hypertrophic reactions have been identified (summarized in Fig. 1). These include a-adrenergic agonists, ET-1, AT-II and various polypeptide growth factors. In addition, cytokines (IL-1b, CT-1) and T3 have recently been recognized as hypertrophic stimuli in cardiomyocyte cell cultures (Palmer et al., 1995; Gosteli-Peter et al., 1996; Wollert et al., 1996). The hypertrophic effects of pressure/volume overload in vivo may depend on mechanical stretch. Stretching of cardiac myocytes cultured on deformable surfaces has indeed been shown to induce the cellular features of hypertrophy, as well as to activate multiple signal transduction pathways (Watson, 1996).
Intracellular Signaling Pathways One general aim, not only in cellular cardiology, is to attribute distinct phenotypic features to defined stimuli and signaling pathways. In contrast to the fast electrochemical signal propagation in nervous and skeletal muscle tissue, the regulation of longer lasting intracellular reactions comprises biochemical and structural alterations, and many operate via reversible covalent protein modifications, such as phosphorylation. Phosphorylation is brought about by two types of protein kinases, i.e. transmembrane receptor kinases (RTK and RSTK in Fig. 1) and a large number of various intracellular kinases. The latter include the non-receptor tyrosine kinases (NRTKs) of the Src and Jak families, which may serve as intermediates in transducing membrane receptor signals to the serine/threonine kinases (PKAs, PKCs, MAP kinases) operating further downstream in the pathways (Figs 1 and 2). Protein phosphorylation can be reversed by two families of protein phosphatases which specifically dephosphorylate phosphoserine/threonine or phosphotyrosine residues. A discussion of the phosphatases is beyond the scope of this review (see Hunter, 1995; Streuli, 1996). A cascade of successive transduction steps allows signal enhancement and diversification at branching points and thus permits combinatorial interactions between multiple pathways (Fig. 1). The pathways discussed in this section have been elucidated using different cell systems, and many have been shown to be operative in cardiac cells too (Sugden and Bogoyevitch, 1995). Cell membrane receptors and intracellular signaling proteins are highly conserved between mammalian species and the triggering events for cellular hypertrophy in
humans are likely to resemble closely those in the various animal models used (Glennon et al., 1995).
Growth Factors and their Signaling Pathways The classic peptide growth factors can be divided into five families: EGFs, FGFs, IGFs, PDGFs and TGFs. Each family comprises multiple members, where individual growth factor properties, besides induction of cell growth and proliferation, are manifold and range from being highly specific to being more general. FGF, TGFb, IGF and PDGF have been shown to cause cardiomyocyte hypertrophy in in vitro systems (Long, 1996). In the heart, PDGF is produced by non-myocytes. FGF and TGFb can be produced by cardiac myocytes and non-myocytes (Speir et al., 1992; Brand and Schneider, 1995) and therefore, these two factors may act in a paracrine or autocrine manner in the heart. IGF is produced predominantly in the liver and circulates in substantial amounts in the blood, where it may be bound to a number of IGF binding proteins (IGFBPs, six members of a homologous protein family ranging in size from 24–45 kDa) (Blakesley et al., 1996). The work on cardiac hypertrophy has concentrated mainly on the effects of FGF, TGFb and IGF and these studies have been done with cultured NRC and ARC. FGF The FGF gene family comprises nine members which share 40–50% sequence homology (Basilico and Moscatelli, 1992). The two prototypes are aFGF (FGF-1, 140 amino acids, IEP of 5.6) and bFGF (FGF-2, 154 amino acids, IEP of 9.6). They are highly conserved among various mammalians and chicken (90% sequence homology). Neither contains a classic signal sequence to direct its secretion through membranes (Friesel and Maciag, 1995). Most of the other FGFs contain such a functional signal sequence and their genes were originally identified as oncogenes. It is assumed that for secretion the prototype FGFs form disulfide bonded homodimers and exocytosis may be involved (Bastagli et al., 1995). In addition, an intracrine mode of action has been proposed where bFGF may be active after nuclear translocation without necessarily exiting the cell (Logan, 1990). aFGF and bFGF also occur as isoforms with higher molecular weights (22 instead of 17 kDa), resulting from alternative initiation of translation. A specific sequence of Lys-Lys-Pro-Lys near the N-terminus
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Hypertrophy in Cultured Cardiomyocytes Mechanical stress
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[Cytokines] IL-1β CT-1
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Figure 1 Cardiac hypertrophic stimuli initiate multiple signal transduction pathways. A whole range of substances and subsequently activated intracellular pathways are involved in mediating the hypertrophic phenotype in cultured cardiomyocytes. Evidence discussed in the text suggests a central role for the Ras-Raf-MEK-ERK pathway in transmitting these stimuli to the nucleus. Arrows indicate the major routes of signal transduction and illustrate some of the interactions between different pathways. Open arrowheads denote pathways to cytosolic substrates. Binding of TGFb to the primary receptor (RSTK with open box) enables this type-II receptor to bind and phosphorylate the type-I receptor (RSTK with black box), which then propagates the signal. A direct pathway linking mechanical stress to gene expression is thought to be operative (dashed arrow). For abbreviations see Table 1.
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Figure 2 Growth factor signaling through receptor tyrosine kinases. Various proteins containing SH2 domains are able to interact with activated growth factor receptors such as FGFR or with IRS to initiate multiple downstream responses. Each of these proteins binds to a different phosphorylated site on the activated receptor, recognizing surrounding features of the polypeptide chain in addition to the phosphotyrosine residue. One downstream event is the activation of Ras by Sos. The relay system downstream from Ras involves three distinct cascades of protein phosphorylation, known as the MAP kinases cascades. As discussed in the text, recent work has shown that many additional effectors can interact with Ras to initiate distinct responses, some of which are depicted here. It is likely that for a given stimulus only a subset of all possible interactions shown here actually take place. The specific effect of a given stimulus may be the result of a complex combinatorial activation of individual pathways depending on spatial and temporal abundance of relevant proteins in the cell in question. For abbreviations see Table 1.
targets these larger peptides to the cell nucleus. Their function within the nucleus is not known. The high molecular weight forms predominate in neonatal rat hearts and the low molecular weight forms in the adult heart (Liu et al., 1993). Their balance seems to depend on thyroid hormone levels, the higher the T3 level the lower the high molecular weight FGF isoforms. Overexpression of both low and high molecular weight forms displays similar effects in cultured embryonic chicken cardiomyocytes (Pasumarthi et al., 1994). All FGFs associate with the glycosaminoglycan heparin and with heparan sulfate proteoglycan of the outer cell surface. The association of FGFs with the extracellular matrix
provides potential storage sites from which they may be released upon injury and activation of matrix degrading enzymes. aFGF and bFGF have identical effects but aFGF is 30–100 times less potent. aFGF and bFGF are both found in the heart in myocytes and in non-myocytes. Their content is higher in atria than in ventricles suggesting a possible correlation with a higher regenerative capacity of the atria (Kardami and Fandrich, 1989). IGF-I IGF-I and IGF-II share structural and biological function homology with pro-insulin and are cleaved to release active monomeric proteins with 70 and
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67 amino acids respectively (Blakesley et al., 1996). The smaller IGF-II is more prominent during fetal development and IGF-I persists during adult life. Most of the functions of both are mediated via IGFI receptors. Complex formation of IGFs with the IGFBPs is required for transport through membranes and may control the interaction of the IGFs with receptors. The IGF-I receptor is structurally similar to the insulin receptor (White and Kahn, 1994). Both are synthesized as single chains that become glycosylated and cleaved, producing a- and b-subunits. The mature receptor is a disulfide-bonded heterotetramer with two 125 kDa a-chains oriented extracellularly, and two 95 kDa b-chains extending through the membrane and containing tyrosine kinase activity. Although pituitary secretion of growth hormone stimulates the production of IGF-I primarily in the liver (endocrine mode), most tissues are able to synthesize IGF-I locally (autocrine/paracrine mode).
TGFb TGFb belongs to a superfamily of over 20 regulatory proteins consisting of factors distantly related (30– 40% sequence homology) including the activins and bone morphogenetic proteins (BMPs). TGFb now designated TGFb1, shares 70–80% sequence homology with its other family members TGFb2, TGFb3 (1, 2 and 3 are mammalian forms), TGFb4 (avian) and TGFb5 (Xenopus). TGFb1 is synthesized as a 390 amino acid precursor protein, which is cleaved to yield an N-terminal remnant and a Cterminal monomeric TGFb segment. The mature TGFb consists of a disulfide bonded dimer (25 kDa) which is secreted as a latent complex non-covalently associated with its processed “latency peptide” (75 kDa) and an additional binding protein (135 kDa) (Sporn and Roberts, 1992; Kingsley, 1994; Long, 1996). Cultured NRC and ARC release TGFb from day 1 on into the culture medium ¨ (Roberts et al., 1992; Schluter et al., 1995). The three mammalian forms of TGFb bind to three types of receptors found on most mammalian cell types. The type-III receptors (200–400 kDa proteoglycan) have no known signaling function (Brand and Schneider, 1995). Type-I and type-II receptors are distantly related Ser/Thr kinases (RSTKs). Ligand binding by the primary receptor (type-II) enables it to bind the type-I receptor. In the resulting heteromeric complex, the type-II receptor phosphorylates the type-I receptor, which then propagates the signal (Fig. 1). Smad proteins have recently been identified as the first family of putative
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´ TGF-b signal transducers (Massague et al., 1997). Upon phosphorylation by activated receptors, Smads form complexes, move into the nucleus, associate with DNA binding proteins and activate gene transcription.
Receptor tyrosine kinase signaling The receptors of most growth factors, with the exception of TGF-b and IGF-II, are transmembrane tyrosine kinases (RTKs) (Ullrich and Schlessinger, 1990). Binding of growth factors such as EGF, PDGF and FGF leads to receptor dimerization which in turn enables the two cytoplasmic domains to crossphosphorylate each other on multiple tyrosine residues (Heldin, 1995). Dimerization is a general mechanism for activating enzyme-linked receptors with a single transmembrane domain. In each case the autophosphorylated tyrosines serve as highaffinity binding sites for a number of intracellular signaling proteins (Fig. 2). Although these proteins have varied structures and functions, they usually share two highly conserved non-catalytic modular binding domains, termed SH2 and SH3 (for Src homology), since they were first found in a tyrosine kinase encoded by the Rous sarcoma virus oncogene src. Each of these proteins binds to a different phosphorylated site on the activated receptor, recognizing surrounding features of the polypeptide chain in addition to the phosphotyrosine (Cohen et al., 1995). The interaction between SH2 domains and the phosphorylated receptors allows the recruitment of cytosolic substrates to the plasma membrane. The IGF-I receptor acts in a slightly different way. This receptor is a tetramer where ligand binding induces an allosteric interaction of the two halves and phosphorylates its catalytic domains. This activates them to phosphorylate separate proteins called IRS on multiple tyrosines (Waters and Pessin, 1996). The phosphotyrosines on IRS then serve as high-affinity binding sites for the docking and activation of intracellular signaling proteins containing SH2 domains, many of which can also bind to activated receptor tyrosine kinases directly (Fig. 2). Once bound, these proteins can be further regulated by phosphorylation or conformational changes. Different RTKs probably bind different combinations of these signaling proteins and therefore initiate different responses. PLC-c binds to receptors of PDGF, EGF and FGF via its SH2 domains. Subsequent phosphorylation stimulates the catalytic activity of PLC-c to hydrolize PIP2, which produces the second messengers IP3
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and DG (Lee and Rhee, 1995). In addition, activated PLC-c1 may associate with actin of the cytoskeleton by its SH3 domain. PIP2 itself has also been shown to interact with a number of actin-binding proteins such as a-actinin, profilin or vinculin. It seems likely, therefore, that PLC-c isoenzymes represent one link from growth factor signaling to the organization of the cytoskeleton. PI3K binds to activated RTKs via SH2 domains in its regulatory p85 subunit. Its catalytic p110 subunit is able to phosphorylate several phosphoinositides at their 3position and, thus, PI3K has been implicated in many intracellular pathways (Carpenter and Cantley, 1996). In addition to PI3K and PLC-c, many other SH2-domain containing proteins bind to RTKs, such as Src (a ubiquitous NRTK), Nck (an adaptor protein containing one SH2 and three SH3 domains), Shp2 (a phosphotyrosine phosphatase also known as Syp) as well as the two adaptor proteins Shc and Grb2 (Pawson, 1995) (Fig. 2).
Ras in Signal Transduction Ras proteins were first discovered as the hyperactive product of mutant ras genes which promote cancer by disrupting the normal controls on cell proliferation and differentiation. Like the other monomeric GTPases and the trimeric G-proteins, Ras proteins function as switches, cycling between two conformational states, active when GTP is bound and inactive when GDP is bound. In cells containing a ras oncogene, the Ras oncoprotein is locked in the GTP-bound state as a result of its defective GTPase and is likely to generate a constitutive signal even in the absence of growth factor stimulation. Two classes of signaling proteins regulate the activity of Ras and its relatives by influencing the transition between the active and inactive states (Boguski and McCormick, 1993). GTPase activating proteins (GAPs) increase the rate of hydrolysis of bound GTP by Ras, thereby inactivating it (Fig. 2). These negative regulators are counteracted by guanine nucleotide exchange factors (GEFs) which promote the exchange of bound GDP for GTP. In mammalian cells, Ras is activated for example by Sos, a GEF named after its Drosophila counterpart “Son-of-sevenless”. Sos is associated with Grb2 via the latter’s two SH3domains. Similarly, phosphorylated Shp2 can serve as a docking site for Grb2 as has been shown for the PDGF receptor (Sun and Tonks, 1994). Phosphorylated Shc has been implicated in the Ras signaling pathway by virtue of its association with the Grb2 adaptor molecule. Interestingly, Shc might
act as a go-between that relays messages from Gprotein coupled receptors to Ras (see below).
Ras effectors Multiple signaling pathways are activated by Ras (Fig. 2). Molecules that interact with GTP-bound Ras and transmit the signal, are referred to as Ras effectors (Marshall, 1996). Each of these effectors may be activated by Ras-GTP via a unique interaction on a particular site on Ras. PI3K seems to be one of these Ras effectors, binding in this case, however, is mediated by the p110 subunit. PKCf and RGL (a dissociation factor similar to Ral-GDS) have also been shown to interact with activated Ras. Nucleotide-exchange factors for Ral-GTPases, such as Ral-GDS, are the newest members of putative Ras effectors (Feig et al., 1996). Ral, in turn, may link growth factor signaling to lipid second messengers via PLD and to two members of the Rho family of small GTPases, namely cdc42 and Rac-1. PLD catalyses the hydrolysis of phosphatidylcholine to PA and choline. PA may be subsequently metabolized to yield DG, an activator of PKC. The two Rho family GTPases cdc42 and Rac-1 are involved in the regulation of the actin cytoskeleton (Symons, 1996) and they have also been shown to stimulate two distinct MAP kinases, the JNK/SAPK and the p38 MAP kinase (Feig et al., 1996). Microinjection experiments in Swiss 3T3 fibroblasts have revealed that cdc42 can activate Rac, which in turn activates Rho (Nobes and Hall, 1995) and recent mutational analysis has revealed that the ability of Rac and cdc42 to activate MAP kinase cascades and to induce cytoskeletal rearrangements is mediated by distinct, independent downstream signals (Lamarche et al., 1996).
MAP Kinases The relay system that transmits signals from Ras to the nucleus involves three distinct cascades of protein phosphorylation, each with three levels of chemically defined reactions: (i) MAPKKKs phosphorylate MAPKKs on serine or threonine residues; (ii) MAPKKs are dual-specificity protein kinases that phosphorylate the MAPKs on a threonine and a tyrosine residue in a Thr-X-Tyr motif; and (iii) once activated, the MAPKs phosphorylate serine or threonine residues in various nuclear and extranuclear substrates (Fig. 2) (Seger and Krebs, 1995; Denhardt, 1996). The three mammalian MAPK families are defined by being phosphorylated in a
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distinct motif. These three motifs are Thr-Glu-Tyr (T-E-Y) for ERKs, Thr-Phe-Tyr (T-P-Y) for the JNK/ SAPKs and Thr-Gly-Tyr (T-G-Y) for p38/RK. The first MAPKs to be identified were p44 (ERK1) and p42 (ERK2). ERK1 and ERK2 are proline-directed kinases, i.e. they generally phosphorylate Ser/Thr residues to the left of proline. Many important substrates for MAPKs are nuclear transcription factors that are thought to be phosphorylated after MAPK translocation to the nucleus (Treisman, 1996). Thus, there appears to be two reasonably discrete pathways under the influence of Ras: (i) the Raf-MEK-ERK pathway activated by growth factors, mitogens and tumor promoters and (ii) the MEKK-SEK-JNK/SAPK pathway that is mainly activated by various stressors and cytokines (Fig. 1). The pathways leading to activation of p38/RK, the third family of MAPKs, are not well understood yet (Denhardt, 1996). Extranuclear substrates for ERKs comprise the MAPs such as MAP-1, MAP-2, MAP-4 and tauprotein (Seger and Krebs, 1995). Their phosphorylation by ERKs is part of the regulation of microtubular rearrangements and cellular morphology. It has been shown that rabbit cardiac titin can be phosphorylated by the proline-directed ERK1 (Sebestyen et al., 1995). Additional substrates for ERKs are downstream serine/threonine kinases termed MAPK-activated protein kinases (MAPKAPK), such as p90 (RSK). ERKs may also phosphorylate upstream components in the signaling cascade that lead to their activation, thus demonstrating feedback regulation. Numerous studies have addressed the role of Ras, Ras effectors and the molecules of the MAPK cascades in cardiac signal transduction. It has been suggested, that in ventricular muscle, Ras activity may regulate “the basic transcriptional machinery” (Abdellatif et al., 1994). Introduction of a constitutively active Ras molecule into NRCs induced both genetic (expression of the c-Fos and ANF genes) and morphological effects (changes in the organization of the contractile apparatus) associated with hypertrophy (Thorburn et al., 1993). Furthermore, expression of a dominant inhibitory Ras mutant could prevent activation of ANF gene expression by PE. However, in a different study, coexpression of an inhibitory Ras mutant, together with a constitutively active Gq a-subunit, did not abolish the latters stimulation of ANF gene expression (La Morte et al., 1994). It has also been shown that Raf-1 and the downstream ERKs are necessary and sufficient for gene expression changes but not for cytoskeletal actin changes associated with PE-induced cardiac myocyte hypertrophy
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(Thorburn et al., 1994). However, activation of Ras is sufficient for both these changes and this may now be explained in view of the multiple pathways leading to extranuclear responses which can be activated by Ras. Adding to complexity, constitutively active MEK1 and ERK2 have been shown to stimulate ANF, b-MHC and a-sk- actin expression, whereas dominant negative mutants of these two molecules inhibited PE-induced ANF expression to a certain extent (Gillespie-Brown et al., 1995). Similarly, antisense oligodeoxynucleotides against the initiation of translation of the p42 and p44 MAPK isoform mRNAs inhibited development of the morphological features of hypertrophy (sarcomerogenesis, and increased cell size) in NRC exposed to PE. PE-induced activation of the ANF promoter and induction of ANF mRNA were also attenuated (Glennon et al., 1996). In a contrasting study, dominant-interfering mutants of p42 and p44 MAPK as well as the use of PD 098059, an inhibitor of MEK, failed to block PE induced ANF expression (Post et al., 1996). Apart from technical differences and the sometimes difficult interpretation of results obtained with mutant molecules and more or less specific inhibitors with respect to the in vivo situation, these findings may also reflect pathway redundancy, i.e. that multiple or different pathways may be potentially activated in response to a hypertrophic stimulus. Many interactions, e.g. concerning the precise interactions of various GTPases of the Ras superfamily are still unclear or have yet to be identified, but are likely to depend on the spatial and temporal abundance of the relevant regulatory proteins, such as the GAPs and GEFs, but also on the specific cell in question. Interestingly, RhoA has been suggested to function in a pathway, separate from, but complementary to Ras and is involved in Gq a-signaling in cardiomyocytes (Sah et al., 1996). Very recently, it has been suggested, that an effector-like function of Ras-GAP may predominate in the cardiac background, since abolishing GAP binding to Ras interfered with Rasdependent expression in transfected NRCs (Abdellatif and Schneider, 1997). Thus, effector-like functions of Ras-GAP, may be at least in part responsible for the global increase of RNA and protein synthesis in cardiac hypertrophy.
Signaling Through G-protein Coupled Receptors Catecholamines and vasoactive peptides (AT-II and ET-1) are all reported to induce anabolic responses
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with cardiac myocyte growth. The specific receptors for all of these mediators belong to the G-protein linked superfamily of homologous heptahelical transmembrane proteins. G-proteins, in turn, are a large family of heterotrimeric GTP-binding regulatory proteins such as Gq, Gi or Gs and are each composed of an a-, b- and c-subunit (Gudermann et al., 1996). G-protein function has historically been attributed to the GTP-bound a-subunit which is released from the bc-dimer upon receptor stimulation. However, recent work has suggested that the bc-subunits may play an equally prominent role in effector regulation. Numerous studies have shown that for instance ET-1 stimulates the PIP2 signaling pathway producing the two messengers DG and IP3 through a phospholipase-Cb catalysed hydrolysis of PIP2 (Fig. 1) (Sugden and Bogoyevitch, 1996). IP3 induces release of Ca2+ from intracellular Ca-stores. DG remains in the plain of the membrane for translocation and activation of members of the PKC family (Jaken, 1996; Spiegel et al., 1996). This family consists of cPKCs (classical, requiring DG and Ca2+), nPKCs (novel, requiring only DG) and aPKCs (atypical, activation mechanism unknown, probably not depending on DG). The tumor-promoting phorbol esters can activate the phospholipid-dependent cPKCs and nPKCs by substituting for DG. Active PKC has a number of nuclear and cytosolic substrates (Nishizuka, 1995). The a-isoform of PKC, for example has been shown to directly activate Raf-1 (Kolch et al., 1993), and thus provides a link to the signaling pathway that is activated by growth factor receptors. PKC-mediated activation of the MEKK-SEK-JNK/SAPK cascade has also been described as a consequence of GPR stimulation (Denhardt, 1996). In addition, signaling from GPR can be directly crosslinked to Ras (Fig. 1). ATII induced Ras activation in NRC involves a pathway linking a member (Fyn) of the Src family to Shc and Grb2-Sos (Sadoshima and Izumo, 1996). Recently, the bc-subunit of G-proteins have been suggested to be involved in a pathway from GPRs to Ras. This has been shown in the established cell lines like COS-7 (fibroblast-like simian kidney cells) and PC12 (phaeochromocytoma cells), where the bc-subunits of Gq, Gs and Gi are involved in this transmission (Crespo et al., 1995; Dikic et al., 1996; Lutrell et al., 1996). Taken together, these findings may offer an explanation for previous studies that reported Ras involvement for example, in the a1adrenergic receptor agonist PE-induced hypertrophic response of cardiac myocytes (Thorburn et al., 1993). The a-subunits of the G-proteins are known to be responsible for activation (Gq) of PLCb
as well as for stimulation (Gs) or inhibition (Gi) of adenylate cyclase, which is one of the main signal routes leading to PKA. PKA activity results directly in nuclear as well as cytosolic events. Some of these effects may be linked to changes in cytosolic levels of Ca2+ (Fig. 1). An interesting inhibitory effect has been demonstrated, in that PKA can interfere with the Ras-Raf-cascade by inhibiting the latter (Sugden and Bogoyevitch, 1995; Denhardt, 1996). Additional crosslinks are known to connect GPR signals to early events of the growth factor (RTK) and cytokine receptor (CR) induced pathways (Fig. 1). In the Rat-1 fibroblast cell line, the EGF receptor becomes rapidly tyrosine phosphorylated upon stimulation with ET-1 (Daub et al., 1996). In these cells, this ligand-independent mechanism of RTK activation is crucial for ET-1 induced activation of ERKs. In vascular smooth muscle cells, AT-II leads to activation of IGF-I receptor by tyrosine phosphorylation, probably via an intermediate NRTK (Delafontaine et al., 1996). It has also been shown that AT-II can modulate cytokine responses directly by activating the JAK-STAT cascade in cultured cardiac fibroblasts (Brecher, 1996).
Endothelin-1 The endothelium-derived vasoconstrictor ET-1 is a peptide of 21 amino acids. Two additional homologues (ET-2 and ET-3) were later identified. After cleavage from a large precursor peptide, ET-1 is released from vascular endothelium and other cells including cardiomyocytes (Suzuki et al., 1993). The ET peptides bind to three receptor subtypes (ET-A, ET-B and ET-C). The ET-A receptor probably mediates the vasoconstrictor effects and the ET-B receptor may induce vasodilation by causing release of NO and/or prostacyclin from endothelial cells. The ET-A receptor is the predominant species in rat hearts and is coupled to both the Gq and Gi species of G-proteins (Hilal-Dandan et al., 1994). In ventricular NRC, ET-1 induces a characteristic pattern of gene expression: (i) immediate early genes (c-fos, c-jun, egr-1); (ii) the early genes ANF, bMHC and a-sk actin, and later (iii) the genes for ventricular MLC-2 and a-cd actin (Sugden and Bogoyevitch, 1996). These transcriptional changes may involve the Gq-Ras-Raf-ERK pathway (Fig. 1). However, ET-1 also affects gene expression and causes hypertrophy via the Ras-MEKK1-SEK-JNK pathway (Bogoyevitch et al., 1996). Beside the effects on the transcriptional level, ET-1 also stimulates protein synthesis on the translational level. The mechanism of a positive inotropic response of
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the heart muscle to ET-1 is not yet clear. It has also been reported that ET-1 causes cellular injury in cardiac myocytes in vivo and, recently, it was shown that the survival rate of rats with heart failure is improved by long-term treatment with the ET-A receptor blocker BQ-123 (Sakai et al., 1996).
Angiotensin-II AT-II is the main effector molecule of the reninangiotensin system which is responsible for pressure and volume homeostasis (Bernstein and Berk, 1993). Renin which is synthesized by the kidney and secreted into the blood, hydrolyses the decapeptide angiotensin-I (AT-I) from the N-terminus of angiotensinogen. AT-I is converted to the octapeptide AT-II by the dipeptidyl carboxypeptidase ACE. In addition, a chymotrypsin-like protease (chymase) has been identified which is though to represent the main pathway for conversion of AT-I to AT-II in the human heart (Urata et al., 1990). ATII is a potent vasoconstrictor, increases aldosterone secretion, enhances central sympathetic outflow, catecholamine release from adrenal medulla and promotes release of vasopressin. Because of its hypertrophic effect on cardiomyocytes, it is also considered to be a growth factor. Most of these biological effects are mediated through the AT-1 receptor, although several other receptor subtypes have been identified (Timmermans et al., 1993). AT-II is able to induce hypertrophy of rat cardiomyocytes and hyperplasia in cardiac non-myocytes, both actions being mediated by the AT-I receptor (Sadoshima and Izumo, 1993). Electron microscopy reveals that immunoreactive AT-II is preferentially localized in what appears to be secretory granules in ventricular myocytes. AT-II may then be secreted from cardiomyocytes and act in an autocrine fashion by binding to the AT-I receptor which in turn associates with Gq and Gi-proteins (Sadoshima and Izumo, 1996). Important effects of AT-II have been demonstrated using cardiac fibroblasts and vascular smooth muscle cells where AT-II interacts with the IGF-I system (Delafontaine et al., 1996). For one, it stimulates cardiac IGF-I expression. However, due to a change in the balance between the various IGFBPs, the level of circulating IGF-I is lowered. This may account for the low metabolic activity which leads to weight loss in the AT-II infused animal. In cardiac fibroblasts, AT-II induced activation of the JAK-STAT-pathway and the MAP kinases, leads to changes in transcription and to cell proliferation.
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Catecholamines The transcriptional alterations typically associated with the hypertrophic reaction which is induced via the Gq-Ras-Raf-ERK or via the PKC signaling pathway in ventricular NRC is usually attributed to a1adrenergic stimulation, e.g. by epinephrine (Simpson et al., 1991). Catecholamine-activated signaling pathways in NRC and their phenotypic features resemble those induced by either AT-II or ET-1. This phenotype induced by a-adrenergic or ET-I stimulation can be inhibited by application of retinoic acid (RA) to NRC (Zhou et al., 1995). NRC express functional retinoic acid receptors which belong to the nuclear receptors of the steroid hormone receptor superfamily. Upon stimulation, these receptors may interfere with the transcription of those genes, which are stimulated by the GPR mediated signals. The a1GPR may couple to different G-proteins, among which are Gq and a 74 kDa Gh species (Terzic et al., 1993). Both b1- and b2-receptors usually couple to Gs when activated. It has recently been demonstrated in NRC that both a- and b-adrenergic agonists are able to activate MEK and ERK, albeit by different signaling pathways (Bogoyevitch et al., 1996). Accordingly, an increase in mRNA for a-sk actin is observed on either a- or b-adrenergic stimulation in NRC (Martin et al., 1996). However, b-adrenergic stimulation of NRC enhances accumulation of a-MHC (Rupp et al., 1991), while b-MHC mRNA and protein accumulation is activated by a-adrenergic stimulation (Waspe et al., 1990). The elevation of intracellular Ca2+ rather than cAMP appears important for the activation of ERK by b-adrenergic agonists, where a direct Gs-mediated coupling of the -type Ca-channel could be operative (Yatani and Brown, 1989; Bogoyevitch et al., 1996). Dual effects of b-adrenergic stimulation on ERK in COS-7 cells are due to a bcsubunit mediated activation of Ras and an inhibitory effect induced by the a-subunit via PKA on Raf further downstream (Crespo et al., 1995). The existence of these two opposing mechanisms (Fig. 1) may explain some of the differences observed between aand b-stimulation in heart cells.
Mechanical Stress Deformation of the cardiomyocyte as it occurs in vivo during contraction, or in vitro under artificial conditions, presents a mechanical stress for the cellular structures. This mechanical loading may present the most important physiological stimulus for cardiac hypertrophy. Cardiomyocytes kept in
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long-term culture spontaneously contract rhythmically, but can also be subjected to passive stretch. Mechanical loading induces a host of responses associated with cellular hypertrophy and with structural remodeling (Watson, 1996). Acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents (Hu and Sachs, 1996). These currents seem to arise from stretch-activated ion channels. Different ion-specific and non-specific mechanosensitive channel types have been described (Sackin, 1995). Stretch-activation is a membrane or membrane-cytoskeleton phenomenon and does not seem to involve soluble cytosolic messengers. However, such ion fluxes may result in lowering or increasing the cytosolic free Ca2+ levels which, in turn, may affect gene expression in the cell nucleus directly (dashed arrow in Fig. 1), or may modulate the signaling by other stimuli. Indeed, stretch was found to induce transient activation of PKC followed by Raf-1 and MEKK (both on the level of MAPKKK) and the successive components of the MAPK signaling cascade in a sequential time order over several minutes in isolated neonatal rat cardiomyocytes (Yamazaki et al., 1995a). Activation of PKC and MAP kinases can also be elicited by ATII and lead to hypertrophy; and AT-II has been reported to be secreted from cardiomyocytes under mechanical stress (Sadoshima et al., 1993). On the one hand, the stretch induced activation of Raf-1 and MAP kinases can only be suppressed partially by an AT-1 receptor inhibitor, and, on the other hand, also the stretch induced activation of MAPK can only partially be attenuated by inhibition of PKC (Yamazaki et al. 1993). In contrast, AT-II evoked activation of Raf-1 and MAP kinases can be blocked completely by the PKC inhibitor staurosporine (Yamazaki et al., 1995a). In adult rat and feline cardiomyocytes in culture, AT-II does not seem to be required for the anabolic processes (increased protein synthesis and expression of Na–Ca-exchanger mRNA) induced by mechanical load (Kent and McDermott, 1996). Taken together, externally applied stretch may activate multiple signaling pathways in ventricular NRC. One component of the stretch-induced effects operates by the release of AT-II, that acts in an autocrine and/or paracrine mechanism. However, AT-II was shown to be only partly responsible for the stretch-induced hypertrophic response involving activation of the MEK-ERK route (Yamazaki et al., 1995b). Thus, an alternative signaling pathway must be mediated by stretch which could involve mechano-sensitive membrane structures resulting in an increase of intracellular Ca2+ as a consequence of membrane deformation.
Cytokines and their Signaling Pathways Cytokines comprise signaling molecules that act as local mediators in cell–cell communication, such as colony-stimulating factors, interferons and interleukins. IL-1b, a 17 kDa protein initially termed endogenous pyrogen, is primarily known as mediator of inflammation and to play a key role in immune responses. Using the NRC culture model, IL-1b has recently been reported to induce a novel form of cardiac myocyte hypertrophy that is characterized by an increase in protein content, but an absence of the fetal program of a-sk actin or bMHC gene expression (Patten et al., 1996). This contrasts with the hypertrophy induced by PE, vasoactive peptides and other growth factors (see Table 2). In fact, IL-1b inhibits PE-induced a-sk actin and b-MHC gene expression. Transient transfection studies with a-sk actin and b-MHC promoter constructs indicate that the repression may occur at the level of transcription via activation of the negative transcription factor YY1. In contrast, an upregulation by IL-1b of mRNA levels for b-MHC and ANF was reported in NRC, concomitant with a down regulation of three Ca-regulatory genes (SERCA2, Ca-release channel and voltage dependent Ca-channel) (Thaik et al., 1995). For IL1b little is known concerning the immediate postreceptor events, although activation of the JNK/ SAPK and p38 MAP kinase cascade has been shown (Saklatvala et al., 1996). IL-1b seems to act in paracrine fashion since it is expressed in cardiac fibroblasts, but not in cardiomyocytes (Long, 1996). Cardiotrophin-1, a 21.5 kDa protein, is a novel member of the interleukin-6 family of cytokines and was isolated by expression cloning based on its ability to induce an increase in cell size in NRC (Pennica et al., 1995). CT-1 activates another form of cardiac myocyte hypertrophy that is different from the phenotype seen after a-adrenergic stimulation with PE. CT-1 induces a distinct set of immediate early genes as well as the ANF gene, but not the a-sk actin gene, and leads to a change in overall cell shape (Wollert et al., 1996). The overall cell shape is significantly elongated in culture with sarcomere assembly in series rather than in parallel. This is reminiscent of the morphological changes observed in hearts under volume as opposed to pressure overload (Gerdes et al., 1988). CT-1 is expressed in cardiomyocytes and acts in an autocrine way. CT-1 has been shown to signal via the gp130-LIFRb (leukaemia inhibitory factor receptor) dependent pathways (Wollert et al., 1996) and continuous activation of gp130 was shown to cause myocardial hypertrophy in vivo (Hirota et al., 1995).
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Hypertrophy in Cultured Cardiomyocytes Table 2 Effects of growth factors and other hypertrophic stimuli on mRNA and/or protein accumulation of markers of hypertrophy. Data for pressure overload (PO) and in part for T3 come from adult rat hearts in vivo. All other data was obtained from cultured neonatal or adult rat cardiomyocytes∗ (see text for details).
a-MHC b-MHC a-skeletal muscle actin a-smooth muscle actin SERCA2 ANF References
PO†
bFGF
TGFb
IGF-I
T3
IL-1b
ET-1
PE
↓ ↑ ↑ ↑ ↓ ↑ 1–6
↓ ↑ ↑ ↑ ↓ ↑ 7–9
↓ ↑ ↑ ↑ ↓ ↑ 10
n§ ↑ ↑ ↓ n ↓ 7–8 and 11–13
↑ ↓ ↑ ↑ ↑ ↑ 7 and 14–21
↓ ↑/↔ ↔ n ↓ ↑ 22–23
↑ ↑ ↑ n n ↑ 24–25
↓/↔ ↑ ↑ n ↓ ↑ 22 and 26–28
∗ Results are taken from refs: (1) Black et al., 1991; (2) Calderone et al., 1995; (3) Izumo et al., 1988; (4) Komuro et al., 1989; (5) Lompre et al., 1991; (6) Schwartz et al., 1986; (7) Gosteli-Peter et al., 1996; (8) Harder et al., 1996; (9) Parker et al., 1990b; (10) Brand and Schneider, 1995; (11) Donath et al., 1994; (12) Florini and Ewton, 1992; (13) Ito et al., 1993; (14) Dubus et al., 1993; (15) Kessler-Icekson, 1988; (16) Ladenson et al., 1988; (17) Lompre et al., 1984; (18) Nag and Cheng, 1986; (19) Rohrer and Dillmann, 1988; (20) Rohrer et al., 1991; (21) Winegrad et al., 1990; (22) Patten et al., 1996; (23) Thaik et al., 1995; (24) Sugden and Bogoyevitch, 1996; (25) Wang et al., 1992; (26) Bishopric et al., 1987; (27) Knowlton et al., 1991; (28) Waspe et al., 1990. † PO, pressure overload in rat heart in vivo induced by aortic constriction. ‡ PE, phenylephrine (a1-adrenergic stimulation). § n, not known at present.
The general features of cytokine signaling briefly discussed here are true also for the IL-6 family, of which CT-1 is a member. Cytokine receptors are not tyrosine kinases themselves, but upon ligand binding interact with associated proteins, the Janus kinases (JAKs), that phosphorylate tyrosine residues on both themselves and the receptors. Thereby, they provide docking sites for various downstream effector proteins: adaptor molecules such as Shc, various NRTKs and STATs (Ihle, 1995; Briscoe et al., 1996; Waters and Pessin, 1996). Thus, the CRs are able to signal via several downstream routes (Fig. 1). In addition, multiple JAK and STAT isoforms provide different specific interactions with particular CRs. The mode of activation for the STAT transcription factors is unique in that they are activated by tyrosine phosphorylation directly at the membrane level. Activated STATs form dimers, translocate to the nucleus and bind response elements to induce transcription.
Thyroid Hormone Signaling Cardiovascular disorders are frequently observed in hyperthyroidism and hypothyroidism. Cardiac performance can be directly correlated to the thyroid hormone serum level (Feldman et al., 1986). Heart rate, contractility, ejection fraction and coronary blood flow are all increased in hyperthyroidism, while peripheral vascular resistance is reduced concomitantly. These changes are accompanied by cardiac hypertrophy and may eventually result in heart failure. On the other hand,
thyroid hormone deficit is associated with decreased contractility and cardiac dilation. Both changes can be reversed by redressing the unbalanced thyroid hormone status (Ladenson et al., 1992). The active thyroid hormone is 3,5,3′-triiodo--thyronine (T3) which arises by deiodination of -thyroxine (T4) in the periphery. About 0.3% of serum T3 is in a free form which can be taken up by cells by an energy dependent process. Although the cell nucleus is the primary site of T3 action, it is still unclear how it is actually transported into the nucleus. Different cytosolic T3 binding proteins (TBP) have been identified which seem to direct translocation of T3 either to mitochondria or to the nucleus in vitro (Ichikawa and Hashizume, 1995). The nuclear T3 receptor (TR) is an acidic non-histone protein. It belongs to the superfamily of hormone responsive nuclear transcription factors, which are similar in structure and mechanism of action, including the receptors for steroid hormones, vitamin-D and RA (Lazar, 1993). Two TR genes exist on two different chromosomes giving rise to TRa (46 kDa) and TRb (55 kDa). At least two alternative mRNA splice products exist for each gene (TRa1, TRa2, TRb1, TRb2). The C-terminal portion is important for ligand binding and for interactions between receptors. The DNA-binding domain of TR extends over 69 amino acids (52–120 in TRa, 106–174 in TRb) and includes two zinc finger motifs. TRa2 does not bind T3. This variant is expressed in most tissues and may inhibit binding of activated TRs to DNA. Transcriptionally active forms include monomers, homodimers and heterodimers with other nuclear
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protein partners, such as the retinoid-X receptor (whose ligand is RA). The T3–TR complexes interact with specific sequences in DNA regulatory regions and modify gene expression. T3 causes both increases and decreases in gene expression and may also influence the stability of mRNA (Brent, 1994). The expression patterns of TR mRNA varies in a developmental and tissue-specific way and are regulated by T3. Cardiac muscle contains TRa1, TRa2 and TRb1. T3 upregulates TRb1 and downregulates TRa1 and TRa2 mRNA. T3 regulates a number of genes specific for cardiac muscle proteins which are directly involved in contractile function and intracellular Ca-handling. The positive chronotropic and inotropic effects of T3 on rat hearts in vivo have been shown to be mediated by catecholamines (Zimmer et al., 1995). This may be explained by the finding that T3 specifically stimulates expression of the b1-adrenergic receptor gene in cultured NRC (Bahouth, 1991). Thus, T3 may be considered to be permissive for the increased cardiac catecholamine responsiveness. On the other hand, the induction of cardiac hypertrophy by T3 (i.e. increased RNA/ DNA ratio and increased ratio of left ventricular weight over body weight) is not prevented by aand b-adrenoceptor blockade and, therefore, seems to represent a direct effect of T3 (Zimmer et al., 1995). This would conform with the notion that T3 regulates several proteins of the contractile apparatus as well as additional markers of hypertrophy such as ANF (see Table 2). In fact, the promoter regions of the genes of a-MHC (Tsika et al., 1990), a-sk actin (Collie and Muscat, 1992) and SERCA2 (Rohrer et al., 1991) have all been shown to contain T3 responsive elements. In contrast to most of the other hypertrophic stimuli, T3 upregulates a-MHC and downregulates b-MHC (Lompre et al., 1984). In addition, T3 specifically upregulates the mRNA of three subunits (a2, a3 and b) of the sarcolemmal Na-K-ATPase in cultured NRC (Orlowski and Lingrel, 1990). It has recently been shown, that T3 represses PKCa and PKCe expression in the neonatal and PKCe in the adult rat heart (Rybin and Steinberg, 1996). Thus, the developmental change in PKC isoform expression may be due to the surge of thyroid hormone levels shortly after birth (Walker et al., 1980).
Individual Hypertrophic Stimuli, Gene Expression and Cross-talk The biochemical profiles of some typical genetic markers of hypertrophy in response to pressure
overload in adult rat hearts in vivo and in response to several individual hypertrophic stimuli on cultured cardiomyocytes are summarized in Table 2. The data is presented as up- or downregulation of mRNA or of the corresponding proteins. Some data for T3 stems from experiments with rats in vivo, while all other data comes from cultured NRC or ARC. Pressure overload (PO) in vivo invariably leads to an increase in heart mass. A significant increase in cell size has been reported for ARC treated with bFGF (Harder et al., 1996) and for NRC treated with either IGF-I (Ito et al., 1993), IL-1b (Palmer et al., 1995), ET-1 (Ito et al., 1991) or a1-adrenergic stimulation (Simpson, 1985). Cell size of ARC treated with IGF-I is not increased in comparison to control cells (Harder et al., 1996). This may represent a difference in response capacity between NRC and ARC. The response profiles for the genetic markers listed in Table 2 are identical for treatment with bFGF, TGFb and also for pressure overload in vivo. All results for TGFb come from NRC in culture (Brand and Schneider, 1995), while results for bFGF come in part from NRC (Parker et al., 1990b) and in part from ARC (Gosteli-Peter et al., 1996, Harder et al., 1996). Treatment with bFGF upregulates mRNA and protein levels of ANF and a-sm actin in both NRC and ARC. A reciprocal regulation of a-sk actin mRNA was reported for aFGF (downregulation) and bFGF (upregulation) in normal NRC as well as in NRC transfected with a promoterdriven reporter gene (Parker et al., 1990a, 1990b). In these two studies, aFGF was employed at the same concentration as bFGF (1.4 n), which is optimal for the latter. However, it is known that aFGF is almost 100 times less potent than bFGF (Gospodarowicz et al., 1987). The data for only four of the listed genetic markers is available for IGF-I. In contrast to the just mentioned stimuli, IGF-I downregulates a-sm actin and ANF in ARC (Donath et al., 1994; Gosteli-Peter et al., 1996; Harder et al., 1996). For a-sm actin, downregulation of both mRNA and protein has been shown. The upregulation of b-MHC (Florini and Ewton, 1992) and a-sk actin in NRC is accompanied by an upregulation of additional proteins such as troponin-I and ventricular MLC-2 (Ito et al., 1993). This upregulation of several sarcomeric proteins seems to correspond with the observed increase in cell size in NRC. For T3 treatment, data for all proteins in Table 2 except a-sm actin have been obtained from rat hearts in vivo: MHC (Lompre et al., 1984), a-sk actin (Winegrad et al., 1990), ANF (Ladenson et al., 1988), and SERCA2 (Rohrer and Dillmann,
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1988). Upregulation of SERCA2 was also shown in cultured NRC (Rohrer et al., 1991). The changes concerning the MHC isoforms in vivo are also observed in NRC (Kessler-Icekson, 1988) and in ARC (Nag and Cheng, 1986; Dubus et al., 1993; GosteliPeter et al., 1996). Upregulation of both mRNA and protein for a-sm actin has been shown in ARC (Gosteli-Peter et al., 1996). The data with IL-1b, ET-1 and PE all comes from NRC (for refs. see Table 2). For b-MHC, either upregulation (Thaik et al., 1995) or no change (Patten et al., 1996) was observed under treatment with IL1b. For a-MHC either downregulation (Patten et al., 1996) or no change (Waspe et al., 1990) was seen with PE. Taken together, each hypertrophic stimulus elicits its own characteristic profile on the molecular level of gene regulation. bFGF and TGFb present an exception, in that they induce the set of selected molecular markers in vitro in the same way as PO does in vivo. Several experimental findings imply that crosstalk between different signaling pathways activated by individual stimuli may be effective. We have shown, recently, that T3 is permissive for the action of bFGF and IGF-I in that these two growth factors require around 1 n T3 in the medium in order to differentially regulate the a-sm actin gene in ARC. With saturating T3 concentrations, bFGF potentiates the effects of T3 alone (Gosteli-Peter et al., 1996; Schaub et al., 1997). Another example concerns TGFb, which on its own does not induce any overall protein synthesis or cell growth in either NRC or ARC (Parker et al., ¨ 1990a; Schluter et al., 1995). In ARC isoproterenol (ISO) alone also fails to induce protein synthesis and only the combination of both TGFb and ISO ¨ leads to cell hypertrophy (Schluter et al., 1995). TGFb is thus considered to represent the causal factor that induces hypertrophic responsiveness to b-adrenoceptor stimulation in ARC. In NRC, ISO has been shown also to stimulate cardiac nonmyocytes to produce TGFb3 which, in turn, provokes hypertrophy in the myocytes (Long et al., 1993). Therefore, this is an example of cross-talk between two signals that involve at least two different types of cells. The a1-adrenergic agonist PE did not operate in this manner. It is likely that in the intact heart tissue even more complex interactions between different cell types take place (Long, 1996). Using the in vitro model of cultured NRC, RA has recently been reported to suppress two hypertrophic markers induced by PE (Zhou et al., 1995). RA inhibits the expression of an ANF-luciferase gene construct under the stimulation of either PE or ET1 and prevents PE induced increase in cell size.
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These results suggest the possibility that a balance may exist in vivo by which cardiac hypertrophy can be counteracted by RA. Another example of antagonistic interaction concerns the IL-1b mediated inhibition of PE induced expression of mRNA for a-sk actin and b-MHC (Patten et al., 1996). In NRC, it has also recently been observed that co-treatment with IL-1b reversed the T3 effects on both MHC isogenes (Patten and Long, 1996). Whereas a-MHC mRNA expression was reduced in the presence of IL-1b and T3, b-MHC mRNA was re-expressed with IL-1b co-treatment. This reversal of T3 effects is suggested to occur at the transcriptional level (C.S. Long, 1997, personal communication). Taken together, the examples presented here illustrate the fact that different hypertrophic stimuli may act in concert to induce a variety of morphological and biochemical phenotypes.
Conclusions Using cell culture models, a whole range of stimuli have been identified as mediators of cardiomyocyte hypertrophy. This in vitro approach has also enabled the molecular dissection of some of the pathways involved in the intracellular transduction of these signals. We have pointed out some recently discovered points of cross-talk between different signaling pathways and the subsequent activation of a characteristic set of genes. So, lateral cross-talk between different signal pathways may occur upon stimulation by more than one factor at a time. These stimuli may then act alone, or in concert in a synergistic, antagonistic or permissive manner. Thus, there is no such thing as a general type of “hypertrophic reaction” or a reversion to the socalled “fetal gene expression program” during growth stimulation. The induction of such a variety of morphological and biochemical phenotypes raises, of course, the question about its physiological significance. The hypertrophic growth of the heart may similarly be the result of a complex combinatorial action of various stimuli, which may also lead to different morphological and biochemical phenotypes with distinct physiological properties.
Acknowledgement The work reported here has been supported by a grant from the Swiss National Science Foundation (31–40694.94) and from the Swiss Heart Foundation (1995) to M.C. Schaub.
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