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Gq Signaling in Cardiac Adaptation and Maladaptation Gerald W. Dorn II* and Joan Heller Brown

Accumulating evidence suggests that cardiac responses to a number of circulating or locally released humoral factors contribute to adaptive responses after hemodynamic stress or myocardial injury. In particular, hormones such as angiotensin II, endothelin 1, norepinephrine and prostaglandin F2a which bind to and activate cardiomyocyte membrane receptors coupled to the Gq class of GTP binding proteins have been implicated in the development and ultimate decompensation of cardiac hypertrophy. Herein we summarize recent developments in cultured cardiomyocyte and transgenic mouse systems which are defining the phenotypes resulting from Gq signaling events in cardiomyocytes, and which are elucidating the critical downstream mediators. Postulated roles for protein kinase C, p38 MAP kinase and jun-N terminal kinase are discussed in relation to Gq-mediated cardiomyocyte hypertrophy and apoptotic signaling. The evidence to date suggests that molecular targeting of Gq or its effectors has the potential to modify cardiac adaptive and maladaptive responses to stress or injury. (Trends Cardiovasc Med 1999; 9:26–34). © 1999, Elsevier Science Inc.

• Cardiac Hypertrophy as a Gq-Mediated Autocrine/ Paracrine Response The common chronic adaptive response of the adult heart to injury or abnormal Gerald W. Dorn II is at the Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; Joan Heller Brown is at the Department of Pharmacology, University of California, San Diego, La Jolla, CA. * Address correspondence to: Gerald W. Dorn II, MD, University of Cincinnati College of Medicine, 231 Bethesda Avenue, ML 0590, Cincinnati, OH 45267. © 1999, Elsevier Science Inc. All rights reserved. 1050-1738/99/$-see front matter

hemodynamic load is increased cardiomyocyte size and contractile protein content (i.e., hypertrophy). Results from the Framingham Heart Study show that myocardial hypertrophy predisposes to early death (Levy et al. 1990). However, the broad categorization of enlarged hearts as “hypertrophied” is a generalization that ignores diverse physiologic or pathologic stimuli for hypertrophy and that fails to distinguish between critical differences in etiology, mechanism, and prognosis. For example, the physiologic hypertrophy that develops in highly trained athletes is not associated with either altered myocardial protein or changes TCM Vol. 9, No. 1/2, 1999

in genetic background, nor does it predispose to heart failure or death. In contrast, pathologic hypertrophy of mechanically overloaded hearts is associated with specific changes in gene and protein expression that recapitulate an embryonic phenotype (Chien et al. 1991, Izumo et al. 1988). In addition, while pathologic hypertrophy initially compensates for increased hemodynamic loading by normalizing wall stress, these functional benefits are transitory and the hypertrophied heart ultimately dilates and fails in a poorly understood process commonly referred to as decompensation (Grossman et al. 1975, Meerson 1961, Osler 1892). Thus, arresting or limiting pathologic hypertrophy is anticipated to prevent decompensation. Physiologic and molecular studies have identified stimuli for and functional consequences of pathologic myocardial hypertrophy and defined a host of accompanying changes in gene expression. Details of how the heart translates the mechanical stimulus of hemodynamic overload into a biochemical signal(s) for hypertrophy are being elucidated, but a complete and coherent biochemical mechanism describing the development and decompensation of myocardial hypertrophy has not yet been achieved. The aforementioned distinction between pathologic and physiologic cardiac hypertrophy supports the existence of unique pathophysiologic processes in these two paradigms. One example is in the role of ANF which modifies intravascular volume and pressure in hemodynamically overloaded hearts. In the physiologic setting ANF is secreted only from cardiac atrial tissue (Oikawa et al. 1984), whereas with pathologic hypertrophy ANF is re-expressed and released from ventricular tissue as well as part of a pattern of gene and gene product expression which reprises that of the fetal heart (Chien et al. 1991, Kroemer et al. 1995, Subramaniam et al. 1991). The notion that cardiac endocrine/ autocrine mechanisms regulate cardiomyocyte growth and development is gaining acceptance. In particular, there is accumulating evidence that the Gqcoupled receptor agonist angiotensin II has direct cardiotrophic effects which may transduce experimental and human pathologic myocardial hypertrophy. Angiotensin converting enzyme (ACE) inhibition with enalapril prevents presTCM Vol. 9, No. 1/2, 1999

sure overload hypertrophy in abdominal aorta coarcted rats, without inhibiting left ventricular hypertrophy stimulated by direct infusion of angiotensin II (Baker et al. 1990, Dostal and Baker 1992). In human studies, the ACE inhibitors enalapril and lisinopril can lead to regression of myocardial hypertrophy in a manner which is at least partially independent of their antihypertensive activity (Dunn et al. 1984, Garavaglia et al. 1988, Nakashima et al. 1984) and they exhibit beneficial effects on mortality and morbidity in heart failure (The SOLVD Investigators 1991). It has been postulated that ACE inhibition limits cardiac hypertrophy by blocking regulatory effects of a myocardial reninangiotensin system which is the source of angiotensin II production under pathological conditions (Schunkert et al. 1990, Urata et al. 1993 and 1990). Further evidence for a cardiotrophic effect of angiotensin II includes the observation that blockade of angiotensin II receptors with AT-II type 1 receptor antagonists attenuates the development of left ventricular hypertrophy in pressure overloaded mice (Rockman et al. 1994) and regresses hypertrophy in spontaneously hypertensive rats (Kojima et al. 1994). Furthermore, direct administration of angiotensin II to isolated perfused rat hearts stimulates new protein synthesis (Schunkert et al. 1995). Thus, multiple avenues of investigation are consistent with a role for angiotensin, possibly released directly from the myocardium, as a transducer of in vivo pathologic cardiac hypertrophy. • Stimulation of Multiple Gq-coupled Receptors Induces Cardiomyocyte Hypertrophy in vitro The most detailed mechanistic investigations of cardiomyocyte signal transduction pathways underlying hypertrophy have been performed using cultures of spontaneously beating neonatal rat ventricular myocytes. Hypertrophy in these stellate cells can be measured as an increase in cell size or surface area, greater organization of myofilaments (visualized by increased myosin light chain staining or phalloidin staining of actin filaments), and augmented atrial natriuretic factor gene or protein expression (Izumo et al. 1988). There is also a pattern of immediate early gene expression and a shift in

contractile protein isoforms similar to that observed with in vivo pathologic hypertrophy. The initial studies demonstrating hypertrophic effects of specific G-protein coupled agonists in cultured neonatal cardiomyocytes were performed by Simpson and colleagues who observed that the alpha adrenergic agonist norepinephrine, but not the beta adrenergic agonist isoproterenol, increased cardiomyocyte cell size in a dose-dependant manner, and further demonstrated that this hypertrophic effect was mediated by stimulation of a1 adrenergic receptors (a1AR) (Simpson 1983, Simpson et al. 1982). Using variations of this tissue culture model, hypertrophic effects of phenylephrine, angiotensin II, endothelin, and prostaglandin F2a (PGF2a) have all been demonstrated (Adams et al. 1996, Knowlton et al. 1993, Sadoshima and Izumo 1993a, Shubeita et al. 1990). A critically important feature of these structurally diverse hypertrophic factors is that, like angiotensin II, they each stimulate a seven transmembranespanning or heptahelical receptor that activates phospholipase C via the Gq class of GTP binding proteins. Hypertrophic effects of Gq-coupled receptor agonists are associated with activation of phospholipase C as demonstrated by receptor-mediated increases in cardiomyocyte inositol phosphate and diacylglycerol content (Adams et al. 1998a, Brown and Martinson 1992, Clerk and Sugden 1997, McDonough et al. 1987, Sadoshima and Izumo 1993b, Shubeita et al. 1990). Overexpression in cardiomyocytes of receptors that couple to Gq/ phospholipase C leads to hypertrophy, whereas expression of receptors with mutations that block Gq coupling or that couple to Gi do not mediate hypertrophic responses (Ramirez et al. 1995). Furthermore, expression of activated Gaq in cardiomyocytes is sufficient to stimulate at least some features of hypertrophy as measured by increased cell size, organization of actin elements, and/or expression of ANF (Adams et al. 1998b, LaMorte et al. 1994). Conversely, inhibition of Gaq signaling with microinjected neutralizing antibodies prevents a1-adrenergic receptor mediated cardiomyocyte hypertrophy (LaMorte et al. 1994). These studies support an obligatory role for Gaq signaling in cardiomyocyte hypertrophy. Of particular interest regarding the

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potential relevance of Gq-coupled receptor agonists in pathologic cardiomyocyte hypertrophy is the observation that angiotensin II and endothelin-1 can be released from mechanically deformed cardiomyocytes (Kojima et al. 1994, Sadoshima et al. 1993, Yamazaki et al. 1996). Accordingly, stretch-mediated secretion of these Gq-stimulating agonists from the heart could constitute an autocrine mechanism regulating hypertrophic responses to mechanical stress. That mechanical stretch could also stimulate autocrine production of Gq-coupled receptor agonists in the intact heart is indicated by a recent study in which isolated perfused guinea pig hearts were subjected to left ventricular diastolic distension by increasing the pressure in a balloon placed within the left ventricular cavity (Paul et al. 1997). This paradigm for ventricular “stretch” increased phospholipase C activity (measured as inositol phosphate formation) and activated protein kinase C. Taken together with the aforementioned in vitro studies of mechanically deformed cultured neonatal cardiomyocytes, this ex vivo study of isolated perfused hearts suggests that the heart responds to mechanical load with production of Gq activating agonists. The observations that Gq-coupled receptor agonists: (a) stimulate hypertrophy of cultured neonatal cardiomyocytes, and (b) are elevated in the myocardium of animals subjected to hypertrophic interventions (Berger et al. 1976, Lai et al. 1996, Rockman et al. 1996) further support a causal relationship between secretion of these autocrine/paracrine factors and the development of pathologic cardiac hypertrophy. One limitation to the in vivo models discussed earlier however, is that they do not distinguish between the direct cardiotrophic effects of Gq-coupled receptor agonists and indirect effects which might result from their ability to increase blood pressure and cardiac mechanical load. • Transgenic Analysis of in vivo Myocardial Gaq Signaling In vivo analysis of cardiac Gq signaling has been greatly advanced by utilization of transgenic techniques to selectively overexpress Gq-coupled receptors, Gaq itself or a dominant negative inhibitor of Gaq in the heart. The seminal transgenic experiment which first established

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that activation of a cardiomyocyte Gqcoupled receptor was sufficient to stimulate cardiac hypertrophy was performed by Milano et al. and utilized overexpression of a mutant constitutively activated a1B adrenergic receptor (AR) in the mouse heart (Milano et al. 1994). This experimental design (i.e., overexpression of a mutant receptor which activated downstream signaling pathways in the absence of bound agonist) represented an attempt to reproduce the effects of chronic a1-adrenergic stimulation of cardiomyocytes in the absence of the confounding extracardiac effects of adrenergic agonist. Expression of a constitutively activated a1BAR at a level three times that of the endogenous wild type a1AR increased phospholipase C-dependent phospholipid metabolism, as demonstrated by a 75% increase in myocardial diacylglycerol content. Myocardial hypertrophy with similarities to that induced by pressure overload developed in these CA-a1BAR overexpressing mice. Specifically, these mice exhibited increased heart weight, increased cardiomyocyte cross-sectional area, and ectopic ventricular atrial natriuretic factor gene expression. Thus, chronic activation of cardiomyocyte a1BAR signaling was sufficient to cause cardiac hypertrophy. Interestingly, in a follow-up study where a nonactivated form of the a1BAR was overexpressed in mouse hearts at levels approximately 40-fold greater than normal (Akhter et al. 1997), myocardial diacylglycerol content was increased by 50%, and atrial natriuretic factor expression was substantially increased, but morphometric or histologic cardiac hypertrophy did not develop. In this report the authors demonstrated dual coupling of a1BAR expressed at these levels to Gi as well as Gq, suggesting a possible biochemical mechanism (concurrent activation of Gi signaling) for the absence of hypertrophy in wild type (WT) a1BAR overexpressors. These two studies not only defined effects of chronic a adrenergic signaling on the heart, but also demonstrated some of the experimental difficulties encountered with transgenic overexpression of G protein coupled receptors. Three-fold overexpression of a constitutively activated a1BAR increased phospholipase C activity by only 75%, suggesting that the transgenic receptors were not efficiently coupled to Gq signaling effectors. In contrast, the more

marked overexpression achieved with the nonactivated form of a1BAR resulted in promiscuous coupling of receptors to Gi and possible activation of atypical effectors. Finally, although phospholipase C activity was apparently increased in both constitutively activated and WT a1BAR overexpressing mice, it is not clear that phospholipase C activation was the cause of myocardial hypertrophy. In fact, the dissociation of phospholipase C activation from development of morphometric or cellular myocardial hypertrophy in WT a1BAR overexpressors raises questions about the correlative versus causal relationship of Gq/ phospholipase C signaling in hypertrophy development. In considering alternate experimental approaches which could clarify the role of Gq/phospholipase C signaling, but avoid promiscuous receptor coupling, we chose overexpression of the alpha subunit of the Gq heterotrimer (D’Angelo et al. 1997). Based on reports by Homcy and Vatner that overexpression of nonactivated alpha subunit of Gs increased adenylyl cyclase signaling in the heart (Gaudin et al. 1995, Iwase et al. 1996), it was anticipated that overexpression of the nonactivated Gaq subunit would similarly enhance myocardial phospholipase C signaling and therefore reveal some effects of chronically activating cardiomyocyte Gaq via the postulated autocrine receptor pathways. The wild type (WT) mouse Gaq cDNA coding region was expressed in mouse hearts under control of the fulllength mouse a myosin heavy chain promoter which has been widely utilized to achieve high level, cardiac-specific postnatal transgene expression. Three independent Gaq transgenic lines were established and designated Gaq-9, -25, and -40 based on estimates of incorporated transgene copy number. As expected, transgene mRNA expression was found at high levels in cardiac tissue only, and immunoblot analysis demonstrated two-, four-, and five-fold increases in Gaq protein levels in the Gaq-9, -25, and -40 hearts, respectively (Adams et al. 1998b, D’Angelo et al. 1997). Mice from the Gaq-25 and -40 lines exhibited nearly identical phenotypes of cardiac hypertrophy and contractile dysfunction, differing only in that Gaq-40 mice manifested myocardial hypertrophy and TCM Vol. 9, No. 1/2, 1999

gene expression which was somewhat exaggerated in comparison to Gaq-25. Hypertrophy in Gaq transgenic mice was similar to pressure overload hypertrophy in hearts of mice after aortic banding in terms of the extent of cardiac hypertrophy, the pattern of fetal gene expression, and the increase in cardiomyocyte cross-sectional area (Dorn et al. 1994, Sakata et al. 1998). However, Gaq expressors also exhibited features distinct from compensated pressure overload hypertrophy such as resting sinus bradycardia and left ventricular contractile depression at matched (atrial paced) heart rates, measured either by echocardiographic or microminiaturized catheterbased hemodynamic techniques. Left ventricular systolic dysfunction in Gaq overexpressors was not solely the consequence of altered ventricular geometry since (as with pressure overload hypertrophy) isolated mechanically unloaded Gaq overexpressing ventricular myocytes had depressed rates of shortening and relengthening (Sakata et al. 1998). This phenotype of hypertrophy and contractile depression in independent lines of Gaq overexpressors indicated that signaling events downstream of Gaq are sufficient to cause cardiac hypertrophy. Interestingly, Gaq-9 mice, which overexpress Gaq at only twice endogenous levels, exhibited no detectable molecular, biochemical, or pathologic phenotype under normal conditions (D’Angelo et al. 1997) or after provocation by surgical pressure overloading (Sakata et al. 1998). The normal phenotype of Gaq-9 mice suggests that a threshold level of WT-Gaq protein expression must be achieved to autonomously activate or facilitate receptor-mediated Gq signaling, and supports the notion that hypertrophy in Gaq-25 and Gaq-40 mice is the result of increased Gaq signaling, rather than a nonspecific effect of increased cardiomyocyte Gaq protein content. In measuring the biochemical consequences of Gaq overexpression on myocardial signaling, it has been possible to identify some putative mediators of Gqstimulated cardiac hypertrophy and to rule out other postulated hypertrophy signaling effectors. As would be predicted from tissue culture studies of Gaq overexpression (Wu et al. 1992a and b), transgenic Gaq overexpression in the heart increased basal and agoniststimulated phospholipase C activity meaTCM Vol. 9, No. 1/2, 1999

sured as inositol phosphate accumulation in 3H-myoinositol labeled atrial sections (unpublished studies). Consistent with the relatively minor role of InsP3 in cardiac Ca11 homeostasis, increased phospholipase C activity and InsP3 formation did not increase resting cytosolic free calcium concentration ([Ca11]i) measured in Fura-2 loaded cardiac myocytes (unpublished studies). While resting [Ca11]i was normal, the duration of the calcium transient in paced ventricular myocytes was significantly prolonged, possibly reflecting downregulation of the sarcoplasmic reticulum calcium ATPase, an alteration which is observed in Gaq overexpressors as well as other forms of pathologic hypertrophy (De la Bastie et al. 1990, Dorn et al. 1998, Komuro et al. 1989). Whereas a pathogenic role for altered calcium signaling in Gaq overexpressors cannot be ruled out, it is more likely that the phenotypic features of Gaq overexpressors are the result of chronic protein kinase C activation. The protein kinase C (PKC) family of kinases are regulated by phospholipid, most commonly by diacylglycerol (DAG) which, along with InsP3, is a product of phospholipase C-mediated phosphatidylinositol hydrolysis. Mouse myocardium expresses several different diacylglycerolresponsive PKC isoforms (PKCa, d, «, h) (D’Angelo et al. 1997, Dorn et al. 1998) which differ in their requirements for activation, and presumably in their subcellular targets (Steinberg et al. 1995). PKCa is a conventional PKC isoform which must bind calcium as well as DAG to be activated. In contrast, the novel PKC isoforms PKCd, «, and h are activated by DAG, but do not require increased calcium. Each of these four PKC isoforms shares the characteristic of translocating from the cytosolic to a particulate membrane subcellular fraction upon activation, a response which has been attributed to binding to anchor proteins termed RACKs (Receptor for Activated C Kinases) (Disatnik et al. 1994, Mochly-Rosen 1995). Translocation is an early step in PKC activation, providing the basis for measuring PKC activation by determining the relative amount of PKC in soluble versus particulate subcellular fractions. Using PKC isoform-specific antibodies and differential centrifugation techniques, Gaq-25 and Gaq-40 mouse hearts exhibited intrinsic activation of PKC«,

but not PKCa (D’Angelo et al. 1997, Gaudin et al. 1995) relative to nontransgenic hearts. Preferential activation of PKC«, but not PKCa, has also been observed in phenylephrine-treated isolated neonatal and adult rat cardiomyocytes (Puceat et al. 1994). In contrast to the observed pattern for activation of PKC, the absolute expression of PKCa in Gaq transgenic hearts was substantially increased at the mRNA (.66%) and protein level (.50%), suggesting transcriptional upregulation of PKCa. Interestingly, Simpson’s laboratory previously demonstrated upregulation of PKC in cardiomyocytes stimulated to hypertrophy through a1-AR activation (Henrich and Simpson 1988). In contrast, PKC« in Gaq overexpressing hearts is downregulated at the protein level, consistent with a mechanism of proteolytic degradation which has previously been described for chronically activated PKC (Young et al. 1987). PKCd and h did not appear to be either activated or regulated in Gaq overexpressors. The observation that PKC« is specifically activated in Gaq overexpressors is consistent with recent observations of PKC« activation in pressure overload hypertrophy, balloon stretched isolated perfused hearts and phenylephrine, endothelin, PGF2a, ATP and angiotensin II treated cardiomyocytes (Bogoyevitch et al. 1993, Gu and Bishop 1994, Paul et al. 1997, Puceat et al. 1994, Sadoshima and Izumo 1993b, Schunkert et al. 1995, Zou et al. 1996). Conventional and inducible cardiacspecific transgenesis has recently been applied to explore the direct effects of PKCb in the in vivo heart. Although there is controversy over whether PKCb is normally expressed in the adult mouse heart (Rybin and Steinberg 1994), the results of PKCb overexpression are interesting and instructive. Wakasaki et al. expressed nonactivated PKCb2 under control of a truncated aMHC promoter and observed a phenotype of hypertrophy, fibrosis, and systolic dysfunction (Wakasaki et al. 1997). In a follow-up study, PKCb-mediated phosphorylation of troponin I was suggested as a mechanism for contractile dysfunction in these mice (Takeishi et al. 1998). Bowman et al., employing inducible expression of a mutationally activated PKCb2 (Bowman et al. 1997), found hypertrophy when the transgene was induced in the adult heart, but a lethal effect of expres-

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sion in the neonatal mouse. These studies have helped to demonstrate that PKCb is sufficient to cause cardiac hypertrophy and/or contractile depression, but the issue of which endogenous PKC isoforms, if any, are important in signaling various cardiac adaptive and maladaptive responses have not been resolved. It is clear that angiotensin II, phenylephrine, and other Gq-coupled receptor agonists also have the potential to activate p42 or p44 MAP kinases (ERKs) in cardiac myocytes as well as other target cell types (Bogoyevitch et al. 1995 and 1994, Post et al. 1996a, Sadoshima et al. 1995, Sugden and Clerk 1998, Zou et al. 1996). Thus, an unexpected finding in hypertrophied Gaq overexpressing hearts was the absence ERK activation (D’Angelo et al. 1997). Involvement of ERK in hypertrophy mediated by G protein coupled receptors in the in vitro cardiomyocyte model is supported by several lines of investigation (Gillespie-Brown et al. 1995, Glennon et al. 1996, Thorburn et al. 1994), but inconsistent with others (Post et al. 1996b, Thorburn et al. 1995), paralleling the dissociation seen in the transgenic Gaq overexpressors. • Physiological Modeling of Gaq Overexpressors The molecular, pathologic, biochemical, and functional changes associated with myocardial hypertrophy in Gaq overexpressors resemble those induced by thoracic aortic constriction. This is consistent with the postulated role for Gq signaling in transducing pressure overload hypertrophy. The recent demonstration by Lefkowitz and Koch that inhibition of receptor-mediated Gaq signaling by expression of an inhibitory peptide in transgenic mice attenuates the hypertrophic response to pressure overload provides convincing proof that Gaq signaling is necessary for the normal development of pressure overload hypertrophy (Akhter et al. 1998). We recently addressed the question of whether enhanced signaling through Gaq expression would modify the hypertrophic response to pressure overload. Possible outcomes were that unusually rapid hypertrophy might occur in aortic banded Gaq overexpressors due to the basal increase in Gq signaling, that hypertrophy in response to

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pressure overload would be attenuated because the required signaling pathways were autonomously active in the Gaq overexpressing heart, or that contractile dysfunction in Gaq overexpressors was of sufficient severity that creation of a .40 mm Hg trans-aortic gradient (Dorn et al. 1994) would be intolerable and the surgically modeled animals would simply die of catastrophic heart failure. To evaluate the effects of intrinsic Gaq signaling activation on cardiac responses to pressure overload, Gaq overexpressors were subjected to transverse aortic banding (Sakata et al. 1998). Left ventricular function and mass were assessed by serial weekly echocardiograms for three weeks after surgical modeling, and cardiac morphometry, histology, and hypertrophy-associated gene expression were assessed at 48 hours, 1 week, and 3 weeks after surgery in identically operated Gaq overexpressors and their nontransgenic siblings. As expected, nontransgenic mice developed fully compensated concentric left ventricular hypertrophy after aortic banding. Cardiomyocyte cross-sectional area in these mice tripled, left ventricular wall thickness nearly doubled, and gravimetric left ventricular mass (normalized to tibial length) increased by approximately 30% after 3 weeks. In Gq overexpressors the absolute magnitude of hypertrophy which developed in response to aortic banding was equivalent to that in aortic banded nontransgenics, but this represented a diminished relative hypertrophic response (measured as percent increase over nonbanded) in pressure overloaded Gaq overexpressors due to their basal hypertrophy. A striking difference between hearts from aortic banded and Gaq overexpressing mice was evident in their ventricular geometry and function. Whereas aortic banded nontransgenic mice developed compensated concentric left ventricular hypertrophy, Gaq overexpressors developed eccentric hypertrophy with progressively declining ventricular function, eventually resulting in overt functional decompensation and pulmonary edema. The hypertrophy associated with Gaq overexpression, while similar to that induced by aortic banding, is therefore of a form which upon further insult, transitions to the decompensated state. Interestingly, similar phenotypic changes are seen in prelimi-

nary analysis of transgenic mice expressing wild type RhoA driven by the aMHC promoter (V.P. Sah et al. personal communication). Rho is a small G protein suggested to function downstream of Gq in regulating cardiac hypertrophy (Sah et al. 1996). The features of hypertrophy in Gaq and Rho overexpressors contrast with the hypertrophy which develops in transgenic mice overexpressing the small GTP protein Ras (Gottshall et al. 1997, Hunter et al. 1995). Clearly there are important differences in design and construction of Gaq and Ras overexpressors, including the use of a myosin light chain promoter for Ras versus the a myosin heavy chain promoter for Gaq, mutationally activated Ras versus nonactivated Gaq, and the development of a phenotype only in homozygous Ras overexpressors versus in heterozygous Gaq overexpressors. Nonetheless, it is instructive to compare the concentric ventricular remodeling and normal contractile function of Ras overexpressors to the eccentric hypertrophy with contractile depression of the Gaq phenotype because Ras overexpression stimulates hypertrophy by activating what appears to be different downstream events than Gaq (Sah et al. 1996). Indeed, in vitro cardiomyocyte hypertrophy induced by activated Ras and Gaq or Ras and Rho are parallel but synergistic pathways and blockade of either pathway by antibodies or dominant interfering proteins attenuates cardiomyocyte hypertrophy induced by aadrenergic receptor stimulation (Hines and Thorburn 1998, Ramirez et al. 1997, Sah et al. 1996). We therefore postulate that pathologic hypertrophy can be an autocrine receptor-mediated response involving simultaneous activation of Gaq and Ras coupled signaling pathways (Figure 1). Based on the differences in Gaq and Ras overexpressor phenotypes, we hypothesize that Gaq signaling events confer the characteristics of hypertrophy which ultimately lead to its decompensation, whereas Ras-mediated events signal the features of hypertrophy which are more truly adaptive. • Gq Signaling, Cardiomyocyte Apoptosis, and Hypertrophy Decompensation Increased Gaq expression and signaling are associated with hypertrophy decompensation in aortic banded Gaq overexTCM Vol. 9, No. 1/2, 1999

Figure 1. Shematic of Gq-coupled receptor, Gq, and small G protein involvement in hypertrophy and failure. The schematic depicts a generic heptahelical, or seven transmembranespanning, receptor that interacts with the heterotrimeric G protein Gaq. Some of the Gqlinked receptors believed to regulate cardiac hypertrophy are listed. Agonist binding to the receptor activates Gaq, increasing its GTP binding, and the phosphoinositide-specific phospholipase C (PLC) is stimulated. Diacylglycerol and inositol trisphosphate, generated from PLC-induced hydrolysis of the membrane phospholipid PIP2, activate protein kinase C (PKC) and regulate intracellular Ca11 release, respectively. The bg subunits of the heterotrimeric G proteins also transduce signals and are in some systems mediators of Ras activation. Other putative proteins involved in Ras activation by Gaq-coupled receptors are not depicted here. Ras activates the upstream MAP kinase kinases (not shown) and subsequently the MAP kinases, which include three families—ERK, JNK, and p38. Other Rasindependent mechanisms are also involved in their activation. The pathways leading to Rho activation by G protein-coupled receptors remain to be elucidated as do the potential Rho effectors regulating hypertrophy; Rho does not regulate MAP kinases in most systems. The schematic further represents the observed involvement of both Rho and Ras/MAP kinase cascades in hypertrophic cardiomyocyte growth; the possible roles of PKC and MAP kinases, especially JNK or p38, in apoptosis; and the hypothesized involvement of apoptosis in the transition from hypertrophy to failure.

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pressors as described earlier in this article and also in dual heterozygous Gaq transgenic mice which express Gaq at high levels in the heart (Adams et al. 1998b, D’Angelo et al. 1997). Recently, increased Gaq expression and phospholipase C signaling were described in the scar and peri-infarcted myocardium of rat hearts 8 weeks after coronary artery ligation (Ju et al. 1998). Thus, transgenic or natural mechanisms which increase expression of and signaling through myocardial Gaq are associated with cardiomyocyte hypertrophy and ventricular failure. A pathophysiologic explanation for the progression of Gaq-mediated myocardial hypertrophy to cardiac failure was suggested by the recent observation that greatly enhanced Gaq signaling in vitro can cause cardiomyocyte apoptosis (Adams et al. 1998b). Cultured neonatal cardiac myocytes infected with adenoviral expression constructs encoding nonactivated wild type Gaq exhibited a 12-fold increase in phospholipase C activity compared to Lac-Z infected cells, and underwent hypertrophy resembling that induced by Gq-coupled receptor agonists. Cardiomyocytes infected with adenovirus encoding a constitutively activated form of Gaq deficient in GTPase activity (Gaq Q209L) showed significantly greater increases in phospholipase C activity (65-fold) and also initially hypertrophied. Unexpectedly however, hypertrophy was not sustained and the cells rapidly and reproducibly underwent apoptotic cell death as determined by in situ nuclear DNA end labeling and in vitro oligonucleosomal DNA fragmentation. In examining potential biochemical mediators of Gaq-stimulated apoptosis, it was found that cJun N-terminal kinase (JNK) and p38 MAP kinase were concomitantly activated in the apoptotic cells expressing activated Gaq, whereas only p38 was significantly increased in the hypertrophic wild typeGaq expressing cardiomyocytes. Thus, consistent with the findings of Althoefer et al. in noncardiac cells transfected with constitutively activated Gaq (Althoefer et al. 1997), high levels of Gaq signaling can stimulate apoptosis in cultured cardiomyocytes. Further studies were carried out using this in vitro model to determine whether exaggeration of signals leading to hypertrophy would induce apoptosis. Cardiomyocytes overexpressing wild type

Gaq, like cardiomyocytes stimulated with PGF2a or other agonists of Gq-coupled receptors, show characteristic hypertrophic responses (Adams et al. 1998b). When PGF2a is added to Gaq expressing cells however, phospholipase C activation is enhanced and the cells undergo apoptotic cell death as determined by DNA laddering and end labeling (Adams et al. submitted). Apoptotic levels of Gq signaling are associated with increased JNK and p38 kinase activities and with activation of an ICE-like protease, probably caspase3 (unpublished studies). Pharmacological inhibition of caspase dramatically reduced myocyte DNA fragmentation and cell death, indicating that caspase activation is required for these responses. An in vivo correlate of Gq-mediated cardiomyocyte apoptosis is the unique peripartum cardiomyopathy which develops in Gaq transgenic mice. Gaq overexpressing mice in the terminal period of pregnancy or immediately following delivery develop an extremely rapid and aggressive cardiomyopathy which is lethal in approximately half of Gaq-25 and 30% of Gaq-40 overexpressors (Adams et al. 1998b). The hearts from animals with this peripartum cardiomyopathy exhibit massive biventricular and biatrial dilatation with frequent atrial mural thrombi (indicative of a low cardiac output state). Pulmonary congestion, pleural effusions, and ascites are nearly universal findings. Histologically, the hearts demonstrate evidence of cardiomyocyte dropout and replacement, without the inflammatory reaction expected with a necrotic process. TUNEL assays demonstrate massive cardiomyocyte-specific nuclear labeling in peripartal cardiomyopathy hearts and electrophoretic DNA separation reveals the characteristic apoptotic DNA fragmentation into 200 base pair ladders. Strikingly, biochemical studies of ventricular tissue from the apoptotic cardiomyopathic peripartum Gaq overexpressors show exaggerated PKC activity and, consistent with in vitro studies in apoptotic cardiomyocytes expressing activated Gaq, selective activation of Jun kinase and p38 (Adams et al. 1998a). Importantly, PKC activation was also implicated in the Gaq-mediated fibroblast apoptosis reported by Althoefer et al. (Althoefer et al., 1997). Thus, in vitro and in vivo studies indicate a role for Gaq signaling not only in stimulating cardiac hyper-

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trophy, but also in decompensation of the hypertrophied heart through stimulation of cardiomyocyte apoptosis. In conclusion, it is intriguing to postulate in light of data presented here, that autocrine or paracrine factors which couple to and enhance Gq signaling pathways are involved in cardiac adaptation to stress or injury (hypertrophy) as well as maladaptive sequelal (apoptotic heart failure). However, the precise nature of the hypothetical Gq enhancing factors remains to be determined and the importance of apoptosis in decompensation of cardiac function remains to be proven. The association of Gq signaling and activation of various downstream effectors underscores the importance of further clarifying pathways by which cardiomyocyte hypertrophy and apoptosis is controlled and consequently delineating sites of potential therapeutic intervention. • Acknowledgments This work was supported in part by National Institutes of Health Grants HL49267, HL58010, HL52318 and HL59888 (GWD) and HL28143 and HL46345 (JHB) and a Veterans Administration Merit Review Grant (GWD).

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PII S1050-1738(99)0004-3

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Negative Regulation of Inflammation by Fas Ligand Expression on the Vascular Endothelium Kenneth Walsh* and Masataka Sata

It is generally believed that the vascular endothelium serves as a barrier to inflammation by providing a nonadherent surface to leukocytes. Recently, we reported that vascular endothelial cells (ECs) express Fas ligand, which functions to actively inhibit inflammation by inducing apoptosis in Fas-bearing leukocytes. The inflammatory cytokine TNF a downregulates Fas ligand expression with an accompanying decrease in EC cytotoxicity toward Fas-bearing cells in co-culture. Endothelial Fas ligand expression in arteries is also downregulated by the local administration of TNFa, and this correlates with robust mononuclear cell infiltration of the subendothelial space. This TNFa-induced mononuclear cell infiltration is inhibited by pre-infecting the endothelium with a replication-defective adenovirus that constitutively expresses Fas ligand. Under these conditions, adherent leukocytes undergo apoptosis rather than extravasation. These findings suggest that Fas ligand expression on the vascular endothelium functions to inhibit inflammatory responses that are often associated with vascular disorders. (Trends Cardiovasc Med 1999;9:34–41) © 1999 Elsevier Science Inc.

Kenneth Walsh and Masataka Sata are affiliated with the Division of Cardiovascular Research, St. Elizabeth’s Medical Center, Tufts University School of Medicine, Boston, Massachusetts and the Program in Cell, Molecular, and Developmental Biology, Sackler School of Biomedical Sciences, Tufts University, Boston, Massachusetts. * Address correspondence to: Dr. Kenneth Walsh, Division of Cardiovascular Research, St. Elizabeth’s Medical Center, 736 Cambridge Street, Boston, MA 02135, USA. © 1999, Elsevier Science Inc. All rights reserved. 1050-1738/99/$-see front matter

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