JOURNAL OF CELLULAR PHYSIOLOGY 186:11±18 (2001)

Neurohormonal Regulation ofMyocardial Cell Apoptosis During the

Development of Heart Failure

KOJI HASEGAWA,* ERI IWAI-KANAI, AND SHIGETAKE SASAYAMA

Department of Cardiovascular Medicine, Graduate School of Medicine,Kyoto University, Sakyo-ku, Kyoto, Japan

Adult cardiac myocytes are terminally differentiated cells that are no longer ableto divide. Accumulating data support the idea that apoptosis in these cells isinvolved in the transition from cardiac compensation to decompensated heartfailure. Since a number of neurohormonal factors are activated in this state, thesefactors may be involved in the positive and negative regulation of apoptosis incardiac myocytes. b1-Adrenergic receptor and angiotensin type 1 receptor path-ways, nitric oxide and natriuretic peptides are involved in the induction ofapoptosis in these cells, while a1- and b2-adrenergic receptor and endothelin-1type A receptor pathways and gp130-related cytokines are antiapoptotic. Themyocardial protection of the latter is mediated, at least in part, through mitogen-activated protein kinase-dependent pathways, compatible with the ®ndings inother cell types. In contrast, signaling pathways leading to apoptosis in cardiacmyocytes are distinct from those in other cell types. The cAMP/PKA pathwayinduces apoptosis in cardiac myocytes and blocks apoptosis in other cell types.The p300 protein, a coactivator of p53, mediates apoptosis in ®broblasts butappears to play a protective role in differentiated cardiac myocytes. The inhibitionof myocardial cell apoptosis in heart failure may be achieved by directly blockingapoptosis signaling pathways or by modulating neurohormonal factors involved intheir regulation. These may provide novel therapeutic strategies in some forms ofheart failure. J. Cell. Physiol. 186:11±18, 2001. ß 2001 Wiley-Liss, Inc.

THE ROLE OF MYOCARDIAL CELLAPOPTOSIS IN THE DEVELOPMENT OF

HEART FAILURE

The heart is a highly differentiated and developedorgan in which contraction and relaxation occur repeat-edly. Cardiac tissue mainly consists of terminally differ-entiated myocardial cells that are no longer able todivide after birth. In response to hemodynamic overloadsuch as hypertension, individual myocardial cells areenlarged, thus maintaining the systolic ability of theentire heart to compensate for the increased workload.That is, cardiac myocyte hypertrophy occurs as anadaptive mechanism during the early phase of over-loading. When overloading persists, this adaptivemechanism fails, resulting in decompensation andsubsequent development of heart failure (systolicdysfunction). Currently, the precise mechanisms thatmediate the transition from adaptation to decompen-sated heart failure are unclear.

Apoptosis, or programmed cell death, is an active,gene-directed process in which cells initiate their owndeaths in response to internal or external stimuli. Thismode of death serves as an orderly means for multi-cellular organisms to eliminate unwanted cells withoutadversely affecting the surrounding tissue. Thus,apoptosis is a key mechanism for normal tissue devel-

opment in the fetus and for cell replacement in certainadult tissues (e.g., the thymus), and it is most oftenencountered in cells that are progressing through thecell cycle (Kerr et al., 1972; Wyllie, 1980; Jacobson et al.,1997; Nagata, 1997). Recent studies have demonstratedthat TUNEL-positive cardiac myocytes are observed invarious animal models of heart failure including modelsof rapid ventricular pacing (Liu et al., 1995; Sharov etal.,1996), and pressure overload due to aortic constriction(Teiger et al., 1996), and aged spontaneously hyperten-sive rats (Li et al., 1997). Since apoptosis is completedwithin several hours, it is not surprising that morpho-logically evident apoptotic cardiac myocytes are seldom

Abbreviations: NE, norepinephrine; MAPK, mitogen-activatedprotein kinase; ERK, extracellularly responsive kinase; PKA,protein kinase A.

Contract grant sponsor: Ministry of Education, Science andCulture of Japan.

*Correspondence to: Koji Hasegawa, Department of Cardiovas-cular Medicine, Graduate School of Medicine, Kyoto University,54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan.E-mail: koj@kuhp.kyoto-u.ac.jp

Received 17 August 2000; Accepted 17 August 2000

Published online in Wiley InterScience, 30 November 2000.

ß 2001 WILEY-LISS, INC.

seen. However, key signal transducers of apoptosis areactivated in failing adult cardiomyocytes (Misao et al.,1996; Narula et al., 1999); these include upregulation ofBax, release of cytochrome C from mitochondria intocytoplasm, upregulation of caspase 3, and cleavage ofcaspase 3 to the active form. Apoptosis is primarily adefense mechanism against excessive cell proliferation.However, if apoptosis occurs in myocardial cells lackingproliferative and regenerative abilities, the number ofmyocardial cells is decreased, thus the stress on theremaining cells is increased, eventually leading to heartfailure. These ®ndings suggest that myocardial cellapoptosis is involved in the transition from compensatedcardiac hypertrophy to heart failure. In addition, it hasbeen reported that in mice lacking membrane proteingp130, considerable myocardial cell apoptosis is inducedby hypertension, which results in the rapid progressionof cardiac dilatation and heart failure (Hirota et al.,1999). This ®nding provides evidence that apoptosissignaling pathways are activated in response to hemo-dynamic overload and that myocardial cell apoptosis issuf®cient to induce decompensated heart failure inadult mice in vivo.

Since a number of neurohormonal factors are acti-vated in heart failure, these factors may positively andnegatively regulate myocardial cell apoptosis. Activa-tion of the sympathetic nervous system, renin-angio-tensin system, and endothelin-1 are closely related toincreased morbidity in heart failure (Packer, 1992;Francis et al., 1993). These factors all participate inthe regulation of myocardial cell apoptosis as well as theincreases in both preload and afterload. On the otherhand, nitric oxide and natriuretic peptides exhibitvasodilative and diuretic effects and have been shownto induce myocardial cell apoptosis. Myocardial cellapoptosis is also regulated by cytokines that act viamembrane protein gp130 such as cardiotrophin-1 (Fujioet al., 1997; Sheng et al., 1997), as well as in¯ammatorycytokines such as tumor necrosis factor-a and inter-leukin-1b. In addition, pathways that activate apoptosisin cardiac myocytes are distinct from those in other celltypes. Norepinephrine (NE) is a representative factor,the elevation of which in plasma closely correlates withthe severity and poor prognosis of heart failure. We andothers have shown that NE induces myocardial cellapoptosis via a b-adrenergic receptor-dependent path-way (Communal et al., 1998; Iwai-Kanai et al., 1999).This might provide further insight into the mechanismsthat explain the preferential effects of b-blockade onchronic congestive heart failure. This article focuses onthe regulation of myocardial cell apoptosis by adrenergicreceptor pathways, angiotensin II, endothelin-1, andgp130-related cytokines. We will also refer to thedifferential regulation of apoptosis by a p300 protein incardiac myocytes and other cell types.

DIFFERENTIAL REGULATION OF MYOCARDIALCELL APOPTOSIS BY a- AND b-ADRENERGIC

RECEPTOR PATHWAYS

The intracellular pathways for adrenergic signalingin cardiac myocytes begin with binding of NE toG-protein-coupled adrenergic receptors on cell mem-branes. At least nine subtypes of adrenergic receptorshave been identi®ed to date. Of these, the receptors

mainly expressed in cardiac myocytes are b1, b2, and a1.Both b1- and b2-adrenergic receptors are coupled with aGas protein that stimulates adenylyl cyclase activity,leading to the production of cAMP, followed by activa-tion of protein kinase A (PKA). The b2-adrenergic rece-ptor is also coupled with Gi, which inhibits adenylylcyclase activity. Thus, the b2-adrenergic receptor path-way negatively regulates the Gs-dependent pathway,which is activated by b1- and b2-adrenergic receptors.On the other hand, the a1-adrenergic receptor is coupledpredominantly with Gaq. Stimulation of Gaq results inan increase in phospholipase C that activates bothprotein kinase C and inositol 1,4,5-triphosphate kinase.These are followed by activation of the mitogen-acti-vated protein kinase (MAPK) cascade.

NE, an activator ofa- andb-adrenergic receptors, is aninducer of apoptosis in cardiac myocytes. Using a systemof cultured rat cardiac myocytes, we and others haveshown that NE-induced apoptosis is mediated throughthe b1-adrenergic receptor pathway. The cAMP/PKApathway is necessary and suf®cient to mediate b1-adre-nergic agonist-induced myocardial cell apoptosis. Inter-estingly, the cAMP/PKA pathway is anti-apoptotic inother cell types such as PC 12 cells, indicating that thispathway acts as an apoptotic messenger in a cell-typespeci®c manner (Iwai-Kanai et al., 1999). The myocar-dial cell apoptosis induced by b1-adrenergic agonists hasalso been shown in adult rat cardiac myocytes in vivo(Shizukuda et al., 1998). Two kinds of transgenic miceprovided further evidence that the induction of myocar-dial cell apoptosis by b1-adrenergic stimulation resultsin the development of heart failure. One is transgenicmice that overexpress b1-adrenergic receptors in theheart, which results in left ventricular dilation and con-tractile dysfunction with age (Liggett et al., 2000). Theother is mice overexpressing Gsa in myocardium (Genget al., 1999). These mice exhibit myocardial cell apo-ptosis and the development of cardiomyopathy. All ofthese studies indicated thatb1-adrenergic stimulation issuf®cient to induce myocardial cell apoptosis, whichresults in the development of heart failure. In contrast,transgenic mice that overexpress b2-adrenergic recep-tors in the heart do not develop heart failure. Singhand co-workers demonstrated that activation of theb2-adrenergic receptor pathway is involved in theinhibition of myocardial cell apoptosis through Gi-protein (Communal et al., 1999).

Thea1-adrenergic receptor is coupled with Gaq, whichleads to the activation of MAPK cascade. This pathwayplays an important role in the stimulation of a hyper-trophic response. In contrast to the induction of myo-cardial cell apoptosis by the b1-adrenergic receptorpathway, we found that stimulation of a1-adrenergicreceptors antagonized b-adrenergic agonist-inducedapoptosis (Iwai-Kanai et al., 1999). The protective ef-fects of a1-adrenergic agonists on cardiac myocytesagainst apoptosis are mediated, at least in part, throughthe activation of extracellular signal-regulated kinases(ERKs). Similarly, the activation of Gaq through otherreceptors such as endothelin-1 type A receptor inhibitsapoptosis in addition to inducing hypertrophy (Arakiet al., 2000). Supporting the role of Gaq in myocardialcell growth, transgenic mice with four- to ®ve-foldincreases in Gq protein levels in the myocardium exhibit

12 HASEGAWA ET AL.

cardiac hypertrophy associated with a normal lifespan(Adams et al., 1998). Interestingly, those with aneightfold-increase in myocardial Gq levels died fromheart failure at an average age of 11 weeks with amarked induction of myocardial cell apoptosis. Thisobservation led to the notion that excessive activationof Gaq-dependent pathways in cardiac myocytesresults in apoptosis, while adequate activation of thispathway mediates both hypertrophic and anti-apoptoticresponses.

ENDOTHELIN-1 IS A SURVIVAL FACTORAGAINST MYOCARDIAL CELL APOPTOSIS

Endothelin-1 is a 21-residue peptide originally islo-lated from supernatants of vascular endothelial cells(Yanagisawa et al., 1988). While endothelin-1 is mainlyproduced by endothelial cells in the basal state, anumber of cell types can synthesize endothelin-1 inresponse to various stimuli (Shichiri et al., 1991; Itoet al., 1993; Kaddoura et al., 1996; Yamazaki et al.,1996). Endothelin-1 expression in cardiac myocytes isinduced by myocardial stretch, angiotensin II andnorepinephrine (Ito et al., 1993; Kaddoura et al., 1996;Yamazaki et al., 1996). The levels of endothelin-1 inplasma and in ventricular myocardium are markedlyincreased in human and animal models of heart failure(Wei et al., 1994; Sakai et al., 1996; Iwanaga et al., 1998).Endothelin-1 exerts diverse physiological effects in-cluding vasoconstriction and growth-promotion. Endo-thelin-1 is suf®cient to induce the myocardial cellhypertrophy associated with reactivation of the fetalgene program (Shubeita et al., 1990; Ito et al., 1991).These various effects are mediated by two distinctsubtypes of G-protein-coupled heptahelical receptors,endothelin-1 type A and type B receptors, expressed in awide variety of tissues (Arai et al., 1990; Sakurai et al.,1990). Endothelin-1 is a survival factor in activelyproliferating cells such as smooth muscle cells(Wu-Wong et al., 1997) and ®broblasts (Shichiri et al.,1998). This survival effect is also seen in post-mitoticcardiac myocytes (Araki et al., 2000). This effect ismediated mainly through the endothelin-1 type Areceptor-dependent pathway, compatible with cardiacmyocytes that predominantly expressed the type Areceptor. Type A receptor is coupled with both Gq and Gi.The antiapoptotic effects of endothelin-1 are mediated,in part, through activation of Gq and MAPK pathways.Because endothelin-1 accumulates within cardiacmyocytes in failing hearts as shown by immunohisto-chemistry (Sakai et al., 1996; Iwanaga et al., 1998),endogenous ET-1 may function in ``self-protection'' by anautocrine mechanism.

Despite the evidence that endothelin-1 is a protectivefactor against myocardial cell apoptosis, upregulatedexpression of ET-1 in cardiac myocytes apparently playsa role in the deterioration of systolic function duringthe development of heart failure in vivo. Endothelin-1receptor antagonists, bosentan or BQ123, preventremodeling of the heart and have been shown to improvesurvival following myocardial infarction and pressureoverload (Sakai et al., 1996; Iwanaga et al., 1998). These®ndings suggest that the cardiac endothelin-1 pathwayis involved in the development of heart failure indepen-dent of myocardial cell apoptosis. At present, the precise

mechanisms that mediate the induction of heart failureby this pathway are unclear. One possible mechanismmight be endothelin-1-mediated decrease in intracellu-lar cAMP levels by coupling with Gi protein (James et al.,1994). Therefore, chronic elevation of endothelin-1 incardiac myocytes could decrease the systolic function ofthe entire heart.

ANGIOTENSIN II-MEDIATED REGULATION OFAPOPTOSIS BY TYPE 1 AND TYPE 2 RECEPTORS

Angiotensin II is synthesized not only by the systemicrenin±angiotensin system but also by the local renin±angiotensin system in the heart. The effects of angio-tensin II are mediated through two types of receptors,namely type 1 and type 2 receptors (Horiuchi et al.,1997). Type 1 receptor is expressed in the heart, smoothmuscle, kidney, brain, adrenal cortex, and liver. Incontrast, type 2 receptor is expressed widely in varioustissues during the fetal stage and downregulated afterbirth. However, the expression of type 2 receptor isre-induced in vascular injury, myocardial infarction,myocardial hypertrophy, and wounded skin. While bothreceptors belong to the seven-transmembrane receptorsuperfamily, recent evidence has shown that thefunctions of type 1 and type 2 receptors are mutuallyantagonistic. Type 1 receptor is coupled with Gq andpromotes vasoconstriction and cell growth. On the otherhand, type 2 receptor mediates vasodilatation, growthinhibition, and cell differentiation, although preciseintracellular signaling pathways for type 2 receptor areunknown at present. Similarly, apoptosis is differen-tially regulated by these two types of receptors. In PC 12cells, in which angiotensin II type 2 receptor is abun-dantly expressed, growth factor-mediated inhibition ofapoptosis is antagonized by angiotensin II (Yamadaet al., 1996). This effect of apoptosis promotion byangiotensin II is blocked by a type 2 receptor antagonistbut not by a type 1 receptor antagonist. The type 2receptor-dependent pathway downregulates the activa-tion of MAPK by the growth factor resulting in theinactivation of bcl-2 (Horiuchi et al., 1997). In contrast,the type 1 receptor-dependent pathway activates MAPKand inhibits apoptosis in vascular smooth muscle cells(Yamada et al., 1998).

In the heart, angiotensin II is stored in cardiacmyocytes and released from these cells in response tomyocardial stretch (Sadoshima et al., 1993). This auto-crine release of angiotensin II is involved in the stretch-induced myocardial cell hypertrophy. In addition,angiotensin II stimulates proliferation of ®broblasts.These effects are mediated via the type 1 receptor. Bothlocal and systemic renin±angiotensin systems areactivated in heart failure in humans as well as variousanimal models of heart falilure (Wollert and Drexler,1999). Pharmacological blockade of the renin±angio-tensin system is bene®cial in patients with heart failure.Despite the strong evidence that angiotensin II type 1and type 2 receptors play different roles in the regula-tion of apoptosis, and that the type 1 receptor inhibitsapoptosis, the regulation of myocardial cell apoptosis byangiotensin II appears to be unique. In contrast to the®ndings in other cell types, the angiotensin II type 1receptor pathways are involved in the promotion ofapoptosis in cardiac myocytes. Stretch-mediated release

NEUROHORMONES IN MYOCARDIAL APOPTOSIS 13

of angiotensin II induces myocyte apoptosis, which isabolished by losartan, a type 1 receptor antagonist(Kajstura et al., 1997; Leri et al., 1998). Angiotensin IIinduces the expression of p53, whose binding to thepromoter of angiotensinogen, type 1 receptor, p53, andBax was also increased. Therefore, it appears that theactivation of p53 is responsible for stretch-mediated andangiotensin II-dependent apoptosis in cardiac myocytes.These ®ndings suggest that activation of the renin±angiotensin system in heart failure is involved inpromoting myocardial cell apoptosis during the devel-opment of heart failure. This hypothesis is supported byangiotensin-converting enzyme inhibitors reducingmyocardial cell apoptosis as well as blocking theremodeling of the heart in animal models of heartfailure (Li et al., 1997; Goussev et al., 1998). Thus,bene®cial effects of these agents in patients with heartfailure might be attributable, in part, to the inhibition ofmyocardial cell apoptosis.

PROTECTION OF CARDIAC MYOCYTES AGAINSTAPOPTOSIS BY gp130-RELATED CYTOKINES

Recent work has demonstrated that members of afamily of structurally related cytokines, including leu-kemia inhibitory factor and cardiotrophin-1 protectagainst apoptosis as well as induce hypertrophy incardiomyocyte culture (Pennica et al., 1995; Wollertet al., 1996). The receptors in this cytokine family aremultimeric and share the class-speci®c transmembranesignal transducing component gp130 (Hibi et al., 1990;Taga et al., 1992; Ip et al., 1992; Yin et al., 1993; Taga,1996). Signaling is triggered through the homodimer-ization of gp130 (Murakami et al., 1993) or the hetero-dimerization of gp130 with a related transmembranesignal transducer, the leukemia inhibitory factor recep-tor subunit b (Gearing et al., 1991; Davis et al., 1993).Overexpression of both interleukin-6 and its receptorresults in constitutive tyrosine phosphorylation ofgp130 (i.e., activation) in the myocardium and leftventricular hypertrophy in vivo (Hirota et al., 1995).Members of the interleukin-6 cytokine family, includingleukemia inhibitory factor have been shown to activatethe JAK/STAT pathway and phosphorylate STAT3(Akira et al., 1994; Kisimoto et al., 1994; Narazakiet al., 1994; Taga, 1996). It is also clear that this family ofcytokines can activate Ras and MAP kinase cascades(Kisimoto et al., 1994; Heim, 1996; Taga, 1996). Domi-nant negative forms of MEK-1 and a MEK-1-speci®cinhibitor PD098059 block the activation of ERK as wellas cardiotrophin-1-mediated protection against myocar-dial cell apoptosis (Sheng et al., 1997). These ®ndingsindicate that MEK-1/ERK pathways are required for theantiapoptotic effects of gp-130-related cytokines. Cardi-otrophin-1-mediated blockade of myocardial cell apop-tosis is associated with up-regulated expression of anantiapoptotic molecule bcl-xl. STAT1 binds to its speci®cbinding site within the bcl-xl promoter and activatesthis promoter in a sequence-speci®c manner (Fujio et al.,1997). Thus, JAK/STAT as well as MEK-1/ERK path-ways are involved in the protective effects of thesecytokines. Protective effects of gp130-dependent signalson cardiac myocytes are demonstrated in the adult heartin vivo. The mice with cardiac-speci®c gp130 de®ciencyexhibit progressive dilated cardiomyopathy with mas-

sive cardiac myocyte apoptosis when subjected topressure overload (Hirota et al., 1999). This observationclearly demonstrates that pressure overload activatesapoptotic signals in the heart and that gp130-dependentpathways are required for antagonizing these signals. Inaddition, this ®nding also provides evidence thatmyocardial cell apoptosis is suf®cient to induce heartfailure in vivo

ROLE OF p300 IN THE REGULATION OFAPOPTOSIS IN CARDIAC MYOCYTES

AND OTHER CELL TYPES

Cardiac myoctes are highly differentiated and lost theability to undergo mitosis soon after birth. Because ofthese unique characteristics, signaling pathways thatactivate apoptosis in these cells are distinct from thosein other cell types. An interesting feature is the opposingeffects of cAMP on apoptosis in two different cell types.In contrast to the ®ndings in primary cardiac myocytesin culture, the administration of 8-Br-cAMP, a cell-permeable cAMP analogue, completely inhibits serumdeprivation-induced apoptosis in PC12 pheochromo-cytoma cells (Iwai-Kanai et al., 1999). Thus, cAMP-mediated apoptosis in cardiac myocytes might involvesome cell type-speci®c signal transduction mechanisms.One of the candidate molecules involved in the regula-tion of apoptosis in terminally differentiated cells is theadenovirus E1A-associated cellular protein p300. Thep300 protein is classi®ed as a transcriptional coactivatorthat communicates between the enhancer DNA and thebasal transcriptional complex formed on the promoternear the transcription initiation site (Eckner et al.,1994). This communication is required for transcrip-tional activation by the transactivators. Consistent withthis view, the p300 family of transcriptional coactivatorshas been noted to modulate many examples of enhancer-mediated transcription (Chrivia et al., 1993; Arias et al.,1994; Eckner et al., 1994, 1996; Kwok et al., 1994; Aranyet al., 1995; Lee et al., 1995; Lundblad et al., 1995;Missero et al., 1995; Bannister and Kouzarides, 1996;Chakravarti et al., 1996; Dai et al., 1996; Janknecht andNordheim, 1996; Kamei et al., 1996; Oelgeschlager et al.,1996; Trouche and Kouzarides, 1996; Yuan et al., 1996;Puri et al., 1997; Sartorelli et al., 1997). The p300 proteininteracts directly with components of the basal tran-scriptional apparatus (e.g., TFIIB and TBP (Abrahamet al., 1993; Kwok et al., 1994; Yuan et al., 1996) anddiverse enhancer binding proteins. In skeletal muscle,p300/CBP are transcriptional adaptors for MyoD andMEF-2, and these interactions are required for myogen-esis (Eckner et al., 1996; Yuan et al., 1996; Puri et al.,1997; Sartorelli et al., 1997). The p300 protein isinvolved in cardiac-speci®c transcription as a coactiva-tor of zinc-®nger proteins GATA-4/5 (Kakita et al.,1999). In addition to their possible ``bridging function,''p300 proteins possess intrinsic histone acetyl-transfer-ase activity (Bannister and Kouzarides, 1996; Ogryzkoet al., 1996), which is hypothesized to promote a locally``open'' and transcriptionally active chromatin con®g-uration. The p300 protein is a homologue of CBP, aprotein that is associated with, and coactivates thetranscription factor CREB, mediating the induction ofcAMP-responsive promoters (Arany et al., 1995; Lund-blad et al., 1995).

14 HASEGAWA ET AL.

The role of p300 in the regulation of apoptosis appearsto be distinct in cardiac myocytes from other cell types.Fibroblasts prepared from p300 knock-out mice aremore resistant to radiation-induced apoptosis thanthose from wild-type mice (Yao et al., 1998; Yuan et al.,1999). The p300 protein forms a speci®c complex withp53. This suggests a mechanism by which p300 activatesthe expression of Bax and mediates apoptosis as well asmodulates the checkpoint function in the G1 phase of thecell cycle (Avantaggiati et al., 1997; Shikama et al.,1999). In contrast to the ®ndings in ®broblasts, p300appears to play a role in the survival of cardiac myocytes.This is suggested by the study on the effects of theadenovirus E1A oncoprotein on these cells. When E1A isexpressed in differentiated cardiac muscle cells, it re-presses muscle-speci®c gene expression (Kirshenbaumand Schneider, 1995; Liu and Kitsis, 1996; Hasegawaet al., 1997) and induces apoptosis. Studies with E1Amutants indicate that interference with the p300 path-

way is suf®cient to repress muscle-speci®c gene exp-ression and induce apoptosis in cardiac myocytes(Kirshenbaum et al., 1995; Liu et al., 1996; Hasegawaet al., 1997). These experiments suggest that p300proteins are involved both in maintaining cardiac-speci®c gene expression and in myocardial cell survival.Our preliminary ®ndings suggest that a forced expres-sion of p300 inhibits the myocardial cell apoptosisinduced by 8-Br-cAMP. The possible involvement ofthe p300/CBP family in cAMP-mediated apoptosis incardiac myocytes requires further investigation.

CAN BLOCKADE OF MYOCARDIAL CELLAPOPTOSIS PREVENT THE DEVELOPMENT

OF HEART FAILURE?

It is apparent that the occurrence of myocardial cellapoptosis leads to heart failure, or at least worsens thestate of heart failure. However, it is unclear at presentwhether the blockade of apoptosis could prevent the

Fig. 1. Regulation of myocardial cell apoptosis by neurohormonal factors activated in heart failure.

Fig. 2. Differential regulation of myocardial cell growth and apoptosis by a1- and b1-adrenergicpathways.

NEUROHORMONES IN MYOCARDIAL APOPTOSIS 15

development of heart failure. In transgenic mice over-expressing an activated form of calcineurin in the heart,myocyte loss following ischemia-reperfusion is less thanin wild-type mice (De-Windt et al., 2000). Calcineurinactivation plays a role in protection against myocardialcell apoptosis. Nevertheless, the transgenic mice heartswith activated calcineurin exhibit marked dilatationand heart failure. These ®ndings suggest that heartfailure can develop independent of myocardial cellapoptosis. In contrast, doxyrubicin is a potent inducerof myocardial cell apoptosis both in culture and in adultrats in vivo. Thus, myocardial cell apoptosis maysigni®cantly contribute to the development of doxy-rubicin-induced cardiomyopathy. In most cases of end-stage heart failure, the number of cardiac myocytes isdecreased compared with a normal heart. One example,in which a loss of cardiac myocytes is prominent, is thedilated phase of hypertrophic cardiomyopathy. In thiscondition, serum levels of cardiac troponin T arecontinuously high (Sato et al., 2000), suggesting thatcardiac myocyte loss occurs during the transition to thedilated phase. The dilated phase of hypertrophic cardio-myopathy is associated with massive ®brosis and amarked decrease in myocyte number. While the con-tribution of myocardial cell apoptosis varies in differenttypes of heart failure, it is possible that blockade ofapoptosis may prevent the development of some types ofheart failure.

Prevention of apoptosis in heart failure may beachieved by directly blocking the apoptotic signals orby modulating neurohormonal factors that regulateapoptosis. The b-adrenergic pathway but not the a1-adrenergic pathway induces cell type-speci®c apoptosisin cardiac myocytes (Iwai-Kanai et al., 1999). Severalbodies of evidence suggest that activation of thesympathetic nervous system exerts a direct deleteriouseffect on the heart that is independent of the hemody-namic actions of these endogenous mechanisms. Thera-peutic intervention with b-adrenergic receptor blockersfavorably alters the natural history of heart failure, andsuch bene®ts cannot be explained solely by the effect ofthese agents on cardiac contractility and ejection frac-tion. These ®ndings might indicate that apoptosis inhi-bition is one mechanism of the bene®cial effects ofb-adrenergic receptor blockers in patients with heartfailure. Further elucidation of precise signaling path-

ways leading to myocardial cell protection and apoptosismay contribute to the establishment of novel strategiesfor heart failure therapy in humans.

ACKNOWLEDGMENTS

This work was supported in part by grants to K.H.from the Ministry of Education, Science and Culture ofJapan.

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