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Targeting survival signaling in heart failure
Targeting survival signaling in heart failure Michael R Morissette and Anthony Rosenzweig Accumulating evidence suggests that apoptosis is not only a common feature of diverse forms of heart failure but also contributes to disease pathogenesis and progression. This contribution of apoptotic signaling to heart failure could reflect not only loss of cardiomyocytes but also dysfunction of surviving cells. The convergence of signaling mechanisms controlling both cardiomyocyte survival and function provides an opportunity for therapeutic strategies that target these pathways. However, significant hurdles must be overcome before the clinical application of these insights becomes possible. Addresses Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Corresponding author: Rosenzweig, A ([email protected])
Current Opinion in Pharmacology 2005, 5:165–170 This review comes from a themed issue on Cardiovascular and renal Edited by Robert Scarborough and George Vlasuk
1471-4892/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2005.01.004
Introduction: cardiomyocyte apoptosis and heart failure In response to specific stimuli, cells can activate intrinsic suicide pathways and undergo programmed cell death or apoptosis, a genetically programmed energy-requiring process mediated in large part through activation of cascades of cysteine-proteases (i.e. caspases). The highly conserved nature of this process has enabled investigators to document biochemical and morphological markers of apoptosis in disease processes. Markers of increased apoptosis have been detected in a wide variety of cardiac conditions, including heart failure [1,2]. As cardiomyocyte regeneration appears generally inadequate to repair injured myocardium, these observations have raised the possibility that apoptosis contributes to the pathogenesis and/or progression of heart failure, and could represent an opportunity for intervention. Support for this perspective is provided by growing evidence from genetically engineered animal models. For example, loss of survival signaling through genetic www.sciencedirect.com
deletion of the glycoprotein 130 (gp130) receptor in a ventricle-specific manner resulted in massive apoptosis and rapid progression to heart failure after aortic constriction [3]. More recently, Wencker et al. [4] demonstrated that cardiac-specific expression of ligand-activatable procaspase-8 was sufficient to induce a lethal dilated cardiomyopathy, which could be prevented by administration of a caspase inhibitor. Importantly, even mice with lowlevel transgene expression and low rates of cardiomyocyte apoptosis comparable to those seen in human heart failure developed cardiomyopathy. In addition, caspase inhibition in outbred rats mediates significant reductions in both apoptosis and infarct size after transient ischemia [5,6]. Finally, enhanced survival signaling through adenoviral gene transfer of the anti-apoptotic Bcl-2 or transgenic overexpression of insulin-like growth factor-I (IGFI) lessens ventricular remodeling after ischemia [7,8]. Thus, activation of apoptotic signaling and even low-level apoptosis can cause cardiac dysfunction, whereas enhanced protective signaling has beneficial effects on cardiac injury and function. These important studies do not address the extent to which these changes might reflect altered function in surviving cells in addition to loss of cardiomyocytes. As discussed below, recent studies highlight the functional effects of these pathways as an interrelated contributor to the phenotypes observed. Moreover, although therapeutic interventions designed to inhibit apoptosis are appealing, significant logistical hurdles exist to clinical application of these approaches. Consideration of the optimal intervention, as well as the delivery, dose, duration and timing of such strategies, will be of critical importance. Here we review the evidence that apoptosis contributes to cardiac dysfunction and heart failure, while focusing on the role of pro-survival signaling via the phosphoinositide 3-kinase (PI3-K)/Akt pathway. Finally, the clinical implications of research in this area are discussed in the context of strategic considerations for delivery, dose, and timing of potential therapeutic interventions.
Cardiac PI3-K and Akt signaling Activation of PI3-K leads to phosphorylation of phosphatidylinositol 4,5-biphosphate to generate phosphatidylinositol 3,4,5-triphosphate, which recruits phosphoinositidedependent kinase-1 to the plasma membrane resulting in the activation of Akt [9]. The PI3-K/Akt pathway was initially shown to promote cellular survival in cell types other than cardiomyocytes [9]. However, many prosurvival cardiomyocyte signaling pathways, such as those regulated by insulin [10], IGF-I [11–13], gp130 cytokines [14], oestrogen [15,16], neuregulin-1 [17] and the heteroCurrent Opinion in Pharmacology 2005, 5:165–170
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Figure 1
Insulin
LIF
IGF-I
CT-1
PE Serotonin
PGF2
17-β Estradiol
Neuregulin
erbB2
Gq-coupled receptors
LIFR gp130
Receptor tyrosine kinases
erbB4
ER Estrogen receptor
PI3-K
Akt
Survival
Current Opinion in Pharmacology
Multiple survival signaling pathways converge on PI3-K/Akt. Agonists that activate Gq-coupled receptors (serotonin, phenylephrine [PE], prostaglandin F2a [PGF2a]), receptor tyrosine kinases (insulin, IGF-I), leukemia inhibitory factor (LIF)/gp 130 receptors (LIF, cardiotrophin-1 [CT-1]), epidermal growth factor erbB2/erbB4 receptors (neuregulin) and oestrogen ERa receptors (17-b estradiol) promote cardiomyocyte survival through the downstream activation of PI3-K and Akt.
trimeric Gq-protein [18], converge on the PI3-K/Akt pathway (Figure 1). We have shown that acute viral overexpression of PI3-K or Akt in cardiomyocytes is sufficient to inhibit hypoxia-induced apoptosis in vitro [19]. Moreover, acute activation of Akt in the heart by in vivo adenoviral gene delivery was sufficient to reduce both apoptosis and infarct size after ischaemic injury [20]. Importantly, acute activation of Akt not only prevented cardiomyocyte apoptosis but also promoted overall cell survival and prevented hypoxia-induced cardiomyocyte dysfunction, thereby resulting in an even greater benefit on overall cardiac function than might otherwise have been predicted [20]. These studies provide proof-of-concept that common signaling pathways contribute not only to apoptosis but also to cardiomyocyte dysfunction, and that a single intervention can mitigate both. Implications and possible explanations for this observation are considered below. Current Opinion in Pharmacology 2005, 5:165–170
Downstream effectors of Akt Many downstream effectors have been shown to mediate the anti-apoptotic effects of Akt (Figure 2) (for review, see [21]); however, we only focus on the ones most relevant to cardiomyocyte biology. The phosphorylation and subsequent inhibition of the pro-apoptotic Bcl-family member, Bad, has been shown to regulate the anti-apoptotic effects of Akt in neonatal cardiomyocytes [22,23] and, more recently, in vivo in the adult heart following gene transfer of kallikrein [24]. However, changes in Bad phosphorylation were not seen after acute gene transfer of Akt to cardiomyocytes or the intact heart [19,20]. The pro-survival effects of nuclear factor-kB (NFkB) can be mediated downstream of Akt via direct phosphorylation of inhibitor of kB kinase (IKKb), which allows NFkB to translocate to the nucleus where it can transcriptionally upregulate anti-apoptotic target genes [25]. Overexpression of IKKb is sufficient to activate NFkB and prevent www.sciencedirect.com
Targeting survival signaling in heart failure Morissette and Rosenzweig
Figure 2
PI3-Kinase
eNOS NO
SGK1
Akt GSK-3β
IKKs
NFκB
FOXO3α
Bad Caspase-9
kallikrein and adrenomedullin in the setting of ischemia/ reperfusion-induced myocardial injury [24,36]. Recently, the importance of PI3-K/Akt-mediated inhibition of GSK-3b in the heart has been underscored by the discovery that it was shown to promote cardiomyocyte survival by inhibiting the mitochondrial transition pore, an important distal regulator of apoptosis [37]. Thus, many downstream effectors of Akt might contribute to its pro-survival effects in cardiomyocytes. It seems likely that the benefits of Akt activation are a result of the combined actions (and interactions) of several of these pathways, rather than one single effector.
Preservation of cardiomyocyte function
Survival
Current Opinion in Pharmacology
Downsteam effectors of Akt and SGK1 that modulate survival. Akt and SGK1 are activated downstream of PI3-K, which leads to enhanced survival through multiple effectors including eNOS, IKKb, Bad, caspase-9, FOXO3a and GSK-3b.
cardiomyocyte death and dysfunction following hypoxic stress [26]. In cardiomyocytes, NFkB was shown to mediate the protective effects of gp130 receptor activation [27] in an Akt-dependent manner and, more recently, downstream of the 5-hydroxytrypamine 2B receptor survival pathway [28]. However, transcriptional upregulation of an NFkB-dependent pattern of genes is not seen during either acute cardiac gene transfer [20] or chronic cardiac-restricted transgenic Akt expression [29], which one would expect with activation of this transcription factor. Akt has been shown to inhibit caspase-9 in immortalized cell lines, but the relevance of this to survival signaling in the heart is not yet known [30]. Akt-mediated phosphorylation of forkhead box O3a (FOXO3a), formally known as forkhead-like protein-1, promotes cell survival via nuclear exclusion and prevention of transcriptional upregulation of a subset of pro-apoptotic genes [31]. Although an increase in FOXO3a phosphorylation was reported in female mouse hearts concomitantly with increased Akt phosphorylation, the contribution of FOXO3a downstream of Akt to cardiomyocyte survival is unclear [15]. Nitric oxide (NO) might also mediate some anti-apoptotic effects of Akt. Akt activates endothelial NO synthase (eNOS), resulting in increased NO release [32,33]; this activation of eNOS has been shown to be critical for insulin-mediated reduction of cardiomyocyte apoptosis following ischemia-reperfusion [34]. Glycogen synthase kinase-3 (GSK-3) is also phosphorylated (and thereby inhibited) by Akt [35]. This pathway has been shown to mediate the anti-apoptotic effects of www.sciencedirect.com
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Inhibition of cardiomyocyte apoptosis would hold little clinical promise if it simply resulted in increased survival of dysfunctional cardiomyocytes. However, as mentioned above, acute Akt activation not only reduced apoptosis but importantly preserved cardiac function [20]. Such studies provide a conceptual demonstration that convergent signaling mechanisms control both cardiomyocyte survival and function, and led us to hypothesize that overtly apoptotic cardiomyocytes may represent a small fraction of the cells in which these signaling mechanisms are deranged and contributing to dysfunction. The implications are that simply counting ‘apoptotic’ cells could underestimate the contribution of these pathways to disease pathogenesis and that restoration of these signaling pathways might have an even greater benefit than anticipated [20]. In this context, it is interesting to note that antibodies to the epidermal growth factor 2 receptor (which can cause heart failure when used as a chemotherapeutic agent [38,39]) cause only a low level of apoptosis but a more dramatic activation of apoptotic signaling and mitochondrial dysfunction [40]. It seems likely that the more dramatic mitochondrial dysfunction observed represents a significant contributor to cardiac dysfunction in addition to the actual loss of cardiomyocytes. Moreover, activation of caspases in cardiomyocytes can itself induce cardiomyocyte dysfunction through cleavage of contractile proteins [41]. Thus, altered apoptotic signaling can mediate significant effects on cardiomyocyte function without culminating in cell death. Support for extrapolation of this model to humans is provided by studies in failing human hearts, in which extensive activation of mitochondrial apoptotic signaling has been seen with relatively little overt apoptosis [42]. Overall, such data from clinical studies and animal models suggest that interventions targeted to deranged apoptotic signaling could have substantial benefits on cardiac function.
Moderation of signaling intensity Despite the encouraging results cited above, caution in pursuing this strategy is prudent as several lines of evidence suggest that excessive activation of pro-survival pathways may have adverse consequences. One example of this is provided by studies of Gaq signaling in cardiCurrent Opinion in Pharmacology 2005, 5:165–170
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omyocytes in vitro, in which moderate activation of Gaq is anti-apoptotic but additional stimulation results in a marked increase in apoptosis [43]. In vivo moderate (fourfold) cardiac overexpression of Gaq leads to stable hypertrophy, whereas higher expression levels (25-fold) result in apoptosis and heart failure during peripartum stress (in pregnant female mice [43]) or pressure overload [44]. Interestingly, high levels of Gaq activation resulted in depletion of phosphatidylinositol 4,5-biphosphate, ultimately leading to a decrease in Akt phosphorylation (below control levels) in cardiomyocytes in vitro and increased apoptosis [45]. However, although acute Akt activation appears consistently beneficial in cardiomyocytes, chronic activation might itself have adverse consequences. Interestingly, during the generation of transgenic mice with cardiacspecific expression of Akt, we observed that the initial three founder mice died with massive cardiac dilation, supporting the idea that high levels of Akt activation in the heart could be maladaptive [46]. Additionally, we have recently found that chronic overexpression of Akt in the heart is functionally maladaptive in the setting of ischemia/reperfusion, owing to negative feedback on the PI3-K survival pathway, suggesting that chronic activation of Akt may be therapeutically undesirable especially during times of increased stress (Nagoshi et al., unpublished). These findings might help explain the otherwise paradoxical observation that phosphorylation of Akt is increased in failing human hearts [47]. Moreover, these data suggest that activation of other PI3-K-dependent but Akt-independent pathways is critical for long-term preservation of cardiomyocyte survival and function. One Akt-independent downstream effector of PI3-K that might play a role in this context is serum and glucocorticoid-responsive kinase-1 (SGK1). We have recently found that SGK1 inhibits cardiomyocyte apoptosis and contributes to the protective effects of IGF-I [48] (Figure 2). Another effector is integrin-linked kinase (ILK), which functions downstream of PI3-K and either upstream of Akt or independently of Akt. ILK has been implicated in cardiac cell survival after coronary ligation in mice downstream of thymosin b4, which preserved in vivo cardiac function [49]. Ultimately, it might be important to simultaneously activate several (if not all) of these PI3-K-dependent pathways to achieve the full cardioprotective phenotype. Support for this approach is provided by transgenic mice overexpressing IGF-I in the heart, which are protected against ischaemic injury and adverse remodeling [8], as well as cardiomyopathy [50], and do not appear to suffer the adverse feedback inhibition seen in Akt transgenics (Nagoshi et al., unpublished).
Therapeutic considerations Although targeting apoptotic signaling could mediate the beneficial effects on cardiomyocyte survival and function, Current Opinion in Pharmacology 2005, 5:165–170
a variety of concerns are raised by consideration of clinical application of these strategies. As noted above, duration and extent of signaling modulation may be critical determinants of outcome. In addition, systemic activation of anti-apoptotic pathways could have pleiotropic effects, including potentially the promotion of cancer. Thus, it might be desirable to limit activation of pro-survival signaling to the heart. One possible solution to this dilemma would be to manipulate survival signaling pathways through local genetic or pharmacological interventions. For example, we have demonstrated recently that acute cardiac gene transfer of IGF-I reduces cardiac injury after ischemia without elevating systemic levels of IGF-I [51]. Moreover, IGF-I secreted after gene transfer protects cardiomyocytes in both an autocrine and paracrine manner [51], and does not induce feedback inhibition of PI3-K signaling (Nagoshi et al., unpublished).
Conclusions Several lines of evidence suggest that apoptosis and alterations in apoptotic signaling contribute to the pathogenesis of heart failure. It seems likely that this occurs both through direct loss of cardiac myocytes and through dysfunction of surviving cells. Although these observations prompt consideration of therapeutic approaches targeting apoptotic pathways in heart failure, significant barriers exist to this goal. These include concerns about the unknown long-term effects of high-level activation of these pathways both in the heart and systemically, as well as the need to identify optimal strategies for modulating cardiomyocyte survival. Ongoing efforts to fully elucidate the mechanisms of cardiomyocyte death and dysfunction in heart failure will provide an essential foundation for consideration of such therapeutic strategies.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Targeting survival signaling in heart failure Morissette and Rosenzweig
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15. Camper-Kirby D, Welch S, Walker A, Shiraishi I, Setchell KD, Schaefer E, Kajstura J, Anversa P, Sussman MA: Myocardial Akt activation and gender: increased nuclear activity in females versus males. Circ Res 2001, 88:020-027.
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16. Patten RD, Pourati I, Aronovitz MJ, Baur J, Celestin F, Chen X, Michael A, Haq S, Nuedling S, Grohe C et al.: 17beta-estradiol reduces cardiomyocyte apoptosis in vivo and in vitro via activation of phospho-inositide-3 kinase/Akt signaling. Circ Res 2004, 95:692-699.
34. Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL: Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation 2002, 105:1497-1502.
17. Fukazawa R, Miller TA, Kuramochi Y, Frantz S, Kim YD, Marchionni MA, Kelly RA, Sawyer DB: Neuregulin-1 protects ventricular myocytes from anthracycline-induced apoptosis via erbB4dependent activation of PI3-kinase/Akt. J Mol Cell Cardiol 2003, 35:1473-1479.
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18. Naga Prasad SV, Esposito G, Mao L, Koch WJ, Rockman HA: Gbetagamma-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy. J Biol Chem 2000, 275:4693-4698. 19. Matsui T, Li L, del Monte F, Fukui Y, Franke T, Hajjar R, Rosenzweig A: Adenoviral gene transfer of activated PI 3-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro. Circulation 1999, 100:2373-2379. 20. Matsui T, Tao J, del Monte F, Lee K-H, Li L, Picard M, Force TL, Franke TF, Hajjar RJ, Rosenzweig A: Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation 2001, 104:330-335. 21. Brazil DP, Park J, Hemmings BA: PKB binding proteins. Getting in on the Akt. Cell 2002, 111:293-303. 22. Aikawa R, Nawano M, Gu Y, Katagiri H, Asano T, Zhu W, Nagai R, Komuro I: Insulin prevents cardiomyocytes from oxidative stress-induced apoptosis through activation of PI3 kinase/ Akt. Circulation 2000, 102:873-879. www.sciencedirect.com
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Current Opinion in Pharmacology 2005, 5:165–170 This review comes from a themed issue on Cardiovascular and renal Edited by Robert Scarborough and George Vlasuk
1471-4892/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2005.01.004
Introduction: cardiomyocyte apoptosis and heart failure In response to specific stimuli, cells can activate intrinsic suicide pathways and undergo programmed cell death or apoptosis, a genetically programmed energy-requiring process mediated in large part through activation of cascades of cysteine-proteases (i.e. caspases). The highly conserved nature of this process has enabled investigators to document biochemical and morphological markers of apoptosis in disease processes. Markers of increased apoptosis have been detected in a wide variety of cardiac conditions, including heart failure [1,2]. As cardiomyocyte regeneration appears generally inadequate to repair injured myocardium, these observations have raised the possibility that apoptosis contributes to the pathogenesis and/or progression of heart failure, and could represent an opportunity for intervention. Support for this perspective is provided by growing evidence from genetically engineered animal models. For example, loss of survival signaling through genetic www.sciencedirect.com
deletion of the glycoprotein 130 (gp130) receptor in a ventricle-specific manner resulted in massive apoptosis and rapid progression to heart failure after aortic constriction [3]. More recently, Wencker et al. [4] demonstrated that cardiac-specific expression of ligand-activatable procaspase-8 was sufficient to induce a lethal dilated cardiomyopathy, which could be prevented by administration of a caspase inhibitor. Importantly, even mice with lowlevel transgene expression and low rates of cardiomyocyte apoptosis comparable to those seen in human heart failure developed cardiomyopathy. In addition, caspase inhibition in outbred rats mediates significant reductions in both apoptosis and infarct size after transient ischemia [5,6]. Finally, enhanced survival signaling through adenoviral gene transfer of the anti-apoptotic Bcl-2 or transgenic overexpression of insulin-like growth factor-I (IGFI) lessens ventricular remodeling after ischemia [7,8]. Thus, activation of apoptotic signaling and even low-level apoptosis can cause cardiac dysfunction, whereas enhanced protective signaling has beneficial effects on cardiac injury and function. These important studies do not address the extent to which these changes might reflect altered function in surviving cells in addition to loss of cardiomyocytes. As discussed below, recent studies highlight the functional effects of these pathways as an interrelated contributor to the phenotypes observed. Moreover, although therapeutic interventions designed to inhibit apoptosis are appealing, significant logistical hurdles exist to clinical application of these approaches. Consideration of the optimal intervention, as well as the delivery, dose, duration and timing of such strategies, will be of critical importance. Here we review the evidence that apoptosis contributes to cardiac dysfunction and heart failure, while focusing on the role of pro-survival signaling via the phosphoinositide 3-kinase (PI3-K)/Akt pathway. Finally, the clinical implications of research in this area are discussed in the context of strategic considerations for delivery, dose, and timing of potential therapeutic interventions.
Cardiac PI3-K and Akt signaling Activation of PI3-K leads to phosphorylation of phosphatidylinositol 4,5-biphosphate to generate phosphatidylinositol 3,4,5-triphosphate, which recruits phosphoinositidedependent kinase-1 to the plasma membrane resulting in the activation of Akt [9]. The PI3-K/Akt pathway was initially shown to promote cellular survival in cell types other than cardiomyocytes [9]. However, many prosurvival cardiomyocyte signaling pathways, such as those regulated by insulin [10], IGF-I [11–13], gp130 cytokines [14], oestrogen [15,16], neuregulin-1 [17] and the heteroCurrent Opinion in Pharmacology 2005, 5:165–170
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Figure 1
Insulin
LIF
IGF-I
CT-1
PE Serotonin
PGF2
17-β Estradiol
Neuregulin
erbB2
Gq-coupled receptors
LIFR gp130
Receptor tyrosine kinases
erbB4
ER Estrogen receptor
PI3-K
Akt
Survival
Current Opinion in Pharmacology
Multiple survival signaling pathways converge on PI3-K/Akt. Agonists that activate Gq-coupled receptors (serotonin, phenylephrine [PE], prostaglandin F2a [PGF2a]), receptor tyrosine kinases (insulin, IGF-I), leukemia inhibitory factor (LIF)/gp 130 receptors (LIF, cardiotrophin-1 [CT-1]), epidermal growth factor erbB2/erbB4 receptors (neuregulin) and oestrogen ERa receptors (17-b estradiol) promote cardiomyocyte survival through the downstream activation of PI3-K and Akt.
trimeric Gq-protein [18], converge on the PI3-K/Akt pathway (Figure 1). We have shown that acute viral overexpression of PI3-K or Akt in cardiomyocytes is sufficient to inhibit hypoxia-induced apoptosis in vitro [19]. Moreover, acute activation of Akt in the heart by in vivo adenoviral gene delivery was sufficient to reduce both apoptosis and infarct size after ischaemic injury [20]. Importantly, acute activation of Akt not only prevented cardiomyocyte apoptosis but also promoted overall cell survival and prevented hypoxia-induced cardiomyocyte dysfunction, thereby resulting in an even greater benefit on overall cardiac function than might otherwise have been predicted [20]. These studies provide proof-of-concept that common signaling pathways contribute not only to apoptosis but also to cardiomyocyte dysfunction, and that a single intervention can mitigate both. Implications and possible explanations for this observation are considered below. Current Opinion in Pharmacology 2005, 5:165–170
Downstream effectors of Akt Many downstream effectors have been shown to mediate the anti-apoptotic effects of Akt (Figure 2) (for review, see [21]); however, we only focus on the ones most relevant to cardiomyocyte biology. The phosphorylation and subsequent inhibition of the pro-apoptotic Bcl-family member, Bad, has been shown to regulate the anti-apoptotic effects of Akt in neonatal cardiomyocytes [22,23] and, more recently, in vivo in the adult heart following gene transfer of kallikrein [24]. However, changes in Bad phosphorylation were not seen after acute gene transfer of Akt to cardiomyocytes or the intact heart [19,20]. The pro-survival effects of nuclear factor-kB (NFkB) can be mediated downstream of Akt via direct phosphorylation of inhibitor of kB kinase (IKKb), which allows NFkB to translocate to the nucleus where it can transcriptionally upregulate anti-apoptotic target genes [25]. Overexpression of IKKb is sufficient to activate NFkB and prevent www.sciencedirect.com
Targeting survival signaling in heart failure Morissette and Rosenzweig
Figure 2
PI3-Kinase
eNOS NO
SGK1
Akt GSK-3β
IKKs
NFκB
FOXO3α
Bad Caspase-9
kallikrein and adrenomedullin in the setting of ischemia/ reperfusion-induced myocardial injury [24,36]. Recently, the importance of PI3-K/Akt-mediated inhibition of GSK-3b in the heart has been underscored by the discovery that it was shown to promote cardiomyocyte survival by inhibiting the mitochondrial transition pore, an important distal regulator of apoptosis [37]. Thus, many downstream effectors of Akt might contribute to its pro-survival effects in cardiomyocytes. It seems likely that the benefits of Akt activation are a result of the combined actions (and interactions) of several of these pathways, rather than one single effector.
Preservation of cardiomyocyte function
Survival
Current Opinion in Pharmacology
Downsteam effectors of Akt and SGK1 that modulate survival. Akt and SGK1 are activated downstream of PI3-K, which leads to enhanced survival through multiple effectors including eNOS, IKKb, Bad, caspase-9, FOXO3a and GSK-3b.
cardiomyocyte death and dysfunction following hypoxic stress [26]. In cardiomyocytes, NFkB was shown to mediate the protective effects of gp130 receptor activation [27] in an Akt-dependent manner and, more recently, downstream of the 5-hydroxytrypamine 2B receptor survival pathway [28]. However, transcriptional upregulation of an NFkB-dependent pattern of genes is not seen during either acute cardiac gene transfer [20] or chronic cardiac-restricted transgenic Akt expression [29], which one would expect with activation of this transcription factor. Akt has been shown to inhibit caspase-9 in immortalized cell lines, but the relevance of this to survival signaling in the heart is not yet known [30]. Akt-mediated phosphorylation of forkhead box O3a (FOXO3a), formally known as forkhead-like protein-1, promotes cell survival via nuclear exclusion and prevention of transcriptional upregulation of a subset of pro-apoptotic genes [31]. Although an increase in FOXO3a phosphorylation was reported in female mouse hearts concomitantly with increased Akt phosphorylation, the contribution of FOXO3a downstream of Akt to cardiomyocyte survival is unclear [15]. Nitric oxide (NO) might also mediate some anti-apoptotic effects of Akt. Akt activates endothelial NO synthase (eNOS), resulting in increased NO release [32,33]; this activation of eNOS has been shown to be critical for insulin-mediated reduction of cardiomyocyte apoptosis following ischemia-reperfusion [34]. Glycogen synthase kinase-3 (GSK-3) is also phosphorylated (and thereby inhibited) by Akt [35]. This pathway has been shown to mediate the anti-apoptotic effects of www.sciencedirect.com
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Inhibition of cardiomyocyte apoptosis would hold little clinical promise if it simply resulted in increased survival of dysfunctional cardiomyocytes. However, as mentioned above, acute Akt activation not only reduced apoptosis but importantly preserved cardiac function [20]. Such studies provide a conceptual demonstration that convergent signaling mechanisms control both cardiomyocyte survival and function, and led us to hypothesize that overtly apoptotic cardiomyocytes may represent a small fraction of the cells in which these signaling mechanisms are deranged and contributing to dysfunction. The implications are that simply counting ‘apoptotic’ cells could underestimate the contribution of these pathways to disease pathogenesis and that restoration of these signaling pathways might have an even greater benefit than anticipated [20]. In this context, it is interesting to note that antibodies to the epidermal growth factor 2 receptor (which can cause heart failure when used as a chemotherapeutic agent [38,39]) cause only a low level of apoptosis but a more dramatic activation of apoptotic signaling and mitochondrial dysfunction [40]. It seems likely that the more dramatic mitochondrial dysfunction observed represents a significant contributor to cardiac dysfunction in addition to the actual loss of cardiomyocytes. Moreover, activation of caspases in cardiomyocytes can itself induce cardiomyocyte dysfunction through cleavage of contractile proteins [41]. Thus, altered apoptotic signaling can mediate significant effects on cardiomyocyte function without culminating in cell death. Support for extrapolation of this model to humans is provided by studies in failing human hearts, in which extensive activation of mitochondrial apoptotic signaling has been seen with relatively little overt apoptosis [42]. Overall, such data from clinical studies and animal models suggest that interventions targeted to deranged apoptotic signaling could have substantial benefits on cardiac function.
Moderation of signaling intensity Despite the encouraging results cited above, caution in pursuing this strategy is prudent as several lines of evidence suggest that excessive activation of pro-survival pathways may have adverse consequences. One example of this is provided by studies of Gaq signaling in cardiCurrent Opinion in Pharmacology 2005, 5:165–170
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omyocytes in vitro, in which moderate activation of Gaq is anti-apoptotic but additional stimulation results in a marked increase in apoptosis [43]. In vivo moderate (fourfold) cardiac overexpression of Gaq leads to stable hypertrophy, whereas higher expression levels (25-fold) result in apoptosis and heart failure during peripartum stress (in pregnant female mice [43]) or pressure overload [44]. Interestingly, high levels of Gaq activation resulted in depletion of phosphatidylinositol 4,5-biphosphate, ultimately leading to a decrease in Akt phosphorylation (below control levels) in cardiomyocytes in vitro and increased apoptosis [45]. However, although acute Akt activation appears consistently beneficial in cardiomyocytes, chronic activation might itself have adverse consequences. Interestingly, during the generation of transgenic mice with cardiacspecific expression of Akt, we observed that the initial three founder mice died with massive cardiac dilation, supporting the idea that high levels of Akt activation in the heart could be maladaptive [46]. Additionally, we have recently found that chronic overexpression of Akt in the heart is functionally maladaptive in the setting of ischemia/reperfusion, owing to negative feedback on the PI3-K survival pathway, suggesting that chronic activation of Akt may be therapeutically undesirable especially during times of increased stress (Nagoshi et al., unpublished). These findings might help explain the otherwise paradoxical observation that phosphorylation of Akt is increased in failing human hearts [47]. Moreover, these data suggest that activation of other PI3-K-dependent but Akt-independent pathways is critical for long-term preservation of cardiomyocyte survival and function. One Akt-independent downstream effector of PI3-K that might play a role in this context is serum and glucocorticoid-responsive kinase-1 (SGK1). We have recently found that SGK1 inhibits cardiomyocyte apoptosis and contributes to the protective effects of IGF-I [48] (Figure 2). Another effector is integrin-linked kinase (ILK), which functions downstream of PI3-K and either upstream of Akt or independently of Akt. ILK has been implicated in cardiac cell survival after coronary ligation in mice downstream of thymosin b4, which preserved in vivo cardiac function [49]. Ultimately, it might be important to simultaneously activate several (if not all) of these PI3-K-dependent pathways to achieve the full cardioprotective phenotype. Support for this approach is provided by transgenic mice overexpressing IGF-I in the heart, which are protected against ischaemic injury and adverse remodeling [8], as well as cardiomyopathy [50], and do not appear to suffer the adverse feedback inhibition seen in Akt transgenics (Nagoshi et al., unpublished).
Therapeutic considerations Although targeting apoptotic signaling could mediate the beneficial effects on cardiomyocyte survival and function, Current Opinion in Pharmacology 2005, 5:165–170
a variety of concerns are raised by consideration of clinical application of these strategies. As noted above, duration and extent of signaling modulation may be critical determinants of outcome. In addition, systemic activation of anti-apoptotic pathways could have pleiotropic effects, including potentially the promotion of cancer. Thus, it might be desirable to limit activation of pro-survival signaling to the heart. One possible solution to this dilemma would be to manipulate survival signaling pathways through local genetic or pharmacological interventions. For example, we have demonstrated recently that acute cardiac gene transfer of IGF-I reduces cardiac injury after ischemia without elevating systemic levels of IGF-I [51]. Moreover, IGF-I secreted after gene transfer protects cardiomyocytes in both an autocrine and paracrine manner [51], and does not induce feedback inhibition of PI3-K signaling (Nagoshi et al., unpublished).
Conclusions Several lines of evidence suggest that apoptosis and alterations in apoptotic signaling contribute to the pathogenesis of heart failure. It seems likely that this occurs both through direct loss of cardiac myocytes and through dysfunction of surviving cells. Although these observations prompt consideration of therapeutic approaches targeting apoptotic pathways in heart failure, significant barriers exist to this goal. These include concerns about the unknown long-term effects of high-level activation of these pathways both in the heart and systemically, as well as the need to identify optimal strategies for modulating cardiomyocyte survival. Ongoing efforts to fully elucidate the mechanisms of cardiomyocyte death and dysfunction in heart failure will provide an essential foundation for consideration of such therapeutic strategies.
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