Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro

BACKGROUND: The intracellular signaling pathways that control cardiomyocyte apoptosis have not been fully defined. Because insulin-like growth factor-1 (IGF-1) prevents cardiomyocyte apoptosis, we examined the role of its downstream signaling molecules in an in vitro model of hypoxia-induced cardiomyocyte apoptosis. METHODS AND RESULTS: Treatment of rat neonatal cardiomyocytes with IGF-1 increased activity of both phosphatidylinositol 3' (PI 3)-kinase and its downstream target, Akt (also known as protein kinase B or PKB). Cardiomyocytes were subjected to hypoxia for 24 hours, and apoptosis was assessed by DNA laddering, TUNEL staining, and ELISA for histone-associated DNA fragments. IGF-1 treatment (100 nmol/L) reduced cardiomyocyte apoptosis, and this effect was inhibited by simultaneous treatment with a PI 3-kinase inhibitor. Cardiomyocytes were infected with either a control adenovirus (Ad.EGFP) or adenoviruses carrying constitutively active forms of PI 3-kinase (Ad.BD110) or Akt (Ad. myr-Akt-HA). Ad.BD110 significantly inhibited apoptosis of hypoxic cardiomyocytes compared with Ad.EGFP (61.0+/-4.6% less DNA fragmentation than in Ad.EGFP-infected cells, P<0.0001). Ad. myr-Akt-HA even more dramatically inhibited apoptosis of hypoxic cardiomyocytes (90.9+/-1.4% less DNA fragmentation than in controls, P<0.0001). CONCLUSIONS: IGF-1 activates PI 3-kinase and Akt in cardiomyocytes. Activated PI 3-kinase and Akt are each sufficient to protect hypoxic cardiomyocytes against apoptosis in vitro. Adenoviral gene transfer provides a useful tool for investigating the role of these signaling pathways in cardiomyocyte apoptosis.     

Urocortin-induced cardiomyocytes hypertrophy is associated with regulation of the GSK-3β pathway

Urocortin-1 (UCN), a member of the corticotropin-releasing factor, is a cardioprotective peptide, and is also involved in cardiac hypertrophy. The involvement of GSK-3β, a pivotal kinase in cardiac hypertrophy, in response to UCN is not yet documented. Cardiomyocytes from adult rats were stimulated for 48 h with UCN. Cell size, protein, and DNA contents were determined. Phosphorylated and total forms GSK-3β and the total amount of β-catenin were quantified by Western immunoblots. The effects of astressin, a UCN competitive receptor antagonist, were also evaluated. UCN increased cell size and the protein-to-DNA ratio, in accordance with a hypertrophic response. This effect was associated with increased phosphorylation of GSK-3β and marked accumulation of β-catenin, a downstream element to GSK-3β. All these effects were prevented by astressin and LY294002, an inhibitor of the phosphatidyl-inositol-3-kinase. UCN-induced cardiomyocytes hypertrophy is associated with regulation of GSK-3β, a pivotal kinase involved in cardiac hypertrophy, in a PI3K-dependent manner. Furthermore, the pharmacological blockade of UCN receptors was able to prevent UCN-induced hypertrophy, which leads to inhibition of the Akt/GSK-3β pathway.     

Tissue kallikrein protects against pressure overload-induced cardiac hypertrophy through kinin B2 receptor and glycogen synthase kinase-3beta activation

OBJECTIVE: We assessed the role of glycogen synthase kinase-3beta (GSK-3beta) and kinin B2 receptor in mediating tissue kallikrein's protective effects against cardiac hypertrophy. METHODS: We investigated the effect and mechanisms of tissue kallikrein using hypertrophic animal models of rats as well as mice deficient in kinin B1 or B2 receptor after aortic constriction (AC). RESULTS: Intramyocardial delivery of adenovirus containing the human tissue kallikrein gene resulted in expression of recombinant kallikrein in rat myocardium. Kallikrein gene delivery improved cardiac function and reduced heart weight/body weight ratio and cardiomyocyte size without affecting mean arterial pressure 28 days after AC. Icatibant and adenovirus carrying a catalytically inactive GSK-3beta mutant (Ad.GSK-3beta-KM) abolished kallikrein's effects. Kallikrein treatment increased cardiac nitric oxide (NO) levels and reduced NAD(P)H oxidase activity and superoxide production. Furthermore, kallikrein reduced the phosphorylation of apoptosis signal-regulating kinase1, mitogen-activated protein kinases (MAPKs), Akt, GSK-3beta, and cAMP-response element binding (CREB) protein, and decreased nuclear factor-kappaB (NF-kappaB) activation in the myocardium. Ad.GSK-3beta-KM abrogated kallikrein's actions on GSK-3beta and CREB phosphorylation and NF-kappaB activation, whereas icatibant blocked all kallikrein's effects. The protective role of kinin B2 receptor in cardiac hypertrophy was further confirmed in kinin receptor knockout mice as heart weight/body weight ratio and cardiomyocyte size increased significantly in kinin B2 receptor knockout mice after AC compared to wild type and B1 receptor knockout mice. CONCLUSIONS: These findings indicate that tissue kallikrein, through kinin B2 receptor and GSK-3beta signaling, protects against pressure overload-induced cardiomyocyte hypertrophy by increased NO formation and oxidative stress-induced Akt-GSK-3beta-mediated signaling events, MAPK and NF-kappaB activation.     

Neuregulin-1 protects ventricular myocytes from anthracycline-induced apoptosis via erbB4-dependent activation of PI3-kinase/Akt

We have found that neuregulin-1beta (NRG-1beta) is expressed in the cardiac microvascular endothelium, and promotes the growth and survival of cardiac myocytes in culture through the activation of erbB2 and erbB4 receptor tyrosine kinases. In this study, we examined the role of NRG-1/erbB signaling in protection of cardiac myocytes from anthracycline-induced apoptosis in vitro to determine the coupling between erbB receptor subtypes and cytoprotective signaling. Treatment of neonatal rat ventricular myocytes with NRG-1beta inhibited daunorubicin-induced apoptosis as shown by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining for DNA fragmentation as well as flow cytometric quantification of apoptotic myocytes. Daunorubicin-induced activation of caspase-3 in cardiomyocytes was similarly inhibited by NRG-1beta. The phosphoinositol-3-kinase (PI3-kinase) inhibitor wortmannin prevented the effects of NRG-1beta on daunorubicin-induced apoptosis and activation of caspase-3. NRG-1beta treatment induced rapid activation of Akt/PKB that was inhibited by wortmannin, and adenoviral-mediated overexpression of a dominant-negative Akt prevented the protective effect of NRG-1beta. Akt activation by NRG-1beta was prevented by the tyrphostin AG1478, which we show inhibits erbB4 activation by NRG-1beta. In contrast, the erbB2-specific tyrphostin AG879 had no effect on NRG-1beta activation of Akt. Myocyte treatment with an activating antibody to erbB2 caused phosphorylation of erbB2, and led to activation of Erk but not Akt. Treatment with the erbB2 antibody had no effect on anthracycline-induced apoptosis. Thus, NRG-1beta protects against anthracycline-induced apoptosis via erbB4-dependent activation of the PI3-kinase/Akt pathway.     

PARP inhibition prevents postinfarction myocardial remodeling and heart failure via the protein kinase C/glycogen synthase kinase-3beta pathway

The inhibition of glycogen synthase kinase-3beta (GSK-3beta) via phosphorylation by Akt or protein kinase C (PKC), or the activation of mitogen-activated protein kinase (MAPK) cascades can play a pivotal role in left ventricular remodeling following myocardial infarction. Our previous data showed that MAPK and phosphatidylinositol-3-kinase/Akt pathways could be modulated by poly(ADP-ribose)polymerase (PARP) inhibition raising the possibility that cardiac hypertrophic signaling responses may be favorably influenced by PARP inhibitors. A novel PARP inhibitor (L-2286) was tested in a rat model of chronic heart failure following isoproterenol-induced myocardial infarction. Subsequently, cardiac hypertrophy and interstitial collagen deposition were assessed; additionally, mitochondrial enzyme activity and the phosphorylation state of GSK-3beta, Akt, PKC and MAPK cascades were monitored. PARP inhibitor (L-2286) treatment significantly reduced the progression of postinfarction heart failure attenuating cardiac hypertrophy and interstitial fibrosis, and preserving the integrity of respiratory complexes. More importantly, L-2286 repressed the hypertrophy-associated increased phosphorylation of panPKC, PKC alpha/betaII, PKC delta and PKC epsilon, which could be responsible for the activation of the antihypertrophic GSK-3beta. This work provides the first evidence that PARP inhibition beneficially modulates the PKC/GSK-3beta intracellular signaling pathway in a rat model of chronic heart failure identifying a novel drug target to treat heart failure.     

Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes

Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility and was shown previously to activate Akt through an undefined mechanism. Here we report Pak1 as a potential PDK2 that is essential for Akt activity in cardiomyocytes. Both Pak1 and Akt can be activated by multiple hypertrophic stimuli or growth factors in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. Pak1 overexpression induces Akt phosphorylation at both Ser473 and Thr308 in cardiomyocytes. Conversely, silencing or inactivating Pak1 gene diminishes Akt phosphorylation in vitro and in vivo. Purified Pak1 can directly phosphorylate Akt only at Ser473, suggesting that Pak1 may be a relevant PDK2 responsible for AKT Ser473 phosphorylation in cardiomyocytes. In addition, Pak1 protects cardiomyocytes from cell death, which is blocked by Akt inhibition. Our results connect two important regulators of cellular physiological functions and provide a potential mechanism for Pak1 signaling in cardiomyocytes.     

Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway

Cardiomyocytes from common experimental animals rapidly exit the cell cycle upon isolation, impeding studies of basic cell biology and applications such as myocardial repair. Here we examined proliferation of cardiomyocytes derived from human and mouse embryonic stem (ES) cells. While mouse ES cell-derived cardiomyocytes showed little proliferation, human cardiomyocytes were highly proliferative under serum-free conditions (15-25% BrdU+/sarcomeric actin+). The cells exhibited only a small serum dose-response, and proliferation gradually slowed with increasing differentiation of the cells. Neither cell density nor different matrix attachment factors affected cardiomyocyte proliferation. Blockade of phosphatidylinositol 3-kinase (PI 3-kinase) and Akt significantly reduced cardiomyocyte proliferation, whereas MEK inhibition had no effect. Antibody blocking of the insulin-like growth factor-1 (IGF-1) receptor significantly inhibited cardiomyocyte proliferation, while addition of IGF-1 or IGF-2 stimulated cardiomyocyte proliferation in a dose-dependent manner. Thus, cardiomyocytes derived from human ES cells proliferate extensively in vitro, and their proliferation appears to be mediated primarily via the PI 3-kinase/Akt signaling pathway, using the IGF-1 receptor as one upstream activator. This system should permit identification of regulatory pathways for human cardiomyocyte proliferation and may facilitate expansion of cardiomyocytes from human ES cells for therapeutic purposes.     

The acute phase protein alpha2-macroglobulin induces rat ventricular cardiomyocyte hypertrophy via ERK1,2 and PI3-kinase/Akt pathways

OBJECTIVE: Alpha2-macroglobulin (alpha2M) is an acute phase protein released to the serum upon challenges such as cardiac hypertrophy and infarction. Here we report on the role of alpha2M in the induction of hypertrophic cell growth, contractile responsiveness of rat ventricular cardiomyocytes, and on the underlying extracellular regulated kinase 1,2 (ERK1,2) and phosphoinositide 3-kinase (PI3-kinase)/Akt pathways. METHODS: Cell volume and cross-sectional areas were assessed as parameters of hypertrophic growth, and real time RT-PCR for the analysis of hypertrophy-related genes was performed. Protein synthesis was analyzed by 14C-phenylalanine incorporation. Activation of ERK1,2, PI3-kinase and Akt was assessed by immunohistochemical analysis of phosphorylated proteins. Contractile responsiveness was investigated by determination of cell shortening following electrical field stimulation. Intracellular calcium concentration [Ca2+]i was determined by fluo-3 microfluorometry. RESULTS: Treatment of ventricular cardiomyocytes for 24 h with alpha2M significantly increased cell volume and protein synthesis as well as expression of hypertrophy-associated genes [brain natriuretic protein (BNP), beta-myosin heavy chain (beta-MHC), myosin light chain-2 (MLC-2), atrial natriuretic factor (ANF), and skeletal alpha-actin]. Comparable effects were achieved by treatment of cells with an antibody directed against the alpha2M-receptor LDL receptor-related protein-1 (LRP-1) and counteracted upon coincubation with receptor-associated protein (RAP), suggesting an involvement of alpha2M-LRP-1 signalling. Furthermore, alpha2M treatment increased sarcoplasmic reticulum Ca2+-ATPase (SERCA-2a) expression, diastolic and systolic [Ca2+]i, and contractile responsiveness after electrical stimulation. Shortly after alpha2M stimulation, activation of ERK1,2, Akt, and PI3-kinase pathways was observed. Consequently, alpha2M-induced protein synthesis was inhibited upon treatment with the ERK1,2 inhibitor UO126 as well as by LY294002 and wortmannin, which inhibit PI3-kinase, and by rapamycin, which inhibits mammalian target of rapamycin (mTOR) downstream of Akt. CONCLUSIONS: Our data show that alpha2M induces hypertrophic cell growth in rat ventricular cardiomyocytes via ERK1,2 and PI3-kinase/Akt and improves cardiac cell function.     

Inhibition of glycogen synthase kinase 3beta during heart failure is protective

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3beta during heart failure could be compensatory.     

Ras, PI3-kinase and mTOR signaling in cardiac hypertrophy

Cardiac hypertrophy involves increased mass (growth) of the heart and a cardinal feature of this condition is increased rates of protein synthesis. Several signaling pathways have been implicated in cardiac hypertrophy including the phosphatidylinositol 3-kinase (PI3K) and Ras/Raf/MEK/Erk pathways. PI3K lies upstream of the mammalian target of rapamycin (mTOR), an important positive regulator of protein synthesis and cell growth. However, recent data suggest that, in response to certain hypertrophic agents, signaling via Ras and MEK/Erk, as well as mTOR, is required for activation of protein synthesis, indicating new connections between these key signaling pathways.     

Distinct roles of GSK-3α and GSK-3β phosphorylation in the heart under pressure overload

Glycogen synthase kinase-3 (GSK-3) is a master regulator of growth and death in cardiac myocytes. GSK-3 is inactivated by hypertrophic stimuli through phosphorylation-dependent and -independent mechanisms. Inactivation of GSK-3 removes the negative constraint of GSK-3 on hypertrophy, thereby stimulating cardiac hypertrophy. N-terminal phosphorylation of the GSK-3 isoforms GSK-3α and GSK-3β by upstream kinases (e.g., Akt) is a major mechanism of GSK-3 inhibition. Nonetheless, its role in mediating cardiac hypertrophy and failure remains to be established. Here we evaluated the role of Serine(S)21 and S9 phosphorylation of GSK-3α and GSK-3β in the regulation of cardiac hypertrophy and function during pressure overload (PO), using GSK-3α S21A knock-in (αKI) and GSK-3β S9A knock-in (βKI) mice. Although inhibition of S9 phosphorylation during PO in the βKI mice attenuated hypertrophy and heart failure (HF), inhibition of S21 phosphorylation in the αKI mice unexpectedly promoted hypertrophy and HF. Inhibition of S21 phosphorylation in GSK-3α, but not of S9 phosphorylation in GSK-3β, caused phosphorylation and down-regulation of G1-cyclins, due to preferential localization of GSK-3α in the nucleus, and suppressed E2F and markers of cell proliferation, including phosphorylated histone H3, under PO, thereby contributing to decreases in the total number of myocytes in the heart. Restoration of the E2F activity by injection of adenovirus harboring cyclin D1 with a nuclear localization signal attenuated HF under PO in the αKI mice. Collectively, our results reveal that whereas S9 phosphorylation of GSK-3β mediates pathological hypertrophy, S21 phosphorylation of GSK-3α plays a compensatory role during PO, in part by alleviating the negative constraint on the cell cycle machinery in cardiac myocytes.     

Adenovirus-mediated overexpression of caveolin-3 inhibits rat cardiomyocyte hypertrophy

Caveolae are omega-shaped organelles of the cell surface. The protein caveolin-3, a structural component of cardiac caveolae, is associated with cellular signaling. To investigate the effect of adenovirus-mediated overexpression of caveolin-3 on hypertrophic responses in cardiomyocytes, we constructed an adenovirus that encoded human wild-type caveolin-3 (Ad.Cav-3), mutant caveolin-3 (Ad.Cav-3Delta), or bacterial beta-galactosidase (Ad.LacZ). This mutant has been reported to cause human limb-girdle muscular dystrophy. It lacks 9 nucleotides in the caveolin scaffolding domain and behaves in a dominant-negative fashion. Rat neonatal cardiomyocytes were infected with the virus and then harvested 36 hours after infection. In noninfected cells, phenylephrine (PE) and endothelin-1 (ET) increased cell size and [3H]leucine incorporation, along with the induction of sarcomeric reorganization and the reexpression of beta-myosin heavy chain, indicating myocyte hypertrophy. Infection with Ad.LacZ had no effect on those parameters. Ad.Cav-3 prevented the PE- and ET-induced increases in cell size, leucine incorporation, sarcomeric reorganization, and reexpression of beta-myosin heavy chain. Ad.Cav-3 also blocked the PE- and ET-induced phosphorylations of extracellular signal-regulated kinases (ERKs) but did not affect c-Jun amino-terminal kinase and p38 mitogen-activated protein kinase activities. In contrast, Ad.Cav-3Delta significantly augmented hypertrophic responses to ET, which were associated with increased ET-induced phosphorylation of ERK1/2. These results suggest that caveolin-3 behaves as a negative regulator of hypertrophic responses, probably through suppression of ERK1/2 activity.     

Putative role of the phosphatidylinositol 3-kinase-Akt signaling pathway in the survival of granulosa cells

Insulin-like growth factor-1 (IGF-1) is an important differentiation and survival factor for granulosa cells. The purpose of this study was to test the hypothesis that IGF-1 promotes survival of porcine granulosa cells by signaling though the phosphatidylinositol (PI) 3-kinase/Akt signal transduction pathway. Treatment with IGF-1 (100 ng/ml) for 10 min stimulated PI 3-kinase and Akt protein kinase activity. IGF-I stimulated the phosphorylation and activation of Akt in a time- and concentration-dependent manner. The PI 3-kinase inhibitors wortmannin and LY294002 blocked IGF-1 induced increases in PI 3-kinase activity and phosphorylation of Akt. Additionally, IGF-1 treatment prevented apoptosis. The survival response to IGF-I was blocked by treatment with either wortmannin or LY294002. These data suggest that IGF-I-induced phosphorylation of Akt is mediated through PI 3-kinase and that inactivation of this pathway results in granulosa cell apoptosis. We conclude that the P1 3-kinase/Akt signaling serves as a functional survival pathway in the ovary.     

Celecoxib modulates hypertrophic signalling and prevents load-induced cardiac dysfunction

In human hearts, the transition from cardiac hypertrophy to advanced heart failure (HF) is accompanied by a tremendous increase in Akt phosphorylation. In non-myocardial tissue, the cyclooxygenase (COX)-2 inhibitor celecoxib has been shown to COX-independently inhibit Akt signalling. We studied the effects of celecoxib on Akt signalling and hypertrophic response in myocardium. In rabbit isolated cardiac myocytes celecoxib concentration-dependently (10-100 micromol/L) inhibited the insulin-induced increase in phosphorylation of Akt and its downstream targets, GSK-3beta and p70 S6 kinase, by reducing the phosphorylation level of the upstream regulator PTEN. Inhibition of Akt signalling was accompanied by a significant suppression of characteristic features of cardiac hypertrophy: Celecoxib concentration-dependently suppressed the agonist-induced enhancement of total protein synthesis and BNP mRNA expression. In mice (C57BL/6NCrl) subjected to left ventricular (LV) pressure overload by aortic banding, celecoxib treatment (50mg x kg-1 x d-1) significantly attenuated LV dilation and contractile dysfunction compared with placebo-treated mice. Moreover, celecoxib significantly reduced mortality 8 weeks after banding. Thus, celecoxib can be used to titrate Akt signalling and hypertrophic response in myocardium. It reduces load-induced LV dilation, contractile dysfunction and mortality in vivo. This may have clinical implications for the prevention and treatment of maladaptive hypertrophy and its progression to HF in humans.