Akt-mediated Cardiomyocyte Survival Pathways Are Compromised by Gαq-induced Phosphoinositide 4,5-Bisphosphate Depletion*

Expression of the wild type α subunit of Gq (GqWT) in cardiomyocytes induces hypertrophy, whereas a constitutively active Gαq subunit (GqQ209L) induces apoptosis. Akt phosphorylation increases with GqWT expression but is markedly attenuated in cardiomyocytes expressing GqQ209L or in those expressing GqWT and treated with agonist. A membrane-targeted Akt rescues GqQ209L-expressing cardiomyocytes from apoptotic cell death. In contrast, leukemia inhibitory factor fails to activate Akt or promote cell survival in these cells. Association of Akt and PDK-1 with the membrane is also diminished in GqQ209L-expressing cardiomyocytes. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the primary regulator of Akt, increases significantly in GqWT-expressing cells but not in cardiomyocytes expressing GqQ209L. Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), the immediate precursor of PIP3, are also markedly lower in GqQ209L-expressing compared to control cells. Expression of a GqQ209L mutant that has diminished capacity to activate phospholipase C does not decrease PIP2 or Akt or induce apoptosis. In transgenic mice with cardiac Gαq overexpression, heart failure and increased cardiomyocyte apoptosis develop during the peripartal period. Akt phosphorylation and PIP2 levels decrease concomitantly. Our findings suggest that an Akt-mediated cell survival pathway is compromised by the diminished availability of PIP2 elicited by pathological levels of Gq activity.

G␣ q signaling plays a key role in the hypertrophic growth of cardiomyocytes. Hormones that act through receptors coupled to the heterotrimeric protein G q (e.g. norepinephrine, PGF 2␣ , 1 endothelin, and angiotensin II) induce cardiomyocyte hypertrophy in vitro (1). Further evidence for a physiological role of G␣ q activation is that blocking G q signaling in vivo prevents pressure-overload hypertrophy (2,3). Hypertrophy can transition to heart failure under conditions of enhanced G␣ q signaling induced by overexpression of G q -coupled receptors, of phospholipase C-␤ (PLC), of constitutively active G␣ q , or of wild type G␣ q in combination with stress (4 -11). Thus studies in isolated cardiomyocytes and in transgenic animals have led to the conclusion that although G␣ q signaling is critical for cardiac hypertrophy, prolonged or marked enhancement of G q activity is deleterious to the heart.
Our laboratory has characterized a model of in vitro cardiomyocyte hypertrophy and apoptosis by expressing the ␣ subunit of G q (8). When the wild type ␣ subunit (G q WT) is expressed by adenoviral infection, neonatal rat ventricular myocytes (NRVMs) undergo hypertrophy (8). In contrast, when the constitutively active ␣ subunit (G q Q209L) is expressed, or G q WTexpressing cardiomyocytes are treated with G q -coupled receptor agonists, the NRVMs undergo apoptosis (8,12). Varying the amount of G␣ q signaling in myocytes therefore results in distinct cellular phenotypes. Although modest levels of signaling induce hypertrophy, at higher levels survival is diminished and apoptosis ensues. This provides a paradigm for elucidating the molecular mechanisms by which G␣ q signaling can transition from stimulation of hypertrophic growth to the development of apoptosis.
We demonstrated previously (12) that expression of G q Q209L leads to apoptosis through mitochondrial permeability (PT) pore opening, cytochrome c release, and caspase activation. Sustained G␣ q signaling, beyond the level that causes hypertrophy, may also compromise normal survival pathways. A well documented cell survival pathway is the PI3K/phosphoinositide-dependent kinase-1/Akt (PI3K/PDK-1/Akt) pathway (13). PI3K generates phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) from phosphatidylinositol 4,5bisphosphate (PIP 2 ) to stimulate the membrane colocalization of PDK-1 and Akt (14 -16). Akt/protein kinase B is phosphorylated by PDK-1 at Thr-308 and subsequently autophosphorylates at Ser-473 (17). In cardiomyocytes, Akt has been shown to be activated by agonists that stimulate tyrosine kinase receptors (e.g. insulin and insulin-like growth factor (15,18)) and by cytokines (e.g. cardiotrophin-1 (CT-1) and leukemia inhibitory factor (LIF) (19 -21)). Akt has been reported to protect cardiomyocytes from apoptosis induced by serum deprivation or hypoxia (14,15). Furthermore, in vivo gene transfer of activated Akt protects against cell death and the development of cardiomyopathies caused by ischemiareperfusion and doxorubicin treatment (22)(23)(24). The critical role of Akt in cardioprotection underscores the need to examine the regulation of PI3K, PIP 3 formation, and PDK-1 activity and determine how these control Akt activation in cardiomyocytes.
We report here that increasing G q signaling to pathological levels results in a loss of the protective PI3K/Akt signaling pathway. This occurs at a time point that precedes the appearance of apoptotic markers such as cytochrome c release and nuclear fragmentation. This contrasts directly with the enhanced Akt phosphorylation seen in cardiomyocytes that undergo hypertrophy in response to expression of G q WT. Our investigation into the pathways leading to the down-regulation of Akt activation in cardiomyocytes expressing G q Q209L revealed an unexpected decrease in cellular levels of PIP 2 , the substrate for both PLC and PI3K, which appeared to be related to the G q -mediated increase in PLC activity. Importantly, in vivo studies also revealed decreased Akt phosphorylation and PIP 2 levels in a G q -expressing model of heart failure. We suggest that PIP 3 generation and Akt activation are influenced by the availability of PIP 2 and that this serves as a nodal point in determining the balance between hypertrophic and apoptotic pathways.

EXPERIMENTAL PROCEDURES
Cardiomyocyte Culture and Adenoviral Infection-NRVMs were prepared and infected with adenoviruses as described previously (8). Briefly, cardiomyocytes were plated at a density of 1.5 ϫ 10 6 cells per 6-cm dish or 3 ϫ 10 5 per well of a 6-well plate in serum-containing media overnight. The cells were then washed, and the medium was replaced with serum-free medium supplemented with insulin/transferrin/selenium (ITS). Cells were infected with adenoviruses at 200 -500 viral particles/cell for 16 -18 h or as indicated. Cells were subsequently washed and maintained in serum-free medium with supplements. The p110WT and p110caax expressing adenoviruses were

FIG. 1. Expression of G q Q209L decreases Akt phosphorylation.
NRVMs were infected with control adenovirus, G q WT, or G q Q209L overnight. The G q WTϩPGF 2␣ dishes were treated with 1 M PGF 2␣ coincident with adenoviral infection. 8 h after washing out the viruses, cell lysates were prepared and analyzed by SDS-PAGE. A, representative Western blots using the phospho-specific Ser-473 antibody (upper panel) and the Akt antibody for total protein levels (bottom panel) are shown. Similar results were obtained with the Thr-308 antibody. B, average Ϯ S.E. of normalized values obtained from densitometric analysis from 4 to 6 experiments using the antibody directed against the phosphorylated Ser-473 site. *, p Ͻ 0.05 compared with control, G q WTϩPGF 2␣ , or G q Q209L; **, p Ͻ 0.01 compared with control or G q WT.
FIG. 2. Activated Akt prevents induction of apoptotic end points by G q Q209L. NRVMs were infected overnight with control adenovirus, G q Q209L, myr-Akt, or G q Q209L and myrAkt viruses together. A, cells were harvested 36 h after removal of viruses and processed for DNA laddering, as described previously (12). DNA fragments were separated on 2% agarose gels, stained with ethidium bromide, and visualized under UV light. Results shown are representative of three independent experiments. B and C, cells grown on laminin-coated chamber slides were fixed with 3.7% formaldehyde 48 h after infection, permeabilized, and stained with rhodamine-conjugated phalloidin and Hoechst 33342, as described under "Experimental Procedures." Phalloidin staining (B) was visualized using an Axiovert fluorescent microscope, and images were captured on a digital SPOT camera. For quantitation of nuclear fragmentation (C), over 300 nuclei were examined per condition, and each condition was tested in quadruplicate. Values are means Ϯ S.E. *, p Ͻ 0.001 compared with G q Q209L. kindly provided by Dr. Jerry Olefsky (University of California, San Diego) (25). The virus encoding myristoylated Akt was obtained from Dr. Kenneth Walsh (Tufts University) (26). The G q WT and G q Q209L adenoviruses have been characterized previously, and the G q Q209LDNE adenovirus was subcloned into adenovirus from a plasmid obtained from Dr. John Exton (Vanderbilt University), by using our method published previously (8).
Apoptosis Measurements-Oligonucleosomal DNA fragmentation was analyzed by DNA laddering as described previously (12). Nuclear condensation was visualized using fluorescence microscopy after staining with Hoechst 33342 as described previously (12).
Western Blot Analysis-Cells were harvested in lysis buffer, and Bradford analysis was performed. Equal micrograms of protein (10 -30 g) were loaded onto SDS-PAGE, run, transferred to an Immobilon membrane, and the resulting blot probed using the following antibodies. The Ser-473 and Thr-308 phosphospecific antibodies for Akt, as well as the Pan-Akt antibody, were purchased from Cell Signaling Technologies. Anti-PTEN and anti-p85 were purchased from Upstate Biotechnology, Inc. Anti-SHIP-2 was purchased from Santa Cruz Biotechnology. The antibody against the cardiac sodium/calcium exchanger was purchased from Swant. p110␣ (Transduction Laboratories), p110␤ (Santa Cruz Biotechnology), and p110␥ (Santa Cruz Biotechnology) antibodies were kind gifts from Dr. Stephanie Watts (Michigan State University). The phospho-PDK-1 antibody (Cell Signaling) was a kind gift from Dr. Alexandra Newton's laboratory (University of California, San Diego). 3 -NRVMs plated at 4 ϫ 10 6 cells on 10-cm plates were labeled with 30 Ci/ml myo-[ 3 H]inositol for 36 h, and then infected with control, G q WT, or G q Q209L adenoviruses overnight, and harvested 24 h later. Cells were extracted with 5% trichloroacetic acid, 5 mM EDTA, 5 mM phytic acid; the lysates were sonicated for 10 s, and lipid-containing material was pelleted by centrifugation. [ 3 H]Inositollabeled phospholipids in the trichloroacetic acid pellet were extracted with chloroform/methanol/hydrochloric acid (200:100:1, v/v) and subsequently deacylated using methylamine/methanol/butanol (42:47:9) for 45 min at 50°C as described previously (27), treated with proteinase K (28), and then separated by anion-exchange HPLC and quantified using an on-line ␤-counter (28).
For the mass measurement of PIP 2 , unlabeled lipids were extracted and deacylated as described above except 5 mM ATP replaced phytic acid in the extraction buffer. Deacylated lipids were deglycerated by treating with periodate (50 mM) for 30 min with the reaction being terminated by adding 10% ethylene glycol followed by 0.3% dimethylhydrazine (27). The resulting Ins(1,4,5)P 3 was assayed by competitive binding assay (BioTrak IP 3 Assay protocol, Amersham Biosciences), as described by the supplier.
G q Transgenic Mouse Studies-Female G q transgenic mice (kindly provided by Dr. G. W. Dorn II, University of Cincinnati Medical Center) and nontransgenic littermate control mice were mated, and 5 days after parturition, hearts were excised and snap-frozen in liquid nitrogen. Age-matched virgin mice were used as controls. Procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. The frozen hearts were powdered and homogenized in lysis buffer, and 30 g of protein was loaded onto SDS-PAGE for Western blot analysis as described above. For PIP 2 mass analysis, 10 mg of powder was homogenized in trichloroacetic acid; then the lipids were deacylated and deglycerated before being subjected to the same mass assay as described above.
Statistical Analysis-All results are reported as mean Ϯ S.E. Comparisons of two groups only were accomplished using unpaired Student's t test. Experiments with more than two groups were compared by one-way analysis of variance followed by the Tukey post-hoc test for comparison between groups.
FIG. 3. LIF does not reduce apoptosis or induce Akt phosphorylation in G q Q209L-expressing NRVMs. A, 1st lane, 123-bp DNA ladder. 2nd to 4th lanes, NRVMs were infected overnight with control adenovirus (2nd lane) or G q Q209L (3rd and 4th lanes), followed by removal of virus. Cells were maintained for 36 h following removal of adenovirus, in the presence (4th lane) or absence (3rd lane) of 1 nM LIF. 5th and 6th lanes show data from NRVMs deprived of ITS supplement to induce apoptosis, and maintained in the presence (6th lane) or absence (5th lane) of 1 nM LIF. Cells were processed for DNA laddering as described previously (12). Data shown are representative of three independent experiments. B, NRVMs were infected overnight with control virus or G q Q209L-expressing adenovirus. 8 h after removal of virus, cells were treated with LIF (1 nM) for 5 min.
Lysates were prepared and separated on 8% SDS-PAGE and blotted with antibody directed against Akt phosphorylated at Ser-473. The blot shown is representative of three independent experiments. C, calyculin (10 M) was added to the appropriate cells for 30 min prior to 5 min of LIF stimulation, and cells were harvested for Western blot analysis. Blots were probed with the P-Akt Ser-473 antibody. Immunoblot is representative of three independent experiments.

Phospho-Akt Levels Are Diminished in G q Q209L-expressing
NRVMs-Expression of a constitutively active mutant of G␣ q (G q Q209L) induces apoptosis in NRVMs, whereas expression of the G q WT protein induces a sustained hypertrophy with no evidence of cell death (8). We hypothesized that this might reflect differential activation of protective pathways such as those initiated by phosphorylation of Akt. NRVMs were infected with viruses expressing G q WT, G q Q209L, or control virus backbone for 16 h followed by washing out of the viruses. Cells were harvested immediately after removal of the virus or after a further 8 or 24 h. All of these time points precede the development of apoptosis (36 -48 h after overnight infection) (12) and exhibited similar results. As shown in Fig. 1, at 8 h after virus removal, G q WT expression caused a 3-fold increase in Akt phosphorylation (Fig 1, A and B) relative to control. Treating G q WT-expressing myocytes with PGF 2␣ to further enhance G q activity abolished this increase in Akt phosphorylation. Enhancing G q activity even further, via expression of the constitutively active G q Q209L, resulted in a marked (45%) decrease in Akt phosphorylation, relative to control. Total Akt levels did not differ in G q WT or G q Q209L-expressing NRVMs versus control (Fig. 1A).
Akt Can Prevent G q Q209L-induced Apoptosis-The observation that levels of phospho-Akt were markedly decreased in G q Q209L-expressing cells suggested that the loss of this survival pathway could be a critical factor in the apoptotic response to G q Q209L. If this is so, then direct activation of Akt should prevent G q Q209L-induced apoptosis. To examine this possibility, NRVMs were coinfected with G q Q209L along with adenovirus expressing a constitutively activated Akt construct in which the c-Src myristoylation sequence was fused to the N terminus of Akt (myr-Akt). This membrane-targeted form of Akt has been reported to prevent serum deprivation-induced apoptosis in cultured cardiomyocytes and to reduce apoptosis caused by ischemia-reperfusion in vivo (15) or hypoxia in vitro (14).
Several independent indices of apoptosis were examined 36 -48 h after infection to establish whether myr-Akt could protect the myocytes from G q Q209L-induced apoptosis. The characteristic laddering pattern resulting from oligonucleosomal DNA fragmentation is prominent in G q Q209L-expressing cells undergoing apoptosis (8), and this was reduced in cells in which myr-Akt was expressed along with G q Q209L (Fig. 2A). In addition, whereas disorganization of myofilaments is evident in cells expressing G q Q209L (12), the expression of myr-Akt along with G q Q209L preserved myofilament organization (Fig. 2B). Finally, Hoechst staining was used to quantitate the number of cells with fragmented nuclei following infection with G q Q209L, myrAkt, or both viruses together. Expression of myr-Akt reduced the number of fragmented nuclei in G q Q209L-infected cells by ϳ60% (Fig. 2C). The effect of enhancing Akt activity supports the notion that decreases in Akt activity play a central role in the development of G q Q209L-induced apoptosis.
LIF Does Not Prevent Apoptosis or Activate Akt in G q Q209Lexpressing NRVMs-Cytokines that act via the gp130 receptor, including LIF and cardiotrophin 1 (CT-1), induce PI3K-dependent Akt phosphorylation (21,30) and can prevent apoptosis induced by serum deprivation in NRVMs (31). When NRVMs were placed in serum-free medium lacking supplementation with ITS, apoptosis ensued. LIF was able to prevent the development of apoptosis as assessed by DNA laddering (Fig. 3A, 5th  and 6th lanes). In contrast, LIF failed to protect against G q Q209L-induced DNA laddering (Fig. 3A, 2nd to 4th lanes).
Because LIF may not be sufficiently stable to signal for the periods needed for protection from cell death, we added additional LIF to the G q Q209L-expressing cells, throughout the course of G q Q209L expression (data not shown). Even under these conditions, LIF failed to rescue the G q Q209L phenotype. In addition, whereas acute treatment with LIF markedly increased Akt phosphorylation in control NRVMs, it failed to do so in G q Q209L-expressing myocytes (Fig. 3B). The lack of Akt activation by LIF would appear to be responsible for the inability of this cytokine to prevent G q Q209L-expressing NRVMs from undergoing apoptosis.
Phosphatase Inhibition Does Not Restore Akt Phosphorylation in G q Q209L-expressing NRVMs-We considered the possibility that G q Q209L expression might diminish Akt phosphorylation by activating phosphatases, such as PP2A, that can dephosphorylate Akt (32). Calyculin, a phosphatase inhibitor with specificity for PP1 and PP2A, was used to test this possibility. In both control-infected and G q Q209L-expressing NRVMs pretreated for 30 min with calyculin, basal Akt phosphorylation was enhanced, demonstrating some endogenous control by phosphatases PP1 and PP2A. However, LIF was unable to increase Akt phosphorylation in G q Q209L-expressing cells even when these phosphatases were inhibited (Fig. 3C). This argues against enhanced dephosphorylation of Akt as the mechanism by which G q Q209L expression limits Akt activation and cell survival.
G q Q209L-expressing Cells Do Not Exhibit an Increase in PIP 3 -Because it was not possible to stimulate Akt phosphorylation using the cytokine LIF, and a calyculin-sensitive phosphatase did not appear to be involved, we turned our focus upstream of Akt phosphorylation. Activation of Akt is often used as a surrogate for changes in PIP 3 levels because Akt phosphorylation by PDK-1 is PIP 3 -dependent. We therefore asked whether there was diminished PIP 3 production in G q Q209L-expressing cells. To our knowledge, there are no published reports of direct PIP 3 measurements in cardiomyocytes, and PIP 3 levels at rest are thought to be extremely low. To establish the feasibility of measuring [ 3 H]PIP 3 in NRVMs, we labeled cells (4 ϫ 10 6 per plate) with 30 Ci/ml of myo-[ 3 H]inositol for 36 h and used an adenoviral vector to express a membrane-targeted, and thus constitutively active, PI3K catalytic subunit (p110caax). The wild type p110 subunit of PI3K (p110WT), which does not target to membranes, served as a control. [ 3 H]Inositol phospholipids were extracted and quantified using HPLC as described under "Experimental Procedures." Chromatographic analysis of extracts from cells expressing p110caax revealed a peak at the expected elution time of PIP 3 . The corresponding peak in p110WT-expressing cells was substantially smaller, confirming the identity of the peak as PIP 3 (Fig. 4). We then examined [ 3 H]PIP 3 levels in cells infected with control, G q WT, and G q Q209L-expressing adenoviruses. HPLC analysis revealed significant levels of [ 3 H]PIP 3 in G q WT-expressing cells (Fig. 5). In contrast, PIP 3 was barely detectable in cells expressing G q Q209L. PIP 3 -dependent Membrane Translocation Is Not Stimulated in G q Q209L-expressing Cardiomyocytes-Akt phosphorylation, and thus activation, occurs when PDK-1 and Akt are colocal-ized at the membrane following PIP 3 generation (33). The localization of these enzymes was therefore examined by fractionation and immunoblotting of cells to determine whether alterations in PIP 3 formation led to diminished membrane associations. The fractionation method was verified by Western blot analysis using an antibody against the sodium/calcium exchanger, an ion transporter found on the plasma membrane (Fig. 6C). The pattern of Akt phosphorylation observed in membrane fractions was similar to that seen in whole cell lysates, with an increase in phospho-Akt observed in cells expressing G q WT, and less phosphorylated Akt at the membrane in cells expressing G q Q209L (Fig. 6A). The pattern of PDK-1 membrane localization was consistent with that of Akt, its substrate (Fig. 6A). When control-infected myocytes were stimulated with LIF for 90 s prior to fractionation, recruitment of both Akt and PDK-1 to the membrane fraction was observed (Fig. 6B). In contrast, but consistent with the inability of LIF to stimulate Akt phosphorylation in G q Q209L-expressing NRVMs, LIF treatment did not cause an increase in membrane-associated Akt or PDK-1 in cells expressing G q Q209L (Fig. 6B). These data imply that limited PIP 3 formation in G q Q209L-expressing cells precludes recruitment of Akt and PDK-1 to the membrane for activation. PIP 3 Regulatory Enzymes Are Not Altered with G q Q209L Expression-We explored the possibility that the differential effects of G q WT and G q Q209L expression on PIP 3 formation and Akt phosphorylation resulted from altered expression of PI3K or PIP 3 phosphatases. No differences in the p85, p110␣, or p110␤ PI3K subunits or in PTEN or SHIP2 expression were evident in control, G q WT, and G q Q209L-expressing cells based on immunoblotting experiments (Fig. 7). The only difference observed was an increase in PI3K␥ expression in G q Q209Lexpressing cells, which would not explain the diminished PIP 3 levels. These findings do not support the concept that changes in expression of PI3Ks or PIP 3 phosphatases are the basis for the altered PIP 3 formation and Akt activation in G q WT-versus G q Q209L-expressing cells.

FIG. 5. Reduced [ 3 H]PIP 3 content in G q Q209L-versus
PIP 2 Is Depleted in Cells Expressing Constitutively Active G␣ q -The substrate for PI3K is PIP 2 . The hydrolysis of PIP 2 is directly stimulated by binding of the activated ␣ subunit of G q to one of the ␤-isoforms of PLC (34,35). We have shown previously that G q WT expression in cardiomyocytes activates PLC and that expression of the constitutively active G q Q209L elicits far greater inositol phosphate formation (8). This led us to consider the possibility that membrane PIP 2 levels might be altered by sustained PLC activation in G q Q209L-expressing NRVMs, and that this might be limiting for PIP 3 formation. Cardiomyocyte PIP 2 levels were measured, at time points that preceded the incidence of apoptosis, in cells labeled with myo-[ 3 H]inositol for 36 h and then infected overnight with control, G q WT, or G q Q209L adenovirus. After overnight infection with G q Q209L there was a marked decrease in [ 3 H]PIP 2 observed at all time points tested (Fig. 8, A and B). This same assay also revealed marked decreases in both [ 3 H]PI and [ 3 H]PIP with G q Q209L expression (data not shown), suggesting that the entire phospholipid pool was diminished in an attempt to replenish the diminished PIP 2 . Decreased PIP 2 was also observed when this was measured by mass assay (Fig. 8C).
To explore further the relationship between G q -mediated PLC activation, PIP 2 hydrolysis, and Akt activation, we compared the effects of G q Q209L with those of a mutant G q Q209LDNE which retains constitutive activity due to diminished GTPase activity but has a reduced ability to activate PLC (36,37). The extent of PLC activation by the adenovirally expressed mutant G q Q209LDNE was less than a third of that observed with G q Q209L (data not shown). Strikingly, PIP 2 was not significantly decreased (Fig. 9A) by G q Q209LDNE expression, and Akt activation paralleled that seen with G q WT expression (Fig. 9B). Phenotypically, the mutant G q Q209LDNE causes cardiomyocyte hypertrophy without observable apoptosis, as seen with G q WT expression (data not shown). On the other hand, when G q and PLC activation are enhanced by the addition of PGF 2␣ to NRVMs expressing G q WT, cellular PIP 2 levels are significantly reduced to ϳ43% of those in control FIG. 6. Alterations in membrane localization and recruitment of PDK-1 and Akt in G q Q209L-expressing NRVMs. NRVMs were infected with control, G q WT-, or G q Q209L-expressing adenoviruses overnight, followed by virus removal. A, cells were harvested using detergent-free buffer and fractionated as described under "Experimental Procedures." The membrane fractions were subjected to Western blot analysis using the Ser-473 P-Akt antibody or the Ser-241 P-PDK1 antibody (this phosphorylation site is phosphorylated regardless of the activation state of PDK-1). B, appropriate cells were stimulated with 1 nM LIF for 90 s at 37°C, and then all cells were harvested in detergent-free buffer and subjected to the fractionation protocol. Western blot analysis was performed on the membrane fractions obtained using either the total Akt antibody or the Ser-241 P-PDK-1 antibody. C, control-infected cells were harvested as in A, and equal volumes of the cytosolic, membrane, and insoluble fractions were subjected to Western blot analysis using the sodium/calcium exchanger antibody as an indicator of membrane purity. All immunoblots are representative of two experiments. FIG. 7. PI3Ks and PIP 3 phosphatases are not differentially expressed in cells expressing G q WT or G q Q209L. NRVMs were infected with control, G q WT, or G q Q209L adenoviruses overnight, the virus was washed out, and 8 h later lysates were prepared for Western blot analysis. Immunoblots pictured (each sample in duplicate) are representative of three independent experiments. cardiomyocytes (data not shown), there is a concomitant decrease in Akt phosphorylation (Fig. 1), and these cells ultimately undergo apoptosis (12).
Akt Phosphorylation and PIP 2 Levels Are Decreased in G q Transgenic Mice-Mice with cardiac-specific G q WT overexpression develop significant hypertrophy associated with modest decreases in ventricular function (38). However, in the peripartum period (after giving birth) these mice develop a cardiomyopathy associated with significant amounts of cardiomyocyte apoptosis (8,39). The changes seen in the peripartal period have been suggested to result from enhanced G q activation and are similar to those induced by G q Q209L expression in cardiomyocytes. To determine whether our in vitro observations had a parallel in vivo, we examined Akt phosphorylation in ventricles from hearts isolated from mice 5 days postpartum. Representative Western blots (Fig. 10A), and pooled data from densitometric analysis from 7 animals per group (Fig. 10B), revealed a significant decrease in Akt phosphorylation in ventricles from the transgenic mice. PIP 2 mass was then assessed. Consistent with our data in isolated cardiomyocytes, a significant decrease in PIP 2 mass was observed in G q transgenic mouse hearts versus nontransgenic littermate control mouse hearts at 5 days postpartum (Fig. 10C).

DISCUSSION
The current study reveals dramatic differences in the level of signaling through the protective PI3K/Akt signaling pathway as a result of increasing G␣ q activity. In contrast to the ability of G q WT expression to increase PIP 3 levels and Akt phosphorylation, G q Q209L expression results in decreased Akt phosphorylation and a reduced ability to generate PIP 3 . The ability of G q Q209L to depress PI3K/Akt activity requires PLC activation, is associated with marked decreases in PIP 2 , and is paralleled by similar changes in Akt and PIP 2 in an in vivo model in which hypertrophy transitions to heart failure.
Although the physiological significance of constitutive G q activation could be questioned, we have also demonstrated that increasing G q activity by stimulating myocytes expressing G q WT with PGF 2␣ enhances PLC activity and results in cardiomyocyte apoptosis (12), whereas either stimulus alone induces hypertrophy. We show here that PIP 2 and Akt phosphorylation also decrease under the same conditions. These data suggest that the increased level of PLC activation achieved under physiological conditions in which G␣ q expression is enhanced, and/or availability of G q -coupled receptor agonists is increased, may be sufficient to lead to diminished PIP 2 and Akt phosphorylation. In this regard, and relevant to cardiac pathophysiology, it is noteworthy that up-regulation of the G␣ q protein has been reported in the border zones of the myocardium after infarct (40), a paradigm in which there is also enhanced release of G␣ q -coupled receptor agonists (41). In addition, increased expression of PLC␤ has been reported to occur in a model of chronic heart failure (42). Furthermore, in two models of heart failure (the cardiomyopathic hamster and post-myocardial infarct), there is evidence for decreased PIP 2 mass and decreased expression of phosphatidylinositol 4-phosphate 5-ki- FIG. 9. Mutant G q Q209LDNE with impaired PLC coupling does not cause a reduction in PIP 2 or Akt phosphorylation. A, NRVMs were labeled with [ 3 H]inositol as described in Fig. 8 and then infected with control, G q Q209LDNE, or G q Q209L adenoviruses. [ 3 H]PIP 2 was extracted and quantified as in Fig. 8. Values shown are averaged data from two independent experiments, performed in duplicate. *, p Ͻ 0.001. B, NRVMs were infected with control, G q WT, G q Q209LDNE, or G q Q209L adenoviruses overnight; viruses were washed out, and lysates were harvested for Western blot analysis using the Ser-473 antibody. Immunoblot shown is representative of three independent experiments.
FIG. 10. G q transgenic mice exhibit reduced Akt phosphorylation and PIP 2 levels after the stress of giving birth. Female G q transgenic mice (Dr. Dorn, University of Cincinnati Medical Center) or nontransgenic (NTG) littermates were bred, and at 5 days postpartum, hearts were excised and ventricles snap-frozen in liquid nitrogen. The hearts were powdered to facilitate further analysis. A, for Western blots, powdered tissue was homogenized in lysis buffer and 30 g of protein run on SDS-PAGE, transferred, and probed with the S473 P-Akt antibody. B, averaged densitometric data from Western blots on 7 animals per group. The Ser-473 P-Akt antibody was used, and values were normalized to total Akt levels from the same samples (which did not change significantly). *, p Ͻ 0.001 compared with nontransgenic peripartum animals. C, for PIP 2 mass analysis, 10 mg of powdered tissue was homogenized in trichloroacetic acid, and the lipids were deacylated and deglycerated as described under "Experimental Procedures" before being subjected to mass assay (BioTrak, Amersham Biosciences). Mass assay was performed twice, in duplicate, with 4 animals per group. *, p Ͻ 0.05 compared with nontransgenic peripartum. nase, the enzyme required to replete stores of PIP 2 (42,43). We show here that in another model of heart failure, the peripartal cardiomyopathy seen in transgenic mice expressing G␣ q , both PIP 2 levels and Akt phosphorylation are decreased compared with nontransgenic littermate controls. Therefore, significant reductions in PIP 2 are detectable in multiple models of cardiomyopathy, and our data are the first to suggest that pathophysiological levels of G q signaling can lead to PIP 2 reduction and concomitant loss of the Akt survival pathway.
The relationship between decreased PIP 2 and loss of Akt phosphorylation has not been directly proven here. However, it is important to note that in vitro reductions in total cellular PIP 2 were evident at early time points following G␣ q expression, prior to the time when we observed appearance of cellular apoptotic markers (e.g. Hoechst staining of fragmented nuclei, oligosomal DNA fragmentation, and cytochrome c release) in our previous studies (12). Thus reduced lipid levels do not appear to be secondary to loss of membrane integrity associated with apoptosis. These data imply that reduced PIP 2 is an early event and a potentially causal factor in the apoptotic response following expression of activated G␣ q . Furthermore, G q Q209L and G q Q209LDNE differ only in their ability to couple to PLC and thus decrease PIP 2 , suggesting that this is the cellular change responsible for the distinct effect of these two G␣ q mutants on Akt phosphorylation. Importantly, decreased PIP 2 accompanies diminished Akt phosphorylation in vitro in cells expressing G q Q209L or G q WT plus PGF 2␣ and in an in vivo model of G q -associated peripartal cardiomyopathy.
The finding that adenoviral expression of myr-Akt was able to protect against G q Q209L-induced apoptosis is consistent with the now well documented role of Akt in cardiomyocyte survival (15,23). Interestingly, whereas expression of membrane-targeted Akt was able to prevent G q Q209L-induced apoptosis, addition of LIF was ineffective. LIF, like CT-1, works through the gp130 receptor pathway to activate PI3K and Akt and thus support cardiomyocyte survival (21,30,44). Whereas LIF was able to prevent apoptosis in response to serum deprivation, it did not prevent that induced by G q Q209L. The ability of LIF to induce Akt phosphorylation and to recruit PDK1 and Akt to the membrane was also compromised in G q Q209L-infected cells. Thus, decreases in PIP 2 or other effects of G q /PLC activation would appear to block protective pathways normally elicited through cytokines or other growth factors. A recent report (45) in heterologous cell lines and fibroblasts demonstrated that G q Q209L could block the ability of insulin or PDGF to stimulate the PI3K/Akt pathway. Although the authors suggested this was through a PLC-independent effect of G q Q209L, based on a pharmacological inhibitor, our findings with the G q Q209LDNE mutant indicate that the inhibition of PI3K signaling in cardiomyocytes requires PLC activation.
Sustained activation of signaling pathways often results in compensatory decreases in components of the downstream signal transduction pathway. Common mechanisms include down-regulation of signaling proteins (e.g. receptors or protein kinase C) or up-regulation of mechanisms for signal termination (e.g. phosphatases). Decreases in PIP 3 formation and Akt activation could conceivably result from alterations in the expression of PI3Ks or PIP 3 phosphatases; however, these mechanisms do not appear to explain the changes that occur in G q Q209L-expressing NRVMs. We suggest, instead, that PIP 2 availability becomes rate-limiting following sustained or heightened PLC activation, pointing to a previously unanticipated relationship with decreases in Akt phosphorylation. Although dramatic decreases in PIP 2 are demonstrated here, it is conceivable that more subtle changes in pools of PIP 2 necessary for hormonal stimulation of PI3K-mediated PIP 3 formation might occur following agonist-induced PLC activation. Several lines of evidence indicate that PIP 2 depletion in selected microdomains reduces downstream responses that depend on generation of second messengers derived from PLC-mediated PIP 2 hydrolysis (46 -50). Our data suggest that such localized depletion of PIP 2 would impact the PIP 3 /Akt signaling pathway, either via diminished substrate availability or through altered recruitment of other signaling proteins, and provide a novel mechanism for shifting the balance between cell survival and apoptosis.