This Article Abstract Full Text (PDF) All Versions of this Article: 90/8/850    most recent 01.RES.0000016165.23795.1Fv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sabri, A. Articles by Steinberg, S. F. Search for Related Content PubMed PubMed Citation Articles by Sabri, A. Articles by Steinberg, S. F. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB HYDROGEN PEROXIDE Related Collections Apoptosis Cell signalling/signal transduction Heart failure - basic studies

(Circulation Research. 2002;90:850.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Dual Actions of the G_q Agonist Pasteurella multocida Toxin to Promote Cardiomyocyte Hypertrophy and Enhance Apoptosis Susceptibility

Abdelkarim Sabri, Brenda A. Wilson, Susan F. Steinberg

From the Departments of Pharmacology and Medicine (A.S., S.F.S.), College of Physicians and Surgeons, Columbia University, New York, NY; and the Department of Microbiology (B.A.W.), University of Illinois at Urbana-Champaign, Urbana, Ill.

Correspondence to Susan F. Steinberg, MD, Associate Professor of Pharmacology and Medicine, Dept of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 West 168 St, New York, NY 10032. E-mail sfs1{at}columbia.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous attempts to delineate the consequences of G _q activation in cardiomyocytes relied largely on molecular strategies in cultures or transgenic mice. Modest levels of wild-type G_q overexpression induce stable cardiac hypertrophy, whereas intense G_q stimulation induces cardiomyocyte apoptosis. The precise mechanism(s) whereby traditional targets of G _q subunits that induce hypertrophy also trigger cardiomyocyte apoptosis is not obvious and is explored with recombinant Pasteurella multocida toxin (rPMT, a G_q agonist). Cells cultured with rPMT display cardiomyocyte enlargement, sarcomeric organization, and increased atrial natriuretic factor expression in association with activation of phospholipase C, novel protein kinase C (PKC) isoforms, extracellular signal-regulated protein kinase (ERK), and (to a lesser extent) JNK/p38-MAPK. rPMT stimulates the ERK cascade via epidermal growth factor (EGF) receptor transactivation in cardiac fibroblasts, but EGF receptor transactivation plays no role in ERK activation in cardiomyocytes. Surprisingly, rPMT (or novel PKC isoform activation by PMA) decreases basal Akt phosphorylation; rPMT prevents Akt phosphorylation by EGF or IGF-1 and functionally augments cardiomyocyte apoptosis in response to H₂O₂. These results identify a G_q-PKC pathway that represses basal Akt phosphorylation and impairs Akt stimulation by survival factors. Because inhibition of Akt enhances cardiomyocyte susceptibility to apoptosis, this pathway is predicted to contribute to the transition from hypertrophy to cardiac decompensation and could be targeted for therapy in heart failure.

Key Words: G_q • protein kinase C • Akt • hypertrophy • apoptosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myocardial hypertrophy is an adaptive response to stresses that increase cardiac work. The increase in tissue mass diminishes systolic wall stress and improves contractile performance in the short term. However, compensated hypertrophy generally progresses to decompensated cardiac failure with chamber dilatation and contractile dysfunction. Because cardiac failure represents a major public health problem with substantial mortality, many laboratories have invested considerable effort to understand the regulatory determinants that contribute to the development of hypertrophy and its transition to heart failure. Recent research has focused on the family of G_q-coupled G protein-coupled receptors (GPCRs), which induce a hypertrophic phenotype with many common molecular and morphological features when stimulated by agonists or overexpressed in the hearts of transgenic mice.¹ G_q-coupled GPCR actions generally are attributed to effector pathways that emanate from _q subunits, which individually have been implicated in cardiomyocyte hypertrophic growth responses (including protein kinase C [PKC], mitogen-activated protein kinase [MAPK] cascades, and tyrosine kinases). However, the precise cellular actions of G_q proteins are not precisely delineated by studies of G_q-coupled GPCRs for 2 major reasons: (1) many GPCRs that couple to G_q also activate other G protein classes, perhaps explaining subtle differences in resultant hypertrophic phenotypes^2,3; (2) activation of heterotrimeric G_q liberates ß dimers (in addition to _q subunits), with independent signaling functions to downstream effector pathways. The prediction that G _q activates only a subset of the signaling machinery recruited by agonist-occupied GPCRs is borne out by experimental evidence that G_q subunits alone are not sufficient to induce the entire spectrum of changes characteristically induced by hypertrophic agonists acting at their cognate GPCRs.¹

Strategies more directly targeted to G_q subunits have been used to resolve their role in cardiomyocyte hypertrophy. Initial studies demonstrated that microinjection of inhibitory antibodies to G_q/11 in rat cardiomyocytes cultures or cardiac-restricted overexpression of a G_q inhibitory peptide in mice (which prevents signal transmission at the receptor-G _q subunit interface) interferes with the acquisition of features of cardiac hypertrophy in response to ₁-adrenergic receptors or pressure overload hypertrophy.^4,5 Subsequent studies involved overexpression of G_q subunits in cardiomyocyte cultures or expression of a G_q transgene in mouse myocardium. These studies provided unanticipated evidence that modest increases in wild-type G_q expression induce stable cardiac hypertrophy, but very intense G _q stimulation (with very high levels of wild-type G _q proteins or constitutively activated G _q mutants) induces dilated cardiomyopathy, with functional decompensation and cardiomyocyte apoptosis.^6–8 These observations suggest that hypertrophy and apoptosis may represent different phases of the same process, initiated by a common G_q-activated biochemical signal. However, few traditional G_q targets simultaneously promote hypertrophy and trigger cardiomyocyte apoptosis. One notable exception is PKC, which displays isoform-selective signaling to MAPK cascades, apoptosis, and cardioprotection.^9,10 Akt, a serine/threonine protein kinase that mediates cardioprotection by receptor tyrosine kinases, also is activated by certain GPCRs (including in cardiomyocytes^3,11), but the role of G_q in this process is disputed; Murga et al¹² identify a G _q-dependent pathway for Akt activation, whereas Bommakanti et al¹³ report Akt inhibition by G_q. Finally, G _q promotes cardiomyocyte apoptosis in genetically engineered systems, where intense stimulation by exogenous proteins may alter the stoichiometry and/or targeting of G_q relative to physiologically relevant binding partners in the plasma membrane. The relevance of studies on exogenous (frequently constitutively activated) proteins to physiological signaling by endogenous G_q in cardiomyocytes remains uncertain.

Pasteuralla multocida toxin (PMT) is a potent mitogen for a variety of cell types. PMT is internalized via receptor-mediated endocytosis and acts intracellularly to activate signaling pathways, including phosphoinositide hydrolysis, mobilization of intracellular calcium, translocation of PKC, activation of the extracellular-regulated kinase [ERK] cascade, and tyrosine phosphorylation of focal adhesion kinase.^14,15 The actions of recombinant PMT (rPMT) are inhibited in Xenopus oocytes by antibodies directed against the subunit of G_q/11 or G_q antisense RNA and in HEK293 cells by overexpression of the C-terminal peptide inhibitor of G_q/11.^14,16 These results identify the free monomeric G _q/11 subunit as the target of rPMT’s actions. Recent studies in fibroblasts deficient in either G_q or G₁₁ further localize rPMT’s actions to G_q (not G₁₁).¹⁷ Accordingly, this study uses rPMT as a pharmacological probe to elucidate the biochemical and functional consequences of endogenous G_q activation in cardiomyocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cultures of neonatal rat ventricular myocytes and cardiac fibroblasts were prepared and assays of inositol phosphate accumulation were performed according to methods described previously.¹⁸ Immunoblotting was according to methods published previously or manufacturer’s instructions with antibodies for total or phosphorylated (activated) signaling proteins from the following sources: phospho-ERK1/2, total and phospho-p38 MAP kinase, phospho-c-Jun-NH₂-terminal kinase (JNK), total and phospho-Akt, phospho-PKC-Pan, and phospho-PKC--Thr-505 (Cell Signaling Technology); ERK1/2 (Santa Cruz Biotechnology); PKC- and PKC- (Gibco-BRL); phospho-PKC--Tyr-311 (Biosource International); and PKC (Dr Doriano Fabbro, CIBA-Geigy, Basel, Switzerland¹⁹). Each panel in each figure represents results from a single gel exposed for a uniform duration, with bands detected by enhanced chemiluminescence and quantified by laser scanning densitometry. Cardiomyocyte growth was quantified by digitized image analysis of cell surface area, with 75 to 150 cells imaged per condition; cells with well-organized myofibrillar staining patterns were scored positive for sarcomeric organization.³ Northern blot analysis of 10 µg of total RNA isolated by Qiagen kit according to manufacturer’s instructions was with a rat atrial natriuretic factor (ANF) cDNA probe (600 bp) labeled with [³²P]dCTP; signals were normalized to GAPDH.³ Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed for detection of apoptotic cells according to manufacturer’s instructions (Boehringer).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Agonist Properties of rPMT in Cardiomyocytes
Consistent with evidence that rPMT gains access to cells slowly, rPMT promotes inositol phosphate accumulation in cardiomyocytes, but with slow kinetics. The response is detectable at 1 hour and increases progressively for at least 48 hours (Figure 1A). Figure 1B shows that maximal activation of inositol phosphate accumulation at 24 hours typically is with 400 ng/mL toxin; the magnitude of the response is comparable to that induced by a 30-minute challenge with the ₁-adrenergic receptor agonist norepinephrine (NE; with individual batches of rPMT displaying some variability).

View larger version (34K): [in this window] [in a new window]   Figure 1. rPMT activates PLC. Media was supplemented with 3 µCi/mL [3H]myoinositol for 96 hours, with 400 ng/mL rPMT for the indicated intervals at the end of this interval (A) or the indicated concentrations of rPMT (B, left) or 400 ng/mL rPMT (B, right) during the final 24 hours. This was followed by 30 minutes in the presence of 10 mmol/L LiCl (alone or plus 10 µmol/L NE; B, right). Results are mean of 3 replicates from a single experiment (representative of 3 separate experiments using different batches of rPMT; A) or mean±SEM from 3 independent experiments (using a single rPMT preparation; B). Levels of IP2 and IP3 increased in parallel in all experiments.

rPMT-dependent activation of phospholipase C (PLC) also leads to the formation of diacylglycerol, the endogenous activator of PKC. There is general consensus that neonatal rat ventricular myocytes coexpress calcium-sensitive PKC (which is largely recovered as a soluble enzyme under basal conditions) and novel PKC and PKC isoforms (which are variably recovered in the particulate fraction even under resting conditions^18–20). The hallmarks of PKC activation include rapid translocation of soluble enzyme to particulate/membrane structures followed by downregulation (proteolysis) of the enzyme. However, preliminary studies indicated that the very protracted PLC activation kinetics for rPMT (which are quite distinct from the rapid kinetics of PLC activation by agonist-occupied GPCRs) does not lead to detectable changes in PKC isoforms partitioning between soluble and particulate fractions (or abundance) at 30 minutes to 2 hours (data not shown). However, persistent rPMT stimulation (400 ng/mL, 24 hours) leads to a substantial reduction in the abundance of PKC and PKC in the soluble fraction; however, PKC and PKC abundance in the particulate fraction is preserved (Figure 2A). Reduced amounts of PKC and PKC, which disproportionately partition with the particulate fraction, recapitulates the changes detected in cardiomyocyte cultures exposed to NE for 24 hours or in ventricles from mice that overexpress G _q.^6,18 PKC and PKC abundance and subcellular distribution are not altered by rPMT or NE (Figure 2A and data not shown). These results provide evidence that persistent G_q stimulation by rPMT or NE results in the activation of only novel PKC and PKC isoforms. rPMT-dependent downregulation of novel PKC isoforms occurs over a dose range that parallels the concentration-response relationship for rPMT-dependent stimulation of PLC (data not shown).

View larger version (53K): [in this window] [in a new window]   Figure 2. rPMT selectively activates nPKC isoforms. Cardiomyocytes were incubated in culture medium without or with NE (10 µmol/L), rPMT (400 ng/mL), or PMA (100 nmol/L) for 24 hours (A) or 5 minutes (B) as indicated. Soluble and particulate extracts (A) or whole cell extracts (B) were separated by SDS-PAGE followed by Western blotting with antibodies specific for individual PKC isoforms, PKC phosphorylated at the hydrophobic motif (with P-PKC-Pan), or PKC phosphorylated at the activation loop or hinge region (with P-PKC -Thr-505 and P-PKC-Tyr-311, respectively) as indicated. The band designated PKC on immunoblots with P-PKC-Pan corresponds to the major immunoreactive species recognized by the antibody to total PKC; the identity of the band with slower mobility in the particulate fraction (that disappears in cells subjected to PMA for 24 hours) is less certain. Electrophoresis was performed according to conditions described by Bornancin and Parker34 (A, bottom), to improve protein separation and detect minor difference in protein mobility; NE- and rPMT-dependent shifts in total PKC mobility also were detected in separate experiments that used these conditions and lower amounts of protein loading (data not shown). GF109203X was included at 5 µmol/L for 30 minutes before stimulation where indicated.

Western blot analysis with anti-phospho-PKC antibodies provided independent evidence of PKC activation by rPMT. Recent studies identify PKC phosphorylation at conserved sites in the C-terminus (the turn and hydrophobic motif, corresponding to Thr641 and Ser660 in PKCßII) as a mechanism to control the maturation and allosterically regulate the activity of PKC isoforms.²¹ Anti-phospho-PKC-Pan, an antibody directed at the conserved hydrophobic motif in conventional and novel PKC isoforms (but not atypical PKC isoforms, where the Ser phospho-acceptor site is replaced by the acidic Glu), identifies major bands that comigrate with PKC, PKC, and PKC (approximately 78-, 82-, and 96-kDa, respectively; Figure 2A, bottom). Immunoreactivity is completely stripped by acid phosphatase treatment (data not shown) and drastically reduced by pharmacological downregulation of PKC protein with phorbol 12-myristate 13-acetate (PMA) (Figure 2A), validating the identification of these bands as phosphorylated PKC isoforms. The phosphorylated form of PKC is abundant in both soluble and particulate fractions of resting cardiomyocytes, although the protein is preferentially recovered from the soluble fraction (although small amounts of PKC are detected in particulate with long exposures). This result suggests that either a greater proportion of PKC in the particulate fraction is phosphorylated at this site (relative to PKC in the soluble fraction) and/or that the antibody to total PKC discriminates between phosphorylated and unphosphorylated conformations of the enzyme (ie, reacts only weakly with phosphorylated PKC^22,23). PKC phosphorylation is not noticeably altered by short-term stimulation with NE/PMA or long-term stimulation with rPMT. In contrast, PKC phosphorylation at the hydrophobic motif is increased by these treatments (Figures 2A and 2B); PKC phosphorylation is detected in the particulate fraction of rPMT-treated cardiomyocytes, along with the bulk of PKC protein. Consistent with evidence that the hydrophobic motif is a site of autophosphorylation in cPKC isoforms,²¹ PKC phosphorylation requires intact PKC kinase activity and is prevented by GF109203X (an inhibitor of cPKC/nPKC isoforms). PKC phosphorylation at the hydrophobic motif also is detected in the particulate (but not soluble) fraction of quiescent and stimulated cardiomyocytes. rPMT reduces PKC mobility, without increasing PKC phosphorylation at the hydrophobic motif. This result is most consistent with rPMT increasing PKC phosphorylation at a site that is distinct from the hydrophobic motif. However, antibodies that specifically recognize PKC phosphorylation at Thr505 in the activation loop and Tyr311 in the hinge region ruled out rPMT-dependent phosphorylation of PKC at these other sites; immunoreactivity with these antibodies is confined to the particulate fraction of control cardiomyocytes, lost during PKC downregulation by PMA, and fails to increase in cells treated with rPMT. Because PKC undergoes complex regulatory phosphorylations at other Ser/Thr or Tyr residues (which can decrease its mobility²⁴), rPMT-dependent phosphorylation of PKC remains a viable mechanism to explain the reduced PKC mobility after rPMT treatment (although other modifications that reduce PKC mobility also are possible). Collectively, these experiments identify persistent PKC activation (with selectivity for novel PKC isoforms) in rPMT treated cardiomyocytes.

The MAPK cascades that lie downstream in G _q-dependent pathways in cardiomyocytes were next studied. Figure 3 shows that rPMT induces dose-dependent increases in signaling through the ERK1/2, p38-MAPK, and JNK cascades, as detected by an increase in the phosphorylation of the terminal kinase of each pathway. For each, activation was observed with slow kinetics (detectable at 1 hour and increasing progressively for 24 hours). Immunoblot analyses with antisera that recognize total (phosphorylated and nonphosphorylated) ERK1/2, p38-MAPK, and JNK show that rPMT does not significantly alter the expression of these proteins (Figure 3 and data not shown). This indicates that the protracted kinetics for MAPK activation by rPMT is not due to changes in terminal kinase protein expression. ERK1/2 activation by rPMT is quite robust (comparable to acute stimulation with PMA), whereas JNK and p38-MAPK activation by rPMT is more modest (relative to the strong JNK and p38-MAPK activation by sorbitol).

View larger version (35K): [in this window] [in a new window]   Figure 3. rPMT promotes the activation of ERK1/2, JNK, and p38-MAPK cascades. Incubations were for 24 hours in the presence of the indicated concentrations of rPMT or 5 minutes with PMA (100 nmol/L) or sorbitol (0.5 mmol/L). Cell lysates were subjected to SDS-PAGE and Western blotting with specific anti-phospho-ERK1/2, anti-p38 MAPK, or anti-JNK antibodies; stripped blots were subsequently analyzed for total ERK1/2 or p38 MAP kinase protein (A and B). Anti-P-p38-MAPK recognizes activated forms of both and ß isoforms of p38-MAPK (although only -p38-MAPK is recognized by the antibody against total protein). Top, Representative autoradiograms (with each lane from a single gel exposed for the same duration). Bottom, Quantification of experiments from separate cultures (n=3).

rPMT displays potent growth stimulatory properties in cells with proliferative potential. Figure 4 shows that rPMT also induces the hallmarks of cardiomyocyte hypertrophy. rPMT mimics the effects of NE to promote myofibrillar organization, increase ANF mRNA expression, promote cellular enlargement, and enhance [³H]phenylalanine incorporation.

View larger version (63K): [in this window] [in a new window]   Figure 4. rPMT mimics the effect of NE to promote cardiomyocyte hypertrophy. Cardiomyocytes were cultured in serum-free medium without or with rPMT (400 ng/mL) or norepinephrine (NE, 10 µmol/L) for 48 hours. Cardiomyocytes grown on fibronectin precoated slides were fixed with cold methanol, permeabilized with 0.1% Triton X-100 and 0.2% BSA, and stained overnight at 4°C with monoclonal anti--actinin sarcomeric (A, magnification x100) or subjected to Northern blot analysis for measurements of steady-state ANF mRNA expression (B). Effects of rPMT (black bars) and NE (gray bars) vs vehicle (white bars) on cell size (C), sarcomeric organization (measured as the percentage of total cells displaying well-developed sarcomeric banding patterns; D), and protein synthesis assessed as [3H]phenylalanine incorporation normalized to protein content (according to methods described previously3; E) are illustrated (n=3 separate culture preparations for each).

ERK Activation by rPMT Does Not Require EGF Receptor Kinase Activity in Cardiomyocytes
GPCR-dependent activation of ERK can follow a Ras-independent pathway involving PKC isoforms or a Ras-dependent pathway involving receptor or nonreceptor tyrosine kinases, depending on cell type and particular GPCR. Seo et al¹⁴ recently reported that rPMT stimulation of ERK is via a Ras-dependent (PKC-independent) pathway that involves EGF receptor transactivation in HEK293 cells. To determine whether this pathway mediates rPMT actions in cardiomyocytes, studies were performed in the presence of the EGF receptor-specific tyrphostin AG1478. Figure 5A shows that AG1478 effectively blocks EGF receptor-mediated ERK phosphorylation in cardiomyocytes, but induces only a very minor reduction in ERK phosphorylation by rPMT. As controls, similar studies were performed in cardiac fibroblasts. Here, rPMT-mediated activation of ERK displays a significant requirement for EGF receptor kinase activity; it is markedly inhibited by AG1478 (Figure 5B). These results indicate that EGF receptor transactivation is necessary for G_q activation of ERK in cardiac fibroblasts, but plays little to no role in G_q signaling to ERK in cardiomyocytes.

View larger version (48K): [in this window] [in a new window]   Figure 5. rPMT requires EGF receptor kinase activity to activate ERK in cardiac fibroblasts, but not in cardiomyocytes. Neonatal rat cardiomyocytes (A) or cardiac fibroblasts (B) were treated with rPMT (400 ng/mL) for 24 hours or EGF (50 ng/mL) for 5 minutes without or with AG1478 (2 µmol/L, starting 30 minutes before stimulations). Western blotting was with the anti-phospho-ERK1/2 antibody, with data representative of results obtained in 3 separate cultures.

rPMT or Activation of PKC Isoforms Negatively Regulates Akt; rPMT Enhances Susceptibility to H₂O₂-Induced Apoptosis
Mechanisms for Akt activation by receptor tyrosine kinases are well established. In contrast, efforts to delineate the mode and mechanism(s) of Akt regulation by GPCRs have yielded seemingly conflicting evidence, with G _q variably reported to activate or inhibit Akt.^12,13 Given the importance of Akt as a mediator of cell survival (and recent evidence that intense G _q signaling enhances cardiomyocyte apoptosis), the studies next compared Akt protein expression and activation (detected as increased regulatory phosphorylation on Ser473 in the C-terminal hydrophobic motif) under control conditions and after stimulation with rPMT.

Figure 6A shows that exposure to rPMT for 24 hours leads to reduced basal Akt phosphorylation (71±5%, n=4, P<0.05); this effect is sustained for at least 48 hours (Figure 6B). Of note, EGF and IGF-1 increase Akt phosphorylation in control cultures, but both responses are severely blunted in rPMT-treated cultures. Reduced levels of phospho-Akt in rPMT-treated cultures do not result from a change in Akt protein expression; total Akt immunoreactivity is similar in control and rPMT-treated cultures. Decreased signaling through the Akt pathway could render cardiomyocytes vulnerable to stresses that induce apoptosis. Indeed, Figure 6C shows that rPMT induces a small increase in the number of TUNEL-positive cells in cardiomyocyte cultures grown in serum-free conditions for 48 hours. Although this effect of rPMT to augment basal apoptosis is not statistically significant, apoptosis induced by H₂O₂ is significantly augmented by rPMT.

View larger version (47K): [in this window] [in a new window]   Figure 6. rPMT decreases basal Akt phosphorylation, prevents its phosphorylation by EGF or IGF-1, and increases H2O2-induced apoptosis in cardiomyocyte cultures. Neonatal rat cardiomyocytes were treated with vehicle, rPMT (400 ng/mL) for 24 hours (A) or 48 hours (B), and EGF or IGF-1 (each at 50 ng/mL for 5 minutes) alone or after rPMT (400 ng/mL for 24 hours; A). Cell lysates were subjected to SDS-PAGE followed by Western blotting with antibodies that recognize phosphorylated or total Akt. Data are from representative gels, with similar results obtained in 4 separate cultures. C, Cells were treated with vehicle or rPMT (400 ng/mL) for 48 hours, without or with H2O2 (500 µmol/L) during the final 24 hours. For each condition, 300 to 500 cardiomyocytes were imaged by fluorescence microscopy, and cells undergoing apoptosis were scored TUNEL positive. Results are reported as the percentage of TUNEL-positive cells (mean±SEM, n=3). H2O2 (but not rPMT) increased apoptosis; H2O2-induced apoptosis is augmented by rPMT (*P<0.05 by paired t test with Bonferroni correction for multiple comparisons). Of note, cross-talk between H2O2 and rPMT signaling pathways was not considered, because the short-term effects of H2O2 to activate Akt are not retained at the termination of the long-term stimulation paradigm (data not shown).

Given recent evidence that certain PKC isoforms interact in a functionally relevant manner with Akt in tumor cells, and that rPMT leads to prominent PKC isoform activation in cardiomyocytes, the studies next examined whether PKC isoforms might represent a mechanism whereby G _q subunits negatively regulate Akt. Figure 7 shows that ₁-adrenergic receptor activation with NE leads to a very modest increase in Akt phosphorylation. In contrast, selective activation of the PKC limb of the phosphoinositide signaling pathway with PMA leads to a marked reduction in basal Akt phosphorylation (79±6%, n=5, P<0.05). PMA-dependent inhibitory modulation of Akt phosphorylation is completely abrogated by GF109203X (a relatively nonselective inhibitor of phorbol ester-sensitive PKC, PKC, and PKC), under conditions where PMA-dependent activation of ERK is effectively abrogated.¹⁸ In contrast, PMA-dependent inhibition of Akt phosphorylation is not influenced by Go6976, a selective inhibitor of cPKC (-, -ß, and -). The MEK inhibitor U0126 also did not influence PMA-dependent repression of Akt phosphorylation (under conditions where PMA-dependent activation of ERK was completely abrogated); these results indicate that nPKC isoform(s) repress Akt phosphorylation through a mechanism that does not require activation of the ERK cascade. Short-term incubations with GF109203X and Go6976 appeared to be well-tolerated. In contrast, chelerytherine (a general inhibitor of all PKC isoforms) and rottlerin (a commonly used PKC inhibitor) impaired cardiomyocyte viability in association with a marked increase in the phosphorylation of Akt (as well as changes in the phosphorylation of a host of other signaling kinases) and were avoided in this study; this is consistent with recent literature that impugns the selectivity/efficacy of several compounds touted to be useful PKC inhibitors.^25,26 A pharmacological approach to explore the role of PKC isoforms in the inhibitory modulation of Akt by rPMT also was not informative, because prolonged incubations with PKC inhibitors alone (ie, the controls required to study rPMT’s actions) reduced cardiomyocyte viability and/or markedly altered Akt phosphorylation. This could reflect a PKC requirement for cell viability or alternatively a nonspecific toxic effect of these compounds that becomes manifest with prolonged incubations; it precludes conclusions from long-term experiments in neonatal cardiomyocyte cultures with pharmacological PKC antagonists. Hence, although these studies cannot exclude a PKC-independent mechanism that contributes to rPMT repression of Akt phosphorylation, the observations that nPKC isoforms are activated in rPMT-treated cardiomyocytes and that nPKC isoforms mediate Akt repression by PMA supports the provisional conclusion that nPKC isoforms contribute to inhibitory modulation of Akt by rPMT.

View larger version (39K): [in this window] [in a new window]   Figure 7. PKC activation represses Akt phosphorylation. Cells were treated for 5 minutes with norepinephrine (NE, 10 µmol/L) or PMA (100 nmol/L) alone or after pretreatment with Go6976 or GF109203X (each at 5 µmol/L) or U0126 (10 µmol/L) as indicated. Blots were probed with anti-P-Akt, stripped, and then re-probed with anti-P-ERK (to validate the inhibitory effects of U1026; B, bottom) and/or anti-Akt to normalize for minor variations in protein loading. Data are from representative gels, with similar results obtained in 3 separate cultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies using molecular strategies to identify the signaling properties of G_q in cardiomyocytes provided unanticipated evidence that G_q induces a continuum of responses, from compensated hypertrophy to decompensated heart failure, as the stimulus strength increases or is maintained over prolonged intervals. Although this suggests that hypertrophy and apoptosis may be initiated by a common G_q-activated biochemical signal, few molecular signals fulfill this requirement. Studies reported herein, using rPMT to identify signals emanating from endogenous cardiomyocyte G _q proteins, emphasize a role for PKC isoforms in this process. The observation that G_q stimulation by rPMT activates PLC, nPKC isoforms, and MAPK cascades (ERK, with lesser activation of JNK and p38-MAPK) and that these pathways act as potent signals for cardiomyocyte hypertrophy was anticipated. However, the evidence that G _q stimulation by rPMT (or direct activation of nPKC isoforms by PMA) represses Akt phosphorylation and prevents its recruitment by ligands that activate receptor tyrosine kinases was unexpected. These results suggest a novel model that places PKC pivotally upstream in pathways that both promote hypertrophy and render cardiomyocytes susceptible to apoptosis (Figure 8). This model provides an appealing explanation for the characteristic progression from hypertrophy to cardiac decompensation observed in clinical syndromes of heart failure in humans. It would explain studies in transgenic mice where modest levels of G_q overexpression generally are tolerated, but stresses (pregnancy, parturition, or aortic banding) result in a lethal dilated cardiomyopathy⁷; stresses or the insults that characterize the progression of most cardiac diseases would be poorly tolerated by cells that cannot recruit the Akt pathway.

View larger version (34K): [in this window] [in a new window]   Figure 8. Schematic of rPMT signaling pathways in cardiomyocytes. rPMT induces sustained Gq activation, which stimulates nPKC isoforms; nPKC stimulates ERK (and to a lesser extent JNK and p38-MAPK) and negatively regulates the Akt survival pathway.

G_q/11-PCRs employ divergent pathways to initiate signaling via the ERK cascade. This heterogeneity has been attributed to differences in the identity of the GPCR as well cell type-specific differences in endogenous signaling machinery. Recent studies identify transactivation of EGF receptors as a prominent mechanism leading to ERK activation by G_q/11-PCRs (and rPMT) in experimental model systems.¹⁴ Studies reported herein identify a similar pathway for rPMT activation of ERK in cardiac fibroblasts. However, consistent with previous evidence that signaling in cardiac fibroblasts and cardiomyocytes differs,²⁷ studies reported herein establish that ERK activation by G_q subunits does not require EGF receptor kinase activity in cardiomyocytes. These studies establish the utility of rPMT as a reagent to define the signaling properties and cellular actions of endogenous G_q subunits in cardiomyocytes and emphasize the need for caution when applying results obtained in model systems to the heart.

There is recent interest in growth factor-dependent pathways leading to PI-3K/Akt activation as a mechanism for cell survival, proliferation, and differentiation. While growth responses typically reflect a dynamic balance between stimulatory and inhibitory pathways, mechanisms that curtail Akt activation are less well understood and there is only very limited information on Akt regulation by phorbol ester-sensitive PKC isoforms. To date, most of the studies have focused on the atypical PKC, which coimmunoprecipitates with Akt and attenuates its phosphorylation in CHO and breast cancer cells.^28,29 A physical association between certain phorbol ester-sensitive PKC isoforms (PKC and PKC) and the PH-domain of Akt has been reported, although the importance of this interaction (or the conditions that favor this pathway) has been questioned.^28,30 Other studies identify a range of effects of phorbol ester-sensitive PKC isoforms on Akt, with PKC overexpression increasing Akt activity (and reducing apoptosis) in myeloid cells,³¹ evidence interpreted as implicating PKC in the repression of IGF-1-dependent activation of Akt in PC12 cells,³² and a kinase-deficient mutant of PKC (but not wild-type PKC or kinase-dead PKC or PKC) inhibiting Akt phosphorylation/activation by insulin, heat shock, or H₂O₂ in L6 myotubes and CHO cells.³³ On the basis of these divergent PKC isoform-dependent effects on Akt, the consequences of PKC activation on Akt phosphorylation in cardiomyocytes could not be predicted. Studies reported herein demonstrate that persistent G _q stimulation with rPMT targets to the selective activation of nPKC isoforms, which repress Akt phosphorylation in cardiomyocytes. The relative importance of individual PKC isoforms in the cellular actions of rPMT could not be resolved with available pharmacological PKC inhibitors because these agents alone impair cell viability in association with changes in the phosphorylation state of various signaling kinases during long-term treatment protocols. The interpretation of studies that use a molecular strategy might be more straightforward; such studies are ongoing, given the potential profound clinical importance of a PKC-dependent pathway for Akt repression that negatively impacts on cardiomyocyte survival.

PKC is a well-recognized mediator of ischemic preconditioning and is generally viewed as cardioprotective. This study suggests that PKC isoforms fulfill a more broad range of cellular functions with very distinct biological consequences. PKC is highly protective when acutely activated in the course of ischemia/reperfusion injury. However, the evidence that PKC represses Akt indicates that prolonged PKC activation (as typically occurs in heart failure with elevated catecholamine levels) also represses survival pathways and would be deleterious to the natural history of heart failure. Evidence that cardioprotection (ischemic preconditioning) and enhanced apoptosis (negative regulation of Akt) reflect the actions of distinct PKC isoforms would provide strong impetus to consider agents targeted to individual PKC isoforms as therapeutic modalities to halt the progression of clinical heart failure syndromes. This would be consistent with recent evidence for PKC isoform-selective regulation of MAPK cascades, modulation of cardiac injury during ischemic insults, and induction of apoptosis.^9,10 Moreover, this type of interplay between receptor tyrosine kinases and PKC activators (that positively and negatively regulate Akt, respectively) could have more broad clinical implications that extend to the pathogenesis and therapy of other diseases, including insulin-resistant diabetes.

	Acknowledgments

 
This study was supported by United States Public Health Service/National Heart, Lung, and Blood Institute grant HL-64639 (to S.F.S.) and NIH/National Institute of Allergy and Infectious Diseases grant AI38396 (to B.A.W.).

Received November 14, 2001; revision received March 12, 2002; accepted March 12, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Dorn GW, Brown JH. G_q signaling in cardiac adaptation and maladaptation. Trends Cardiovasc Med. 1999; 9: 26–34.
2. Hilal-Dandan R, Ramirez MT, Villegas S, Gonzalez A, Endo-Mochizuki Y, Brown JH, Brunton LL. Endothelin ET _A receptor regulates signaling and ANF gene expression via multiple G protein-linked pathways. Am J Physiol. 1997; 272: H130–H137.
3. Sabri A, Muske G, Zhang H, Pak E, Darrow A, Andrade-Gordon P, Steinberg SF. Signaling properties and functions of two distinct cardiomyocyte protease-activated receptors. Circ Res. 2000; 86: 1054–1061.
4. LaMorte VJ, Thorburn J, Absher D, Spiegel AM, Brown JH, Chien KR, Feramisco JR, Knowlton KU. G_q- and ras-dependent pathways mediate hypertrophy of neonatal rat ventricular myocytes following ₁-adrenergic stimulation. J Biol Chem. 1994; 269: 13490–13496.
5. Akhter SA, Luttrell LM, Rockman HA, Iaccarino G, Lefkowitz RJ, Koch WJ. Targeting the receptor-G_q interface to inhibit in vivo pressure overload myocardial hypertrophy. Science. 1998; 280: 574–577.
6. D’Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, Dorn GW. Transgenic Gq overexpression induces cardiac contractile failure in mice. Proc Natl Acad Sci U S A. 1997; 94: 8121–8126.
7. Adams JW, Sakata Y, Davis MG, Sah VP, Wang Y, Liggett SB, Chien KR, Brown JH, Dorn GW. Enhanced Gq signaling: a common pathway mediates cardiac hypertrophy and apoptotic heart failure. Proc Natl Acad Sci U S A. 1998; 95: 10140–10145.
8. Mende U, Kagen A, Meister M, Neer EJ. Signal transduction in atria and ventricles of mice with transient cardiac expression of activated G protein _q. Circ Res. 1999; 85: 1085–1091.
9. Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Banci L, Guo Y, Bolli R, Dorn GW, Mochly-Rosen D. Opposing cardioprotective actions and parallel hypertrophic effects of and PKC. Proc Natl Acad Sci U S A. 2001; 98: 11114–11119.
10. Heidkamp MC, Bayer AL, Martin JL, Samarel AM. Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C and in neonatal rat ventricular myocytes. Circ Res. 2001; 89: 882–890.
11. Chesley A, Lundberg MS, Asai T, Xiao RP, Ohtani S, Lakatta EG, Crow MT. The ß₂-adrenergic receptor delivers an anti-apoptotic signal to cardiac myocyte through G_i-dependent coupling to phosphatidylinositol 3'-kinase. Circ Res. 2000; 87: 1172–1179.
12. Murga C, Laguinge L, Wetzker R, Cuadrado A, Gutkind JS. Activation of Akt/protein kinase B by G protein-coupled receptors. J Biol Chem. 1998; 273: 19080–19085.
13. Bommakanti RK, Vinayak S, Simonds WF. Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits. J Biol Chem. 2000; 275: 38870–38876.
14. Seo B, Choy EW, Maudsley S, Miller WE, Wilson BA, Luttrell LM. Pasteurella multocida toxin stimulates mitogen-activated protein kinase via G_q/11-dependent transactivation of the epidermal growth factor receptor. J Biol Chem. 2000; 275: 2239–2245.
15. Lacerda HM, Lax AJ, Rozengurt E. Pasteurella multocida toxin, a potent intracellularly acting mitogen, induces p125^FAK and paxillin tyrosine phosphorylation, actin stress fiber formation, and focal contact assembly in Swiss 3T3 cells. J Biol Chem. 1996; 271: 439–445.
16. Wilson BA, Zhu X, Ho M, Lu L. Pasteurella multocida toxin activates the inositol triphosphate signaling pathway in Xenopus oocytes via G_q-coupled phospholipase C-ß₁. J Biol Chem. 1997; 272: 1268–1275.
17. Zywietz A, Gohla A, Schmelz M, Schultz G, Offermanns S. Pleiotropic effects of Pasteurella multocida toxin are mediated by G_q- dependent and -independent mechanisms: involvement of G_q but not G₁₁. J Biol Chem. 2001; 276: 3840–3845.
18. Rohde S, Sabri A, Kamasamudran R, Steinberg SF. The ₁-adrenergic receptor subtype- and protein kinase C isoform-dependence of norepinephrine’s actions in cardiomyocytes. J Mol Cell Cardiol. 2000; 32: 1193–1209.
19. Rybin VO, Steinberg SF. Protein kinase C isoform expression and regulation in the developing rat heart. Circ Res. 1994; 74: 299–309.
20. Pucéat M, Hilal-Dandan R, Strulovici B, Brunton LL, Brown JH. Differential regulation of protein kinase C isoforms in isolated neonatal and adult cardiomyocytes. J Biol Chem. 1994; 269: 16938–16944.
21. Parekh DB, Ziegler W, Parker PJ. Multiple pathways control protein kinase C phosphorylation. EMBO J. 2000; 19: 496–503.
22. Keranen LM, Dutil EM, Newton AC. Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr Biol. 1995; 5: 1394–1403.
23. Hansra G, Garcia-Paramio P, Prevostel C, Whelan RD, Bornancin F, Parker PJ. Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes. Biochem J. 1999; 342: 337–344.
24. Gschwendt M. Protein kinase C-. Eur J Biochem. 1999; 259: 555–564.
25. Soltoff SP. Rottlerin is a mitochondrial uncoupler that decreases cellular ATP levels and indirectly blocks PKC tyrosine phosphorylation. J Biol Chem. 2001; 276: 37986–37992.
26. Clerk A. Death by protein kinase C inhibitor: a stressful event. J Mol Cell Cardiol. 2001; 33: 1773–1776.
27. Zou Y, Komuro I, Yamazaki T, Aikawa R, Kudoh S, Shiojima I, Hiroi Y, Mizuno T, Yazaki Y. Protein kinase C, but not tyrosine kinases or Ras, plays a critical role in angiotensin II-induced activation of Raf-1 kinase and extracellular signal-regulated protein kinases in cardiac myocytes. J Biol Chem. 1996; 271: 33592–33597.
28. Doornbos RP, Theelen M, van der Hoeven PC, van Blitterswijk WJ, Verkleij AJ, van Bergen en Henegouwen PM. Protein kinase C is a negative regulator of protein kinase B activity. J Biol Chem. 1999; 274: 8589–8596.
29. Mao M, Fang X, Lu Y, Lapushin R, Bast RC, Mills GB. Inhibition of growth-factor-induced phosphorylation and activation of protein kinase B/Akt by atypical protein kinase C in breast cancer cells. Biochem J. 2000; 352: 475–482.
30. Konishi H, Matsuzaki H, Tanaka M, Ono Y, Tokunaga C, Kuroda S, Kikkawa U. Activation of RAC-protein kinase by heat shock and hyperosmolarity stress through a pathway independent of phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A. 1996; 93: 7639–7643.
31. Li W, Zhang J, Flechner L, Hyun T, Yam A, Franke TF, Pierce JH. Protein kinase C- overexpression stimulates Akt activity and suppresses apoptosis induced by interleukin-3 withdrawal. Oncogene. 1999; 18: 6564–6572.
32. Zheng WH, Kar S, Quirion R. Stimulation of protein kinase C modulates insulin-like growth factor-1-induced Akt activation in PC12 cells. J Biol Chem. 2000; 275: 13377–13385.
33. Matsumoto M, Ogawa W, Hino Y, Furukawa K, Ono Y, Takahashi M, Ohba M, Kuroki T, Kasuga M. Inhibition of insulin-induced activation of Akt by a kinase-deficient mutant of the epsilon isozyme of protein kinase C. J Biol Chem. 2001; 276: 14400–14406.
34. Bornancin F, Parker PJ. Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase C-. Curr Biol. 1996; 6: 1114–1123.

This article has been cited by other articles:

				T. Niizeki, Y. Takeishi, T. Kitahara, T. Arimoto, M. Ishino, O. Bilim, S. Suzuki, T. Sasaki, O. Nakajima, R. A. Walsh, et al. Diacylglycerol kinase-{varepsilon} restores cardiac dysfunction under chronic pressure overload: a new specific regulator of G{alpha}q signaling cascade Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H245 - H255. [Abstract] [Full Text] [PDF]

				N. Ozgen, M. Obreztchikova, J. Guo, H. Elouardighi, G. W. Dorn II, B. A. Wilson, and S. F. Steinberg Protein Kinase D Links Gq-coupled Receptors to cAMP Response Element-binding Protein (CREB)-Ser133 Phosphorylation in the Heart J. Biol. Chem., June 20, 2008; 283(25): 17009 - 17019. [Abstract] [Full Text] [PDF]

				J. O-Uchi, H. Sasaki, S. Morimoto, Y. Kusakari, H. Shinji, T. Obata, K. Hongo, K. Komukai, and S. Kurihara Interaction of {alpha}1-Adrenoceptor Subtypes With Different G Proteins Induces Opposite Effects on Cardiac L-type Ca2+ Channel Circ. Res., June 6, 2008; 102(11): 1378 - 1388. [Abstract] [Full Text] [PDF]

				L. R. Aminova, S. Luo, Y. Bannai, M. Ho, and B. A. Wilson The C3 domain of Pasteurella multocida toxin is the minimal domain responsible for activation of Gq-dependent calcium and mitogenic signaling Protein Sci., May 1, 2008; 17(5): 945 - 949. [Abstract] [Full Text] [PDF]

				F. Cosentino, P. Francia, G. G. Camici, P. G. Pelicci, M. Volpe, and T. F. Luscher Final Common Molecular Pathways of Aging and Cardiovascular Disease: Role of the p66Shc Protein Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 622 - 628. [Abstract] [Full Text] [PDF]

				D. M. Harris, H. I. Cohn, S. Pesant, R.-H. Zhou, and A. D. Eckhart Vascular smooth muscle Gq signaling is involved in high blood pressure in both induced renal and genetic vascular smooth muscle-derived models of hypertension Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3072 - H3079. [Abstract] [Full Text] [PDF]

				K. Y. Chung and J. W. Walker Interaction and Inhibitory Cross-Talk between Endothelin and ErbB Receptors in the Adult Heart Mol. Pharmacol., June 1, 2007; 71(6): 1494 - 1502. [Abstract] [Full Text] [PDF]

				J. H. C. Orth, K. Aktories, and K. F. Kubatzky Modulation of Host Cell Gene Expression through Activation of STAT Transcription Factors by Pasteurella multocida Toxin J. Biol. Chem., February 2, 2007; 282(5): 3050 - 3057. [Abstract] [Full Text] [PDF]

				J. Guo, A. Sabri, H. Elouardighi, V. Rybin, and S. F. Steinberg {alpha}1-Adrenergic Receptors Activate AKT via a Pyk2/PDK-1 Pathway That Is Tonically Inhibited by Novel Protein Kinase C Isoforms in Cardiomyocytes Circ. Res., December 8, 2006; 99(12): 1367 - 1375. [Abstract] [Full Text] [PDF]

				M. Obreztchikova, H. Elouardighi, M. Ho, B. A. Wilson, Z. Gertsberg, and S. F. Steinberg Distinct Signaling Functions for Shc Isoforms in the Heart J. Biol. Chem., July 21, 2006; 281(29): 20197 - 20204. [Abstract] [Full Text] [PDF]

				J. H. C. Orth, S. Lang, M. Taniguchi, and K. Aktories Pasteurella multocida Toxin-induced Activation of RhoA Is Mediated via Two Families of G{alpha} Proteins, G{alpha}q and G{alpha}12/13 J. Biol. Chem., November 4, 2005; 280(44): 36701 - 36707. [Abstract] [Full Text] [PDF]

				N. Wettschureck and S. Offermanns Mammalian G Proteins and Their Cell Type Specific Functions Physiol Rev, October 1, 2005; 85(4): 1159 - 1204. [Abstract] [Full Text] [PDF]

				K. Vijayan, E. L. Szotek, J. L. Martin, and A. M. Samarel Protein kinase C-{alpha}-induced hypertrophy of neonatal rat ventricular myocytes Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2777 - H2789. [Abstract] [Full Text] [PDF]