Published online before print March 3, 2008, doi: 10.1161/CIRCULATIONAHA.107.752618
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(Circulation. 2008;117:1378-1387.)
© 2008 American Heart Association, Inc.
Heart Failure |
Targeted Inhibition of Cardiomyocyte Gi Signaling Enhances Susceptibility to Apoptotic Cell Death in Response to Ischemic Stress
From the Center for Translational Medicine (B.R.D., E.G., M.B., L.E.V., J.S.M., P.W.R., J.K.C., D.M.H., G.W.K., S.S., A.D.E., W.J.K.), George Zallie and Family Laboratory of Cardiovascular Gene Therapy (B.R.D., E.G., M.B., L.E.V., J.S.M., P.W.R., J.K.C., G.W.K., S.S., W.J.K.), and Eugene Feiner Laboratory of Vascular Biology and Thrombosis (D.M.H., A.D.E.), Thomas Jefferson University, Philadelphia, Pa, and Institute for Surgical Research, University of Oslo, Oslo, Norway (L.E.V.).
Correspondence to Walter J. Koch, PhD, FAHA, W.W. Smith Professor of Medicine and Director, Center for Translational Medicine and George Zallie and Family Laboratory of Cardiovascular Gene Therapy, Thomas Jefferson University, 1025 Walnut St, Room 317, Philadelphia, PA 19107. E-mail walter.koch{at}jefferson.edu
Received November 14, 2007; accepted January 22, 2008.
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Abstract |
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Background— A salient characteristic of dysfunctional myocardium progressing to heart failure is an upregulation of the adenylyl cyclase inhibitory guanine nucleotide (G) protein
subunit, Gi2. It has not been determined conclusively whether increased Gi activity in the heart is beneficial or deleterious in vivo. Gi signaling has been implicated in the mechanism of cardioprotective agents; however, no in vivo evidence exists that any of the G subunits are cardioprotective. We have created a novel molecular tool to specifically address the role of Gi proteins in normal and dysfunctional myocardium.
Methods and Results— We have developed a class-specific Gi inhibitor peptide, GiCT, composed of the region of Gi2 that interacts specifically with G protein–coupled receptors. GiCT inhibits Gi signals specifically in vitro and in vivo, whereas Gs and Gq signals are not affected. In vivo expression of GiCT in transgenic mice effectively causes a "functional knockout" of cardiac Gi2 signaling. Inducible, cardiac-specific GiCT transgenic mice display a baseline phenotype consistent with nontransgenic mice. However, when subjected to ischemia/reperfusion injury, GiCT transgenic mice demonstrate a significant increase in infarct size compared with nontransgenic mice (from 36.9±2.5% to 50.9±4.3%). Mechanistically, this post-ischemia/reperfusion phenotype includes increased myocardial apoptosis and resultant decreased contractile performance.
Conclusions— Overall, our results demonstrate the in vivo utility of GiCT to dissect specific mechanisms attributed to Gi signaling in stressed myocardium. Our results with GiCT indicate that upregulation of Gi2 is an adaptive protective response after ischemia to shield myocytes from apoptosis.
Key Words: apoptosis • heart diseases • myocardial infarction • signal transduction
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Introduction |
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Apoptosis has been proposed as a mechanism for a significant amount of cell death occurring during acute myocardial infarction1–6 and is thought to contribute significantly to the progressive loss of functional myocardium in the failing heart.7,8 A variety of cardioprotective agents used in clinical practice to manage patients with acute myocardial infarction such as β-adrenergic receptor (β-AR) blockers, adenosine, and morphine are thought to be cardioprotective because of altered signaling through their cognate G protein–coupled receptor (GPCR). Importantly, these ligands have been demonstrated to reduce myocardial apoptotic cell death in animal models of ischemic injury,9–11 a finding that supports their clinical use as cardioprotective agents. Of note, the GPCRs acted on by these drugs can couple to the adenylyl cyclase inhibitory G protein
subunit (Gi).12,13
Clinical Perspective p 1387
Heterotrimeric G proteins exist as G subunits linked to Gβ
subunits. On ligand binding, GPCRs undergo a conformational change, which facilitates an interaction with the G protein heterotrimer, facilitating the dissociation of G and Gβ subunits that transduce intracellular signaling pathways, leading to specific GPCR-mediated cellular events.14 The G subunits can be used to classify heterotrimeric G proteins into 4 primary families: Gs, Gi/o, Gq/11, and G12/13. Gi proteins are ubiquitously expressed and have been classically associated with the inhibition of adenylyl cyclase, whereas Gβ has a variety of cellular effectors, including phosphoinositol-3 kinase/Akt, Ras, GPCR kinases, and phospholipase C-β.15 Of the Gi isoforms (Gi1, Gi2, Gi3), Gi2 is the predominant isoform in cardiac myocytes; along with Gi3, it has been shown to couple to cardiac β2-ARs.16
The role of Gi signaling in the context of myocardial ischemia and the progression toward heart failure is not straightforward.17,18 One salient characteristic of the failing heart is upregulation of Gi2 at the protein and transcript levels19; to date, however, there has been controversy surrounding the beneficial or maladaptive nature of this upregulation.16,20 To address Gi signaling in this regard, we have developed a Gi-selective inhibitory peptide to "functionally knock out" all GPCR-Gi–induced signals in the heart. This peptide, called GiCT, comprises the carboxyl-terminal 63 amino acids of Gi2, which shares >90% similarity with other Gi subunit family members, and is modeled after specific Gq and Gs inhibitory peptides previously described by our laboratory.21,22 The carboxy-terminal region of Gi2 facilitates the interaction of the ligand-activated receptor and G protein.23 Therefore, because GiCT is composed of this region, targeting the intracellular GPCR and G protein interface, both Gi and Gβ signals are inhibited. Mice harboring the GiCT transgene specifically in myocytes have allowed us to directly address the role of Gi signaling in response to stress in the form of myocardial ischemia, a causative agent in the development of heart failure, without knocking out or overexpressing any endogenous genes.
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Methods |
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For an expanded Methods section, please refer to the online-only Data Supplement.
Construction of Plasmid, Adenovirus, and Transgenic Mice
The carboxyl-terminal 63 amino acids of human Gi2 (Gi2 293 through 355) were inserted into the pCMV-HA vector (Clontech, Mountain View, Calif) to generate GiCT. Recombinant GiCT adenoviruses were generated with the Adeasy XL Adenoviral Vector System (Stratagene, La Jolla, Calif). For transgenic mouse generation, the bitransgenic system initially described by Sanbe and colleagues24 was used. The stable tetracycline analog doxycycline was administered to mice at 300 mg/kg of mouse diet (Bio-Serv, Frenchtown, NJ) to repress GiCT transcription as described.25
RNA Isolation and Semiquantitative Polymerase Chain Reaction
Quantitative polymerase chain reaction was carried out with iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, Calif) and the following gene-specific oligonucleotides: 28s, Gi2, Gs, Gq, and HA-GiCT (sequences available on request).
cAMP Production Assay
Mouse cardiac myocytes were isolated using a standard retrograde perfusion method from GiCT transgenic (Tg-GiCT) and nontransgenic mice. Myocytes were treated with 0.5 mmol/L 3-isobutyl-1-methylxanthine to inhibit phosphodiesterases and then stimulated as indicated. cAMP accumulation was quantified with a cAMP ELISA-based assay according to the manufacturer’s protocol (Sigma-Aldrich, St Louis, Mo).
Transthoracic Echocardiographic Analysis
Transthoracic 2-dimensional echocardiography in mice anesthetized with an intraperitoneal dose of Avertin (8 µL/g) with spontaneous respiration was performed with a 12-MHz probe as described previously.26
Ischemia/Reperfusion Injury Model
Surgical procedures were carried out according to National Institutes of Health Guidelines on the Use of Laboratory Animals, and all procedures were approved by the Thomas Jefferson University Committee on Animal Care. The ischemia/reperfusion (I/R) injury model was performed as previously described with minor modifications.27
Hemodynamic Analysis of Cardiac Function
Hemodynamic analysis was conducted as described previously.26
Determination of Left Ventricular Infarct Size and Area at Risk
Left ventricular (LV) infarct size and area at risk were assessed as previously described with slight modifications.27
Assessment of Myocardial Apoptosis and Apoptotic Signaling
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed as previously described.27 DNA fragmentation was assessed with the Gentra Puregene Tissue DNA Isolation Kit (QIAgen, Valencia, Calif). Caspase-3 cleavage was assessed by immunoblotting with cleaved caspase-3 antibody (Calbiochem, San Diego, Calif).
Isolation and Primary Culture of Neonatal Rat Ventricular Cardiomyocytes
Ventricular cardiomyocytes from 1- to 2-day–old rat neonatal hearts (neonatal rat ventricular myocytes [NRVMs]) were prepared as published in detail elsewhere.28 On day 2 of culture, NRVMs were infected with the indicated adenoviruses at a multiplicity of infection of 100, and experiments were performed within 24 hours of infection.
Mitochondrial Membrane Potential
To assess mitochondrial membrane potential (
m), we used tetramethylrhodamine ethyl ester (Molecular Probes, Eugene, Ore), which accumulates in mitochondria in response to the m. Experiments were performed as described elsewhere.29
Statistical Analysis
All values in the text and figures are presented as mean±SEM from independent experiments from given numbers. Statistical significance of multiple treatments was determined by 1-way or 2-way ANOVA followed by the Bonferroni post hoc test when appropriate. Statistical significance between 2 groups was determined by use of the 2-tailed Student t test. Values of P<0.05 were considered significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Expression and Gi Protein Selectivity of GiCT
The 63 amino acids of the G
i2 carboxyl-terminus were ligated in frame into pCMV-HA to generate an amino-terminus hemaglutinin-tagged GiCT construct (Figure 1A), which, on transient transfection into COS-7 cells, produced an 
10-kDa product (Figure 1B). To demonstrate the G protein–selective nature of GiCT on cellular Gi signaling, COS-7 cells were transiently transfected with pCMV-HA-GiCT or an empty vector control in addition to the GPCRs, β1-AR, or 1-AR. The overexpressed β1-AR and 1-AR were able to direct signaling through Gs and Gq, respectively, whereas Gi signals in COS-7 cells were assessed through endogenous lysophosphatidic acid receptors. We stimulated these cells with the β-AR agonist isoproterenol, the 1-AR agonist phenylephrine, or lysophosphatidic acid and measured the activation of p42/44 extracellular signal–regulated kinase (ERK) to assess activation of Gs-, Gq-, and Gi-coupled signaling to mitogen-activated protein kinases. Cells expressing GiCT demonstrated a significant reduction in ERK phosphorylation (ie, activation) in response to lysophosphatidic acid, whereas signals in response to isoproterenol or phenylephrine were not affected (Figure 1C and 1D), indicating inhibition of Gi-mediated signals but not of Gs or Gq.
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Generation and Functional Characterization of Mice Expressing GiCT Specifically in Cardiomyocytes
Founder transgenic mice that express GiCT under the control of the cardiac-specific tetracycline-responsive -myosin heavy chain promoter described by Sanbe et al24 were crossbred with homozygous mice possessing an -myosin heavy chain–driven tetracycline transactivator construct. The resultant pups from this crossing consisted of mice possessing both the GiCT and tetracycline transactivator transgenes, which will express the GiCT construct when doxycycline is removed from the food supply. These mice are referred to as Tg-GiCT mice, whereas mice possessing only the tetracycline transactivator transgene are referred to as nontransgenic mice and are used as control for functional and biochemical characterization. Tg-GiCT mice demonstrated robust expression of the 10-kDa HA epitope-tagged transgene at both the transcript and protein levels specifically in the heart (Figure 1E and 1F).
To assess the baseline functional consequences of our Gi inhibitor peptide at the whole-heart level, 6- to 8-week–old mice were subjected to transthoracic echocardiography under Avertin anesthesia. Tg-GiCT mice demonstrated no significant differences with respect to heart rate, wall thickness as assessed by LV posterior wall thickness and LV septal wall thickness, chamber dilatation as assessed by LV end-diastolic diameter, or contractile function as assessed by ejection fraction and fractional shortening (the Table). Tg-GiCT mice demonstrated no significant differences in organ morphometry as assessed by the ratios of heart weight to body weight and heart weight to tibial length compared with nontransgenic control mice (the Table). Furthermore, Tg-GiCT hearts were sectioned and subjected to staining with hematoxylin and eosin to assess gross structure, morphology, and individual cardiomyocyte size and to staining with Gomori trichrome to assess cardiac fibrosis. None of these parameters were significantly altered by expression of the GiCT transgene (data not shown).
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cAMP Accumulation in Isolated Tg-GiCT Cardiomyocytes
To establish the ex vivo biochemical phenotype of cardiomyocytes from Tg-GiCT mice, cAMP levels were measured from isolated myocytes from 8- to 10-week–old mice at baseline in response to the Gi-coupled agonist carbachol, which will inhibit cAMP production; the Gs-coupled agonist isoproterenol alone, which will stimulate cAMP production; and isoproterenol after carbachol pretreatment. In nontransgenic myocytes, carbachol pretreatment nearly completely inhibited cAMP accumulation in response to isoproterenol, an effect that was significantly blocked in Tg-GiCT myocytes, which demonstrate a significant reduction in Gi-mediated response to carbachol pretreatment (113.7±19.4% for nontransgenic compared with 201.9±26.9% for Tg-GiCT myocytes; Figure 2). Furthermore, as expected, Tg-GiCT mice demonstrated a trend toward a smaller reduction in baseline cAMP in response to carbachol treatment alone, and no significant difference was observed between cAMP accumulation in nontransgenic and Tg-GiCT mice in response to the Gs-coupled agonist isoproterenol, further confirming the Gi-selective nature of the GiCT transgene ex vivo.
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P<0.05 vs nTg; n=4.
Altered G Subunit Expression After I/R Injury
To explore the potential for I/R injury to regulate the mRNA expression of G protein subunits, we isolated mRNA from the area at risk of wild-type mice subjected to 30 minutes of ischemia followed by 72 hours of reperfusion and sham-operated mice. Semiquantitative reverse-transcription polymerase chain reaction conducted on these samples demonstrated a 3-fold upregulation of cardiac Gi2 compared with sham-operated mice (Figure 3A), whereas no significant change occurred in the mRNA levels of Gs or Gq (Figure 3B and 3C). Levels of Gi1 and Gi3 also were investigated. Gi1 mRNA levels were not detectable, whereas Gi3 mRNA expression was increased after I/R, but the expression was significantly (20-fold) less compared with Gi2 (data not shown). These data indicate that stress in the form of I/R injury results in changes in the signal transduction milieu, ultimately resulting in the regulation of Gi signaling at the mRNA level.
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Effect of Myocyte-Specific Inhibition of Gi Signaling on Susceptibility to I/R Injury
Given that Gi is regulated in this model of myocardial dysfunction, we investigated the role of Gi signal inhibition on susceptibility to myocardial I/R injury. We subjected 8- to 10-week–old Tg-GiCT and nontransgenic mice maintained on a standard or doxycycline-supplemented diet to 30 minutes of myocardial ischemia followed by 24 hours of reperfusion, at which time infarct sizing was conducted by a combined Evan’s Blue/triphenyltetrazolium chloride staining method (Figure 4B). It is important to note that Tg-GiCT mice maintained on a doxycycline-supplemented diet do not express a detectable amount of GiCT transgene at the protein level (Figure 4A). The infarct size of Tg-GiCT mice, represented as the percentage of the area at risk, was significantly increased by 14.2% (36.9±2.5% for nontransgenic and 50.9±4.3% for Tg-GiCT; Figure 4C), although the area at risk was not significantly changed between groups (53.2±1.5% for nontransgenic and 51.5±2.1% for Tg-GiCT; Figure 4D), thus indicating that Gi signal inhibition, specif-ically in myocytes, results in increased susceptibility to I/R injury. Importantly, the addition of doxycycline completely ameliorates the susceptibility for I/R injury of Tg-GiCT mice (Figure 4B through 4D). Tg-GiCT and nontransgenic mice maintained on doxycycline were subjected to the same I/R protocol, which resulted in a normalization of infarct sizes between groups consistent with that of nontransgenic mice subjected to I/R, thus indicating that "turning off" the expression of the GiCT transgene normalizes susceptibility to I/R injury.
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Enhanced Myocardial Apoptosis in Tg-GiCT Mice in Response to I/R Injury
To ascertain the mechanism for the increased infarct size in response to ischemic injury in Tg-GiCT mice, we subjected 8- to 10-week–old mice to 30 minutes of ischemia followed by 3 hours of reperfusion, at which point hearts were embedded in paraffin and processed for TUNEL staining. Tg-GiCT mice demonstrate an 2.5-fold increase in the number of TUNEL-positive nuclei compared with nontransgenic control mice (Figure 5A and 5B), indicating increased apoptotic cell death in Tg-GiCT mice in response to ischemic stress. No difference between groups was observed with respect to baseline TUNEL-positive nuclei because positive TUNEL staining was rarely observed in either sham-operated group.
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To independently confirm the finding of increased apoptotic cell death in mice with cardiomyocyte-specific expression of GiCT, we investigated DNA fragmentation and caspase-3 cleavage in these mice. Preparation of genomic DNA from the area at risk demonstrated apparent DNA fragmentation in nontransgenic control mice after I/R; however, the degree of DNA fragmentation was markedly more evident in Tg-GiCT mice (Figure 5C). Furthermore, caspase-3 cleavage was significantly increased, as evidenced by the increased detection of the 17-kDa caspase-3 large cleavage product in the myocardial extracts from the area at risk of Tg-GiCT mice subjected to 30 minutes of ischemia followed by 1.5 hours of reperfusion compared with nontransgenic control mice (Figure 5D).
Effect of Myocardial Expression of GiCT on Cardiac Function in Response to I/R Injury
To measure cardiac function in Tg-GiCT and nontransgenic control mice subjected to stress in the form of I/R injury or sham-operative procedure, hemodynamic parameters were recorded after LV catheterization at baseline and in response to increasing doses of isoproterenol. Under sham conditions, both at baseline and in response to β-AR stimulation, no difference was detectable between Tg-GiCT and nontransgenic mice with respect to heart rate, cardiac contractility as assessed by LV±dP/dt, LV end-diastolic pressure, and mean arterial blood pressure (Figure 6). However, Tg-GiCT mice subjected to 30 minutes of ischemia and 72 hours of reperfusion demonstrated a significant reduction in cardiac contractile performance compared with both sham-operated mice and I/R-operated nontransgenic control mice (Figure 6). In addition, Tg-GiCT mice exhibited a trend toward increased LV end-diastolic pressure. These data indicate an overall worsening of contractile performance in Tg-GiCT in response to stress in the form of I/R injury and represent a functional correlate to our data indicating increased infarct size and apoptosis in Tg-GiCT mice.
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Effect of GiCT on Determining Mitochondrial Response to Oxidative Stress In Vitro
To assess the effect of in vitro blockade of Gi signaling on cardiomyocyte response to oxidative stress, a key component of ischemia/reperfusion injury, we measured m. Loss of m represents an initial step in the mitochondrial apoptotic cascade, followed by release of cytochrome c and activation of caspases. NRVMs infected with the GiCT adenovirus demonstrated no difference in m compared with LacZ-infected NRVMs at baseline, and expression of GiCT in NRVMs was confirmed by immunoblot (Figure 7A). In response to 100 µmol/L H2O2, both LacZ- and GiCT-infected NRVMs displayed a significant loss of m; however, this effect was significantly enhanced in GiCT-infected NRVMs (Figure 7B), indicating that the Gi pathway protects cardiomyocytes from cell death at the level of the mitochondria.
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Importantly, differential survival and apoptotic signals have been reported for cardiac β-ARs, with β1-ARs being proapoptotic (via Gs) and β2-ARs being primarily prosurvival via the apparent ability of this receptor to couple to Gi proteins.17 In fact, data have shown that stimulation of β2-AR signaling can prevent reactive oxygen species–induced cell death through its ability to couple to Gi.30 Thus, in these experiments, we stimulated myocytes with the β2-AR agonist zinterol. Zinterol alone resulted in a significant increase in m in LacZ-infected NRVMs (Figure 7B). However, cells expressing the GiCT showed no response to zinterol from baseline m (Figure 7B). Importantly, the addition of zinterol in the absence of tetramethylrhodamine ethyl ester did not alter baseline fluorescence (data not shown). In addition, expression of GiCT blocked the protective effect of zinterol pretreatment in response to H2O2-induced cell death. This indicates that the Gi component of β2-AR signaling provides survival signals that are blocked by GiCT, resulting in a loss of m followed by release of deleterious factors from mitochondria, activation of the caspase cascade, and death of cardiomyocytes. Blockade of prosurvival pathways by expression of GiCT in NRVMs was confirmed by assessment of Akt phosphorylation. Short-term exposure to reactive oxygen species has previously been shown to increase the phosphorylation and thus activation of the serine/threonine kinase Akt, leading to decreased apoptotic cell death.31,32 Exposure to 100 µmol/L H2O2 for 60 minutes resulted in a significant increase in Akt phosphorylation in LacZ-infected NRVMs that was significantly inhibited in GiCT-infected NRVMs (Figure 7C and 7D), further establishing that Gi signals are a key component to survival signaling in response to reactive oxygen species, a key contributor to myocardial injury in response to I/R.32
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Discussion |
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Here, we have developed a novel molecular tool, GiCT, for selectively inhibiting signals induced by Gi activation. We have found that Gi class–specific inhibition in vivo in the heart via GiCT expression worsens outcome after myocardial ischemia via increased myocyte apoptosis. Thus, GiCT serves as a tool to delineate the role of Gi upregulation in dysfunctional myocardium, and we have created inducible transgenic mice that effectively represent a functional knockout of cardiac Gi proteins without deleting a gene or overexpressing endogenous genes. Our findings reveal that after I/R injury the increased Gi signaling resulting from Gi upregulation appears to be adaptive and promotes cellular protection after ischemia.
We have previously demonstrated the utility of specific G protein subunit carboxyl-terminal peptides to serve as molecular tools to dissect G protein–selective intracellular signaling in vitro and in vivo.21,22 Indeed, using a peptide (GqI) specific for GPCR-Gq activation, we determined that Gq signals are the final common trigger for induction of pressure-overload ventricular hypertrophy.21 Moreover, we developed a similar tool for the specific inhibition of Gs-coupled receptor signaling in cells.22 These studies have revealed that class-specific G protein inhibition is possible and may have therapeutic potential for combating certain cardiovascular diseases such as maladaptive hypertrophy or hypertension.33,34 In this study, we find that it is possible to selectively inhibit Gi-mediated signals in vivo with GiCT expression, and using this tool, we have begun to study more in depth the role of Gi in dysfunctional myocardium.
Importantly, we found no basal cardiac phenotype resulting from transgenic GiCT expression, showing that Gi inhibition in the adult heart leads to no overt structural or functional myocardial alterations. This is similar to what was observed in mice expressing a Gq-inhibitory peptide in myocytes21 and is encouraging for any potential therapeutic applications of these peptides. We did confirm that cardiac Gi signals were blocked in Tg-GiCT mice because myocytes were isolated and inhibition of cAMP production resulting from the Gi-coupled receptor agonist carbachol was attenuated compared with myocytes from control mice. Moreover, apoptotic signaling was enhanced in vivo after ischemia via I/R injury and in vitro in myocytes exposed to oxidative stress when Gi signals were blocked by GiCT expression. Thus, GiCT appears to be effective at Gi blockade, and signaling via Gi activation after myocardial ischemia appears to be prosurvival. This is apparently enhanced even when myocardium is stressed with I/R; we found enhanced Gai2 transcript levels after I/R, which indicates that myocytes adapt to injury with signaling molecules promoting survival. Thus, GiCT expression blocks even enhanced levels of GI, and targeted inhibition of Gi results in increased myocardial infarct size and myocardial apoptosis, as evidenced by enhanced TUNEL staining, DNA fragmentation, and activation of caspases. This translated into Tg-GiCT mice having worsened in vivo LV contractile function after I/R, most probably as a result of an increased loss of viable myocardium.
The cell death induced by I/R injury is a combination of apoptotic and necrotic processes. Our goal in the present work was to assess the role of Gi signaling in vivo on the susceptibility to apoptosis in response to stress. In doing so, however, we are not excluding a potential role of necrosis in the increased infarct size seen in Tg-GiCT mice. Importantly, we have assessed necrosis in vitro in NRVMs after exposure to H2O2 at concentrations and times that we have shown to elicit enhanced apoptosis in GiCT-infected NRVMs, and GiCT expression did not appear to alter necrotic cell death (data not shown).
The use of GiCT as a molecular tool to study the functional consequences of in vivo myocyte-specific Gi blockade represents an advancement over previous approaches using pertussis toxin (PTx) or global Gi isoform knockout mice. Conclusions derived from in vivo application of PTx, derived from Bordetella pertussis, the causative agent of whooping cough, are difficult to establish definitively given the array of noncardiac side effects of intraperitoneal injection of the toxin.35 Furthermore, ex vivo application of PTx through a modified Langendorff procedure may affect cardiac function; however, the respective contribution of cardiac myocytes compared with nonmyocytes is difficult to ascertain.36,37 Importantly, in cells, we found that GiCT inhibited Gi signaling as effectively as PTx (data not shown). Thus, GiCT appears to have key advantages over PTx because it can be expressed in a myocyte-specific manner and is nontoxic to basal cellular homeostasis.
Global Gi knockout mice also are subject to similar concerns. Gi2 knockout mice display a phenotype resembling histopathological features of ulcerative colitis and adenocarcinoma of the colon with premature death, potentially masking any gross cardiac phenotype.38 In addition, the effect of Gi2 or any Gi subunit knockout does not identify any specific signals attributed to Giβ dissociation. Because GiCT competes with endogenous Gi proteins with agonist-occupied GPCRs, preventing G activation and subsequent G and Gβ dissociation, it is a blocker of total Gi signaling, much like PTx. Therefore, GiCT has an advantage over classic methods of knocking out a Gi gene. Moreover, Tg-GiCT mice express this Gi inhibitor in a manner temporally controlled by doxycycline, and importantly, we demonstrate that the worsening of myocardial cell death after I/R is absent in these mice if they are maintained on doxycycline and GiCT expression is silenced.
The utility of GiCT allowed us to specifically investigate in myocytes the potential mechanistic role of Gi in the prosurvival signaling that has been attributed to β2-AR activation. Previously, using PTx and the βARKct peptide as a Gβ inhibitor has implicated Giβ-mediated phosphoinositol-3 kinase activation in promoting antiapoptotic pathways in myocytes.17 In this study, we were able to use a single molecular tool, GiCT, to attenuate the β2-AR–mediated protective effects on m. As mentioned, GiCT effectively inhibits both Gi and Giβ signals, and these results are consistent with that notion.
Overall, our results indicate that the upregulation of Gi2 in ischemic myocardium appears to be protective in inducing cell survival pathways to prevent myocyte death in response to I/R injury. This may also provide insight into the role of upregulation of cardiac Gi2 in human heart failure, which may provide survival signals that prevent apoptotic cell death induced by the toxic levels of catecholamines and calcium overload present in chronically dysfunctional myocardium.39
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Acknowledgments |
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The bitransgenic system used for transgenic mouse generation was a kind gift from Dr Jeffrey Robbins of the Univeristy of Cincinnati.
Sources of Funding
This research was supported in part by grants from the Pennsylvania–Delaware Affiliate of the American Heart Association (predoctoral fellowship to B.R. DeGeorge Jr) and National Institutes of Health/National Heart, Lung, and Blood Institute grants R01 HL61690, R01 HL56205, and P01 HL075433 (project 2) (to Dr Koch). B.R. DeGeorge was supported in part by the Dubbs Scholar Fellowship Award and Percival E. and Ethel Brown Foerderer Foundation Fellowship. This work also was supported in part by grant A75301 from the Commonwealth of Pennsylvania Department of Health. Dr Koch is the W.W. Smith Professor of Medicine.
Disclosures
None.
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References |
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1. Buja LM, Eigenbrodt ML, Eigenbrodt EH. Apoptosis and necrosis: basic types and mechanisms of cell death. Arch Pathol Lab Med. 1993; 117: 1208–1214.[Medline] [Order article via Infotrieve]
2. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest. 1994; 94: 1621–1628.[Medline] [Order article via Infotrieve]
3. Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res. 1996; 79: 949–956.
4. Foo RS, Mani K, Kitsis RN. Death begets failure in the heart. J Clin Invest. 2005; 115: 565–571.[CrossRef][Medline] [Order article via Infotrieve]
5. James TN. Normal and abnormal consequences of apoptosis in the human heart: from postnatal morphogenesis to paroxysmal arrhythmias. Circulation. 1994; 90: 556–573.
6. Kitsis RN, Mann DL. Apoptosis and the heart: a decade of progress. J Mol Cell Cardiol. 2005; 38: 1–2.[CrossRef][Medline] [Order article via Infotrieve]
7. Abbate A, Bussani R, Amin MS, Vetrovec GW, Baldi A. Acute myocardial infarction and heart failure: role of apoptosis. Int J Biochem Cell Biol. 2006; 38: 1834–1840.[CrossRef][Medline] [Order article via Infotrieve]
8. Sabbah HN. Apoptotic cell death in heart failure. Cardiovasc Res. 2000; 45: 704–712.
9. Barrere-Lemaire S, Combes N, Sportouch-Dukhan C, Richard S, Nargeot J, Piot C. Morphine mimics the antiapoptotic effect of preconditioning via an Ins(1,4,5)P3 signaling pathway in rat ventricular myocytes. Am J Physiol. 2005; 288: H83–H88.
10. Funakoshi H, Zacharia LC, Tang Z, Zhang J, Lee LL, Good JC, Herrmann DE, Higuchi Y, Koch WJ, Jackson EK, Chan TO, Feldman AM. A1 adenosine receptor upregulation accompanies decreasing myocardial adenosine levels in mice with left ventricular dysfunction. Circulation. 2007; 115: 2307–2315.
11. Tilley DG, Rockman HA. Role of beta-adrenergic receptor signaling and desensitization in heart failure: new concepts and prospects for treatment. Exp Rev Cardiovasc Ther. 2006; 4: 417–432.[CrossRef]
12. Hamad E, Mather PJ, Srinivasan S, Rubin S, Whellan DJ, Feldman AM. Pharmacologic therapy of chronic heart failure. Am J Cardiovasc Drugs. 2007; 7: 235–248.[CrossRef][Medline] [Order article via Infotrieve]
13. Salazar NC, Chen J, Rockman HA. Cardiac GPCRs: GPCR signaling in healthy and failing hearts. Biochim Biophys Acta. 2007; 1768: 1006–1018.[Medline] [Order article via Infotrieve]
14. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev. 2002; 3: 639–650.[CrossRef]
15. Wettschureck N, Offermanns S. Mammalian G proteins and their cell type specific functions. Physiol Rev. 2005; 85: 1159–1204.
16. Xiao RP, Avdonin P, Zhou YY, Cheng H, Akhter SA, Eschenhagen T, Lefkowitz RJ, Koch WJ, Lakatta EG. Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ Res. 1999; 84: 43–52.
17. Zhu WZ, Zheng M, Koch WJ, Lefkowitz RJ, Kobilka BK, Xiao RP. Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes. Proc Natl Acad Sci U S A. 2001; 98: 1607–1612.
18. Leineweber K, Bohm M, Heusch G. Cyclic adenosine monophosphate in acute myocardial infarction with heart failure: slayer or savior? Circulation. 2006; 114: 365–367.
19. Feldman AM, Cates AE, Veazey WB, Hershberger RE, Bristow MR, Baughman KL, Baumgartner WA, Van Dop C. Increase of the 40,000-mol wt pertussis toxin substrate (G protein) in the failing human heart. J Clin Invest. 1988; 82: 189–197.[Medline] [Order article via Infotrieve]
20. Baker AJ, Redfern CH, Harwood MD, Simpson PC, Conklin BR. Abnormal contraction caused by expression of G(i)-coupled receptor in transgenic model of dilated cardiomyopathy. Am J Physiol. 2001; 280: H1653–H1659.
21. Akhter SA, Luttrell LM, Rockman HA, Iaccarino G, Lefkowitz RJ, Koch WJ. Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy. Science. 1998; 280: 574–577.
22. Feldman DS, Zamah AM, Pierce KL, Miller WE, Kelly F, Rapacciuolo A, Rockman HA, Koch WJ, Luttrell LM. Selective inhibition of heterotrimeric Gs signaling: targeting the receptor-G protein interface using a peptide minigene encoding the Galpha(s) carboxyl terminus. J Biol Chem. 2002; 277: 28631–28640.
23. Oldham WM, Van Eps N, Preininger AM, Hubbell WL, Hamm HE. Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins. Nat Struct Mol Biol. 2006; 13: 772–777.[CrossRef][Medline] [Order article via Infotrieve]
24. Sanbe A, Gulick J, Hanks MC, Liang Q, Osinska H, Robbins J. Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter. Circ Res. 2003; 92: 609–616.
25. Funakoshi H, Chan TO, Good JC, Libonati JR, Piuhola J, Chen X, MacDonnell SM, Lee LL, Herrmann DE, Zhang J, Martini J, Palmer TM, Sanbe A, Robbins J, Houser SR, Koch WJ, Feldman AM. Regulated overexpression of the A1-adenosine receptor in mice results in adverse but reversible changes in cardiac morphology and function. Circulation. 2006; 114: 2240–2250.
26. Most P, Seifert H, Gao E, Funakoshi H, Volkers M, Heierhorst J, Remppis A, Pleger ST, DeGeorge BR Jr, Eckhart AD, Feldman AM, Koch WJ. Cardiac S100A1 protein levels determine contractile performance and propensity toward heart failure after myocardial infarction. Circulation. 2006; 114: 1258–1268.
27. Gao E, Boucher M, Chuprun JK, Zhou RH, Eckhart AD, Koch WJ. Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart. Am J Physiol. 2007; 293: H60–H68.
28. Most P, Boerries M, Eicher C, Schweda C, Ehlermann P, Pleger ST, Loeffler E, Koch WJ, Katus HA, Schoenenberger CA, Remppis A. Extracellular S100A1 protein inhibits apoptosis in ventricular cardiomyocytes via activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2). J Biol Chem. 2003; 278: 48404–48412.
29. Oubrahim H, Stadtman ER, Chock PB. Mitochondria play no roles in Mn(II)-induced apoptosis in HeLa cells. Proc Natl Acad Sci U S A. 2001; 98: 9505–9510.
30. El Jamali A, Freund C, Rechner C, Scheidereit C, Dietz R, Bergmann MW. Reoxygenation after severe hypoxia induces cardiomyocyte hypertrophy in vitro: activation of CREB downstream of GSK3beta. FASEB J. 2004; 18: 1096–1098.
31. Brunt KR, Fenrich KK, Kiani G, Tse MY, Pang SC, Ward CA, Melo LG. Protection of human vascular smooth muscle cells from H2O2-induced apoptosis through functional codependence between HO-1 and AKT. Arterioscler Thromb Vasc Biol. 2006; 26: 2027–2034.
32. Kwon SH, Pimentel DR, Remondino A, Sawyer DB, Colucci WS. H(2)O(2) regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways. J Mol Cell Cardiol. 2003; 35: 615–621.[CrossRef][Medline] [Order article via Infotrieve]
33. Esposito G, Rapacciuolo A, Naga Prasad SV, Takaoka H, Thomas SA, Koch WJ, Rockman HA. Genetic alterations that inhibit in vivo pressure-overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation. 2002; 105: 85–92.
34. Keys JR, Greene EA, Koch WJ, Eckhart AD. Gq-coupled receptor agonists mediate cardiac hypertrophy via the vasculature. Hypertension. 2002; 40: 660–666.
35. Schultz JE, Hsu AK, Barbieri JT, Li PL, Gross GJ. Pertussis toxin abolishes the cardioprotective effect of ischemic preconditioning in intact rat heart. Am J Physiol. 1998; 275: H495–H500.[Medline] [Order article via Infotrieve]
36. Lawson CS. G proteins and preconditioning. Cardiovasc Res. 1993; 27: 689–690.[Medline] [Order article via Infotrieve]
37. Piacentini L, Wainwright CL, Parratt JR. The antiarrhythmic effect of ischaemic preconditioning in isolated rat heart involves a pertussis toxin sensitive mechanism. Cardiovasc Res. 1993; 27: 674–680.
38. Rudolph U, Finegold MJ, Rich SS, Harriman GR, Srinivasan Y, Brabet P, Boulay G, Bradley A, Birnbaumer L. Ulcerative colitis and adenocarcinoma of the colon in G alpha i2-deficient mice. Nat Genet. 1995; 10: 143–150.[Medline] [Order article via Infotrieve]
39. Miyamoto S, Howes AL, Adams JW, Dorn GW 2nd, Brown JH. Ca2+ dysregulation induces mitochondrial depolarization and apoptosis: role of Na+/Ca2+ exchanger and AKT. J Biol Chem. 2005; 280: 38505–38512.
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The online-only Data Supplement, which contains expanded Methods, can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107. 752618/DC1.
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