Cellular Biology

Cardiomyocyte Apoptosis Induced by Gαq Signaling Is Mediated by Permeability Transition Pore Formation and Activation of the Mitochondrial Death Pathway

John W. Adams, Amy L. Pagel, Christopher K. Means, Donna Oksenberg, Robert C. Armstrong, Joan Heller Brown
https://doi.org/10.1161/01.RES.87.12.1180
Circulation Research. 2000;87:1180-1187
Originally published December 8, 2000

Jump to

Abstract

Abstract—Expression of the wild-type α subunit of Gq stimulates phospholipase C and induces hypertrophy in cardiomyocytes. Addition of Gq-coupled receptor agonists additionally activates phospholipase C, as does expression of a constitutively active mutant form of Gαq. Under these conditions, hypertrophy is rapidly succeeded by apoptotic cellular and molecular changes, including myofilament disorganization, loss of mitochondrial membrane potential, alterations in Bcl-2 family protein levels, DNA fragmentation, increased caspase activity (≈4-fold), cytochrome c redistribution, and nuclear chromatin condensation in ≈12% of the cells. We used various interventions to define the molecular relationships between these events and identify potential sites at which these features of apoptosis could be rescued. Treatment with caspase inhibitors prevented DNA fragmentation and promoted myocyte survival; however, cytochrome c release and loss of mitochondrial membrane potential still occurred. In contrast, treatment with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore, not only prevented DNA fragmentation and reduced nuclear chromatin condensation but also preserved mitochondrial membrane potential and limited cytochrome c redistribution to only ≈2% of cells. These data demonstrate the central role of mitochondrial membrane potential in initiation of caspase activation and downstream apoptotic events and suggest that preservation of mitochondrial integrity is crucial for prolonging the life and function of cardiomyocytes exposed to pathological levels of stress.

In the last several years, in vitro and in vivo experiments have established a pivotal role for the heterotrimeric G protein, Gq, in initiating activation of intracellular growth-signaling pathways in cardiac myocytes. For example, studies using cultured neonatal rat ventricular myocytes have demonstrated that many hormones or growth factors that couple to Gq to activate phospholipase C stimulate cardiomyocyte hypertrophy.1 2 3 4 We showed previously that adenovirus-mediated overexpression of the wild-type α subunit of Gq can also induce cardiomyocyte hypertrophy.5

Transgenic mice expressing a modestly increased (≈4-fold) level of Gαq in the heart manifest a stable cardiac hypertrophy, whereas transgenic mice with higher levels of Gαq overexpression (8-fold) develop a dilated cardiomyopathy.6 In addition, the hypertrophy seen in mice overexpressing Gαq at modest levels rapidly transitioned into a lethal dilated cardiomyopathy in females during the stress associated with pregnancy and parturition5 and in Gαq-expressing mice subjected to aortic banding (J.W. Adams, J. Ross, Jr, unpublished data, 2000). Expression of a constitutively activated form rather than a wild-type form of Gαq (GαqWT) in the hearts of transgenic mice was also found to result in a dilated cardiomyopathy.7 Cardiomyocyte apoptosis occurs in all of these models in which it was examined, suggesting that it contributes to the observed cardiomyopathies. Consistent with this, expression of activated Gαq or stimulation of the Gq-coupled angiotensin II receptor induces apoptosis in isolated cardiomyocytes.5 8

It has been hypothesized that loss of contractile function in the overloaded heart may result, at least in part, from myocyte dropout attributable to apoptotic cell death. However, despite substantial evidence for a role of apoptosis in the pathogenesis of heart failure, its biochemical triggers have not been identified. Recently, it was demonstrated that moderate heart failure after myocardial infarction in rats is associated with upregulation of the expression of Gαq and phospholipase C (PLC)-β.9 Additionally, many of the neurohumoral activators of Gq signaling (eg, catecholamines, endothelin, prostaglandin F2α [PGF2α], and angiotensin II) are also elevated in the failing myocardium.10 These observations led us to examine mechanisms by which Gq signaling might trigger apoptosis and thereby contribute to heart failure.

Apoptosis is distinguished from necrosis by several morphological and biochemical criteria, including proteolytic activation of caspases. Recent evidence points to the mitochondria as a critical trigger for caspase activation in mammalian cells.11 In response to a variety of stimuli, proapoptotic signals converge on the mitochondria to provoke the release of cytochrome (cyto) c and other factors, which combine in the cytoplasm to initiate caspase activation.11 Cyto c release has been associated with changes in mitochondrial membrane permeability secondary to loss of mitochondrial membrane potential (ΔΨm). Recently, a pivotal role for the mitochondrial permeability transition (PT) pore as a mechanism for loss of ΔΨm was shown.12

Mitochondrial cyto c release has been observed in several models of cardiomyocyte apoptosis,13 14 15 16 17 but the importance of mitochondria as a target for preserving cardiomyocyte function has not been clearly examined. In this study, we demonstrate the involvement of the mitochondrial pathway as an upstream event in apoptotic signaling induced by activation of Gq and present the first evidence that inhibition of the mitochondrial PT rescues cardiomyocytes from apoptotic cell death.

Materials and Methods

More detailed methods are described in the online data supplement, available at http://www.circresaha.org, and in published studies, as referenced.

Neonatal cell culture,18 19 phosphoinositide hydrolyisis,20 generation of recombinant adenoviruses,5 21 DNA fragmentation,5 electron microscopy,22 and immunoblotting3 were performed as described previously. Methods for assessment of caspase activity and inhibition experiments are described in the online data supplement.

Adenoviral Constructs

Adenoviral constructs were prepared in our laboratory from expression plasmids encoding wild-type, constitutively active Gαq (GαqQ209L), generated by Dr Gary Johnson (University of Colorado), and a mutant form of activated Gαq (GαqDNE/AAA), obtained from Dr John Exton (Vanderbilt University, Nashville, Tenn).23 AdBcl-xl was obtained from Dr Gianluigi Condorelli (Thomas Jefferson University, Philadelphia, Pa).

Inhibitors and Antibodies

Idun 1965 was synthesized and characterized at Idun Pharmaceuticals.24 ZVAD-fmk and bongkrekic acid (BA) were obtained commercially (Calbiochem). Antibodies for Bcl-2 and Bcl-xl were obtained from Transduction Laboratories; Bad and Bax antibodies were obtained from Santa Cruz.

Immunocytochemistry and Cyto c Redistribution

Cells were fixed, permeabilized, blocked, and incubated sequentially with a mouse monoclonal antibody against cyto c (Pharmingen) and an Alexa 594–conjugated goat anti-mouse IgG (Molecular Probes). Myocyte nuclei and sarcomeres (F-actin) were stained concurrently with Hoechst 33342 (Molecular Probes) and FITC-conjugated phalloidin (Molecular Probes). Cyto c localization, sarcomere organization, and nuclear structure were visualized on a Zeiss Axiovert fluorescence microscope. Alternatively, multispectral digital images of fluorescent cellular structures were visualized using a deconvolution microscope (Nikon).

Measurement of ΔΨm

ΔΨm was assessed using JC-125 (10 μmol/L) and MitoTracker Red CMXRos (200 nmol/L) staining at 37°C for 10 minutes. Cover slips with attached live (unfixed) cells were inverted onto glass slides for microscopy. Multispectral digital images of fluorescent cellular structures were visualized by deconvolution microscopy. Alternatively, myocytes stained with MitoTracker Red were collected by trypsinization (0.05% Trypsin, 0.53 mmol/L EDTA) and prepared for quantitative analysis of ΔΨm by flow cytometry.

Statistical Analysis

Results are reported as mean±SEM. Statistical significance was determined using ANOVA with Newman-Keuls correction for multiple comparisons. A P value of <0.05 was considered statistically significant.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

Results

Adenovirus-Mediated Expression of Gαq Activates PLC

We previously reported that constitutively activated Gαq expression leads to a marked increase in PLC activity and subsequent myocyte apoptosis.5 Stimulating endogenous Gq-coupled receptors in cells overexpressing GαqWT elicited a comparable level of PLC activation (Figure 1⇓). Thus, although overexpression of GαqWT or stimulation with PGF2α caused a 10- to 15-fold increase in inositol phosphate formation, addition of PGF2α (or phenylephrine; data not shown) to GαqWT-overexpressing myocytes increased PLC activation to the level induced by constitutively activated Gαq (GαqQ209L).

Figure 1.
Figure 1.

Figure 1⇓. Phosphoinositide hydrolysis is increased by constitutively activated Gαq or agonist-enhanced Gαq signaling. Myocytes were infected with indicated adenovirus constructs and labeled with 2 μCi/mL [3H]-myoinositol for 24 hours in serum-free medium and then washed and switched to HEPES-buffered medium containing 10 mmol/L LiCl with or without 1 μmol/L PGF2α for 30 minutes. Cell lysates were run over ion exchange columns, and total inositol phosphates were eluted and incorporated label measured with a liquid scintillation counter. Data represent average fold stimulation±SEM from 3 experiments, each done in triplicate. *P<0.05 vs control adenovirus (AdCtrl); #P<0.01 versus AdCtrl/PGF2α.

Enhanced Gαq Signaling Causes Cardiomyocyte Apoptosis

To correlate the level of PLC activation with cardiomyocyte apoptosis, we measured oligonucleosomal DNA fragmentation in myocytes infected with either GαqWT or activated Gαq adenoviruses in the presence or absence of PGF2α. A striking increase in DNA fragmentation was seen with GαqQ209L expression or when PGF2α was added to GαqWT-overexpressing cells (Figure 2A⇓). To determine whether coupling to PLC was required for Gαq-induced apoptosis, we infected myocytes with an adenovirus encoding an activated Gαq mutated to inhibit coupling to PLC.23 Significantly reduced hydrolysis of inositol phospholipids (10-fold above control versus 60-fold for GαqQ209L) was seen when this mutant (GαqDNE/AAA) was expressed in myocytes at levels similar to GαqQ209L. Importantly, no apoptosis was observed in cells infected with this construct (data not shown). Furthermore, expression of a constitutively active mutant of the α subunit of the heterotrimeric G protein, Gi, does not activate PLC, nor did it induce DNA fragmentation (Figure 2A⇓). Thus, stimuli that cause high-level PLC activation are associated with apoptotic cell death, evidenced by the formation of oligonucleosomal-sized DNA fragments.

Figure 2.
Figure 2.

Figure 2⇓. DNA fragmentation and caspase activation are increased by constitutively activated Gαq or agonist-enhanced Gαq signaling. A, DNA was isolated from myocytes infected with the indicated adenovirus constructs after incubation in the presence or absence of 1 μmol/L PGF2α for 48 hours. DNA samples were separated on 2% agarose gels, and fragmentation was detected by staining with ethidium bromide under ultraviolet light. Data are representative of at least 3 separate experiments. B, Myocytes infected with indicated adenovirus constructs were incubated in serum-free medium for 48 hours with or without addition of 1 μmol/L PGF2α. Caspase activity was measured in cell lysates using the fluorogenic substrate DEVD-amc. Values represent the mean±SEM of triplicate reactions from 3 separate experiments and are expressed as the change in fluorescence units per minute per milligram of cellular protein. *P<0.01 vs AdCtrl.

Caspases Are Activated and Required for Gαq-Induced Apoptosis

To determine if enhanced Gq signaling activated caspases, we measured cleavage of a caspase-specific fluorogenic substrate DEVD-amc. Caspase activity did not increase in cells that hypertrophied in response to PGF2α or GαqWT overexpression (Figure 2B⇑). In contrast, a significant (≈4-fold) increase in caspase activity was seen in response to PGF2α treatment of GαqWT-overexpressing myocytes. A similar increase in caspase activity was seen in myocytes infected with GαqQ209L.

To examine the involvement of caspase activation in Gαq-induced cardiomyocyte apoptosis, we used Idun 1965, a broad-spectrum caspase inhibitor. Idun 1965 (10 μmol/L) was able to completely abolish caspase activation (DEVD-amc cleavage) induced by overexpression of GαqQ209L in cardiac myocytes (data not shown). Notably, Idun 1965 completely blocked DNA fragmentation and dramatically reduced cell death induced by either constitutively active Gαq or activation of GαqWT by PGF2α (Figures 3A⇓ and 3B⇓). This inhibitor did not prevent increased myocyte size (Figure 3A⇓) or sarcomere organization (not shown) in response to GαqWT overexpression, demonstrating integrity of Gαq signaling pathways and specificity for events in the apoptotic pathway rather than the hypertrophic pathways activated by Gαq. ZVAD-fmk, another caspase inhibitor, also inhibited Gαq-induced DNA fragmentation and cell death (not shown).

Figure 3.
Figure 3.

Figure 3⇓. Caspase inhibitors block Gαq-induced DNA fragmentation and cell death. Myocytes were infected with indicated adenovirus constructs in the absence or presence of 10 μmol/L Idun 1965. After washing, cells were incubated in serum-free medium with fresh inhibitor for an additional 48 hours. A, Before DNA isolation, cells were photographed using phase contrast to demonstrate increased cell survival in GαqQ209L-infected myocytes and hypertrophy in GαqWT-infected myocytes treated with Idun 1965. Bar=25 μm. B, DNA was isolated, separated by electrophoresis, and analyzed for oligonucleosomal fragmentation under ultraviolet light.

Enhanced Gαq Signaling Causes Mitochondrial Damage and Loss of ΔΨm

Electron microscopy revealed pronounced mitochondrial abnormalities in myocytes infected with GαqQ209L. In the presence of intact plasma membranes, mitochondria of myocytes infected with GαqQ209L appeared focally dilated and showed disrupted cristae (Figure 4A⇓, white arrow) compared with normal mitochondria in myocytes infected with GαqWT. In addition, the cytoplasm of virtually all myocytes expressing GαqQ209L contained numerous large membrane-bound vacuoles (Figure 4A⇓, black arrow). The origin and function of the vacuoles remain unknown, but lack of staining with either osmium tetroxide or Oil red-O suggests that they do not contain lipid (data not shown).

Figure 4.
Figure 4.

Figure 4⇓. Mitochondrial abnormalities and vacuole formation in cardiac myocytes expressing activated Gαq. A, Myocytes infected with indicated adenovirus constructs were incubated in serum-free medium for 48 hours and then fixed and prepared for electron microscopy. Healthy mitochondria (M) shown in GαqWT-infected cells are contrasted with damaged mitochondria (white arrows) in GαqQ209L-infected myocytes. Nuclear (N) changes and cytoplasmic vacuoles (black arrows) are also prominent in myocytes infected with GαqQ209L. B, Myocytes infected with GαqWT adenovirus (left) show red fluorescent JC-1 aggregates indicative of an intact ΔΨm. In contrast, expression of GαqQ209L (right) results in a reduction of JC-1 aggregate formation indicative of loss of ΔΨm.

In light of the observed mitochondrial abnormalities, we asked whether GαqQ209L expression also caused a decrease in ΔΨm. These studies were carried out using 2 potential sensitive mitochondrial dyes, JC-1 and MitoTracker Red. Hypertrophied myocytes expressing GαqWT (Figure 4B⇑), like AdCtrl-infected cells (not shown), showed punctate red staining, demonstrating formation of JC-1 aggregates and thus normal membrane potential in virtually every cell. Although it is difficult to quantitate JC-1 staining in live adherent cells, we reproducibly found a significant proportion of GαqQ209L-expressing myocytes in which JC-1 aggregate formation was decreased, indicative of a loss of ΔΨm. Similarly, many myocytes observed after infection with GαqQ209L demonstrated a decrease in fluorescence intensity after staining with the potential sensitive dye MitoTracker Red. This effect was confirmed by flow cytometry, where a loss of ΔΨm in myocytes infected with GαqQ209L is indicated by the decrease in the population of myocytes with high-fluorescence intensity CMXRos staining (Figure 6A⇓, right peak), as discussed in more detail below. Thus, enhanced Gαq signaling stimulated by GαqQ209L expression results in loss of ΔΨm in cardiomyocytes.

Enhanced Gαq Signaling Causes Mitochondrial Cyto c Release

Redistribution of cyto c from the mitochondria to the cytosol was examined by immunocytochemical staining for cyto c. In virtually all cells infected with GαqWT, cyto c fluorescence was contained within an extensive threadlike network surrounding the nucleus but extending deeply into the cytosol (Figure 5A⇓). An identical pattern of staining was seen when MitoTracker Red was added to the culture medium, indicating that the localization of cyto c mirrors that of cardiomyocyte mitochondria (data not shown). In contrast, the association of cyto c with mitochondria was reduced in myocytes expressing GαqQ209L. The immunocytochemical data are shown quantitatively in Figure 5B⇓ and indicate that ≈14% of the GαqQ209L-expressing myocytes showed diffuse cyto c staining, with fluorescence visible throughout the cytoplasm and nucleus (Figure 5A⇓, white arrow) rather than localized to the mitochondria. The same GαqQ209L-infected myocytes that showed cytosolic redistribution of cyto c consistently demonstrated condensed nuclear chromatin (Hoechst) and loss of actin (phalloidin) organization (Figure 5A⇓, white arrows). A similar effect on cyto c redistribution and nuclear condensation was seen in GαqWT-overexpressing cells stimulated with PGF2α (not shown).

Figure 5.
Figure 5.

Figure 5⇓. Caspase-independent redistribution of cyto c from mitochondria to cytoplasm occurs in response to enhanced Gαq signaling. A, Myocytes infected with indicated adenovirus constructs were incubated for 48 hours in serum-free medium then fixed and stained for cyto c (top), nuclear chromatin (middle), and F-actin (bottom). Fluorescent micrographs were obtained using 3 different filters without changing the viewing field. Note diffuse cyto c staining throughout the cytoplasm and nucleus (white arrow) in GαqQ209L-infected cell and the corresponding nuclear chromatin condensation (Hoechst 33342) and loss of sarcomere organization (phalloidin) in the same cell. Similar redistribution of cyto c to the cytoplasm is seen in GαqQ209L-infected cells treated with 10 μmol/L Idun 1965 (white arrowheads). Bar=10 μm. B, Myocytes were infected with indicated adenovirus constructs and incubated in the absence or presence of 10 μmol/L Idun 1965 for 48 hours. Cells were stained as in panel A, and ≈300 cells per treatment group were scored for cyto c release. Values represent mean±SEM of values from 3 separate experiments. *P<0.05 vs GαqWT; **P<0.01 vs GαqQ209L.

To determine whether caspase activation participates in the control of cyto c release, we examined the effect of Idun 1965 on cyto c localization in myocytes infected with GαqQ209L. Idun 1965 did not decrease the percentage of myocytes with diffuse cytoplasmic cyto c staining. In fact, there was a significant increase in the number of myocytes with cytosolic cyto c in the presence of Idun 1965 (Figure 5A⇑, arrowheads on cyto c panel, and Figure 5B⇑). This can be explained by the fact that the GαqQ209L-infected myocytes normally die and detach after cyto c release, whereas cells that release cyto c in the presence of the inhibitor remain attached and accumulate, contributing to the increased number of cells with cytosolic cyto c that can be counted. Surviving myocytes seem to have a normal nuclear appearance (Figure 5A⇑, arrowheads on Hoechst stain) but lack organized myofilaments (Figure 5A⇑, arrowheads on phalloidin stain). These observations demonstrate that caspase activation, although necessary for the nuclear changes leading to cell death, is not required for release of cyto c from mitochondria. Studies using MitoTracker Red also demonstrated that Idun 1965 had no effect on GαqQ209L-induced disruption of ΔΨm (see below). Thus, our data demonstrate that activated caspases participate downstream of, but do not mediate, the mitochondrial permeability changes or cyto c release that occurs in response to enhanced G protein activation.

Bongkrekic Acid (BA) Prevents Gαq-Induced Cyto c Release and Loss of ΔΨm and Inhibits Cardiomyocyte Apoptosis

Cyto c release has been attributed to loss of mitochondrial integrity initiated by opening of the mitochondrial PT pore. To examine the role of the PT pore in Gαq-induced apoptosis, we tested the effects of BA, which has been shown to block mitochondrial PT.12 As mentioned above, flow cytometry of cells stained with MitoTracker Red (CMXRos) revealed that GαqQ209L led to a decrease in the number of myocytes with high-intensity fluorescence, indicating a subpopulation of cells in which membrane potential is diminished (Figure 6A⇓). Although BA largely prevented the decreased fluorescence in this subpopulation of cells, inhibition of caspase activation with Idun 1965 had no protective effect (Figure 6B⇓). Thus, the membrane potential change seems to be caused by PT activation but independent of caspase activation.

Figure 6.
Figure 6.

Figure 6⇓. Gαq-induced loss of ΔΨm is blocked by inhibitors of PT pore but not by caspase inhibition. A, Myocytes were infected with indicated adenovirus constructs in the presence or absence of 50 μmol/L BA. After an additional 48-hour incubation in serum-free medium (pH 6.8)±fresh inhibitor, myocytes were stained for ΔΨm with 200 nmol/L MitoTracker Red for 10 minutes, trypsinized, pelleted, and then resuspended and prepared for flow cytometry as described in Materials and Methods. Data are representative examples of at least 3 separate experiments. B, Myocytes were infected with GαqQ209L adenovirus in the presence or absence of either 50 μmol/L BA or 10 μmol/L Idun 1965 (Idun). Overlay of data from an experiment in which the effects of BA or Idun on GαqQ209L-induced loss of ΔΨm were directly compared.

Blockade of PT and ΔΨm with BA was highly effective at preventing GαqQ209L-induced cyto c release, reducing the fraction of cells demonstrating loss of mitochondrial cyto c from 10.4±3.0% to 1.9±0.9% (Figures 7A⇓ and 7B⇓). The effect of BA was additionally assessed by examining changes in Gαq-induced nuclear damage with Hoechst 33342. Consistent with its effect on cyto c release, BA treatment reduced the percentage of GαqQ209L-infected myocytes with nuclear chromatin condensation from 12.2±3.8% to 2.2±1.0% (Figure 7B⇓). Loss of myocyte attachment induced by GαqQ209L was also blocked by BA treatment, indicating that cell death was prevented (data not shown). Finally, BA was able to reduce some of the early events associated with Gαq-induced cardiomyocyte apoptosis, including vacuole formation and cell shrinkage (not shown). Another inhibitor of PT pore formation, cyclosporin A (5 to 50 μmol/L), was also able to reduce Gαq-induced vacuole formation and cell shrinkage (data not shown). However, in contrast to what was observed with BA, the protective effect of cyclosporin A on cardiac myocytes was not sustained and death was not prevented.

Figure 7.
Figure 7.

Figure 7⇓. BA blocks Gαq-induced cyto c redistribution. A, Myocytes were infected with indicated adenovirus constructs in the presence or absence of 50 μmol/L BA in serum-free medium (pH 6.8). After an additional 48-hour incubation in serum-free medium (pH 6.8)±fresh BA, myocytes were fixed, permeabilized, and incubated with cyto c antibody, phalloidin, and Hoechst 33342, as described in Materials and Methods. Subcellular localization of cyto c (red), nuclear chromatin structure (blue), and sarcomere organization (green) were visualized for either cyto c and nuclear chromatin (A through C) or all 3 combined (D through F). Mitochondrial cyto c redistribution to the cytoplasm (white arrow) is best seen in the absence of FITC-phalloidin fluorescent staining (A through C). B, Myocytes were infected with indicated adenovirus constructs in the presence or absence of 50 μmol/L BA. After 48 hours of treatment, myocytes were fixed, stained, and scored for cyto c localization, chromatin condensation, and sarcomere organization, as described in Materials and Methods. Values represent averages±SEM for 3 separate experiments, where ≈300 cells were counted per treatment group. *P<0.05 vs Q209L without BA.

Gαq-Induced Changes in Bcl-2 Family Protein Levels

To determine if Bcl-2 family protein levels are altered by increased Gαq activity, we performed Western blots using antibodies specific for Bcl-2, Bcl-xl, Bax, and Bad. As shown in Figure 8A⇓, expression of GαqQ209L in myocytes dramatically decreased the protein levels of Bcl-2 and Bcl-xl, whereas Bad levels were increased. To determine if decreased levels of Bcl-xl were responsible for Gαq-induced apoptosis, we infected myocytes with an adenovirus encoding Bcl-xl (AdBcl-xl). Bcl-xl protein levels were dramatically increased in myocytes infected with AdBcl-xl (Figure 8B⇓). DNA fragmentation analysis demonstrated that overexpression of Bcl-xl completely blocked cardiomyocyte apoptosis induced by 2-deoxyglucose (Figure 8C⇓). In contrast, Bcl-xl expression did not block Gαq-induced DNA fragmentation. Similarly, overexpression of Bcl-2 by adenovirus exhibited no protective effect on Gαq-induced apoptosis (data not shown). These results suggest that decreased cellular levels of Bcl-xl or Bcl-2 do not play a causal role in Gαq-induced cardiomyocyte apoptosis. The role of increased Bad and potential changes in other Bcl-2 family proteins in Gαq-induced apoptosis are presently under investigation.

Figure 8.
Figure 8.

Figure 8⇓. Role of Bcl-2 family proteins in Gαq-induced cardiomyocyte apoptosis. A, Cell lysates were prepared from myocytes infected with the indicated adenovirus constructs, and equal amounts of protein (30 μg) were separated by electrophoresis. Relative levels of Bcl-2 family proteins were assessed by Western blot analysis. B, Myocytes were infected with control adenovirus (AdCtrl) or adenovirus expressing Bcl-xl at the indicated multiplicity of infection expressed in viral particles per cell (vp/cell). Lysates were harvested and analyzed by Western blotting for Bcl-xl expression 24 hours after infection. C, Myocytes were coinfected with Bcl-xl and GαqQ209L (Q209L) or control adenovirus (AdCtrl) or were infected with Bcl-xl 16 hours before treatment with 2 mmol/L 2-deoxyglucose (2-DOG). DNA was isolated after 24 hours of treatment, and oligonucleosomal fragmentation was assessed. Blots and gels are representative of at least 3 separate experiments.

Discussion

Recent studies have identified a role for caspases in cardiomyocyte apoptosis induced by staurosporine, reperfusion after ischemia, and serum and glucose deprivation.14 26 27 Our findings demonstrate that cardiomyocyte apoptosis induced by enhanced Gαq signaling is likewise associated with a marked increase in caspase activity. To test the functional importance of the caspase pathway, we examined the effect of the broad-spectrum caspase inhibitor Idun 1965. Our studies clearly indicate that caspase inhibition markedly attenuates the nuclear changes associated with Gαq-induced apoptosis, as assessed by nuclear chromatin condensation and internucleosomal DNA cleavage. We also found that caspase inhibitors block apoptosis induced by the addition of PGF2α and ligands for other endogenous Gq-coupled receptors to cardiomyocytes overexpressing GαqWT. Thus, caspases are essential mediators of apoptosis elicited by enhanced stimulation of Gq-signaling pathways.

Caspase inhibitors have been shown to reduce cardiomyocyte apoptosis and attenuate ischemia/reperfusion injury in rats.28 29 However, the functional rescue of surviving cardiomyocytes and its effect on sustained cardiac function was not thoroughly examined in these studies. Caspases would be viable therapeutic targets for heart failure only if they could successfully prevent or delay the functional changes associated with cardiac decompensation. Thus, although inhibiting caspases may significantly attenuate the ultimate apoptotic nuclear events induced by a variety of stimuli, it is not clear that the surviving myocytes would maintain normal cell function.

Our experiments provide a basis for questioning the ability of caspase inhibitors to preserve myocyte function in vivo. Specifically, we demonstrate that in GαqQ209L-induced apoptosis, redistribution of cyto c to the cytosol, indicative of mitochondrial dysfunction, occurs despite inactivation of caspases. We have also noted that collapse of the ΔΨm is not prevented by caspase inhibition. This contrasts with what has been observed for Fas-induced cyto c release from mitochondria, which is prevented by inhibition of caspases and thereby seems to require caspase activity.30 The limited effect of caspase inhibitors also demonstrates that the mitochondrial apoptotic cascade is proximal to and a potential mediator of caspase activation in Gq protein–induced apoptosis.

Cardiac muscle contains the highest volume density of mitochondria of any mammalian tissue. Therefore, it is unlikely that cardiomyocytes could maintain sufficient levels of ATP for extended periods of time without intact mitochondria. Apoptotic triggers upstream of caspase activation were analyzed for their role in Gαq-induced cardiomyocyte apoptosis. Mitochondrial release of cyto c is a particularly attractive mechanism for caspase activation and induction of apoptosis. A critical element involved in the mitochondrial apoptotic pathway is a change in the permeability of the outer mitochondrial membrane. This may be regulated by the pore-forming capabilities of Bcl-2 family proteins or secondary to loss of ΔΨm. Loss of ΔΨm is proposed to occur in response to opening of the PT pore, a large nonselective channel in the inner membrane.11 Loss of ΔΨm has been correlated in time with the point at which apoptosis can no longer be reversed by withdrawal of the stimulus.31 Thus, its disruption seems to lead to unalterable molecular events predisposing to cell death. Our studies demonstrate that this same nodal event occurs in response to enhanced Gαq signaling.

The molecular triggers downstream of Gαq and responsible for the catastrophic collapse in ΔΨm are currently unknown. Using BA, an inhibitor of PT pore formation, we demonstrated that this agent prevents the loss of ΔΨm and blocks cyto c release. Although the precise composition and structure of the PT pore are still undetermined, one of its components is the adenine nucleotide translocase. BA stabilizes the closed conformation of adenine nucleotide translocase and thereby inhibits PT pore opening.32 These data suggest that opening of the PT pore and the associated loss of ΔΨm are initiating apoptotic events caused by enhanced Gαq signaling. The observation that BA also prevents cyto c release, cell shrinkage, and nuclear chromatin condensation confirms that these are sequelae of PT pore opening. Cyclosporin A has also been observed to prevent mitochondrial PT.12 33 34 However, in contrast to BA, cyclosporin A showed only a transient ability to block apoptotic responses in cardiac myocytes. This is consistent with findings by others demonstrating that cyclosporin A can prevent loss of ΔΨm in short-term experiments (30 to 60 minutes) but fails to maintain ΔΨm over longer time periods.12

Bcl-2 family proteins have also been implicated as regulators of mitochondrial function and PT pore formation.33 35 We find dramatic changes in expression of several Bcl-2 family proteins in myocytes expressing activated Gαq, and it is likely that some of these changes play a significant role in the apoptotic process. However, expression of Bcl-xl or Bcl-2 at high levels did not prevent apoptosis in response to enhanced Gαq signaling. The role of increased Bad and possible changes in other Bcl-2 family proteins in Gαq-induced cardiomyocyte apoptosis are currently under additional investigation.

In conclusion, the evidence presented here demonstrates a pivotal role for the mitochondria in cardiac myocyte apoptosis induced by enhanced Gαq signaling. Our studies with BA indicate that interruption of the apoptotic cascade at proximal points, such as the mitochondrial PT pore, not only promotes cell survival but preserves mitochondrial integrity, as assessed by cyto c release and maintenance of ΔΨm. Maintenance of mitochondrial oxidative metabolism in cardiac myocytes is essential to sustaining function. Therefore, the mitochondria are critical targets for development of therapeutic strategies to attenuate myocyte loss while preserving cardiac function and preventing heart failure. The cellular model described here provides a basis for understanding how stress-induced stimuli might alter mitochondrial permeability and allows us to examine the ability of potential therapeutic interventions to rescue cardiomyocyte function.

Acknowledgments

This study was supported by National Institutes of Health grants HL-28143 and HL46345 (to J.H.B.) and an American Heart Association Beginning Grant-in-Aid (to J.W.A.). We thank Dennis Young for help with flow cytometry, Dr Jim Feramisco of the UCSD Cancer Center Imaging Core for help with image capture, and Alex DeCastro, Jon Genetti, and David R. Nadeau of the SDSC for 3-D volume rendering.

  • Received June 30, 2000.
  • Revision received October 19, 2000.
  • Accepted October 19, 2000.

References

  1. ↵
    LaMorte VJ, Thorburn J, Absher D, Spiegel AM, Brown JH, Chien KR, Feramisco JR, Knowlton KU. Gq- and Ras-dependent pathways mediate hypertrophy of neonatal rat ventricular myocytes following α1-adrenergic stimulation. J Biol Chem. 1994;269:13490–13496.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Sadoshima J, Qiu Z, Morgan JP, Izumo S. Angiotensin II and other hypertrophic stimuli mediated by G protein-coupled receptors activate tyrosine kinase, MAP kinase and 90-kD S6 kinase in cardiac myocytes: the critical role of Ca2+-dependent signaling. Circ Res. 1995;76:1–15.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Adams JW, Sah VP, Henderson SA, Brown JH. Tyrosine kinase and Jun NH2-terminal kinase mediate hypertrophic responses to prostaglandin F2α in cultured neonatal rat ventricular myocytes. Circ Res. 1998;83:167–178.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, Chien KR. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes: a paracrine mechanism for myocardial cell hypertrophy. J Biol Chem. 1990;265:20555–20562.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Adams JW, Sakata Y, Davis MG, Sah VP, Wang Y, Liggett SB, Chien KR, Brown JH, Dorn GW. Enhanced Gαq signaling: a common pathway mediates cardiac hypertrophy and apoptotic heart failure. Proc Natl Acad Sci U S A. 1998;95:10140–10145.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    D’Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, Dorn GW. Transgenic Gαq overexpression induces cardiac contractile failure in mice. Proc Natl Acad Sci U S A. 1997;94:8121–8126.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Mende U, Kagen A, Cohen A, Aramburu J, Schoen FJ, Neer EJ. Transient cardiac expression of constitutively active Gαq leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways. Proc Natl Acad Sci U S A. 1998;95:13893–13898.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Kajstura J, Cigola E, Malhotra A, Li P, Cheng W, Meggs LG, Anversa P. Angiotensin II induces apoptosis of adult ventricular myocytes in vitro. J Mol Cell Cardiol. 1997;29:859–870.
    OpenUrlCrossRefPubMed
  9. ↵
    Ju H, Zhao S, Tappia PS, Panagia V, Dixon IMC. Expression of Gqα and PLC-β in scar and border tissue in heart failure due to myocardial infarction. Circulation. 1998;97:892–899.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Hunter JJ, Grace A, Chien, KR, Molecular and cellular biology of cardiac hypertrophy and failure. In: Chien KR, ed. Molecular Basis of Cardiovascular Disease. Philadelphia, Pa: WB Saunders; 1999:211–250.
  11. ↵
    Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;281:1309–1312.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Zamzami N, Marchetti P, Castedo M, Hirsch T, Susin SA, Masse B, Kroemer G. Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett. 1996;384:53–57.
    OpenUrlCrossRefPubMed
  13. ↵
    Narula J, Pandey P, Arbustini E, Haider N, Narula N, Kolodgie FD, Dal Bello B, Semigran MJ, Bielsa-Masdeu A, Dec GW, Israels S, Ballester M, Virmani R, Saxena S, Kharbanda S. Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci U S A. 1999;96:8144–8149.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Bialik S, Cryns VL, Drincic A, Miyata S, Wollowick AL, Srinivasan A, Kitsis RN. The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes. Circ Res. 1999;85:403–414.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Malhotra R, Brosius FC. Glucose uptake and glycolysis reduce hypoxia-induced apoptosis in cultured neonatal rat cardiac myocytes. J Biol Chem. 1999;274:12567–12575.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Cook SA, Sugden PH, Clerk A. Regulation of Bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes. Circ Res. 1999;85:940–949.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    von Harsdorf R, Li P-F, Dietz R. Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation. 1999;99:2934–2941.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Iwaki K, Sukhatme VP, Shubeita HE, Chien KR. α and β Adrenergic stimulation induce distinct patterns of immediate early gene expression in neonatal rat myocardial cells: fos/jun expression is associated with sarcomere assembly; Egr-1 induction is primarily an α1 mediated response. J Biol Chem. 1990;265:13809–13817.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Shubeita HE, Martinson EA, Van Bilsen M, Chien KR, Brown JH. Transcriptional activation of the cardiac myosin light chain 2 and atrial natriuretic factor genes by protein kinase C in neonatal rat ventricular myocytes. Proc Natl Acad Sci U S A. 1992;89:1305–1309.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    McDonough PM, Brown JH, Glembotski CC. Phenylephrine and endothelin differentially stimulate cardiac PI hydrolysis and ANF expression. Am J Physiol. 1993;264:H625–H630.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Graham FL, Prevec L. Manipulation of adenovirus vectors. In: Murray EJ, ed. Methods in Molecular Biology: Gene Transfer and Expression Protocols. Vol 7. Clifton, NJ: Humana Press; 1991:109–128.
  22. ↵
    Huang S, Deerinck TJ, Ellisman MH, Spector DL. In vivo analysis of the stability and transport of nuclear poly(A)+ RNA. J Cell Biol. 1994;126:877–899.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Venkatakrishnan G, Exton JH. Identification of determinants in the α-subunit of Gq required for phospholipase C activation. J Biol Chem. 1996;271:5066–5072.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Natori S, Selzner M, Valentino KL, Fritz LC, Srinivasan A, Clavian PA, Gores GJ. Apoptosis of sinusoidal endothelial cells occurs during live preservation injury by a caspase-dependent mechanism. Transplantation. 1999;68:89–96.
    OpenUrlCrossRefPubMed
  25. ↵
    Reers M, Smiley S, Mottola-Hartshorn C, Chen A, Lin M, Chen L. Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol. 1995;260:406–417.
    OpenUrlCrossRefPubMed
  26. ↵
    Yue T-L, Wang C, Rominic A, Kikly K, Keller P, DeWolf W, Hart T, Thomas H, Storer B, Gu J-L, Wang X, Feuerstein G. Staurosporine-induced apoptosis in cardiomyocytes: a potential role of caspase-3. J Mol Cell Cardiol. 1998;30:495–507.
    OpenUrlCrossRefPubMed
  27. ↵
    Black S, Huang J, Rezaiefar P, Radinovic S, Eberhart A, Nicholson D, Rodger I. Co-localization of the cysteine protease caspase-3 with apoptotic myocytes after in vivo myocardial ischemia and reperfusion in the rat. J Mol Cell Cardiol. 1998;30:733–742.
    OpenUrlCrossRefPubMed
  28. ↵
    Yaoita H, Ogawa K, Maehara K, Maruyama Y. Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation. 1998;87:276–281.
    OpenUrl
  29. ↵
    Holly TA, Drincic A, Byun Y, Nakamura S, Harris K, Klocke FJ, Cryns VL. Caspase inhibition reduces myocyte cell death induced by myocardial ischemia and reperfusion in vivo. J Mol Cell Cardiol. 1999;31:1709–1715.
    OpenUrlCrossRefPubMed
  30. ↵
    Vander-Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB. Bcl-xl regulates the membrane potential and volume homeostasis of mitochondria. Cell. 1997;91:627–637.
    OpenUrlCrossRefPubMed
  31. ↵
    Kroemer G, Dallaporta B, Resche-Rignon M. the mitochondrial death/life regulator in apoptosis, and necrosis. Annu Rev Physiol.. 1998;60:619–642.
    OpenUrlCrossRefPubMed
  32. ↵
    Scheffler IE. Mitochondrial electron transport and oxidative phosphorylation. In: Mitochondria. New York, NY: Wiley-Liss; 1999:141–245.
  33. ↵
    Marzo I, Brenner C, Zamzami N, Jürgensmeier JM, Susin SA, Vieira HLA, Prevost M-C, Xie Z, Matsuyama S, Reed JC, Kroemer G. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science. 1998;281:2027–2031.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Narita M, Shimizu S, Ito T, Chittenden T, Lutz RJ, Matsuda H, Tsujimoto Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci U S A. 1998;95:14681–14686.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Brenner C, Cadiou H, La Vieira H, Zanzami N, Marzo I, Xie Z, Leber B, Andrews D, Duclohier H, Reed JC, Kroemer G. Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oncogene. 2000;19:329–336.
    OpenUrlCrossRefPubMed
View Abstract

This Issue

Jump to

Article Tools

Share this Article

  • Email
  • Cardiomyocyte Apoptosis Induced by Gαq Signaling Is Mediated by Permeability Transition Pore Formation and Activation of the Mitochondrial Death Pathway
    John W. Adams, Amy L. Pagel, Christopher K. Means, Donna Oksenberg, Robert C. Armstrong and Joan Heller Brown
    Circulation Research. 2000;87:1180-1187, originally published December 8, 2000
    https://doi.org/10.1161/01.RES.87.12.1180
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects