Cardiomyocyte Apoptosis Induced by Gαq Signaling Is Mediated by Permeability Transition Pore Formation and Activation of the Mitochondrial Death Pathway
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 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.
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.
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).
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.
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).
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).
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).
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.
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.
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.
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.
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.
- © 2000 American Heart Association, Inc.
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