Arc Protein Levels Research Articles

Apoptosis repressor with caspase recruitment domain, a multifunctional modulator of cell death

Apoptosis repressor with caspase recruitment domain (ARC) is a highly potent and multifunctional inhibitor of apoptosis that is physiologically expressed predominantly in post-mitotic cells such as cardiomyocytes, skeletal muscle cells and neurons. ARC was also found to be up-regulated in many forms of malignant tumours. ARC impairs the cellular apoptotic responsiveness to a wide range of stresses and insults, including extrinsic apoptosis initiation via death receptor ligands, dysregulation of cellular Ca2+ homeostasis and endoplasmatic reticulum (ER) stress, genotoxic drugs, ionizing radiation, oxidative stress and hypoxia. ARC is subject to both transcriptional and post-translational regulation and exhibits its function through a multitude of molecular interactions with upstream transducers of apoptosis signals. This review summarizes, structures and comments on the published knowledge regarding ARC and its roles in modulating apoptotic cell death responsiveness in physiological and pathophysiological contexts.

The role of amygdaloid brain‐derived neurotrophic factor, activity‐regulated cytoskeleton‐associated protein and dendritic spines in anxiety and alcoholism

Innate anxiety appears to be a robust factor in the promotion of alcohol intake, possibly due to the anxiolytic effects of self-medication with alcohol. Brain-derived neurotrophic factor (BDNF) and its downstream target, activity-regulated cytoskeleton-associated (Arc) protein, play a role in the regulation of synaptic function and structure. In order to examine the role of the BDNF-Arc system and associated dendritic spines in the anxiolytic effects of ethanol, we investigated the effects of acute ethanol exposure on anxiety-like behaviors of alcohol-preferring (P) and -nonpreferring (NP) rats. We also examined changes in the expression of BDNF and Arc, and dendritic spine density (DSD), in amygdaloid brain regions of P and NP rats with or without ethanol exposure. It was found that in comparison with NP rats, P rats displayed innate anxiety-like behaviors, and had lower mRNA and protein levels of both BDNF and Arc, and also had lower DSD in the central amygdala (CeA) and medial amygdala (MeA), but not in the basolateral amygdala (BLA). Acute ethanol treatment had an anxiolytic effect in P, but not in NP rats, and was associated with an increase in mRNA and protein levels of BDNF and Arc, and in DSD in the CeA and MeA, but not BLA. These results suggest that innate deficits in BDNF-Arc levels, and DSD, in the CeA and MeA may be involved in the anxiety-like and excessive alcohol-drinking behaviors of P rats, as ethanol increased these amygdaloid synaptic markers and produced anxiolytic effects in P rats, but not NP rats.

Apoptosis repressor with caspase recruitment domain is dramatically reduced in cardiac, skeletal, and vascular smooth muscle during hypertension

Apoptosis repressor with caspase recruitment domain (ARC) is a unique anti-apoptotic protein with a distinct tissue distribution. In addition, unlike most anti-apoptotic proteins which act on one pathway, ARC can inhibit apoptosis mediated by both the death-receptor and mitochondrial signaling pathways. In this study, we confirm previous reports showing high levels of ARC protein in rat heart and skeletal muscle, but demonstrate for the first time that ARC is also expressed in rat aorta. Immunoblot analysis on endothelium-denuded aorta as well as immunohistochemical analysis on intact aorta demonstrated that ARC was highly expressed in smooth muscle. Immunoblot analysis also found that ARC protein was severely downregulated in skeletal muscle (−82%; P < 0.001), heart (−80%; P < 0.001), and aorta (−71%; P < 0.001) of spontaneously hypertensive rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats. Decreased ARC levels were also confirmed in tissues of hypertensive animals by immunohistochemical analysis. Collectively, this data suggests that ARC protein is expressed in vascular smooth muscle and is significantly reduced in several target tissues during hypertension.

Expression of Apoptosis Repressor with Caspase Recruitment Domain (ARC), An Antiapoptotic Protein, Is Strongly Prognostic in Acute Myeloid Leukemia (AML).

Abstract 400▪▪This icon denotes an abstract that is clinically relevant.The identification of relevant targets to induce apoptosis and sensitize to chemotherapy is a focus in AML research. Reverse-phase protein array (RPPA) is a validated and robust proteomic system which can quantitatively determine protein expression and facilitate the selection of molecular-targeted therapies. ARC, located on chromosome 16q21-q23, is an antiapoptotic protein. It inhibits both the intrinsic and extrinsic apoptosis pathways by suppressing the activity of caspase-2 and -8 and by blocking Bax activation. ARC also negatively regulates p53 by inhibiting its tetramerization and triggering its cytoplasmic localization and degradation of ARC depends on the p53-induced ubiquitin E3 ligase, MDM2. ARC has only recently been demonstrated to be expressed in various cancer cells and to confer both chemo- and radio-resistance. To determine its expression and assess its importance in AML, we profiled ARC expression in 511 newly diagnosed patients with AML using RPPA. Similar levels of ARC expression were found in 140 paired blood and bone marrow samples and no significant difference was detected between diagnosis and relapse in 49 paired samples from the same patients. ARC levels were inversely correlated with the protein levels of Bad (p<0.00001), cleaved caspase-9 (p<0.00001), cIAP1 (p<0.00001), caspase-9 (p<0.00001), XIAP (p<0.00001), and Bcl-2 (p=0.00001). Further, ARC levels are significantly higher in FAB M4 (p<0.00001) and M3 (p=0.012) and lower in FAB M2 (p=0.002) AML, but it is neither associated with favorable, intermediate, or unfavorable cytogenetics nor with FLT-3 mutation status. ARC levels do not correlate with bone marrow blast counts, but shows an inverse correlation with blood blast counts (p<0.00001). Importantly, AML patients with low or mid range ARC protein levels had significantly longer overall survival (Median 53 or 61 vs. 38 weeks, p=0.004, A) and remission duration (Median 75 vs 45 vs 29 weeks, p=0.0008, B) (see the figure below) than patients with high levels of ARC. Multivariate analysis indicated that ARC is a statistically significant independent predictor of survival in AML. The final model included: Gender (p=0.008), age at diagnosis (p<0.00001), cytogenetic groups (p<0.00001), FLT-3ITD (p=0.001), WBC (p<0.00001), platelet (p=0.009), albumin (p=0.00006), and ARC (p=0.00006). Results suggest that the expression levels of ARC are highly prognostic in AML and that ARC could be a potential therapeutic target in AML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Effects of Skilled Training on Sleep Slow Wave Activity and Cortical Gene Expression in the Rat

The best characterized marker of sleep homeostasis is the amount of slow wave activity (SWA, 0.5-4 Hz) during NREM sleep. SWA increases as a function of previous waking time and declines during sleep, but the underlying mechanisms remain unclear. We have suggested that SWA homeostasis is linked to synaptic potentiation associated with learning during wakefulness. Indeed, studies in rodents and humans found that SWA increases after manipulations that presumably enhance synaptic strength, but the evidence remains indirect. Here we trained rats in skilled reaching, a task known to elicit long-term potentiation in the trained motor cortex, and immediately after learning measured SWA and cortical protein levels of c-fos and Arc, 2 activity-dependent genes involved in motor learning. Intracortical local field potential recordings and training on reaching task. Basic sleep research laboratory. Long Evans adult male rats. N/A. SWA increased post-training in the trained cortex (the frontal cortex contralateral to the limb used to learn the task), with smaller or no increase in other cortical areas. This increase was reversible within 1 hour, specific to NREM sleep, and positively correlated with changes in performance during the prior training session, suggesting that it reflects plasticity and not just motor activity. Fos and Arc levels were higher in the trained relative to untrained motor cortex immediately after training, but this asymmetry was no longer present after 1 hour of sleep. Learning to reach specifically affects gene expression in the trained motor cortex and, in the same area, increases sleep need as measured by a local change in SWA.

Expression pattern of neural synaptic plasticity marker-Arc in different brain regions induced by conditioned drug withdrawal from acute morphine-dependent rats

The immediate early gene Arc (activity-regulated cytoskeletal-associated protein) mRNA and protein are induced by strong synaptic activation and rapidly transported into dendrites, where they localize at active synaptic sites. Thus, the Arc mRNA and protein are proposed as a marker of neuronal reactivity to map the neural substrates that are recruited by various stimuli. In the present study, we examined the expression of Arc protein induced by conditioned naloxone-precipitated drug withdrawal in different brain regions of acute morphine-dependent rats. The objective of the present study was to address the specific neural circuits involved in conditioned place aversion (CPA) that has not yet been well characterized. Place aversion was elicited by conditioned naloxone-precipitated drug withdrawal following exposure to a single dose of morphine. An immunohistochemical method was employed to detect the expression of Arc, which was used as a plasticity marker to trace the brain areas that contribute to the formation of the place aversion. Marked increases in Arc protein levels were found in the medial and lateral prefrontal cortex, the sensory cortex, the lateral striatum and the amygdala. This effect was more pronounced in the basolateral amygdala (BLA), the central nucleus of the amygdala (CeA), and the bed nucleus of the striatal terminals (BNST) when compared with the control group. Our results suggest that these brain regions may play key roles in mediating the negative motivational component of opiate withdrawal.

ARC is a critical cardiomyocyte survival switch in doxorubicin cardiotoxicity

Despite its complexity of action, doxorubicin (Dox)-induced cardiomyopathy eventually results in loss of cardiac myocytes which further contributes to the development of overt heart failure. In the present study, we examined the relevance of the apoptosis repressor with caspase recruitment domain (ARC) on cardiac myocyte survival and its underlying mechanisms in a model of Dox-induced cardiotoxicity. Exposure of neonatal rat ventricular cardiomyocytes with Dox resulted in a downregulation of ARC mRNA and protein levels that occurred in a pre-translational and post-translational manner and led to a significant induction of apoptosis. Proteasomal inhibitors partially rescued both Dox-induced downregulation of ARC protein and induction of apoptosis. Knockdown of endogenous ARC sensitised cardiomyocytes to undergo apoptosis upon treatment with Dox. In contrast, enforced expression of ARC by adenoviral-mediated gene transfer dramatically increased the resistance of cardiomyocytes to undergo apoptotic cell death following Dox administration. In response to Dox, Bax translocated from cytosol to mitochondria where it resulted in dissipation of the mitochondrial membrane potential, cytochrome c release and activation of caspases -3 and -9. ARC prevented Bax translocation to the mitochondrium and thereby blocked the activation of the mitochondrial apoptotic death pathway in a t-Bid and caspase-8-independent manner. In this study, we provide evidence for the protective role of anti-apoptotic ARC in Dox-induced cardiotoxicity, which makes this molecule an interesting target for future therapies.

The cardioprotective effect of postconditioning is mediated by ARC through inhibiting mitochondrial apoptotic pathway

Postconditioning protects the heart against ischemia/reperfusion injury by inhibiting cardiomyocyte apoptosis. However, the molecular mechanism by which postconditioning suppresses apoptosis remains to be fully understood. Apoptosis repressor with caspase recruitment domain (ARC) has been demonstrated to possess the ability to protect cardiomyocytes from apoptosis induced by ischemia/reperfusion. It is not yet clear as to whether ARC contributes to the inhibitory effect of postconditioning against cardiomyocyte apoptosis. The cultured cardiomyocytes from 1-day old male Sprague-Dawley rats were exposed to 3 h hypoxia followed by 3 h of reoxygenation. Cells were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia before 3 h of reoxygenation. Hypoxia/reoxygenation led to a decrease of endogenous ARC protein levels. In contrast, postconditioning could block the reduction of endogenous ARC protein levels. Interestingly, inhibition of endogenous ARC expression by ARC antisense oligodeoxynucleotides reduced the inhibitory effect of postconditioning against apoptosis. Furthermore, our data showed that postconditioning suppressed the loss of mitochondrial membrane potential, Bax activation and the release of mitochondrial cytochrome c to cytosol. However, these inhibitory effects of postconditioning disappeared upon knockdown of endogenous ARC. Our data for the first time demonstrate that ARC plays an essential role in mediating the cardioprotective effect of postconditioning against apoptosis initiated by the mitochondrial pathway.

Hypothesis

Hypothesis

Arc/Arg3.1 Translation Is Controlled by Convergent N-Methyl-D-aspartate and Gs-coupled Receptor Signaling Pathways

Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-D-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through G(s)-coupled dopamine and beta-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and G(s)-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, G(s)-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of G(s)-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.

P53 Initiates Apoptosis by Transcriptionally Targeting the Antiapoptotic Protein ARC

p53 plays an important role in regulating apoptosis. However, the molecular mechanism by which it initiates the apoptotic program still remains to be fully understood. Here, we report that p53 can transcriptionally target the antiapoptotic protein, apoptosis repressor with caspase recruitment domain (ARC). Our results show that reactive oxygen species and anoxia lead to the up-regulation of p53 expression. Concomitantly, ARC is down-regulated at both the protein and mRNA levels. Knockdown of p53 expression can attenuate the decreases in ARC protein and mRNA levels, indicating that ARC down-regulation is a consequence of p53 activation. Strikingly, p53-induced ARC repression occurs in a transcription-dependent manner. We further demonstrate that the p53 up-regulated modulator of apoptosis (PUMA) and Bad are up-regulated in response to the stimulation with reactive oxygen species or anoxia, and p53 is responsible for their up-regulation. ARC can interact with PUMA or Bad via its N terminus. Such an interaction displaces the association of PUMA or Bad with Bcl-2. ARC repression by p53 leads to its failure to counteract the proapoptotic activity of PUMA and Bad. Thus, our data reveal a novel p53 apoptotic pathway in which it initiates apoptosis by transcriptionally repressing ARC.

ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis

Inhibition of cardiomyocyte apoptosis plays a key role in preconditioning-triggered cardioprotection. However, the molecular mechanism(s) by which preconditioning inhibits apoptosis is not fully understood. Apoptosis repressor with caspase recruitment domain (ARC) possesses the ability to block hypoxia-induced cardiomyocyte apoptosis. We tested whether ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis. Cardiomyocytes from 1-day-old male Sprague-Dawley rats were preconditioned by exposing to 10 min of hypoxia, followed by 30 min of reoxygenation. Then, the preconditioned and non-preconditioned cardiomyocytes were exposed to 90 min of hypoxia followed by 120 min of reoxygenation. The results showed that preconditioning inhibited cell death induced by hypoxia and reoxygenation. Hypoxia and reoxygenation could induce a decrease of ARC protein levels. Intriguingly, preconditioning could maintain ARC protein levels. Inhibition of endogenous ARC expression by ARC antisense oligonucleotides reduced the inhibitory effect of preconditioning on apoptosis. Furthermore, preconditioning-induced suppression of the release of mitochondrial cytochrome c to cytosol and caspase-3 activation could be abolished by the inhibition of endogenous ARC expression using ARC antisense oligonucleotides. These data indicate that ARC participates in preconditioning-triggered cardioprotection by interfering with cytochrome c release and caspase-3 activation.

Effect of chronic treadmill running on apoptotic protein expression in soleus muscle of normotensive and hypertensive rats

Augmented apoptotic signaling in skeletal muscle has been well documented in several conditions including aging, chronic heart failure, disuse, and neuromuscular disorders. Recently, we have found increased DNA fragmentation and altered apoptotic protein levels in skeletal muscle of spontaneously hypertensive rats (SHR) relative to normotensive controls (WKY). Current evidence suggests that exercise may alter apoptosis-related proteins in skeletal muscle; however, the effect of exercise on skeletal muscle apoptosis in hypertensive animals is unclear. Male WKY (n=24) and SHR (n=24) rats were assigned to a sedentary (SED) condition or exercise (EX) consisting of progressive treadmill running 5 days/wk for 6 wks. Compared to WKY controls, soleus muscle of SHR had decreased anti-apoptotic Bcl-2, XIAP and ARC protein levels as well as elevated pro-apoptotic caspase 3 activity. Further, the levels of Bcl-2, MnSOD and Hsp70 protein in soleus muscle were significantly elevated following EX compared to the SED condition. In contrast, EX training had no effect on ARC, XIAP and CuZnSOD protein levels as well as caspase 3 activity. These results suggest that exercise can alter the expression of several proteins which may provide protection against skeletal muscle apoptosis in both SHR and WKY rats; however, exercise may not influence caspase signaling. Research supported by the Heart and Stroke Foundation of Ontario.

Apoptosis Repressor With Caspase Recruitment Domain Is Required for Cardioprotection in Response to Biomechanical and Ischemic Stress

Ischemic heart disease and heart failure are associated with an increased loss of cardiomyocytes due to apoptosis. Whether cardiomyocyte apoptosis plays a causal role in the pathogenesis of heart failure remains enigmatic. The apoptosis repressor with caspase recruitment domain (ARC) is a recently discovered antiapoptotic factor with a highly specific expression pattern in striated muscle and neurons. ARC is a master regulator of cardiac death signaling because it is the only known factor that specifically inhibits both the intrinsic and extrinsic apoptotic death pathway. In this study we attempted to elucidate the physiological role of ARC and to understand pathophysiological consequences resulting from its deletion. We generated ARC-deficient mice, which developed normally to adulthood and had no abnormality in cardiac morphology and function under resting conditions. On biomechanical stress induced by aortic banding, ARC-deficient mice developed accelerated cardiomyopathy compared with littermate controls, which was characterized by reduced contractile function, cardiac enlargement, and myocardial fibrosis. Likewise, ischemia/reperfusion injury of ARC-deficient mice resulted in markedly increased myocardial infarct sizes. Although in both instances a significant increase in apoptotic cardiomyocytes could be observed in ARC-deficient mice, neither in vitro nor in vivo studies revealed any effect of ARC on classic hypertrophic cardiomyocyte growth responses. The pathophysiological relevance of downregulated ARC levels was underscored by specimens from failing human hearts showing markedly reduced ARC protein levels. Our study identifies a tissue-specific antiapoptotic factor that is downregulated in human failing myocardium and that is required for cardioprotection in pressure overload and ischemia.

Immediate-Early Gene-Encoded Protein Arc Is Associated With Synaptic Delivery of GluR4-containing AMPA Receptors During In Vitro Classical Conditioning

The immediate-early gene Arc is rapidly expressed in response to neuronal activity and is thought to be involved in mechanisms of synaptic plasticity. The function of Arc in these processes remains unknown. The present study demonstrates that during an in vitro neural correlate of eyeblink classical conditioning, there is a rapid and transient increase in levels of Arc protein that require activation of N-methyl-d-aspartate receptors. In the early phase of conditioning during conditioned response (CR) acquisition, there is significantly greater colocalization of Arc protein and GluR4-containing AMPA receptors at synaptic sites, however, colocalization of Arc and GluR4 was not observed after later stages of conditioning during CR expression. There was also significantly enhanced coimmunoprecipitation of Arc with GluR4 subunits and actin early in conditioning but not of Arc with NR1 subunits, and these associations declined to control levels in later stages of conditioning. These data suggest a role for Arc protein in the synaptic delivery of GluR4-containing AMPA receptors by interactions with cytoskeletal protein complexes during the acquisition phase of in vitro classical conditioning.

Memory-influencing intra-basolateral amygdala drug infusions modulate expression of Arc protein in the hippocampus

Activation of beta-adrenoceptors in the basolateral complex of the amygdala (BLA) modulates memory storage processes and long-term potentiation in downstream targets of BLA efferents, including the hippocampus. Here, we show that this activation also increases hippocampal levels of activity-regulated cytoskeletal protein (Arc), an immediate-early gene (also termed Arg 3.1) implicated in hippocampal synaptic plasticity and memory consolidation processes. Infusions of the beta-adrenoreceptor agonist, clenbuterol, into the BLA immediately after training on an inhibitory avoidance task enhanced memory tested 48 h later. The same dose of clenbuterol significantly increased Arc protein levels in the dorsal hippocampus. Additionally, posttraining intra-BLA infusions of a memory-impairing dose of lidocaine significantly reduced Arc protein levels in the dorsal hippocampus. Increases in Arc protein levels were not accompanied by increases in Arc mRNA, suggesting that amygdala modulation of Arc protein and synaptic plasticity in efferent brain regions occurs at a posttranscriptional level. Finally, infusions of Arc antisense oligodeoxynucleotides into the dorsal hippocampus impaired performance of an inhibitory avoidance task, indicating that the changes in Arc protein expression are related to the observed changes in memory performance.

Experience-Dependent Regulation of the Immediate-Early Gene Arc Differs across Brain Regions

Previously, we demonstrated that initial acquisition of a lever-press task resulted in higher levels of activity-regulated cytoskeleton-associated protein (Arc) mRNA induction than did overtrained performance (Kelly and Deadwyler, 2002). The present study extends this finding by characterizing (1) the behavioral regulation of Arc protein expression, (2) the time course of decay of Arc mRNA signal in different brain regions immediately after the initial acquisition session, and (3) the persistence of Arc mRNA induction in those same brain regions across sessions. Rats killed after initial acquisition of a simple lever-press response demonstrated significantly elevated levels of Arc protein. Interestingly, of the brain regions that demonstrated Arc mRNA induction 30 min after the acquisition session, there was a differential rate in signal decay, with only half of the regions continuing to demonstrate elevated levels of Arc at 60 min. Similarly, the extent to which Arc mRNA induction persisted across days also varied across brain regions. An unexpected outcome was that areas such as CA1 and CA3 that showed the least persistence in Arc activation immediately after the initial acquisition session showed the greatest perseverance of induction across days of training. Finally, animals less proficient at the task expressed higher levels of Arc mRNA than animals that acquired the task more quickly. Taken together, the results show that Arc mRNA and protein were regulated in an experience-dependent manner; however, the fact that the time course of Arc mRNA expression differed across brain structures suggests a differential rate of consolidation of the newly acquired behavior across specific brain regions.

Insulin-induced expression of the activity-regulated cytoskeleton-associated gene (ARC) in human neuroblastoma cells requires p21ras, mitogen-activated protein kinase/extracellular regulated kinase and src tyrosine kinases but is protein kinase C-independent

We examined the effect of insulin on the expression of the activity-regulated cytoskeleton-associated gene (ARC), an effector immediate early gene with a proposed role in memory formation. In human SH-SY5Y neuroblastoma cells, application of insulin leads to dramatic increase in ARC mRNA and protein levels. Inhibition experiments reveal, that p21ras, mitogen-activated protein kinase/extracellular regulated kinase and tyrosine kinase (src) activity are required for the insulin-induced ARC expression in SH-SY5Y cells, whereas protein kinase C is not involved in the signal transduction pathway. Our data indicated for the first time a correlation of the insulin-controlled signal cascade and the induction of synaptic plasticity-associated immediate early genes.

Ischemia elicits a coordinated expression of pro-survival proteins in mouse myocardium.

Cardiomyocytes are post-mitotic, long-lived cells until disruptions to pro-survival factors occur after myocardial ischemia. To gain an understanding of the factors involved with ischemic injury, we examined expression changes in pro-survival and opposing pro-apoptotic signals at early and chronic periods of ischemia using an in vivo murine model. Alterations of pro-survival proteins such as the inhibitor of apoptosis protein on chromosome X (xIAP) and the apoptotic repressor protein (ARC) have not been evaluated in a murine model of cardiac ischemia. Early ischemia (1 day) resulted in a 50% reduction in ARC protein levels relative to sham-operated left ventricles, without significant changes in the expression of xIAP or other pro-survival factors. In contrast, a deficiency of xIAP expression was found in cardiac infarcts starting after 1 week, concomitant with significant evidence of apoptotic cell death and an up-regulation of pro-apoptotic signals including Bax, tumor necrosis factor-a, and caspase-8 activation. Chronic ischemia (after 2 weeks) was associated with elevated levels of other pro-survival factors such as Bcl-xL and the phosphorylated form of Akt, as part of the adaptive remodeling of the myocardium. Altogether, these findings suggest that strategies to increase IAP expression may promote myocyte survival after chronic ischemia.

ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells.

Ischemia induces apoptosis as well as necrosis of cardiac myocytes. We recently reported the cloning of a cDNA that encodes an apoptotic inhibitor, ARC, that is expressed predominantly in cardiac and skeletal muscle. In the present study, we examined the ability of ARC to protect rat embryonic heart-derived H9c2 cells from apoptosis induced by hypoxia, a component of ischemia. We found that H9c2 cells express ARC and that exposure to hypoxia substantially reduces ARC expression while inducing apoptosis. Transfected H9c2 cells in which cytosolic ARC protein levels remain elevated during hypoxia were significantly more resistant to hypoxia-induced apoptosis than parental H9c2 cells or H9c2 cells transfected with a control vector. Loss of endogenous ARC in the cytosol of H9c2 cells was associated with translocation of ARC from the cytosol to intracellular membranes, release of cytochrome c from the mitochondria, activation of caspase-3, poly(ADP-ribose)polymerase (PARP) cleavage, and DNA fragmentation. All of these events were inhibited in H9c2 cells overexpressing ARC when compared with control cells. In contrast, caspase inhibitors prevented PARP cleavage but not cytochrome c release, suggesting that exogenously expressed ARC acts upstream of caspase activation in this model of apoptosis. These results demonstrate that ARC can protect heart myogenic H9c2 cells from hypoxia-induced apoptosis, and that ARC prevents cytochrome c release by acting upstream of caspase activation, perhaps at the mitochondrial level.