p27 protein protects metabolically stressed cardiomyocytes from apoptosis by promoting autophagy.

p27(Kip1) (p27), a key regulator of cell division, has been implicated in autophagy of cancer cells. However, its role in autophagy, the evolutionarily conserved catabolic process that enables cells to remove unwanted proteins and damaged organelles, had not been examined in the heart. Here we report that ectopic delivery of a p27 fusion protein (TAT-p27) was sufficient to induce autophagy in neonatal rat ventricular cardiomyocytes in vitro, under basal conditions and after glucose deprivation. Conversely, lentivirus-delivered shRNA against p27 successfully reduced p27 levels and suppressed basal and glucose-deprived levels of autophagy in cardiomyocytes in vitro. Glucose deprivation mimics myocardial ischemia and induces apoptosis in cardiomyocytes. During glucose deprivation, TAT-p27 inhibited apoptosis, whereas down-regulation of p27 decreased survival of cardiomyocytes. However, inhibition of autophagy by pharmacological (3-methyladenine, chloroquine, or bafilomycin A1) or genetic approaches (siRNA-mediated knockdown of Atg5) sensitized cardiomyocytes to glucose deprivation-induced apoptosis, even in the presence of TAT-p27. TAT-p27 was also able to provoke greater levels of autophagy in resting and fasting cardiomyocytes in vivo. Further, TAT-p27 enhanced autophagy and repressed cardiomyocytes apoptosis, improved cardiac function, and reduced infarct size following myocardial infarction. Again, these effects were lost when cardiac autophagy in vivo was blocked by chloroquine. Taken together, these data show that p27 positively regulates cardiac autophagy in vitro and in vivo, at rest and after metabolic stress, and that TAT-p27 inhibits apoptosis by promoting autophagy in glucose-deprived cardiomyocytes in vitro and in post-myocardial infarction hearts in vivo.

Anti-apoptotic function of the E2F transcription factor 4 (E2F4)/p130, a member of retinoblastoma gene family in cardiac myocytes.

The E2F4-p130 transcriptional repressor complex is a cell-cycle inhibitor in mitotic cells. However, the role of E2F4/p130 in differentiated cells is largely unknown. We investigated the role of E2F4/p130 in the regulation of apoptosis in postmitotic cardiomyocytes. Here we demonstrate that E2F4 can inhibit hypoxia-induced cell death in isolated ventricular cardiomyocytes. As analyzed by chromatin immunoprecipitation, the E2F4-p130-repressor directly blocks transcription of essential apoptosis-related genes, E2F1, Apaf-1, and p73α through recruitment of histone deacetylase 1 (HDAC1). In contrast, diminution of the E2F4-p130-HDAC1-repressor and recruitment of E2F1 and histone acetylase activity to these E2F-regulated promoters is required for the execution of cell death. Expression of kinase-dead HDAC1.H141A or HDAC-binding deficient p130ΔHDAC1 abolishes the antiapoptotic effect of E2F4. Moreover, histological examination of E2F4(-/-) hearts revealed a markedly enhanced degree of cardiomyocyte apoptosis. Taken together, our genetic and biochemical data delineate an essential negative function of E2F4 in cardiac myocyte apoptosis.

Post-myocardial infarct p27 fusion protein intravenous delivery averts adverse remodelling and improves heart function and survival in rodents.

AIMS: P27Kip1 (p27) blocks cell proliferation through the inhibition of cyclin-dependent kinase 2 (cdk-2). Despite robust expression in the heart, little is known about the regulation and function of p27 in this terminally differentiated tissue. Previously, we demonstrated that p27 exerts anti-apoptotic and growth-inhibitory effects through interaction with casein kinase 2 (ck2) in neonatal rat cardiomyocytes. Here, we test the hypothesis that delivery of a transactivator of transcription (TAT)-p27 fusion protein (TAT.p27) will improve cardiac function and survival in a rat model of myocardial infarction (MI). METHODS AND RESULTS: Fisher rats underwent permanent left anterior descending ligation-induced MI followed by iv injection of TAT.p27 or TAT.LacZ (20 mg/kg) on Days 1 and 7 post-MI. Delivery of TAT.p27 was evaluated by western blot (WB) and immunofluorescence microscopy. Heart function was assessed by echocardiography and pressure-volume catheter. Apoptosis, hypertrophy, and fibrosis were detected by histochemistry and morphometry. WB confirmed gradual reduction in endogenous cardiac p27 levels following MI, with immunohistochemistry demonstrating successful delivery of TAT.p27 to the heart. At 48 h post-MI, cardiac apoptosis was decreased in rats treated with TAT.p27 when compared with saline- and TAT.LacZ-treated controls. At 28 days post-MI, rats treated with TAT.p27 manifested less cardiomyocyte hypertrophy and fibrosis, less diminished cardiac function, and greater survival. Additionally, p27KO mice undergoing experimental MI suffered an early increase in apoptosis with a larger infarct size and markedly reduced survival when compared with wild-type (WT) controls. CONCLUSION: These gain- and loss-of-function studies reveal a critical role for p27 in cardiac remodelling post-MI.

The adverse long-term impact of renal impairment in patients undergoing percutaneous coronary intervention in the drug-eluting stent era.

BACKGROUND: An observational study determining the long-term impact of chronic kidney disease (CKD) on patients undergoing percutaneous coronary intervention at a tertiary cardiac referral center. CKD is associated with poor in-hospital outcomes after percutaneous coronary intervention, but its effect beyond 1 year, particularly in the drug-eluting stent (DES) era, has not been reported. METHODS AND RESULTS: Baseline creatinine was available for 11,953 patients entered into a prospective registry (April 2000 to September 2007). Patients were stratified: those with or without at least moderate CKD (creatinine clearance, <60 mL/min). Follow-up data were obtained through linkage to a provincial registry. Kaplan-Meier analysis was performed. Cox multiple-regression analysis identified independent predictors of late mortality and major adverse cardiac events (MACE) and examined the association between DES use and late outcomes in the presence or absence of CKD. CKD was present in 3070 patients (25.7%). In-hospital mortality and MACE were significantly increased in CKD (3.34% versus 0.44%, P<0.001 and 5.73% versus 2.2%, P<0.001). Survival and MACE-free survival at 7 years were reduced (64.5+/-1.4% versus 89.4+/-0.5%, P<0.001; 44.0+/-1.4% versus 63.4+/-0.8%, P<0.001). CKD was an independent predictor of late mortality and MACE (hazard ratio [HR]: 2.18, CI: 1.90 to 2.49, P<0.0001; HR: 1.37, CI: 1.25 to 1.49, P<0.0001). DES use was associated with a significant reduction in both (HR: 0.71, CI: 0.60 to 0.83, P<0.0001; HR: 0.70, CI: 0.63 to 0.78, P<0.0001). In patients with CKD, DES use was associated with reduced revascularization (HR: 0.68, CI: 0.53 to 0.88, P=0.004) and reduced MACE (HR: 0.81, CI: 0.69 to 0.95, P=0.011) but not reduced mortality (HR: 0.85, CI: 0.69 to 1.05, P=0.1). CONCLUSIONS: In a large registry of "all comers" for percutaneous coronary intervention, CKD was an independent predictor of adverse late outcomes. DES use may be associated with improved long-term outcomes in this high-risk cohort, but further prospective studies are required.

Protein kinase CK2 links extracellular growth factor signaling with the control of p27(Kip1) stability in the heart.

p27(Kip1) (p27) blocks cell proliferation through the inhibition of cyclin-dependent kinase-2 (Cdk2). Despite its robust expression in the heart, little is known about both the function and regulation of p27 in this and other nonproliferative tissues, in which the expression of its main target, cyclin E-Cdk2, is known to be very low. Here we show that angiotensin II, a major cardiac growth factor, induces the proteasomal degradation of p27 through protein kinase CK2-alpha'-dependent phosphorylation. Conversely, unphosphorylated p27 potently inhibits CK2-alpha'. Thus, the p27-CK2-alpha' interaction is regulated by hypertrophic signaling events and represents a regulatory feedback loop in differentiated cardiomyocytes analogous to, but distinct from, the feedback loop arising from the interaction of p27 with Cdk2 that controls cell proliferation. Our data show that extracellular growth factor signaling regulates p27 stability in postmitotic cells, and that inactivation of p27 by CK2-alpha' is crucial for agonist- and stress-induced cardiac hypertrophic growth.

Phosphatidylinositol 3-Akt-kinase-dependent phosphorylation of p21(Waf1/Cip1) as a novel mechanism of neuroprotection by glucocorticoids.

The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). In primary cortical neurons, corticosterone leads to a dose- and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21(Waf1/Cip1) at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21(Waf1/Cip1) preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21(Waf1/Cip1) and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3- and Akt-kinase. Both germline and somatically induced p21(Waf1/Cip1) deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21(Waf1/Cip1) suffices to protect neurons from apoptosis. We identify p21(Waf1/Cip1) as a novel antiapoptotic factor for postmitotic neurons and implicate p21(Waf1/Cip1) as the molecular target of neuroprotection by high-dose glucocorticoids.

Critical role for FoxO3a-dependent regulation of p21CIP1/WAF1 in response to statin signaling in cardiac myocytes.

Statins are widely used clinical drugs that exert beneficial growth-suppressive effects in patients with cardiac hypertrophy. We investigated the role of the cell cycle inhibitor p21(CIP1/WAF1) (p21) in statin-dependent inhibition of hypertrophic growth in postmitotic cardiomyocytes. We demonstrate that lovastatin fails to inhibit cardiac hypertrophy to angiotensin II in p21(-/-) mice and that reconstitution of p21 function by TAT.p21 protein transduction can rescue statin action in these otherwise normally developed animals. Lovastatin specifically recruits the forkhead box FoxO3a transcription factor to the p21 promoter, mediating transcriptional transactivation of the p21 gene as analyzed in isolated primary cardiomyocytes. Lovastatin also stimulates protein kinase B/Akt kinase activity, and Akt-dependent phosphorylation forces p21 in the cytoplasm, where it inhibits Rho-kinases contributing to the suppression of cardiomyocyte hypertrophy. Loss of p21 or FoxO3a by RNA interference causes a general inhibition of lovastatin signal transduction. These results suggest that p21 functions as FoxO3a downstream target to mediate an statin-derived anti-hypertrophic response. Taken together, our genetic and biochemical data delineate an essential function of p21 for statin-dependent inhibition of cardiac myocyte hypertrophy.

Apoptosis repressor with caspase recruitment domain is required for cardioprotection in response to biomechanical and ischemic stress.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

E2F transcription factors and pRb pocket proteins in cell cycle progression.

The E2F-family of transcripion factors exerts fascinating and contrasting functions in transcriptional repression and activation of genes regulating proliferation, apoptosis, and differentiation. E2F is principally regulated by its temporal association with retinoblastoma pocket protein (pRb) family members. In turn, pRb is regulated through phosphorylation by cyclin-dependent kinase (cdk). The activity of cdk is negatively regulated by cdk-inhibitors, exemplified by p16INK4a, p21CIP1, and p27KIP1. Therefore, positive and negative signaling events converge on E2F activity resulting in distinct growth-controling and apoptotic activities. Here we describe the immunocytochemical detection of E2F, genomic DNA, BrdU-incorporation, and mitosis in cardiomyoctes. A detailed protocol is given to illustrate this technique in primary heart muscle cells.

Regenerative capacity of the myocardium: implications for treatment of heart failure.

Research into myocardial regeneration has an exciting future, shown by the results of experimental and clinical work challenging the dogma that the heart is a postmitotic non-regenerating organ. Such studies have initiated a lively debate about the feasibility of novel treatment approaches leading to the recovery of damaged myocardial tissue. The possibility of reconstituting dead myocardium by endogenous cardiomyocyte replication, transplantation, or activation of stem cells--or even cloning of an artificial heart--is being advanced, and will be a major subject of future research. Although health expenditure for heart failure in the industrial world is high, we are still a long way from being able to treat the cause of reduced myocardial contractility. Despite the hopes of some people, conventional treatment for heart failure does not achieve myocardial regeneration. We present a virtual case report of a patient with acute myocardial infarction; we discuss treatment options, including strategies aimed at organ regeneration.

p21(CIP1) Controls proliferating cell nuclear antigen level in adult cardiomyocytes.

Cell cycle withdrawal associated with terminal differentiation is responsible for the incapability of many organs to regenerate after injury. Here, we employed a cell-free system to analyze the molecular mechanisms underlying cell cycle arrest in cardiomyocytes. In this assay, incubation of S phase nuclei mixed with cytoplasmic extract of S phase cells and adult primary cardiomyocytes results in a dramatic reduction of proliferating cell nuclear antigen (PCNA) protein levels. This effect was blocked by the proteasome inhibitors MG132 and lactacystin, whereas actinomycin D and cycloheximide had no effect. Immunodepletion and addback experiments revealed that the effect of cardiomyocyte extract on PCNA protein levels is maintained by p21 but not p27. In serum-stimulated cardiomyocytes PCNA expression was reconstituted, whereas the protein level of p21 but not that of p27 was reduced. Cytoplasmic extract of serum-stimulated cardiomyocytes did not influence the PCNA protein level in S phase nuclei. Moreover, the hypertrophic effect of serum stimulation was blocked by ectopic expression of p21 and the PCNA protein level was found to be upregulated in adult cardiomyocytes derived from p21 knockout mice. Our data provide evidence that p21 regulates the PCNA protein level in adult cardiomyocytes, which has implications for cardiomyocyte growth control.

Inhibition of hypoxia-induced apoptosis by modulation of retinoblastoma protein-dependent signaling in cardiomyocytes.

Apoptotic cell death is an important mode of cell loss contributing to heart dysfunction. To analyze the importance of the E2F-dependent regulation of gene transcription in cardiomyocyte apoptosis, the function of cell cycle factors impinging on the retinoblastoma protein (pRb)/E2F pathway was investigated. In isolated neonatal ventricular myocytes, apoptotic cell death induced by hypoxia (deferoxamine, 100 micro mol/L) specifically activated cyclin-dependent kinases (cdks) 2 and 3. Apoptotic cell death was inhibited by ectopic expression of cdk inhibitors p21(CIP) and p27(KIP1) but not p16(INK4). In addition, apoptosis was also abrogated by forced expression of kinase dead mutant proteins of cdk2/3 but not of cdk4/6. Introduction of cdk inhibitors or dominant-negative cdk2/3 blocked pRb hyperphosphorylation and abrogated E2F-dependent gene transcription, including that of the E2F-responsive genes of proapoptotic caspase 3 and caspase 7. Moreover, introduction of constitutively active pRb and transcriptionally inert mutant E2F1/DP1 efficiently protected cardiomyocytes from apoptosis. In conclusion, these data demonstrate that cdk-specific inactivation of pRb and the subsequent activation of E2F-dependent gene transcription are required for cardiomyocyte apoptosis.

Phosphorylation by protein kinase CK2: a signaling switch for the caspase-inhibiting protein ARC.

Caspases play a central role in apoptosis, but their activity is under the control of caspase-inhibiting proteins. A characteristic of caspase-inhibiting proteins is direct caspase binding. It is yet unknown how the localization of caspase-inhibiting proteins is regulated and whether there are upstream signals controlling their function. Here we report that the function of ARC is regulated by protein kinase CK2. ARC at threonine 149 is phosphorylated by CK2. This phosphorylation targets ARC to mitochondria. ARC is able to bind to caspase-8 only when it is localized to mitochondria but not to the cytoplasm. Our results reveal a molecular mechanism by which a caspase-inhibiting protein requires phosphorylation in order to prevent apoptosis.

Regulation of E2F1-dependent gene transcription and apoptosis by the ETS-related transcription factor GABPgamma1.

The E2F family of transcription factors comprises six related members which are involved in the control of the coordinated progression through the G(1)/S-phase transition of cell cycle or in cell fate decision. Their activity is regulated by pocket proteins, including pRb, p107, and p130. Here we show that E2F1 directly interacts with the ETS-related transcription factor GABPgamma1 in vitro and in vivo. The binding domain interacting with GABPgamma1 was mapped to the C-terminal amino acids 310 to 437 of E2F1, which include its transactivation and pRb binding domain. Among the E2F family of transcription factors, the interaction with GABPgamma1 is restricted to E2F1. DNA-binding E2F1 complexes containing GABPgamma1 are characterized by enhanced E2F1-dependent transcriptional activity. Moreover, GABPgamma1 suppresses E2F1-dependent apoptosis by mechanisms other than the inhibition of the transactivation capacity of E2F1. In summary, our results provide evidence for a novel pRb-independent mechanism regulating E2F1-dependent transcription and apoptosis.