Over-expression of a modified bifunctional apoptosis regulator protects against cardiac injury and doxorubicin-induced cardiotoxicity in transgenic mice.

AIMS: Bifunctional apoptosis regulator (BAR) is an endoplasmic reticulum protein that interacts with both the extrinsic and intrinsic apoptosis pathways. We hypothesize that over-expression of BAR Delta RING prevents apoptosis and injury following ischaemia/reperfusion (I/R) and attenuates doxorubicin (DOX)-induced cardiotoxicity. METHODS AND RESULTS: We generated a line of transgenic mice that carried a human BAR Delta RING transgene under the control of the mouse alpha-myosin heavy chain promoter. The RING domain, which binds ubiquitin conjugating enzymes, was deleted to prevent auto-ubiquitination of BAR and allow accumulation of the BAR protein, which binds apoptosis-regulating proteins. High levels of human BAR Delta RING transcripts and 42 KDa BAR Delta RING protein were expressed in the hearts of transgenic mice. When excised hearts were reperfused ex vivo for 45 min as Langendorff preparations after 45 min of global ischaemia, the functional recovery of the hearts, expressed as left ventricular developed pressure x heart rate, was 23 +/- 1.7% in the non-transgenic hearts compared with 51.5 +/- 4.3% in the transgenic hearts (P < 0.05). For in vivo studies, mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by 4 h of reperfusion. The infarct sizes following I/R injury, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (29 +/- 4 vs. 55 +/- 4%, P < 0.05). In hearts of mice subjected to cardiac I/R injury, BAR transgenic hearts had significantly fewer in situ oligo-ligation-positive cardiac cells (5.0 +/- 0.4 vs. 13.4 +/- 0.5%, P < 0.05). Over-expression of BAR Delta RING also significantly attenuated DOX-induced cardiac dysfunction and apoptosis. CONCLUSION: Our results demonstrate that over-expression of BAR Delta RING renders the heart more resistant to I/R injury and DOX-induced cardiotoxicity, and this protection correlates with reduced cardiomyocyte apoptosis.

Glutathione peroxidase 1-deficient mice are more susceptible to doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxide anion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reduction of superoxide generates H(2)O(2), which diffuses throughout the cell and potentially contributes to oxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1) primarily functions to eradicate H(2)O(2). In this study, we hypothesize that Gpx1 plays a pivotal role in the clearance of H(2)O(2) generated by DOX. To test this hypothesis, we compared DOX-induced cardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wild type mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acute functional derangements than wild type hearts, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impaired the mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated with DOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratio relative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidative phosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantly increased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest that Gpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiac dysfunction in the acute phase.

Neuroprotective effect of humanin on cerebral ischemia/reperfusion injury is mediated by a PI3K/Akt pathway.

Humanin (HN) is an anti-apoptotic peptide that suppresses neuronal cell death induced by Alzheimer's disease, prion protein fragments, and serum deprivation. Recently, we demonstrated that Gly14-HN (HNG), a variant of HN in which the 14th amino acid serine is replaced with glycine, can decrease apoptotic neuronal death and reduce infarct volume in a focal cerebral ischemia/reperfusion mouse model. In this study, we postulate that the mechanism of HNG's neuroprotective effect is mediated by the PI3K/Akt pathway. Oxygen-glucose deprivation (OGD) was performed in cultured mouse primary cortical neurons for 60 min. The effect of HNG and PI3K/Akt inhibitors on OGD-induced cell death was examined at 24 h after reperfusion. HNG increased cell viability after OGD in primary cortical neurons, whereas the PI3K/Akt inhibitors wortmannin and Akti-1/2 attenuated the protective effect of HNG. HNG rapidly increased Akt phosphorylation, an effect that was inhibited by wortmannin and Akti-1/2. Mouse brains were injected intraventricularly with HNG before being subjected to middle cerebral artery occlusion (MCAO). HNG treatment significantly elevated p-Akt levels after cerebral I/R injury and decreased infarct volume. The protective effect of HNG on infarct size was attenuated by wortmannin and Akti-1/2. Taken as a whole, these results suggest that PI3K/Akt activation mediates HNG's protective effect against hypoxia/ischemia reperfusion injury.

Overexpression of IAP-2 attenuates apoptosis and protects against myocardial ischemia/reperfusion injury in transgenic mice.

Inhibitors of apoptosis proteins (IAPs) are key intrinsic regulators of caspases-3 and -7. During ischemia, IAP-2 is upregulated dramatically, while the other IAPs show little or no change. To test whether IAP-2 prevents cardiac apoptosis and injury following ischemia/reperfusion, we generated a line of transgenic mice that carried a mouse IAP-2 transgene. High levels of mouse IAP-2 transcripts and 70 kDa IAP-2 were expressed in the hearts of transgenic mice, whereas IAP-1 and XIAP levels remained the same. Immunohistochemical studies revealed more intense staining of IAP-2 in the myocytes of transgenic mouse hearts. To assess the role of IAP-2 in I/R injury, the transgenic mice were subjected to ligation of the left descending anterior coronary artery ligation followed by reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (30+/-2% vs. 44+/-2%, respectively, P<0.05). This protection was accompanied by a decrease of the serum level of troponin I in the transgenic mice. IAP-2 transgenic hearts had significantly fewer TUNEL-positive cardiac cells, which indicated an attenuation of apoptosis. Our results demonstrate that overexpression of IAP-2 renders the heart more resistant to apoptosis and I/R injury.

Multiple actions of pifithrin-alpha on doxorubicin-induced apoptosis in rat myoblastic H9c2 cells.

Doxorubicin (Dox) is a chemotherapeutic agent that causes significant cardiotoxicity. We showed previously that Dox activates p53 and induces apoptosis in mouse hearts. This study was designed to elucidate the molecular events that lead to p53 stabilization, to examine the pathways involved in Dox-induced apoptosis, and to evaluate the effectiveness of pifithrin-alpha (PFT-alpha), a p53 inhibitor, in blocking apoptosis of rat H9c2 myoblasts. H9c2 cells that were exposed to 5 muM Dox had elevated levels of p53 and phosphorylated p53 at Ser15. Dox also triggered a transient activation of p38, p42/p44ERK, and p46/p54JNK MAP kinases. Caspase activity assays and Western blot analysis showed that H9c2 cells treated with Dox for 16 h had marked increase in the levels of caspases-2, -3, -8, -9, -12, Fas, and cleaved poly(ADP ribose) polymerase (PARP). There was a concomitant increase in p53 binding activity, cytochrome c release, and apoptosis. These results suggest that Dox can trigger intrinsic, extrinsic, and endoplasmic reticulum-associated apoptotic pathways. Pretreatment of cells with PFT-alpha followed by Dox administration attenuated Dox-induced increases in p53 levels and p53 binding activity and partially blocked the activation of p46/p54JNK and p42/p44ERK. PFT-alpha also led to decreased levels of caspases-2, -3, -8, -9, -12, Fas, PARP, cytochrome c release, and apoptosis. Our results suggest that p53 stabilization is a focal point of Dox-induced apoptosis and that PFT-alpha interferes with multiple steps of Dox-induced apoptosis.

Dexamethasone induces caspase activation in murine osteoblastic MC3T3-E1 cells.

Glucocorticoids are widely used as anti-inflammatory and chemotherapeutic agents. However, prolonged use of glucocorticoids leads to osteoporosis. This study was designed to examine the mechanism of dexamethasone (DEX)-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 10(-7) M DEX for 6 h. DEX exerted a variety of effects on apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that DEX upregulated mRNA levels of caspases-1, -3, -6, -8, -11, -12, and bcl-XL. Western blot analysis showed enhanced processing of these caspases, with the appearance of their activated enzymes 8 h after DEX treatment. In addition, DEX also induced the activation of caspase-9. DEX elevated the levels of cleaved poly(ADP-ribose) polymerase and lamin A, a caspase-3 and a caspase-6 substrate, respectively. Expression of bcl-XL protein level was upregulated by DEX. Cytochrome c release was detected in the cytosol of DEX-treated cells. Furthermore, caspase-3 enzyme activity was elevated by 2-fold after DEX treatment for 7 h. Finally, early apoptotic cells were detected in cells treated with DEX for 3 h. Our results demonstrate that DEX-induced apoptosis involves gene activation of a number of caspases.

Protective effect of melatonin on myocardial infarction.

The dose- and time dependence of melatonin and the effective window of melatonin administration were determined in a mouse model of myocardial infarction. When mouse hearts were subjected to 60 min of occlusion of the left anterior descending artery (LAD) followed by 4 h of reperfusion, melatonin pretreatment for 30 min significantly reduced the infarct size/risk area. The most effective dose was found to be 150 microg/kg intraperitoneally, and the effective period of protection lasted up to 2 h after melatonin administration. Melatonin administration 45 min after LAD ligation or right before reperfusion was as effective as administration 30 min before ligation; however, melatonin administered after the release of occlusion was not protective. Melatonin's effect was still present in mice deficient for the Mel1a melatonin receptor. 8-Methoxy-2-propionamidotetralin, a melatonin receptor agonist with no antioxidant activity, offered no protection, suggesting a lack of involvement of melatonin receptors. Finally, the effects of melatonin were similar in rats and mice. Our results demonstrate that melatonin is an effective cardioprotective agent when administered either before or during coronary occlusion at a very low dose.

TGF-beta1 inhibits multiple caspases induced by TNF-alpha in murine osteoblastic MC3T3-E1 cells.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Transforming growth factor beta1 (TGF-beta1) is an abundant growth factor that is known to stimulate bone formation. This study was designed to examine the role of TGF-beta1 on TNF-alpha-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 20 ng/ml of TNF-alpha, 10 ng/ml of TGF-beta1, or combination, for 6 h. TNF-alpha exerted a variety of effects on the apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that TNF-alpha upregulated the mRNA levels of caspase-1, -7, -11, -12, and FAS. Western blot analysis showed enhanced processing of caspase-1, -7, -11, and -12, with the appearance of their activated enzymes 24 h after TNF-alpha treatment. In addition, caspase-3-like activity was significantly activated following TNF-alpha treatment. Levels of cleaved poly(ADP-ribose) polymerase and FAS protein were also elevated by TNF-alpha. Finally, Hoechst staining, terminal deoxynucleotidyl-transferase nick-end labeling (TUNEL) assay, and oligonucleosome ELISA all indicated that TNF-alpha induced apoptosis. In contrast, the addition of TGF-beta1 attenuated all of the aforementioned effects of TNF-alpha. Our results demonstrate that TGF-beta1 can decrease TNF-alpha-induced apoptosis in murine osteoblasts at least in part by attenuating TNF-alpha-induced caspase gene expression.

Melatonin as an effective protector against doxorubicin-induced cardiotoxicity.

The present study was designed to explore the protective effects of melatonin and its analogs, 6-hydroxymelatonin and 8-methoxy-2-propionamidotetralin, on the survival of doxorubicin-treated mice and on doxorubicin-induced cardiac dysfunction, ultrastructural alterations, and apoptosis in mouse hearts. Whereas 60% of the mice treated with doxorubicin (25 mg/kg ip) died in 5 days, almost all the doxorubicin-treated mice survived when melatonin or 6-hydroxymelatonin (10 mg/l) was administered in their drinking water. Perfusion of mouse hearts with 5 microM doxorubicin for 60 min led to a 50% suppression of heart rate x left ventricular developed pressure and a 50% reduction of coronary flow. Exposure of hearts to 1 microM melatonin or 6-hydroxymelatonin reversed doxorubicin-induced cardiac dysfunction. 8-Methoxy-2-propionamidotetralin had no protective effects on animal survival and on in vitro cardiac function. Infusion of melatonin or 6-hydroxymelatonin (2.5 microg/h) significantly attenuated doxorubicin-induced cardiac dysfunction, ultrastructural alterations, and apoptosis in mouse hearts. Neither melatonin nor 6-hydroxymelatonin compromised the antitumor activity of doxorubicin in cultured PC-3 cells. These results suggest that melatonin protect against doxorubicin-induced cardiotoxicity without interfering with its antitumor effect.