Increased autophagy leads to decreased apoptosis during ß-thalassaemic mouse and patient erythropoiesis.

ß-Thalassaemia results from defects in ß-globin chain production, leading to ineffective erythropoiesis and subsequently to severe anaemia and other complications. Apoptosis and autophagy are the main pathways that regulate the balance between cell survival and cell death in response to diverse cellular stresses. Herein, the death of erythroid lineage cells in the bone marrow from both ßIVS2-654-thalassaemic mice and ß-thalassaemia/HbE patients was investigated. Phosphatidylserine (PS)-bearing basophilic erythroblasts and polychromatophilic erythroblasts were significantly increased in ß-thalassaemia as compared to controls. However, the activation of caspase 8, caspase 9 and caspase 3 was minimal and not different from control in both murine and human thalassaemic erythroblasts. Interestingly, bone marrow erythroblasts from both ß-thalassaemic mice and ß-thalassaemia/HbE patients had significantly increased autophagy as shown by increased autophagosomes and increased co-localization between LC3 and LAMP-1. Inhibition of autophagy by chloroquine caused significantly increased erythroblast apoptosis. We have demonstrated increased autophagy which led to minimal apoptosis in ß-thalassaemic erythroblasts. However, increased PS exposure occurring through other mechanisms in thalassaemic erythroblasts might cause rapid phagocytic removal by macrophages and consequently ineffective erythropoiesis in ß-thalassaemia.

p53 Regulates oxidative stress-mediated retrograde signaling: a novel mechanism for chemotherapy-induced cardiac injury.

The side effects of cancer therapy on normal tissues limit the success of therapy. Generation of reactive oxygen species (ROS) has been implicated for numerous chemotherapeutic agents including doxorubicin (DOX), a potent cancer chemotherapeutic drug. The production of ROS by DOX has been linked to DNA damage, nuclear translocation of p53, and mitochondrial injury; however, the causal relationship and molecular mechanisms underlying these events are unknown. The present study used wild-type (WT) and p53 homozygous knock-out (p53(-/-)) mice to investigate the role of p53 in the crosstalk between mitochondria and nucleus. Injecting mice with DOX (20 mg/kg) causes oxidative stress in cardiac tissue as demonstrated by immunogold analysis of the levels of 4-hydroxy-2'-nonenal (4HNE)-adducted protein, a lipid peroxidation product bound to proteins. 4HNE levels increased in both nuclei and mitochondria of WT DOX-treated mice but only in nuclei of DOX-treated p53((-/-)) mice, implicating a critical role for p53 in causing DOX-induced oxidative stress in mitochondria. The stress-activated protein c-Jun amino-terminal kinase (JNKs) was activated in response to increased 4HNE in WT mice but not p53((-/-)) mice receiving DOX treatment, as determined by co-immunoprecipitation of HNE and pJNK. The activation of JNK in DOX treated WT mice was accompanied by Bcl-2 dissociation from Beclin in mitochondria and induction of type II cell death (autophagic cell death), as evidenced by an increase in LC3-I/LC-3-II ratio and γ-H2AX, a biomarker for DNA damage. The absence of p53 significantly reduces mitochondrial injury, assessed by quantitative morphology, and decline in cardiac function, assessed by left ventricular ejection fraction and fraction shortening. These results demonstrate that p53 plays a critical role in DOX-induced cardiac toxicity, in part, by the induction of oxidative stress mediated retrograde signaling.

Mrp1 localization and function in cardiac mitochondria after doxorubicin.

Multidrug resistance-associated protein 1 (Mrp1; Abcc1) is expressed in sarcolemma of murine heart, where it probably protects the cardiomyocyte by mediating efflux of endo- and xenobiotics. We used doxorubicin (DOX), a chemotherapeutic drug known to induce oxidative stress and thereby cardiac injury, as a model cardiotoxic compound and observed changes in the Mrp1 expression pattern in cardiac tissue of DOX-versus saline-treated mice. Confocal immunofluorescent and immunogold electron microscopy, together with subcellular fractionation followed by immunoblot analyses and transport measurements, localized functional Mrp1 to mitochondria after DOX. Expressions of Mrp1 in heart homogenate, sarcolemma, and submitochondrial particles (SMP) were increased 1.6-, 2-, and 3-fold, respectively, at 24 h after DOX. Mitochondrial Mrp1 expression was markedly increased 72 h after DOX, whereas transport of Mrp1 substrates in SMP was maximal at 24 h. ATP-dependent transport in SMP occurred into an osmotically sensitive space and was inhibited by the anti-MRP1 antibody QCRL3. Adduction of a 190-kDa protein with the reactive lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was detected in SMP and was maximal at 72 h after DOX; immunoprecipitation confirmed Mrp1-HNE adduction. In vitro, HNE (10 muM) inhibited mitochondrial respiration and transport activity in SMP, suggesting that Mrp1 is adversely affected by oxidative stress. These data demonstrate that after DOX, functional Mrp1 is detected in mitochondria in addition to that in sarcolemma; however, adduction with HNE inhibits Mrp1 activity. Mrp1 may serve to protect the heart by mediating the efflux of toxic products of oxidative stress from mitochondria and cardiomyocytes.

Phenylbutyrate, a histone deacetylase inhibitor, protects against Adriamycin-induced cardiac injury.

Cardiac injury is a major complication for oxidative-stress-generating anticancer agents exemplified by Adriamycin (ADR). Recently, several histone deacetylase inhibitors (HDACIs) including phenylbutyrate (PBA) have shown promise in the treatment of cancer with little known toxicity to normal tissues. PBA has been shown to protect against oxidative stress in normal tissues. Here, we examined whether PBA might protect heart against ADR toxicity in a mouse model. The mice were i.p. injected with ADR (20 mg/kg). PBA (400 mg/kg/day) was i.p. injected 1 day before and daily after the ADR injection for 2 days. We found that PBA significantly decreased the ADR-associated elevation of serum lactate dehydrogenase and creatine kinase activities and diminished ADR-induced ultrastructural damages of cardiac tissue by more than 70%. Importantly, PBA completely rescued ADR-caused reduction of cardiac functions exemplified by ejection fraction and fraction shortening, and increased cardiac manganese superoxide dismutase (MnSOD) protein and activity. Our results reveal a previously unrecognized role of HDACIs in protecting against ADR-induced cardiac injury and suggest that PBA may exert its cardioprotective effect, in part, by the increase of MnSOD. Thus, combining HDACIs with ADR could add a new mechanism to fight cancer while simultaneously decrease ADR-induced cardiotoxicity.

Mitochondrial and nuclear p53 localization in cardiomyocytes: redox modulation by doxorubicin (Adriamycin)?

Reactive oxygen (ROS) and nitrogen species (RNS) generation have been proposed to be an important mechanism of doxorubicin (Adriamycin; ADR)-induced cardiotoxicity and cardiomyocyte apoptosis, processes that may be mediated by p53 protein. We note that ADR treatment resulted in increased levels of p53 protein in cardiomyocyte mitochondria and nuclei. Modulation of the cardiomyocyte redox state in genetically engineered mice by modulation of enzymes involved in metabolism of ROS/RNS, manganese superoxide dismutase (MnSOD), or inducible nitric oxide synthase (iNOS), or a combination of these, regulated levels of mitochondrial/nuclear p53 in cardiomyocytes after ADR administration. These observations led to the hypothesis that mitochondrial/nuclear p53 localization and function in the cardiomyocyte response to ADR may be regulated through redox-dependent mechanism(s).

Evidence for p53 as guardian of the cardiomyocyte mitochondrial genome following acute adriamycin treatment.

The present study is an initial analysis of whether p53 may function as guardian of the cardiomyocyte mitochondrial genome, with mitochondrial p53 localization proposed to be involved in both mitochondrial DNA (mtDNA) repair and apoptosis. Subcellular distribution, protein levels, and possible function(s) of p53 protein in the response of cardiomyocytes to adriamycin (ADR) were analyzed. Levels and subcellular localization of proteins were determined by Western blot and immunogold ultrastructural analysis techniques. Here we demonstrate that stress caused by ADR induced upregulation of p53 protein in cardiomyocyte mitochondria and nuclei between 3 and 24 hr. Increased expression of PUMA and Bax proteins, pro-apoptotic targets of p53, was documented following ADR treatment and was accompanied by increased levels of apoptotic markers, with elevation of cytosolic cytochrome c at 24 hr and subsequent caspase-3 cleavage at 3 days. Mitochondrial p53 levels correlated with mtDNA oxidative damage. Loss of p53 in knockout mouse heart resulted in a significant increase in mtDNA vulnerability to damage following ADR treatment. Our results suggest that mitochondrial p53 could participate in mtDNA repair as a first response to oxidative damage of cardiomyocyte mtDNA and demonstrate an increase of apoptotic markers as a result of mitochondrial/nuclear p53 localization.

Tumor necrosis factor receptor deficiency exacerbated Adriamycin-induced cardiomyocytes apoptosis: an insight into the Fas connection.

Cardiomyopathy is a major dose-limiting factor for applications of Adriamycin, a potent chemotherapeutic agent. The present study tested the hypothesis that increased tumor necrosis factor (TNF)-alpha signaling via its receptors protects against Adriamycin-induced cardiac injury. We used mice in which both TNF receptor I and II have been selectively inactivated (DKO) with wild-type mice as controls. Morphometric studies of cardiac tissue following Adriamycin treatment revealed greater ultrastructural damage in cardiomyocyte mitochondria from DKO mice. Biochemical studies of cardiac tissues showed cytochrome c release and the increase in proapoptotic protein levels, suggesting that lack of TNF-alpha receptor I and II exacerbates Adriamycin-induced cardiac injury. The protective role of TNF receptor I and II was directly confirmed in isolated primary cardiomyocytes. Interestingly, following Adriamycin treatment, the levels of Fas decreased in the wild-type mice. In contrast, DKO mice had an increase in Fas levels and its downstream target, mitochondrial truncated Bid. These results suggested that TNF-alpha receptors play a critical role in cardioprotection by suppression of the mitochondrial-mediated associated cell death pathway.