Exposure to the non-phthalate plasticizer di-heptyl succinate is less disruptive to C57bl/6N mouse recovery from a myocardial infarction than DEHP, TOTM or related di-octyl succinate.

Phthalate plasticizers are incorporated into plastics to make them soft and malleable, but are known to leach out of the final product into their surroundings with potential detrimental effects to human and ecological health. The replacement of widely-used phthalate plasticizers, such as di-ethylhexyl phthalate (DEHP), that are of known toxicity, by the commercially-available alternative Tris(2-ethylhexyl) tri-mellitate (TOTM) is increasing. Additionally, several newly designed "green" plasticizers, including di-heptyl succinate (DHPS) and di-octyl succinate (DOS) have been identified as potential replacements. However, the impact of plasticizer exposure from medical devices on patient recovery is unknown and, moreover, the safety of TOTM, DHPS, and DOS is not well established in the context of patient recovery. To study the direct effect of clinically based chemical exposures, we exposed C57bl/6 N male and female mice to DEHP, TOTM, DOS, and DHPS during recovery from cardiac surgery and assessed survival, cardiac structure and function, immune cell infiltration into the cardiac wound and activation of the NLRP3 inflammasome. Male, but not female, mice treated in vivo with DEHP and TOTM had greater cardiac dilation, reduced cardiac function, increased infiltration of neutrophils, monocytes, and macrophages and increased expression of inflammasome receptors and effectors, thereby suggesting impaired recovery in exposed mice. In contrast, no impact was detected in female mice and male mice exposed to DOS and DHPS. To examine the direct effects in cells involved in wound healing, we treated human THP-1 macrophages with the plasticizers in vitro and found DEHP induced greater NLRP3 expression and activation. These results suggest that replacing current plasticizers with non-phthalate-based plasticizers may improve patient recovery, especially in the male population. In our assessment, DHPS is a promising possibility for a non-toxic biocompatible plasticizer.

Acute exposure to phthalates during recovery from a myocardial infarction induces greater inflammasome activation in male C57bl/6N mice.

Plasticizers escape from medical devices used in cardiac surgery into patient blood and tissues. Increased di-ethylhexyl phthalate (DEHP) exposure is correlated with chronic inflammation in vivo and increased cytokine release in exposed monocytes in vitro. To determine if acute phthalate exposure enhanced inflammation in a model of cardiac damage, we measured immune cell infiltration, inflammasome expression and cardiac function in male C57bl/6 N mice exposed to phthalates during recovery from a surgically-induced myocardial infarction (MI). Phthalate exposed mice had greater neutrophil and pro-inflammatory macrophage infiltration, greater cardiac dilation and reduced cardiac function when compared with control mice. The greater expression of NLRP3 and NLRP6, but not AIM2 or P2xR7, in the infarcts of phthalate exposed versus control mice suggests a selectivity in pattern recognition receptor activation. Treatment of human THP-1 macrophages with phthalates revealed increased NLRP3 and NLRP6 expression and induction of a pro-inflammatory macrophage population. Pre-treatment with the PPARγ antagonist GW9662 reduced these increases. An increase in expression of IL-1R, MyD88 and IRAK4 in infarcts of phthalate exposed mice and THP-1 cells argues for greater priming downstream of IL-1R signaling and increased susceptibility for inflammasome activation. Importantly, these effects were moderated in vivo when phthalate exposure was reduced by 90% and when the NLRP3 antagonist MCC950 was co-administered. Our study suggests that reductions in phthalate exposure, which might be realized using plasticizers with a reduced ability to leach out from plastic, or short-term treatment with an anti-inflammasome may improve healing post-surgery.

Recovery From a Myocardial Infarction Is Impaired in Male C57bl/6 N Mice Acutely Exposed to the Bisphenols and Phthalates That Escape From Medical Devices Used in Cardiac Surgery.

Bisphenols and phthalates leach from medical devices, and this exposure is likely to increase in postcardiac surgery patients. Previous studies suggest that such chemical exposure may impact recovery and wound healing, yet the direct effects of bisphenols and phthalates are unknown in this context. To study the direct effect of clinically based chemical exposures, we measured the metabolites representative of 6 bisphenols and 10 phthalates in men before and after cardiac surgery and then replicated this exposure in a mouse model of cardiac surgery and assessed survival, cardiac function and inflammation. Bisphenol A (BPA), di-ethyl hexyl phthalate (DEHP), butylbenzyl phthalate, di-isodecyl phthalate, and di-n-butyl phthalate metabolites were increased after surgery. DEHP exposure predominated, was positively correlated with duration on the cardiopulmonary bypass machine and exceeded its tolerable daily intake limit by 37-fold. In vivo, C57bl/6 N male mice treated with BPA+phthalates during recovery from surgery-induced myocardial infarction had reduced survival, greater cardiac dilation, reduced cardiac function and increased infiltration of neutrophils, monocytes and macrophages suggesting impaired recovery. Of interest, genetic ablation or estrogen receptor beta (ERß) antagonism did not improve recovery and replacement of DEHP with tri-octyl trimellitate or removal of BPA from the mixture did not ameliorate these effects. To examine the direct effects on inflammation, treatment of human THP-1 macrophages with BPA and phthalates induced a dysfunctional proinflammatory macrophage phenotype with increased expression of M1-type macrophage polarization markers and MMP9 secretion, yet reduced phagocytic activity. These results suggest that chemicals escape from medical devices and may impair patient recovery.

From the Cover: Lifelong Exposure of C57bl/6n Male Mice to Bisphenol A or Bisphenol S Reduces Recovery From a Myocardial Infarction.

Bisphenol A (BPA) leaches from plastics to contaminate foodstuffs. Analogs, such as bisphenol S (BPS), are now used increasingly in manufacturing. Greater BPA exposure has been correlated with exacerbation of cardiovascular disease, including myocardial infarction (MI). To test the hypothesis that bisphenol exposure impairs cardiac healing, we exposed C57bl/6n mice to water containing 25ng/ml BPA or BPS from conception and surgically induced an MI in adult male progeny. Increased early death and cardiac dilation, and reduced cardiac function were found post-MI in BPA- and BPS-exposed mice. Flow cytometry revealed increased monocyte and macrophage infiltration that correlated with increased chemokine C-C motif ligand-2 expression in the infarct. In vitro BPA and BPS addition increased matrix metalloproteinase-9 (MMP) protein and secreted activity in RAW264.7 macrophage cells suggesting that invivo increases in MMP2 and MMP9 in exposed infarcts were myeloid-derived. Bone marrow-derived monocytes isolated from exposed mice had greater expression of pro-inflammatory polarization markers when chemokine stimulated indicating an enhanced susceptibility to develop a pro-inflammatory monocyte population. Chronic BPA exposure of estrogen receptor beta (ERß) deficient mice did not worsen early death, cardiac structure/function, or expression of myeloid markers after an MI. In contrast, BPS exposure of ERß-deficient mice resulted in greater death and expression of myeloid markers. We conclude that lifelong exposure to BPA or BPS augmented the monocyte/macrophage inflammatory response and adverse remodeling from an MI thereby reducing the ability to survive and successfully recover, and that the adverse effect of BPA, but not BPS, is downstream of ERß signaling.

Early growth response-1-mediated down-regulation of drebrin correlates with loss of dendritic spines.

Post-synaptic dendritic spines are structurally composed of actin cytoskeleton, which undergoes dynamic morphological changes to accommodate incoming synaptic activity. Drebrin is an actin-binding protein highly expressed in dendritic spines that serves an important role in regulating spine morphology. Functionally, loss of drebrin directly correlates with deficits in learning and memory, as is the case observed in Alzheimer's disease. Despite these findings, the regulatory factor responsible for drebrin loss remains unclear. Here, we show that early growth response-1 (Egr-1), an inducible zinc finger transcription factor, down-regulates drebrin expression. Chromatin immunoprecipitation analyses identified Egr-1 binding sites upstream of the drebrin start site in neuronal cells. Over-expression of Egr-1 in vitro in primary hippocampal neurons or in vivo in homogenates prepared from the hippocampi of an inducible mouse model of Egr-1 show reduced drebrin mRNA and protein levels. Conversely, increased drebrin was detected in hippocampal samples isolated from Egr-1-deficient brain. These data demonstrate that Egr-1 interacts with the drebrin promoter and negatively regulates drebrin expression. Furthermore, immunocytochemical and Golgi staining analyses revealed reduced drebrin protein and dendritic spine density as well as reduced expression of synaptic markers in in vitro hippocampal neurons over-expressing Egr-1 and in vivo inducible mouse model of Egr-1. In contrast, increased drebrin expression correlated with increased dendritic spine density was detected in samples from Egr-1-deficient mice. These data provide evidence that Egr-1 is a novel regulator of drebrin expression, which is linked to changes in dendritic spine density.

Characterization of the NPC1L1 gene and proteome from an exceptional responder to ezetimibe.

BACKGROUND: Strategies to reduce LDL-cholesterol involve reductions in cholesterol synthesis or absorption. We identified a familial hypercholesterolemia patient with an exceptional response to the cholesterol absorption inhibitor, ezetimibe. Niemann-Pick C 1-like 1 (NPC1L1) is the molecular target of ezetimibe. METHODS AND RESULTS: Sequencing identified nucleotide changes predicted to change amino acids 52 (L52P), 300 (I300T) and 489 (S489G) in exceptional NPC1L1. In silico analyses identified increased stability and cholesterol binding affinity in L52P-NPC1L1 versus WT-NPC1L1. HEK293 cells overexpressing WT-NPC1L1 or NPC1L1 harboring amino acid changes singly or in combination (Comb-NPC1L1) had reduced cholesterol uptake in Comb-NPC1L1 when ezetimibe was present. Cholesterol uptake was reduced by ezetimibe in L52P-NPC1L1, I300T-NPC1L1, but increased in S489G-NPC1L1 overexpressing cells. Immunolocalization studies found preferential plasma membrane localization of mutant NPC1L1 independent of ezetimibe. Flotillin 1 and 2 expression was reduced and binding to Comb-NPC1L1 was reduced independent of ezetimibe exposure. Proteomic analyses identified increased association with proteins that modulate intermediate filament proteins in Comb-NPC1L1 versus WT-NPC1L1 treated with ezetimibe. CONCLUSION: This is the first detailed analysis of the role of NPC1L1 mutations in an exceptional responder to ezetimibe. The results point to a complex set of events in which the combined mutations were shown to affect cholesterol uptake in the presence of ezetimibe. Proteomic analysis suggests that the exceptional response may also lie in the nature of interactions with cytosolic proteins.

Cardiac structure/function, protein expression, and DNA methylation are changed in adult female mice exposed to diethylstilbestrol in utero.

The detrimental effects of in utero exposure to the non-steroidal estrogen diethylstilbestrol (DES) are particularly marked in women. Fetal hearts express estrogen receptors, making them potentially responsive to DES. To examine whether gestational exposure to DES would impact the heart, we exposed pregnant C57bl/6n dams to DES (0.1, 1.0, and 10.0 µg·(kg body mass)(-1)·day(-1)) on gestation days 11.5-14.5, and examined the measured cardiac structure/function and calcium homeostasis protein expression in adult females. At baseline, echocardiography revealed eccentric hypertrophy in mice treated with 10.0 µg·(kg body mass)(-1)·day(-1) DES, and immunoblots showed increased SERCA2a in all DES-treated mice. Mice were swim-trained to assess cardiac remodeling. Swim-trained vehicle-treated mice developed eccentric hypertrophy without changing SERCA2 or calsequestrin 2 expression. In contrast, no DES-treated mice hypertrophied, and all increased in SERCA2a and calsequestrin 2 expression after training. To determine whether DES-induced changes in DNA methylation is part of the mechanism for its long-term effects, we measured DNA methyltransferase expression and DNA methylation. Global DNA methylation and DNA methyltransferase 3a expression were unchanged. However, DES-treated mice had increased DNA methylation in the calsequestrin 2 promoter. Thus, gestational exposure to DES altered female ventricular DNA, cardiac structure/function, and calcium homeostasis protein expression. We conclude that gestational exposure to estrogenizing compounds may impact cardiac structure/function in adult females.

Gestational exposure to diethylstilbestrol alters cardiac structure/function, protein expression and DNA methylation in adult male mice progeny.

Pregnant women, and thus their fetuses, are exposed to many endocrine disruptor compounds (EDCs). Fetal cardiomyocytes express sex hormone receptors making them potentially susceptible to re-programming by estrogenizing EDCs. Diethylstilbestrol (DES) is a proto-typical, non-steroidal estrogen. We hypothesized that changes in adult cardiac structure/function after gestational exposure to the test compound DES would be a proof in principle for the possibility of estrogenizing environmental EDCs to also alter the fetal heart. Vehicle (peanut oil) or DES (0.1, 1.0 and 10.0µg/kg/da.) was orally delivered to pregnant C57bl/6n dams on gestation days 11.5-14.5. At 3months, male progeny were left sedentary or were swim trained for 4weeks. Echocardiography of isoflurane anesthetized mice revealed similar cardiac structure/function in all sedentary mice, but evidence of systolic dysfunction and increased diastolic relaxation after swim training at higher DES doses. The calcium homeostasis proteins, SERCA2a, phospholamban, phospho-serine 16 phospholamban and calsequestrin 2, are important for cardiac contraction and relaxation. Immunoblot analyses of ventricle homogenates showed increased expression of SERCA2a and calsequestrin 2 in DES mice and greater molecular remodeling of these proteins and phospho-serine 16 phospholamban in swim trained DES mice. DES increased cardiac DNA methyltransferase 3a expression and DNA methylation in the CpG island within the calsequestrin 2 promoter in heart. Thus, gestational DES epigenetically altered ventricular DNA, altered cardiac function and expression, and reduced the ability of adult progeny to cardiac remodel when physically challenged. We conclude that gestational exposure to estrogenizing EDCs may impact cardiac structure/function in adult males.

The impact of doxorubicin and dexrazoxane injection of prepubertal female rats on pregnancy outcome and cardiac function postpartum.

Childhood cancer survivors can develop significant cardiac dysfunction in adulthood as a consequence of their cancer treatment. Studies have linked heart failure during pregnancy to childhood doxorubicin (DOX) exposure. We hypothesized that DOX injection would reduce cardiac function peripartum and that DOX-treated dams would show greater cardiac remodeling postweaning. Weanling female Sprague-Dawley rats were injected with phospate-buffered saline, DOX (3 mg/kg), or DOX plus the cardioprotectant dexrazoxane (DEX; 60 mg/kg) and followed for 2 pregnancies. DOX and DOX:DEX dams were fertile, but had fewer pups and more pup losses. Echocardiography, 1-day postpartum after each pregnancy, revealed greater increases in cardiac mass and eccentric hypertrophy in DOX-treated dams and early dilation in DOX:DEX dams. The expression of calcium homeostasis proteins can change after DOX treatment and cardiac remodeling. SERCA2a expression did not change. Reductions in phospholamban and phospho-serine 16-specific phospholamban expression in DOX dams were not relieved by DEX coinjection. DOX binds and inactivates calsequestrin 2 expression so increased calsequestrin 2 expression in DOX:DEX-treated dams suggests some DEX compensation. The eccentric hypertrophy and dilation development, despite compensatory changes in proteins controlling calcium cycling, suggest DOX damage with repeat pregnancy that was not alleviated fully by DEX.

Cardiac response to doxorubicin and dexrazoxane in intact and ovariectomized young female rats at rest and after swim training.

The impact of cancer therapies on adult cardiac function is becoming a concern as more children survive their initial cancer. Cardiovascular disease is now a significant problem to adult survivors of childhood cancer. Specifically, doxorubicin (DOX) may be particularly harmful in young girls. The objective of this study was to characterize DOX damage and determine the ability of dexrazoxane (DEX) to reduce DOX-mediated cardiac damage in sedentary and swim-trained female rats. Female Sprague-Dawley rats were left intact or ovariectomized (OVX) at weaning then injected with DEX (60 mg/kg) before DOX (3 mg/kg), DOX alone, or PBS. Rats were separated into sedentary and swim cohorts. Body weight was reduced in DOX:DEX- but not PBS- or DOX-treated rats. Echocardiographic parameters were similar in sedentary rats. Swim training revealed greater concentric remodeling in DOX-treated rats and reduced fractional shortening in DOX:DEX-treated rats. Calsequestrin 2 was reduced with DOX and increased with DOX:DEX postswim. Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a was reduced and calsequestrin 2 reduced further by swim training only in intact rats. OVX rats were heavier and developed eccentric remodeling post-swim with DOX and eccentric hypertrophy with DOX:DEX. Changes in SERCA2a and calsequestrin 2 expression were not observed. Ovariectomized DOX- and DOX:DEX-treated rats stopped growing during swim training. DEX coinjection did not relieve DOX-mediated cardiotoxicity in intact or hormone-deficient rats. DOX-mediated reductions in growth, cardiac function, and expression of calcium homeostasis proteins were exacerbated by swim. DEX coadministration did not substantially relieve DOX-mediated cardiotoxicity in young female rats. Ovarian hormones reduce DOX-induced cardiotoxicity.

Sex hormone control of left ventricular structure/function: mechanistic insights using echocardiography, expression, and DNA methylation analyses in adult mice.

Calcium flux into and out of the sarco(endo)plasmic reticulum is vitally important to cardiac function because the cycle of calcium entry and exit controls contraction and relaxation. Putative estrogen and androgen consensus binding sites near to a CpG island are present in the cardiac calsequestrin 2 (CSQ2) promoter. Cardiomyocytes express sex hormone receptors and respond to sex hormones. We hypothesized that sex hormones control CSQ2 expression in cardiomyocytes and so affect cardiac structure/function. Echocardiographic analysis of male and female C57bl6n mice identified thinner walled and lighter hearts in females and significant concentric remodeling after long-term gonadectomy. CSQ2 and sodium-calcium exchanger-1 (NCX1) expression was significantly increased in female compared with male hearts and decreased postovariectomy. NCX1, but not CSQ2, expression was increased postcastration. CSQ2 expression was reduced when H9c2 cells were cultured in hormone-deficient media; increased when estrogen receptor-α (ERα), estrogen receptor-ß (ERß), or androgen agonists were added; and increased in hearts from ERß-deficient mice. CSQ2 expression was reduced in mice fed a diet low in the methyl donor folic acid and in cells treated with 5-azadeoxycytidine suggesting an involvement of DNA methylation. DNA methylation in CpG in the CSQ2 CpG island was significantly different in males and females and was additionally changed postgonadectomy. Expression of DNA methyltransferases 1, 3a, and 3b was unchanged. These studies strongly link sex hormone-directed changes in CSQ2 expression to DNA methylation with changed expression correlated with altered left ventricular structure and function.

Atorvastatin modulates matrix metalloproteinase expression, activity, and signaling in abdominal aortic aneurysms.

Statins may reduce abdominal aortic aneurysm (AAA) progression. We sought to measure how atorvastatin (AT) treatment might modulate matrix metalloproteinase (MMP) expression and/or activity in human AAA. Tissue from human AAAs at surgical repair was obtained from patients who were either not on statins (NST, n = 19) or treated with AT (n = 19). Immunoblots measured expression and zymography measured activity. Expression of most proteins was greater in the central compared with distal AAA region. Matrix metalloproteinase 1, MMP2, MMP3, MMP9, Tissue Inhibitor of Metalloproteinase (TIMP2), TIMP3, TIMP4, or total Sma Mothers Against Decapentaplegia (SMAD2) expression did not differ with treatment. There was a trend toward reduced MMP8 and TIMP1 expression and MMP2 zymographic activity in the AT-treatment group. In contrast, AT-treated samples had significantly reduced MMP13 (P = .02), latent-transforming growth factor (TGF)-beta (P = .02), and phospho-SMAD2 (P = .029) expression than NST-treated samples. We conclude that the AT-mediated decrease in MMP expression and activity reduces TGF-beta signaling in the central region of human AAAs.

Mesenchymal stromal cells genetically engineered to overexpress IGF-I enhance cell-based gene therapy of renal failure-induced anemia.

We previously demonstrated that erythropoietin (EPO)-secreting mesenchymal stromal cells (MSC) can be used for the long-term correction of renal failure-induced anemia. The present study provides evidence that coimplantation of insulin-like growth factor I (IGF-I)-overexpressing MSC (MSC-IGF) improves MSC-based gene therapy of anemia by providing paracrine support to EPO-secreting MSC (MSC-EPO) within a subcutaneous implant. IGF-I receptor RNA expression in murine MSC was demonstrated by RT-PCR. Functional protein expression was confirmed by immunoblots and MSC responsiveness to IGF-I stimulation in vitro. IGF-I was also shown to improve MSC survival following staurosporin-induced apoptosis in vitro. A cohort of C57Bl/6 mice was rendered anemic by right kidney electrocoagulation and left nephrectomy. MSC-EPO were subsequently admixed in a bovine collagen matrix and implanted, in combination with MSC-IGF or MSC null, by subcutaneous injection in renal failure mice. In mice receiving MSC-EPO coimplanted with MSC-IGF, hematocrit elevation was greater and enhanced compared with control mice; heart function was also improved. MSC-IGF coimplantation, therefore, represents a promising new strategy for enhancing MSC survival within implanted matrices and for improving cell-based gene therapy of renal anemia.

Ubiquinol-cytochrome-c reductase 7.2 kDa protein of mitochondrial complex III is steroid-responsive and increases in cardiac hypertrophy and hypertension.

Women and men do not respond identically to cardiac insults; premenopausal women are somewhat protected from cardiovascular disease. Our objective was to isolate and characterize hormone-responsive genes in the heart. Differential display identified an estrogen-inducible fragment that was found to encode the ubiquinol-cytochrome-c reductase (UCCR) 7.2 kDa protein of the mitochondrial respiratory complex III. We found UCCR7.2 mRNA to be highly expressed in the heart, and this expression increased in hearts of 4-, 10-, and 28-week-old spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto rats. Oral hydralazine treatment to reduce hypertension reduced SHR UCCR7.2 expression. Cardiac UCCR7.2 mRNA expression was also increased significantly after a 5/6 nephrectomy compared with mock surgery. Cardiac expression after ovariectomy was 50% that of intact rats. Supplementation of ovariectomized rats with estrogen had no effect, whereas progesterone increased cardiac expression, although not to intact levels. No change in cardiac UCCR7.2 expression was found when intact rats were treated with either tamoxifen or ICI 182780. Thus, UCCR7.2 expression is reduced in the absence of ovarian hormones, but is not directly regulated by estrogen in the heart. We conclude that UCCR7.2 is a steroid hormone-responsive gene in the heart, with expression increased in cardiac hypertrophy and in response to hypertension.

Phosphorylation of protein phosphatase 1 by cyclin-dependent protein kinase 5 during nerve growth factor-induced PC12 cell differentiation.

The transcription factor Egr-1 activates cyclin-dependent protein kinase 5 (Cdk5) during nerve growth factor (NGF)-induced differentiation of PC12 cells into neurons (Harada, T. Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459). The downstream target of Cdk5 in the Egr-1/Cdk5 pathway is not clear. In this study, we observed that phosphorylation of protein phosphatase 1 (PP1) on Thr(320) is reduced in brain extracts from Egr-1(-/-) mice, indicating that a kinase downstream of Egr-1 phosphorylates PP1. In HEK 293 cells co-transfected with PP1 and Cdk5, Cdk5 phosphorylates PP1. In vitro, Cdk5 purified from bovine brain phosphorylates bacterially expressed recombinant PP1. In NGF-treated PC12 cells, inhibition of Cdk5 by olomoucine or silencing Cdk5 expression by small interfering RNA strategy, suppresses PP1 phosphorylation. Silencing Cdk5 expression by small interfering RNA also blocks NGF-induced neurite outgrowth. Overexpression of PP1 (wild type) promotes NGF-induced differentiation of PC12 cells, whereas that of PP1 (T320A) has no effect. Our data indicate that PP1 is a downstream target of the NGF/Egr-1/Cdk5 pathway during NGF-induced differentiation of PC12 cells and suggest that PP1 phosphorylation promotes neuronal differentiation.

TBP-associated factor 1 overexpression induces tolerance to Doxorubicin in confluent H9c2 cells by an increase in cdk2 activity and cyclin E expression.

Doxorubicin (DOX) is a DNA topoisomerase II inhibitor widely used in anticancer treatment, however, it can lead to irreversible cardiac damage with severe debilitation. TBP-binding associated factor 1 (TAF1) is increased in DOX damaged hearts in vivo and in cardiomyocytes in vitro. To identify the functional role for TAF1 in DOX-treated heart we overexpressed wild type and mutant TAF1 in H9c2 cells. Overexpression of wild-type TAF1, but not N-terminal kinase domain mutants, increased tolerance to DOX in confluent cells. DOX treatment can cause prolonged G1 arrest. We found increased cdk2 activity coupled to increased cyclin E protein and decreased p21(waf1Cip1) and p27(Kip1) protein to correlate only with increased DOX tolerance and wild-type TAF1. DOX sensitivity was restored when the cdk2-inhibitor Roscovitine was co-administered with DOX. Overexpression of cdk2-alone increased resistance to DOX. Thus, TAF1 induced DOX tolerance in confluent cells through an increase in cdk2 activity is directed by the TAF1 N-terminal domain. These studies suggest new avenues for myocardial protection against DOX toxicity and suggest a role for cdk2 in chemorefractory cells.

Chronic beta-adrenoreceptor stimulation in vivo decreased Bcl-2 and increased Bax expression but did not activate apoptotic pathways in mouse heart.

Prolonged activation of the sympathetic nervous system is deleterious to heart function. In vitro beta1-adrenergic activation promotes apoptosis, whereas beta2-adrenergic activation reduces apoptosis in cultured adult cardiomyocytes. To determine the effect of chronic catecholamine infusion in vivo, we measured apoptosis marker expression in C57Bl/6 and catecholamine-sensitive Egr-1 deficient mice after treatment with the nonspecific beta-adrenergic agonist, isoproterenol, the beta1-specific agonist, dobutamine, or the beta2-specific agonist, metaproterenol. Antiapoptotic and proapoptotic protein expression, cytochrome c release and caspases 3, 9, and 12 activation products were measured on immunoblots. Catecholamine-treated mice had decreased Bcl-2 and increased Bax and BNIP1 expression, suggesting mitochondria-dependent apoptosis pathway activation. However, cytosolic cytochrome c or caspase 3 or 9 activation products were not detected. In mice, increased molecular chaperone expression and caspase 12 activation characterize endoplasmic-reticulum-driven apoptosis. Clusterin expression was increased in catecholamine-treated mice, but GRP78 expression was not increased, and caspase 12 activation products were not detected. Thus, neither the mitochondrial nor the endoplasmic apoptotic pathway was fully activated. Further, Egr-1 deficiency did not increase cardiac apoptosis. We conclude that although chronic in vivo infusion of beta1- or beta2-adrenergic receptor agonists partially activates the apoptosis program, full activation of the caspase cascade requires more, or other, cardiac insults.

Dexrazoxane does not protect against doxorubicin-induced damage in young rats.

Doxorubicin (DOX), an anticancer drug, causes a dose-dependent cardiotoxicity. Some evidence suggests that female children have an increased risk for DOX-mediated cardiac damage. To determine whether the iron chelator dexrazoxane (DXR) could reduce DOX-induced cardiotoxicity in the young, we injected day 10 neonate female and male rat pups with a single dose of saline or DOX, DXR, or DXR + DOX (20:1). We followed body weight gain with growth, measured cardiac hypertrophy after a 2-wk swim exercise program, markers of apoptosis (Bcl-2, BAX, BNIP1, caspase 3 activation), oxidative stress (heme oxygenase 1, protein carbonyl levels), the chaperone protein clusterin, and the transcriptional activator early growth response gene-1 (Egr-1) in hearts of nonexercised and exercised rats on neonate day 38. All DOX-alone and DXR + DOX-treated rats showed decreased weight gain, with female rats affected earlier than male rats. DXR-alone, DOX-alone, and DXR + DOX-treated rats had an increased heart weight-to-body weight (heart wt/body wt) ratio after the exercise program with female rats showing the largest increase in heart wt/body wt. Drug-treated females also showed increased cardiac apoptosis, as measured by the increased expression of the proapoptotic proteins BAX and BNIP1 and the appearance of caspase 3 activation products, and oxidative stress, as measured by increased heme oxygenase 1 expression, and reduced Egr-1 and clusterin expression when compared with the similarly treated male rats. We conclude that DXR preinjection did not reduce DOX-induced noncardiac and cardiac damage and that young female rats were more susceptible to DXR and DOX toxicities than age-matched male rats.