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.

BAK1 gene variation and abdominal aortic aneurysms.

We sought to examine the role of genetics in the multifactorial disease, abdominal aortic aneurysm (AAA), by studying sequence variation in the BAK1 gene (BAK1) that codes for an apoptotic-promoting protein, as chronic apoptosis activation has been linked to AAA development and progression. BAK1 abdominal aorta cDNA from AAA patients and nondiseased individuals were compared with each other, as well as to the BAK1 genomic sequence obtained from matching blood samples. We found specific BAK1 single nucleotide polymorphism (SNP) containing alleles in both aneurysmic (31 cases) and healthy aortic tissue (5 cases) without seeing them in the matching blood samples. These same BAK1 SNPs have been reported, although rarely (average frequency <0.06%), in reference BAK1 DNA sequences. Based on this and other similar observations, we propose a novel hypothesis postulating that multiple variants of genes may preexist in "minority" forms within specific nondiseased tissues and be selected for, when intra- and/or extracellular conditions change. Therefore, the fact that different BAK1 variants can exist in both diseased and nondiseased AA tissues compared to matching blood samples, together with the rare occurrence of these same SNPs in reference sequences, suggests that selection may be a significant factor in AAA ontogeny.

Tumor necrosis factor-alpha-induced anterior pituitary folliculostellate TtT/GF cell uncoupling is mediated by connexin 43 dephosphorylation.

The anterior pituitary folliculostellate (FS) cells are key elements of the paracrine control of the pituitary function. These cells are the source and the target of growth factors and cytokines, and are connected to other pituitary cells via Cx43-mediated gap junctions. Here, we show that acute treatment of the FS TtT/GF cell line with TNF-alpha caused a transient cell uncoupling that was accompanied by the dephosphorylation of Cx43 in Ser368. These TNF-alpha-evoked effects were dependent on protein phosphatase 2A (PP2A) and protein kinase C (PKC) activities. TNF-alpha did not affect total cell Cx43-PP2A catalytic subunit interaction, but it did induce PP2A catalytic subunit recruitment to the Triton X-100 insoluble subcellular fraction, in which Cx43-gap junction plaques are recovered. This recruitment temporally coincided with Cx43 phosphorylated in Ser368-Cx43 dephosphorylation. Cx43 did not interact with the conventional PKC-alpha, but it did interact with the atypical PKC-zeta. Moreover, this interaction was weakened by TNF-alpha. Cx43 dephosphorylation in Ser368 was followed by the tyrosine phosphorylation of the protein. The temporary closure of gap junctions during acute TNF-alpha challenge may constitute a protective mechanism to limit or confine the spread of inflammatory signals among the FS cells.

Modulation of GJA1 turnover and intercellular communication by proinflammatory cytokines in the anterior pituitary folliculostellate cell line TtT/GF.

Our previous studies have advanced the idea that the folliculostellate cell GJA1 (gap junction membrane channel protein alpha1; previously known as connexin 43)-mediated gap junctions contribute to the establishment of an intercellular network that regulates the paracrine messages and the endocrine response within the anterior pituitary. The folliculostellate cells are targets for growth factors and cytokines that modulate hormone secretion. Proinflammatory cytokines modulate the cell-to-cell communication in many tissues of the body. The present study measured the effect of the proinflammatory cytokines tumor necrosis factor and interleukin-1 on the GJA1-mediated intercellular communication, specifically the expression, localization, degradation, and phosphorylation status of GJA1 in the folliculostellate cell line TtT/GF. The GJA1 localized to the plasma membrane and to minute cytoplasmic vesicles in the perinuclear area. Using different antibodies that recognize distinctly the nonphosphorylated from the phosphorylated forms of GJA1, we showed that nonphosphorylated GJA1 in Ser-368 (NP-GJA1) localized chiefly in the cytoplasm, whereas GJA1 phosphorylated in Ser-368 (P-GJA1) localized to the plasma membrane in controls. The cytokine treatment transiently increased 1) GJA1, NP-GJA1, and P-GJA1 levels; 2) NP-GJA1 and P-GJA1 degradation by both the lysosomal and proteasomal pathways; and 3) cell-to-cell communication in TtT/GF cells. The results suggest that the cytokine-evoked, transient enhancement of folliculostellate cell-mediated intercellular communication contributes to the coordination of the response among folliculostellate cells.