Cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells are the major cellular constituents of the heart. The aim of this study was to observe alterations in myocardial cell populations during early neonatal development in the adult animal and to observe any variations of the cardiac cell populations in different species, specifically, the rat and mouse. Whole hearts were isolated from either mice or rats during the neonatal and adult stages of development, and single cell suspensions were prepared via sequential collagenase digestion. Heterogeneous cell populations were immunolabeled for specific cell types and analyzed using fluorescence-activated cell sorting (FACS). In addition, the left ventricle, right ventricle, and septa were isolated, fixed, and sectioned for morphometric analyses. These same cardiac regions were also analyzed using FACS. We observed that the adult murine myocardium is composed of approximately 56% myocytes, 27% fibroblasts, 7% endothelial cells, and 10% vascular smooth muscle cells. Moreover, our morphometric and FACS data demonstrated similar percentages in the three regions examined. During murine neonatal cardiac development, we observed a marked increase in numbers of cardiac fibroblasts and a resultant decrease in percentages of myocytes in late neonatal development (day 15). Finally, FACS analyses of the rat heart during development displayed similar results in relation to increases in cardiac fibroblasts during development; however, cell populations in the rat differed markedly from those observed in the mouse. Taken together, these data enabled us to establish a homeostatic model for the myocardium that can be compared with genetic and cardiac disease models.

Background & Aims During tumorigenesis, loss of rapid messenger RNA (mRNA) decay allows for overexpression of cancer-associated genes. The RNA-binding proteins Hu antigen R (HuR) and tristetraprolin (TTP) bind AU-rich elements in the 3′ untranslated region of many cancer-associated mRNAs and target them for stabilization or rapid decay, respectively. We examined the functions of HuR and TTP during colon tumorigenesis and their ability to regulate cyclooxygenase (COX-2), a mediator of prostaglandin synthesis that increases in the colon tumor microenvironment. Methods We evaluated expression of HuR and TTP during colorectal tumorigenesis and in colon cancer cells and associated them with COX-2 expression. HuR and TTP-inducible cells were created to investigate HuR- and TTP-mediated regulation of COX-2. Results In normal colon tissues, low levels of nuclear HuR and higher levels of TTP were observed. By contrast, increased HuR expression and cytoplasmic localization were observed in 76% of adenomas and 94% of adenocarcinomas, and TTP expression was lost in >75% of adenomas and adenocarcinomas. Similar results were obtained for HuR and TTP mRNA levels in normal and staged tumor samples. In both adenomas and adenocarcinomas, COX-2 overexpression was associated with increased HuR and decreased TTP (P < .0001); similar associations were observed in colon cancer cells. HuR overexpression in cells up-regulated COX-2 expression, whereas overexpression of TTP inhibited it; limited TTP expression antagonized HuR-mediated COX-2 overexpression. Conclusions Increased expression of the mRNA stability factor HuR and loss of the decay factor TTP occurs during early stages of colorectal tumorigenesis. These changes promote COX-2 overexpression and could contribute to colon tumorigenesis.

BackgroundMuscle protein turnover regulation during cancer cachexia is being rapidly defined, and skeletal muscle mitochondria function appears coupled to processes regulating muscle wasting. Skeletal muscle oxidative capacity and the expression of proteins regulating mitochondrial biogenesis and dynamics are disrupted in severely cachectic ApcMin/+ mice. It has not been determined if these changes occur at the onset of cachexia and are necessary for the progression of muscle wasting. Exercise and anti-cytokine therapies have proven effective in preventing cachexia development in tumor bearing mice, while their effect on mitochondrial content, biogenesis and dynamics is not well understood. The purposes of this study were to 1) determine IL-6 regulation on mitochondrial remodeling/dysfunction during the progression of cancer cachexia and 2) to determine if exercise training can attenuate mitochondrial dysfunction and the induction of proteolytic pathways during IL-6 induced cancer cachexia.MethodsApcMin/+ mice were examined during the progression of cachexia, after systemic interleukin (IL)-6r antibody treatment, or after IL-6 over-expression with or without exercise. Direct effects of IL-6 on mitochondrial remodeling were examined in cultured C2C12 myoblasts.ResultsMitochondrial content was not reduced during the initial development of cachexia, while muscle PGC-1α and fusion (Mfn1, Mfn2) protein expression was repressed. With progressive weight loss mitochondrial content decreased, PGC-1α and fusion proteins were further suppressed, and fission protein (FIS1) was induced. IL-6 receptor antibody administration after the onset of cachexia improved mitochondrial content, PGC-1α, Mfn1/Mfn2 and FIS1 protein expression. IL-6 over-expression in pre-cachectic mice accelerated body weight loss and muscle wasting, without reducing mitochondrial content, while PGC-1α and Mfn1/Mfn2 protein expression was suppressed and FIS1 protein expression induced. Exercise normalized these IL-6 induced effects. C2C12 myotubes administered IL-6 had increased FIS1 protein expression, increased oxidative stress, and reduced PGC-1α gene expression without altered mitochondrial protein expression.ConclusionsAltered expression of proteins regulating mitochondrial biogenesis and fusion are early events in the initiation of cachexia regulated by IL-6, which precede the loss of muscle mitochondrial content. Furthermore, IL-6 induced mitochondrial remodeling and proteolysis can be rescued with moderate exercise training even in the presence of high circulating IL-6 levels.

BackgroundAryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family, is well characterized to regulate the biochemical and toxic effects of environmental chemicals. More recently, AhR activation has been shown to regulate the differentiation of Foxp3+ Tregs as well as Th17 cells. However, the precise mechanisms are unclear. In the current study, we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand, on epigenetic regulation leading to altered Treg/Th17 differentiation, and consequent suppression of colitis.Methodology/Principal FindingsDextran sodium sulphate (DSS) administration induced acute colitis in C57BL/6 mice, as shown by significant weight loss, shortening of colon, mucosal ulceration, and increased presence of CXCR3+ T cells as well as inflammatory cytokines. Interestingly, a single dose of TCDD (25 µg/kg body weight) was able to attenuate all of the clinical and inflammatory markers of colitis. Analysis of T cells in the lamina propria (LP) and mesenteric lymph nodes (MLN), during colitis, revealed decreased presence of Tregs and increased induction of Th17 cells, which was reversed following TCDD treatment. Activation of T cells from AhR+/+ but not AhR -/- mice, in the presence of TCDD, promoted increased differentiation of Tregs while inhibiting Th17 cells. Analysis of MLN or LP cells during colitis revealed increased methylation of CpG islands of Foxp3 and demethylation of IL-17 promoters, which was reversed following TCDD treatment.Conclusions/SignificanceThese studies demonstrate for the first time that AhR activation promotes epigenetic regulation thereby influencing reciprocal differentiation of Tregs and Th17 cells, and amelioration of inflammation.

Cardiac fibroblasts are organized into a three-dimensional network in the heart. This organization follows the endomysial weave network that surrounds groups of myocytes. Reverse transcriptase-polymerase chain reaction, Western blots, and immunohistochemistry were used to show that discoidin domain receptor 2 (DDR2) was specific for cardiac fibroblasts and not expressed on endothelial cells, smooth muscle cells, or cardiac myocytes. DDR2 is expressed early in development and in the adult heart. High voltage electron microscopy (HVEM), scanning electron microscopy, and laser scanning confocal microscopy document the three-dimensional organization of fibroblasts in the heart. Antibodies against connexin 43 and 45 showed different patterns but confirmed, along with HVEM, that fibroblasts are connected to each other as well as cardiac myocytes. The implications of this arrangement of fibroblasts can be important to cardiac function. The signaling of DDR2 and the expression of matrix metalloproteinase 2 in relation to collagen turnover and remodeling is discussed. Developmental Dynamics 230: 787-794, 2004.