Predictive value of matrix metalloprotease 9 on surgical outcomes after pericardiectomy.

BACKGROUND: The effects of matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) expressions on the patients with constrictive pericarditis undergoing pericardiectomy remain unclear. This study explored the associations of MMPs and TIMPs expressions with postoperative outcomes in these patients. METHODS: Pericardial specimens were obtained during pericardiectomy from the patients with constrictive pericarditis. The levels of MMP1, MMP2, MMP9 and TIMP1 in pericardium were analyzed by quantitative real-time polymerase chain reaction. The enrolled patients were divided into two groups according to the optimal cutoff value of gene expression predicting postoperative complications. Postoperative outcomes were compared between the two groups. Binary logistic regression analysis was performed to determine the degree of contribution of gene expression on postoperative outcomes. RESULTS: A total of 22 patients and their pericardial specimens were included. The level of MMP9 was significantly associated with postoperative complications and the optimal cutoff value predicting postoperative complications was 3.67. The patients with low level of MMP9 (< 3.67) had lower incidence of postoperative complications (P = 0.002), shorter postoperative intensive care unit (P = 0.040) and hospital stay (P = 0.043) in comparison to those with high level of MMP9 (≥ 3.67). Binary logistic regression analysis showed that high level of MMP9 increased the risk of postoperative complications (OR 27.096, 95% CI 1.166-629.886, P = 0.040). CONCLUSIONS: High level of MMP9 in the pericardium was associated with poor postoperative outcomes and was the independent risk factor of postoperative complications. The level of MMP9 could be used as a potential marker for prediction of surgical outcomes.

Dual role for angiotensin-converting enzyme 2 in Severe Acute Respiratory Syndrome Coronavirus 2 infection and cardiac fat.

Angiotensin-converting enzyme 2 (ACE2) has been an increasingly prevalent target for investigation since its discovery 20 years ago. The finding that it serves a counterregulatory function within the traditional renin-angiotensin system, implicating it in cardiometabolic health, has increased its clinical relevance. Focus on ACE2's role in cardiometabolic health has largely centered on its apparent functions in the context of obesity. Interest in ACE2 has become even greater with the discovery that it serves as the cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), opening up numerous mechanisms for deleterious effects of infection. The proliferation of ACE2 within the literature coupled with its dual role in SARS-CoV-2 infection and obesity necessitates review of the current understanding of ACE2's physiological, pathophysiological, and potential therapeutic functions. This review highlights the roles of ACE2 in cardiac dysfunction and obesity, with focus on epicardial adipose tissue, to reconcile the data in the context of SARS-CoV-2 infection.

Paracardial fat remodeling affects systemic metabolism through alcohol dehydrogenase 1.

The relationship between adiposity and metabolic health is well established. However, very little is known about the fat depot, known as paracardial fat (pCF), located superior to and surrounding the heart. Here, we show that pCF remodels with aging and a high-fat diet and that the size and function of this depot are controlled by alcohol dehydrogenase 1 (ADH1), an enzyme that oxidizes retinol into retinaldehyde. Elderly individuals and individuals with obesity have low ADH1 expression in pCF, and in mice, genetic ablation of Adh1 is sufficient to drive pCF accumulation, dysfunction, and global impairments in metabolic flexibility. Metabolomics analysis revealed that pCF controlled the levels of circulating metabolites affecting fatty acid biosynthesis. Also, surgical removal of the pCF depot was sufficient to rescue the impairments in cardiometabolic flexibility and fitness observed in Adh1-deficient mice. Furthermore, treatment with retinaldehyde prevented pCF remodeling in these animals. Mechanistically, we found that the ADH1/retinaldehyde pathway works by driving PGC-1α nuclear translocation and promoting mitochondrial fusion and biogenesis in the pCF depot. Together, these data demonstrate that pCF is a critical regulator of cardiometabolic fitness and that retinaldehyde and its generating enzyme ADH1 act as critical regulators of adipocyte remodeling in the pCF depot.

Cellular Identification and Quantification of Senescence-Associated ß-Galactosidase Activity In Vivo.

Senescence-associated ß-galactosidase (hereafter SA-ß-gal) staining has now been employed for more than 20 years to identify the presence of senescent cells (Dimri et al., Proc Natl Acad Sci U S A 92:9363-9367, 1995). These cells, characterized by a permanent cell-cycle arrest (Hayflick and Moorhead, Exp Cell Res 25:585-621, 1961) and the production of a distinct secretory phenotype of cytokines, chemokines, and proteases (Coppe et al., PLoS Biol 6:2853-2868, 2008), have received much attention in recent years for their impacts on diverse biological processes. Here we describe a method to identify and quantify the specific cells that become senescent in vivo using transmission electron microscopy after SA-ß-gal staining that can be used in countless scenarios.

Loss of BAP1 Expression in Atypical Mesothelial Proliferations Helps to Predict Malignant Mesothelioma.

Distinguishing reactive mesothelial proliferation from malignant mesothelioma (MM) can be difficult, particularly on small biopsies. In this scenario, a diagnosis of atypical mesothelial proliferation might be rendered. However, the distinction between a reactive process and MM is important for prognosis and treatment. Recently, loss of BRCA1-associated protein 1 (BAP1) expression and/or homozygous deletion of CDKN2A were identified in some MM, but not in reactive mesothelial proliferations. We studied 34 cases of atypical mesothelial proliferation from our institutional files (1993 to 2016) for BAP1 expression, deletion of CDKN2A, and clinical outcome. Fifteen of 34 patients (44%) were subsequently diagnosed with MM. BAP1 expression was lost in 6 of these 15 (40%) patients. Ten of 15 (67%) patients died of disease within a median time of 18.2 months. BAP1 expression was also lost in 1 case of probable MM. In this case atypical mesothelial proliferation was identified in the pleura during a lobectomy procedure for lung adenocarcinoma. Follow-up of 57.0 months was remarkable for visceral and parietal pleural thickening with continued unilateral effusion identified on imaging studies but no subsequent definitive diagnosis of MM. CDKN2A studies by fluorescence in situ hybridization (performed in 31 cases) found no homozygous deletion of that gene in any case. In conclusion, loss of BAP1 expression in atypical mesothelial proliferation helps to predict MM and is a useful adjunct test in these cases. Homozygous deletion of CDKN2A in mesothelial cell proliferations did not prove to be useful to predict MM in cases of atypical mesothelial proliferation.

[Difficulties in classifying body cavity fluids into transudate/ exudate depending on the various diagnostic criteria]. / Trudnosci w klasyfikacji plynów z jam ciala na przesiek/wysiek na podstawie róznych kryteriów diagnostycznych.

Disease processes may impair the production and reabsorption of fluid from in the body cavities, which results in its excessive accumulation. AIM: The aim of the study was the evaluation of difficulties regarding the classification of fluids from the body cavities into transudate/exudate observing the following: Light's criteria, total fluid protein concentration, and total protein ratio (TP ratio) and lactate dehydrogenase ratio (LDH ratio). MATERIALS AND METHODS: Retrospective analysis was conducted on pleural (N=314), peritoneal (N=114) and pericardial (N=10) fluids, which were tested for the total protein concentration and LDH activity both in fluid and serum and calculated on TP ratio and LDH ratio. RESULTS: Based on the total protein concentration, 278 fluids from pleural cavity were classified as an exudate; 36 as a transudate. Applying the Light's criteria 240 fluids were classified as an exudate; the remaining 74 fluids were classified as a transudate. Based on TP and LDH ratios, 229 fluids from pleural cavity were classified as an exudate; 85 as a transudate. Depending on the total protein concentration, 35 fluids from the peritoneal cavity were classified as an exudate; 79 as a transudate. Applying the Light's criteria 54 fluids were classified as an exudate; the remaining 60 fluids were classified as a transudate. Based on TP and LDH ratios, 22 fluids from peritoneal cavity were classified as an exudate; 92 as a transudate. Analysis of pericardial fluids, depending on the total protein concentration classified 9 of them as an exudate and 1 as a transudate. The same results were obtained by applying Light's criteria. Based on TP and LDH ratios, 7 fluids from pericardial cavity were classified as an exudate; 3 - as a transudate. CONCLUSIONS: Applying the Light's criteria or the total protein concentration in differential diagnostics of fluids from the body cavities resulted in qualification more of them as an exudates as compared to the analysis of the same fluids depending on the TP and LDH ratios. It can be assumed that some of the transudative/exudative fluids were incorrectly classified. Performed analysis suggest that more adequate criteria of the classification of fluids from the body cavities into transudate/exudate are of great importance.

Pericardial Parietal Mesothelial Cells: Source of the Angiotensin-Converting-Enzyme of the Bovine Pericardial Fluid.

BACKGROUND: Angiotensin II (Ang II), the primary effector hormone of the renin-angiotensin system (RAS), acts systemically or locally, being produced by the action of angiotensin-converting-enzyme (ACE) on angiotensin I. Although several tissue RASs, such as cardiac RAS, have been described, little is known about the presence of an RAS in the pericardial fluid and its possible sources. Locally produced Ang II has paracrine and autocrine effects, inducing left ventricular hypertrophy, fibrosis, heart failure and cardiac dysfunction. Because of the difficulties inherent in human pericardial fluid collection, appropriate experimental models are useful to obtain data regarding the characteristics of the pericardial fluid and surrounding tissues. OBJECTIVES: To evidence the presence of constituents of the Ang II production paths in bovine pericardial fluid and parietal pericardium. METHODS: Albumin-free crude extracts of bovine pericardial fluid, immunoprecipitated with anti-ACE antibody, were submitted to electrophoresis (SDS-PAGE) and gels stained with coomassie blue. Duplicates of gels were probed with anti-ACE antibody. In the pericardial membranes, ACE was detected by use of immunofluorescence. RESULTS: Immunodetection on nitrocellulose membranes showed a 146-KDa ACE isoform in the bovine pericardial fluid. On the pericardial membrane sections, ACE was immunolocalized in the mesothelial layer. CONCLUSIONS: The ACE isoform in the bovine pericardial fluid and parietal pericardium should account at least partially for the production of Ang II in the pericardial space, and should be considered when assessing the cardiac RAS.

Pericardial Parietal Mesothelial Cells: Source of the Angiotensin-Converting-Enzyme of the Bovine Pericardial Fluid / Células Mesoteliais Pericárdicas Parietais: Fonte da Enzima Conversora de Angiotensina Existente no Líquido Pericárdico Bovino

Abstract Background: Angiotensin II (Ang II), the primary effector hormone of the renin-angiotensin system (RAS), acts systemically or locally, being produced by the action of angiotensin-converting-enzyme (ACE) on angiotensin I. Although several tissue RASs, such as cardiac RAS, have been described, little is known about the presence of an RAS in the pericardial fluid and its possible sources. Locally produced Ang II has paracrine and autocrine effects, inducing left ventricular hypertrophy, fibrosis, heart failure and cardiac dysfunction. Because of the difficulties inherent in human pericardial fluid collection, appropriate experimental models are useful to obtain data regarding the characteristics of the pericardial fluid and surrounding tissues. Objectives: To evidence the presence of constituents of the Ang II production paths in bovine pericardial fluid and parietal pericardium. Methods: Albumin-free crude extracts of bovine pericardial fluid, immunoprecipitated with anti-ACE antibody, were submitted to electrophoresis (SDS-PAGE) and gels stained with coomassie blue. Duplicates of gels were probed with anti-ACE antibody. In the pericardial membranes, ACE was detected by use of immunofluorescence. Results: Immunodetection on nitrocellulose membranes showed a 146-KDa ACE isoform in the bovine pericardial fluid. On the pericardial membrane sections, ACE was immunolocalized in the mesothelial layer. Conclusions: The ACE isoform in the bovine pericardial fluid and parietal pericardium should account at least partially for the production of Ang II in the pericardial space, and should be considered when assessing the cardiac RAS.

Resumo Fundamentos: Angiotensina II (Ang II), o hormônio efetor primário do sistema renina-angiotensina (SRA), atuando em níveis sistêmicos ou locais, é produzida pela ação da enzima conversora de angiotensina (ECA) sobre a angiotensina I. Embora diversos SRAs teciduais, como o SRA cardíaco, tenham sido descritos em muitos estudos, dados de um SRA no líquido pericárdico e sua origem não são ainda disponíveis. A Ang II localmente produzida tem efeitos parácrinos e autócrinos, induzindo a hipertrofia ventricular esquerda, fibrose, insuficiência e disfunção cardíacas. Devido às dificuldades inerentes à obtenção de líquido pericárdico humano, modelos experimentais apropriados são muito úteis para obter dados relativos às suas características bem como dos tecidos contíguos. Objetivos: Obter evidências da presença de constituintes das vias de produção de Ang II no líquido pericárdico e no pericárdio parietal bovinos. Métodos: Extratos brutos de líquido pericárdico bovino sem albumina (sobrenadantes), imunoprecipitados com anticorpo anti-ECA, foram submetidos a eletroforese (SDS-PAGE) e os géis corados com Coomassie Blue. Duplicatas dos géis foram sondadas com anticorpo anti-ECA. A detecção de ECA nas membranas pericárdicas foi realizada por imunofluorescência. Resultados: A imunodetecção sobre as membranas de nitrocelulose mostrou uma isoforma de ECA com 146 KDa no líquido pericárdico bovino. Nas secções de membrana pericárdica, a ECA foi imunolocalizada na camada mesotelial. Conclusões: A isoforma de ECA do líquido pericárdico bovino e do pericárdio parietal deve ser, pelo menos em parte, responsável pela produção de Ang II no espaço pericárdico, devendo ser considerada quando o SRA cardíaco for avaliado.

Common pathways regulate Type III TGFß receptor-dependent cell invasion in epicardial and endocardial cells.

Epithelial-Mesenchymal Transformation (EMT) and the subsequent invasion of epicardial and endocardial cells during cardiac development is critical to the development of the coronary vessels and heart valves. The transformed cells give rise to cardiac fibroblasts and vascular smooth muscle cells or valvular interstitial cells, respectively. The Type III Transforming Growth Factor ß (TGFßR3) receptor regulates EMT and cell invasion in both cell types, but the signaling mechanisms downstream of TGFßR3 are not well understood. Here we use epicardial and endocardial cells in in vitro cell invasion assays to identify common mechanisms downstream of TGFßR3 that regulate cell invasion. Inhibition of NF-κB activity blocked cell invasion in epicardial and endocardial cells. NF-κB signaling was found to be dysregulated in Tgfbr3(-/-) epicardial cells which also show impaired cell invasion in response to ligand. TGFßR3-dependent cell invasion is also dependent upon Activin Receptor-Like Kinase (ALK) 2, ALK3, and ALK5 activity. A TGFßR3 mutant that contains a threonine to alanine substitution at residue 841 (TGFßR3-T841A) induces ligand-independent cell invasion in both epicardial and endocardial cells in vitro. These findings reveal a role for NF-κB signaling in the regulation of epicardial and endocardial cell invasion and identify a mutation in TGFßR3 which stimulates ligand-independent signaling.

Activity of MMP-9 after repair of abdominal wall defects with acellular and crosslinked bovine pericardium in rabbit.

This study was undertaken for the identification of matrix metalloproteinases (MMPs) in extracts obtained from native, acellular and crosslinked bovine pericardium (in vitro), as well as in the plasma after implantation of these biomaterials in rabbits (in vivo). Native pericardium (NP) expressed a 72 kDa (MMP-2) band; whereas, in acellular pericardium (AP) two bands (10 kDa and 92 kDa) of MMPs were observed of which, 92 kDa band was very faint. AP crosslinked with glutaraldehyde did not show any gelatinase activity and thus reflects the creation of new additional chemical bonds between the collagen molecules which has been effectively removed. Gelatin zymography showed only one major band of 92 kDa in all the implanted and untreated rabbit plasma, but the relative amount of 92 kDa was 1-2 times higher in acellular bovine pericardium implanted rabbits as compared to crosslinked and native groups. In NP group, the 92 kDa band was the dullest among the three groups. This indicated that the level of MMP-9 corresponds to the degree of collagen degradation.

11ß-hydroxysteroid dehydrogenase type 1 gene expression is increased in ascending aorta tissue of metabolic syndrome patients with coronary artery disease.

11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1) activity and mRNA levels are increased in visceral and subcutaneous adipose tissues of metabolic syndrome subjects. We analyzed 11ß-HSD-1 expression in human epicardial adipose (EA) and ascending aorta (AA) tissues of metabolic syndrome patients and examined their contribution to the development of coronary atherosclerosis. The 11ß-HSD-1 expression was evaluated by qRT-PCR in EA and AA tissues of 20 metabolic syndrome patients with coronary artery disease (metabolic syndrome group) and 10 non-metabolic syndrome patients without coronary artery disease (controls). 11ß-HSD-1 expression was increased in EA and AA tissues of the metabolic syndrome group (4.1- and 5.5-fold, respectively). A significant positive correlation was found between 11ß-HSD-1 expression in EA tissue and waist hip ratio and 11ß-HSD-1 expression in AA tissue and body mass index, while a negative correlation was found between 11ß-HSD-1 expression in EA tissue and HDL. Expression of CD68, a macrophage marker, was significantly increased in both tissues of the metabolic syndrome group; it was 2-fold higher in AA tissue compared to EA tissue in the metabolic syndrome group. Our findings of increased expression of 11ß-HSD-1 and CD68 in AA tissue of the metabolic syndrome group lead us to suggest that they contribute to coronary atherosclerosis in metabolic syndrome. This positive correlation between obesity markers and 11ß-HSD-1 in AA and EA tissues strengthens the evidence that 11ß-HSD-1 has a role in metabolic syndrome. To the best of our knowledge, this is the first report showing 11ß-HSD-1 and CD68 expression in AA tissue of metabolic syndrome patients. We suggest that there is tissue-specific expression of 11ß-HSD-1 in metabolic syndrome and associated cardiovascular disorders.

A dominant negative zebrafish Ahr2 partially protects developing zebrafish from dioxin toxicity.

The toxicity by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is thought to be caused by activation of the aryl hydrocarbon receptor (AHR). However, our understanding of how AHR activation by TCDD leads to toxic effects is poor. Ideally we would like to manipulate AHR activity in specific tissues and at specific times. One route to this is expressing dominant negative AHRs (dnAHRs). This work describes the construction and characterization of dominant negative forms of the zebrafish Ahr2 in which the C-terminal transactivation domain was either removed, or replaced with the inhibitory domain from the Drosophila engrailed repressor protein. One of these dnAhr2s was selected for expression from the ubiquitously active e2fα promoter in transgenic zebrafish. We found that these transgenic zebrafish expressing dnAhr2 had reduced TCDD induction of the Ahr2 target gene cyp1a, as measured by 7-ethoxyresorufin-O-deethylase activity. Furthermore, the cardiotoxicity produced by TCDD, pericardial edema, heart malformation, and reduced blood flow, were all mitigated in the zebrafish expressing the dnAhr2. These results provide in vivo proof-of-principle results demonstrating the effectiveness of dnAHRs in manipulating AHR activity in vivo, and demonstrating that this approach can be a means for blocking TCDD toxicity.

MicroRNA-processing enzyme Dicer is required in epicardium for coronary vasculature development.

The epicardium is a sheet of epithelial cells covering the heart during early cardiac development. In recent years, the epicardium has been identified as an important contributor to cardiovascular development, and epicardium-derived cells have the potential to differentiate into multiple cardiac cell lineages. Some epicardium-derived cells that undergo epithelial-to-mesenchymal transition and delaminate from the surface of the developing heart subsequently invade the myocardium and differentiate into vascular smooth muscle of the developing coronary vasculature. MicroRNAs (miRNAs) have been implicated broadly in tissue patterning and development, including in the heart, but a role in epicardium is unknown. To examine the role of miRNAs during epicardial development, we conditionally deleted the miRNA-processing enzyme Dicer in the proepicardium using Gata5-Cre mice. Epicardial Dicer mutant mice are born in expected Mendelian ratios but die immediately after birth with profound cardiac defects, including impaired coronary vessel development. We found that loss of Dicer leads to impaired epicardial epithelial-to-mesenchymal transition and a reduction in epicardial cell proliferation and differentiation into coronary smooth muscle cells. These results demonstrate a critical role for Dicer, and by implication miRNAs, in murine epicardial development.

Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) resembles Antley-Bixler syndrome.

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.

Involvement of the MEKK1 signaling pathway in the regulation of epicardial cell behavior by hyaluronan.

During embryonic development, cells comprising the outermost layer of the heart or epicardium play a critical role in the formation of the coronary vasculature. Thus, uncovering the molecular mechanisms that govern epicardial cell behavior is imperative to better understand the etiology of cardiovascular diseases. In this study, we investigated the function of hyaluronan (HA), a major component of the extracellular matrix, in the modulation of epicardial signaling. We show that stimulation of epicardial cells with high molecular weight HA (HMW-HA) promotes the association of MEKK1 with the HA receptor CD44 and induces MEKK1 phosphorylation. This leads to the activation of two distinct pathways, one ERK-dependent and another NFkappaB-dependent. Furthermore, HMW-HA stimulates epicardial cells to differentiate and invade, as suggested by increased vimentin expression and enhanced invasion through a collagen matrix. Blockade of CD44, transfection with a kinase-inactive MEKK1 construct or the use of ERK1/2 and NFkappaB inhibitors significantly abrogates the invasive response to HMW-HA. Together, these findings suggest an important role for HA in the regulation of epicardial cell fate via activation of MEKK1 signaling cascades.

Secretory type II phospholipase A2 is produced and secreted by epicardial adipose tissue and overexpressed in patients with coronary artery disease.

CONTEXT: Epicardial adipose tissue (EAT) is a visceral adipose tissue in close contact with coronary vessels, the excess of which is associated with coronary artery disease (CAD). OBJECTIVE: Our goal was to identify candidate molecule(s) characterizing EAT that could intervene in the pathogenesis of CAD. DESIGN: An approach combining microarrays and bioinformatic sequence analysis tools for predicting secreted proteins (TargetP) was applied to paired biopsies of sc adipose tissue (SAT) and EAT, obtained from patients with or without CAD (NCAD). RESULTS were validated in three independent groups of subjects by quantitative RT-PCR, Western blot, immunohistochemistry, and explant secretion. RESULTS: Secretory type II phospholipase A2 (sPLA2-IIA) ranked as the highest gene coding for potentially secreted proteins with the highest overexpression in EAT in both CAD and NCAD. Quantitative RT-PCR confirmed its increased expression in EAT (P < 0.01) as well as EAT from CAD as compared with NCAD (49.3 +/- 13 vs. 17.4 +/- 9.7 P < 0.01). sPLA2-IIA protein levels were higher in EAT than SAT (P < 0.001). EAT explants also showed significantly higher sPLA2-IIA secretion levels than SAT ones (4.37 +/- 2.7 vs. 0.67 +/- 0.28 ng/ml to 1 per gram tissue per 24 h, P < 0.03). sPLA2-IIA labeling was seen in the stroma vascular fraction between adipocytes and in connective capsules in EAT, with no immunostaining of the adipocytes. SAT was weakly labeled following the same process. CONCLUSION: We have shown for the first time an increased expression of sPLA2-IIA in EAT in patients with CAD. sPLA2-IIA is a phospholipase, which has been shown to be an independent risk factor for CAD. These findings suggest that EAT has a potentially pathophysiological role in CAD.

Structural and molecular mechanisms of gap junction remodeling in epicardial border zone myocytes following myocardial infarction.

Lateralization of the ventricular gap junction protein connexin 43 (Cx43) occurs in epicardial border zone myocytes following myocardial infarction (MI) and is arrhythmogenic. Alterations in Cx43 protein partners have been hypothesized to play a role in lateralization although mechanisms by which this occurs are unknown. To examine potential mechanisms we did nuclear magnetic resonance, yeast 2-hybrid, and surface plasmon resonance studies and found that the SH3 domain of the tyrosine kinase c-Src binds to the Cx43 scaffolding protein zonula occludens-1 (ZO-1) with a higher affinity than does Cx43. This suggests c-Src outcompetes Cx43 for binding to ZO-1, thus acting as a chaperone for ZO-1 and causing unhooking from Cx43. To determine whether c-Src/ZO-1 interactions affect Cx43 lateralization within the epicardial border zone, we performed Western blot, immunoprecipitation, and immunolocalization for active c-Src (p-cSrc) post-MI using a canine model of coronary occlusion. We found that post-MI p-cSrc interacts with ZO-1 as Cx43 begins to decrease its interaction with ZO-1 and undergo initial loss of intercalated disk localization. This indicates that the molecular mechanisms by which Cx43 is lost from the intercalated disk following MI includes an interaction of p-cSrc with ZO-1 and subsequent loss of scaffolding of Cx43 leaving Cx43 free to diffuse in myocyte membranes from areas of high Cx43, as at the intercalated disk, to regions of lower Cx43 content, the lateral myocyte membrane. Therefore shifts in Cx43 protein partners may underlie, in part, arrhythmogenesis in the post-MI heart.

Thymosin beta4 mediated PKC activation is essential to initiate the embryonic coronary developmental program and epicardial progenitor cell activation in adult mice in vivo.

Hypoxic heart disease is a predominant cause of disability and death worldwide. Since adult mammalian hearts are incapable of regeneration after hypoxia, attempts to modify this deficiency are critical. As demonstrated in zebrafish, recall of the embryonic developmental program may be the key to success. Because thymosin beta4 (TB4) is beneficial for myocardial cell survival and essential for coronary development in embryos, we hypothesized that it reactivates the embryonic developmental program and initiates epicardial progenitor mobilization in adult mammals. We found that TB4 stimulates capillary-like tube formation of adult coronary endothelial cells and increases embryonic endothelial cell migration and proliferation in vitro. The increase of blood vessel/epicardial substance (Bves) expressing cells accompanied by elevated VEGF, Flk-1, TGF-beta, Fgfr-2, Fgfr-4, Fgf-17 and beta-Catenin expression and increase of Tbx-18 and Wt-1 positive myocardial progenitors suggested organ-wide recall of the embryonic program in the adult epicardium. TB4 also positively regulated the expression and phosphorylation of myristoylated alanine-rich C-kinase substrate (Marcks), a direct substrate and indicator of protein kinase C (PKC) activity in vitro and in vivo. PKC inhibition significantly reduced TB4 initiated epicardial thickening, capillary growth and the number of myocardial progenitors. Our results demonstrate that TB4 is the first known molecule capable of organ-wide activation of the embryonic coronary developmental program in the adult mammalian heart after systemic administration and that PKC plays a significant role in the process.

Role of activated CaMKII in abnormal calcium homeostasis and I(Na) remodeling after myocardial infarction: insights from mathematical modeling.

Ca(2+)/calmodulin-dependent protein kinase II is a multifunctional serine/threonine kinase with diverse cardiac roles including regulation of excitation contraction, transcription, and apoptosis. Dynamic regulation of CaMKII activity occurs in cardiac disease and is linked to specific disease phenotypes through its effects on ion channels, transporters, transcription and cell death pathways. Recent mathematical models of the cardiomyocyte have incorporated limited elements of CaMKII signaling to advance our understanding of how CaMKII regulates cardiac contractility and excitability. Given the importance of CaMKII in cardiac disease, it is imperative that computer models evolve to capture the dynamic range of CaMKII activity. In this study, using mathematical modeling combined with biochemical and imaging techniques, we test the hypothesis that CaMKII signaling in the canine infarct border zone (BZ) contributes to impaired calcium homeostasis and electrical remodeling. We report that the level of CaMKII autophosphorylation is significantly increased in the BZ region. Computer simulations using an updated mathematical model of CaMKII signaling reproduce abnormal Ca(2+) transients and action potentials characteristic of the BZ. Our simulations show that CaMKII hyperactivity contributes to abnormal Ca(2+) homeostasis and reduced action potential upstroke velocity due to effects on I(Na) gating kinetics. In conclusion, we present a new mathematical tool for studying effects of CaMKII signaling on cardiac excitability and contractility over a dynamic range of kinase activities. Our experimental and theoretical findings establish abnormal CaMKII signaling as an important component of remodeling in the canine BZ.

Biochemical markers of myocardial injury in the pericardial fluid of patients undergoing heart surgery.

The purpose of this study was to compare cardiac markers in the pericardial fluid and serum in order to evaluate preoperative myocardial injury. Thirty patients were divided into three groups. The first group (AVR; n=10) received an aortic valve replacement. The second group (SA; n=10) included patients with stable angina who underwent elective coronary artery bypass grafting (CABG). The third group (ACS; n=10) included patients with acute coronary syndrome who underwent urgent CABG. Pericardial fluid and venous samples were taken after opening the pericardium and 24 h postoperatively. Serum and pericardial concentration of troponin I (cTnI), creatine kinase (CK), its MB isoenzyme (CK-MB) and myoglobin were determined. Preoperative pericardial cTnI was significantly (P<0.01) higher than in serum in all groups. Preoperative pericardial CK, CK-MB and myoglobin were significantly (P<0.01) lower than in serum in groups AVR and SA. Preoperative pericardial and serum cTnI were significantly higher in the ACS than in AVR and SA groups (P<0.01). Postoperative pericardial concentration of all markers was significantly higher (P<0.01) than in serum in all groups. We conclude that preoperative pericardial accumulation of cTnI may reflect subclinical injury which may not be demonstrated by the usual laboratory tests.