Ultrastructural evidence of intercalated disc remodelling in arrhythmogenic right ventricular cardiomyopathy: an electron microscopy investigation on endomyocardial biopsies.

AIMS: The ultrastructural features of the myocardium in arrhythmogenic right ventricular cardiomyopathy (ARVC) have not been systematically investigated so far. The recent discovery of gene mutations encoding intercalated disc proteins prompted us to perform a transmission electron microscopy study on endomyocardial biopsies. METHODS AND RESULTS: Twenty-one ARVC probands who fulfilled the international Task Force diagnostic criteria underwent right ventricular endomyocardial biopsy and screening of desmosome (D) protein encoding genes. Myocyte intercalated discs were analysed by transmission electron microscope and the data were compared with those of 10 controls and 10 patients with idiopathic dilated cardiomyopathy. Extensive fibro-fatty replacement with a residual myocardium of 59+/-23% was found in ARVC biopsy samples. Pathogenic D gene mutations were identified in 10 (48%): desmoglein-2 in four, desmoplakin in three and plakophilin-2 in three. Mean D length and D percent length of intercalated disc were significantly higher, D number was significantly lower and D gap was widened in ARVC. Moreover, abnormally located D in 75%, abnormal small junctions in 52%, and pale internal plaques in 32% of ARVC patients were found in the presence of a normal intercalated disc convolution index. CONCLUSION: The ultrastructural evidence of intercalated discs remodelling in ARVC, together with the positive screening of D protein encoding genes in half of probands, are in keeping with an intercellular junction cardiomyopathy.     

Myocardial gene expression of angiogenic factors in human chronic ischemic myocardium: influence of acute ischemia/cardioplegia and reperfusion

OBJECTIVE: Angiogenic therapies in animals have demonstrated the development of new blood vessels within ischemic myocardium. However, results from clinical protein and gene angiogenic trials have been less impressive. The present study aimed to investigate the expression of angiogenic genes in human chronic ischemic myocardium and the influence of acute ischemia/cardioplegia and reperfusion on their expression. METHODS: Myocardial biopsies were taken from chronic ischemic and nonischemic myocardium in 15 patients with stable angina pectoris during coronary bypass surgery. Tissue samples were evaluated by oligonucleotide microarray and quantitative real-time PCR for the expression of angiogenic factors. RESULTS: There was identical baseline expression of VEGF-A and VEGF-C mRNA in chronic ischemic myocardium compared with nonischemic myocardium. Reperfusion increased the gene expression of VEGF-A and VEGF-C mRNA both in nonischemic and ischemic myocardium. VEGF-A protein was detected mainly in the extracellular matrix around the cardiomyocytes in ischemic myocardium. CONCLUSION: These data suggest that the nonconclusive VEGF gene therapy trials chronic coronary artery disease was not due to a preexisting upregulation of VEGF in chronic ischemic myocardium. There might be room for further therapeutic angiogenesis in chronic ischemic myocardium.     

P-ERK、P-p38及iNOS、GLUT4在冠心病患者冬眠心肌中表达及意义

目的探讨冬眠心肌（HM）细胞内磷酸化ERK（P-ERK）、磷酸化p38（P-p38）、葡萄糖转运因子4（GLUT4）、诱导型一氧化氮合酶（iNOS）的变化和意义,探讨P-ERK、P-p38与GLUT4、iNOS的关系。方法选择行冠脉搭桥手术的冠心病患者10例,术前1周用多巴酚丁胺超声负荷试验结合多普勒组织成像确定HM及正常心肌（NM）的存在部位,术中根据检测结果取材,用免疫印迹法检测P-ERK、P-p38、iNOS、GLUT4的表达情况,分析HM与NM的P-ERK、P-p38、iNOS、GLUT4含量;分析四者之间的相关性。结果HM细胞内P-ERK、P-p38、GLUT4、iNOS水平较正常心肌高;P-ERK与GLUT4呈正相关（r=0.665,P〈0.05）,P-p38与GLUT4、iNOS呈正相关（r=0.708、0.676,P〈0.05）。结论心肌缺血缺氧可触发ERK、p38活化,活化的ERK、p38促使心肌细胞增加GLUT4及iNOS表达,促进HM形成。     

A recessive mutation in desmoplakin causes arrhythmogenic right ventricular dysplasia,skin disorder,and woolly hair

OBJECTIVES: The goal of this study was to analyze the genetic disorder of a family with cardiomyopathy, skin disorder, and woolly hair. BACKGROUND: Arrhythmogenic right ventricular dysplasia (ARVD) is a heart muscle disorder causing arrhythmia and sudden cardiac death. We report a patient with familial autosomal recessive ARVD, woolly hair, and a pemphigous-like skin disorder with a new mutation in the desmoplakin gene. METHODS: Genomic deoxyribonucleic acid was extracted from the patient's blood and 12 first- and second-degree family members, and was amplified by polymerase chain reaction. Linkage analysis with polymorphic microsatellites was performed for 11 genes that code for structural desmosomal proteins. The genetic locus of the disease in this family was mapped to the chromosomal region 6p24 that contains the desmoplakin gene. Exons of the desmoplakin gene were analyzed by single-strand conformational polymorphism and direct sequencing. Confirmation of the mutation was carried out by restriction enzyme analysis. RESULTS: We identified in the patient a homozygous missense mutation in exon 24 of the desmoplakin gene, leading to a Gly2375Arg substitution in the C-terminal of the protein where the binding site to intermediate filaments is located. Eight of 12 family members without hair or skin abnormalities were heterozygous for this mutation. The remaining 4, as well as 90 unrelated healthy control individuals of the same ethnic origin, were homozygous for the normal allele. CONCLUSIONS: We have described a new mutation in the desmoplakin gene that causes familial ARVD. These findings suggest that desmosomal proteins play an important role in the integrity and function of the myocardium. Dysfunction of these proteins can lead to the development of cardiomyopathies and arrhythmias.     

Gene expression profiling of human hibernating myocardium: increased expression of B-type natriuretic peptide and proenkephalin in hypocontractile vs normally-contracting regions of the heart

A greater understanding of the molecular basis of hibernating myocardium may assist in identifying those patients who would most benefit from revascularization. Paired heart biopsies were taken from hypocontractile and normally-contracting myocardium (identified by cardiovascular magnetic resonance) from 6 patients with chronic stable angina scheduled for bypass grafting. Gene expression profiles of hypocontractile and normally-contracting samples were compared using Affymetrix microarrays. The data for patients with confirmed hibernating myocardium were analysed separately and a different, though overlapping, set (up to 380) of genes was identified which may constitute a molecular fingerprint for hibernating myocardium. The expression of B-type natriuretic peptide (BNP) was increased in hypocontractile relative to normally-contracting myocardium. The expression of BNP correlated most closely with the expression of proenkephalin and follistatin 3, which may constitute additional heart failure markers. Our data illustrate differential gene expression in hypocontractile and/hibernating myocardium relative to normally-contracting myocardium within individual human hearts. Changes in expression of these genes, including increased relative expression of natriuretic and other factors, may constitute a molecular signature for hypocontractile and/or hibernating myocardium.     

Increased myocardial expression of angiopoietin-2 in patients undergoing urgent surgical revascularization for acute coronary syndromes

BACKGROUND:

Myocardial ischemia triggers the expression of multiple angiogenic factors including vascular endothelial growth factor and its receptors. However, vascular endothelial growth factor does not act in isolation.

OBJECTIVE:

To identify other genes important in the angiogenic response to clinically relevant myocardial ischemia.

METHODS AND RESULTS:

Paired intraoperative biopsies of ischemic and nonischemic myocardium were obtained from 12 patients with acute coronary syndromes (ACS) undergoing urgent coronary artery bypass graft surgery. Real-time polymerase chain reaction demonstrated significant upregulation of angiopoietin-2 (Ang-2) in ischemic myocardium, to a greater extent than other classical angiogenic factors. Microarray gene profiling identified Ang-2 to be among the top 10 differentially upregulated genes, in addition to genes involved in inflammation, cell signalling, remodelling and apoptosis.

CONCLUSIONS:

The present document is the first report of microarray analysis of patients with ACS, and supports an important role for Ang-2 in the angiogenic response to severe ischemia in the human heart. Common gene expression patterns in ACS may provide opportunities for targeted pharmacological and cellular intervention.     

Lipid accumulation in the myocardium during acute regional ischaemia in cats

Accumulation of lipid material in the myocardium was studied in cat hearts with acute regional ischaemia of 3 h duration. The fractional volume of lipid droplets in cytosol was analysed by electron microscopy of myocardial biopsies using a quantitative stereologic technique. Ischaemic and normally perfused myocardium were identified by fluoresceine injection, and tissue blood flow measurements were performed with labelled microspheres. In normal myocardium only small amounts of lipid droplets were found. A marked accumulation of lipid droplets occurred in borderline tissue between the two types of myocardium, whereas lipid accumulation in ischaemic myocardium was less pronounced. The arterial concentration of nonesterified fatty acids was clearly increased during the 3 h coronary artery occlusion period. Increased triglyceride synthesis from arterial fatty acids, or redistribution of intracellular lipids, are suggested as possible explanations for lipid accumulation during acute myocardial ischaemia.     

c-Jun N-terminal kinase activation mediates downregulation of connexin43 in cardiomyocytes

Loss of gap junctions and impaired intercellular communication are characteristic features of pathological remodeling in heart failure as a result of stress or injury, yet the underlying regulatory mechanism has not been identified. Here, we report that in cultured myocytes, rapid loss of the gap junction protein connexin43 (Cx43) occurs in conjunction with the activation of c-Jun N-terminal kinase (JNK), a stress-activated protein kinase, on stress stimulation. To investigate the specific role of JNK activation in the regulation of connexin in cardiomyocytes, an activated mutant of mitogen-activated protein kinase kinase 7 (mutant D), a JNK-specific upstream activator, was expressed in myocytes by adenovirus-mediated gene transfer. JNK activation in infected cardiomyocytes resulted in significant reduction of Cx43 expression at both mRNA and protein levels and impaired cell-cell communication. To evaluate the role of JNK in the regulation of Cx43 expression and gap junction structure in vivo, a Cre-LoxP-mediated gene-switch system was used to establish a transgenic animal model with targeted activation of JNK in ventricular myocardium. The transgenic hearts exhibited significant downregulation of Cx43 expression and loss of gap junctions in myocardium that may contribute to the cardiac dysfunction and premature death phenotype. Our report represents the first evidence, both in vitro and in vivo, implicating JNK as an important mediator of stress-induced Cx43 downregulation and impaired intercellular communication in the failing heart.     

Endothelial dysfunction of peripheral arteries in patients with immunohistologically confirmed myocardial inflammation correlates with endothelial expression of human leukocyte antigens and adhesion molecules in myocardial biopsies

OBJECTIVES: The aim of this study was to investigate whether myocardial inflammation (MC) and endothelial activation are associated with clinically detectable endothelial dysfunction. BACKGROUND: In patients with MC, immunohistologic evaluation of myocardial biopsies demonstrates a cellular infiltrate of lymphocytes in the myocardium and endothelial activation, as indicated by enhanced expression of human leukocyte antigen (HLA)-1, HLA-DR and intercellular adhesion molecule (ICAM)-1. This chronic inflammatory process may be associated with endothelial dysfunction. METHODS: In 65 patients with suspected MC, endothelial function of the radial artery was noninvasively assessed. By means of high-resolution ultrasound, diameter changes in response to reactive hyperemia (endothelium-dependent), as compared with glyceroltrinitrate (endothelium-independent), were analyzed. In the myocardial biopsies, MC was confirmed by immunohistology in 53 patients; 12 patients with normal myocardial biopsies served as controls. Endothelial expression of HLA-1, HLA-DR and ICAM-1 was semiquantitatively evaluated by immunohistology. To minimize other factors influencing endothelial function, patients with coronary artery disease, diabetes, severely impaired left ventricular function or more than one arteriosclerotic risk factor were excluded from this study. RESULTS: Endothelial function, as determined by flow-mediated vasodilation (FMD), in patients with MC was impaired (FMD(MC) 4.28%), as compared with controls (FMD(Co) 10.10%). The severity of endothelial dysfunction in patients with MC correlated significantly with the extent of endothelial expression of HLA-1, HLA-DR and ICAM-1 in myocardial biopsies. Endothelium-independent vasodilation was not affected by MC or endothelial activation. CONCLUSIONS: Myocardial inflammation is associated with endothelial dysfunction of peripheral arteries. The severity of endothelial dysfunction correlates with the extent of endothelial activation.     

Systemic and cell type-specific gene expression patterns in scleroderma skin

We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20+ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease.     

Extension of borderzone myocardium in postinfarction dilated cardiomyopathy

This study tests the hypothesis that hypocontractile, borderzone myocardium adjacent to an expanding infarct becomes progressively larger and more hypocontractile as remodeling continues.Early infarct expansion following anteroapical myocardial infarction (MI) is associated with progressive ventricular dilation and heart failure. The contribution of perfused, hypocontractile, borderzone myocardium to this process is unknown.Using a sheep model of anteroapical infarction, sonomicrometry array localization and serial microsphere injections were used to track changes in regional myocardial contractility, geometry, and perfusion. Eight sheep were studied before and after infarction and two, five, and eight weeks later. Thirty intertransducer chord lengths were analyzed to measure regional contractility and serial changes in regional geometry at end systole.Beginning as a narrow band of fully perfused hypocontractile myocardium adjacent to the infarction, borderzone myocardium extends to involve additional contiguous myocardium that progressively loses contractile function as the heart remodels. Three distinct myocardial zones develop as a result of transmural MI: infarct, borderzone (perfused but hypocontractile), and remote (perfused and normally functioning).This study demonstrates that hypocontractile, fully perfused borderzone myocardium extends to involve contiguous normal myocardium during postinfarction remodeling. This borderzone myocardium is a unique type of perfused, hypocontractile myocardium, which is distinct from hibernating or stunned myocardium. Preventing extension of borderzone myocardium by medical or surgical means offers the prospect of preventing late-onset heart failure following transmural expanding MIs.     

Regional myocardial blood flow during cardiac tamponade in hearts with chronic coronary artery occlusion

The effects of increased pericardial pressure on blood flow to collateral dependent and normal myocardium were investigated and the mechanisms responsible for these effects evaluated in 10 anaesthetised dogs after collateral inducement by gradual occlusion of a coronary artery. Regional myocardial blood flows were measured with radioactive microspheres during control conditions, mild tamponade, severe tamponade, and severe tamponade with aortic blood pressure held at the control value by blood volume expansion. Mild tamponade increased heart rate by 10% and decreased aortic blood pressure by 15%. Left atrial and central venous blood pressures were moderately increased, and indices of cardiac function were reduced. Blood flow to collateral dependent and normally perfused myocardium was not significantly altered, but the endocardial to epicardial flow ratio was significantly decreased in collateral dependent myocardium. Severe tamponade decreased aortic blood pressure by 45% and cardiac index by 62%. Left atrial and central venous blood pressures were appreciably increased and cardiac function indices considerably depressed. Blood flow to collateral dependent and normally perfused myocardium was decreased similarly (by 54-57%), but the endocardial to epicardial flow ratio was decreased by a greater degree in collateral dependent myocardium. During severe tamponade at control aortic blood pressure, left atrial and central venous blood pressures were further increased, but blood flow to collateral dependent and normally perfused myocardium returned to within 84% of control and endocardial to epicardial flow ratios were normal. Total peripheral vascular resistance increased during severe tamponade, but coronary vascular resistance remained constant. Thus blood flow to collateral dependent and normally perfused myocardium varied according to net coronary perfusion pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.     

Pulsatile stretch remodels cell-to-cell communication in cultured myocytes

Mechanical stretch is thought to play an important role in remodeling atrial and ventricular myocardium and may produce substrates that promote arrhythmogenesis. In the present work, neonatal rat ventricular myocytes were cultured for 4 days as confluent monolayers on thin silicone membranes and then subjected to linear pulsatile stretch for up to 6 hours. Action potential upstrokes and propagation velocity (theta) were measured with multisite optical recording of transmembrane voltage of the cells stained with the voltage-sensitive dye RH237. Expression of the gap junction protein connexin43 (Cx43) and the fascia adherens junction protein N-cadherin was measured immunohistochemically in the same preparations. Pulsatile stretch caused dramatic upregulation of intercellular junction proteins after only 1 hour and a further increase after 6 hours (Cx43 signal increased from 0.73 to 1.86 and 2.02% cell area, and N-cadherin signal increased from 1.21 to 2.11 and 2.74% cell area after 1 and 6 hours, respectively). This was paralleled by an increase in theta from 27 to 35 cm/s after 1 hour and 37 cm/s after 6 hours. No significant change in the upstroke velocity of the action potential or cell size was observed. Increased theta and protein expression were not reversible after 24 hours of relaxation. Nonpulsatile (static) stretch produced qualitatively similar but significantly smaller changes than pulsatile stretch. Thus, pulsatile linear stretch in vitro causes marked upregulation of proteins that form electrical and mechanical junctions, as well as a concomitant increase in propagation velocity. These changes may contribute to arrhythmogenesis in myocardium exposed to acute stretch.