Gene expression of antioxidative enzymes in the human heart: increased expression of catalase in the end-stage failing heart

BACKGROUND: An increase in oxidative stress is suggested to be intimately involved in the pathogenesis of heart failure. However, gene expression of enzymes that metabolize reactive oxygen metabolites has not been investigated in the human heart. METHODS AND RESULTS: Myocardial tissue homogenates of the left ventricular wall from hearts in end-stage failure due to dilated (DCM) or ischemic (ICM) cardiomyopathy (n=12 each), as well as from nonfailing donor hearts (n=12), were analyzed for mRNA levels of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), glutathione peroxidase (GPX), and catalase by Northern blot analyses. Protein levels of MnSOD, CuZnSOD, and catalase were determined by Western blot or ELISA. MnSOD, CuZnSOD, and GPX mRNA levels were similar in all 3 groups. In contrast, catalase mRNA levels were found to be increased by 123+/-23% in DCM hearts and by 93+/-10% in ICM hearts (P<0.01 each) compared with control hearts. Likewise, catalase protein levels were found to be increased in failing hearts (DCM by 90+/-10%, ICM by 90+/-13%; P<0. 05 each) compared with control hearts. In addition, the observed upregulation of catalase mRNA and protein in failing hearts was attended by an increased catalase enzyme activity (DCM by 124+/-16%, ICM by 117+/-15%; P<0.01 each), whereas MnSOD, CuZnSOD, and GPX enzyme activity levels were unchanged in failing compared with nonfailing myocardium. CONCLUSIONS: Increased oxidative stress in human end-stage heart failure may result in a specific upregulation of catalase gene expression as a compensatory mechanism, whereas SOD and GPX gene expression remain unaffected.     

Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium

Compromised SERCA 2a activity is a key malfunction leading to the Ca(2+) cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) but alterations of the CaM-kinase pathway regarding SERCA 2a in heart failure are unresolved. Therefore we investigated the CaM-kinase and phosphatase calcineurin mediated regulation of SERCA 2a in failing and non-failing human myocardium. We studied human myocardial preparations from explanted hearts from non-failing organ donors (NF, n=8) and from patients with terminal heart failure undergoing cardiac transplantation (dilated cardiomyopathy, DCM, n=8). SERCA 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by(45)Ca(2+) uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/microg protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma -(32)P] (expressed as pmol(32)P/mg). Maximal(45)Ca(2+) uptake (in pmol/mg/min) (NF: 3402+/-174; DCM: 2488+/-189) and maximal SERCA 2a activity were reduced in DCM compared to NF (V(max): NF: 125+/-9; DCM: 98+/-5). The V(max) reduction could be mimicked by calcineurin in vitro in NF (NF(control): 72.1+/-3.7; NF(+calcineurin): 49.8+/-2.9) and restored in DCM by CaM-kinase in vitro (DCM(control): 98+/-5; DCM(+CaM-kinase): 120+/-6). Protein expression of SERCA 2a, phospholamban and calsequestrin remained similar, but calcineurin expression was significantly increased in failing human hearts (NF: 11.6+/-1.5 v DCM: 17.1+/-1.6). Although the capacity of endogenous CaM-kinase to phosphorylate PT17 was significantly higher in DCM (DCM(control): 128+/-36; DCM(+endogenous CaM-kinase): 205+/-20) compared to NF myocardium (NF(control): 273+/-37; NF(+endogenous CaM-kinase): 254+/-31), net phosphorylation at threonine-17 phospholamban was significantly lower in DCM (DCM 130+/-11 v NF 170+/-11). A calcineurin-dependent dephosphorylation of phospholamban could be mimicked in vitro by incubation of NF preparations with calcineurin (NF(control) 80.7+/-4.4 v NF(+calcineurin) 30.7+/-4.1, P<0.05). In human myocardium, the V(max) of SERCA 2a and the phosphorylation of phospholamban is modulated by CaM-kinase and calcineurin, at least in vitro. In failing human myocardium, despite increased CaM-kinase activity, calcineurin dephosphorylation leads to decreased net phosphorylation of threonine-17 phospholamban in vivo. Increased calcineurin activity contributes to the impaired V(max) of SERCA 2a in failing human myocardium and the disorder in Ca(2+)-handling in heart failure.     

Increase of Gi alpha in human hearts with dilated but not ischemic cardiomyopathy

In myocardial membranes from hearts with dilated cardiomyopathy (DCM), there was a 37% increase of the Gi alpha-protein as measured by 32P-ADP-ribosylation of a approximately 40 kDa pertussis toxin substrate. Immunoblotting techniques also showed increased amounts of Gi alpha in DCM. In hearts with ischemic cardiomyopathy (ICM), Gi alpha was not altered compared with nonfailing myocardium (NF). Basal and Gpp(NH)p-stimulated adenylate cyclase activity was reduced in DCM but not in ICM. The number of beta-adrenoceptors was similarly reduced both in DCM and ICM compared with NF. Alterations of m-cholinoceptors or A1-adenosine receptors did not occur. Consistently, "indirect" negative inotropic effects of the m-cholinoceptor agonist carbachol and the A1-adenosine receptor agonist R-PIA were not different in ICM, DCM, and nonfailing myocardium. In ICM and DCM, there was a marked reduction of the positive inotropic responses to isoprenaline and milrinone. However, there was a further reduction in DCM compared with ICM. It is concluded that the increase of Gi alpha is accompanied by a reduction of basal and guanine-nucleotide-stimulated adenylate cyclase activity. Alterations of m-cholinoceptors and A1-adenosine receptors do not appear to be involved. The further decrease of the positive inotropic effects of isoprenaline and milrinone in DCM provides evidence that the increase of Gi alpha is functionally relevant in DCM but not ICM and hence might contribute to the reduced effects of endogenous catecholamines and exogenous cAMP-dependent positive inotropic agents in the former but not the latter condition.     

Endotoxin induces desensitization of cardiac endothelin-1 receptor signaling by increased expression of RGS4 and RGS16.

OBJECTIVE: Endotoxin (LPS)-induced acute cardiac failure during sepsis is associated with alterations in G protein mediated signal transduction. We therefore examined the expression of the G proteins G(i), G(q), and G(s) and of four 'regulators of G protein signaling' (RGS) proteins, RGS1, RGS4, RGS5, and RGS16 in rat hearts. METHODS: For in vivo experiments, Wistar rats were treated with 600 microg/day E. coli LPS, intravenously) and hearts were excised after 6, 24 and 72 h. Cultured neonatal rat cardiomyocytes were treated with 4 microg/ml LPS for 24 and 72 h. Isolated membrane proteins were used for Western blot analysis and for evaluation of phospholipase C (PLC) activity. RGS16 mRNA was detected by RNAse protection. RESULTS: LPS induced G(i) protein 1.4-fold 72 h after in vivo administration of LPS, whereas expression of G(s) and G(q) was unaltered. After 6 h of LPS treatment, RGS16 mRNA was transiently up-regulated 3.7-fold, followed by transient protein induction (24 h: 2.5-fold; 72 h: 1.5-fold). Similarly, RGS4 protein was transiently induced (24 h: 3.1-fold; 72 h: 1.5-fold), whereas expression of RGS1 and RGS5 was not altered. Similar to the LPS-treated rat hearts, RGS16 expression was transiently induced by LPS in cultured neonatal rat cardiomyocytes (24 h: 1.6-fold, 72 h: 0.9-fold). To determine the functional consequences of the RGS protein induction phospholipase C (PLC) activity was analyzed in membranes obtained from solvent and LPS-treated hearts. Basal and endothelin-1-stimulated PLC activity was transiently repressed by LPS with a maximum after 24 h although no apparent changes in PLCbeta1 or endothelin receptor expression could be detected. CONCLUSION: These data suggest that the rapid up-regulation of cardiac RGS4 and RGS16 is associated with a desensitization of endothelin-1 receptor signaling. Up-regulation of these RGS proteins may thus be involved in the early onset of cardiac failure during sepsis.     

Increased functional importance of the Na,Ca-exchanger in contracting failing human myocardium but unchanged activity in isolated vesicles

The present study aimed to investigate the hypothesis that the function of the Na,Ca-exchanger (NCX) is of higher importance for contractility and Ca(2+)-homeostasis in left ventricle from terminally failing than from nonfailing human hearts. The effect of decreasing extracellular [Na](e) (140 to 25 mmol/L) on force of contraction in isolated left ventricular papillary muscle strips was studied as a reflection of NCX function in multicellular preparations (terminally failing, DCM, dilated cardiomyopathy, NYHA IV, n = 13; nonfailing, NF, donor hearts, n = 10). Decreasing [Na](e) has previously been shown to increase contractility in vitro secondary to a decreased Ca(2+)-extrusion by the NCX. In addition, the NCX activity was measured as Na(+)-dependent (45)Ca(2+)-uptake into isolated myocardial vesicles as a function of time and Ca(2+)-concentration (DCM n = 8, NF n = 8). Decreasing [Na](e) enhanced the contractility of papillary muscle strips in both DCM and NF, but the contractility of DCM was increased at smaller reductions of [Na](e) than NF. The NCX activity in isolated myocardial vesicles was unchanged as a function of time (T(1/2): DCM 2.4 +/- 0.3 s versus NF 2.5 +/- 0.3 s) and as a function of Ca(2+) (DCM 0.99 +/- 0.08 versus NF 0.96 +/- 0.07 nmol/mg protein x 3 s, K(1/2): DCM 39.2 microM versus NF 38.3 microM). These results demonstrate a higher sensitivity of the failing human myocardium towards Na,Ca-exchanger mediated positive inotropic effects, suggesting a higher significance of the Na,Ca-exchanger for the extrusion of Ca(2+)-ions in intact failing versus nonfailing human myocardium. Since the activity and the Ca (2+)-affinity of the Na,Ca-exchanger in isolated vesicles was unchanged, we propose that alterations in Ca(2+)-and Na(+)-homeostasis (due to impaired function of the sarcoplasmic reticulum and the Na(+), K(+)-ATPase) or the prolonged action potential are the reason for this observation.     

Regulation of cardiomyocyte signaling by RGS proteins: differential selectivity towards G proteins and susceptibility to regulation

Many signals that regulate cardiomyocyte growth, differentiation and function are mediated via heterotrimeric G proteins, which are under the control of RGS proteins (Regulators of G protein Signaling). Several RGS proteins are expressed in the heart, but so far little is known about their function and regulation. Using adenoviral gene transfer, we conducted the first comprehensive analysis of the capacity and selectivity of the major cardiac RGS proteins (RGS2-RGS5) to regulate central G protein-mediated signaling pathways in adult ventricular myocytes (AVM). All four RGS proteins potently inhibited Gq/11-mediated phospholipase C beta stimulation and cell growth (assessed in neonatal myocytes). Importantly, RGS2 selectively inhibited Gq/11 signaling, whereas RGS3, RGS4 and RGS5 had the capacity to regulate both Gq/11 and Gi/o signaling (carbachol-induced cAMP inhibition). Gs signaling was unaffected, and, contrary to reports in other cell lines, RGS2-RGS5 did not appear to regulate adenylate cyclase directly in AVM. Since RGS proteins can be highly regulated in their expression by many different stimuli, we also tested the hypothesis that RGS expression is subject to G protein-mediated regulation in AVM and determined the specificity with which enhanced G protein signaling alters endogenous RGS expression in AVM. RGS2 mRNA and protein were markedly but transiently up-regulated by enhanced Gq/11 signaling (alpha1-adrenergic stimulation or Galphaq* overexpression), possibly by a negative feedback mechanism. In contrast, the other negative regulators of Gq/11 signaling (RGS3-RGS5) were unchanged. Endogenous RGS2 (but not RGS3-RGS5) expression was also up-regulated in cells with enhanced AC signaling (beta-adrenergic or forskolin stimulation). Taken together, these findings suggest diverse roles of RGS proteins in regulating myocyte signaling. RGS2 emerged as the only selective and highly regulated inhibitor of Gq/11 signaling that could potentially become a promising target for ameliorating Gq/11-mediated signaling and growth.     

Regulation of Connective Tissue Growth Factor Gene Expression and Fibrosis in Human Heart Failure

BackgroundHeart failure (HF) is associated with excessive extracellular matrix (ECM) deposition and abnormal ECM degradation leading to cardiac fibrosis. Connective tissue growth factor (CTGF) modulates ECM production during inflammatory tissue injury, but available data on CTGF gene expression in failing human heart and its response to mechanical unloading are limited.Methods and ResultsLeft ventricle (LV) tissue from patients undergoing cardiac transplantation for ischemic (ICM; n = 20) and dilated (DCM; n = 20) cardiomyopathies and from nonfailing (NF; n = 20) donor hearts were examined. Paired samples (n = 15) from patients undergoing LV assist device (LVAD) implantation as “bridge to transplant” (34–1,145 days) also were analyzed. There was more interstitial fibrosis in both ICM and DCM compared with NF hearts. Hydroxyproline concentration was also significantly increased in DCM compared with NF samples. The expression of CTGF, transforming growth factor (TGF) β1, collagen (COL) 1-α1, COL3-α1, matrix metalloproteinase (MMP) 2, and MMP9 mRNA in ICM and DCM were also significantly elevated compared with NF samples. Although TGF-β1, CTGF, COL1-α1, and COL3-α1 mRNA levels were reduced by unloading, there was only a modest reduction in tissue fibrosis and no difference in protein-bound hydroxyproline concentration between pre- and post-LVAD tissue samples. The persistent fibrosis may be related to a concomitant reduction in MMP9 mRNA and protein levels following unloading.ConclusionsCTGF may be a key regulator of fibrosis during maladaptive remodeling and progression to HF. Although mechanical unloading normalizes most genotypic and functional abnormalities, its effect on ECM remodeling during HF is incomplete.     

Myocardial dysfunction in donor hearts. A possible etiology.

BACKGROUND: Potential cardiac donors show various degrees of myocardial dysfunction, and the most severely affected hearts are unsuitable for transplantation. The cause of this acute heart failure is poorly understood. We investigated whether alterations in calcium-handling proteins, beta-adrenoceptor density, or the inhibitory G protein Gialpha could account for this phenomenon in unused donor hearts (n=4 to 8). We compared these with end-stage failing hearts (n=14 to 16) and nonfailing hearts (n=3 to 12). METHODS AND RESULTS: Myocardial samples were obtained from unused donor hearts displaying ejection fractions <30%. Both trabeculae and isolated myocytes responded as poorly as those from the group of failing hearts to increasing stimulation frequency with regard to inotropic function in vitro. Immunodetectable abundance of sarcoplasmic reticulum calcium-ATPase and sodium calcium exchanger were greater (177%; P<0.01) and smaller (29%; P<0.01), respectively, in the unused donor hearts relative to the failing group, which suggests that alterations of these proteins are not a common cause of contractile dysfunction in the 2 groups. Myocytes from the unused donor group were desensitized to isoprenaline to a similar degree as those from the failing heart group. However, beta-adrenoceptor density was reduced in the failing (P<0.001) but not in the unused donor heart group (P=0.37) relative to the nonfailing heart group (n=5). Gialpha activity was increased in samples from unused donor and failing hearts relative to nonfailing hearts (P<0.05). CONCLUSIONS: Increased activity of the inhibitory G protein Gialpha is a significant contributory factor for impaired contractility in these acutely failing donor hearts.     

Loss of β<Subscript>1</Subscript>D-integrin function in human ischemic cardiomyopathy

Integrins play a pivotal role in cardiomyocyte survival and function, with integrin loss leading to myocyte apoptosis and heart failure. The aim of this study was to characterize whether regulation of integrins may contribute to cardiac remodeling in human ischemic cardiomyopathy (ICM). Myocardial tissues of the left ventricle were obtained from patients with ICM (n = 8) undergoing cardiac transplantation and from unused donor hearts (NF, n = 8). In addition, tissue samples from patients with dilated cardiomyopathy (DCM, n = 5) were analyzed. Expression of integrins β₁D and β₃, the effector proteins focal adhesion kinase (FAK) and melusin, and FAK phosphorylation were examined by Western blotting, real-time-PCR and immunofluorescence analysis, respectively. β₁D-integrin protein was decreased in ICM vs. NF by 36%. β₁D-integrin mRNA levels and β₁D-integrin shedding were unchanged. Corresponding to β₁D-integrin regulation, FAK and phosphorylated FAK were decreased in ICM vs. NF by 54% and 49%, respectively. β₃-integrin and melusin were not altered in ICM. As a mediator of integrin effects, AKT kinase activity was examined. In parallel to β₁D-integrin and FAK, AKT activity was decreased in ICM by 44%. In contrast, none of the proteins were significantly altered in DCM compared to NF. Integrins and integrin signaling are regulated differentially in ICM and DCM with a decrease of β₁D-integrin and FAK in ICM. The loss of the β₁Dintegrin-FAK-complex in ICM was paralleled by a reduced AKT activity supporting in vitro data which demonstrate the pivotal role of intact integrin function in anti-apoptotic signaling and cell survival.     

Circulation of CD34+ progenitor cell populations in patients with idiopathic dilated and ischaemic cardiomyopathy (DCM and ICM).

AIMS: This study aimed at analysing the endogenous stem cell circulation in patients suffering from idiopathic dilated cardiomyopathy (DCM) and ischaemic cardiomyopathy (ICM). METHODS AND RESULTS: Cytokines in peripheral blood were analysed using enzyme-linked immunosorbent assay and circulating CD34(+) stem cell populations (CD34(+)CD133(+), CD34(+)CD31(+), CD34(+)CXCR-4(+)) were measured by flow cytometry in DCM patients (n = 25), ICM patients (n = 15), and controls (n = 10). Explanted DCM (n = 5), ICM (n = 4) and normal hearts (n = 5) were analysed for the expression of several homing factors [stromal cell-derived factor-1 (SDF-1), Stem cell factor (SCF), HIF-1a, vascular cell adhesion molecule (VCAM), and Hepatocyte growth factor] by quantitative real-time polymerase chain reaction (PCR). SDF-1 was significantly elevated and positively correlated with brain natriuretic peptide (BNP) in peripheral blood of DCM and ICM patients showing the same New York heart association- (NYHA) class. In DCM patients circulating CD34(+) cell populations were significantly increased in comparison to ICM patients and controls. mRNA of SDF-1, SCF, HIF-1a, and VCAM related to glyceraldehyde-3-phosphate dehydrogenase was significantly upregulated in ICM hearts when compared with DCM hearts and controls. CONCLUSION: Myocardial homing factors are upregulated in ICM when compared with DCM hearts. Reduced homing of stem cells might therefore explain the increased number of CD34(+) cells in DCM patients. These findings may open a new insight into the pathology and the treatment of idiopathic DCM.     

Diminished posttetanic potentiation in failing human myocardium

In heart failure a decreased function of SERCA2 has been demonstrated. The present study aimed at investigating the relation between sarcoplasmic reticulum-Ca²⁺-load (SR-Ca²⁺-load) and the activity of the SERCA2. SR-Ca²⁺ load was evaluated by measuring posttetanic potentiation (PTP) in human nonfailing (NF, n=10) and endstage failing myocardium (DCM, n=11). In addition, the effect of cyclopiazonic acid (CPA), a specific inhibitor of SERCA2, on PTP was studied in both NF and DCM. In crude membrane preparations from the same hearts the maximal SERCA2 activity was determined and correlated with the PTP. In failing myocardium the PTP was significantly reduced compared to nonfailing myocardium (13.7±0.75 mN/mm² vs. 17.1±1.55 mN/mm², p<0.05, ±SEM). When PTP was studied in the presence of increased extracellular Ca²⁺-concentrations, the difference between NF and DCM was further pronounced. CPA decreased PTP in both nonfailing and failing human tissue. The maximal SERCA2 activity was significantly reduced in failing myocardium (NF 267±18.5 nmol ATP/mg protein · min⁻¹ vs. DCM 191±13.4 nmol ATP/mg protein · min⁻¹, p<0.05, ± SEM). Correlation of the PTP and maximal SERCA2 activity revealed a close correlation between both parameters in NF and DCM. In summary, the presented results suggest that reduced SERCA2 activity in DCM influences posttetanic force potentiation probably through a reduced SR-Ca²⁺-load. Received: 30 July 1999 Returned for 1. revision: 9 September 1999 1. Revision received: 24 November 1999 Returned for 2. revision: 26 January 2000 2. Revision received: 26 April 2000 Accepted: 9 May 2000     

Selective upregulation of endothelin converting enzyme-1a in the human failing heart

BACKGROUND: Increased plasma levels of endothelin-1 (ET-1) occur with congestive heart failure (CHF), but the components of the enzymatic activation of ET-1 in the myocardium remain to be defined. Accordingly, endothelin converting enzyme-1 (ECE-1) activity and expression in normal and failing heart were examined. METHODS AND RESULTS: Left ventricular (LV) tissue samples were obtained from patients undergoing heart transplantation because of dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM) and from normal donor hearts. The gene expression of ET-1 precursor and ECE-1a was upregulated 4- and 3-fold, respectively, in the failing heart. ECE-1 activity (fmol/mg protein per hour) was augmented from 2,291+/-257 in normal tissue samples to 5,507+/-666 in DCM samples and to 7,435+/-682 in ICM samples (P < .05). Phosphoramidon and a specific ECE-1 inhibitor, FR901533, inhibited ECE-1 activity by over 90%. However, inhibitors of neutral endopeptidase (thiorphan) and matrix metalloproteases (batimistat) did not affect the conversion of big ET-1 to ET-1. CONCLUSIONS: This study showed that the biosynthetic pathway of ET-1 is activated in LV myocardium in the failing heart, and the myocardial processing of big ET-1 is highly specific for ECE-1.     

Microarray-based screening of common pathophysiological processes in dilated cardiomyopathy and ischemic cardiomyopathy

Background Heart failure is the end stage of various heart diseases,which has different underlying mechanisms but somehow has similar features among different etiologies. Dilated cardiomyopathy(DCM) and ischemic cardiomyopathy(ICM)are two major subtypes. Unlike widely explored mechanisms on these heart diseases,their shared gene expression alterations in heart failure have been rarely investigated. Methods We first obtained genetic profiles of DCM and ICM as well as their non-failing(NF)control from two datasets in GEO database,GSE3585 and GSE1869. Comparing to NF,the differentially expressed genes(DEGs)of DCM and ICM were screened out and used for Gene Ontology(GO)analysis by DAVID. The two sets of DEGs were compared and the overlap was selected out. Protein-protein interaction(PPI)network of the encoded proteins of shared DEGs was constructed and visualized via STRING and Cytoscape respectively. Gene annotation interactions were analyzed using Bi NGO. The closely linked genes were sorted out as hub genes and accounted for common processes of DCM and ICM. Results There were 99 DEGs in DCM vs. NF,while 1317 DEGs in ICM vs. NF.29 DEGs were shared in two comparisons,which were mainly enriched in regulation of protein catabolic process,Golgi to endosome transport,protein targeting to Golgi,inactivation of MAPK activity,Golgi to plasma membrane protein transport,cytosol,and protein binding. There were 13 genes linked in the PPI network and thus they were selected as hub genes. Functions of these hub genes were mainly focused on energetic metabolism disorder,apoptosis and cardiac fibrosis. Conclusion Energetic metabolism disorder,apoptosis and cardiac fibrosis are likely the shared pathophysiological processes for dilated cardiomyopathy and ischemic cardiomyopathy.[S Chin J Cardiol 2019;20(3):190-197]     

Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1

AIMS: Remodelling of gap junctions, involving reduction of total gap junction quantity and down-regulation of connexin43 (Cx43), contributes to the arrhythmic substrate in congestive heart failure. However, little is known of the underlying mechanisms. Recent studies from in vitro systems suggest that the connexin-interacting protein zonula occludens-1 (ZO-1) is a potential mediator of gap junction remodelling. We therefore examined the hypothesis that ZO-1 contributes to reduced expression of Cx43 gap junctions in congestive heart failure. METHODS AND RESULTS: Left ventricular myocardium from healthy control human hearts (n = 5) was compared with that of explanted hearts from transplant patients with end-stage congestive heart failure due to idiopathic dilated cardiomyopathy (DCM; n = 5) or ischaemic cardiomyopathy (ICM; n = 5). Immunoconfocal and immunoelectron microscopy showed that ZO-1 is specifically localized to the intercalated disc of cardiomyocytes in control and failing ventricles. ZO-1 protein levels were significantly increased in both DCM and ICM (P = 0.0025), showing a significant, negative correlation to Cx43 levels (P = 0.0029). There was, however, no significant alteration of ZO-1 mRNA (P = 0.537). Double immunolabelling demonstrated that a proportion of ZO-1 label is co-localized with Cx43, and that co-localization of Cx43 with ZO-1 is significantly increased in the failing ventricle (P = 0.003). Interaction between the two proteins was confirmed by co-immunoprecipitation. The proportion of Cx43 that co-immunoprecipitates with ZO-1 was significantly increased in the failing heart. CONCLUSION: Our findings suggest that ZO-1, by interacting with Cx43, plays a role in the down-regulation and decreased size of Cx43 gap junctions in congestive heart failure.     

Immunohistochemical study of remodeling of myocardial lymphatic and blood microvascular structures in terminal heart failure: differences between ischemic and dilated cardiomyopathy

This study investigated (cardiac) remodeling of the myocardial microvasculature in patients with terminal heart failure due to ischemic (ICM) and dilative (DCM) cardiomyopathy. Seventeen transmural left-ventricular (LV) biopsies (9 ICM and 8 DCM), taken from heart transplant recipients at transplantation (n=4) or during ventricular assist device implantation (n=13) were investigated by immunohistostaining for VEGFR-1 and VEGFR-2 as capillary markers and VEGFR-3, D2-40, PROX-1 and LYVE-1 as lymphatic markers. Results were compared to LV biopsies from 7 donor hearts (control). Compared to control, DCM hearts showed a significantly higher density of LYVE-1 positive lymphatics (p < 0.05), whereas no difference was seen for other markers. ICM hearts showed a significantly higher density of D2-40 positive lymphatics (p < 0.01) and a lower density of VEGFR-2 capillaries compared to control (p < 0.05). In comparison to normal donor hearts, ICM and DCM hearts showed a significantly different pattern of microvascular receptor expression. As distinct patterns were seen in ICM and DCM, the effect of microvascular remodeling may be substantially different between two clinically important causes of cardiomyopathy. Further research should be aimed at defining the impact of extracellular matrix composition and VEGF-related angiogenesis on the myocardial microvasculature at various stages of heart failure.