Wolfgang Poller

The Cardiac Mechanical Stretch Sensor Machinery Involves a Z Disc Complex that Is Defective in a Subset of Human Dilated Cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.

Interferon-β Treatment Eliminates Cardiotropic Viruses and Improves Left Ventricular Function in Patients With Myocardial Persistence of Viral Genomes and Left Ventricular Dysfunction

Background—Viral infections are important causes of myocarditis and may induce cardiac dysfunction and finally lead to dilated cardiomyopathy. We investigated whether interferon (IFN)-&bgr; therapy is safe and may achieve virus clearance and prevent deterioration of left ventricular (LV) function in patients with myocardial virus persistence. Methods and Results—In this phase II study, 22 consecutive patients with persistence of LV dysfunction (history of symptoms, 44±27 months) and polymerase chain reaction–proven enteroviral or adenoviral genomes were treated with 18×106 IU/week IFN-&bgr; (Beneferon) subcutaneously for 24 weeks. Histological and immunohistological analysis of endomyocardial biopsies was used to characterize myocardial inflammation. LV diameters and ejection fraction were assessed by echocardiography and angiography, respectively. During the treatment period, IFN-&bgr; was well tolerated by all patients. No patient deteriorated. Clearance of viral genomes was observed in 22 of 22 of patients after antiviral therapy. Virus clearance was paralleled by a significant decrease of LV end diastolic and end systolic diameters, decreasing from 59.7±11.1 to 56.5±10.0 mm (P <0.001) and 43.2±13.6 to 39.4±12.1 mm (P <0.001), respectively. LV ejection fraction increased from 44.6±15.5% to 53.1±16.8% (P <0.001). Conclusions—A 6 months, IFN-&bgr; treatment was safe in patients with myocardial enteroviral or adenoviral persistence and LV dysfunction and resulted in elimination of viral genomes (22 of 22 patients) and improved LV function (15 of 22 patients).

Dilated Cardiomyopathy Is Associated With Significant Changes in Collagen Type I/III ratio

BACKGROUNDnIt is controversial whether myocardial fibrosis in end-stage dilated cardiomyopathy (DCM) is associated with altered collagen type I/type III (Col I/Col III) ratio.nnnMETHODS AND RESULTSnPatients with DCM (ejection fraction [EF] <50%, n=12) and with mild global left ventricular dysfunction (EF >50%, n=18) were examined. Col I, Col III, and transforming growth factors-beta1 (TGF-beta1) and -beta2 (TGF-beta2) gene expression in endomyocardial biopsies was evaluated by quantitative competitive reverse transcriptase-polymerase chain reaction (qRT-PCR). Collagen content was quantified after picrosirius red and immunohistological staining and by hydroxyproline assay. In patients with EF <50%, there was a pronounced 2- to 6-fold increase of myocardial Col I mRNA abundance (P<0.01), with a corresponding 1.6-fold increase at the protein level versus that found in patients with EF >50%. The Col III mRNA abundance showed a 2.0-fold increase (P<0.04). There was a relevant shift in the Col I/Col III mRNA ratio for DCM patients (Col I/Col III, 8.2) compared with patients with an EF >50% (Col I/Col III, 6. 4). In addition, total collagen content was increased in patients with EF <50% (n=3) (4.3+/-0.1%) compared with patients with EF >50% (n=8) (2.7+/-0.9%) (P<0.004). The biochemically determined ratio of hydroxyproline/total protein (n=12) was correlated to the Col I mRNA abundance (P<0.05, r=0.77). TGF-beta1 and TGF-beta2 showed elevated myocardial mRNA abundances (1- to 7-fold and 4- to 5-fold, respectively) in DCM patients.nnnCONCLUSIONSnDifferential increase of Col I and Col III leads to an increased Col I/Col III ratio in DCM myocardium. Because Col I provides substantial tensile strength and stiffness, this may contribute to systolic and in particular diastolic dysfunction in DCM.

Regulation of Human Endothelial Cell Focal Adhesion Sites and Migration by cGMP-dependent Protein Kinase I

cGMP-dependent protein kinase type I (cGK I), a major constituent of the atrial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathway, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins involved in regulation of the actin cytoskeleton. Here we demonstrate that stimulation of human umbilical vein endothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3′:5′-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes detachment of VASP and its known binding partner (zyxin) from focal adhesions in >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effects, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK Iβ mutant (cGK Iβ-K405A) was incapable of mediating these effects. VASP mutated (Ser/Thr to Ala) at all three of its established phosphorylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not phosphorylated by cGK I and was resistant to detaching from HUVEC focal adhesions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK Iβ-K405A, caused a 1.5–2-fold inhibition of HUVEC migration, a dynamic process highly dependent on focal adhesion formation and disassembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could contribute to cGK alteration of cytoskeleton-regulated processes such as cell migration.

Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction

OBJECTIVEnMyocardial collagen degradation is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs). The possible relevance of MMPs in association with the inflammatory induction was investigated in a murine coxsackievirus B3 myocarditis model.nnnMETHODSnHearts from viral infected and sham-infected BALB/c mice were analyzed by semi-quantitative RT-PCR, picrosirius red staining, Western blot analysis, and immunohistochemistry.nnnRESULTSnIn viral infected mice, both mRNA and protein abundance for collagen type I remained unaltered. In addition, picrosirius red staining showed the unchanged total collagen content. However, degraded soluble fraction of collagen type I protein was increased. Moreover, the mRNA abundance for MMP-3 and MMP-9 was upregulated, whereas the mRNAs for TIMP-1 and TIMP-4 were downregulated, respectively. The upregulation of MMP-3/MMP-9 and downregulation of TIMP-4 were confirmed at the protein level, and were associated with significantly increased mRNA levels of interleukin 1beta, tumor necrosis factor-alpha, transforming growth factor-beta1 and interleukin-4.nnnCONCLUSIONnThe increment of MMPs in the absence of counterbalance by TIMPs may lead to a functional defect of the myocardial collagen network by posttranslational mechanisms. This may contribute significantly to the development of left ventricular dysfunction in murine viral myocarditis. The inflammatory response with induction of cytokines may mediate the dysregulation of the myocardial MMP/TIMP systems.

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl− secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels was analyzed after expression of cGK II or cGK Iβ in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I− efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Iβ or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Iβ enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e.g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Iβ nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells

To develop new therapeutic options for the treatment of disc degeneration we tested the possibility of overexpression of active growth and differentiation factor (GDF) 5 and of transforming growth factor (TGF) β1 by adenoviral gene transfer and characterized its effect on cell proliferation and matrix synthesis of cultured rabbit and human intervertebral disc cells. Recombinant adenovirus encoding for GDF-5 or TGF-β1 was developed and transgene expression characterized by RT-PCR, western blot and ELISA. Growth and matrix synthesis of transduced cells was measured by [3H]thymidine or [35S]sulfate incorporation. Disc cells expressed the receptors BMPR1A, BMPR1B, and BMPR2, which are relevant for GDF-5 action. Adenovirus efficiently transferred the GDF-5 gene or the TGF-β1 gene to rabbit and human intervertebral disc cells. About 50xa0ng GDF-5 protein/106 cells per 24xa0h or 7xa0ng TGF-β1 protein/106 cells per 24xa0h was produced. According to western blotting, two GDF-5 forms, with molecular weights consistent with the activated GDF-5 dimer and the proform, were secreted over the 3xa0weeks following gene transfer. Overexpressed GDF-5 and TGF-β1 were bioactive and promoted growth of rabbit disc cells in monolayer culture. Our results suggest that ex vivo gene delivery of GDF-5 and TGF-β1 is an attractive approach for the release of mature and pre-GDF-5 in surrounding tissue. This leads us to hope that it will prove possible to improve the treatment of degenerative disc disease by means of ex vivo gene transfer of single or multiple growth factors.

Highly variable expression of virus receptors in the human cardiovascular system

Coxsackieviren und Adenoviren sind weitverbreitete Erreger viraler Herzmuskelerkrankungen. Bei der überwiegenden Zahl der Exponierten verursachen sie jedoch keine myokardiale Erkrankung, da sie nicht a priori kardiotrop sind. Die molekularen Grundlagen ihres ungewöhnlichen Tropismus bei Patienten, die eine virale Herzmuskelerkrankung entwickeln, waren bis vor kurzem unbekannt. Ein bedeutender Fortschritt wurde hier erzielt durch die Klonierung eines Rezeptors, der die beiden strukturell unverwandten Viren bindet. Dieser Coxsackievirus-Adenovirus-Rezeptor (CAR) ist eine wesentliche Determinante für die zelluläre Aufnahme beider Viren und für die molekulare Pathogenese von Coxsackievirus- und Adenovirus-Infektionen. Bei der Kartierung der CAR-Expression in menschlichen Herzen fanden wir hochvariable Expressionsmuster. In gesunden Donor-Herzen war die CAR-Expression niedrig, während explantierte Herzen von Patienten mit Dilatativer Cardiomyopathie (DCM) eine hohe CAR-Expression im Myokard aufwiesen. Bemerkenswerterweise war jedoch nicht die Herzinsuffizienz perse mit hoher CAR-Expression assoziiert, da bei non-DCM Herzinsuffizienz keine Induktion gefunden wurde. Zusätzliche Untersuchungen über die molekularen Mechanismen der CAR-Induktion in Kardiomyozyten wiesen auf die Existenz eines Zell-Zell-Kontakt-abhängigen molekularen Regulationsmechanismus für CAR hin, während die zelluläre Aufnahme und eine low level Replikation von Virus keinen Effekt auf die Rezeptor-Expression hatten. Rekombinante Expression von humanem CAR in Kardiomyozyten-Kulturen führte zu einer starken Zunahme der Virus-Aufnahme in diese Zellen. Rezeptor-Induktion in vivo sollte daher die myokardiale Vulnerabilität für Viren wesentlich erhöhen, während gesundes Myokard weitgehend resistent wäre. Sie könnte auch den klinischen Krankheitsverlauf aggravieren, sodass die Blockade der Rezeptor-Expression oder von Rezeptor-Virus Interaktionen neue therapeutische Perspektiven eröffnet. Die Aufklärung des molekularen Mechanismus der CAR-Induktion bei DCM, jedoch nicht bei Herzinsuffizienz perse, könnte möglicherweise zur Identifikation eines spezifischen pathogenetischen Prozesses bei DCM führen. Eine umfassendere Analyse der kardiovaskulären Expression von Rezeptoren auch für andere potentiell kardiotrope Viren (CMV, EBV, HIV, HHV-6, Parvo-B19 etc.) sollte zu einem besseren Verständnis individueller Risikofaktoren für virale Herzmuskelerkrankungen und deren hochvariabler klinischer Verläufe führen, und neue therapeutische Optionen eröffnen. Coxsackieviruses and adenoviruses are common agents of viral heart disease. In the majority of exposed individuals they do not cause myocardial disease, however, since they are not primarily cardiotropic. Until recently the molecular basis of their anomalous tropism in patients who develop viral heart disease was unknown. An important step towards clarification of the molecular basis of cardiotropic viral infections was achieved in 1997, when a common receptor for the two structurally unrelated viruses was cloned. This coxsackievirus-adenovirus receptor (CAR) is a key determinant for the cellular uptake of both viruses and for the molecular pathogenesis of coxsackievirus and adenovirus diseases. We have mapped the CAR expression in human hearts and observed highly variable expression patterns. Healthy donor hearts had low CAR expression levels, whereas explanted hearts of patients with dilated cardiomyopathy (DCM) displayed high CAR expression in the myocardium. Remarkably, however, heart failure per se was not associated with CAR induction, since in heart failure of non-DCM origin no induction was found. Additional studies on the molecular mechanisms of CAR induction in cardiomyocytes indicated the existence of a cell-cell contact-dependent molecular mechanism regulating CAR expression, whereas cellular virus uptake and low level replication had no effect. Recombinant expression of human CAR in cardiomyocytes strongly increased their virus uptake rate suggesting that CAR induction enhances cardiac vulnerability to viral disease, whereas healthy myocardium is rather resistant to CAR-dependent viruses. Receptor induction may significantly aggravate the clinical course of viral heart disease, so that the blockade of receptor expression or receptor-virus interactions opens new therapeutic perspectives. Elucidation of the molecular mechanism of CAR induction in DCM, but not in heart failure per se, may reveal a particular pathogenetic process in this disease. A broader analysis of the cardiovascular expression patterns of receptors for other potentially cardiotropic viruses (CMV, EBV, HIV, HHV-6, Parvo-B19, etc.) should lead to a better understanding of individual risk factors for viral heart diseases and of their highly variable clinical courses, and offer new therapeutic options.

Molecular characterisation of the defective α1-antitrypsin alleles PI Mwürzburg ( Pro369Ser ), Mheerlen ( Pro369Leu ), and Q0lisbon ( Thr68lle )

Deficiency of the serine proteinase inhibitor (serpin) α1-antitrypsin (α1AT) is the most common autosomal recessive genetic disorder in Northern Europe. α1AT is the physiological regulator of the proteolytic enzyme neutrophil elastase and severe deficiency states are associated with an increased risk of developing chronic obstructive pulmonary disease (COPD) as a consequence of chronic proteolytic damage to the lungs. Among the known mutations of the α1AT gene causing severe α1AT deficiency and COPD a few alleles are also associated with liver disease. When expressed in cell cultures, all these particular alleles cause intracellular α1AT accumulation which appears to be a prerequisite for the development of hepatic injury. Liver disease is seen in only a small fraction of all patients carrying such alleles, however. The reason for this is not completely clear, but there is evidence that PI ZZ individuals ‘susceptible’ to liver disease carry an additional defect affecting protein degradation in the endoplasmic reticulum (ER). We characterise a newly identified defective α1AT allele PI Mwürzburg (Pro369 [CCC] to Ser [TCC]) associated with a complete intracellular transport block in cell cultures in vitro. The allele PI Mheerlen, a previously described different amino acid substitution in the same position as PI Mwürzburg (Pro369 [CCC] to Leu [CTC]) is shown to cause complete retention of the mutant α1AT in the ER, too, whereas in the recently described mutant allele PI Q0lisbon (Thr68 [ACC] to Ile [ATC]) a significantly reduced α1AT secretion from the cells was observed. Adenovirus-mediated recombinant expression of mutant Mwürzburg and Mheerlen, and of wild-type α1AT in mouse liver in vivo showed that the mutant human proteins were not secreted into the mouse plasma, in contrast with human wild-type α1AT which circulated at high concentrations over several weeks. In summary, all transportation deficient α1ATs analysed have the potential to cause lung disease in the homozygous state or in heterozygous carriers of another deficiency allele, and they may also cause liver disease in certain patients. The mutant PI Mwürzburg and Mheerlen α1ATs are completely retained within synthesising cells, and the molecular defect of transportation in these two alleles may be similar to that in the common PI Z allele. The molecular defect in the PI Q0lisbon allele (Thr68Ile) shows similarity with the immediately neighbouring Mmineral springs mutation (Gly67Glu).

CAR-diology--a virus receptor in the healthy and diseased heart.

The interplay of diverse cell-contact proteins is required for normal cardiac function and determines the mechanical and electrical properties of the heart. A specialized structure between cardiomyocytes—the intercalated disk—contains a high density of these proteins, which are assembled into adherens junctions, desmosomes, and gap junctions. The Coxsackievirus–adenovirus receptor (CAR) as a tight junction protein of the intercalated disk has recently been implied in cardiac remodeling and electrical conductance between atria and ventricle. This review summarizes recent in vivo studies that relate CAR to heart disease and how they could translate to improved diagnosis and therapy of viral myocarditis and arrhythmia.