H.-P. Schultheiss

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

BACKGROUND It 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. METHODS AND RESULTS Patients 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. CONCLUSIONS Differential 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.

Prospektive Untersuchung zu Häufigkeit, Pathogenese und Therapie spontaner, koronarangiographisch diagnostizierter Koronararteriendissektionen

Die spontane Koronararteriendissektion ist eine seltene Ursache einer ischämischen Herzkrankheit, über deren genaue Inzidenz, Ätiologie und optimale Therapie bisher wenig bekannt ist. In einer prospektiven Datenerhebung ermittelten wir 42 spontane Koronararteriendissektionen (36 Männer, sechs Frauen, mittleres Alter 59±12 Jahre) in einem Kollektiv von 3803 konsekutiv koronarangiographisch untersuchten Patienten, bei denen zwischen Juli 1995 und Dezember 1997 erstmals die Diagnose einer koronaren Herzkrankheit gestellt worden war (Inzidenz: 1,1%). Die Häufigkeit spontaner Koronararteriendissektionen war in der Teilgruppe von 450 Patienten mit akutem Myokardinfarkt (13 Fälle; 2,9%) und in der Teilgruppe von 501 Patienten mit instabiler Angina pectoris bzw. Postinfarktangina (21 Fälle; 4,2%) signifikant höher als im übrigen Patientenkollektiv mit stabiler Angina pectoris (8 Fälle; 0,3%). Am häufigsten war der Ramus interventricularis anterior der linken Herzkranzarterie von der Dissektion betroffen (19 Fälle), gefolgt von der rechten Herzkranzarterie (15 Fälle) und dem Ramus circumflexus der linken Herzkranzarterie (8 Fälle). Zur eindeutigen Diagnosestellung war bei 13 Patienten die zusätzliche Durchführung einer intravaskulären Ultraschalluntersuchung erforderlich. Als Auslöser der spontanen Koronararteriendissektionen waren in 35 Fällen eine arteriosklerotische Plaqueruptur, in vier Fällen eine schwere körperliche Anstrengung und in einem Fall hormonelle Einflüsse durch Schwangerschaft und Einnahme oraler Kontrazeptiva anzunehmen. In zwei Fällen ließ sich keine offensichtliche Ursache finden. Die Therapie umfaßte eine intrakoronare Stentimplantation bei 24 Patienten (davon zehn Patienten mit akutem Myokardinfarkt), eine Bypassoperation bei acht Patienten und eine Ballonangioplastie (PTCA) bei sieben Patienten. Drei Patienten wurden konservativ behandelt. Während einer mittleren Nachbeobachtungsdauer von 13,5±9,9 Monaten verstarben zwei Patienten. 31 Patienten blieben vollständig asymptomatisch, darunter alle operierten Patienten. Eine Rezidivstenose trat bei drei Patienten nach Stentimplantation (Rezidivstenoserate: 12,5%) auf. Nach primärer PTCA kam es bei zwei Patienten zu einer rezidivierenden Dissektion, an deren Folgen ein Patient verstarb. Spontaneous coronary artery dissection is a rare cause of ischemic heart disease. Incidence, etiology and optimal treatment are ill-defined. Between July 1995 and December 1997, we prospectively identified 42 patients (36 men, six women, mean age 59±12 years) with spontaneous coronary artery dissection among 3803 consecutive angiographic examinations in which the diagnosis of coronary artery disease was established for the first time (incidence 1.1%). In comparison to the remaining study population with stable angina pectoris (8 cases of spontaneous coronary artery dissection among 2852 patients; incidence: 0.3%), the incidence of spontaneous coronary artery dissection was significantly higher in the patient subgroups with acute myocardial infarction (13/450; 2.9%) and with unstable angina pectoris or postinfarction angina (21/501; 4.2%). Dissection was most frequently located in the left anterior descending coronary artery (19 cases), followed by the right coronary artery (15 cases) and the left circumflex coronary artery (8 cases). Because of an ambiguous angiographic lesion appearance intravascular ultrasound imaging was performed in 13 patients to confirm the diagnosis. The presumed etiology of spontaneous coronary artery dissection was atherosclerotic plaque rupture in 35 cases, heavy physical exercise in four cases and hormonal influences related to pregnancy and contraception in one case. In two cases, no obvious risk factor could be identified. Therapy consisted of intracoronary stenting in 24 patients (including ten patients with acute myocardial infarction), coronary artery bypass grafting (CABG) in 8 patients and balloon angioplasty (PTCA) in seven patients. Three patients were treated conservatively. During a mean follow-up period of 13.5±9.9 months, two patients died and 31 patients remained entirely asymptomatic, including all patients who were treated with CABG. Restenosis developed in three patients after stent implantation (restenosis rate: 12.5%). Following primary PTCA, spontaneous coronary artery dissection recurred in two patients, one of whom subsequently died.

Tissue-specific transcription pattern of the adenine nucleotide translocase isoforms in humans

Three adenine nucleotide translocase isoforms (ANT1, ANT2 and ANT3) are coded by different genes. The relative amounts of the three ANT isoform mRNAs were determined in detail in various human tissues. ANT isoforms were co‐expressed in all tested tissues revealing tissue‐specific transcription patterns. The highest ANT1 mRNA proportions were found in terminally differentiated tissues like skeletal muscle, heart and brain, whereas ANT2 was mainly expressed in tissues capable of proliferation and regeneration as in the kidneys, spleen, liver, fibroblasts and lymphocytes. The ANT3 mRNA proportion was not prominently expressed in any of the tissues tested. In conclusion, tissue‐specific expression of ANT isoforms is strongly related to the state of cellular differentiation.

Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells.

Gene therapy of cancer requires high-level expression of therapeutic transgenes in the target cells. Poor gene transfer is an important limitation to adenovector-mediated cancer gene therapy. We investigated two fundamentally different approaches to improve transgene expression in poorly permissive cancer cells. First, overexpression of the adenovirus attachment receptor CAR to facilitate receptor-mediated adenovector (AdV) uptake into the target cells; second, co-infection of this vector together with traces of replication competent adenovirus (RCA) accidentally arising by back-recombination during large-scale vector preparation. Among eight gastrointestinal cancer cell lines, the colorectal cancer lines showed particularly poor vector-mediated transgene expression (down to 67-fold lower than in HeLa cells). Expression of the adenovirus receptors CAR, αvβ5- and αvβ3-integrin were highly variable between cell lines. AdV uptake was significantly associated with CAR levels on the cell surface, but not with those of the integrins. AdV-mediated CAR overexpression increased CAR density on the surface of all investigated tumor cells and led to enhancement of transgene expression by 1.8- to 6.7-fold. The other principle to enhance transgene expression was ‘trans-complementation’ of the therapeutic vector, ie induction of its replication within the target cells. Traces of RCA in a vector preparation, as well as purified RCA were found to provide sufficient E1-region transcripts to induce replication of the therapeutic vector genome. The number of adenovector-based transgene expression cassettes was greatly amplified by this principle, notably without any influence on the rate of vector entry. Co-infection of four colorectal cancer cell lines with marker vector plus RCA (at around 240:1 particle ratio) resulted in far stronger enhancement of transgene expression (up to 46-fold) as compared with CAR overexpression, even in cancers almost refractory to standard adenovector-mediated gene transfer. Whereas RCAs need to be strictly avoided in gene therapy of non-malignant diseases for safety reasons, the magnitude of helper virus-induced therapeutic transgene expression could possibly warrant application of this principle to overcome the resistance of highly malignant cancers against gene therapy.

Myocardial bradykinin B2-receptor expression at different time points after induction of myocardial infarction.

Objective To characterize the regulation of the myocardial bradykinin B2 receptor after induction of myocardial infarction (MI), we studied its expression at different time points in the left ventricle (LV), right ventricle (RV) and interventricular septum (S) of the heart. Design Male Sprague-Dawley rats were submitted to permanent occlusion of the left descending coronary artery. Six hours, 24 h or 6 days after MI induction or a sham operation, a Millar-tip catheter was placed in the LV. Left ventricular pressure (LVP) and contractility [(dP/dt)max] were measured. The LV, RV and S of all animals were isolated, and total RNA was extracted. B2-receptor expression was analysed by an RNase-protection assay. In addition, Western blot analysis was used to determine protein levels of the B2 receptor in the infarcted area of the LV. Results We observed a decrease in LVP and contractility at all time points after MI in comparison with sham-operated animals. Basal B2-receptor expression was detected in the LV and RV, but not in the S of sham-operated rats. In the LV of infarcted hearts, we found a time-dependent up-regulation of the B2-receptor expression, which was increased twofold and fivefold, respectively, 6 h and 24 h after induction of MI compared with controls. This increase was maintained for at least 6 days. Similarly, we also found an up-regulation of the B2-receptor expression in the RV and S. Both reached a peak 24 h after induction of MI. The protein level of the receptor gradually increased up to day 6. Conclusion We conclude that myocardial ischaemia triggers B2-receptor up-regulation in both the infarcted and non-infarcted areas of the heart.

Coxsackievirus B3 and adenovirus infections of cardiac cells are efficiently inhibited by vector-mediated RNA interference targeting their common receptor.

As coxsackievirus B3 (CoxB3) and adenoviruses may cause acute myocarditis and inflammatory cardiomyopathy, isolation of the common coxsackievirus–adenovirus-receptor (CAR) has provided an interesting new target for molecular antiviral therapy. Whereas many viruses show high mutation rates enabling them to develop escape mutants, mutations of their cellular virus receptors are far less likely. We report on antiviral efficacies of CAR gene silencing by short hairpin (sh)RNAs in the cardiac-derived HL-1 cell line and in primary neonatal rat cardiomyocytes (PNCMs). Treatment with shRNA vectors mediating RNA interference against the CAR resulted in almost complete silencing of receptor expression both in HL-1 cells and PNCMs. Whereas CAR was silenced in HL-1 cells as early as 24 h after vector treatment, its downregulation in PNCMs did not become significant before day 6. CAR knockout resulted in inhibition of CoxB3 infections by up to 97% in HL-1 cells and up to 90% in PNCMs. Adenovirus was inhibited by only 75% in HL-1 cells, but up to 92% in PNCMs. We conclude that CAR knockout by shRNA vectors is efficient against CoxB3 and adenovirus in primary cardiac cells, but the efficacy of this approach in vivo may be influenced by cell type-specific silencing kinetics in different tissues.

Transcription of the adenine nucleotide translocase isoforms in various types of tissues in the rat

Two different isoforms of the adenine nucleotide translocase (ANT1 and ANT2) have been identified in the rat. In order to obtain enhanced knowledge of the ANT isoform expression, we analyzed the transcription pattern of both isoforms and their mRNA levels in various tissues of the rat using the PCR technique. A predominant ANT1 mRNA percentage was recorded in the skeletal muscle, heart and brain, ranging from 81 to 58%. In contrast to these tissues, the percentages of ANT2 were dominant with a range from 59 to 75% in the kidney, lung, spleen and liver. The level of total ANT mRNA varied markedly in the various organs. Tissues with a dominant ANT1 percentage simultaneously showed a high level of total ANT transcription (24-41 attomol/ng total RNA). In comparison to the latter, tissues with a prevalent ANT2 transcription were shown to have an even lower ANT transcription level (2-5 attomol/ng total RNA). The predominance of the ANT1 expression appeared to be restricted to tissues with an inability to regenerate by means of mitotic division, whereas a prevalent ANT2 transcription is found in cell types able to proliferate. The level of total ANT transcription but not the individual ANT isoform expression depends to a great extent on the energy requirements of the tissue.

Adenine nucleotide translocator in dilated cardiomyopathy: Pathophysiological alterations in expression and function

Several findings pointed to an insufficient energy supply in heart muscle tissue of patients suffering from dilated cardiomyopathy (DCM). We found a lowered ANT transport capacity of the adenine nucleotide translocator (ANT), the only transport system for ATP and ADP in eucaryotic cells, in explanted hearts of DCM patients. The reduced ANT transport rate was accompanied by a marked elevation in total ANT protein caused by an increase in ANT 1 isoform protein. Simultaneously, a reduction in ANT 2 transcripts and an unchanged ANT 3 expression was observed. In contrast, patients with ischemic or valvular heart disease showed no alteration in ANT function or expression, which indicates the disease-specificity of these findings.

[Acute myocardial infarct caused by a muscle bridge of the anterior interventricular ramus: complicated course with vascular perforation after stent implantation].

Ein 47jähriger Patient wurde mit einem akuten Vorderwandinfarkt eingewiesen. Eine dringlich durchgeführte koronarangiographische Untersuchung zeigte einen proximalen Verschluß des Ramus interventricularis anterior (RIVA). Nach mechanischer Rekanalisation stellte sich eine Kaliberreduktion des Gefäßes an der Verschlußstelle dar; es wurde eine Ballondilatation (PTCA) mit konsekutiver Stent-Implantation aufgrund einer Dissektion durchgeführt. Danach verblieb bei normalem Koronarfluß (TIMI 3) eine langstreckige Lumenreduktion des gesamten Gefäßsegments distal des Stents ohne erkennbare Stenose. Der RIVA wies am Beginn des mittleren Drittels eine Muskelbrücke auf, die auch einen abgehenden Diagonalast systolisch subtotal verschloß. Eine Dipyridamol-Thallium-Szintigraphie zeigte eine inkomplette anteroseptale Vernarbung und eine reversible anterolaterale Myokardischämie. Wir entschlossen uns daraufhin zur Implantation eines 3,0 mm-Stents im Bereich der Muskelbrücke. Die Wahl der Ballongröße wurde anhand der quantitativen koronarangiographischen Bestimmung des proximalen Referenzdurchmessers vorgenommen; es resultierte ein Mißverhältnis zwischen Gefäß- und Ballondurchmesser im distalen Dilatationssegment mit konsekutiver Koronararterienperforation in den rechtsventrikulären Ausflußtrakt. Der Patient blieb im Verlauf auch unter Ergometrie asymptomatisch und hämodynamisch stabil. In einer Kontroll-Koronarangiographie nach einer Woche war die Koronarfistel unverändert nachweisbar; der RIVA wurde rasch und vollständig angefärbt (TIMI 3). Innerhalb von drei Monaten schloß sich die Fistel spontan. Schlußfolgerungen: Muskelbrücken sind eine selten berichtete Ursache transmuraler Myokardinfarkte. Bei einer PTCA und Stent-Implantation kann ein Mißverhältnis zwischen Gefäß- und Ballondurchmesser (“balloon oversizing”) im Bereich der Muskelbrücke zu einer Koronararterienperforation führen, so daß diese Therapieoption besonders kritisch geprüft werden muß und ggf. die Wahl der Ballongröße aufgrund einer Durchmesserbestimmung des proximalen und distalen Referenzsegments mit intravaskulärem Ultraschall erfolgen sollte. Myokardseitige Koronarperforationen und Fistelbildungen können bei asymptomatischen Patienten mit hoher Wahrscheinlichkeit eines Spontanverschlusses konservativ behandelt werden. A 47-year-old male patient was admitted to our hospital with acute anterior myocardial infarction. Immediate coronary angiography was carried out, which showed proximal occlusion of the left anterior descending artery (LAD). After mechanical recanalization, a reduction in vessel caliber at the site of occlusion was visible, and balloon angioplasty with consecutive stent implantation because of vessel wall dissection was performed. After the procedure, diameter reduction of the entire vessel segment distal to the stent and muscular bridging with subtotal systolic obliteration of the LAD and one diagonal branch were demonstrated. Diastolic coronary flow did not appear to be limited (TIMI 3). Dipyridamole-thallium cardiac imaging revealed an incomplete perfusion defect of the anteroseptal region and a reversible perfusion reduction of the anterolateral region. For definitive treatment, we decided to implant a 3.0 mm-stent at the site of muscular bridging. Although balloon sizing was adapted to the diameter of the proximal reference segment, measured by quantitative coronary angiography, coronary perforation into the right ventricular outflow tract due to balloon oversizing in the distal dilation segment occurred. The patient remained asymptomatic at rest as well as under exercise testing, and hemodynamics remained stable. Coronary re-angiography after 1 week demonstrated a persistent fistula with complete opacification of the LAD and normal coronary flow (TIMI 3). Within the following 3 months, the coronary fistula closed spontaneously. Conclusions: Muscular bridging is a rare cause of acute myocardial infarction. Balloon angioplasty and stent implantation in the bridged segment may be complicated by coronary artery perforation due to balloon oversizing. Risks and benefits of this therapeutic option, therefore, have to be critically evaluated, and careful selection of balloon size using measurements of proximal and distal reference diameter assessed by intravascular ultrasound is recommended. Coronary artery perforation into the myocardium with subsequent development of a fistula my be treated conservatively as long as the patient remains asymptomatic. The frequency of spontaneous closure of the fistula is high.

Highly variable expression of virus receptors in the human cardiovascular system. Implications for cardiotropic viral infections and gene therapy.

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.