Henryk Dreger

Nrf2-dependent upregulation of antioxidative enzymes: a novel pathway for proteasome inhibitor-mediated cardioprotection.

AIMS We have shown previously that non-toxic inhibition of the ubiquitin-proteasome system upregulates antioxidative defence mechanisms and protects endothelial cells from oxidative stress. Here, we have addressed the question whether the induction of antioxidative enzymes contributes to cardioprotection by non-toxic proteasome inhibition. METHODS AND RESULTS Treatment with 0.5 micromol/L MG132 for 48 h proved to be non-toxic and protected neonatal rat cardiac myocytes against H(2)O(2)-mediated oxidative stress in lactate dehydrogenase assays. This correlated with reduced levels of intracellular reactive oxygen species as determined by loading myocytes with dichlorofluorescein. Immunoblots showed significant upregulation of superoxide dismutase 1 (SOD1), haem oxygenase 1, and catalase upon proteasome inhibition. Luciferase assays using a reporter driven by the SOD1 promoter revealed proteasome inhibitor-mediated induction of luciferase activity. Deletion and mutation analyses identified an antioxidant response element (ARE) in the SOD1 promoter to be not only essential but also sufficient for transcriptional upregulation by proteasome inhibition. An essential role for the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)-which was stabilized by proteasome inhibition-in ARE-mediated transcriptional activation was revealed in cardiac myocytes from Nrf2 wild-type and knockout mice: proteasome inhibition upregulated antioxidative enzymes and conferred protection against H(2)O(2)-mediated oxidative stress in Nrf2 wild-type cells. In contrast, the induction of antioxidative enzymes and cytoprotection were completely abolished in cardiac myocytes from Nrf2 knockout mice. CONCLUSION Non-toxic proteasome inhibition upregulates antioxidative enzymes via an Nrf2-dependent transcriptional activation of AREs and confers cardioprotection.

Suppression of Cardiomyocyte Hypertrophy by Inhibition of the Ubiquitin-Proteasome System

Inhibitors of the proteasome interfere with transcriptional regulation of growth signaling pathways and block cell cycle progression of mitotic cells. As growth signaling pathways are highly conserved between mitotic and postmitotic cells, we hypothesized that proteasome inhibition might also be a valuable approach to interfere with hypertrophic growth of postmitotic cardiomyocytes. To test this hypothesis, we analyzed the effects of proteasome inhibition on hypertrophic growth of neonatal rat cardiomyocytes. Partial inhibition of the proteasome effectively suppressed cardiomyocyte hypertrophy as determined by reduced cell size, inhibition of hypertrophy-mediated induction of RNA and protein synthesis, reduced expression of several hypertrophic marker genes, and diminished transcriptional activation of the BNP promotor. Importantly, suppression of hypertrophic growth was independent of the hypertrophic agonist used. Expressional profiling and subsequent Western blot and kinase assays revealed that proteasome inhibition induced a cellular stress response with reduced expression of conserved growth signaling mediators and impaired G1/S phase transition of cardiomyocytes. In hypertensive Dahl-salt sensitive rats, inhibition of the proteasome with low doses of the FDA approved proteasome inhibitor Velcade significantly reduced hypertrophic heart growth. Our data provide important insight into the suppressive effects of proteasome inhibitors on hypertrophic growth of cardiomyocytes and establish low-dose proteasome inhibition as a new and broad-spectrum approach to interfere with cardiac hypertrophy.

Protection of vascular cells from oxidative stress by proteasome inhibition depends on Nrf2

AIMS Increased levels of reactive oxygen species cause oxidative stress and severely damage lipids, proteins, and DNA. We have previously shown that partial proteasome inhibition induces an antioxidative gene pattern in endothelial cells. Here, we elucidate the mechanisms of proteasome inhibitor-mediated upregulation of antioxidative enzymes and cytoprotection. METHODS AND RESULTS Non-toxic proteasome inhibition upregulated mRNA and protein expression of superoxide dismutase 1 (SOD1) and haem oxygenase 1 (HO1) in several human endothelial and vascular smooth muscle cell types. Transcriptional activation of these enzymes was shown by inhibition of RNA polymerase II and nuclear run-on assays. Transfection of endothelial cells with luciferase reporter constructs revealed that upregulation can be largely confined to an antioxidant response element (ARE), which proved to be sufficient for transcriptional activation of SOD1 and HO1. Co-transfection studies and bandshift analyses confirmed binding of the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)-which was stabilized by proteasome inhibition as shown by immunoblots-to the ARE site of HO1. Experiments with aortic endothelial and smooth muscle cells from Nrf2 wild-type and knockout mice revealed an essential role of Nrf2: in wild-type cells, proteasome inhibitor-mediated induction of SOD1 and HO1 was accompanied by protection of vascular cells against oxidative stress as determined by lactate dehydrogenase release assays. In contrast, proteasome inhibitor-mediated induction of antioxidative enzymes and cytoprotection were completely lost in cells from Nrf2 knockout mice. CONCLUSION Nrf2-dependent transcriptional activation of antioxidative enzymes is crucial for proteasome inhibitor-mediated protection of vascular cells against oxidative stress.

Early right ventricular systolic dysfunction in patients with systemic sclerosis without pulmonary hypertension: a Doppler Tissue and Speckle Tracking echocardiography study

BackgroundIsovolumetric acceleration (IVA) is a novel tissue Doppler parameter for the assessment of systolic function. The aim of this study was to evaluate IVA as an early parameter for the detection of right ventricular (RV) systolic dysfunction in patients with systemic sclerosis (SSc) without pulmonary hypertension.Methods22 patients and 22 gender- and age-matched healthy subjects underwent standard echocardiography with tissue Doppler imaging (TDI) and speckle tracking strain to assess RV function.ResultsTricuspid annular plane systolic excursion (TAPSE) (23.2 ± 4.1 mm vs. 26.5 ± 2.9 mm, p < 0.006), peak myocardial systolic velocity (Sm) (11.6 ± 2.3 cm/s vs. 13.9 ± 2.7 cm/s, p = 0.005), isovolumetric contraction velocity (IVV) (10.3 ± 3 cm/s vs. 14.8 ± 3 cm/s, p < 0.001) and IVA (2.3 ± 0.4 m/s2 vs. 4.1 ± 0.8 m/s2, p < 0.001) were significant lower in the patient group. IVA was the best parameter to predict early systolic dysfunction with an area under the curve of 0.988.ConclusionIVA is a useful tool with high-predictive power to detect early right ventricular systolic impairment in patients with SSc and without pulmonary hypertension.

Characteristics of Catechin- and Theaflavin-Mediated Cardioprotection

Catechins and theaflavins–the main polyphenolic substances of green and black tea, respectively–exert a plethora of beneficial effects on the cardiovascular system. In a model of H2O2-mediated oxidative stress, we investigated the effects of epigallocatechin-3-gallate (EGCG) and theaflavin-3,3′-digallate (TF3) on neonatal rat cardiomyocytes. Pretreatment with EGCG or TF3 1 hr prior to induction of oxidative stress by H2O2 effectively protected cardiac myocytes as determined by measuring release of lactate dehydrogenase after 24 hrs. Longer pre-incubation times resulted in significant loss of protection. To enable further mechanistic insight, we investigated expression of antioxidative enzymes and activation of prosurvival signaling cascades. Whereas mRNA levels of glutathione peroxidase 3, superoxide dismutase 1, and catalase were not influenced by both polyphenols, heme oxygenase (HO-1) was selectively upregulated by EGCG—but not by TF3. However, inhibition of HO-1 did not diminish polyphenol-mediated cardioprotection. While EGCG and TF3 activated Akt, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase, inhibition of these kinases did not attenuate polyphenol-mediated protection. Loading of cardiomyocytes with dichlorofluorescein revealed that intracellular levels of reactive oxygen species were significantly reduced after treatment with EGCG or TF3 as early as 30 mins after induction of oxidative stress. In conclusion, activation of prosurvival signaling kinases and upregulation of antioxidative enzymes do not play a major role in tea polyphenol-mediated cardioprotection.

Two-dimensional speckle tracking of the left ventricle in patients with systemic sclerosis for an early detection of myocardial involvement

AIMS Myocardial involvement is associated with poor prognosis in patients with systemic sclerosis (SSc). Two-dimensional speckle-tracking echocardiography (STE) is a powerful novel modality for the assessment of subclinical cardiac left ventricular (LV) dysfunction that, so far, has not been investigated in SSc patients. The aim of this study was to evaluate deformation analyses derived from STE for early detection of LV systolic dysfunction in patients with SSc having preserved left ventricular ejection fraction (LVEF). METHODS AND RESULTS Twenty-two patients with SSc (57.1 ± 13.3 years, LVEF 64 ± 3.1%, mean time of 5.4 ± 4.6 years from diagnosis) and 22 gender- and age-matched healthy subjects (57.4 ± 14.0 years, LVEF 65 ± 2.7%) underwent echocardiography with STE to assess global and regional LV function. The global longitudinal 2D peak systolic strain (PSS) of the left ventricle was significantly lower in the SSc group compared with controls: -19.0 ± 2.4 vs. -21.1 ± 2.5% (P = 0.008). This was mainly driven by a reduced strain in the basal segments. Strain in the medial segments and in the apex did not differ significantly. In addition, there was a significant difference between both groups regarding the global longitudinal PSS rate of the left ventricle (-1.19 ± 0.18 vs. -1.43 ± 0.26 s(-1), P = 0.001). CONCLUSION LV deformation analysis by STE is a sensitive method to detect early LV systolic impairment primarily in the basal segments in patients with SSc having preserved LVEF.

Pacing-induced cardiomyopathy in patients with right ventricular stimulation for >15 years.

AIMS The prevalence of pacing-induced cardiomyopathy (PiCMP) has been reported to be 9% 1 year after implantation. As long-term data are sparse, the aim of our study was to evaluate the prevalence of PiCMP in a cohort of patients with at least 15 years of right ventricular (RV) pacing. METHODS AND RESULTS Inclusion criteria were RV stimulation for at least 15 years due to atrioventricular block III° and absence of structural heart disease at the time of initial implantation. All patients were examined by echocardiography and spiroergometry. Pacing-induced cardiomyopathy was pre-defined as left ventricular (LV) ejection fraction (LVEF) ≤45%, dyskinesia during RV pacing and absence of other known causes of cardiomyopathy. Twenty-six patients from our outpatient department met the inclusion criteria. Pacing-induced cardiomyopathy was diagnosed in four patients (15.4%). Echocardiography showed significant LV remodelling in PiCMP patients [LVEF 41.0 ± 4.5%, LV end-diastolic diameter (LVEDD) 54.0 ± 2.7 mm] compared with patients with preserved LVEF (LVEF 61.2 ± 5.8%, P = 0.002, LVEDD 45.6 ± 4.0 mm, P= 0.004). There were no significant differences regarding age, gender, duration of RV pacing, heart rate, interventricular mechanical delay, QRS duration or prevalence of sinus rhythm, and arterial hypertension between both groups. The longest intraventricular delay was significantly shorter in patients with preserved LVEF (65.5 ± 43.0 ms) compared with PiCMP patients (112.5 ± 15.0 ms, P= 0.043). Exercise capacity and quality of life did not differ significantly between both groups. CONCLUSION Considering the very long duration of RV stimulation in our study population (24.6 ± 6.6 years), the prevalence of PiCMP was remarkably low. Pacing-induced cardiomyopathy was associated with more pronounced intraventricular dyssynchrony.

Transesophageal echocardiography in patients with cryptogenic cerebral ischemia

BackgroundIn about one third of all patients with cerebral ischemia, no definite cause can be identified (cryptogenic stroke). In many patients with initially suspected cryptogenic stroke, however, a cardiogenic etiology can eventually be determined. Hence, the aim of this study was to describe the prevalence of abnormal echocardiographic findings in a large number of these patients.MethodPatients with cryptogenic cerebral ischemia (ischemic stroke, IS, and transient ischemic attack, TIA) were included. The initial work-up included a neurological examination, EEG, cCT, cMRT, 12-lead ECG, Holter-ECG, Doppler ultrasound of the extracranial arteries, and transthoracic echocardiography. A multiplane transeophageal echocardiography (TEE, including i.v. contrast medium application [Echovist], Valsalva maneuver) was performed in all patientsResults702 consecutive patients (380 male, 383 IS, 319 TIA, age 18–90 years) were included. In 52.6% of all patients, TEE examination revealed relevant findings. Overall, the most common findings in all patients were: patent foramen ovale (21.7%), previously undiagnosed valvular disease (15.8%), aortic plaques, aortic valve sclerosis, atrial septal aneurysms, regional myocardial dyskinesia, dilated left atrium and atrial septal defects. Older patients (> 55 years, n = 291) and patients with IS had more relevant echocardiographic findings than younger patients or patients with TIA, respectively (p = 0.002, p = 0.003). The prevalence rates of PFO or ASD were higher in younger patients (PFO: 26.8% vs. 18.0%, p = 0.005, ASD: 9.6% vs. 4.9%, p = 0.014).ConclusionA TEE examination in cryptogenic stroke reveals contributing cardiogenic factors in about half of all patients. Younger patients had a higher prevalence of PFO, whereas older patients had more frequently atherosclerotic findings. Therefore, TEE examinations seem indicated in all patients with cryptogenic stroke – irrespective of age – because of specific therapeutic consequences.

Is antiarrhythmia device implantation safe under dual antiplatelet therapy

Background: Device implantations in patients on dual antiplatelet‐therapy (DA‐therapy) continue to rise. The aim of our study was to compile and analyze data on complications of antiarrhythmia device implantation under DA‐therapy.

Epigenetic Regulation of Cell Adhesion and Communication by Enhancer of Zeste Homolog 2 in Human Endothelial Cells

The histone methyltransferase enhancer of zeste homolog 2 (Ezh2) mediates trimethylation of lysine 27 in histone 3, which acts as a repressive epigenetic mark. Ezh2 is essential for maintaining pluripotency of stem cells, but information on its role in differentiated cells is sparse. Whole-genome mRNA expression arrays identified 964 genes that were regulated by >2-fold 72 hours after small interfering RNA-mediated silencing of Ezh2 in human umbilical vein endothelial cells. Among them, genes associated with the gene ontology terms cell communication and cell adhesion were significantly overrepresented, suggesting a functional role for Ezh2 in the regulation of angiogenesis. Indeed, adhesion, migration, and tube formation assays revealed significantly altered angiogenic properties of human umbilical vein endothelial cells after silencing of Ezh2. To identify direct target genes of Ezh2, we performed chromatin immunoprecipitation experiments followed by whole-genome promoter arrays (chromatin immunoprecipitation-on-chip) and identified 5585 genes associated with trimethylation of lysine 27 in histone 3. Comparative analysis with our mRNA expression data identified 276 genes that met our criteria for putative Ezh2 target genes, upregulation by >2-fold after Ezh2 silencing and association with trimethylation of lysine 27 in histone 3. Notably, we observed a striking overrepresentation of genes involved in wingless-type mouse mammary tumor virus integration site (WNT) signaling pathways. Epigenetic regulation of several of these genes by Ezh2 was specifically confirmed by polymerase chain reaction analysis of DNA enrichment after chromatin immunoprecipitation using an antibody specific for trimethylation of lysine 27 in histone 3. Combining mRNA expression arrays and chromatin immunoprecipitation-on-chip analysis, we identified 276 Ezh2 target genes in endothelial cells. Ezh2-dependent repression of genes involved in cell adhesion and communication contributes to the regulation of angiogenesis.