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The clinical efficiency of the highly potent antitumor agent doxorubicin is limited by cardiotoxic effects. In a murine doxorubicin cardiotoxicity model, increased endothelin-1 (ET-1) expression and cardioprotective effects of the dual ET-1 blocker bosentan were demonstrated. To date it is unclear if combined blocking of endothelin A/B receptors is necessary or whether selective inhibition of one of the ET-1 receptors is sufficient for the observed cardioprotection. Therefore, we investigated the impact of dual (bosentan) and single endothelin receptor antagonism through sitaxentan (receptor A blocker) or BQ788 (receptor B blocker) in a murine doxorubicin cardiotoxicity model (C57BL/6N). Simultaneous administration of each endothelin receptor antagonist (ERA) with doxorubicin resulted in a significantly improved hemodynamic performance in comparison to the impaired cardiac function in control mice with bosentan being most effective but closely followed by sitaxentan and also BQ788. This cardioprotection was not caused by diminished doxorubicin levels in heart since the doxorubicin content in cardiac tissue was not altered by ERAs significantly. However, whole transcript expression profiling showed partly different effects of the ERAs on doxorubicin-modulated cardiac gene expression of genes involved in signal transduction (e.g. Stat3, Pim1, Akt1, Plcb2), fibrosis (e.g. Myl4), energy production (e.g. Ant1) or oxidative stress (e.g. Aox1). Furthermore, doxorubicin-mediated gene regulations were verified in the murine cardiomyocyte model HL-1 showing partly reversed expression patterns after co-administration of the ERAs. In summary, our results demonstrate strong cardioprotective effects of blocking ET-1 receptors against the doxorubicin-related cardiomyopathy and provide evidence to potential underlying signaling pathways., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cardiotoxicity, which may result from intense cardiac oxidative stress and inflammation, is the main limiting factor of the anticancer therapy using doxorubicin. Because statins might exert beneficial pleiotropic cardiovascular effects, among other things, by anti-inflammatory and antioxidative mechanisms, we investigated whether or not fluvastatin pretreatment can attenuate doxorubicin-induced cardiotoxicity. Five days after a single injection of doxorubicin (20 mg/kg; i.p.), left ventricular (LV) function was measured in fluvastatin-treated (DoxStatin; 100 mg/kg/day, p.o.) and saline-treated (doxorubicin) mice (n = 8 per group) by a micro conductance catheter. Untreated mice served as controls (placebo; n = 8 per group). After measurement of cardiac function, LV tissues were analyzed by molecular biological and immunohistologic methods. Injection resulted in significantly impaired LV function (LV pressure, -29%; dp/dtmax, -45%; cardiac output, -68%; P < 0.05) when compared with placebo. This was associated with a significant increase in cardiac oxidative stress, inflammation and apoptotic mechanisms, as indicated by significant increased cardiac lipid peroxidation activity, protein expression of nitrotyrosine, tumor necrosis factor alpha and Bax (P < 0.05). In contrast, DoxStatin mice showed improved LV function (LV pressure, +24%; dp/dtmax, +87%; cardiac output, +87%; P < 0.05) when compared with untreated doxorubicin mice. This was associated with reduced cardiac expression of nitrotyrosine, enhanced expression of the mitochondrial located antioxidative SOD 2, attenuated mitochondrial apoptotic pathways, and reduced cardiac inflammatory response. Statin pretreatment attenuates doxorubicin-induced cardiotoxicity via antioxidative and anti-inflammatory effects.

Doxorubicin is a frequently used anticancer drug, but its therapeutic benefit is limited by acute and chronic cardiotoxicity, often leading to heart failure. The mechanisms underlying doxorubicin-induced cardiotoxicity remain unclear. It was previously shown in men that doxorubicin leads to increased endothelin-1 plasma levels. In addition, cardiac-specific overexpression of endothelin-1 in mice resulted in a cardiomyopathy resembling the phenotype following doxorubicin administration. We therefore hypothesized that endothelin-1 is involved in the pathogenesis of doxorubicin cardiotoxicity. In mice (C57Bl/10), we found that doxorubicin (20 mg/kg body weight, i.p.) impaired cardiac function with decreased ejection fraction, diminished cardiac output, and decreased end-systolic pressure points recorded by a microconductance catheter. This impaired function was accompanied by the up-regulation of endothelin-1 expression on mRNA and protein level. In vitro investigations confirmed the regulation of endothelin-1 by doxorubicin and indicated that the doxorubicin-mediated increase of endothelin-1 expression involves epidermal growth factor receptor signaling via the MEK1/2-ERK1/2 cascade, which was further confirmed by immunoblotting studies in the left ventricle of treated animals. Pretreatment of mice with the endothelin receptor antagonist bosentan (100 mg/kg body weight, p.o.) strikingly inhibited doxorubicin-induced cardiotoxicity with preserved indices of contractility. Moreover, bosentan pretreatment resulted in reduced tumor necrosis factor-alpha content, lipid peroxidation, and Bax expression, as well as increased GATA-4 expression. Thus, endothelin-1 plays a key role in mediating the cardiotoxic effects of doxorubicin and its inhibition may be of therapeutic benefit for patients receiving doxorubicin.

Abstract: Background: Cardiac inflammation and generation of oxidative stress are known to contribute to doxorubicin (Dox)-induced cardiomyopathy. Toll-like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signalling, we investigated whether or not TLR4 is involved in Dox-induced cardiotoxicity. Methods and results: Five days after a single injection of Dox (20 mg/kg; i.p.), left ventricular pressure–volume loops were measured in wild-type and TLR4-deficient mice (TLR4−/−) Dox-treated and control mice. Analyses of possible pathophysiological mechanisms were performed in left ventricular tissue and isolated myocytes, respectively. Dox injection resulted in an impairment of left ventricular function and neurohumoral activation, indexed by increased ET-1 expression. This was further associated with an increase in cardiac oxidative stress, inflammation and apoptosis, as indicated by an up-regulation of cardiac lipid peroxidation, TNF-α expression and enhanced content of TUNEL-positive cells. In contrast, TLR4−/−Dox mice showed improved left ventricular function with reduced oxidative and inflammatory stress response including reduced cardiac apoptosis. These results were found to be associated with an increase of GATA-4 expression. Conclusions: TLR4 deficiency improves left ventricular function and attenuates pathophysiological key mechanisms in Dox-induced cardiomyopathy. [Copyright &y& Elsevier]

Cardiac autoantibodies play a pathogenic role in dilated cardiomyopathy (DCM). Removal of antibodies by immunoadsorption (IA) induces hemodynamic improvement in DCM patients. The present study investigated the effects of IA on myocardial gene expression of the intermediate cytoskeletal filament desmin, which is upregulated in heart failure. RNA was isolated from five explanted non-failing hearts and five explanted failing hearts of DCM patients, and myocardial gene expression of desmin was estimated by real-time polymerase chain reaction (PCR). In a case-control study in six DCM patients (LVEF < 40%, NYHA II-III), IA and subsequent IgG substitution were performed at monthly intervals until month 3. Endomyocardial biopsies (EMBs) were obtained before and after IA (after 3–6 months). From six DCM patients without IA therapy (controls), EMBs were also obtained over a comparable time interval. Expression of the desmin gene was analyzed in these EMBs by real-time PCR. In failing explanted hearts, expression of desmin was significantly increased (0.88 ± 0.12 vs 0.45 ± 0.15 in non-failing hearts, P < 0.05). After IA, myocardial gene expression of desmin was significantly decreased (from 0.26 ± 0.05 [baseline] to 0.14 ± 0.04 [ P < 0.05] vs baseline and controls). Removal of antibodies by IA modulates myocardial gene expression of desmin in DCM patients. [ABSTRACT FROM AUTHOR]

Background: Growing evidence suggests participation of autoimmune mechanisms in the pathogenesis of dilated cardiomyopathy (DCM). Methods: Patients with heart failure (left ventricular ejection fraction ≤50%) due to DCM (n = 98) or ischemic cardiomyopathy (ICM, n = 49) and controls with normal left ventricular function (n = 98) were included. Immunoglobulin G antibodies were purified from plasma by affinity chromatography and analyzed by surface plasmon resonance analysis. We analyzed the distribution of autoantibodies against Kv channel-interacting protein (KChIP) 2.6, cardiac troponin I (cTnI), and the β1-adrenergic receptor (second extracellular loop, cardiac β1-adrenergic receptor [SEL-β1-AR])—two other known autoantibodies involved in heart failure. Effects of antibodies against KChIP2 on cell death of isolated rat cardiomyocytes were assessed by flow cytometry. Results: We detected autoantibodies against KChIP2.6 in 14.3% (P < .015 vs controls, P = .286 vs ICM) of the DCM samples, in 8.2% of the ICM samples (P = .304 vs controls), and in 4.1% of the control samples. Virus persistence was significantly associated with detection of autoantibodies against KChIP2.6 in DCM patients (P = .025). Antibodies against SEL-β1-AR were more frequent in DCM samples (34.7%, P < .001 vs controls, P = .02 vs ICM) and ICM samples (16.3%, P = .083 vs control) than in control samples (7.1%). Antibodies against cTnI were more frequent in DCM samples (20.4%, P < .001 vs controls, P = .769 vs ICM) and in ICM samples (18.4%, P < .01 vs controls) than in control samples (4.1%). Antibodies against rat KChIP2 enhanced cell death in isolated rat cardiomyocytes. Immunofluorescence indicated cell surface expression of KChIP2. Conclusions: Autoantibodies against KChIP2.6, SEL-β1-AR, and cTnI appear to be associated with DCM. Antibodies against KChIP2 may enhance cell death of rat cardiomyocytes. [Copyright &y& Elsevier]

The clinical course of patients with dilated cardiomyopathy (DCM) varies from cardiac recovery to end stage heart failure. The etiology of this variability is largely unknown. In this study, we investigated the impact of coding polymorphisms of the innate immune protein Toll-like receptor 4 (TLR4) on left ventricular performance in patients with DCM. Two variants of TLR4 (rs4986790, TLR4 c.1187A→G, p.299D→G and rs4986791,TLR4 c.1487C→T, p.T399I) were investigated in 158 patients with DCM. Other reasons for heart failure were excluded by coronary angiography, myocardial biopsy, and echocardiography. Risk factors, age, gender, or treatment did not differ among the groups. At the follow-up evaluation (median 4.0 -5.4 months), patients carrying the TLR4 wild type gene displayed cardiac recovery under intense medical heart failure therapy indexed by reduced left ventricular dilation, improved left ventricular ejection fraction, and reduced NT-probrain natriuretic peptide blood level when compared with the initial evaluation. In contrast, patients carrying both the rs4986790 and the rs4986791 variant showed significantly reduced improvement of left ventricular ejection fraction (p = 0.006) and left ventricular dilation (p = 0.015) at the follow-up evaluation when compared with carriers of the wild type gene under the same treatment conditions. In addition, NT-probrain natriuretic peptide level in carriers of both TLR4 variants did not change significantly at the follow up when compared with the first evaluation. Among patients with DCM, the presence of the TLR4 variants rs4986790 and rs4986791 predicts impaired cardiac recovery independently of medical treatment or cardiac risk factors. [ABSTRACT FROM AUTHOR]