We describe a sterile pericarditis model in minipigs for the study of atrial myopathy and atrial fibrillation (AF). We present surgical and anesthetic techniques, strategies for vascular access, and a protocol to study the inducibility of AF.
We describe a sterile pericarditis model in minipigs for the study of atrial myopathy and atrial fibrillation (AF). We present surgical and anesthetic techniques, strategies for vascular access, and a protocol to study the inducibility of AF.
Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Dehousse fellowship Introduction Although progress has been made in the treatment of heart failure, morbidity and mortality remain high, requiring new therapeutic targets. The neuregulin-1 (NRG1)/ERBB4 axis is cardioprotective and antifibrotic when activated in the myocardium, and therefore a possible target for therapy. Phase 2 and 3 clinical trials with NRG1 are ongoing, but require intravenous administration regimens, limiting applicability and efficacy. Purpose To develop small-molecule ERBB4 agonists with cardioprotective and antifibrotic properties. Methods A high-throughput screening (HTS) of 10,240 compounds was performed on a ERBB4/ERBB4 dimerization assay. Hit compounds were co-administered with NRG1 or fluorescently labeled NRG1 to determine competitive binding. Selectivity, receptor phosphorylation, cell proliferation and toxicity were determined using Luminex RTK phosphoprotein, ERBB2/ERBB3 dimerization, WST-1 colorimetric, and adenylate kinase assays. Antifibrotic effects were studied in vitro on TGF-β-induced collagen synthesis in human dermal and atrial fibroblasts, and in a mouse model of angiotensin II (AngII, 1000 ng/kg/min)-induced left ventricular (LV) myocardial fibrosis with selected compounds (83 µg/kg/h), administrated with osmotic minipumps (N=4–5/group). mRNA expression was evaluated after 7 days; LV myocardial fibrosis area, cardiomyocyte cross sectional area (CSA), echocardiographic parameters and heart- to bodyweight ratio (HW:BW) were analyzed at 28 days. Antiapoptotic effects were studied on rat atrial cardiomyocytes (AM) after hydrogen peroxide (H2O2)-induced cardiotoxicity. Results The HTS (Z’=0.7) resulted in 8 similar pyrimidine derivatives (EF-1–8) inducing ERBB4/ERBB4 dimerization (Emax 9–33% relative to NRG1, EC50 6E-6 to 2E-7 M). Competition assays indicate allosteric binding. The compounds also significantly potentiated NRG1-induced ERBB4 receptor dimerization up to 2.7 fold. Two compounds were excluded because of in vitro toxicity. The other 6 compounds were non-toxic and induced ERBB4, but neither ERBB1, ERBB2 or ERBB3 phosphorylation, nor tumor growth–inducing ERBB2/ERBB3 dimerization. Selected compounds showed significant dose-dependent antiapoptotic properties on H2O2-stimulated AM, and antifibrotic effects on human atrial and dermal fibroblasts. In vivo, compound EF-1 significantly decreased myocardial fibrosis (by 76±26%) and Col1a1, Col3a1 (-70±17%; -61±20%), and Nppa (-78±32%) mRNA expression, and significantly enhanced cardiomyocyte CSA (+24±8%). No differences were observed in cardiac function or HW:BW ratio. Conclusion We identified novel pyrimidine derivative small-molecule ERBB4 agonists with cardiomyocyte protective effects and antifibrotic properties in vitro and in AngII-induced myocardial fibrosis in vivo.
Introduction: Atrial fibrillation (AF) results from electrical and structural remodeling of the atria, in which inflammation and fibrosis play an important role. Current therapy is limited to antiarrhythmic drugs and ablations, but does not target the structural problem. Recent studies showed that neuregulin-1 (NRG1), an epidermal growth factor family member, has anti-fibrotic and anti-inflammatory effects in the myocardium. Purpose: To test the effects of JK07, a NRG1 antibody fusion comprising an ERBB3 antagonistic antibody which selectively signals through ERBB4 preferentially over ERBB3, on atrial fibrosis and AF inducibility. Methods: Atrial samples were harvested from male rats (Wistar Han, 10 weeks old), cut into small pieces (1-2mm 2 ) and kept in low serum medium in the presence or absence of JK07 (5nM). Col1a1 and Col3a1 mRNA was quantified after 24-72 hours. AF inducibility was tested in a first AF model in which male mice (C57BL/6N, 12-15 weeks old) were treated with angiotensin-II (Ang-II, 4 weeks, osmotic mini-pumps, 3000 ng/kg/min), and in a second AF model in which mice were fed with a high fat diet (HFD, 8 weeks, 60% Kcal fat) inducing severe weight gain (56±3% increase compared to 23±4% with regular chow). In both models, AF inducibility was tested by 5 runs of programmed electrical stimulation (PES) with a trans-jugular octapolar catheter. AF inducibility (% mice inducible by ≥3 PES-runs) and duration of PES-induced AF (AF duration) were recorded. Mice were randomized for treatment with vehicle or JK07 (2x/week, 1mg/kg, IV, n=5-7/group). Results: In cultured atrial samples, Col1a1 and Col3a1 mRNA expression gradually increased up to 2-3 fold over 3 days. JK07 robustly attenuated this effect by 59±17% (p<0.05). In mice, both Ang-II and HFD significantly increased AF inducibility and AF duration. In Ang-II mice, JK07 attenuated AF inducibility (from 57% to 20%) and AF duration (from 33.3 ± 15.1 to 1.5 ± 1s). In HFD mice, JK07 significantly attenuated AF inducibility (from 57% to 0%) and AF duration (from 10.9±3.2s to 0.76±0.5s, p<0.05). Conclusions: These results show anti-fibrotic effects by selective ERBB4 stimulation with JK07 in atrial tissue in vitro, together with AF-preventive effects in two unrelated mouse models.
Introduction: Morbidity and mortality of heart failure remain high, mandating new therapeutic approaches. The neuregulin-1 (NRG1)/ERBB4 axis is cardioprotective and an attractive target for treatment. Clinical trials with recombinant NRG1 are ongoing, but require intravenous administration, limiting applicability and efficacy. Purpose: To develop selective small-molecule ERBB4 agonists with cardioprotective effects. Methods: A high-throughput screening of 10,240 compounds (cpds) was performed on a ERBB4/ERBB4 dimerization assay. Hit cpds were co-administered with NRG1 or fluorescently labeled NRG1 to determine competitive binding. Selectivity, receptor phosphorylation, toxicity and metabolic stability were determined using Luminex RTK phosphoprotein, ERBB2/ERBB3 dimerization and adenylate kinase assays or LC-MS/MS. Apoptotic and hypertrophic effects of cpds (4-32μM) on cultured cardiomyocytes were studied after exposure to 100μM H 2 O 2 and 100nM angiotensin II (AngII). Antifibrotic effects (4-32μM) were studied on TGF-β-induced collagen synthesis in cultured human fibroblasts, and in mice (n=9-10/group) treated with AngII (1000 ng/kg/min) or cpd (83 μg/kg/h) using osmotic pumps. Results: We identified 8 similar pyrimidine derivatives inducing ERBB4/ERBB4 dimerization (Emax 9-33% relative to NRG1, EC50 6E-6 to 2E-7M). Competition assays indicated allosteric receptor binding and potentiation of NRG1-induced ERBB4 receptor dimerization, up to 2.7 fold (P<0.0001). Six were non-toxic and induced ERBB4 phosphorylation, but ERBB1, ERBB2 or ERBB3 phosphorylation remained unaffected and cpds did not induce ERBB2/ERBB3 dimerization, showing ERBB4 selectivity. Cpds showed a t1/2 of 170min in human liver microsomes. Cpds attenuated hypertrophic and apoptotic effects of AngII or H 2 O 2 (P<0.05), and decreased collagen upregulation in vitro (P<0.05). In vivo , a selected cpd attenuated AngII-induced myocardial interstitial fibrosis (-76±26%, P<0.01), and cardiac Col1a1, Col3a1 (-64±22%; -71±25%, P<0.01) and Nppa (-77±22%, P<0.01) mRNA expression. Conclusion: We identified selective novel pyrimidine derivative small-molecule ERBB4 agonists with cardioprotective effects in vitro and in vivo .
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