Chronic pressure overload induced mortality.

<p>Highest survival rate was observed in placebo treated animals after TAC (92.3%; 1 out of 13). Treatment with E2 or 16α-LE2 led to a significant lower survival rate compared to placebo groups (both groups: 58.3%; 5 out of 13). Log rank Test; p<0.05.</p

Myocyte hypertrophy after nine weeks of chronic pressure overload.

<p>According to LVM/TL raloxifene treated animals developed a significant larger myocyte area compared to placebo and 16α-LE2 treated animals. ANOVA post-hoc Scheffé; p-value p<0.05.</p

Development of cardiac hypertrophy and LV function two weeks (grey bars) and nine weeks (black bars) after TAC.

<p>(A) Left ventricular mass to tibia length ratio (LVM/TL) was significantly reduced by E2 treatment compared to placebo or raloxifene treated animals two weeks after TAC. After nine weeks, raloxifene treated animals showed a significant higher LVM/TL than E2 treated mice. ANOVA post-hoc Scheffé; p-value p<0.05. (B) Ejection fraction (EF) showed a significant reduction of left ventricular function two weeks after TAC in placebo treated mice compared to all treated groups. Nine weeks after TAC surgery exclusively mice with ERα agonist treatment showed a significant higher EF. ANOVA post-hoc Scheffé; p-value p<0.05. (C) Factor analysis underlined the positive influence E2 and 16α-LE2 treatment on the progression of MH and loss of LV function. Broken line: factor <i>LV function</i>; solid line: <i>LV morphology</i>; Friedman test; p-value p<0.05.</p

Echocardiographic parameter for all measured time points.

*<p>p<0,05 vs. Placebo;</p>†<p>p<0,05 vs. E2;</p>‡<p>p<0,05 vs. ERα agonist</p

Additional file 1: Figure S1. of Reduction of apoptosis and preservation of mitochondrial integrity under ischemia/reperfusion injury is mediated by estrogen receptor β

Raw data of cardiac function during ischemia and reperfusion. Data are shown as mean ± SEM. Significances were calculated by ANOVA followed by post hoc Dunnet and defined as significant with p < 0.05. Figure S2: Representative western blots of ACAA2, Bcl2, TIM23, NDUFB8 and cytochrome c in cytosolic (F1) and mitochondrial (F2) fractions and corresponding controls (tubulin for F1, complex II for F2). Figure S3: Representative western blots of whole tissue lysates for caspase 9, caspase 9 cleaved and corresponding loading controls (tubulin and GAPDH). Figure S4: Representative western blots of whole tissue lysates for MLC/pMLC and ERK/pERK. (DOCX 876 kb

LV hypertrophy and function 2 and 9 weeks after sham or TAC surgery.

<p>T-test;</p>#<p>p<0.05 vs. ShamOVX+ShamTAC.</p>*<p>p<0.05 vs. OVX+ShamTAC.</p

Comparative Proteomic Analysis Reveals Sex and Estrogen Receptor β Effects in the Pressure Overloaded Heart

In pressure overload (PO), sex differences in humans and rodents have been well documented and estrogen receptor (ER) β is considered cardioprotective. However, the underlying mechanisms are poorly understood. Our aim was to investigate sex- and ERβ-specific effects in protein abundance in PO employing a 2-dimensional gel electrophoresis/mass spectrometry-based proteomics approach. We hypothesized major sex differences and ERβ-specific alterations consistent with cardioprotection in females. Two-month old male and female wild-type (WT) and ERβ knockout (BERKO) mice were subjected to transverse aortic constriction (TAC) for 9 weeks (<i>n</i> = 4/group). In WT mice, hypertrophy was significantly more pronounced in males than females, while this sex difference was abolished in BERKO mice. We found 82 protein spots modulated between TAC and sham in WT males, 31 in WT females, 114 in BERKO males, and 87 in BERKO females (<i>P</i> ≤ 0.05). Our analysis revealed in WT and BERKO females an altered pattern of various proteins involved in structure and suggests a link between female sex and cytoskeletal integrity. In males, a set of proteins was identified that associate with mitochondrial bioenergetics and energy supply. We confirmed protein regulation by immunoblotting analysis. In conclusion, the proteomic response of the heart to PO is significantly modulated by ERβ and sex. We put forward that the observed differences may identify sex-specific targets for the treatment of heart failure, contributing toward more personalized medical care

CYP2J2 Overexpression Protects against Arrhythmia Susceptibility in Cardiac Hypertrophy

<div><p>Maladaptive cardiac hypertrophy predisposes one to arrhythmia and sudden death. Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) promote anti-inflammatory and antiapoptotic mechanisms, and are involved in the regulation of cardiac Ca²⁺-, K⁺- and Na⁺-channels. To test the hypothesis that enhanced cardiac EET biosynthesis counteracts hypertrophy-induced electrical remodeling, male transgenic mice with cardiomyocyte-specific overexpression of the human epoxygenase CYP2J2 (CYP2J2-TG) and wildtype littermates (WT) were subjected to chronic pressure overload (transverse aortic constriction, TAC) or β-adrenergic stimulation (isoproterenol infusion, ISO). TAC caused progressive mortality that was higher in WT (42% over 8 weeks after TAC), compared to CYP2J2-TG mice (6%). In vivo electrophysiological studies, 4 weeks after TAC, revealed high ventricular tachyarrhythmia inducibility in WT (47% of the stimulation protocols), but not in CYP2J2-TG mice (0%). CYP2J2 overexpression also enhanced ventricular refractoriness and protected against TAC-induced QRS prolongation and delocalization of left ventricular connexin-43. ISO for 14 days induced high vulnerability for atrial fibrillation in WT mice (54%) that was reduced in CYP-TG mice (17%). CYP2J2 overexpression also protected against ISO-induced reduction of atrial refractoriness and development of atrial fibrosis. In contrast to these profound effects on electrical remodeling, CYP2J2 overexpression only moderately reduced TAC-induced cardiac hypertrophy and did not affect the hypertrophic response to β-adrenergic stimulation. These results demonstrate that enhanced cardiac EET biosynthesis protects against electrical remodeling, ventricular tachyarrhythmia, and atrial fibrillation susceptibility during maladaptive cardiac hypertrophy.</p></div

Chronic pressure overload-induced alterations in Cx43 localization.

<p>(A) Representative immunofluorescence staining of left ventricular cryosections prepared from WT and CYP2J2-TG mice 4 weeks after TAC surgery. The sections were co-stained for detecting Cx43 (green fluorescent signal) and N-cadherin (red). Cx43 and N-cadherin are colocalized (yellow) to the intercalated disks (indicated by white arrows). This normal Cx43 localization was largely preserved in CYP2J2-TG mice, whereas WT mice featured TAC- induced redistribution of Cx43 to the cytoplasm and lateral borders of the cardiomyocytes (pink arrows). Nuclei were stained with DAPI (blue). Scale bar: 50 µm. (B) Quantitative analysis of Cx43 and N-cadherin colocalization. Results represent mean±SEM based on the analysis of 5 sections per heart and 4–6 animals per group; ANOVA, Post-Hoc Tukey; *p<0.05 vs. WT-Sham; ^‡p<0.05 vs. WT-TAC.</p

Electrophysiological parameters of WT and CYP2J2-TG mice four weeks after TAC or sham operation.

<p>WT – Wildtype; CYP – CYP2J2 overexpressing mice; TAC – Transverse aortic constriction; HR – Heart rate; bpm – Beats per minute; ms – milliseconds; P – P-wave duration; PR – PR interval; QRS – QRS interval; QTc – QT interval (corrected for heart rate); AV WB – 1∶1 Atrioventricular node conduction capacity ( = Wenckebach point); AV 2∶1 - 2∶1 Atrioventricular node conduction capacity; AVNERP – Atrioventricular node effective refractory period; AERP – Atrial effective refractory period; VERP - Ventricular effective refractory period.</p><p>p<0.05 * vs. WT Sham,</p>†<p>vs. CYP Sham,</p>‡<p>vs. WT TAC.</p