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

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

Gene-level association analysis of genes identified in the SNP-level analysis

<p>Gene-level association analysis of genes identified in the SNP-level analysis</p

Forest Plots of odd-ratios (log) in the different populations.

<p>The results show that the associations were largely homogeneous across populations (See also heterogeneity column in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172995#pone.0172995.t002" target="_blank">Table 2</a>)</p

DCM gene set association analysis

<p>DCM gene set association analysis</p

Manhattan Plot of association P-values.

<p>95,499 variants were investigated for association with DCM by logistic regression analysis. Associations are summarized in a Manhattan plot (R/qqman package) which displays the eleven SNVs significantly associated with DCM (Q-values < 0.01) as green dots. Note that the applied logistic model assumed an additive mode of inheritance. For variants on chromosome 15 in the <i>ALPK3</i> region, a dominant mode of inheritance was better supported by the data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172995#pone.0172995.t001" target="_blank">Table 1</a> for corresponding P-values)</p

Variants associated with DCM.

<p>Variants associated with DCM.</p

BAG3 interacts with HspB7.

<p>(A) GST Pull-Down showing interaction of GFP-BAG3 expressed in HEK293 cells and recombinant GST-HSPB7. GFP-BAG3 was expressed in HEK293 cells (cell extract panel) and and GST-HSPB7 was produced in a bacterial expression system. GST-HSPB7 co-sediment with GFP-BAG3 but not with GFP alone indicating specific BAG3/GST-HSPB7 interaction (Pull-Down panel). (B) Co-immunoprecipitation experiment showing interaction of Flag-HSPB7 and GFP-BAG3 in HEK293 cells. GFP alone or GFP-BAG3 were co-expressed together with Flag-HSPB7 in HEK293 cells (cell extract panel) and subjected to immunoprecipitation with an antibody against GFP. Only GFP-BAG3 immunoprecipitated with FLAG-HSPB7 (IP:GFP panel). Western blottings in (A) and (B) used HSPB7 (for GST-HSPB7), GFP (for GFP and GFP-BAG3), and α-tubulin specific antibodies.</p