Sex-specific pathways in early cardiac response to pressure overload in mice

Pressure overload (PO) first causes cardiac hypertrophy and then heart failure (HF), which are associated with sex differences in cardiac morphology and function. We aimed to identify genes that may cause HF-related sex differences. We used a transverse aortic constriction (TAC) mouse model leading to hypertrophy without sex differences in cardiac function after 2 weeks, but with sex differences in hypertrophy 6 and 9 weeks after TAC. Cardiac gene expression was analyzed 2 weeks after surgery. Deregulated genes were classified into functional gene ontology (GO) categories and used for pathway analysis. Classical marker genes of hypertrophy were similarly upregulated in both sexes (α-actin, ANP, BNP, CTGF). Thirty-five genes controlling mitochondrial function (PGC-1, cytochrome oxidase, carnitine palmitoyl transferase, acyl-CoA dehydrogenase, pyruvate dehydrogenase kinase) had lower expression in males compared to females after TAC. Genes encoding ribosomal proteins and genes associated with extracellular matrix remodeling exhibited relative higher expression in males (collagen 3, matrix metalloproteinase 2, TIMP2, and TGFβ2, all about twofold) after TAC. We confirmed 87% of the gene expression by real-time polymerase chain reaction. By GO classification, female-specific genes were related to mitochondria and metabolism and males to matrix and biosynthesis. Promoter studies confirmed the upregulation of PGC-1 by E2. Less downregulation of metabolic genes in female hearts and increased protein synthesis capacity and deregulation of matrix remodeling in male hearts characterize the sex-specific early response to PO. These differences could contribute to subsequent sex differences in cardiac function and HF

Geschlechterunterschiede bei drucklast-induzierter Myokardhypertrophie im Mausmodell

Die Entwicklung und der Verlauf einer Myokardhypertrophie (MH) und Herzinsuffizienz (HF) unterscheiden sich deutlich zwischen Frauen und Männern. Diese Geschlechterunterschiede können zumindest partiell auf Sexualhormone, insbesondere Östrogen, zurückgeführt werden. Östrogene wirken biologisch über zwei verschiedene Östrogenrezeptoren (ER): ERalpha und ERbeta. Viele Befunde weisen auf eine positiv modulierende Wirkung der Östrogene und speziell auf eine besondere Rolle des ERbeta bei der Entwicklung der druckinduzierten MH und HF hin. Diese Studie befasst sich mit der Untersuchung der Geschlechterunterschiede in der Ausprägung einer pathologischen MH und den myokardialen Veränderungen unter dem Einfluss des ERbeta. Grundlage der Arbeit bildete ein Mausmodell mit ERbeta-/-- und Wildtyp- Mäusen, denen durch eine transversale Aortenkonstruktion (TAC) eine artifizielle MH induziert wurde. Zur Dokumentation der Progression und den damit verbundenen Veränderungen wurden zwei Zeitpunkte gewählt, welche die adaptive und maladaptive kardiale Antwort darstellen. Die Entwicklung der Myokardhypertrophie wurde sowohl durch Echokardiographie als auch hämodynamischen Messungen charakterisiert und durch biochemische und molekulare Techniken sowie den Einsatz von Mikroarrays untersucht. Es konnte der Einfluss des Geschlechts auf die Entwicklung der MH bei andauernder Druckbelastung nachgewiesen werden. ERbeta modulierte dabei die kardiale Funktion sowie die molekulare Antwort in hypertrophie- assoziierten Prozessen, wie dem kardialen Metabolismus, der kardialen Fibrose und der Apoptose in weiblichen und männlichen Tieren. ERbeta trug in den weiblichen Tieren zum Erhalt des kardialen Energiehaushaltes bei und limitierte dadurch die Entwicklung einer kardialen Fibrose und Apoptose. Ein positiver Einfluss des ERbeta konnte in auch in den männlichen Tieren beobachtet werden: Ein starker Funktionsverlust des Herzens und apoptotische Prozesse wurden durch ERbeta verlangsamt oder inhibiert.Development and progress of myocardial hypertrophy (MH) and the transition to heart failure (HF) differ between the sexes in humans. There is evidence that sex- related differences are mediated through sex hormones, especially the sex hormone estrogen. Effects of estrogen are mediated by two different estrogen receptors (ER): ERalpha und ERbeta. Many studies indicate a beneficial role of estrogen and particularly for ERbeta in the development of pressure overload induced MH and HF. This study investigates sex differences in the development of pathological MH and accompanying myocardial changes under the influence of ERbeta. For this purpose wildtype and estrogen receptor beta lacking (ERbeta-/-) mice were investigated. Myocardial hypertrophy was induced by using the method of transversal aortic constriction (TAC). To determine the progression of MH to HF two time points were studied, which describe the adaptive and the maladaptive response to cardiac pressure overload. The development of MH was characterized by echocardiography and hemodynamic measurements in vivo. Additionally microarrays, molecular and biochemical analyses were performed in left ventricles. We identified sex differences in the development of MH induced by chronic pressure overload. ERbeta modulated the cardiac function and the molecular response in hypertrophy associated processes like cardiac metabolism, fibrosis and apoptosis in female and male animals. ERbeta contribute to the maintenance of energy homeostasis in female mice and limits the development of maladaptive cardiac hypertrophy, fibrosis and apoptosis in female and in male mice and slows the progression to heart failure consequently down

Sex-specific regulation of cardiac microRNAs targeting mitochondrial proteins in pressure overload

Abstract Background Maladaptive remodeling in pressure overload (PO)-induced left ventricular hypertrophy (LVH) may lead to heart failure. Major sex differences have been reported in this process. The steroid hormone 17β-estradiol, along with its receptors ERα and ERβ, is thought to be crucial for sex differences and is expected to be protective, but this may not hold true for males. Increasing evidence demonstrates a major role for microRNAs (miRNAs) in PO-induced LVH. However, little is known about the effects of biological sex and ERβ on cardiac miRNA regulation and downstream mitochondrial targets. We aimed at the analysis of proteins involved in mitochondrial metabolism testing the hypothesis that they are the target of sex-specific miRNA regulation. Methods We employed the transverse aortic constriction model in mice and assessed the levels of five mitochondrial proteins, i.e., Auh, Crat, Decr1, Hadha, and Ndufs4. Results We found a significant decrease of the mitochondrial proteins primarily in the male overloaded heart compared with the corresponding control group. Following computational analysis to identify miRNAs putatively targeting these proteins, our in vitro experiments employing miRNA mimics demonstrated the presence of functional target sites for miRNAs in the 3′-untranslated region of the messenger RNAs coding for these proteins. Next, we assessed the levels of the functionally validated miRNAs under PO and found that their expression was induced only in the male overloaded heart. In contrast, there was no significant effect on miRNA expression in male mice with deficient ERβ. Conclusion We put forward that the male-specific induction of miRNAs and corresponding downregulation of downstream protein targets involved in mitochondrial metabolism may contribute to sex-specific remodeling in PO-induced LVH

Effects of estrogen, an ERα agonist and raloxifene on pressure overload induced cardiac hypertrophy.

The aim of this study was to investigate the effects of 17β-estradiol (E2), the selective ERα agonist 16α-LE2, and the selective estrogen receptor modulator (SERM) raloxifene on remodeling processes during the development of myocardial hypertrophy (MH) in a mouse model of pressure overload. Myocardial hypertrophy in ovariectomized female C57Bl/6J mice was induced by transverse aortic constriction (TAC). Two weeks after TAC, placebo treated mice developed left ventricular hypertrophy and mild systolic dysfunction. Estrogen treatment, but not 16α-LE2 or raloxifene reduced TAC induced MH compared to placebo. E2, 16α-LE2 and raloxifene supported maintenance of cardiac function in comparison with placebo. Nine weeks after induction of pressure overload, MH was present in all TAC groups, most pronounced in the raloxifene treated group. Ejection fraction (EF) was decreased in all animals. However, 16α-LE2 treated animals showed a smaller reduction of EF than animals treated with placebo. E2 and 16α-LE2, but not raloxifene diminished the development of fibrosis and reduced the TGFβ and CTGF gene expression. Treatment with E2 or 16α-LE2 but not with raloxifene reduced survival rate after TAC significantly in comparison with placebo treatment. In conclusion, E2 and 16α-LE2 slowed down the progression of MH and reduced systolic dysfunction after nine weeks of pressure overload. Raloxifene did not reduce MH but improved cardiac function two weeks after TAC. However, raloxifene was not able to maintain EF in the long term period

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

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

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

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

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