Corticotropin releasing hormone receptor 2 exacerbates chronic cardiac dysfunction

Abstract

Heart failure occurs when the heart is unable to effectively pump blood and maintain tissue perfusion. Despite numerous therapeutic advancements over previous decades, the prognosis of patients with chronic heart failure remains poor, emphasizing the need to identify additional pathophysiological factors. Here, we show that corticotropin releasing hormone receptor 2 (Crhr2) is a G protein-coupled receptor highly expressed in cardiomyocytes and continuous infusion of the Crhr2 agonist, urocortin 2 (Ucn2), reduced left ventricular ejection fraction in mice. Moreover, plasma Ucn2 levels were 7.5-fold higher in patients with heart failure compared to those in healthy controls. Additionally, cardiomyocyte-specific deletion of Crhr2 protected mice from pressure overload-induced cardiac dysfunction. Mice treated with a Crhr2 antagonist lost maladaptive 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent signaling and did not develop heart failure in response to overload. Collectively, our results indicate that constitutive Crhr2 activation causes cardiac dysfunction and suggests that Crhr2 blockade is a promising therapeutic strategy for patients with chronic heart failure.

Figure 1.

Sustained Crhr2 activation induces cardiac dysfunction. (A) G-protein–coupled receptor (GPCR) gene expression analysis in isolated cardiomyocytes 2 wk after transverse aortic constriction (TAC) using qRT-PCR greater than five copies per ng of RNA. The results are representative of two independent experiments. (B) Protein expression of Crhr2, adrenoceptor β-1 receptor (Adrb1), and prostaglandin E receptor 1 (Ptger1) in left ventricles 2 wk after sham or TAC was determined by immunoblotting analysis. (C) Statistical evaluation of (B; sham set to 1; n = 3; two-tailed Student’s t test). (D) Plasma Ucn2 concentration 2 wk after sham or TAC (n = 17; two-tailed Student’s t test). (E) Crhr2 tissue distribution in humans. (F and G) Plasma Ucn2 concentration (F), and cardiac morphology, left ventricular weight/tibia length (LVW/TL) ratio (G) after 4 wk of sustained Ucn2 infusion (n = 5; two-way ANOVA and Bonferroni post-hoc test). (H) Left ventricular fractional shortening determined by echocardiogram after 4 wk of sustained Ucn2 infusion (n = 5; two-tailed Student’s t test). (I) Systolic blood pressure after 4 wk of sustained Ucn2 infusion (n = 5; two-tailed Student’s t test). (J) Plasma BNP concentration after 4 wk of sustained Ucn2 infusion (n = 5, 2-tailed Student’s t test). Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ns, not significant. The results are representative of three independent experiments (B–J).

Figure 2.

Cardiomyocyte-specific, Crhr2-deficient mice exhibit suppressed pressure overload–induced cardiac dysfunction. (A) Protein extracts from isolated cardiomyocytes of vehicle- or tamoxifen-treated α-myosin heavy chain (αMHC)-CreERT2 (Cre)-negative and -positive Crhr2^flox/flox mice were blotted with antibodies against Crhr2 and actin. (B and C) LVW/TL ratio (B) and left ventricular fractional shortening (C) 4 wk after continuous infusion of Ucn2 or vehicle (sham) through an osmotic pump (n = 6; two-way ANOVA and Bonferroni post-hoc test). (D) LVW/TL ratio 4 wk after transverse aortic constriction (TAC; n = 6–10; two-way ANOVA and Bonferroni post-hoc test). (E) Fibrotic changes in left ventricles 4 wk after TAC were assessed by PicroSirius red staining (n = 4; two-way ANOVA and Bonferroni post-hoc test). (F and G) Left ventricular fractional shortening (F) and left ventricular end-diastolic dimension (G) were assessed by echocardiogram before and 4, 12, and 24 wk after TAC (n = 6; two-way ANOVA and Bonferroni post-hoc test). (H) Survival plot for control mice and cmc-Crhr2-KOs up to 8 mo after TAC (n = 36–37; Log-rank test). Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ns, not significant. The results are representative of three independent experiments (A–G).

Figure 3.

Crhr2 antagonist treatment suppressed pressure overload–induced cardiac dysfunction in preexisting hypertrophy. (A) Experimental design. (B) Systolic blood pressure in sham mice with Crhr2 antagonist treatment (n = 5; two-way ANOVA and Bonferroni post-hoc test). (C) Echocardiogram analysis of left ventricular fractional shortening was performed before and 7, 14, 21, and 35 d after TAC surgery (n = 5; two-way ANOVA and Bonferroni post-hoc test). (D–F) Plasma BNP (D), LVW/TL (E), and fibrotic area (determined by PicroSirius red staining; F) were measured 35 d after TAC surgery (n = 8–10; two-way ANOVA and Bonferroni post-hoc test). (G and H) Immunoblotting showed PKA, CREB, CaMKII, RyR2, and AKT phosphorylation in response to TAC in the heart with or without Crhr2 antagonist treatment (n = 4; two-way ANOVA and Bonferroni post-hoc test). (I) Gene expression 4 wk after Crhr2 antagonist treatment as determined by qRT-PCR in whole hearts (n = 4–6; two-way ANOVA and Bonferroni post-hoc test). Error bars indicate SEM. *, P < 0.05; **, P < 0.01; ns, not significant. The results are representative of two independent experiments (B–I).