Serelaxin treatment promotes adaptive hypertrophy but does not prevent heart failure in experimental peripartum cardiomyopathy

Abstract

Aims: Peripartum cardiomyopathy (PPCM) is a systolic left ventricular dysfunction developing in the peripartum phase in previously healthy women. Relaxin-2 is a pregnancy hormone with potential beneficial effects in heart failure patients. We evaluated Relaxin-2 as a potential diagnostic marker and/or a therapeutic agent in PPCM.

Methods and results: In healthy peripartum women, serum Relaxin-2 levels (measured by ELISA in the second half of pregnancy) were elevated showing a decreasing trend in the first postpartum week and returned to non-pregnant levels thereafter. In PPCM patients diagnosed in the first postpartum week, serum Relaxin-2 levels were lower compared to healthy postpartum stage-matched controls. In PPCM patients diagnosed later (0.5-10 months postpartum) Relaxin-2 levels were in the range of non-pregnant controls and not different from healthy postpartum stage-matched controls. In mice, serum Relaxin-1 (functional equivalent of human Relaxin-2) was increased late in pregnancy and rapidly cleared in the first postpartum week. In mice with PPCM due to a cardiomyocyte-specific knockout of STAT3 (CKO) neither low nor high dose of recombinant Relaxin-2 (serelaxin, sRlx-LD: 30 µg/kg/day; sRlx-HD: 300 µg/kg/day) affected cardiac fibrosis, inflammation and heart failure but sRlx-HD increased capillary/cardiomyocyte ratio. sRlx-HD significantly increased heart/body weight ratio and cardiomyocyte cross-sectional area in postpartum CKO and wild-type mice without changing the foetal gene expression program (ANP or β-MHC). sRlx-HD augmented plasma Prolactin levels in both genotypes, which induced cardiac activation of STAT5. In vitro analyses showed that Prolactin induces cardiomyocyte hypertrophy via activation of STAT5.

Conclusion: Although Relaxin-2 levels seemed lower in PPCM patients diagnosed early postpartum, we observed a high pregnancy-related variance of serum Relaxin-2 levels peripartum making it unsuitable as a biomarker for this condition. Supplementation with sRlx may contribute to angiogenesis and compensatory hypertrophy in the diseased heart, but the effects are not sufficient to prevent heart failure in an experimental PPCM model.

Keywords: Biomarker; Heart failure; Hypertrophy; Peripartum cardiomyopathy; Relaxin; STAT3; STAT5; Serelaxin.

Figure 1

Serum Relaxin levels in healthy peripartum women and in PPCM. (A) Serum Relaxin-2 levels in healthy non-pregnant (NP, n = 9) women, pregnant women (Prg, n = 10) and postpartum women (1 day n = 12, 2d n = 17, 3d n = 5, 0.5–10 months n = 11), **P < 0.01 vs. NP; ^#P < 0.05, ^##P < 0.01 PP vs. Prg), data are median with interquartile range, one-way ANOVA, Bonferroni’s Multiple Comparison Test. (B) Serum Relaxin-2 levels in PPCM patients vs. healthy postpartum controls with serum collected in the first postpartum week (PPCM: n = 15 vs. Control PP: n = 36, **P < 0.01) and (C) measured in later diagnosed patients and postpartum stage-matched controls where serum was obtained after the first postpartum week (PPCM: n = 40 and Control PP: n = 11). Data are shown as median with interquartile range, please note that patients and controls with Relaxin-2 levels below the limit of detection are not included within the scatter plot due to logarithmical scaling, but were used for median and statistical calculation, normality test by D’Agostino–Pearson, Mann-Whitney test for PPCM vs. controls. (D) Serum NT-proBNP levels in PPCM patients vs. healthy control postpartum women (PPCM: n = 34 vs. Control PP: n = 16, **P < 0.01, data are mean ± SD, unpaired, two-tailed t-test. (E) Improvement of left ventricular ejection fraction (ΔEF: LVEF at follow-up–LVEF at diagnosis) of PPCM patients with serum Relaxin-2 levels at diagnosis <20 pg/ml (n = 37) or >20 pg/ml (n = 7), data are mean ± SD.

Figure 2

Kinetics of mouse serum Relaxin-1 and serum levels of Relaxin-2 reached by sRlx injection. (A) Relaxin-1 levels in WT female mice, nulli pari (NP), pregnant (Prg), early (early PP) and late (late PP) postpartum mice (**P < 0.01 vs. NP, n = 5–6), one-way ANOVA, Bonferroni’s Multiple Comparison Test. (B) Serum Relaxin-2 levels in WT-NP mice after daily subcutaneous injection of sRlx-HD and NaCl for 2 days, 6h after the last injection (**P < 0.01 vs. NaCl, n = 3–4 each), unpaired, two-tailed t-test. All data are presented as mean ± SD.

Figure 3

Effect of sRlx treatment on WT and CKO mice after two subsequent pregnancies with regards to cardiac hypertrophy and capillary density. (A) LV cryosections stained with isolectin B4 (blood vessels, green), WGA (cell membranes, red) and nuclei (DAPI, blue) scale bar: 50 μm. (B) Bar graph summarizing cardiomyocyte cross-sectional area (CSA) (**P < 0.01 vs. NaCl-treated WT-PP, ^##P < 0.01 sRlx vs. NaCl-treated CKO-PP n = 6–7), two-way ANOVA, Bonferroni’s Multiple Comparison Test. (C–E) Bar graphs summarizing qRT-PCR results for (C) ANP, (D) β-MHC, and (E) α-MHC mRNA expression in WT-PP and CKO-PP mice injected with NaCl or sRlx-HD (mean of NaCl WT-PP was set at 100%, *P < 0.05, **P < 0.01 vs. NaCl WT-PP, n = 6–14), two-way ANOVA, Bonferroni’s Multiple Comparison Test. (F) Bar graph summarizing capillaries to cardiomyocyte (CAP/CM) ratio (**P < 0.01 vs. NaCl-treated WT-PP, ^##P < 0.01 vs. NaCl-treated CKO-PP, n = 6), two-way ANOVA, Bonferroni’s Multiple Comparison Test. All data are presented as mean ± SD.

Figure 4

Effect of sRlx treatment of CKO-PP mice with regards to cardiac fibrosis and inflammation (A) Representative sections with haematoxylin and eosin staining (upper panels), Sirius red staining in bright field (middle panels) and in polarized light (bottom panels) depicting fibrosis and collagen fibres in WT-PP (n = 6), CKO-PP treated with NaCl (n = 6) or sRlx-HD (n = 6) with at least four sections per mouse analysed, scale bars: 50 μm. (B) Bar graph summarizing qRT-PCR results of mRNA for COL1A1 (mean of WT-PP was set at 100%, *P < 0.05 vs. WT-PP (n = 8), CKO-PP n = 11 (NaCl), and n = 10 (sRlx-HD), data are means±SD, one-way ANOVA, Bonferroni’s Multiple Comparison Test. (C) Western blot showing protein levels of MMP2 and -3 and membrane stained with Ponceau S (PS) for loading control, (D) Western blot showing protein levels of pro- and active MMP9, membrane stained with Ponceau S (PS) for loading control. Western blots in C-D are representative for data from CKO-PP treated with NaCl or sRlx-HD with n = 6 for each condition. (E) Representative immunohistochemical staining with the pan-inflammatory marker CD45 (brown staining; counterstained with eosin, red), in CKO-PP treated with NaCl (n = 6) or sRlx-HD (n = 6), at least four sections per mouse were analysed, scale bars: 50 μm.

Figure 5

sRlx upregulated Prolactin in WT and CKO mice and Prolactin promotes via STAT5 cardiomyocyte hypertrophy. (A) Plasma Prolactin measured in WT and CKO female mice 2h after sRlx-HD injection, n = 7–8 each genotype and condition, *P < 0.05, **P < 0.01 vs. NaCl-treated WT mice, ^##P < 0.01 vs. NaCl-treated CKO mice; data are means±SD, two-way ANOVA, Bonferroni’s Multiple Comparison Test (B) Western Blot showing STAT5 (phospho-Tyrosine 694) activation in LV tissue from WT mice 2 h after sRlx-HD injection and (C) Western Blot depicting activation state of STAT3 (phospho-Tyrosine 705) and STAT5 in WT and CKO LV tissue 30 min after injection with Prolactin (Prl). Western blots in B and C are representative for n = 4–6 mice per condition and time point. (D) Fluorescence microscopy with α-actinin staining (red) showing cell surface area in NRCM stimulated with or without Prl (0.2 iU/ml) or rRelaxin-2 (100 ng/ml) for 48h, STAT5 inhibitor (100 μM) were added 1h prior stimulation and (E) bar graphs summarizing cardiomyocyte surface area in these NRCM after 48h (**P < 0.01, vs. control) All experiments are representative for three different cell isolations and are performed in duplicates or triplicates, data are means±SD, two-way ANOVA, Bonferroni’s Multiple Comparison Test.

Figure 6

sRlx does not enhance 16kDa Prolactin-mediated pathophysiology in post-partum CKO mice (A) Western blots of ErbB4 and ErbB2 and (B) bar graphs summarizing protein levels of ErbB4 and ErbB2 in WT-PP, and CKO-PP with NaCl or sRlx-HD treatment. Membranes were stained with Ponceau S (PS) for loading control. All data are mean±SD. Mean of WT-PP was set at 100%. *P < 0.05, **P < 0.01 vs. WT-PP, n = 7–9 each, one-way ANOVA, Bonferroni’s Multiple Comparison Test.