Multiparity induces persistent myocardial structural, functional and transcriptomic remodelling in mice

Abstract

Pregnancy poses unique hemodynamic and metabolic demands on the heart. Recent data suggests that a history of multiple pregnancies may be associated with a late-term increased risk of heart failure with preserved ejection fraction (HFpEF), however, the mechanistic basis is unknown. We postulated that the repetitive biological and hemodynamic impact of multiple pregnancies could cause persistent myocardial remodelling providing a substrate for subsequent HFpEF. Multiparous (MP) mice (C57Bl/6J) aged 24 months were compared to age-matched non-parous (NP) mice (n = 8) in a blinded analysis. We evaluated blood pressure, body composition, gross weight and histologic cardiac structure. Cardiac function was assessed by echocardiography together with transcriptomic analysis of the left ventricular myocardium. At 15 months post oestrous cycle disruption, MP mice continued to demonstrate a constellation of myocardial alterations including higher heart to tibial length ratio (P < 0.05), body weight (P < 0.01), and fat mass (P < 0.01) compared to NP mice. MP mice had increased isovolumetric relaxation time (P < 0.01), increased end-diastolic (P < 0.05) and systolic volumes (P < 0.005), and mildly reduced left ventricular ejection fractions (LVEF) (P < 0.005) compared to non-parous mice. This was accompanied by marked elevations in mRNA levels of cardiac markers; Myh6 (P < 0.01) and Nppa (P < 0.01). mRNA levels of Il18 (P < 0.05) and the basal extracellular matrix protein-fibronectin (P < 0.05) were also increased, with a significant increase in interstitial fibrosis (P < 0.05). Bulk RNA sequencing (RNA Seq) further revealed persistent differential expression of 128 genes with over/under-represented pathways involved in extracellular matrix (ECM) regulation and organic anion/ion transport, respectively. Multiparity alters cardiac gene profile and causes myocardial remodelling that persists into later life, providing a potential foundation for the HF phenotypes such as HFpEF. Further studies are required to investigate the nature of the interaction between pregnancy-associated remodelling and other factors known to be associated with HFpEF development.

Keywords: Cardiac remodelling; Fibrosis; HFpEF; Hypertrophy; Multiparity.

Fig. 1

Morphometric, metabolic, and physiological differences between age matched multiparous (MP) and nulliparous (NP) mice. MP mice had increased (A) body weight. EchoMRI analysis of body composition showed increased (B) fat mass and (C) total water (D). Quantitative analysis of heart weight-HW and (E) lung weight-LW normalised to tibia length-TL. Metabolic caging data showed increased (F) food, and (G) water intake and (H) urine output for MP mice within 24 h. MP, n = 8, NP, n = 8. *P < 0.05, **P < 0.005, ***P < 0.001, MP vs NP. Unpaired two tailed t-test, with Mann–Whitney test for post hoc analysis. Data presented as mean ± standard error of mean (SEM).

Fig. 2

MP mice had elevated systolic blood pressure (SBP), chronic hypertension coupled with myocyte hypertrophy vs. NP mice. (A) MP mice had elevated SBP. Echocardiography analysis was suggestive of a chronic hypertension phenotype as indicated by (B) increased end diastolic volume- EDV, (C) end systolic volume-ESV, (D) reduced ejection fraction—EF%, (E) and fractional shortening-FS %, and (F) prolonged isovolumetric relaxation time- IVRT, in MP mice. (G) Myocyte size was increased with increased gene expression of (H) alpha myosin heavy chain- Myh6, and (I) atrial natriuretic peptide-Nppa. There were no significant changes in mRNA expression of (J) β myosin light chain- Myh7, and (K) brain natriuretic peptide- Nppb in MP mice. MP, n = 8, NP, n = 8. *P < 0.05, **P < 0.005, ns = P > 0.05, MP vs NP. Unpaired two tailed t-test, with Mann–Whitney test for post hoc analysis. Data presented as ± SEM.

Fig. 3

MP mice had increased interstitial fibrosis & basal fibronectin mRNA, and IL-18 mRNA expression. (A) Blinded masson’s trichrome staining analysis of X2 sections on Aperio Image Scope showed increased % of interstitial fibrosis in the heart in MP compared to NP mice, (B) with no differences in perivascular staining as % of fibrosis to area of vessel wall. (C) RT-qPCR analysis showed increased basal fibronectin (Fn1), with no alteration in (D–G) Collagen (Col) 1a1, 3a1, alpha smooth muscle actin (acta2) & transforming growth factor β1 (Tgfb1) (mRNA expression. The inflammatory marker (H) Interleukin 18 (Il18) was increased, while there were no significant shift in (I) Il1b (J) Nod like receptor pryin domain containing 3 (Nlrp3), (K) tumor necrosis factor α (Tnfa), (L) Il6 & (M) Il10 mRNA expression in the mid-section of the heart. MP, n = 8, NP, n = 8. *P < 0.05, ns = P > 0.05 vs. NP group. Unpaired two tailed t-test, with Mann–Whitney test for post hoc analysis. Data are presented as mean ± SEM.

Fig. 4

128 genes were differentially expressed in the MP mice compared to NP mice. (A,B) multi-dimensional (MDS) and scree plot showing 42% of the variance between MP (n = 8) and NP (n = 8) mice were due to dimension 1 and 2. (C) Heat map showing log CPM values of the top 100 differentially expressed genes (DEGS) ranked through hierarchical cluster profiling. Only 1 of the NP mice grouped into MP group. (D) Volcano plot analysis of DEGs showing 64 upregulated (red) and 64 down regulated genes (blue) in MP mice with P < 0.05, and false discovery rate (FDR) < 0.05, at logFC. (E) Significantly enriched gene ontology (GO) terms associated with biological processes (BP), cellular components (CC), and molecular function (MF) analysed through ShinyGO 0.8 ranked by FDR and fold change. (F) Chord Plot showing of CC GO terms and associated genes and their fold change. Graph generated through SR Plot.

Fig. 5

Cardiomyopathy related pathways were enriched by DE genes associated with ECM, cardiac contraction and membrane transport in MP mice. (A) Top 10 cellular signalling pathway analysis through Bio-Planet, 2019 for up regulated (red) and down regulated (blue) genes, P < 0.05, less stringent FDR of < 0.2. Applying stringent FDR of < 0.05 only produced top 2 pathways. (B,C) Sankey plot of genes involved in each of the top 10 upregulated and downregulated enriched Bio-Planet pathways within the 64 upregulated and 64 downregulated genes, respectively. (D) 5 of the top 10 disease perturbations in GEO UP cardiomyopathy related (red nodes). (E) Cluster map of top 40 genes associated with the top 10 disease perturbations, ranked by P value, FDR < 0.05. Enrichr was used for these enrichment analysis. Data can be found on https://maayanlab.cloud/Enrichr/enrich?dataset=cebccb1381901ed9014bc6aea9eabb9f.