A rat model of metabolic syndrome-related heart failure with preserved ejection fraction phenotype: pathological alterations and possible molecular mechanisms

Abstract

Background: Heart failure with preserved ejection fraction (HFpEF) represents a syndrome involving multiple pathophysiologic disorders and clinical phenotypes. This complexity makes it challenging to develop a comprehensive preclinical model, which presents an obstacle to elucidating disease mechanisms and developing new drugs. Metabolic syndrome (MetS) is a major phenotype of HFpEF. Thus, we produced a rat model of the MetS-related HFpEF phenotype and explored the molecular mechanisms underpinning the observed pathological changes.

Methods: A rat model of the MetS-related HFpEF phenotype was created by feeding spontaneously hypertensive rats a high-fat-salt-sugar diet and administering streptozotocin solution intraperitoneally. Subsequently, pathological changes in the rat heart and their possible molecular mechanisms were explored.

Results: The HFpEF rats demonstrated primary features of MetS, such as hypertension, hyperglycemia, hyperlipidemia, insulin resistance, and cardiac anomalies, such as left ventricular (LV) remodeling and diastolic impairment, and left atrial dilation. Additionally, inflammation, myocardial hypertrophy, and fibrosis were observed in LV myocardial tissue, which may be associated with diverse cellular and molecular signaling cascades. First, the inflammatory response might be related to the overexpression of inflammatory regulators (growth differentiation factor 15 (GDF-15), intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial cell adhesion molecule-1 (VCAM-1)). Secondly, phosphorylated glycogen synthase kinase 3β (GSK-3β) may stimulate cardiac hypertrophy, which was regulated by activated -RAC-alpha serine/threonine-protein kinase (AKT). Finally, the transforming growth factor-β1 (TGF-β1)/Smads pathway might regulate collagen production and fibroblast activation, promoting myocardial fibrosis.

Conclusion: The HFpEF rat replicates the pathology and clinical presentation of human HFpEF with MetS and may be a reliable preclinical model that helps elucidate HFpEF pathogenesis and develop effective treatment strategies.

Keywords: AKT/GSK-3β; GDF-15; ICAM-1; TGF-β1/Smad; VCAM-1; heart failure with preserved ejection fraction; metabolic syndrome.

Figure 1

Experimental protocol and MetS in HFpEF rat. (A) Experimental protocol. Comparison of blood pressure (SBP and DBP) (B,C), plasma lipids (TC, TG, and LDL-C) (D–F), blood glucose (GSP) (G), HOMA-IR (H), and TyG (I) between the three groups. Data are presented as mean ± standard deviations, analyzed with one-way ANOVA, *P < 0.05 vs WKY;**P < 0.01 vs WKY; ***P < 0.001 vs WKY; ▴P < 0.05 vs. SHR, ▴▴P < 0.01 vs. SHR, ▴▴▴P < 0.001 vs SHR. WKY, Wistar Kyoto rats; SHR, spontaneously hypertensive rats; HFpEF, heart failure with preserved ejection fraction; MetS, metabolic syndrome; STZ, streptozotocin; TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; GSP, glycated serum protein; HOMA-IR, homeostasis model assessment index for insulin resistance; TyG, fasting triglycerides and glucose index.

Figure 2

Cardiac structure and function by echocardiography in HFpEF rat. (A) Representative images of M-mode echocardiography (top), four-chambered heart (bottom). (B) pulsed-wave Doppler parameters (top) and tissue Doppler imaging (bottom). (C) Comparison of LV structure (LV Mass, LVAWd, LVPWd, LVEDD, and EDV), LA structure (LAAPD and LALRD), and LV systolic (LVEF) and diastolic function (IVRT, e′, a′, and E/e′) between the three groups. Data are presented as mean ± standard deviations, analyzed with one-way ANOVA, *P < 0.05 vs. WKY;**P < 0.01vs WKY; ***P < 0.001 vs WKY; ▴P < 0.05 vs. SHR, ▴▴P < 0.01 vs. SHR, ▴▴▴P < 0.001 vs SHR. LV, left ventricle; LVAWd, LV end-diastolic anterior wall; LVPWd, LV end-diastolic posterior wall thickness; LVEDD, LV end-diastolic internal diameter; EDV, end-diastolic volume; LA, left atrial; LAAPD, LA anterior-posterior diameter; LALRD, LA left-right diameter; LVEF, LV ejection fraction; IVRT, isovolumic relaxation time; E, peak mitral inflow velocity during early diastole; e′, peak early diastolic mitral annular velocities; a′, peak late diastolic mitral annular velocities.

Figure 3

Inflammation in HFpEF rat. (A–C) Comparison of hs-CRP, IL-1β and IL-6 between the three groups. (D) Representative images of HE-stained sections of LV myocardial tissue from each group (40×): HFpEF rats developed chronic myocardial inflammation, with local inflammatory cell infiltration, predominantly lymphocytes, monocytes, and macrophages, compared to WKY and SHR rats. (E–G) Comparison of protein expression of GDF-15, ICAM-1, and VCAM-1 between the three groups. Data are presented as mean ± standard deviations, analyzed with one-way ANOVA, *P < 0.05 vs. WKY;**P < 0.01 vs WKY; ***P < 0.001 vs WKY; ▴P < 0.05 vs. SHR, ▴▴P < 0.01 vs. SHR, ▴▴▴P < 0.001 vs SHR. hs-CRP, high-sensitivity C-protein; IL-1β, interleukin-1β; IL-6, interleukin-6; GDF-15, growth differentiation factor 15; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular endothelial cell adhesion molecule-1.

Figure 4

Myocardial hypertrophy in HFpEF rat. (A,B) Comparison of ANP and BNP between the three groups. (C) Representative images of HE-stained sections of LV myocardial tissue (top, 1×) and cross-sectional view of the LV cardiomyocytes (bottom, 40×): HFpEF rats exhibited significant LV hypertrophy compared with WKY and SHR rats, as evidenced by increased LV wall thickness and cardiomyocyte size compared to WKY and SHR rats. (D,E) Comparison of LVWT and CSA between the three groups. (F) AKT/GSK-3β signaling pathway in cardiac hypertrophy: On growth factor stimulation, PI3K phosphorylates PIP2 to PIP3, which recruits AKT to the plasma membrane, where AKT is activated in a phosphorylation-dependent manner. Activated AKT phosphorylates GSK-3β (Ser 9), leading to the inactivation of GSK3 activity and removing its inhibitory effect on cardiac hypertrophy. (G–I) Comparison of PI3K protein expression and the ratio of P-AKT/AKT and P-GSK-3β/GSK-3β protein between the three groups. Data are presented as mean ± standard deviations, analyzed with one-way ANOVA, *P < 0.05 vs. WKY;**P < 0.01vs WKY; ***P < 0.001vs WKY; ▴P < 0.05 vs. SHR, ▴▴P < 0.01 vs. SHR, ▴▴▴P < 0.001 vs SHR. ANP, atrial natriuretic peptide; BNP, B-type brain natriuretic peptide; LV, left ventricle; LVWT, LV wall thickness; CSA, cross-sectional area of LV cardiomyocytes; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5- triphosphate; AKT, RAC-alpha serine/threonine-protein kinase; P-AKT, phosphorylated AKT; GSK3β, glycogen synthase kinase 3β; P-GSK3β, phosphorylated GSK3β. NFAT, nuclear factor of activated T-cells; GATA4, GATA-binding protein 4.

Figure 5

Myocardial fibrosis in HFpEF rat. (A,B) Comparison of serum sST2 and Gal3 between the three groups. (C) Representative images of Masson-stained sections of perivascular fibrosis (top, 20×) and LV interstitial (bottom, 40×): Masson trichrome staining showed significantly increased interstitial and perivascular fibrosis in HFpEF rats than in WKY and SHR rats. (D,E) Comparison of PFR and CVF between the three groups. (F) TGF-β1/Smads signaling pathway in cardiac fibrosis: TGF-β1 binds to its receptor and subsequently induces phosphorylation of the receptor-activated Smads (Smad2 and Smad3), which form trimeric complexes with the common-partner Smad (Smad4). The trimeric complexes translocate to the nucleus and then regulate the transcription of fibrotic genes, thereby modulating collagen synthesis and fibroblast activation, resulting in myocardial fibrosis. (G–L) Comparison of protein expression of Coll I, Coll III, α-SMA, TGF-β1, Smad2/3, and P-Smad2/Smad3 between the three groups. Data are presented as mean ± standard deviations, analyzed with one-way ANOVA, *P < 0.05 vs. WKY;**P < 0.01vs WKY; ***P < 0.001vs WKY; ▴P < 0.05 vs. SHR, ▴▴P < 0.01 vs. SHR, ▴▴▴P < 0.001 vs SHR. sST2, Soluble growth stimulated expression gene 2 protein, Gal-3, galectin-3; PFR, perivascular fibrosis; CVF, collagen volume fraction; α-SMA, a-Smooth muscle actin; Coll I, collagen type I; Coll III, collagen type III; TGF-β1, transforming growth factor-β1; P-Smad2/Smad3, phosphorylated Smad2/Smad3. ACTA2, actin alpha 2, smooth muscle; COL1A1, collagen type I alpha 1 chain; COL3A1, collagen type III alpha 1 chain.

Figure 6

Cardiac pathological alterations and possible molecular mechanisms in HFpEF rat. A rat model of the MetS-associated HFpEF phenotype was established by feeding SHR rats a high-fat-salt-sugar diet and administering STZ solution intraperitoneally. The HFpEF rats demonstrated the primary features of MetS and HFpEF-related structural and functional cardiac abnormalities. Additionally, inflammation, myocardial hypertrophy, and fibrosis are observed in HFpEF rats, which may be associated with diverse cellular and molecular signaling cascades. MetS, metabolic syndrome; HFpEF, heart failure with preserved ejection fraction; SHR, spontaneously hypertensive rats; STZ: streptozotocin; hs-CRP, high-sensitivity C-protein; IL-1β, interleukin-1β; IL-6, interleukin-6; ANP, atrial natriuretic peptide; BNP, B-type brain natriuretic peptide; sST2, Soluble growth stimulated expression gene 2 protein, Gal-3, Galectin-3; LV, left ventricle; LA, left atrial; GDF-15, growth differentiation factor 15; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular endothelial cell adhesion molecule-1. PI3K, phosphoinositide 3-kinase; AKT, serine/threonine protein kinase; GSK3β, glycogen synthase kinase 3β; TGF-β1, transforming growth factor-β1.