Quercetin alleviates diastolic dysfunction and suppresses adverse pro-hypertrophic signaling in diabetic rats

Abstract

Introduction: Quercetin (Que) is a potent anti-inflammatory and antioxidant flavonoid with cardioprotective potential. However, very little is known about the signaling pathways and gene regulatory proteins Que may interfere with, especially in diabetic cardiomyopathy. Therefore, we aimed to study the potential cardioprotective effects of Que on the cardiac phenotype of type 2 diabetes mellitus (T2DM) accompanied by obesity.

Methods: For this experiment, we used Zucker Diabetic Fatty rats (fa/fa) and their age-matched lean controls (fa/+) that were treated with either vehicle or 20 mg/kg/day of Que for 6 weeks. Animals underwent echocardiographic (echo) examination before the first administration of Que and after 6 weeks.

Results: After the initial echo examination, the diabetic rats showed increased E/A ratio, a marker of left ventricular (LV) diastolic dysfunction, in comparison to the control group which was selectively reversed by Que. Following the echo analysis, Que reduced LV wall thickness and exhibited an opposite effect on LV luminal area. In support of these results, the total collagen content measured by hydroxyproline assay was decreased in the LVs of diabetic rats treated with Que. The follow-up immunoblot analysis of proteins conveying cardiac remodeling pathways revealed that Que was able to interfere with cardiac pro-hypertrophic signaling. In fact, Que reduced relative protein expression of pro-hypertrophic transcriptional factor MEF2 and its counter-regulator HDAC4 along with pSer²⁴⁶-HDAC4. Furthermore, Que showed potency to decrease GATA4 transcription factor, NFAT3 and calcineurin, as well as upstream extracellular signal-regulated kinase Erk5 which orchestrates several pro-hypertrophic pathways.

Discussion: In summary, we showed for the first time that Que ameliorated pro-hypertrophic signaling on the level of epigenetic regulation and targeted specific upstream pathways which provoked inhibition of pro-hypertrophic signals in ZDF rats. Moreover, Que mitigated T2DM and obesity-induced diastolic dysfunction, therefore, might represent an interesting target for future research on novel cardioprotective agents.

Keywords: diabetes; diastolic dysfunction; hypertrophy; quercetin; remodeling.

Figure 1

The assessment of LV diastolic function at two different time points – pre-treatment (week 0 – prior to the administration of Que) and post-treatment (week 6 – at the end of the Que treatment course). (A) E/A ratio of transmitral inflow from early (E wave) to late (A wave) diastole. (B) Representative echocardiographic image of E/A ratio in DQ group pre-treatment vs. post-treatment. Data are presented as mean ± SEM and statistical differences as *p < 0.05, **p < 0.01 (two-way ANOVA paired with Holm-Sidak´s multiple comparisons test).

Figure 2

The assessment of LV structural parameters at two different time points – pre-treatment (week 0 – prior to the administration of Que) and post-treatment (week 6 – at the end of the Que treatment course). (A) Interventricular septal thickness in end-diastole (IVSd). (B) Left ventricular posterior wall thickness in end-diastole (LVPWd). (C) Relative wall thickness. (D) Left ventricular internal diameter in end-diastole (LVIDd). (E) Representative echocardiographic image of LV wall structure captured in Motion-mode in DQ group pre-treatment vs. post-treatment. Data are presented as mean ± SEM and statistical differences as *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA paired with Holm-Sidak´s multiple comparisons test).

Figure 3

(A) LV total collagen content determined by hydroxyproline assay. (B–F) Western blot analysis of relative protein expression of (B) myocyte enhancer factor-2 (MEF2), (C) pro B-type natriuretic peptide (proBNP) (D) histone deacetylase 4 (HDAC4), (E) pSer²⁴⁶-HDAC4 and (F) pSer²⁴⁶-HDAC4/HDAC4 ratio. (G) Representative Western blots of detected proteins with estimated molecular weight (kDa) compared to total protein staining with Ponceau S. Data are presented as mean ± SEM and statistical differences as *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA paired with Tukey´s multiple comparisons test).

Figure 4

Western blot analysis of relative protein expression of (A) calcineurin A (B) nuclear factor of activated T cells 3 (NFAT3), (C) GATA4 transcription factor, (D) serum response factor (SRF). (E) Representative Western blots of detected proteins with estimated molecular weight (kDa) compared to total protein staining with Ponceau S. (F) The linear regression analysis of correlation between relative protein expression of MEF2 and GATA4 in DQ group. Data are presented as mean ± SEM and statistical differences as: *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA paired with Tukey´s multiple comparisons test).

Figure 5

Western blot analysis of relative protein expression of (A) total extracellular signal-regulated kinase 1/2 (Erk1/2), (B) Phospho Erk1/2 (Thr²⁰²/Tyr²⁰⁴), (C) Phospho Erk1/2 (Thr²⁰²/Tyr²⁰⁴)/total Erk1/2 ratio (D) extracellular signal-regulated kinase 5 (Erk5), (E) Protein Phosphatase 1 Catalytic Subunit Beta (PPP1CB), (F) Protein Phosphatase 2A C Subunit (PP2A), (G) calcium/calmodulin-dependent protein kinase II (CaMKII), (H) pThr²⁸⁶-CaMKII, (I) pThr²⁸⁶-CaMKII/CaMKII ratio, (J) pan-Akt (protein kinase B), (K) pThr³⁰⁸-Akt, (L) pThr³⁰⁸-Akt/pan Akt ratio, (M–O) Representative Western blots of detected proteins with estimated molecular weight (kDa) compared to total protein staining with Ponceau S. Data are presented as mean ± SEM and statistical differences as: *p < 0.05, **p < 0.01 (one-way ANOVA paired with Tukey´s multiple comparisons test).

Figure 6

Diagrammatic representation of epigenetic and transcriptional control of hypertrophic signaling in heart. (A) Generally accepted protein pathways mediating cardiac hypertrophy. (B) Pathways targeted by quercetin in ZDF (Zucker Diabetic Fatty) rats. Quercetin attenuated expression of transcription factors MEF2, GATA4 and NFAT3 and downregulated upstream effectors HDAC4, calcineurin and Erk5.