Cardioprotective Action of Ginkgo biloba Extract against Sustained β-Adrenergic Stimulation Occurs via Activation of M2/NO Pathway

Abstract

Ginkgo biloba is the most popular phytotherapic agent used worldwide for treatment of several human disorders. However, the mechanisms involved in the protective actions of Ginkgo biloba on cardiovascular diseases remain poorly elucidated. Taking into account recent studies showing beneficial actions of cholinergic signaling in the heart and the cholinergic hypothesis of Ginkgo biloba-mediated neuroprotection, we aimed to investigate whether Ginkgo biloba extract (GBE) promotes cardioprotection via activation of cholinergic signaling in a model of isoproterenol-induced cardiac hypertrophy. Here, we show that GBE treatment (100 mg/kg/day for 8 days, v.o.) reestablished the autonomic imbalance and baroreflex dysfunction caused by chronic β-adrenergic receptor stimulation (β-AR, 4.5 mg/kg/day for 8 days, i.p.). Moreover, GBE prevented the upregulation of muscarinic receptors (M₂) and downregulation of β₁-AR in isoproterenol treated-hearts. Additionally, we demonstrated that GBE prevents the impaired endothelial nitric oxide synthase activity in the heart. GBE also prevented the pathological cardiac remodeling, electrocardiographic changes and impaired left ventricular contractility that are typical of cardiac hypertrophy. To further investigate the mechanisms involved in GBE cardioprotection in vivo, we performed in vitro studies. By using neonatal cardiomyocyte culture we demonstrated that the antihypertrophic action of GBE was fully abolished by muscarinic receptor antagonist or NOS inhibition. Altogether, our data support the notion that antihypertrophic effect of GBE occurs via activation of M₂/NO pathway uncovering a new mechanism involved in the cardioprotective action of Ginkgo biloba.

Keywords: Ginkgo biloba; cardiac hypertrophy; cholinergic signaling; chronic β-adrenergic stimulation; endothelial nitric oxide synthase.

FIGURE 1

Ginkgo biloba extract restores the impaired cardiovascular autonomic modulation and baroreflex sensitivity of hypertrophic hearts. (A) low frequency band (LF), (B) high frequency band (HF), (C) LF/HF ratio, and (D) baroreceptor reflex sensitivity (BRS). Data are represented as means ± SEM, (n = 5). ^∗p < 0.05 and ^∗∗p < 0.01 vs. CTR, ^#p < 0.05 and ^##p < 0.01 vs. ISO, one-way ANOVA followed by Bonferroni’s post-test.

FIGURE 2

Ginkgo biloba extract modulates cardiac muscarinic receptor (M₂) and β-adrenergic receptors (β₁-AR) in hypertrophied hearts. (A,B, top) Representative western blot and quantitative analysis of M₂ (A) and β₁-AR (B) protein levels. Data are represented as means ± SEM, (n = 3–6). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. CTR, ^##p < 0.01 vs. ISO, one-way ANOVA followed by Bonferroni’s post-test.

FIGURE 3

Ginkgo biloba extract restores endothelial nitric oxide synthase (eNOS) protein expression and activity of hypertrophic hearts. (A–D, top) Representative western blot and quantitative analysis of eNOS (A), peNOS^ser1177(B), nNOS (C), and pnNOS^ser852 (D) protein levels. Data are represented as means ± SEM, (n = 4–6). ^∗p < 0.05 vs. CTR, ^#p < 0.05 vs. ISO, one-way ANOVA followed by Bonferroni’s post-test.

FIGURE 4

Ginkgo biloba extract prevents the development of cardiac hypertrophy induced by chronic β-adrenergic stimulation. (A) Histological sections of the left ventricle stained with hematoxylin-eosin in each group. The arrows highlight the cellular nucleus for each group. (B) Quantitative analysis of mean nuclear area in all assessed groups. Data are represented as means ± SEM, (n = 3). ^∗∗p < 0.01 vs. CTR, ^##p < 0.01 vs. ISO, one-way ANOVA followed by Bonferroni’s post-test.

FIGURE 5

Ginkgo biloba extract prevents pathological myocardial remodeling induced by chronic β-adrenergic stimulation. Histological sections of the left ventricle stained with hematoxylin-eosin in all of the groups evaluated. The CTR and Ginkgo biloba extract (GBE) groups demonstrate the cardiac striated muscle morphology. Areas of parenchymal replacement by fibrocellular connective tissue are highlighted by arrows. The ISO group demonstrates areas of parenchyma replacement by intense cardiac spindle cell proliferation (fibroblasts). Note that the ISO + GBE group presented a smaller area of parenchymal replacement by fibrocellular tissue (n = 3).

FIGURE 6

Ginkgo biloba extract prevents typical electrocardiographic changes of cardiac hypertrophy. (A) Representative ECG recordings of four experimental groups. Asterisk indicates ST segment depression with a negative T-wave. Bar graph shows that GBE prevented increases in the (B) QRS complex, (C) QTc, and (D) intrinsicoid deflection (ID) interval. Data are represented as means ± SEM, (n = 14). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. CTR; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 vs. ISO, one-way ANOVA followed by Bonferroni’s Test.

FIGURE 7

Ginkgo biloba extract restores the impaired ventricular contractility induced by chronic β-adrenergic stimulation. (A) Representative left ventricular pressure recordings. Bar graph shows (B) left ventricular developed pressure measurements, (C) coronary perfusion pressure, (D) representative western blot and quantitative analysis of SERCA2. Data are represented as means ± SEM, (n = 6). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 vs. CTR; ^#p < 0.05 and ^###p < 0.001 vs. ISO, one-way ANOVA followed by Bonferroni’s post-test.

FIGURE 8

Ginkgo biloba extract suppresses cellular hypertrophy induced by isoproterenol via M₂/NO pathway in rat neonatal cardiomyocytes. (A) Representative immunofluorescence images from phalloidin/DAPI stained neonatal cardiomyocytes in control (CTR), Ginkgo biloba extract (GBE, 100 μg/mL), isoproterenol (ISO, 10 μM), isoproterenol plus Ginkgo biloba extract (ISO + GBE), isoproterenol plus Ginkgo biloba extract and muscarinic receptor antagonist, atropine (ISO + GBE + AT), isoproterenol plus Ginkgo biloba extract and nitric oxide synthase inhibitor, L-NAME (ISO + GBE + L-NAME) treated cells. (B) Quantification of cardiomyocyte surface area from experiments shown in (A). Scale bar = 20 μm. Data are represented as means ± SEM, (n = 44–82 cells analyzed). ^∗p < 0.05 vs. CTR and ^#p < 0.05 vs. ISO, one-way ANOVA followed by Bonferroni’s post-test.