Vinpocetine Attenuates Pathological Cardiac Remodeling by Inhibiting Cardiac Hypertrophy and Fibrosis

Abstract

Purpose: Pathological cardiac remodeling, characterized by cardiac hypertrophy and fibrosis, is a pathological feature of many cardiac disorders that leads to heart failure and cardiac arrest. Vinpocetine, a derivative of the alkaloid vincamine, has been used for enhancing cerebral blood flow to treat cognitive impairment. However, its role in pathological cardiac remodeling remains unknown. The aim of this study is to examine the effect of vinpocetine on pathological cardiac remodeling induced by chronic stimulation with angiotensin II (Ang II).

Methods: Mice received Ang II infusion via osmotic pumps in the presence of vehicle or vinpocetine. Cardiac hypertrophy and fibrosis were assessed by morphological, histological, and biochemical analyses. Mechanistic studies were carried out in vitro with isolated mouse adult cardiac myocytes and fibroblasts.

Results: We showed that chronic Ang II infusion caused cardiac hypertrophy and fibrosis, which were all significantly attenuated by systemic administration of vinpocetine. In isolated adult mouse cardiomyocytes, vinpocetine suppressed Ang II-stimulated myocyte hypertrophic growth. In cultured cardiac fibroblasts, vinpocetine suppressed TGFβ-induced fibroblast activation and matrix gene expression, consistent with its effect in attenuating cardiac fibrosis. The effects of vinpocetine on cardiac myocyte hypertrophy and fibroblast activation are likely mediated by targeting cyclic nucleotide phosphodiesterase 1 (PDE1).

Conclusions: Our results reveal a novel protective effect of vinpocetine in attenuating pathological cardiac remodeling through suppressing cardiac myocyte hypertrophic growth and fibroblast activation and fibrotic gene expression. These studies may also shed light on developing novel therapeutic agents for antagonizing pathological cardiac remodeling.

Keywords: Cardiac fibrosis; Cardiac hypertrophy; Cardiac remodeling; PDE1; Vinpocetine.

Fig. 1

Vinpocetine attenuated cardiac hypertrophy induced by Ang II infusion in vivo. a Representative gross heart images showing the effect of vinpocetine on global heart enlargement induced by Ang II. Scale bars 3 mm. Mice were infused with saline (control) or Ang II (800 ng/min/kg) for 14 days and systemically administrated with vehicle or vinpocetine (5 mg/kg/day) daily via IP injection for 17 days (3 days prior and 14 days during Ang II infusion). b, c Quantified results of Ang II-infused model: heart/body weight (HW/BW) ratio (mg/g) or heart weight/tibia length (HW/TL) ratio (mg/mm). Data represent means ± SEM from control (n = 8), Ang II (n = 10), and Ang II + vinpocetine (n = 10). *P < 0.05 vs. control group and ^#P < 0.05 vs. Ang II group

Fig. 2

Vinpocetine inhibited cardiomyocyte hypertrophy induced by Ang II in vivo. a Representative images of heart section stained for cell membrane with oregon green 488 or fluorescein isothiocyanate-conjugated wheat germ agglutinin (WGA). Myocyte cross-sectional area, the area encircled by a yellow dotted line, was calculated. Scale bars 20 μm. b Quantified results of cross-sectional area (averaged from 200 random myocytes per section per animal; n = 8 for the control group, n = 10 for the Ang II, and n = 10 for the Ang II + vinpocetine group. c Hypertrophic marker BNP mRNA expression normalized to GAPDH analyzed by quantitative real-time RT-PCR (n = 5). *P < 0.05 vs. the control group and ^#P < 0.05 vs. the Ang II group

Fig. 3

Vinpocetine inhibited cardiac fibrosis induced by Ang II in vivo. a Representative images showing collagen deposition (blue) in left ventricle of mouse hearts depicted by Masson’s trichrome staining. b Quantification of percentage fibrosis (n = 8 for the control group, n = 10 for the Ang II group, and n = 10 for the Ang II + vinpocetine group). Scale bar 50 μm. *P < 0.05 vs. the control group and ^#P < 0.05 vs. the Ang II group

Fig. 4

Vinpocetine inhibited cardiomyocyte hypertrophy in vitro. a Representative bright-field images of isolated cardiomyocytes treated Ang II (100 nM) or saline (control) for 72 h to induce hypertrophic growth. Scale bar 50 μm. b Vinpocetine dose-dependently inhibited car-diomyocyte hypertrophic growth. Adult mouse myocytes were pre-treated with indicated dose of vinpocetine, followed by Ang II (100 nM) or saline (control) stimulation for 72 h. Quantification of cell area (the area encircled by yellow dotted line in a. Data represent the averages of a minimum of 100 cardiomyocytes from each animal heart preparation and n = 5 independent animal hearts per condition. c IC86340 dose-dependently inhibited cardiomyocyte hypertrophic growth. Adult mouse myocytes were pre-treated with indicated dose of IC86340, followed by Ang II (100 nM) or saline (control) stimulation for 72 h. d Effects of vinpocetine and IC86340 together on cardiomyocyte hypertrophy. Adult mouse myocytes were pre-treated with vehicle or IC86340 (15 μM) with different doses of vinpocetine as indicated, followed by Ang II (100 nM) or saline (control) stimulation for 72 h. e Effects of vinpocetine on intracellular cGMP levels in adult mouse cardiomyocytes (n = 3). *P < 0.05 vs. the control group. *P < 0.05 vs. the control group and ^#P < 0.05 vs. the Ang II group. ns no significant difference

Fig. 5

Vinpocetine inhibited cardiac fibroblast activation and ECM synthesis in vitro. a Representative immunofluorescent microscopic images showing the effects of vinpocetine on α-SMA protein levels in cultured mouse adult cardiac fibroblasts. Cardiac fibroblasts at passage 1 were seeded and cultured overnight in DMEM supplemented with 10% FBS, serum-starved for 48 h, and pretreated with vehicle of vinpocetine (10 μM), then stimulated with 10 ng/mL of TGFβ for 24 h. α-SMA protein was immunostained (green) and nucleus was stained by DAPI (blue). b–d qRT-PCR results showing effects of the vinpocetine on TGFβ stimulated pro-fibrotic marker gene expression, including α-SMA (b), type I collagen (Col1) (c), and fibronectin 1 (Fn 1) (d). e Effects of vinpocetine and PDE1 inhibition together on cardiac fibroblast activation. Adult mouse cardiac fibroblasts were pre-treated with vehicle, IC86340 (15 μM), or both of vinpocetine (10 μM) and IC86340 (15 μM), followed by stimulation with TGFβ (10 ng/mL) for 24 h. The similar results were obtained from at least three independent experiments. *P < 0.05 vs. the control group and ^#P < 0.05 vs. the Ang II group. ns no significant difference

Fig. 6

Schematic diagram illustrating a proposed mechanism by which vinpocetine antagonizes pathological cardiac remodeling including cardiac hypertrophy and fibrosis by attenuating myocyte hypertrophic growth and fibroblast activation and matrix production. The potential molecular target is PDE1