Cardioprotective mechanism of ω-3 fatty acid icosapent ethyl (IPE) in cardiomyocytes: role in high glucose and shear stress-induced mechano-transduction dysregulation

Abstract

Background: Omega-3 fatty acids (FAs) are long-chain fatty acids that have shown cardioprotective effects through lipid lowering, anti-inflammatory, and membrane-stabilizing properties. In this study we investigated the molecular mechanism underlying the cardioprotective effects of icosapent ethyl (IPE), an ethyl ester of omega-3 fatty (EPA), focusing on its role on mechano-transduction, a process linking cardiac contractility to intracellular signaling, that becomes dysregulated in hyperglycaemia or disturbed blood flow, both major contributors to cardiovascular diseases.

Methods: We conducted in vivo meta-analyses to assess the beneficial effects of omega-3 fatty acids on cardiac contractility and inflammation in patients with cardiovascular and cardiometabolic diseases. We investigated the effects of IPE on mechano-transduction, assessing the activation of the YAP/TAZ signalling pathway, in cardiomyocyte cells AC16 exposed to normal (NG) or high glucose (HG) conditions. We defined the role of IPE against hyperglycaemia-induced inflammation, oxidative stress, metabolism, and apoptosis by evaluating key biomarkers by Western Blot and Real-time PCR. We evaluated IPE's impact on YAP/TAZ activation and on gene expression and protein levels of primary markers related to oxidative stress, inflammation, and metabolism in a dynamic flow model of AC16 cardiomyocytes, to mimic in vivo shear stress.

Results: In vivo meta-analyses showed a significant increase of left ventricular ejection fraction (LVEF%) (mean: 0.5, 95% CI: 0.1-0.9) and a significant reduction of inflammatory markers (mean: - 1.24, 95% CI: 2.05-0.44) in patients treated with omega-3. IPE treatment reduced the activation of YAP/TAZ pathway induced by HG exposure in AC16 cells. IPE partially reversed HG-induced changes in markers of inflammation, oxidative stress, metabolism and apoptosis (p < 0.05). Similarly, in a dynamic model of shear stress, IPE treatment mitigated the turbulent flow-mediated changes in YAP/TAZ pathway, inflammation, oxidative stress and metabolism.

Conclusions: Our results demonstrate a cardioprotective role of IPE through modulation of hyperglycaemia-induced mechano-transduction dysregulation, inflammation, and oxidative stress. Additionally, our results on a shear stress model showing that IPE restores upstream regulators of YAP/TAZ and reduces disturbed flow-induced activation of pro-inflammatory pathways, suggest that IPE may exert a therapeutic effect on cardiovascular disorders associated with disturbed blood flow and hemodynamic stress.

Keywords: Cardiovascular protection; Diabetes; Hyperglycaemia; IPE; Mechano-transduction; Omega-3 fatty acids; Shear stress.

Fig. 1

Meta-analysis of beneficial effects of omega-3 fatty acids in patients with Cardiovascular and cardiometabolic diseases. A Forest plot of CVD patients treated with omega-3 fatty acids or placebo. Data are shown as mean change + SD. The graph was created using Stata software (version 16.0, Stata Corp., College Station, TX). B Forest plot of inflammation markers (IL-6, TNF-α, CRP) in patients with cardiometabolic diseases treated with omega-3 fatty acids or placebo. Data are shown as Mean + SD. The graph was created using Stata software (version 16.0, Stata Corp., College Station, TX)

Fig. 2

IPE effects on mechano-transduction in AC16 cells exposed to hyperglycemia. Western blot analysis for p-MST1 (A), p-LATS1 (B), active YAP (C) and TAZ (D) in AC16 cells exposed to NG concentration (NG), cells exposed to high glucose concentration (HG), and cells co-treated with HG and 40 µM IPE (HG + IPE). The histograms show the densitometric analysis of 3 separate experiments representing the relative expression of proteins; NG value was set as 1. Data are mean ± SEM. * P < 0.05 versus NG; ** P < 0.05 versus HG 7 days

Fig. 3

Effects of IPE on inflammation and oxidative stress in response to HG treatment

Fig. 4

IPE effects on metabolism in a static model of cardiomyocytes exposed to HG. Western blot for p-AMPK (A), PPAR-α (B, right panel) and PPAR-γ (C, right panel) in AC16 exposed to NG, HG, HG + IPE. The histograms show the densitometric analysis of 3 separate experiments representing the relative expression; NG value was set as 1. Data are mean ± SEM. * P < 0.05 versus NG; ** P < 0.05 versus HG 7 days. qRT-PCR for PPAR-α (B, left panel) and PPAR-γ (C, left panel) in AC16 exposed to NG, HG, HG + IPE. β-Actin was used as internal control. The fold increase of mRNA expression compared with NG was calculated using the 2^−ΔΔCt method. Data are mean ± SEM. * P < 0.05 vs NG; ** P < 0.05 vs HG 7 days

Fig. 5

Effects of IPE on apoptosis in cardiomyocytes exposed to hyperglycemia: A, B Bcl-2 and Bax mRNA and protein expression levels in AC16 cells exposed to NG, HG, HG with 40 µM of IPE. For q-RT PCR, β-Actin was used as internal control. The fold increase of mRNA expression compared with NG was calculated using the 2^−ΔΔCt method. Data are mean ± SEM. * P < 0.05 vs NG; ** P < 0.05 versus HG 7 days. For Western blot, the histograms show the densitometric analysis of 3 separate experiments representing the relative expression being NG value set as 1. C BAX/Bcl-2 protein expression ratio in AC16 in response to NG, HG, HG + IPE. Data are mean ± SEM. *p < 0.05 versus NG; **p < 0.05 versus HG 7 days

Fig. 6

Effects of IPE on mechano-transduction in a dynamic shear stress model of cardiomyocytes. Western blot for p-MST1 (A), p-LATS1 (B), YAP (C), TAZ (F), Integrin β3 (G), in cardiomyocytes AC16 in static normal glucose condition (NG), cells exposed to turbulent flow in normal glucose condition (NG FLOW) and cells exposed to turbulent flow in normal glucose condition in presence of IPE (NG FLOW + IPE). The histograms show the densitometric analysis of 3 separate experiments representing the relative expression; NG value was set as 1. * P < 0.05 vs NG; ** P < 0.05 vs NG FLOW. D Immunofluorescence staining for active YAP (green) and nuclei (blue) in Ac16 in NG, NG FLOW, NG FLOW + IPE. Merge columns images show overlapping signals. Scale bar = 100 µm; E) YAP intensity ratio (nuclear/cytoplasmic) from cells randomly selected from 3 independent experiments. *P < 0.05 vs. NG; ** P < 0.05 vs NG FLOW

Fig. 7

IPE effects on inflammation and oxidative stress in a dynamic shear stress model of cardiomyocytes. A, B, E qRT-PCR for NF-kB (A, left panel), IL-6 (B, left panel), SOD2 (E, left panel) in AC16 cells exposed to NG, NG FLOW, NG FLOW + IPE. β-Actin was used as internal control. The fold increase of mRNA expression compared with NG was calculated using the 2^−ΔΔCt method. Data are mean ± SEM. * P < 0.05 vs NG; ** P < 0.05 vs NG FLOW. Western blot for p-NF-kB (A, right panel), IL-6 (B, right panel), Catalase (D), SOD2 (E, right panel) in cardiomyocytes AC16 in NG, NG FLOW, NG FLOW + IPE. The histograms show the densitometric analysis of 3 separate experiments representing the relative expression; NG value was set as 1. * P < 0.05 vs NG; ** P < 0.05 vs NG FLOW

Fig. 8

IPE effects on metabolism in a dynamic model of shear stress of cardiomyocytes. A Western blot for p-AMPK in AC16 exposed to NG, NG FLOW, NG FLOW + IPE. B-C) qRT-PCR and Western blot for PPAR-α (B) and PPAR-γ (C) in AC16 exposed under NG, NG FLOW, NG FLOW + IPE conditions. For qRT-PCR, β-Actin was used as internal control. The fold increase of mRNA expression compared with NG was calculated using the 2^−ΔΔCt method. Data are mean ± SEM. * P < 0.05 vs NG; ** P < 0.05 vs NG FLOW. For Western blot analysis, the histograms show the densitometric analysis of 3 separate experiments representing the relative expression; NG value was set as 1. * P < 0.05 vs NG; ** P < 0.05 vs NG FLOW