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Entresto protected the cardiomyocytes and preserved heart function in cardiorenal syndrome rat fed with high-protein diet through regulating the oxidative stress and Mfn2-mediated mitochondrial functional integrity - 13/11/21

Doi : 10.1016/j.biopha.2021.112244 
Jui-Ning Yeh a, Ya Yue b, Yi-Ching Chu c, d, Chi-Ruei Huang c, d, Chih-Chao Yang e, John Y. Chiang f, g, Hon-Kan Yip c, d, h, i, j, k, ⁎ , Jun Guo a, ⁎
a Department of Cardiology, The First Affiliated Hospital, Jinan University, 613W. Huangpu Avenue, Guangzhou 510630, China 
b Department of Nephrology, The First Affiliated Hospital of Jinan University, Guangzhou, China 
c Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, 123, Dapi Road, Niaosung Dist., Kaohsiung City 83301, Taiwan 
d Center for Shockwave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial Hospital Kaohsiung, Taiwan 
e Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan 
f Department of Computer Science and Engineering, National Sun Yat-Sen University, Kaohsiung, Taiwan 
g Department of Healthcare Administration and Medical Informatics, Kaohsiung Medical University, Kaohsiung, Taiwan 
h Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital Kaohsiung, Taiwan 
i Department of Nursing, Asia University Taichung, Taiwan 
j Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan 
k Division of Cardiology, Department of Internal Medicine, Xiamen Chang Gung Hospital, Xiamen, Fujian, China 

⁎Corresponding author at: Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, 123, Dapi Road, Niaosung Dist., Kaohsiung City 83301, Taiwan.Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine123, Dapi Road, Niaosung Dist.Kaohsiung City83301Taiwan⁎⁎Correspondence to:The First Affiliated Hospital, Jinan University, 613 W. Huangpu Avenue, Guangzhou 510630, China.The First Affiliated Hospital, Jinan University613 W. Huangpu AvenueGuangzhou510630China

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Abstract

This study tested the hypothesis that Entresto (En) therapy protected the cardiomyocytes and heart function in cardiorenal syndrome (CRS) rats fed with high-protein diet (HPD) through regulating the oxidative-stress and Mfn2-mediated mitochondrial functional integrity. En (12.5 μM for the in-vitro study) protected the H9C2-cells against H2O2-induced cell apoptosis, whereas stepwise-increased H2O2 concentrations induced a significant increase in protein expressions of Mfn2/phosphorylated (p)-DRP1/mitochondrial-Bax in H9C2-cells. En downregulated H2O2-induced mitochondrial fission/upregulated mitochondrial fusion and deletion of Mfn2 gene (i.e., shMfn2) to significantly reduce H2O2-induced ROS production. En significantly suppressed and shMfn2 further significantly suppressed both H2O2-reduced mitochondrial-membrane potential and H2O2-induced ROS production/cell apoptosis/mitochondrial damage/mitochondrial-Bax released from mitochondria in H9C2 cells. En significantly reduced protein expressions of Mfn2 and p-DRP1. Additionally, En significantly suppressed and shMfn2 further significantly suppressed the protein expressions of mitochondrial-damaged (DRP1)/oxidative-stress (NOX-1/NOX-2)/apoptosis (mitochondrial-Bax/caspase-3/PARP)/autophagic (LC3B-II/LC3B-I) biomarkers (all p < 0.01). Rats were categorized into group 1 [sham-control + high-protein-diet (HPD)], group 2 (CRS + HPD) and group 3 (CRS+ HPD + En/100 mg/kg/day). By day 63 after CRS induction, the LVEF was significantly lower in group 3 and more significantly lower in group 2 than in group 1, whereas the protein expressions of oxidative-stress (NOX-1/NOX-2/p22phox/oxidized protein)/apoptotic (mitochondrial-Bax/caspase-3/PARP), fibrotic (Smad-3/TGF-ß), autophagic (Beclin-1/Atg5/ratio of LC3B-II/LC3B-I) and mitochondrial-damaged (DRP1/cyclophilin-D/cytosolic-cytochrome-C) biomarkers exhibited an opposite pattern of LVEF among the groups. Downregulation of Mfn2 by En or shMfn2 in cardiomyocytes avoided H2O2 damage and En improved the cardiac function in HPD-feeding CRS rat via adjusting Mfn2-mediated mitochondrial functional integrity.

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Graphical Abstract

The proposed underlying mechanism of entresto therapy in protecting the heart and kidney against cardiorenal syndrome (CRS). The in vitro results demonstrated that downregulation of Mfn2 by entresto or shMfn2 ameliorated H2O2-caused mitochondrial and cardiomyocyte damage. Additionally, entresto preserved the heart function in high-protein diet-feeding CRS rat via adjusting Mfn2-mediated mitochondrial functional integrity, downregulating oxidative stress, cell-stress signaling and mitophagy.



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Highlights

•
Entresto (En) regulated mitochondrial fusion via downregulating Mfn2.
•
Entresto suppressed oxidative-stress & cell-stress signaling via regulating Mfn2.
•
Downregulated Mfn2 was more effective than En on ROS suppression.
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En-mediated Mfn2 preserved mitochondrial and heart functions.

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Keywords : Entresto, Mitofusin 2, Oxidative stress, Mitochondrial damage, Left ventricular ejection fraction (LVEF), Cardiorenal syndrome


Plan

Introduction
Materials and methods
Ethics statement
H9C2 cell culture
Cell transfection and Mfn2 gene knockdown
H2O2-treated H9C2 for assessment of the cytotoxicity and cell survival rate
Cell model for determining the therapeutic impact of sacubitril (Sac)/Valsartan (Val) treatment on protecting H9C2 cell line against oxidative stress
MTT cell viability assay
Flow cytometric analysis for H9C2 cells apoptosis, intracellular ROS and mitochondrial membrane potential
In-vitro study design for examining the cardiomyocyte-protective effect of En against p-Cresol-induced damage
Animal model of dilated cardiomyopathy (DCM)
Animal model of CKD
Rationale of high-protein diet (HPD) for all CKD animals
Transthoracic echocardiographic examination for left ventricular ejection fraction (LVEF)
Western blot analysis of LV specimens
Immunohistochemical (IHC) and immunofluorescent (IF) studies
Histological quantification of myocardial and kidney fibrosis
Assessment of oxidative stress
Statistical analysis
Results
To assess the suitable H2O2 concentration as an oxidative stress tool for the study and determine the optimal therapeutic dose of En ()
To assess the suitable therapeutic dose of Sac/Val on cell proliferation and protection of H9C2 cells against H2O2 damage ()
Lower and higher doses of Val/Sac offered an identical effect on protecting the H2O2-induced H9C2 cell apoptosis ()
Impact of a lower and higher dose of Sac/Val on regulating the mitochondrial fusion and fission undergoing the oxidative stress ()
Deletion of Mfn2 gene expression (shMfn2) and Sac/Val treatment inhibited H2O2-induced ROS and reversed H2O2-induced downregulation of mitochondrial membrane potential (MMP) in H9C2 cells ()
Deletion of Mfn2 gene expression (shMfn2) and Sac/Val treatment inhibited H2O2-induced H9C2 cell apoptosis ()
Deletion of Mfn2 gene expression (shMfn2) and Sac/Val treatment inhibited H2O2-induced cytochrome C released from mitochondria to cytosol in H9C2 cells ()
Deletion of Mfn2 gene expression (shMfn2) and Sac/Val treatment suppressed H2O2-induced translocation of Bax from the cytoplasm to mitochondria in H9C2 cells ()
Mitochondrial-associated protein expressions were proportional to a stepwise increase in H2O2 concentrations and shMfn2 stirred Val/Sac on regulating the protein expressions of mitochondrial fusion and fission ()
Impact of shMfn2 and Sac/Val on suppressing H2O2-induced protein expressions of fusion, fission, oxidative stress, apoptosis and autophagy in H9C2 cells ()
Time courses of cardiac function, and cardiomyocyte size, the ratio of heart weight to body weight, creatinine level and protein expressions of mitochondrial damaged and autophagic biomarkers in LV specimen by day 63 after CRS induction ()
The protein expressions of oxidative stress, apoptosis, fibrosis and angiotensin II receptor in LV specimen by day 63 after CRS ()
Time courses of circulating inflammatory and heart failure biomarkers and protein expressions of cell stress signaling, mitochondrial biogenesis markers and oxidative phosphorylation in LV specimen by day 63 after CRS ()
En treatment markedly suppressed fibrosis in heart and kidney and cellular expression of BNP in LV myocardium by day 63 after CRS induction ()
En therapy suppressed p-Cresol-induced oxidative stress in H9C2 cells ()
En therapy downregulated p-Cresol-induced apoptosis, autophagy, mitochondrial damage, and DNA damage and preserved mitochondrial biogenesis in H9C2 cells ()
En therapy suppressed the cellular and mitochondrial levels of ROS and γ-H2AX and preserved mitochondrial membrane potential (MMP) in H9C2 cells ()
Discussion
Study limitation
CRediT authorship contribution statement

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