Korean J Physiol Pharmacol.  2019 Jan;23(1):37-45. 10.4196/kjpp.2019.23.1.37.

Nicorandil alleviated cardiac hypoxia/reoxygenation-induced cytotoxicity via upregulating ketone body metabolism and ACAT1 activity

• Bai YP
• Han LS

• KMID: 2443614
• DOI: http://doi.org/10.4196/kjpp.2019.23.1.37

Abstract

To study the effect of nicorandil pretreatment on ketone body metabolism and Acetyl-CoA acetyltransferase (ACAT1) activity in hypoxia/reoxygenation (H/R)-induced cardiomyocytes. In our study, we applied H9c2 cardiomyocytes cell line to evaluate the cardioprotective effects of nicorandil. We detected mitochondrial viability, cellular apoptosis, reactive oxygen species (ROS) production and calcium overloading in H9c2 cells that exposed to H/R-induced cytotoxicity. Then we evaluated whether nicorandil possibly regulated ketone body, mainly β-hydroxybutyrate (BHB) and acetoacetate (ACAC), metabolism by regulating ACAT1 and Succinyl-CoA:3-keto-acid coenzyme A transferase 1 (OXCT1) protein and gene expressions. Nicorandil protected H9c2 cardiomyocytes against H/R-induced cytotoxicity dose-dependently by mitochondria-mediated anti-apoptosis pathway. Nicorandil significantly decreased cellular apoptotic rate and enhanced the ratio of Bcl-2/Bax expressions. Further, nicorandil decreased the production of ROS and alleviated calcium overloading in H/R-induced H9c2 cells. In crucial, nicorandil upregulated ACAT1 and OXCT1 protein expressions and either of their gene expressions, contributing to increased production of cellular BHB and ACAC. Nicorandil alleviated cardiomyocytes H/R-induced cytotoxicity through upregulating ACAT1/OXCT1 activity and ketone body metabolism, which might be a potential mechanism for emerging study of nicorandil and other K(ATP) channel openers.

Keyword

Acetyl-CoA acetyltransferase; Hypoxia reoxygenation; Ketone body; Nicorandil; OXCT1

MeSH Terms

Acetyl-CoA C-Acetyltransferase
Apoptosis
Calcium
Cell Line
Coenzyme A
Gene Expression
Metabolism*
Myocytes, Cardiac
Nicorandil*
Reactive Oxygen Species
Transferases
Acetyl-CoA C-Acetyltransferase
Calcium
Coenzyme A
Nicorandil
Reactive Oxygen Species
Transferases

Figure

• 

  Fig. 1 Effects of nicorandil on cell and mitochondrial viability in H/R-induced H9c2 cells. (A) Hypoxia/reoxygenation protocol and drug treatment. Nicorandil was pretreatment 24 h before H/R. H/R was induced by incubating cells in KRB buffer as hypoxia for 4 h followed by incubating cells in cultural medium as reoxygenation for 12 h. (B, D) Effects of Nicorandil (25, 50, 100 µM) on cell viability under normoxia or hypoxia conditioning determined by MTT assay. Data (n=3) were shown as the mean±SEM, NS: no significance. (C, E) Effects of nicorandil (25, 50, 100 µM) on mitochondrial viability under normoxia or hypoxia conditioning determined by mitochondrial viability assay. Data (n=3) were shown as the mean±SEM, ###p<0.001 vs. control group, **p<0.01 vs. H/R group, ***p<0.001 vs. H/R group.

• 

  Fig. 2 Effects of nicorandil on cellular apoptosis, ROS production and calcium overloading in H/R-induced H9c2 cells. (A, B) Effects of nicorandil (25, 50, 100 µM) on cellular apoptosis was detected by PI and Annexin V double staining evaluated by flow cytometry. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, **p<0.01 vs. H/R group, ***p<0.001 vs. H/R group. (C) ROS production was detected by H2DCFDA probe staining and evaluated by flow cytometry. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, **p<0.01 vs. H/R group, ***p<0.001 vs. H/R group. (D) Calcium concentration was detected by Fluo-4 AM staining and evaluated by flow cytometry. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, *p<0.05 vs. H/R group, ***p<0.001 vs. H/R group. (E, F) The expression of Bcl-2 and Bax were detected by western blotting. β-Actin as a loading control. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, *p<0.05 vs. H/R group, ***p<0.001 vs. H/R group.

• 

  Fig. 3 The effects of nicorandil on ACAT1 and OXCT1 protein expressions in H/R-induced H9c2 cells. (A) The expression of ACAT1 and OXCT1 were detected by western blotting. β-Actin as a loading control. (B) The relative protein expressions were calculated by normalizing to β-Actin. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, **p<0.01 vs. H/R group, ***p<0.001 vs. H/R group. (C) mRNA levels were quantified by real-time RT-PCR. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, **p<0.01 vs. H/R group, ***p<0.001 vs. H/R group.

• 

  Fig. 4 Effects of nicorandil on ketone bodies productions in H/R-induced H9c2 cells. (A) The concentration of β-hydroxybutyrate (BHB) in cell extractions. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, **p<0.01 vs. H/R group, ***p<0.001 vs. H/R group. (B) The concentration of acetoacetate (ACAC) in cell extractions. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, ***p<0.001 vs. H/R group.

• 

  Fig. 5 Effects of nicorandil on productions of acetyl-CoA and succinate in H/R-induced H9c2 cells. (A) The concentration of acetyl-CoA in cell extractions. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, **p<0.01 vs. H/R group. (B) The concentration of succinate in cell extractions. Data (n=3) were presented as mean±SEM. ###p<0.001 vs. control group, **p<0.01 vs. H/R group, ***p<0.001 vs. H/R group.

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