Ischemic preconditioning/ischemic postconditioning alleviates anoxia/reoxygenation injury via the Notch1/Hes1/VDAC1 axis

Abstract

Ischemic preconditioning (IPC), and ischemic postconditioning (IPost) have a significant protective effect on myocardial ischemia/reperfusion (MI/R) injury by alleviating oxidative stress and mitochondrial disturbances, although the underlying molecular mechanisms are unclear. The study was to demonstrate that cardioprotection against anoxia/reoxygenation (A/R) injury is transduced via the Notch1/Hes1/VDAC1 signaling pathway. Using mass spectrometry and tandem affinity purification (TAP), to screen for differentially expressed proteins associated with Hes1, followed by standard bioinformatics analysis. The co‐immunoprecipitation (Co‐IP) assay confirmed an interaction between Hes1 and VDAC1 proteins. H9c2 cells were transfected with Hes1 adenoviral N‐terminal TAP vector (AD‐NTAP/Hes1) and Hes1‐short hairpin RNA adenoviral vector (AD‐Hes1‐shRNA) to establish A/R injury, IPC, and IPost models, respectively. The expression of Hes1 and VDAC1 proteins were measured by western blot analysis, while the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and apoptosis were evaluated by flow cytometry. AD‐NTAP/Hes1 can activate the exogenous protein expression of Hes1, thus decreasing creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activity and promoting cell viability. The study found that VDAC1 was a potential target protein for Hes1 and the overexpression of Hes1 protein expression downregulated protein expression levels of VDAC1, reduced ROS production, stabilized ΔΨm, and inhibited apoptosis in H9c2 cells. Additionally, downregulation of Hes1 protein expression also upregulated VDAC1 protein expression, increased ROS production, imbalanced ΔΨm, promoted cell apoptosis, and attenuated the cardioprotection afforded by IPC and IPost. The Notch1/Hes1 signaling pathway activated by IPC/IPost can directly downregulate the protein expression of VDAC1 and consequently relieve A/R injury.

Activation of PPAR-α attenuates myocardial ischemia/reperfusion injury by inhibiting ferroptosis and mitochondrial injury via upregulating 14-3-3η

“…As presented in a previous study 21 , an in vitro MIRI model (anoxia/reoxygenation injury model) was established as follows: First, H9c2 cardiomyocytes were cultured in anoxia lipid (CaCl 2 1.0 mM, HEPES 20 mM, KCl 10 mM, MgSO 4 1.2 mM, NaCl 98.5 mM, NaH 2 PO 4 0.9 mM, NaHCO 3 6 mM, and sodium lactate 40 mM, pH 6.8) at 95% N 2 and 5% CO 2 for 3 h. Subsequently, reoxygenation lipid (CaCl 2 1.0 mM, glucose 5.5 mM, HEPES 20 mM, KCl 5 mM, MgSO 4 1.2 mM, NaCl 129.5 mM, NaH 2 PO 4 0.9 mM, and NaHCO 3 20 mM, pH 7.4) was applied to mimic reperfusion, and H9c2 cardiomyocytes were cultured in 95% O 2 and 5% CO 2 for 2 h (normal conditions).…”

Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

“…As described in previous studies ( 20 , 21 ), the cell A/R liquid method was applied to establish an in vitro A/R model. H9c2 cells were incubated in anoxia medium (CaCl 2 1.0, HEPES 20.0, KCl 10.0, MgSO 4 1.2, NaCl 98.5, NaH 2 PO 4 0.9, NaHCO 3 6.0 and sodium lactate 40.0 mM, pH 6.8) and 95% N 2 and 5% CO 2 at 37°C for 3 h. Reoxygenation medium (CaCl 2 1.0, glucose 5.5, HEPES 20.0, KCl 5.0, MgSO 4 1.2 mM, NaCl 129.5, NaH 2 PO 4 0.9 and NaHCO 3 20.0 mM, pH 7.4) was used to simulate IRI and cells were incubated under 95% O 2 and 5% CO 2 at 37°C for 2 h.…”

“…As a key component of the outer membrane of mitochondria, VDAC1 participates in the transportation of metabolites, nucleotides and ions ( 16 ). Moreover, previous studies have shown that expression of VDAC1 is upregulated and indicates more severe myocardial damage after anoxia/reoxygenation (A/R) treatment ( 18 , 20 ).…”

Role of the Notch1 signaling pathway in ischemic heart disease (Review)

“…Notch1 also mediates cardioprotection due to IPC by regulating STAT3 phosphorylation (94). The Notch1/Hes1 signaling pathway is activated by IPC/IPost and has been confirmed to directly downregulate the expression of voltage-dependent anion channel 1 and relieve MI/RI (95).…”