Dataset Information

Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK.

ABSTRACT: A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody subset, is pathogenic in mesenteric I/R injury. Furthermore, it has previously been shown that the immunodominant epitope for the majority of circulating pathogenic aPLs resides in the N-terminal domain I (DI) of beta-2 glycoprotein I (?2GPI). This study describes the enhanced pathogenic effect of purified IgG derived from patients with lupus and/or the antiphospholipid syndrome in a cardiomyocyte H/R in vitro model. Furthermore, we have demonstrated a pathogenic role for aPL containing samples, mediated via aPL-?2GPI interactions, resulting in activation of the pro-apoptotic p38 MAPK pathway. This was shown to be inhibited using a recombinant human peptide of domain I of ?2GPI in the fluid phase, suggesting that the pathogenic anti-?2GPI antibodies in this in vitro model target this domain.

SUBMITTER: Bourke LT

PROVIDER: S-EPMC5386347 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK.

Bourke Lauren T LT McDonnell Thomas T McCormick James J Pericleous Charis C Ripoll Vera M VM Giles Ian I Rahman Anisur A Stephanou Anastasis A Ioannou Yiannis Y

Cell death & disease 20170112 1

A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody ...[more]

PMID: 28079888

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Project description:BackgroundAnesthetic postconditioning is a cellular protective approach whereby exposure to a volatile anesthetic renders a tissue more resistant to subsequent ischemic/reperfusion event. Sevoflurane postconditioning (SPostC) has been shown to exert cardioprotection against ischemia/reperfusion injury, but the underlying mechanism is unclear. We hypothesized that SPostC protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury by maintaining/restoring mitochondrial morphological integrity, a critical determinant of cell fate.MethodsPrimary cultures of neonatal rat cardiomyocytes (NCMs) were subjected to H/R injury (3 h of hypoxia followed by 3 h reoxygenation). Intervention with SPostC (2.4% sevoflurane) was administered for 15 min upon the onset of reoxygenation. Cell viability, Lactate dehydrogenase (LDH) level, cell death, mitochondrial morphology, mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) opening were assessed after intervention. Mitochondrial fusion and fission regulating proteins (Drp1, Fis1, Mfn1, Mfn2 and Opa1) were assessed by immunofluorescence staining and western blotting was performed to determine the level of protein expression.ResultsCardiomyocyte H/R injury resulted in significant increases in LDH release and cell death that were concomitant with reduced cell viability and reduced mitochondrial interconnectivity (mean area/perimeter ratio) and mitochondrial elongation, and with reduced mitochondrial membrane potential and increased mPTP opening. All the above changes were significantly attenuated by SPostC. Furthermore, H/R resulted in significant reductions in mitochondrial fusion proteins Mfn1, Mfn2 and Opa1 and significant enhancement of fission proteins Drp1 and Fis1. SPostC significantly enhanced Mfn2 and Opa1 and reduced Drp1, without significant impact on Mfn1 and Fis1.ConclusionsSevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury (HRI) by restoring mitochondrial fusion/fission balance and morphology.

Project description:ObjectivesTo explore the effects of miR-16-5p and PTPN4 on the apoptosis and autophagy of AC16 cardiomyocytes after hypoxia/reoxygenation treatment.MethodsAC16 cells were divided into the control group (NC), hypoxia/reoxygenation group (H/R), knockdown miR-16-5p negative control group (NC inhibitor), knockdown miR-16-5p group (miR-16-5p inhibitor), overexpression miR-16-5p negative control group (NC mimics), overexpression miR-16-5p group (miR-16-5p mimics), silent PTPN4 negative control group (sh-NC), silent PTPN4 group (sh-PTPN4), and silent PTPN4 + knockdown miR-16-5p group (sh-PTPN4 + miR-16-5p inhibitor). Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting (WB) were used to measure the expression level of miR-16-3p, miR-16-5p, protein tyrosine phosphatase nonreceptor type 4 (PTPN4), and autophagy-related proteins (beclin-1, LC3 II/I, and P26) in AC16 cells. The apoptosis level of AC16 cells in each group was measured by flow cytometry and TUNEL. The dual-luciferase reporter gene experiment was also used to verify the targeting relationship between miR-16-5p and PTPN4.ResultsAfter H/R treatment, the levels of myocardial injury markers including LDH and CK-MB in AC16 cells were increased significantly (P < 0.05), and the levels of cell apoptosis and autophagy also increased significantly (P < 0.05). The level of miR-16-3p in AC16 cells did not change significantly after H/R treatment, whereas the level of miR-16-5p was increased significantly (P < 0.05). After miR-16-5p was knocked down, the levels of LDH and CK-MB in AC16 cells treated with H/R were significantly reduced (P < 0.05), and the rates of cell apoptosis and autophagy were also significantly reduced (P < 0.05). miR-16-5p negatively regulated the expression level of PTPN4 protein in AC16 cells (P < 0.05), and the dual-luciferase reporter gene experiment confirmed that PTPN4 was the downstream target of miR-16-5p. Silencing of PTPN4 significantly increased the damage of AC16 cells induced by H/R treatment (P < 0.05), but simultaneously inhibiting the expression of PTPN4 and miR-16-5p reversed the protective effect of miR-16-5p knockdown on AC16 cells (P < 0.05).ConclusionsThe expression of miR-16-5p is upregulated in AC16 cells after H/R treatment and the knockdown which can protect AC16 cells from H/R-induced cell damage that may be due to its regulation on the expression of PTPN4.

Dataset Information

Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK.

Publications

Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK.

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