The Protective Role of Paraoxonase 2 in Cardiomyocytes Against Myocardial Ischemia‐Reperfusion Injury

Acute myocardial infarction (AMI) induced ischemic injury results in ion accumulation, particularly calcium overload, ATP depletion and cell death. Following AMI, restoration of blood flow, or reperfusion, to the myocardium exacerbates cell death, primarily through the generation of reactive oxygen species (ROS) and the opening of the mitochondrial permeability transition pore (mPTP). Paraoxonase 2 (PON2), an intracellular protein with antioxidant, anti‐inflammatory properties, is localized in the mitochondria and is known to protect against mitochondrial oxidative stress and dysfunction. Hypothesis PON2 protects cardiomyocytes against myocardial ischemia‐reperfusion (IR) injury by mitigating mitochondrial oxidative stress and dysfunction. Methods Male PON2 deficient (PON2‐def) and C57BL6/J (WT) mice were subjected to in vivo IR injury via left anterior descending coronary artery occlusion (30‐minute) and reperfusion (24‐hours), and PON2 gene expression and infarct sizes were evaluated. Excised PON2‐def and WT hearts were subjected to ex vivo Langendorff IR injury (20‐minute ischemic episode, 5‐minute reperfusion). Following ex vivo IR injury, cardiomyocytes were isolated and mitochondrial calcium retention capacity (CRC), generation of ROS and cell apoptosis (TUNEL assay) were measured (n=3–4/group). Stable cell lines were generated in H9c2 cells, a rat ventricular cardiomyocyte cell line, which overexpressed human PON2 (H9c2‐hPON2) and empty vector (H9c2‐EV). H9c2 cell lines were subjected to hypoxia‐reoxygenation (HR) injury [3‐hours in 2% O 2 (hypoxia), followed by 2‐hours in normoxic conditions (reperfusion)]. The cells were also subjected to HR injury with and without LY294002, a potent PI3K inhibitor. Following HR, CRC and ROS generation were measured (n=3–4/group). Data is expressed as mean±SEM. T‐test and two‐way ANOVA are used for statistical analysis, with probability values less than 0.05 considered statistically significant. Results Following in vivo IR injury, PON2‐def mice exhibited a ~2‐fold larger infarct than WT mice, and PON2 gene expression was ~2‐fold upregulated in the WT mice. Cardiomyocytes from PON2‐def mice subjected to ex vivo IR injury had reduced CRC (160±32 in WT, 80±0 in PON2‐def, nmoles calcium/6.0×10 4 cells, p<0.05), 2‐fold increased ROS production (p<0.05) and increased apoptosis compared to WT controls (25% of TUNEL positive nuclei in PON2‐def vs 15% in WT, p<0.05). H9c2‐hPON2 cells subjected to HR injury exhibited increased CRC compared to H9c2‐EV cells (141.7±16.67 in H9c2‐EV, 200±14.43 in H9c2‐hPON2, nmoles calcium/1.5×10 6 cells, p<0.05), indicating that PON2 overexpression increases the threshold for the opening of the mPTP in response to calcium overload. H9c2‐hPON2 cells demonstrated a 40% reduction in ROS generation versus the H9c2‐EV control cells post HR injury (p<0.05). Treatment with LY294002 abolished the protective effects of overexpressing PON2 with significantly reduced CRC in the H9c2‐hPON2 group (105±5 in H9c2‐EV+PI3Ki, 60±10 in H9c2‐hPON2+PI3Ki, nmoles calcium/1.5×10 6 cells, p<0.05) and a 60% increase in ROS (p<0.05) production versus H9c2‐EV. Conclusion PON2 plays a protective role in cardiomyocytes against myocardial IR injury by inhibiting mPTP opening and reducing ROS generation and functions by way of the PI3K/Akt/GSK‐3β pathway. Our results suggest that PON2 is a potential therapeutic candidate to ameliorate myocardial IR injury. Support or Funding Information This work is supported by NHLBI grant R01 HL071776‐01. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .