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BACKGROUND:Mitochondrial oxidation is a major source of reactive oxygen species (ROS) and mitochondrial dysfunction plays a central role in development of heart failure (HF). Paraoxonase 2 deficient (PON2-def) mitochondria are impaired in function. In this study, we tested whether PON2-def aggravates HF progression. METHODS AND RESULTS:Using qPCR, immunoblotting and lactonase activity assay, we demonstrate that PON2 activity was significantly decreased in failing hearts despite increased PON2 expression. To determine the cardiac-specific function of PON2, we performed heart transplantations in which PON2-def and wild type (WT) donor hearts were implanted into WT recipient mice. Beating scores of the donor hearts, assessed at 4 weeks post-transplantation, were significantly decreased in PON2-def hearts when compared to WT donor hearts. By using a transverse aortic constriction (TAC) model, we found PON2 deficiency significantly exacerbated left ventricular remodeling and cardiac fibrosis post-TAC. We further demonstrated PON2 deficiency significantly enhanced ROS generation in heart tissues post-TAC. ROS generation was measured through dihydroethidium (DHE) using high-pressure liquid chromatography (HPLC) with a fluorescent detector. By using neonatal cardiomyocytes treated with CoCl2 to mimic hypoxia, we found PON2 deficiency dramatically increased ROS generation in the cardiomyocytes upon CoCl2 treatment. In response to a short CoCl2 exposure, cell viability and succinate dehydrogenase (SDH) activity assessed by MTT assay were significantly diminished in PON2-def cardiomyocytes compared to those in WT cardiomyocytes. PON2-def cardiomyocytes also had lower baseline SDH activity. By using adult mouse cardiomyocytes and mitochondrial ToxGlo assay, we found impaired cellular ATP generation in PON2-def cells compared to that in WT cells, suggesting that PON2 is necessary for proper mitochondrial function. CONCLUSION:Our study suggests a cardioprotective role for PON2 in both experimental and human heart failure, which may be associated with the ability of PON2 to improve mitochondrial function and diminish ROS generation.

QQ plots of different tests in detecting DE isoforms.

Comparison of isoform expression estimates from Cufflinks, RSEM and MMSEQ.

To directly assess the physiological roles of sarcoplasmic reticulum (SR) and miitochondria (MT), we have utilized energy dispersive electron probe microanalysis (EPMA) on ultrathin freeze-dried cryosections from isolated papillary muscles, rapidly frozen at precise time points of the contractile cycle. Using this approach, we can detect redistribution of subcellular Ca2+ during the cardiac contractile cycle. Changes in Ca2+ of less than 1.0 mmol/kg dry wt can be detected. By determining the variability of the Ca2+ measurements in preliminary experiments, we have also demonstrated that it is possible to optimize experimental design, i.e., to predict the number of animals per treatment group and the number of X-ray spectra per animal that are required in order to detect a specified Ca2+ difference. Quantitative EPMA of rapidly frozen contracting papillary muscle has also allowed us to correlate the Ca2+ content of SR and MT with the contractile state of the muscle. Our results show a decrease of 40% in the amount of Ca2+ stored in the junctional SR during a cardiac muscle twitch, thus providing direct evidence for a role of the SR as a primary site of Ca2+ release. In addition, we have demonstrated dissociation between MT Ca2+ uptake and activation of regulatory enzymes, such as pyruvate dehydrogenase, indicating that MT Ca2+ uptake is not required for activation of MT metabolism.