e of ROS in the heart and although superoxide is the first moiety generated by NADPH oxidase, the signaling effects of ROS appear to be mediated by the more stable and diffusible hydrogen peroxide. As we and others have shown, physiologically increased levels of H2O2 activates ROS-mediated signaling pathways including MAPK members and NF-B and induces compensated left ventricular hypertrophy . 
We recently showed that APN mediates cardioprotective effects against physiologically increased ROS 19571414 in cardiomyocytes by regulating an AMPK/ERK/NF-B signaling axis. However, pathophysiological levels of ROS causes both apoptosis and autophagy in the cardiovascular system, eventually impairing myocardial function. Although, autophagy is upregulated during periods of stress, such as cell starvation it has a “Janus-like” role in degenerative diseases such as Alzheimer’s and cancer, where it might be both deleterious or protective. In chronic cardiac stress, such as HF and hypertension, oxidative stress induces maladaptive autophagy, possibly resulting in compensated LVH transitioning to decompensated HF. Despite pathophysiological levels of 15930314 H2O2 increasing oxidative stress and cardiomyocyte apoptosis, little is known about the effects of APN on pathophysiological levels of ROS-induced autophagy. We thus sought to test the hypothesis that the cardioprotective actions of APN extends to modulating pathophysiological levels of ROS-induced autophagy in cardiomyocytes and to define the intracellular signaling pathways involved. acids 15-247) was cloned into the bacterial expression vector pTrcHisB. The histidine-tagged proteins were purified using nickel ion-agarose column, mono Q column, and for removal of lipopolysaccharide, Detoxi-Gel Affinity Pak column . Isolation and treatment of adult rat cardiac myocytes Adult rat ventricular myocytes were isolated as follows. Briefly, ARVM were harvested from adult male Sprague-Dawley rats and plated non-confluently on laminin-coated plastic culture dishes at a density of 30-50 cells/mm2. Cells were maintained at 37 C prior to treatment, in Dulbecco’s Modified Eagle Medium containing, 2mg/mL BSA, 2mmol/L L-carnitine, 5mmol/L creatinine, 5mmol/L taurine, 100IU/mL penicillin, and 10g/mL streptomycin. ARVM were subjected to various concentrations of H2O2 and the final concentration of 1mM was used, which has been described by others to induce cell death. In some experiments ARVM were pre-treated with recombinant APN for 18hrs prior to H2O2 treatment. In vivo murine model Male WT and APN-KO mice in a C57/BL6 background were used as previously described. The Institutional Animal Care and Use Committee at Boston University School of Medicine approved all study procedures related to the handling and surgery of the mice. WT and APN-KO were subjected to subcutaneous Ang-II or saline infusion using an implanted osmotic minipump. Tail cuff blood pressure, non-invasive heart rate, morphology and MedChemExpress ML-128 echocardiography measurements were performed as previously described. Fourteen days after Ang-II infusion, mice were sacrificed, hearts were dissected and the LV was snap-frozen in liquid nitrogen. Echocardiography determinants for LV dimensions Transthoracic echocardiography was performed in conscious mice using an Acuson Sequoia C-256 echocardiography machine and a 15-MHz probe as previously described. Materials and Methods All of the animals were treated according to the guidelines of the Guide for the Care and Use of Laboratory An