Roles Of Autophagy In Myocardial Damage Soon After Severe Burns And Exploration On Its Regulation Mechanisms

Background: The prompt myocardial damage and cardiac dysfunction are key pointsto initiate the ischemic/hypoxic injuries to other organs at early stages in severe burns.However, the exact mechanisms of the prompt myocardial damage are still not totallyclarified. Autophagy acts as a highly conservative mechanism of self-protection bydigesting the long-life proteins and damaged organelles to recycle nutrients in eukaryocytes.And the autophagic cell death is described as a second type of programmed cell deathwhich differs from apoptosis and necrosis. The present study is designed to investigate theroles of autophagy in the myocardial damage after severe burns and further to explore theregulation mechanisms of autophagy under the conditions of hypoxia and Ang IIstimulation.Methods: Firstly, based on the burn model of30%TBSA third degree scald rats, wedetected the cardiac function in vivo and in vitro on a Langendorff apparatus at1,3,6, and12h post-burn. Western blotting was used to determine the expressions of LC3and Beclin1proteins which indicated the autophagic activity in myocardium after burns.Immunofluorescence staining was performed to label myocardial cell death includingautophagic cell death (ubiquitin labeling), apoptosis (TUNEL labeling), and oncosis (C5b9labeling). For the perfusion hearts isolated at6h post-burn, the autophagy activator andinhibitor, ACEI, AT1blocker, and ROS inhibitor were added to the K-H perfusion bufferand the following changes in myocardial autophagy and cardiac function were thendetermined. Further, transmission electron microscope was used to confirm the changes ofautophagic status in the cultured neonatal cardiomyocytes of S-D rats under hypoxia and/orAng II stimulation. Based on the cultured cell model, in vivo fluorescence staining of DHE(ROS detection) and MDC (autophagosome vacuole labeling) were performed to explorethe relationship between ROS and autophagy in the setting of ROS inhibition. Expression of PKCδ and PKCε in the cultured cardiomyocytes under stress of hypoxia and Ang II werequantified by an ELISA method. Using the same method, we confirmed the downregulationeffect of the recombined lentivirus with siRNA on PKCε. In order to shed light on the rolesof PKCδ and PKCε under these stress conditions, we investigated the changes of cellularROS and autophagy in the setting of PKCδ specific inhibition and PKCε RNA interferingby the recombined lentivirus.Results: Myocardial autophagy was remarkably enhanced early after severe burns.Autophagic cardiomyocyte death was found3h post-burn, preceding apoptosis andnecrosis with a higher incidence rate, which indicated that autophagic cell death play animportant role in cardiomyocyte loss in severe burns. At the decompensation stage, e.g.after3h post-burn, autophagy was detrimental and its inhibition resulted in improvement ofheart function. ROS and Ang II participated in arousing myocardial autophagy. Resultsfrom cultured cardiomyocytes also indicated that autophagy was significantly enhancedunder the conditions of hypoxia and Ang II stimulation, accompanied with the increase ofcellular ROS. Inhibition of ROS caused to a sharp decrease of autophagic activity,suggesting that ROS may induce myocardial autophagy in a direct way. Under hypoxia orcombined with Ang II stimulation, PKCδ specific inhibition decreased cellular ROS contentand autophagic activity, while with Ang II stimulation alone, inhibition of PKCδ did not affectROS and autophagy. Interestingly, downregulation of PKCε by siRNA interfering decreased ROSamount and autophagic activity under Ang II stimulation or combined hypoxia, but it failed withhypoxia alone.Conclusion: In severe burns, hypoxia and Ang II stimulation cause to an increase of ROSin cardiomyocytes, and ROS enhance autophagy by damage to cellular macromolecules andorganelles. However, hypoxia induces autophagy through a PKCδ/NADPH oxidase/ROSapproach, while Ang II inducing autophagy depends on an AT1/PKCε/NADPHoxidase/ROS way. Before decompensation, the internal environment can be maintained in arelatively stable level. Under moderate hypoxia and Ang II stimulation, autophagy isaroused to protect cardiomyocytes, which is also a part of cellular compensationmechanisms. However, with the hypoxia prolonged and Ang II accumulated, the internalenvironment homeostasis is broken, and autophagy turns to be detrimental, causingautophagic cell death and being an important mechanism of the prompt myocardial damage and cardiac dysfunction soon after a severe burn.