Role Of Sumoylation In Regulating TFEB Biological Function And Autophagy In Aged Heart

BackgroundModern medicine prove that the incidence of cardiovascular disease rises with aging. Average annual rate of first cardiovascular events increases 25 times from 85-94 years of age to 35-44 years of age, and cardiovascular disease becomes the leading cause of death in ≥65 years of age according to AHA 2015. From both clinical observations and animal experiments demonstrate that the heart is highly susceptible to additional stress, such as ischemia, with aging, but it is unclear about the concrete mechanisms. So explore the mechanisms and effective interventions of aging-related vulnerability to myocardial ischemia reperfusion injury will make a big difference.Myocardium is one of the terminally differentiated cells, which made self-control more important to deal with cellular abnormal event, so protein and organelle quality control is critical to cardiac structure and function. Autophagy is the major intracellular proteolytic systems in degradation of impaired organelles, such as mitochondria, and proteins. Growing basic evidences shows that aging-dependent decline of autophagy contributes tocardiac dysfunction and injury intolerance, while upregulation of the autophagic pathway may be beneficial for the aged heart and maintenance endogenous protection against irreversible injury. Autophagic activity is regulated by many factors, recently, transcription factor EB(TFEB), a new transcription-dependent mechanism regulating autophagy has been identified. TFEB is one of the Mi T/TFE subfamily members, normally localized to the cytosol but mobilizes into the nucleus under starvation or stress. Previously studies implicated that translocation to the nucleus is important to TFEB in charge of the autophagic transcriptional program by driving expression of autophagy and lysosomal genes. More importantly, TFEB could be modulated by post-translational modifications. It was reported that In vitro ERK-dependent phosphorylation inhibits nuclear transport of TFEB. However, the existing studies do not address what transcriptional modification contributes to TFEB stabilization and transcriptional activity in nucleus, especially in aged cardiomyocyte nuclei. Targeting posttranslational of TFEB is novel strategy to regulate autophagy in aged heart.The small ubiquitin-related modifier(SUMO) can be conjugated to lysine residues of nuclear proteins and is involved in many cellular processes, such as protein subcellular location, interaction and activity. SUMO modification, or SUMOylation, as a critical regulatory event of both cardiac development and functional disorders is newly emerging. Considerable evidences reported that dysfunction of SUMOylation contributed to aging-dependent cardiac injury intolerance. SUMO-1 is added to substrates at lysine residues in a consensus sequence ψ KXE( where ψ is large and hydrophobic amino acid, K is lysine and X is any amino acid), we also identified that examination of the amino acid sequences of mouse and human TFEB possesses a conserved SUMOylation site(347VKQE350) by bioinformatics analysis. So, our research pay more attention to role of SUMOylation in aging-dependent decline of autophagy, and connection with transcriptional regulator of autophagy TFEB.AimThe aims of present research were:(1) to determine SUMOylation is the critical post-translational modification for TFEB regulating autophagy;(2) to investigate the role of TFEB-SUMOylation in aging-related cardiac autophagic decrease;(2) to explorewhether aging-related myocardial ischemic intolerance could be improved by regulating autophagy through TFEB-SUMOylation.MethodsYoung adult(4-6 months) and aged(22-24 months) C57BL/6 mice were euthanized and analyzed. Co-immunoprecipitation was used to investigate whether TFEB indeed SUMOylated in hearts. Western blotting and RT-PCR were used to evaluate TFEB、SUMO1、LC3、Atg5、Beclin1、LAMP1 expression level in heart homogenates. Rapamycin(0.25 mg/kg) or an equivalent volume of vehicle were administered to young and aged mice for 8 weeks, then evaluated TFEB、SUMO1、LC3、Atg5、Beclin1、LAMP1 expression level in heart homogenates. Next, delivered cardiotropic recombinant adeno-associated viruses, serotype 9(r AAV9) to downregulate or overexpress SUMO1 in young or aged mice, Adenoviruses to downregulate TFEB in heart, Western blotting and RT-PCR were used to evaluate TFEB、SUMO1、LC3、Atg5、Beclin1、LAMP1 expression level in heart homogenates. The mice were then subjected to myocardial I/R, TUNEL staining was uesd for the detection of myocardial apoptosis, Evans blue/TTC double staining for myocardial infarct size.Results1. Co-immunoprecipitation show only 80 k D nuclear TFEB, and not 68 k D TFEB, is SUMOylated. The results demonstrated that TFEB expressed predominantly as a SUMOylated form in cardiomyocyte nuclei.2. Compared with young, the level of TFEB and nuclear sumoylated TFEB were significantly reduced in aged heart.3. Compared with young, aged heart exhibited a overtly reduced autophagy as evidenced by decreased autophagosomes markers including Beclin-1, Atg5, LC3-II levels(LC3-II/LC3-I ratio), lysosomes proteins LAMP-1, and accumulated of the autophagy substrate protein p62.4. Systemic treatment with rapamycin(0.25 mg/kg) for 8 weeks facilitated TFEB shuttling to the nucleus and improved autophagy in young heart, whereas no significant augment occured in the nuclear TFEB of aged hearts in response torapamycin. To investigate the role of TFEB-SUMOylation in cardiac autophagic change, we delivered cardiotropic recombinant adeno-associated viruses, serotype 9(r AAV9) that express SUMO1-directed sh RNA(r AAV9–sh Sumo1), eight weeks after tail-vein injection of viruses. As expected, r AAV9–sh Sumo1 injected mice showed depressed SUMO1 in the myocardium, while nuclear TFEB levels were decreased in r AAV9–sh Sumo1-injected hearts. Moreover, hearts from r AAV9–sh Sumo1-injected mice showed overtly reduced autophagy as evidenced by decreased autophagosomes LC3-II levels, decreased lysosomes LAMP1 levels and accelerated p62 protein level. These results support the premise that SUMOylation is an important foundation in maintain TFEB stability and transcription activity in cardiomyocyte nuclei.5. On the contrary, we delivered r AAV9–SUMO1 to increase SUMO1 in the aged-myocardium, consistently, the nuclear TFEB level was significantly increased, as well as the levels of cardiac autophagy.6. However, knockdown of cardiac TFEB by shRNA, blocked the protective effect of upregulation of SUMO1 in aged hearts. These reaults demonstated SUMOylation of TFEB is the main regulator of autophagy in the senescent heart.7. Stimulated TFEB SUMOylation enhanced autophagy under unstressed conditions, ultimately promoted tolerance to cardiac stress in aged heart, reduced infarct size and myocardial apoptosis.Conclusions1. The present study newly demonstrates that SUMOylation of TFEB is a critical post-translational modification that regulates cardiac autophagy, and impaired SUMOylation of TFEB leads to decline of autophagy in the aged heart. Improve SUMOylation of TFEB is a effective measure to enhance autophagy in the aged heart.2. Targeting SUMOylation of TFEB will be effective not only in sustaining the autophagy under unstressed conditions, but also in promotion tolerance to cardiac stress in aged heart.