Chaperone-mediated Autophagy Attenuates H₂O₂-induced Cardiomyocytes Apoptosis By Depredating PARP1

Background:PARP1（Poly（ADP-Ribose）Polymerase 1）is a typical representative of the PRRP enzyme family,which is mainly related to DNA repair,gene transcription regulation,inflammation,and oxidative stress.Studies found that PARP1 is involved in the pathophysiological process of a variety of cardiovascular diseases.But so far,it has only been found that PARP1 can be degraded through the ubiquitin-dependent proteasome pathway,and It is not clear whether there are other degradation pathways.In recent years,researchers have discovered that chaperone-mediated autophagy（CMA）is involved in the molecular regulation of various diseases including cardiovascular diseases.The molecular chaperone Hsc70（Heat shock protein 70）specifically targets the substrate protein with the sequence of“KFERQ”,and delivers the unfolded substrates to LAMP2A（lysosome-associated membrane protein 2A）.Finally,the substrates were degraded in the lumen of lysosomes.Therefore,the inhibition or over-activation of CMA will cause abnormal cell function and ultimately lead to the occurrence of diseases.Therefore,based on the particularity of the substrate protein,our team discovered for the first time that PARP1 has the“KFERQ”sequences.However,the relationship between PARP1 and CMA in cardiomyocytes remains largely unclear.Objectives:The objective of this study is to investigate whether CMA is involved in the regulation of PARP1 and to further clarify the specific molecular mechanisms.Methods:1.CMA is involved in the regulation of PARP1:NRCMs were treated with autophagy activator EBSS,autophagy inhibitor CQ and protein synthesis inhibitor CHX to detect the expression of PARP1 and its upstream and downstream proteins by Western Blot,which determines whether PARP1 was degraded by lysosome.The interaction between PARP1 and Hsc70 was detected by Co-IP and immunofluor-escence confocal microscopy experiments.2.To determine the mechanism of CMA on the regulation of PARP1:The LAMP2A overexpression and interference adenovirus were used as the gain-of-functional and loss-of-functional approaches to study the effects of CMA in NRCMs.And the expression of PARP1 and related proteins were detected by Western Blot to determine whether PARP1was degraded by CMA.3.To explore the effect of CMA on PARP1 under oxidative stress injury:Cell injury model was established by H₂O₂ and using TUNEL assay to detect the apoptosis;The relationship between protein expression and stress injury was detected by Western Blot experiments;Immunofluorescence confocal assay was used to determine the changes of protein co-localization after treating by H₂O_2.Results:1.PARP1 was degraded by the lysosomal pathway.In NRCMs,we found after the treatment of EBSS,western blot results showed a decrease in PARP1 expression;however,when cells were treated together with autophagy inhibitor（CQ）and CHX,Western blot results showed that the decrease of PARP1 protein is alleviated.Besides,CQ also significantly inhibited the decreased expression included by CHX.2.PARP1 interacted with Hsc70.Co-immunoprecipitation（Co-IP）and immunofluorescence were used to confirm the endogenous interaction of PARP1 and Hsc70;The amino acid sequence of the PARP1 gene conforms to the characteristics of the“KFERQ”motif,which is located at 353-357 and 670-674.3.PARP1 was degraded by chaperone-mediated autophagy（CMA）.To further clarify the regulation of PARP1 by CMA in NRCMs,we found after the treatment of EBSS,Western Blot results showed a decrease in PARP1,PAR and AIF expression,while PARP1 expression did not change significantly after interfering with 3-MA.Besides,after interfering with LAMP2A,PARP1 and related protein expression increased,while in the EBSS treatment group,PARP1expression did not change significantly after interfering with LAMP2A.In contrast,PARP1expression decreased after overexpressing with LAMP2A,which was alleviated by the addition of CQ.4.Activation of CMA inhibited apoptosis induced by oxidative stress by inhibiting the expression of PARP1（cleaved）.4.1 Activating CMA can inhibit cell apoptosis caused by oxidative stress.The results of the TUNEL experiment showed that H₂O₂ caused cell apoptosis in a concentration-dependent manner;activating CMA can effectively inhibit the number of cell apoptosis.Conversely,inhibiting CMA aggravated cell apoptosis.4.2 Activation of CMA affects the protein expression of PARP1（cleaved）.Western Blot showed that low concentrations of H₂O₂can activate CMA and reduce the expression of PARP1（cleaved）;While CMA will be severely damaged,resulting in a reduction in LAMP2A expression and a significant increase in PARP1（cleaved）expression when the concentration of H₂O₂ was more than 400mmol/L.Then activating CMA reduced the expression of PARP1（full length）and PARP1（cleaved）and also alleviated the increased expression of PARP1（cleaved）caused by H₂O₂;Conversely inhibiting CMA increased PARP1（full length）and PARP1（cleaved）expression.Conversely,after inhibiting CMA,PARP1（full length）and PARP1（cleaved）expression increased,also aggravated the increase of PARP1（cleaved）expression after treatment with H₂O₂.Co-IP results showed that the Co-localization of PARP1 and Hsc70 changed significantly after treatment with H₂O₂.This included the transfer of distribution from nuclear transfer to the cytoplasm and the co-localization of PARP1and Hsc70 were increased.Conclusion:1.PARP1 can be recognized by Hsc70 and interacted with it.2.PARP1 was degraded by chaperone-mediated autophagy（CMA）in NRCMs3.CMA inhibits the apoptosis of cardiomyocytes by reducing the expression level of PARP1（cleaved）protein.