CYLD Exaggerates Pressure Overload-induced Cardiomyopathy Via Suppressing Autolysosome Efflux In Cardiomyocytes

Background:The ubiquitin proteasome system(UPS)consists of ubiquitin-activating enzymes(Els),ubiquitin-conjugating enzymes(E2s),ubiquitin ligases(E3s),proteasomes,and deubiquitinating enzymes(DUBs).Since ubiquitin was discovered in the earlier 1970's,UPS has been implicated in virtually all aspects of cell biology.Protein ubiquitination,a process in which ubiquitin(Ub)is covalently conjugated by its terminal glycine(G76)onto a lysine(K)residue of a substrate protein by the sequential action of an E1,E2 and E3,is a reversible posttranslational modification A single Ub molecule may be attached,which is defined as monoubiquitination Several lysine residues can be tagged with single Ub molecules,giving rise to multiple monoubiquitination,also referred to as multiubiquitination.Since Ub has seven lysine residues(K6,K11,K27,K29,K33,K48,K63)itself,Ub molecules can form different types of chains in an iterative process,known as polyubiquitination.In general,K48-linked poly-Ub chains represent a signal for proteasomal degradation of modified substrates,whereas mono-Ub and K63-linked poly-Ub chain modifications function as signaling devices for establishing protein-protein interactions and regulating cellular functions.The hydrolysis of ubiquitin linkage is conducted by DUBs.Eight Els,a dozen different types of E2s,hundreds of E3s,and approximately 100 functional DUBs have been identified in humansDuring the past decades,an important role of Ub,Els,several E2s and E3s,and proteasome in cardiac homeostasis and dysfunction has been well documented However,the myocardial function of DUBs is less well understood.Studies have demonstrated that A20(also known as TNFAIP3)is a negative regulator of cardiac maladaptive remodeling and dysfunction and indicated a mediator role of ubiquitin-specific protease(USP)15 in cardiomyopathy.Notably,our data showed that Cylindromatosis(CYLD)which has DUB activity highly specific for K63-linked Ub chains and is capable of suppressing NF-?B,MAPK and TGF?signaling,is likely a crucial mediator of pressure overload(PO)-induced cardiomyopathy via a mechanism independent of the NF-?B pathway.These findings underscore the functional specificity of individual DUBs in the heart,thereby supporting the notion that the association of DUBs with substrate adaptors,scaffolds and inhibitors may result in regulatory interactions that drive specificity.Nevertheless,the functional significance of individual DUBs in cardiac homeostasis and disease remains poorly understoodAutophagy,an evolutionarily conserved catabolic process,targets cytoplasmic components such as organelles,protein aggregates or individual proteins to the lysosome for degradation.It is a highly dynamic process that leads to formation of a double-membrane bound vesicle termed the autophagosome,which sequesters the targeted cargos,followed by autophagosome fusion with lysosomes to form the autolysosome,resulting in autolysosomal degradation(autolysosome efflux).The role of autophagy in the heart remains controversial.Depending on the nature of stresses as well as the timing of assessments,activation of cardiac autophagy has been shown to be either adaptive or maladaptive.In PO-hearts,recent studies have revealed that autophagy activation is most likely adaptive.However,the definitive proof of autophagy-mediated cardiac protection in adult PO-hearts is still missingRecently,ubiquitination of substrates and protein components of autophagy machinery has emerged as a central regulatory mechanism of autophagy acting at various steps from autophagy induction to termination.Therefore,it is not surprising that DUBs regulate autophagy.However,it is still not fully elucidated how the Ub code regulates autophagy in terms of altering protein signaling and targeting regulatory proteins for degradation.In addition,the crosstalk between E3s which add K63-or K48-linked poly-Ub chains to the components of autophagic machinery and DUBs that counteract their actions in the control of autophagy remains unclear.To date,the pathophysiological relevance of DUB-mediated regulation of autophagy in the heart has not yet been studiedIn the present study,we demonstrated that CYLD suppresses autophagy at the stage of autolysosome efflux in cardiomyocytes,thereby exaggerating cardiac pathological remodeling and dysfunction in the setting of PO.These results uncovered a novel aspect of CYLD acting as an inhibitor of autophagy in the stressed hearts,thus providing the first evidence directly linking DUB-mediated autophagy regulation with cardiomyopathyAims:1.To investigate the impact of cardiomyocyte-restricted(CR)overexpression of CYLD(CR-CYLD)in pressure overload-induced cardiac remodeling and dysfunction;2.To investigate the impact of CR-CYLD in ubiquitinated proteins accumulation and autophagy function in PO-hearts;3.To verify that activation of cardiac autophagy protects against PO-induced cardiomyopathy;4.To figure out the precise mechanism by which CYLD regulates autophagy thereby contributing to cardiac remodeling and dysfunction in the setting of PO.Methods:1.The CR-Cyld Tg,CR-Atg7 × tTA Tg,CR-Cyld and CR-Atg7 × tTA Tg(Duo Tg),Global Cyld knockout(CYLD KO)and wild type mouse were subject to sham or transverse aortic arch constriction(TAC)operation to establish pressure overload-induced cardiomyopathy and heart failure in vivo.The survival rate after the operation was assessed by Kaplan-Merier survival analysis.The cardiac function was monitored by echocardiography,and the cardiac remodeling was evaluated by pathological analysis,including Evens blue labeling,qPCR,WGA staining,Masson staining and TUNEL assay;2.The accumulation of ubiquitinated proteins,autophagic flux and certain proteins expression were measured by Western blot in hearts of CR-Cyld Tg and wild type mice after TAC;The autophagic vacuoles in the heart were determined by transmission electronic microscopy;3.The autophagic flux in control and Cyld knockdown cells was measured by Western blot,and the autophagosome-lysosome fusion and autolysosome efflux assessments were carried out using H9C2 cells transiently expressing mCherry-GFP LC3 reporter and HA-Flag-tagged Cyld plasmids;4.The role of CYLD in regulating cell death was evaluated by PI staining and LDH assay in vitro.Results:1.CR-CYLD overexpression exacerbates PO-induced cardiomyopathy;2.CR-CYLD overexpression worsens PO-induced accumulation of K48-linked ubiquitinated proteins while deteriorating PO-induced autophagy insufficiency in the heart;3.Activation of cardiac autophagy via CR-ATG7 overexpression protects against PO-induced cardiomyopathy;4.Upregulation of myocardial CYLD turns on ATG7-mediated cardiomyopathy while switching off ATG7-dependent cardiac protection in the setting of PO;5.CYLD suppresses autophagy at a stage of autolysosome efflux in cardiomyocytes;6.CYLD inactivates mechanistic target of rapamycin complex 1(mTORC1)reactivation,upregulates Ras genes from rat brain 7(Rab7)and enhances cardiomyocyte death in pressure overloaded hearts.Conclusions1.CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes.2.Mechanistically,it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.