FAT10 Attenuates Ischemia/hypoxia-induced Cardiomyocyte Apoptosis By Stabilizing Caveolin-3

Background:Caveolins(Cavs)are the fundamental components of Caveolae,which compartmentalize and modulate signal transduction in many cell types.Cav-3 is a muscle-specific Caveolin of Caveolins family.Increasing studies paid attention to the role of Cav-3 in cardiovascular diseases,it was demonstrated that Cav-3 plays an important role against apoptosis in cardiomyocyte.But it is still unclear that the regulatory mechanism of Cav-3 in cardiomyocytes under ischemia/hypoxia condition.The ubiquitin-proteasome system(UPS)was confirmed associated with ischemia heart disease.FAT10(F-Adjacent Transcript-10,FAT10)is one of the ubiquitin-like proteins,which arranged in two in-tandem ubiquitin-like domains and participated in posttranslational modification in a series of proteins.Our previous studies also demonstrated for the first time that FAT10 stabilizes specific substrates by antagonizing their ubiquitination.We also reported that FAT10 protectes cardiomyocytes against apoptosis in response to hypoxic/ischemic injury.However,the mechanism by which FAT10 suppresses cardiomyocyte apoptosis remains largely unknown.Objective:This study explored whether FAT10 inhibits hypoxia-induced cardiomyocyte apoptosis by regulating Cav-3 expression,and whether the expression of Cav-3 is stabilized by inhibiting ubiquitin-mediated cardiomyocyte degradation.Together,these findings revealed the novel role of FAT10 in protection against ischemiainduced injury via stabilization of Cav-3,providing evidence that the FAT10/Cav-3 axis may be a potential therapeutic target for patients with ischemic heart conditions.Methods:1.FAT10 knockout(FAT10 KO)rats were generated using the CRISPR-Cas9 technique in a Sprague Dawley(SD)background,and confirmed by gene sequencing.Myocardial infarction(MI)was induced via ligation of the left anterior descending artery(LAD).All rats were divided into four groups: Wild-type Sham(WT-Sham),FAT10-KO-Sham,WT-MI and FAT10-KO-MI groups.2.ECG monitoring(TA11CTA-F40,Data Sciences International)were performed on rats.3.Neonatal rat cardiomyocytes(NRCMs),HEK-293 T cells and H9C2 cells were prepared in this experiment.We constructed FAT10 lentivirus and shRNA lentivirus(Lv-FAT10 and Lv-shFAT10)to regulate the expression of FAT10 in cells.And constructed Cav-3 lentivirus and shRNA lentivirus(Lv-Cav-3 and Lv-sh Cav-3)to regulate the expression of Cav-3 in cells.4.Detection of the expression of FAT10,Cav-3 and Caspase-3: qRT-PCR and Western Blot were used to detect the mRNA and protein level of FAT10,Cav-3 and Caspase-3 in heart tissue or cardiomyocytes under ischemia/hypoxia condition.5.Detection of the apoptosis of cardiomyocytes: flow cytometry and Tunel assay were used to detect the apoptosis of cardiomyocytes.6.Heart tissue fibrosis determination: The deposition of collagen in the cardiac tissue was detected by Masson trichrome.7.GST(Glutathione-S-transferase)pulldown assay: to detect FAT10 affects Cav-3 level by competing with Ub.8.Ubiquitination experiment in vitro: to detect FAT10 affects Cav-3 ubiquitination level.9.All experiments were independently repeated at least 3 times.The data were expressed as the mean ± SEM and were analyzed by one-way ANOVA and Student's t-test using GraphPad Prism.Statistical significance was defined as P< 0.05.Results:1.FAT10 and Cav-3 were both upregulated in cardiomyocytes exposed to ischemic injury.The results showed that the FAT10 and Cav-3 protein levels were significantly elevated in the ischemic myocardium.The NRCMs were subjected to hypoxia treatment.The mRNA and protein levels of FAT10 and Cav-3 were significantly upregulated in cardiomyocytes subjected to hypoxic stress compared to normoxic conditions.2.FAT10 regulated Cav-3 protein expression and suppressed cardiomyocyte apoptosis.To investigate the effects of Cav-3 on cardiomyocyte apoptosis following hypoxia stress.Western blotting and TUNEL assays results showed that downregulation of Cav-3 enhanced cardiomyocyte apoptosis in response to hypoxic stress compared with the shNC groups,and demonstrated that FAT10 was positively correlated with the expression of Cav-3 protein.To further determine it,we overexpression of Cav-3 in FAT10-knockdown cardiomyocytes and the results suggest that under hypoxia stress,compared with the negative control,Cleaved–caspase3 significantly reduced after the overexpression of Cav-3.3.Cav-3 was the key protein for FAT10 protected cardiomyocytes against ischemic injury.We further investigated the effects of FAT10 deficiency on Cav-3 expression and cardiomyocytes apoptosis in the ischemia heart.In neonatal rat cardiomyocytes,transferred Lv-FAT10 and Lv-shCav-3 together,Western Blot results showed the elevated expression of Cleaved-caspase3,and Tunel results demonstrated increased cell apoptosis;then transferred Lv-shFAT10 and Lv-Cav-3 together,Western Blot results showed the decreased expression of Cleaved-caspase3,and Tunel results demonstrated reduced cell apoptosis.These data provided direct evidence that FAT10 protected cardiomyocytes against ischemic injury by increasing Cav-3 expression and suggested the involvement of FAT10 in MI-induced cardiomyocyte apoptosis.4.FAT10-KO rats show exacerbated cardiac dysfunction and increased cardiomyocyte apoptosis after MI.Tunel staining of tissue slices revealed the increase of cardiomyocyte apoptosis in FAT10-KO-MI group,the analysis of fiber staining also revealed that myocardial fibrosis of FAT10-KO rats were more obvious after LAD(*p<0.05 vs WT-MI).5.Cav-3 was modified by ubiquitin.To explore the mechanism by which FAT10 regulates Cav-3 expression in cardiomyocytes.Because of the similar structure and function between FAT10 and Ub,we initially wanted to observe whether Cav-3 was degraded by ubiquitination.In neonatal rat cardiomyocytes,we found after the treatment of cycloheximide(CHX),western blot results showed significant decreased of Cav-3 expression;but when cells treated with proteasome inhibitor(MG132)and CHX together,western blot results show no change of Cav-3 expression.These findings indicated that Cav-3 was modified by ubiquitin.6.Cav-3 protein was degraded by the UPS in cardiomyocytes.To further validate Cav-3 was degraded by the UPS system,we examined the ubiquitination level of Cav-3 following dysregulation the Ub levels in H9C2 cells.The Co-IP and in vivo ubiquitination assay results revealed that the Cav-3 protein was degraded by the UPS in cardiomyocytes.7.FAT10 stabilized the expression of Cav-3 by reducing its ubiquitination in cardiomyocytes.The above experiments revealed that Cav-3 was degraded through the UPS in cardiomyocytes,and our previous study reported that FAT10 regulates substrate expression by antagonizing its ubiquitination.Thus,we speculated that FAT10 regulates Cav-3 expression by affecting its ubiquitination.As expected,Co-IP results confirmed that the expression of ub-Cav-3 was reduced and the expression of Cav-3 was increased after overexpression of FAT10.8.FAT10 and Ub binding to the same sites of Cav-3.We speculated that FAT10 affects Cav-3 ubiquitination by competing with Ub for binding the same binding sites of Cav-3 in cardiomyocytes.Thus,we constructed three different fragment plasmids according to the functional domains of Cav-3,CoIP results showed that FAT10 and Ub only binding to domain I,next,the 4 lysines in the binding region of Cav-3 were mutated to arginine,and mutant plasmids(Cav-3 mut)were constructed.Co-IP experiments revealed that the binding of Cav-3 to Ub and FAT10 disappeared when all 4 of the lysine sites were mutated.9.FAT10 competing with Ub for binding to Cav-3 and subsequently reducing Cav-3 ubiquitination degradation in cardiomyocytes.To further confirm that FAT10 competed with Ub for binding to Cav-3 and subsequently influences its expression,we analyzed the binding of Cav-3 with FAT10 or Ub during the course of competition by using a GST pulldown experiment.Our data demonstrated that FAT10 stabilization of Cav-3 was due to FAT10 competing with Ub for binding to Cav-3 and subsequently reducing Cav-3 ubiquitination degradation in cardiomyocytes.Conclusion:1.Both FAT10 and Caveolin-3 were upregulated in ischemic myocardium tissues and hypoxic cardiomyocytes.2.Overexpression of FAT10 increases Cav-3 expression and inhibits hypoxiainduced cardiomyocyte apoptosis.3.Cav-3 was a crucial effector of FAT10 in hypoxia-induced cardiomyocyte apoptosis.4.FAT10 KO aggravated cardiac injury and increased cardiomyocyte apoptosis by reducing Cav-3 expression in the ischemic rat heart.5.Cav-3 was degraded by the ubiquitin–proteasome system(UPS)in cardiomyocytes.6.FAT10 stabilized Cav-3 expression by antagonizing its ubiquitinationmediated degradation in cardiomyocytes.