Function And Mechanism Of PRMT5 In Cardiomyocyte Autophagy

Autophagy is a lysosome-dependent programmed degradation pathway of cellular components that is highly conserved in evolution.By degrading damaged organelles and misfolded proteins,autophagy can eliminate the adverse effects of these damaged organelles or proteins.On the other hand,the material and energy metabolism needs of cells can be met through the reuse of these substances.Mammalian adult cardiomyocytes are generally considered as a kind of terminally differentiated cells with little self-renewal capacity.Self-renewal of cellular proteins and organelles through autophagy is important for maintaining cardiomyocyte survival and homeostasis.Over the past decade,a series of studies have revealed the important function of autophagy in the maintenance of cardiac homeostasis by using gene knockout or overexpression transgenic mice.However,the understanding of the regulatory mechanism of cardiomyocyte autophagy is far from enough.Exploring the key genes regulating cardiomyocyte autophagy will make it possible to screen and identify potential therapeutic targets,which is of great significance for the treatment of clinical heart diseases.Protein arginine methyltransferase 5（PRMT5）mainly mediates the symmetrical dimethylation of arginine residues of proteins.PRMT5 has important physiological functions in the heart.In vitro studies have shown that PRMT5 can inhibit the hypertrophic growth of cardiomyocytes.A previous study by our team showed that PRMT5 gene knockout in cardiomyocytes led to dilated cardiomyopathy.However,whether PRMT5 can regulate cardiomyocyte autophagy has not been reported.In order to study the function of PRMT5 in the maintenance of adult cardiac homeostasis,we treated inducible cardiomyocyte Prmt5 gene knockout mice with tamoxifen（100 mg/kg/d）for 7 days at the age of one month,and detected the cardiac function by echocardiography at the age of 5 months.Compared with control mice,Prmt5 knockout mice had significantly thinner left ventricular end systolic anterior wall thickness（LVAW;s）and left ventricular end systolic posterior wall thickness（LVPW;s）,significantly increased left ventricular end systolic diameter（LVID;s）and left ventricular end systolic volume（LVVol;s）,and left ventricular ejection fraction（EF）and systolic index（FS）which reflect systolic function,were also significantly decreased.The results showed that the cardiac function of Prmt5 gene knockout mice was seriously impaired at the age of 5 months.H＆E staining and Masson staining were performed on the cardiac tissue sections of 7 month old mice.The results showed that Prmt5 gene knockout mice had enlarged hearts with dilated left ventricles and myocardial fibrosis.In order to study the effect of Prmt5 gene deletion on cardiomyocyte autophagy,we detected the expression of autophagy related proteins by western blot.Compared with control mice,the expression of autophagy substrate protein p62 was up-regulated in Prmt5 gene knockout mice,and the ratio of autophagy marker LC3BⅡ/Ⅰ was significantly decreased,indicating that Prmt5 deletion inhibited cardiomyocyte autophagy in mice.Immunofluorescence staining and transmission electron microscope observation confirmed that the autophagy level of cardiomyocytes was decreased in Prmt5 gene knockout mice.We used adenovirus sh RNA interference vector targeting Prmt5（Ad-sh Prmt5）to knock down Prmt5 gene in rat cardiomyocyte H9C2 cell line,and bafilomycin A1（Baf）to block the fusion of autophagosomes and lysosomes,and then detected the expression of autophagy related proteins.The results showed that LC3BⅡ was significantly decreased in Prmt5 knockdown cells regardless of whether they were treated with Baf or not,indicating that the reduction of autophagy level caused by Prmt5 deletion was not due to the inhibition of autophagolysosome degradation,but due to reduced autophagosome production.To further reveal the molecular mechanism by which PRMT5 regulates autophagy in cardiomyocytes,we examined one of the most important regulatory pathways of autophagy,the m TOR signaling pathway.The results of western blot showed that phosphorylated m TOR（p-m TOR）was significantly increased in Prmt5 knockout cardiomyocytes,and phosphorylated UNC-51-like autophagy-activated kinase 1（p-ULK1）downstream of m TOR was also significantly increased,indicating that the absence of PRMT5 in cardiomyocytes may inhibit autophagy by activating the m TOR signaling pathway.Next,we analyzed differential alternative splicing using the mRNA-seq data of Prmt5 knockout mice,and identified a series of negative regulatory molecules upstream of m TOR,including tuberous sclerosis complex1（TSC1）,TSC2,DEP domain containing 5（depdc5）and seizure threshold 2（szt2）.Reverse transcription PCR confirmed variable splicing abnormalities of these genes in the hearts of Prmt5 knockout mice,suggesting that the regulation of m TOR by Prmt5 may be achieved by maintaining the alternative splicing of its upstream molecule.In summary,we revealed for the first time the important function of PRMT5 in cardiomyocyte autophagy using in vitro and in vivo systems.We found that Prmt5 loss may activate m TOR by affecting the alternative splicing of upstream molecules of m TOR to inhibit the autophagy of cardiomyocytes.Our study provides new insights into the function of PRMT5 in maintaining cardiac homeostasis,and targeting PRMT5-regulated autophagy may be a potential strategy for the treatment of cardiac diseases.