Macrophage KLF5 Regulates Cardiomyocyte Senescence

Objective:To study the effect of macrophage KLF5 on cardiomyocyte senescence and its molecular mechanism.KLF5,a member of the KLFs family,plays a key role in regulating a variety of cellular biological processes,including proliferation,migration,differentiation,growth,survival and apoptosis.As a key transcription factor of the heart,KLF5 is highly expressed in the cardiovascular system during embryonic development,and mediates the expression of genes regulated structure and function of the heart.CircRNA,a very stable new type of non-coding RNA,is a ring structure with 5'end cap or 3'end?A?tail continuous.CircRNA is more stable than linear RNA and not affected by RNA exonuclease?capping?or adenylation.It had been proved that some circRNA could act as effective mi RNA sponges and had potential regulatory functions on mi RNAs.circRNA could also be used as a signal molecule participated in information transmission among cells and gene expression controlling.Cardiomyocytes,myocardial macrophages and cardiac fibroblasts are the main cell types of myocardial tissue.Our study found that overexpression of KLF5 in smooth muscle cells promoted the migration of macrophages,thus promoting the formation of aneurysms dominated by smooth muscle lesions.Therefore,we speculated that macrophage KLF5 could affect cardiomyocytes function by some way,such as circRNA.Therefore,this study mainly focused on whether KLF5 in macrophages is involved in hypoxia-induced cardiomyocyte senescence and its molecular mechanism.Methods:1. The macrophage derived from bone marrow was divided into normoxia group and hypoxia group.The proliferation of macrophages was detected by CCK-8.2.The cardiomyocytes were divided into four groups:normoxia cardiomyocyte in a mixed medium(the ratio of 10%FBS medium to KLF5^wtor Ad-GFP normoxic macrophage culture medium was 4:1);normoxia cardiomyocyte in a mixed medium(the ratio of 10%FBS medium to KLF5^lysm-/-or Ad-KLF5 normoxic macrophage culture medium was 4:1);anoxic cardiomyocytes in a mixed medium(the ratio of 10%FBS medium to KLF5^wtor Ad-GFP hypoxic macrophage culture medium was 4:1);anoxic cardiomyocytes in a mixed medium(the ratio of 10%FBS medium to KLF5^lysm-/-or Ad-KLF5 hypoxic macrophage medium was 4:1).The cell viability of cardiomyocytes was detected by CCK-8;the aging of cardiomyocytes was stained by?-galactosidase;the myofilament in cardiomyocytes was stained by phalloidine;the expression of genes involved in senescence and apoptosis were detected by Western blot or q RT-PCR;and the expression levels of circ-KHK,circ-dnmt3a,circ-HIF1?,circ-FOXO1were detected by RT-PCR.Results:1. Hypoxia promoted the proliferation of bone marrow-derived mouse macrophages in the early stage.Bone marrow-derived macrophages were divided into normoxic group and anoxic group.CCK-8 results indicated that hypoxia significantly promoted the proliferation of macrophages with the extension of time.But the proliferation activity of macrophages was significantly decreased after 48hours of hypoxia,indicating that hypoxia promoted the proliferation of bone marrow-derived macrophages in the early stage.2. Knockdown of KLF5 in macrophage retarded the aging process and apoptosis of cardiomyocyte.The results of CCK-8 showed that knockdown of KLF5 in macrophages could significantly enhance cardiomyocytes viability under normoxia and hypoxia.?-galactosidase stain revealed that knockdown of KLF5 in macrophages could delay the senescence of cardiomyocytes.Consistent with the above results,the results of phalloidine stain showed that knockdown KLF5 in macrophages stabilized the uniform distribution of myocardial myofilament and inhibited the depolymerization of cardiomyocyte cytoskeleton.These results indicated that the knockdown of KLF5 could delay the senescence and apoptosis of cardiomyocytes.3. Knockdown of KLF5 in macrophage inhibited cardiomyocyte senescence and apoptosis related gene expression.Under normoxia and hypoxia,knockdown of KLF5 in macrophages decreased the protein expression of P16^INK4a in KLF5^lysm-/-mice compared with wild mice.The protein expression of P21^cip1 was consistent with that of P16^INK4a under hypoxia,but up-regulated under normoxia.PT-PCR showed that the knockdown of KLF5 in macrophages significantly decreased the protein expression of P16^INK4a,Caspase and P21^cip1.4.Overexpression of KLF5 in macrophages promoted cardiomyocyte senescence and apoptosis.Western Blot showed that the overexpression of KLF5 in macrophages was significant,indicating the overexpression was successful.The results of CCK-8 showed that the overexpression of KLF5 in macrophages inhibited the cell viability of cardiomyocytes under hypoxia.The results of?-galactosidase stain showed that overexpression of KLF5 in macrophages could accelerate the senescence of cardiomyocytes.The results of phalloidine stain further identified that overexpression of KLF5 in macrophages could destroy the uniform distribution of cytoskeleton of cardiomyocytes.Western Blot showedcardiomyocytes was significantly increased in the co-culture of the macrophage overexpression KLF5 medium with cardiomyocytes.The RT-PCR results showed that the expression of p21^cip1 and caspase3 was significantly up-regulated while p16^INK4a was down-regulated when macrophages overexpressed KLF5.These results suggest that overexpression of KLF5 in macrophages promoted cardiomyocyte senescence and apoptosis.5.CircRNA participated in the interaction between macrophages and cardiomyocytes.RT-PCR results showed that the expression of circ-KHK,circ-HIF1?,circ-FOXO1 in cardiomyocytes decreased significantly after knockdown of KLF5 in macrophages,while the expression of circ-dnmt3a increased significantly.On the contrary,the expression of circ-KHK,circ-HIF1?,circ-FOXO1 and circ-dnmt3a was decreased significantly in cardiomyocytes after KLF5 was overexpressed in macrophages.It suggests that macrophages KLF5 may regulate the senescence of cardiomyocytes by affecting the expression of circRNA in cardiomyocytes.Conclusions:1.Hypoxia promoted the proliferation of bone marrow-derived mouse macrophages in the early stage.2.Knockdown of KLF5 in macrophages could delay cardiomyocyte senescence.3.Knockdown of KLF5 in macrophages could delay cardiomyocyte senescence by inhibiting the expression of p16^INK4a and P21^cip1.4.The overexpression of KLF5 in macrophages accelerated cardiomyocyte senescence.5.Circ-dnmt3a may be involved in the interaction between macrophages and cardiomyocytes.