The Role Of CaMKK2 In Myocardial Fibrosis And The Underlying Mechanism

Background Myocardial fibrosis is one of the major global public problems for its involvement in the development of almost all cardiovascular diseases.At present,the mechanism of cardiac fibrosis has not been fully elucidated and there are no effective strategies for the diagnosis and treatment of cardiac fibrosis.It is particularly important to further explore the mechanisms of myocardial fibrosis and to find suitable intervention targets.TGF-β/Smad signaling pathway is the most classic signal pathway in myocardial fibrosis.But unfortunately direct targeting TGF-p/Smad pathway is unworkable to treat myocardial fibrosis.Studies have confirmed that other optional targets could effectively inhibit TGF-β/Smad pathway activation without affecting their physiological regulation functions,of which AMPK has been shown to inhibit myocardial fibrosis by inhibiting TGF-β/Smad3/p38MAPK pathway activation.CaMKK2 is a member of the calmodulin kinase family.It has been demonstrated that calcium dependent CaMKK2 activation specifically regulates AMPK,involving a variety of important biological functions.Only a few studies indirectly pointed out that CaMKK2 may be an important kinase involved in cardiac pathophysiology.Objective To investigate CaMKK2 expression in the heart under normal physiological condition and pathological remodeling and its cellular localization,and to elucidate the role of CaMKK2 in the process of myocardial remodeling and underlying mechanisms.Meanwhile,we aim to discuss whether CaMKK2 could be an effective intervention target for myocardial fibrosis treatment.Research programs and methods:The first part:Animal experiments:male C57BL6 mice(8-10 weeks,23.5-25.5 g)were selected for aortic banding or continuous Ang Ⅱ pump to establish myocardial remodeling models.Hearts were harvested on the 3rd day,1 W,2W,4W and 8 weeks after AB and on the3rd days,1W and 2W after AngⅡ pump.Western blotting was used to test CaMKK2 protein expression.Cell experiments:Isolated rat and mouse cardiomyocytes and fibroblasts were treated with PE(50μM)for 15 min,30 min,1h,2h,3h,6h,and CAMKK2 expression levels were detected by western-blot.Human specimens:Dilated cardiomyopathy and unsuccessful donor heart specimens were collected.The expression and localization of CAMKK2 were detected by immunofluorescence,and the difference of CaMKK2 between normal heart and heart failure specimens was compared.The second part:Animal experiments:healthy male C57BL6 and CaMKK2-/-mice(8-10 W,23.5-24.5 g)were selected.AngⅡ was pumped to establish the myocardial remodeling model.Cardiac function was measured by echocardiography at 1W and 2W.The surgery of aortic banding(AB)was performed to establish pressure overload induced cardiac remodeling model,and cardiac function was detected by echocardiography at 4W and 8W.The hearts were randomly divided into two groups.One group was saved at-80 ℃ for molecular biology.The other group was used for pathological staining.The third part:Left ventricule tissue from C57BL6 and CaMKK2-/-(AngⅡ continuously pumping for 7d)mice were collected.Total RNA was extracted and reversely transcribed to establish a DNA library and then analysising by the Illumina NextSeq 500 machine.The morphological gene changes were plotted by DEseq(version 1.18.0),and the change of gene expression was carried out by using the Rg ggplots 2 software package.The R-language Pheatmap software was used to analyze the gene and the samples were analyzed by two-way cluster analysis.The GO function enrichment analysis showed that the gene set was significantly enriched and the related biological functions were obtained.The number of differentially expressed genes included in the different levels of KEGG Pathway was determined and the major pathway which the major genes take part in.RT-PCR was used to test the consistency of test results.The fourth part:In vivo experiments:Westemblot was performed to detect the relevant signaling pathways;In vitro experiments:Isolate left ventricular fibroblasts from eight-week-old C57BL6 and CaMKK2-/-mice.Flow cytometry was used to detect the fibroblasts differentiation.The proliferation and migration of fibroblasts were observed.The CaMKK2 associated signal transduction pathway was confirmed by the addition of AICAR,rapamycin and adenovirus overexpressing P21.ResultsThe first part:1.After Three days of AngⅡ pump,CaMKK2 expression reached peak,and fell under normal levels seven days later;while CaMKK2 expression peaked at 1 week after AB and fell under normal level at two weeks;2.Co-localization showed that CaMKK2 was located in the interstitium of the heart,and its expression was downregulated in heart failure.3.The expression of CaMKK2 in cardiac fibroblasts was significantly higher than that in cardiomyocytes,and the expression of CaMKK2 in cardiac fibroblasts was significantly higher after PE stimulation.The second part:1.The heart weight,heart weight/body weight and heart weight/tibial length ratio in CaMKK2-/-mice were significantly increased at 1w or 2w after AngⅡ pump and 4w or 8W after AB.2.Echocardigraphy showed that the cardiac function of CaMKK2-/-mice was significantly worse than that of C57BL6 group at 4w or 8W after AB and at 1w or 2w after AngⅡ pump.3.Cross-sectional area of CaMKK2-/-cardiomyocyte was significantly increased;the interstitial fibrosis of CaMKK2-/-mice was significantly increased;interstitial and perivascular a-SMA expression was significantly increased in CaMKK2-/-mice;4.The expression ANP and BNP was significantly increased in CaMKK2-/-mice,and collagen Ⅰ and collagen Ⅲ was also significantly increased in CaMKK2-/-mice.The third part:1.Compared with C57BL6 group,234 genes was upregulated and 79 genes was downregulated in CaMKK2-/-mice heart.2.Gene Ontology enrichment analysis showed significant differences of 292 Term,the former 20 significant changes were mostly associated with cell cycle;KEGG pathway analysis also found that the most significant changes are related to cell cycle.3.RT-PCR was performed to verify the expression of genes,the experimental results were consistent with sequencing results.The fourth part:1.The proliferation,differentiation and collagen secretion of isolated fibroblasts from CaMKK2-/-mice heart were significantly enhanced;2.The activity of AMPKa in cardiac tissue and fibroblasts of was decreased,the mTOR signal pathway was activated and the p53/p21 signal pathway was inhibited.After AICAR treatment,the signal pathways was reversed;3.Rapamycin or adenovirus overexpressing p21(Ad-p21)could markedly inhibit the proliferation,differentiation and collagen secretion of cardiac fibroblasts;combining of rapamycin and Ad-p21 could completely inhibited the proliferation,differentiation and Collagen secretion in cardiac fibroblasts from CaMKK2-/-mouse heart.Conclusion CaMKK2 is mainly expressed in fibroblasts in human,rat and mouse heart.Cardiac fibrosis is aggravated in CaMKK2-/-mice after AngⅡ pump or AB.The sequencing and in vivo and vitro experiments showed that CaMKK2 could stabilize P53 and P21 and inhibit mTORCl phosphorylation by activating AMPKal Targeting CaMKK2 may be a potential strategy for cardiac fibrosis.