The Role of Mir-21 in Mediating Diabetes Associated Cardiac Fibrosis By Regulating Akt/PKB Signaling

Presentation Number: LB SUN 82
Date of Presentation: April 2nd, 2017

Amit Kumar*1, Akhilesh Kumar2 and Madhu Khullar3
1POSTGRADUTE INSITUTE OF MEDICAL EDUCATION AND RESEARCH, Chandigarh, INDIA, 2postgraduate institute of medical education and research, CHANDIGARH, IN, 3Postgraduate Institute of Medical Education and Research, Chandigarh (India), CHANDIGARH, India

Abstract

Akt/PKB signaling is cell survival pathway involved in tissue fibrosis, however, its role in diabetic cardiomyopathy (DCM) associated cardiac fibrosis is not known. The aim of this study was to examine the role of mir-21 in mediating the diabetes induced cardiac fibrosis via regulating the Akt/PKB pathway. We developed an animal model of DCM [Diabetic (n=13) & Control(n=5)] by high-fat diet (HFD) and two low-doses Streptozotocin (STZ). The development of DCM was characterized by echocardiography (two-dimensional M-mode echocardiography), tissue histology staining, myocardial mRNA and protein expression analysis of fibrotic genes (Col-1a, Col-3a,CTGF, FGF-b, TGF-β & α-SMA) and Hypertrophic genes (ANP & β-MHC) was done. Formalin fixed paraffin embedded (FFPE) human myocardial archived tissues from DCM patients/group (n=6) and control group (n=6) were included in this study. Primary adult rat cardiac fibroblasts (CFs) were isolated from the heart of 6 to 8-week old male wistar rats. The isolated CFs were characterized on the basis of immune positivity with anti-vimentin, immune negativity for anti-desmin and ultrastructure examination by TEM imaging having abundant RER, granular cytoplasm and absence of striations. CFs was grown to 60-70% confluency in DMEM complete media. After respective time period, treated CFs was harvested for microRNAs, mRNA and protein expression analysis. miR-21 levels were modulated by miR-21 mimic and miR-21 inhibitor in normal glucose or high glucose treated CFs, using Opti-MEM I reduced serum medium and lipofectamine 2000 transfection reagent. Cy™3 labeled Anti-miR negative control was used as scrambled oligos to monitor the transfection efficiency of miR-21 mimic or inhibitor in CFs. Transfected cells treated with normal and high glucose were used for microRNA, mRNA and protein expression analysis. In present study, cardiac p-Akt (Ser473) expression was significantly increased in DCM rats and high glucose treated cardiac fibroblasts . Cardiac expression of PTEN, PPARα, PDCD4 and Fas-L genes involved in Akt/PKB signaling, was decreased in HG treated CFs and DCM rats (PTEN & PDCD4). RT-PCR analysis showed increased miR-21 in myocardium of DCM rats, FFPE myocardial tissue from DCM patients and HG treated CFs. miR-21 inhibition suppressed HG induced activation of Akt/PKB signaling and reversed PTEN, PPARα, PDCD4 and Fas-L expression in HG treated CFs. miR-21 inhibitor resulted in decreased expression of fibrotic genes (Col-1a, Col-3a, Col-4a, CTGF & TGF-β), decreased myofibroblasts differentiation (decreased α-SMA expression) and decreased CFs proliferation during hyperglycemia, suggesting attenuation of cardiac fibrosis. Akt/PKB signaling is activated in diabetic hearts and miR-21 mediates its profibrotic activity by regulating expression of genes of Akt/PKB pathway in myocardium of DCM rats and in high glucose treated Cardiac fibroblasts.

 

Nothing to Disclose: AK, AK, MK