Effect Of ACE2Transfection On Endoplasmic Reticulum Stress In Diabetic Cardiomyopathy Rats

[Background]:Recent studies revealed that ER stress played a crucial role in the development of diabetic cardiomyopathy(DCM). But the mechanisms of endoplasmic reticulum stress(ERS) in DCM are still poorly understood. Renin-angiotensin system(RAS) plays a pivotal role in the development of DCM. Angiotensin-converting enzyme2(ACE2) is a newly discovered member of the RAS. Recently, the ACE2-Ang1-7-Mas axis has been challenged with RAS. In this axis, angiotensin converting enzyme-2(ACE-2) is a key component of RAS and catalyzes the conversion of angiotensin-II (Ang-II) to angiotensin1-7(Ang1-7) with high efficiency and stimulates a G-protein coupled receptor termed MAS, which has been shown to prevent Angll induced cardiovascular hypertrophy and remodeling associated with blockade of mitogen-activated protein kinases (MAPK) signaling.An understanding of the regulation of these enzymes is clinically relevant in view of recent studies showing that ACE-2/Ang1-7expression is altered in pathological conditions such as diabetes, hypertension, and cardiovascular diseases.C-Jun NH2terminal protein Kinase(JNK) and p38MAPK signal transduction pathways play an important role in inflammation and apoptosis in stress response.We hypothesized that overexpression of ACE2may decrease ERS via MAPK signaling pathaway.[Objective]:DCM animal models were established and the effects of ACE2transfection on endoplasmic reticulum stress in diabetic cardiomyopathy rats were explored.[Methods]:Sixty-four male Wistar rats (weight200-220g) were randomly divided into2groups: control group (n=12)and model group(n=52). After one week’s standard rat diet feeding and12h fasting.Diabetes in the model group were induced by a single intraperitoneal injection of streptozotocin at the dose of65mg/kg(dissolved in0.1mol/1citrate buffer,pH4.2). Control rats received the same dose of citrate buffer. After one week, measured the rats’ fasting plasma glucose level.And rats with plasma glucose at least two times higher than16.7mmol/L were considered as successful diabetic models. All rats were fed for12weeks after STZ or citrate buffer injections and had free access to standard rat diet and water. The DCM rats were randomly divided into3groups including DCM group,EGFP group and ACE2group.The DCM group, the EGFP group, the ACE2group were respectively injected with Sterile saline, recombinant adenovirus carrying enhanced green fluorescent protein gene(AdV5.EGFP)and recombinant adenovirus carrying mouse ACE2gene(AdV5.ACE2) into the myocardium. Before and after STZ injection one week, we detected the levels of fast blood-glucose.After15days of gene transfection, we use western blotting to detect the expression of myocardial ACE2protein. At the end of the study, left ventricular function, myocardial AngⅡ level were measured. We used TUNEL staining to detect the expression of cell apoptosis in diabetic cardiomyopathy. Immunofluorescence was used for the detection of GRP78and caspase12protein expression, and immunohistochemistry for IL-1β and VCAM-1protein expression. Western blotting was used to analyze the protein expression of CHOP,P-JNK,T-JNK.[Results]:1. General features of the experimentDuring the experiment, the rats in the DCM group showed the symptoms of diabetes while the control group had no diabetic symptoms. At the end of the experiment,12control rats,16ACE2rats,17EGFP rats and16DCM rats completed the study.2. TUNEL stainingTo assess the apoptosis levels in diabetic myocardium,the tissue sections were labeled with an in situ TUNEL assay. The TUNEL staining showed apoptosis cells local in both the cardiomyocyte and endothelium of the diabetic heart. The ACE2group showed fewer apoptosis cells than that in the DCM and EGFP group(P<0.01).3. ImmunofluorescenceImmunofluorescence studies showed that GRP78and caspase-12protein in DCM and EGFP groups was abundantly expressed in the myocardium. Compared to the EGFP and DCM groups,the ACE2group exhibited weaker immunoreactivity for GRP78 (P<0.01) and caspase-12(P<0.01) proteins.4. ImmunohistochemistryImmunofluorescence studies showed that IL-1β and VCAM-1protein in DCM and EGFP groups were abundantly expressed in the myocardium.Compared to the EGFP and DCM groups, the ACE2group exhibited weaker immunoreactivity for IL-1β (P<0.01) and VCAM-1(P<0.01) proteins.5. The results of Western blottingThe expression of ACE2protein in ACE2group was higher than that of DCM and EGFP group(P<0.01).There was no significant difference between DCM group and EGFP group in ACE2protein expression (P>0.05).The densitometric analysis of bands for CHOP, P-JNK revealed a significant increase in myocardium of DCM and EGFP groups.This means CHOP was induced and JNK pathway were activated in the diabetic heart.The expression of CHOP, andP-JNK proteins in ACE2group were lower than that in DCM and EGFP groups (P<0.01).There was no significant difference between DCM group and EGFP group (P>0.05).6. The level of Ang ⅡThe level of AngⅡ in ACE2group was decreased compared to DCM and EGFP group (P<0.05);The level of Ang Ⅱ in DCM and EGFP groups were increased compared to control group (P<0.01).7. Echocardiographic examinationAt the end of the study, we detected left ventricular function including left ventricular end diastolic diameter(LVEDD), left ventricular end systolic diameter (LVESD),ejection fraction (EF), fractional shortening (FS). Compared with DCM and EGFP rats echocardiographic changes were detected in ACE2rats(P<0.05).8. Fast blood-glucoseAfter one week of STZ injection,rats’fast blood-glucose’s level in model group were significantly different from control group’s(P<0.01).[Conclusions]:1. DCM animal models were established after3months of STZ intraperitoneal injection which provided a reliable platform for the study.2. Increased expression of GRP78in diabetic heart, proved that endoplasmic reticulum stress played an important role in the occurrence and development of DCM.3. TUNEL staining showed that the apoptosis cells increased significantly in DCM myocardial tissue, revealing cell apoptosis was involved in the progress of DCM.4. ACE2gene transfection was able to increase the expression of ACE2protein..Overexpression of ACE2gene had therapeutic potential in cardiac function by inhibiting the expression of IL-1β and VCAM-1protein.5. Overexpression of ACE2gene had protective effect on diabetic cardiomyopathy by attenuating ERS.