Systemic Delivery of siRNA Specific for Silencing TLR4 Gene Expression Reduces Diabetic Cardiomyopathy in a Mouse Model of Streptozotocin-Induced Type1 Diabetes.

Abstract

IntroductionDiabetic cardiomyopathy is a cardiac dysfunction in patients with diabetes which may lead to overt heart failure and death. Toll-like receptor (TLR) signaling triggers diabetic cardiomyopathy through various mechanisms, one of which is the upregulation of TLR4 expression. The aim of this study was to delineate the role of TLR4 in diabetic cardiomyopathy.MethodsC57BL/6 mice were injected with streptozotocin to induce diabetes. The experimental and control groups were treated with 5 μg of TLR4 small interfering RNA (siRNA) or scrambled siRNA. Cardiac histopathology was evaluated by hematoxylin and eosin, Sirius red, and immunofluorescence staining after treatment with TLR4 siRNA. The myocardial fibrosis and inflammatory factors were detected by quantitative real-time polymerase chain reaction after treatment with TLR4 siRNA. The myocardial function was evaluated by echocardiography after treatment with TLR4 siRNA.ResultsCompared with non-diabetic mouse hearts, hypertrophy, fibrosis, inflammation of cardiomyocytes, and myocardial dysfunction were significantly increased in diabetic mice (p < 0.05). Knockdown of TLR4 decreased hypertrophy, fibrosis, inflammation of cardiomyocytes, and myocardial dysfunction (p < 0.05). Cardiomyocytic cross-sectional areas in hearts of TLR4 siRNA-treated diabetic mice were similar to those of the sham-treated mice (p > 0.05). The induction of expression of cardiac fetal genes, beta-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP), which are two markers of cardiac hypertrophy, was significantly reduced in TLR4 siRNA-treated hearts compared with controls (p < 0.05). Moreover, siRNA-mediated silencing of TLR4 reduced diabetes-induced collagen deposition (p < 0.05). Paralleled with changes in collagen deposition and the expression of collagen I and collagen III, knockdown of TLR4 also reduced the expression of transforming growth factor-β1 (TGFβ1) mRNA (p < 0.05). The increased expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) was significantly attenuated by TLR4 siRNA treatment in the hearts of diabetic mice (p < 0.05). Furthermore, both fractional shortening (FS) and ejection fraction (EF) values were preserved in TLR4 siRNA-treated diabetic mice compared with control siRNA-treated mice (31.80% ± 2.82% vs. 28.50% ± 5.83% for FS, p < 0.05) (57.95% ± 6.48% vs. 45.34% ± 4.25% for EF, p < 0.05).ConclusionOur study used siRNA to specifically silence TLR4 gene expression in the diabetic mouse heart in vivo and to investigate the role that TLR4 plays in diabetic cardiomyopathy. It is likely that silencing of the TLR4 gene through siRNA could prevent the development of diabetic cardiomyopathy.