Prevention of hyperglycemia-induced myocardial apoptosis by gene silencing of Toll-like receptor-4

Abstract

Background Apoptosis is an early event involved in cardiomyopathy associated with diabetes mellitus. Toll-like receptor (TLR) signaling triggers cell apoptosis through multiple mechanisms. Up-regulation of TLR4 expression has been shown in diabetic mice. This study aimed to delineate the role of TLR4 in myocardial apoptosis, and to block this process through gene silencing of TLR4 in the myocardia of diabetic mice. Methods Diabetes was induced in C57/BL6 mice by the injection of streptozotocin. Diabetic mice were treated with 50 μg of TLR4 siRNA or scrambled siRNA as control. Myocardial apoptosis was determined by TUNEL assay. Results After 7 days of hyperglycemia, the level of TLR4 mRNA in myocardial tissue was significantly elevated. Treatment of TLR4 siRNA knocked down gene expression as well as diminished its elevation in diabetic mice. Apoptosis was evident in cardiac tissues of diabetic mice as detected by a TUNEL assay. In contrast, treatment with TLR4 siRNA minimized apoptosis in myocardial tissues. Mechanistically, caspase-3 activation was significantly inhibited in mice that were treated with TLR4 siRNA, but not in mice treated with control siRNA. Additionally, gene silencing of TLR4 resulted in suppression of apoptotic cascades, such as Fas and caspase-3 gene expression. TLR4 deficiency resulted in inhibition of reactive oxygen species (ROS) production and NADPH oxidase activity, suggesting suppression of hyperglycemia-induced apoptosis by TLR4 is associated with attenuation of oxidative stress to the cardiomyocytes. Conclusions In summary, we present novel evidence that TLR4 plays a critical role in cardiac apoptosis. This is the first demonstration of the prevention of cardiac apoptosis in diabetic mice through silencing of the TLR4 gene.

Pattern recognition receptor‐mediated inflammation in diabetic vascular complications

“…It is generally accepted that TLRs are indispensable for inflammation driven by hyperglycemia, hyperlipidemia, and insulin resistance in diabetic hearts. 117,118 TLR2/3/4/6 are expressed in cardiomyocytes, 77,119,120 cardiofibroblasts, 121 and macrophages, 122 and contribute to inflammatory responses. In diabetic mice, activation of TLR2 increases the production of proinflammatory cytokines such as IL-6 and IL-1β, which subsequently cause altered calcium responses, 122,123 elevated calcineurin A, 123 as well as oxidation and phosphorylation of CaMKII.…”

Cellular death, reactive oxygen species (ROS) and diabetic complications

“…Apparently, diabetic complications have an increment of ROS and oxidative stress, leading to cellular death by several mechanisms, and, then finally, tissular damage. In different models, the diabetic complications that are induced by hyperglycemia appear to be due to an imbalance between the oxidizing species (ROS), leading to oxidative stress and cellular death 10 – 14 . Therefore, the downregulation of ROS generation may have a pivotal role in controlling diabetic complications.…”

Systemic Delivery of siRNA Specific for Silencing TLR4 Gene Expression Reduces Diabetic Cardiomyopathy in a Mouse Model of Streptozotocin-Induced Type 1 Diabetes

“…A TLR4 siRNA expression vector was constructed to express hairpin shRNA, which was demonstrated in detail in our previous study [15].…”

“…The Q-RT-PCR reaction conditions were 95°C for 10 min, 95°C for 30 s, 58°C for 1 min, and 72°C for 30 s (40 cycles). More details of Q-RT-PCR reactions were reported in our previous study [15]. Data were analyzed using MX4000 (Stratagene), Microsoft Excel2010, and GraphPad Prism software.…”

“…Apoptosis of cardiomyocytes contributes to diabetic cardiomyopathy [14]. Recently, we have shown that cardiomyocytic apoptosis is mediated through the TLR4-dependent pathway under diabetic conditions [15]. The current study was undertaken to investigate whether systemic delivery of siRNA specific for gene silencing of TLR4 would reduce cardiomyopathic changes in a type 1 diabetic mouse model.…”