Introduction
Diabetic cardiomyopathy was first proposed in 1972 by Rubler and is a disorder of the heart muscle in patients with diabetes [
1,
2]. It can result in an insufficiency of general blood circulation, reaching a state called heart failure, along with pulmonary or peripheral edemas [
3‐
5]. Cardiomyopathy may occur throughout diabetes, and its major clinical manifestations are hypertrophy, fibrosis, inflammation of cardiomyocytes, and myocardial dysfunction. There might be a close relationship between hypertrophy, fibrosis, inflammation of cardiomyocytes, myocardial dysfunction, and ventricular remodeling [
6,
7]. At present, there is no specific method to diagnose diabetic cardiomyopathy. The main problem to be resolved is how to delay and prevent the development of this heart disease [
8,
9].
Toll-like receptor 4 (TLR4), a proximal signaling receptor in innate immune responses to lipopolysaccharide (LPS) of Gram-negative pathogens, is expressed in the heart and vasculature. TLR4 binds to LPS from the Gram-negative bacteria and autogenous ligands, such as heat shock proteins and fibronectin, which are released during oxidative stress. Increased TLR4 expression has been observed in patients with heart failure and in ischemic hearts [
10‐
13].
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.
Methods
Animals
Male adult C57BL/6 mice, aged from 6 to 8 weeks, were purchased from the Jackson Laboratory (Bar Harbor ME, USA). All mice were anesthetized through injection in the abdominal cavity (1% pentobarbital sodium, 80 mg/kg), they were killed by carbon dioxide inhalation of CO2 at a flow rate of more than 30% of the chamber volume per minute, and death was confirmed by cervical dislocation. The were incinerated after the experiment. All experimental procedures were approved by the Animal Use Subcommittee at Sichuan University.
siRNA Expression Vectors
Three target sequences of the TLR4 gene were selected. The oligonucleotides containing sequences specific for TLR4 (5′-GATCCCGTATTAGGAACTACCTCTATGCTTGATATCCGGCATAGAGGTAGTTCCTAATATTTTTTCCAAA-3′ and 5′-AGCTTTTGGAAAAAATATTAGGAACTACCTCTATGCCGGATATCAAGCATAGAGGTAGTTCCTAATACGG-3′; 5′-GATCCCGTTGAAACTGCAATCAAGAGTGTTGATATCCGCACTCTTGATTGCAGTTTCAATTTTTTCCAAA-3′ and 5′-AGCTTTTGGAAAAAATTGAAACTGCAATCAAGAGTGCGGATATCAACACTCTTGATTGCAGTTTCAACGG-3′; 5′-GATCCCATTCGCCAAGCAATGGAACTTGATATCCGGTTCCATTGCTTGGCGAATTTTTTTCCAAA-3′ and 5′-AGCTTTTGGAAAAAAATTCGCCAAGCAATGGAACCGGATATCAAGTTCCATTGCTTGGCGAATGG-3′) were synthesized and annealed.
TLR4 siRNA Expression Vector Construction
A TLR4 siRNA expression vector was constructed to express hairpin shRNA, which was demonstrated in detail in our previous study [
15].
Hyperglycemic Mouse Model
Low dose streptozotocin (STZ) can maintain insulin secretory function and produce a diabetes state. The diabetes state models the partial and continuous loss of β cells in type 2 diabetes mellitus. The metabolic characteristics of STZ-induced diabetic mouse induced by low dose STZ are closer to those of type 2 diabetes in humans compared with the gene rodent models [
16,
17]. So, in the study, we chose low dose STZ for type 2 diabetes induction. Consecutive peritoneal injections of streptozotocin (STZ) (50 mg/kg/day) were utilized to induce diabetes in 14 2-month-old adult male mice for each group. Whole blood was gathered from the mouse tail vein 72 h after the last STZ injection. The data of random glucose levels were measured through the One Touch Ultra 2 blood glucose monitoring system (Life Scan Inc, CA, USA). The mice would be used for the study only if they were considered diabetic and had hyperglycemia (fasting blood glucose at least 16.7 mM) at 72 h after the STZ injections [
18]. Citrate buffer-treated mice were used as the non-diabetic control. Two months after the induction of diabetes, all the mice were subjected to the following experiments.
Treatment of TLR4 siRNA
In accordance with the manufacturer’s instructions, TLR4 siRNA or scrambled siRNA (5 µg) was mixed with 40 µl of transfection reagent NANOPARTICLE (Altogen Biosystems, Las Vegas, NV, USA) in a total volume of 100 µl of 5% glucose (w/v). Then, 30 STZ-induced hyperglycemic C57BL/6 mice were randomly assigned into three groups of ten mice. The STZ-induced hyperglycemic mice were untreated or intravenously injected with the TLR4 siRNA or scrambled mixture via the tail vein. In addition, ten non-diabetic mice were used as control. Four weeks after the first treatment, the diabetic mice were injected again with the same dose of the TLR4 siRNA or scrambled siRNA.
Histological Analysis
Hearts were excised from the mice and washed with saline solution. They were then fixed with 10% formalin. To observe the left and right ventricle, hearts were cut transversely. To detect collagen deposition, heart sections with a thickness of 5 µm were stained with hematoxylin and eosin (H&E) and a saturated solution of picric acid containing 1% Sirius red. Thes sections were then observed under light microscopy using computer-assisted morphometry (Image-Pro Plus Version 6.0). All available fields (more than 30 fields) were measured in each sample, including the septum, the right ventricles, and the left ventricles (all fields were analyzed using a ×40 objective lens). For the cross-sectional area of cardiomyocytes, sections were stained with fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (WGA; Invitrogen) to detect membranes and with 4,6-diamidino-2-phenylindole (DAPI) to detect nuclei. Individual cardiomyocytes were measured with a quantitative digital image analysis system (NIH Image version 1.6). The outline of 200 cardiomyocytes was traced in each section.
Quantitative Real-Time Polymerase Chain Reaction (Q-RT-PCR)
Total RNA was extracted from heart tissues through Trizol reagent (Gibco-BRL) according to the manufacturer’s instruction. RNA was then reverse-transcribed using an oligo-(dT) primer and reverse transcriptase (Invitrogen). Quantitative real-time polymerase chain reaction (Q-RT-PCR) was performed to analyze mRNA expression for beta-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), caspase-3, collagen I and III (Col I and III), transforming growth factor-β1 (TGFβ1), interferon alpha (INFα), intercellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as previously described. The primers used are as follows.
ANP: 5′-GGCTCCTTCTCCATCACCAA-3′ (forward) and 5′-CGAGAGCACCTCCATCTCTC-3′ (reverse).
β-MHC: 5′-GTCAAGCTCCTAAGTAATCTGTT-3′ (forward) and 5′-GAAAGGATGAGCCTTTCTTTGC-3′ (reverse).
TGFβ1: 5′-AAGAAGTCACCCGCGTGCTA-3′ (forward) and 5′-TGTGTGATGTCTTGGTTTTGTCA-3′ (reverse).
Col I: 5-TCCTGGCAACAAAGGAGACA-3 (forward) and 5-GGGCTCCTGGTTTTCCTTCT-3 (reverse).
Col III: 5-ACGTAGATGAATTGGGATGCAG-3 (forward) and 5-GGGTTGGGGCAGTCTAGTC-3 (reverse).
VCAM-1: 5-TACCAGCTCCCAAAATCCTG-3 (forward) and 5-TCTGCTAATTCCAGCCTCGT-3 (reverse).
ICAM-1: 5-GTGATCCCTGGGCCTGGTG-3 (forward) and 5-GGAAACGAATACACGGTGATGG-3 (reverse).
TNFα: 5-CAGCCGATGGGTTGTACCTT-3 (forward) and 5-TGTGGGTGAGGAGCACGTAGT-3 (reverse).
IL-1β: 5-CAGTTCTGCCATTGACCATC-3 (forward) and 5-TCTCACTGAAACTCAGCCGT-3 (reverse).
GAPDH: 5′-ACTCCACTCACGGCAAATTC-3’(forward) and 5′-TCTCCATGGTGGTGAAGACA-3′ (reverse).
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.
Echocardiography
The mice were anesthetized by intraperitoneal injection of ketamine (120 mg/kg). Body temperature was maintained at 36.5–37.5 °C. Mice were imaged in a warm handing platform using a 40-MHz linear array transducer attached to a cardiovascular ultrasound system (Vivo 2100, Visual Sonics). M-mode and 2-D parasternal short-axis scans at the level of the papillary muscles were used to assess changes in left ventricle fractional shortening (FS) and ejection fraction (EF).
Statistical Analysis
All data are expressed as the mean ± SE. One-way ANOVA with Newman Keuls test was used to compare two groups. Statistical analyses were conducted through SPSS 25.0 (IBM Corp., Armonk, NY, USA). Differences were considered to be significant at a two-tailed p < 0.05.
Discussion
Diabetic cardiomyopathy, one of the most common complications in patients with diabetes mellitus, is defined as ventricular dysfunction independent of hypertension and coronary artery disease [
19‐
25]. At present, the morbidity of diabetic cardiomyopathy is gradually increasing with the growing number of individuals with diabetes [
9]. Although the mechanism of diabetic cardiomyopathy is poorly understood, hypertrophy, fibrosis, and the inflammation of cardiomyocytes are reported as the predominant major factors causing myocardial dysfunction [
6]. Moreover, diabetes increases hypertrophy, fibrosis, and inflammation of cardiomyocytes in diabetic animals and patients [
6,
7,
26]. TLRs are the proximal signaling receptors in innate immune responses. Among the 10 identified human TLRs, at least two of them, TLR2 and TLR4, exist abundantly in the heart [
27,
28]. However, the role of TLR4 in myocardial dysfunction has not been characterized. We also found that TLR4 plays a crucial role in cardiac apoptosis. The level of TLR4 mRNA was significantly elevated in myocardial tissue after 7 days of hyperglycemia. However, after TLR4 siRNA treatment, cell apoptosis in myocardial tissues was attenuated significantly, which was associated with caspase-3 inactivation [
14]. In this study, we provide more evidence that hyperglycemia is capable of triggering hypertrophy, fibrosis, and cardiomyocytic inflammation. Furthermore, we demonstrate that TLR4 expression in the myocardia of STZ-treated mice is significantly increased. The hypertrophy, fibrosis, inflammation of cardiomyocytes, and myocardial dysfunction can be attenuated by knockdown of the TLR4 gene.
Hypertrophy of cardiomyocytes is one of the features of diabetic cardiomyopathy. Fibroblasts, a type of cardiac cell, were present in significantly higher numbers in myocardial tissue of diabetic cardiomyopathy and could further induce the synthesis and metabolism of collagen. Type I and type III collagens are the main components of the extracellular matrix. Increased collagen in myocardial tissue can lead to a decrease of myocardial compliance and ventricular elasticity in diabetic mice. Visually, the disorders and changes of collagens I and III in the fibroblast hypertrophy of diabetic cardiomyopathy were the major factors that caused the accumulation of extracellular matrix and cardiac diastolic dysfunction. In our study, we showed that TLR4 siRNA reduced the myocardial fibrosis in diabetic mice.
ANP is a hormone synthesized and secreted by the cardiac tissue. Structural and functional changes in the heart will cause disorders of ANP secretion. There is a significant relationship between ANP secretion and myocardial hypertrophy. Our study showed that silencing TLR4 prevented the development of cardiac hypertrophy in diabetic mice.
Studies have shown that inflammation has a major effect on diabetic cardiomyopathy. TNFα antagonism attenuates the development of diabetic cardiomyopathy associated with a reduction of intramyocardial inflammation and cardiac fibrosis. Cardiac cytokines, such as IL-1 and TGFβ, are involved in the development of cardiac fibrosis and heart failure. β-MHC is associated with chronic hyperglycemia-induced oxidative stress. New methods are being developed to prevent diabetic cardiomyopathy by blocking inflammation associated with these factors. There is a close relationship between TLR4 expression and inflammation. Our study revealed that silencing TLR4 prevented the development of inflammation in diabetic mice.
New studies suggest that diabetes is related to cardiomyocyte dysfunction. Moreover, the morbidity of diabetic cardiomyopathy is increasing [
29,
30]. However, the causes of diabetic cardiomyopathy are unknown, and effective methods to treat this disease are lacking. To clarify the role of TLR4 played in diabetic cardiomyopathy, we silenced the TLR4 gene using siRNA. Our study showed that TLR4 expression is related to myocardium trauma in STZ-induced diabetic mice. Silencing TLR4 can prevent hypertrophy, fibrosis, and inflammation in myocardial hearts to improve heart function. These results are consistent with previous studies [
31‐
33].
Much research about TLR4 has been published in recent years mainly addressing its association with inflammation, sepsis, cancer, obesity, and atherosclerosis [
34‐
38]. This article and our previous research highlight its effect on diabetic cardiomyopathy, an important potentially fatal complication of diabetes, possibly through upregulation of IFN and TNF and inducement of NADPH oxidase activation and ROS production in cardiomyocytes, a different mechanistic pathway compared with previous research. Moreover strong evidence has been provided about TLR4 accessory proteins MD2 and myocardial inflammatory injuries which sheds new light on our future research direction [
28].
siRNA could be designed to target and silence virtually all mRNA. However, the major obstacle in clinical application of siRNA lies in developing safe and effective methods of delivering siRNA to target cells. siRNA requires specific vehicles that could facilitate its intracellular uptake and cytosolic delivery for bioactivity. In a later study, we have attempted to develop tissue- and cell-specific siRNA delivery carriers.