Silencing of Tenascin-C inhibits hypoxia reoxygenation injury of cardiomyocyte through TLR4 (cid:0) NF-κB pathway

Background: Myocardial infarction (MI) is one of the leading causes of morbidity and mortality worldwide, and its main pathophysiological mechanism is myocardial ischemia/reperfusion (I/R) injury. TNC is an extracellular matrix glycoprotein, and high TNC expression was associated with MI and ventricular remodeling. The present study aimed to investigate the effect and the underlying mechanisms of TNC on myocardial I/R injury. Methods: Cardiomyocyte H9c2 cells was used to establish an in vitro hypoxia/reoxygenation (H/R) model, which were under hypoxia for 4 h and reoxygenation for 12 h. TNC was silenced by small interfering RNA (siRNA) in H9c2 cells. Results: TNC mRNA and protein expressions were increased by H/R. TNC knockdown by siRNA improved the cell viability in H/R-stimulated H9c2 cells. TNC knockdown reversed the H/R-induced increase in the apoptosis, as evidenced by reduced TUNEL+ cells and caspase-3 activity, and inhibited oxidative stress and inammatory cytokine production. TNC silencing inhibits TLR4 mRNA and protein expressions and NF-kappa B signals, as evidenced by reduced nuclear NF-κB p65 and increased cytoplasmic I-κBα in H/R-stimulated H9c2 cells. All these cardioprotection against H/R by siTNC was reversed by TLR4 overexpression. Conclusions: TNC silencing prevents H/R-induced injury by inhibiting apoptosis and oxidative stress by inhibition of TLR4-NF-κB pathway.


Background
Myocardial ischemia/reperfusion (I/R) injury is a complex pathological mechanism of myocardium tissue that often induced by cardiac surgery or coronary artery disease. [1,2]. Myocardial I/R injury is composed of two phase, the early myocardial ischemia caused by blockade of the coronary artery, and the late myocardial injury caused by restoration of coronary blood ow [3]. Multiple biological mechanisms involve myocardial I/R injury, including apoptosis, oxidative stress and in ammatory response in cardiomyocytes, which further aggravate myocardium tissue infarction [4,5]. Myocardial I/R injury is an important factor that contributes to high disability and mortality. However, the effective treatments for the disease are limited. Therefore, it is important to explore new molecules and uncover the precise mechanisms underlying myocardial I/R injury, so as to provide basis for the development of potential agents for therapeutic and preventive strategies.
Toll-like receptor 4 (TLR4) is a primary receptor of the innate immune system and can induce in ammatory response in myocardial ischemia through activating signaling pathways, like nuclear factor kappa B (NF-κB) [6]. Activation of NF-κB promotes production of proin ammatory cytokines, such as interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) [7]. It is reported that TLR4 plays a pivotal role in the initiation and progression of atherosclerosis, and contributes myocardial I/R injury of cardiomyocytes [8,9]. Therefore, blockade of excessive TLR4 expression is an potential therapeutic strategy for protection of myocardial I/R injury.
Tenascin-C (TNC) is an extracellular matrix (ECM) protein and is abundantly expressed during embryogenesis and is normally undetectable in most healthy adult tissues. TNC is rapidly induced in adult tissues in response to pathological stress, such as in ammation, wound healing, and various cancers [10][11][12]. TNC is rarely detected in heart of healthy adults, but is highly expressed in the myocardium during the acute post-infarction stage [13]. TNC accelerates ventricular remodeling after MI, and high expression of TNC is associated with poor long-term outcome of MI patients [14]. However, it has not been fully elucidated about the detailed molecular mechanisms of TNC in myocardial ischemia/reperfusion injury.
To further understand the role and mechanisms of TNC in myocardial I/R injury, H9C2 cardiac cell line was applied in this study to establish in vitro hypoxia/reoxygenation (H/R) model. We hypothesized that TNC silencing protects cardiomyocytes against H/R injury by inhibiting TLR4-NF-κB signaling.

Cell viability assay
For cell viability assay, H9c2 cells were seeded in a 96-well plate (5 × 10 3 cells/well). After 18 h of various treatments, cells were incubated with CCK-8 solution (10 µL) for each well, and then kept at 37˚C for 2 h. The absorbance at 450 nm was measured using a microplate reader. lactate dehydrogenase (LDH) release assay The damaged cardiomyocytes release LDH into extracellular uid, and LDH content was measured to evaluate cell injury. Culture medium was collected after 18 h of various treatments, and the concentration of LDH was determined by spectrophotometry using an LDH assay kit (Jiancheng, Nanjing, China).

Measurement of caspase-3 activity
Caspase-3 activity was determined by colorimetric assay using a commercialized assay kit (Biovision, Inc.). H9c2 cells were harvested and lysed,followed by centrifugation at 10000 g for 10 min at 4 ℃. The supernatant (30 µL) was incubated with synthetic peptide substrate Ac-DEVD-pNA (10 µL, 0.2 mM, Sigma, St. Louis, MO, USA) at 37 °C for 2 h. Caspase-3 activity was evaluated by measuring optical absorbance at 405 nm in a spectrophotometer.

TUNEL Assay
H9c2 cells were xed with 4% paraformaldehyde in PBS at room temperature for 1 h, and after washing with PBS solution, cells were blocked by 3% H 2 O 2 in 100% methanol for 10 min at room temperature.
Samples were then incubated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) on ice for 2 min. Cells were added with TUNEL reagent at 37 °C in the dark for 1 h. Cells were then washed with PBS, and incubated with DAPI solution for 30 min at room temperature in the dark. Finally, the cells were observed under uorescence microscope (detection range: 515-565 nm). The number of TUNEL + cell were counted and normalized to that of total cells (DAPI + cells).
Determination of malondialdehyde (MDA) and superoxide dismutase (SOD) After 18 h of various treatments, cells were harvested and lysed by ultrasonic pyrolysis. After centrifuged at 3000 × g for 10 min at 4˚C, the supernatant was collected and incubated with detection working uid for MDA (Cat. No. A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) or superoxide dismutase (SOD; Cat. No. A001-1) at 37˚C for 15 min. Then the reaction mixture was used to measure absorbance values at 532 nm (MDA) or 520 nm (SOD) by a microplate reader. All experiments were repeated three times independently.

Measurement of pro-in ammatory cytokines
After 18 h of various treatments, supernatant was collected and ELISA assay was performed to measure the IL-6 level by a ELISA test kit (Cat. No. R6000B, R&D Systems, Minneapolis, MN, USA). The concentrations of IL-6 in each group was determined based on the standard curve, and experiments were repeated three times independently.

Statistical Analysis
Data were expressed as mean ± standard deviation, and were analyzed by SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). One-way ANOVA was used to analyze the differences among multiple groups, followed by Bonferroni-test. P < 0.05 was considered as statistically signi cant.

H/R treatment induced TNC expression in cardiomyocytes
To investigate the potential relevance of TNC in H/R injury, we rst investigated whether H/R treatment affected TNC expression in cardiomyocytes. Rat cardiomyocytes H9c2 were subjected to hypoxia for 4 h, followed by reoxygenation for 4, 8, 12, or 16 h under normoxic conditions. The results showed that after reoxygenation cell viability was markedly reduced compared to cells with hypoxia alone (Fig. 1A). Moreover, H/R treatment signi cantly increased mRNA and protein expressions of TNC in H9c2 cells (Fig. 1B, 1C). This results suggests that TNC may involve pathological process of H/R injury.

Knockdown of TNC attenuated H/R-induced injury in cardiomyocytes
siRNA was performed to speci cally targeting TNC expression, so as to investigate the biological function of TNC in H/R injury of cardiomyocytes. The results showed that TNC mRNA and protein expressions were signi cantly decreased in cells transfected with TNC siRNAs (Fig. 1D, 1E). Moreover, knockdown of TNC signi cantly increased the cell viability and reduced LDH release in supernatant of H9c2 cells with H/R treatment (Fig. 1F, 1G). These results suggest that TNC plays a protective effect on H/R injury of cardiomyocytes.

Knockdown of TNC ameliorated H/R-induced cardiomyocytes apoptosis
To evaluate the effect of TNC on H/R-induced apoptosis, TUNEL staining was performed detect apoptosis. Representative photomicrographs of H9c2 cardiomyocytes are shown (magni cation, × 200). Apoptotic nuclei were stained with TUNEL (green), total nuclei were stained with DAPI (blue) (Fig. 2A).
Quanti cation of TUNEL staining showed that H/R exposure led to a signi cant increase in the TUNELpositive cells, however, TNC knockdown reduced TUNEL-positive cells in comparison to H/R group (P < 0.05) (Fig. 2B). Consistently, H/R also increased the caspase-3 activity, which indicates the induction of apoptosis. This effect was obviously suppressed by knockdown of TNC (Fig. 2C). These ndings indicated the anti-apoptotic actions of siTNC on H/R-induced cardiomyocytes.

Knockdown of TNC inhibited oxidative stress in H/R-induced cardiomyocytes
To test whether the oxidative stress is modulated by TNC, we assessed the lipid oxidation product MDA level and ROS-scavenging enzyme SOD activity in H9c2 cells. H/R stimulation signi cantly increased MDA content and decreased SOD activity in H9c2 cells compared with the control cells, while these changes were markedly attenuated by knockdown of TNC (Fig. 2D, 2E). In addition, RT-qPCR and ELISA showed that IL-6 mRNA level and content in culture media were signi cantly increased in the H/R group compared to the control, and knockdown of TNC attenuated the increase in this in ammatory factor (Fig. 2F, 2G). Collectively, these ndings indicate that knockdown of TNC has substantial suppression on oxidative stress and in ammatory responses in cardiomyocytes induced by H/R.

Knockdown of TNC on TLR4 expression and NF-κB translocation in cardiomyocytes undergoing H/R
RT-qPCR showed that H/R stimulation signi cantly increased the expression of TLR4 mRNA (P < 0.05). However, compared with the H/R group, cells with siTNC showed reduced TLR4 mRNA expression (P < 0.05) (Fig. 3A). Western blot also showed that the TLR4 protein level was signi cantly lower in siTNC group compared with the H/R group (P < 0.05) (Fig. 3B, 3C). Moreover, H/R stimulation induced nuclear translocation of NF-κB p65 protein in H9c2 cells, and this effect was markedly reversed by siTNC (P < 0.05) (Fig. 3D). H/R also reduced cytoplasmic I-κBα protein levels (P < 0.05), which was reversed by siTNC ( Figure. 3E). These results indicate that knockdown of TNC reversed H/R induced NF-κB nuclear translocation and degradation of cytoplasmic I-κBα, thus inhibiting NF-κB signaling pathway.

Overexpression of TLR4 reverses the protective Effects of siTNC
We then investigated the role of TLR4 in siTNC-mediated cardioprotection by overexpression of TLR4 using pcDNA3.1 vector. The e ciency of gene transfection was evaluated by western blot (Fig. 4A). siTNC increased cell viability and decreased caspase-3 activity in H9c2 cells exposed to H/R. However, these effects were reversed by TLR4 overexpression (Fig. 4B, 4C). Similarly, transfection with pcDNA-TLR4 markedly reversed the siTNC-mediated decrease in MDA and increase in SOD in H/R-exposed cells (Fig. 4D, 4E). Furthermore, TLR4 overexpression also attenuated the reduction in supernatant IL-6 by siTNC in H/R-exposed cells (Fig. 4F). These results indicate that TLR4 signaling pathway mediates siTNC-induced cardioprotection against H/R injury.

Discussion
In the present study, we explored the effect of TNC in H9c2 cells subjected with H/R, in vitro model of myocardial I/R injury. The principal ndings were that (1) TNC silencing by siRNA markedly attenuated the H/R-induced decrease in cell viability and increase in culture medium LDH content in H9c2 cells; (2) TNC silencing decreased TUNEL + cells, caspase-3 activity and attenuated the level of oxidative stress and proin ammatory cytokine (IL-6) induced by H/R in H9c2 cells; (3) TNC silencing suppressed TLR4 expression and blocked NF-κB translocation from the cytoplasm to the nucleus. Our data showed that TNC silencing play a protective role by inhibiting cell death and in ammation in H/R cardiomyocytes, which may be related to TLR4/NF-κB pathway.
In ischemic heart disease, myocardial blood reperfusion is important therapeutic method, but this method often aggravates myocardial injury. Therefore, it is urgent to explore the more detailed mechanisms underlying myocardial IR injury, so as to nd new therapeutic strategies to protect cardiomyocytes during treatment. In this study, TNC was found to increase in a time-dependent manner in H9c2 induced with H/R, and TNC inhibition increased cell viability and decreased LDH release and apoptotic cells. These results are consistent with a previous report that TNC mRNA expression was increased by oxygen and glucose deprivation, and TNC content was elevated in plasma and myocardial tissue in patients 7 days post MI [15]. Moreover, the aggravating effect of TNC on ischemic injury is also reported in other tissues. For instance, ischemia enhanced TNC expression in the optic nerve [16]. TNC de cient mice were more resistant to liver I/R injury [17].
In myocardial H/R injury is a complex pathological process, hypoxia leads to cardiomyocyte apoptosis through destroying microenvironment homeostasis [18]. Further, cardiomyocyte apoptosis promotes the major pathological processes of myocardial H/R injury, such as myocardial injury, cardiac dysfunction and eventually heart failure [19]. Cardiomyocytes under myocardial IR injury also show excessive production of reactive oxygen species (ROS), thus inducing oxidative stress. IR injury can induce cardiomyocyte apoptosis by triggering oxidative stress [20]. Therefore, inhibition of oxidative stress has become a major preventing and therapeutic strategy for myocardial IR injury [21]. TNC expression was upregulated by oxidative damage in lung artery endothelial cells and lung epithelial cells [22,23], which is consistent with our results. Moreover, our data showed that TNC might aggravate the oxidative damage of cardiomyocytes under myocardial IR injury, as evidenced by decreased MDA and increased SOD in cells with siTNC. Excessive oxidative stress is often associated with in ammation in cardiovascular disease, including myocardial I/R injury, with myeloperoxidase (MPO) as link of these two mechanisms [24]. Interestingly, TNC markedly upregulated MPO activity in neutrophils, and potentiated the neutrophilmediated injury in steatotic livers [17]. However, whether TNC also regulate MPO in myocardial IR injury and remain further in vivo investigation.
The present study showed that TNC increased TLR4 expression and promoted NF-κB translocation from cytoplasm to nucleus of cardiomyocytes with H/R injury, which is dependent on IκBα. In response to MI, TLR4 is activated and induce cardiomyocytes express proin ammatory cytokines and initiate a local in ammatory response [6]. TLR4 induced the NF-κB dependent expression of proin ammatory cytokines, such as TNF-α,IL-1β IL- 6 [25]. Recent report showed that inhibition of TLR4/NF-κB signaling pathway protected cardiomyocytes from H/R-induced injury [26]. TNC accelerated left ventricular remodeling after MI by modulating M1/M2-macrophage polarization [27]. TNC could activate in ammasomes in epicardium-derived cells (EPDCs) via TLR4, thus promoting various proin ammatory factors production from EPDCs [28]. These also support our experiment results that siTNC-mediated protection on myocardial IR injury was reversed by TLR4 overexpression. Therefore, our study propose a TNC-TLR4-NF-κB signaling pathway in myocardial IR injury. This pathway has been recently reported in promotion of in ammation in white adipose tissue [29]. TLR4 also mediated the increased mRNA expression and protein secretion of IL-6 in cultured human cardiac myo broblasts [30]. Given the fact that TNC-TLR4 pathway links between in ammation and ECM remodeling of visceral adipose tissue [31], whether it also contribute to the cardiac hypertrophy after MI remains further investigate [32]. Conclusion silencing TNC by siRNA protects cardiomyocytes from H/R injury through inhibiting apoptosis, oxidative stress and in ammatory response. Moreover, TNC-TLR4-NF-κB signaling mediate this process. Future in vivo studies is needed to con rm the protective role and mechanisms of TNC silencing for myocardial I/R injury. The data in support of the results are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This is an in vitro study based on cell line, and no formal ethics approval was required in this particular in vitro study.

Consent for publication
Not applicable.

Competing interests
The authors declared no con ict of interest with other people or organizations.