Inhibition of TGFβ‐activated protein kinase 1 ameliorates myocardial ischaemia/reperfusion injury via endoplasmic reticulum stress suppression

Abstract Transforming growth factor β‐activated protein kinase 1 (TAK1) involves in various biological responses and is a key regulator of cell death. However, the role of TAK1 on acute myocardial ischaemia/reperfusion (MI/R) injury is unknown. We observed that TAK1 activation increased significantly after MI/R and hypoxia/reoxygenation (H/R), and we hypothesized that TAK1 has an important role in MI/R injury. Mice (TAK1 inhibiting by 5Z‐7‐oxozeaenol or silencing by AAV9 vector) were exposed to MI/R injury. Primary cardiomyocytes (TAK1 silencing by siRNA; and overexpressing TAK1 by adenovirus vector) were used to induce H/R injury model in vitro. Inhibition of TAK1 significantly decreased MI/R‐induced myocardial infarction area, reduced cell death and improved cardiac function. Mechanistically, TAK1 silencing suppressed MI/R‐induced myocardial oxidative stress and attenuated endoplasmic reticulum (ER) stress both in vitro and in vivo. In addition, the inhibition of ROS by NAC partially reversed the damage of TAK1 in vitro. Our study presents the first direct evidence that inhibition of TAK1 mitigated MI/R injury, and TAK1 mediated ROS/ER stress/apoptosis signal pathway is important for the pathogenesis of MI/R injury.


| INTRODUC TI ON
Ischaemic heart disease is a common clinical cardiovascular disease that poses a serious threat to human health. 1 Myocardial hypoxia is the basic pathological process of ischaemic cardiomyopathy. Long-term hypoxia and malnutrition in the heart can result in cardiomyocyte death, leading to myocardial remodelling and heart failure. 1,2 In the clinical practice of ischaemic heart disease, myocardial ischaemia/reperfusion (MI/R) can improve blood supply to the ischaemic myocardium but can also lead to severe arrhythmia, myocardial dysfunction and myocardial stunning, and myocardial necrosis caused by cell necrosis or apoptosis can result in tissue necrosis. [3][4][5][6] In recent years, the incidence of MI/R injury has increased year by year. 2 The underlying mechanisms of MI/R injury include free radical damage, calcium overload, energy metabolism disorder, leukocyte activation and microvascular MKK3/6, which activate p38 MAPK and JNK, respectively. In addition, TAK1 activates the NF-κB pathway by interacting with TRAF6 and phosphorylating the NF-κB inducing kinase. 26 Tissue-specific deletion of TAK1 results in severe cell death and tissue damage in liver, epidermis, endothelium and intestinal epithelial cells. [27][28][29] Our previous study has also shown that TAK1 signalling pathway is involved in the regulation of cardiac hypertrophy. [23][24][25] It has been reported that notoginsenoside R1 inhibits the activation of TGF-β1-TAK1 signalling pathway and protects the heart from rabbit lung remote ischaemia/reperfusion (I/R) injury. 30 At the same time, Dusp14 prevents hepatic I/R injury by inhibiting TAK1. 31 These results suggest that TAK1 plays an important role in the regulation of cardiomyocyte death and I/R injury. However, the role of TAK1 on MI/R injury in mice has not been fully determined.
In tumour cells, ablation of TAK1 in keratinocytes and Molt-4 cells causes hypersensitivity to ROS-induced cell apoptosis, while significantly increasing hyperthermia-induced CHOP expression . [32][33][34] Kazuhito et al found that after treating Tak1-deficient mouse fibroblasts and keratinocytes with ER stress inducers Tak1 deficiency increased cell survival, attenuated proteolytic cleavage of caspase 3 and Chop expression during ER stress. 17 Furthermore, Daisuke et al reported that the inhibition of p38 MAPK activity resulted in a significant decrease in the production of ROS in cardiomyocytes. 35 Therefore, we investigated the role of TAK1 in the regulation of MI/R and the effects of TAK1 on MI/R-induced oxidative stress and ER stress.

| Methods
Male C57/B6 mice (8-12 weeks of age) were purchased from the

HIGHLIGHTS
• TAK1 has an important role in myocardial ischaemia/ reperfusion injury.
• Inhibition of TAK1 mitigates oxidative stress and ER stress to protect against myocardial ischaemia/reperfusion injury.
• The TAK1/ROS/ER stress pathway is important for the pathogenesis of myocardial ischaemia/reperfusion injury.

| Echocardiography
Transthoracic echocardiography was accomplished with an M-mode transducer (15-MHz linear transducer, Vevo1100, Visual Sonics, Toronto, Canada), as previously described. 25 At the papillary muscle level, the left ventricular shortening (LVFS) and left ventricular ejection fraction (LVEF) were measured by recording the short-axis view of M-type traces on the left anterior and posterior walls.

| Evans blue/TTC double-staining
The myocardial infarct size was determined by Evan's blue and triph-

| Determination of cardiac troponin I (cTnI) levels
The cTnI levels in mice plasm were measured using commercial ELISA kits (Shanghai Westang Bio-Tech Co., Ltd., Shanghai, China) following the instructions.

| TUNEL staining
The heart tissues were then embedded in OCT (Optimum Cutting
Then, the cells were grown under normal culture conditions for 2, 4 or 6 hours to induce the reoxygenation injury. 22

| Determination of apoptosis ratio
The CMs were collected and incubated with FITC annexin V Apoptosis detection kit (cat#556547, BD Biosciences, Franklin Lakes, NJ, USA) or PE annexin V Apoptosis detection kit (cat#559763, BD Biosciences, USA).The flow cytometry was used to detect the apoptosis, and BD FACS software was to quantify the apoptosis ratio; the experiments were repeated three times.

| ROS detection assay
After different treatments, the CMs were exposed to 50 μM DCFDA (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at 37°C in darkness, and then cells were collected and washed twice with PBS.
The green fluorescence of cells was analysed by a flow cytometry within 1 hour. The above experiments were repeated three times.

| Immunofluorescence staining
The heart tissues were embedded in OCT compound and cut approximately 5 µm thick sections. For immunofluorescence, slides were permeated with 0.5% Triton X-100 for 10 minutes and blocked with goat serum at room temperature for 1 hour. Then, they were incubated with an antibody targeting anti-p-TAK1 (1:100, Cell Signaling Technology).
The slides were washed and followed by a further incubation at room

| Real-time quantitative PCR
Total RNA was isolated from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
The reverse transcription PCR was performed by using TransScript

| Western blot
The total protein was extracted and purified, and then the concentra-

| TAK1 activation is associated with MI/R injury
To investigate the potential role of TAK1 in MI/R injury, we meas-

| Inhibition of TAK1 ameliorates myocardial dysfunction and myocardial injury
In order to investigate the role of TAK1 in MI/R, the specific TAK1 inhibitor 5Z-7-ox was intraperitoneally injected into mice 30 minutes before ischaemia (Figure 2A). 31 To further clarify the role of TAK1 in the MI/R injury, we employed AAV9 vector with a cTnT promoter to selectively silence TAK1 in cardiomyocytes. As shown in Figure 3B, five weeks after AAV9 vector delivery, mice were then subjected to MI/R operation. The inhibitory effect of Sh-TAK1 on cardiac TAK1 was confirmed by PCR and Western blot ( Figure S2). To determine cardiac function, non-invasive echocardiography was employed.
MI/R induced remarkable left ventricular dysfunction, as evidenced by decreased LVEF and LVFS, whereas 5Z-7-ox treatment significantly improved LVEF and LVFS ( Figure 2C-E). Furthermore, compared with MI/R+Sh-Con group, selectively silence TAK1 in cardiomyocytes also improved of LVEF and LVFS ( Figure 2C-E). Moreover, the AAV9-cTNT-eGFP vector carrying shTAK1 displayed no additional effect on cardiac performance in the basic state. Myocardial infarct size was measured by TTC staining. As shown in Figure 2I, the red area represented ischaemic myocardial tissue, while the white represented infarction region. Obviously, no infarction was found in myocardial tissue slices in the sham group and 5Z-7-ox group. The myocardial infarction area pretreated with 5Z-7-ox was obviously smaller than that of the MI/R group ( Figure 2J), but the ischaemic area was similar ( Figure 2K). Furthermore, compared with MI/R+Sh-Con group, administration of Sh-TAK1 significantly reduced infarct size, but the ischaemic area was similar ( Figure 2L-N). These findings corroborated that TAK1 silencing ameliorated myocardial dysfunction and alleviated myocardial injury.

| TAK1 silencing alleviates cell damage induced by MI/R
Cardiomyocyte loss caused by MI/R injury has been considered to be due to necrotic cell death. But in the past decades, studies Furthermore, compared with MI/R+Sh-Con group, TAK1 silencing reduced TUNEL-positive cardiomyocytes ( Figure 2H,I) and decreased serum level of cTnI ( Figure 3J). These findings confirmed that TAK1 silencing alleviated cardiac apoptosis and MI/R injury.

| TAK1 silencing mitigates oxidative stress and dissipates ER stress
MI/R injury affects the ER integrity and promotes ER stress. 16 To further determine the mechanisms of TAK1 silencing reducing cardiac apoptosis, the influences of TAK1 silencing on CHOP (index of ER stress apoptosis) and caspase 12 (index of ER stress-required apoptosis) were F I G U R E 3 TAK1 silencing alleviated MI/R injury. A, Western blot analysing the cardiac caspase 3 activity level in the border zone 4 hours post perfusion from mice subjected (n = 6/group); (B) the quantitative analysis of (A); (C) Western blot analysing the cardiac caspase 3 activity level (n = 6/group); (D) the quantitative analysis of (C); (E) representative cardiac TUNEL staining (green) to reveal cardiac apoptosis in the border zone 4 hours post perfusion(n = 6/group), magnification, ×400; (F) the apoptosis index (TUNEL positivity) in cardiac section, a quantification of (E); (G) cardiac troponin I (cTnI) level in mice serum 4 hours post perfusion (n = 6/group); (H) representative cardiac TUNEL staining (red) to reveal cardiac apoptosis (n = 6/group), magnification, ×400; (I) the apoptosis index in cardiac section, a quantification of (H); (J) cTnI leaves in mice serum (n = 6/group). Data are shown as means ± SEM; *P < 0.05, between the indicated groups examined. As shown in Figure 4A-C, CHOP and cleaved caspase 12 expression were significantly up-regulated after MI/R when compared to Sh-Con group (P < 0.05). However, TAK1 silencing attenuated CHOP and cleaved caspase 12 expression (P < 0.05). Importantly, TAK1 silencing did not change the expression of such proteins under baseline condition. Three main sensor proteins in the ER were also examined.
And oxidative stress has been proven to be involved in metabolic syndrome and numerous cardiovascular diseases. To decipher further the molecular mechanisms lying behind the ER-mediated protective actions of TAK1 silencing, we investigated the effects of TAK1 silencing on oxidative stress. The level of MDA ( Figure 4K, index of lipid oxidative product) was markedly increased after MI/R, while TAK1 silencing significantly reduced the content of MDA in cardiac tissue.
On the contrary, the level of SOD was decreased after MI/R. Moreover, the content of SOD in TAK1 silencing group was higher than that in the MI/R+Sh-Con group and had a tendency to return to the normal ( Figure 4L). DHE staining was performed to evaluate the content of ROS in cardiac tissue. As shown in Figure 4M,N, ROS production increased after MI/R, whereas TAK1 silencing disrupted MI/R-induced ROS production. Taken together, these results suggested that TAK1 silencing attenuated oxidative stress in the reperfused myocardium and subsequently mitigated the apoptotic pathways mediated by ER stress.

| Down-regulation of TAK1 activation attenuates H/R injury in neonatal cardiomyocytes
In order to further explore roles of TAK1 in MI/R injury, we employed hypoxia/reoxygenation (H/R)-injured neonatal cardiomyocytes.  ( Figure 5A). Inhibitory effect of si-TAK1 was confirmed by PCR and Western blot ( Figure S3). The CMs morphology was shown in Figure 5B. As shown in Figure 5C, silencing TAK1 attenuated H/R injury as evidenced by increased cell viability. PI and annexin V-FITC staining were performed on cells, and then, FACS was used to quantify total apoptotic cells (annexin V+). The H/R-induced increases in the proportions of total apoptotic cells were significantly inhibited by the transfection with si-TAK1 ( Figure 5D,E). Moreover, immunofluorescence demonstrated that H/R group exhibited more TUNEL-positive cells than control group, but H/R+si-TAK1 group obviously had fewer TUNEL-positive cells than H/R+si-NC group ( Figure 5F,G). Western blot was performed to analyse the caspase 3 activity level in CMs ( Figure 5H). Cleaved caspase 3 expression was up-regulated by H/R. Compared with H/R+si-NC group, TAK1 silencing significantly suppressed caspase 3 activity ( Figure 3I). Our results suggested that decreased TAK1 activation attenuated H/Rinduced injury and may play a protective role in apoptosis in CMs.

F I G U R E 5
Transfecting cells with si-TAK1 attenuated hypoxia/reoxygenation (H/R) injury in neonatal cardiomyocytes. A, Schematic illustration of the protocol used for H/R injury in neonatal cardiomyocytes (CMs); (B) representative morphology image after H/R in CMs allocated to si-NC, si-TAK1, H/R+si-NC, H/R+si-TAK1 groups, magnification, ×100; (C) cell viability was assessed by the MTS assay (n = 6/ group); (D) CMs were collected and stained with annexin V-FITC and PI for flow cytometry (n = 6/group); (E) quantification of total apoptotic (annexin V+) cells after FACS analysis (n = 6/group); (F) representative images of TUNEL in CMs after H/R injury (n = 6/group), magnification, ×100; (G) the quantitative analysis of (F). G, Western blot analysing caspase 3 activity level in CMs (n = 6/group); (H) the quantitative analysis of (G). Data are shown as means ± SEM; *P < 0.05, between the indicated groups

| Decreased TAK1 activation attenuates ROS production and ER stress-mediated apoptosis induced by H/R
To elucidate the detailed mechanism of TAK1 on cardiomyocyte apoptosis and MI/R, we examined the level of ER stress-related protein activity. As shown in Figure 6A-J, compared with the si-NC group, the expression of CHOP, cleaved caspase 12, ATF4, p-eIF2α, p-JNK, p-PERK, p-IRE1α, cleaved ATF-6 and GRP-78 was higher in H/R group. TAK1 silencing significantly inhibited the activation of ER stress-related proteins induced by H/R. Importantly, the expression of such proteins in si-TAK1 group was similar to si-NC group. Immunofluorescence showed that H/R induced CHOP translocation from the cytoplasm to the nucleus ( Figure 6K). The H/R group exhibited more CHOP-positive cells than the si-NC group, but the H/R+si-TAK1 group had obviously fewer CHOP-positive cells than the H/R group. As shown in Figure 6L,M, the fluorescence intensity of ROS in the H/R group was significantly higher than that in the si-NC group, while that in the H/R+si-TAK1 group was significantly decreased. These data suggested that ROS and the ER stress pathway were involved in the damage effect of TAK1 in CMs.

F I G U R E 6
Decreased TAK1 activation attenuates ROS generation and ER stress-mediated apoptosis in H/R-injured cardiomyocytes (CMs). A through J, The protein expression levels and optical density analysis of CHOP, cleaved caspase 12, ATF4, p-eIF2α, p-JNK, p-PERK, p-IRE1α, cleaved ATF-6 and GRP-78 in CMs (n = 6/group); (K) representative immunofluorescent image of CHOP (green) and F-ACTIN (red) in CMs, all nuclei were stained with DAPI (blue) (n = 6/group), magnification, ×400; (L) ROS generation was evaluated with flow cytometry using DCF fluorescence (n = 6/group); (M) the fluorescence intensity was calculated. Data are shown as means ± SEM; *P < 0.05, between the indicated groups

| Inhibition of ROS partially reverses the damage effect of TAK1
To further confirm the relationship between the TAK1 and ROS generation, TAK1-overexpressed neonatal CMs were exposed to H/R injury and pretreated with ROS inhibitor NAC ( Figure 7A). As shown in Figure 7B-E, compared with the H/R+si-NC group, TAK1 overexpression increased caspase 3 activity and total apoptotic CMs that had been induced by H/R. Notably, ROS inhibition significantly protected CMs from the injury and normalized the H/R injury aggravated by TAK1 overexpression, which was manifested as suppressed caspase 3 activity and decreased total apoptotic CMs.
Signal transduction pathways involving CHOP and caspase 12 are known to mediate ER stress-associated apoptosis. As shown in Figure 7F

| D ISCUSS I ON
Coronary heart disease is a global health problem involving blood flow recovery and acute myocardial infarction. 1 MI/R injury is closely related to the activation of oxygen free radicals. 37 Daisuke et al reported that the inhibition of p38 MAPK activity resulted in a significant decrease in the production of ROS in cardiomyocytes. 35 Furthermore, TAK1 regulates Nrf2 through modulation of Keap-p62/SQSTM1 interaction in the intestinal epithelium, thus affecting the production of ROS as described by Kazunori et al. 47 Therefore, we also evaluate the role of TAK1 in oxidative stress regulation in MI/R. This study suggested that decreased TAK1 expression attenuated ROS production which was induced by MI/R. In addition, the ROS inhibitor NAC revealed the function of ROS in the process of TAK1 damage effect in H/R. The results indicated that the inhibition of ROS generation reversed the TAK1-induced intracellular apoptosis and ER stress activation.
Importantly, the ROS inhibitor NAC did not change the expression of TAK1. Moreover, TAK1 up-regulated the expression of ER stress-related proteins including CHOP and cleaved caspase 12, and they were partially diminished by the ROS inhibitor, indicating a potential relationship between ROS and ER stress-induced apoptosis.
In summary, the inhibition of TAK1 activation reduces the activation of ROS and ER stress, effectively prevents myocardial apoptosis and thus ameliorates MI/R injury. The TAK1/ROS/ER stress pathway may be an essential mechanism of MI/R injury, and modulation of this signalling may be a novel strategy to prevent or interfere with this pathological process.

CO N FLI C T O F I NTE R E S T
All the authors declare that they had no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.