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

Figures

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  FIGURE 1. P‐TAK1 up‐regulation coincides with MI/R injury. A Western blot analysing the p‐TAK1 protein level in hearts from mice subjected to sham treatment or ischaemic for 0.5 hour followed by reperfused for oh, 1, 2, 4 hours (n = 6 per time point). *Indicates P < 0.05 compared with the sham group; (B) the quantitative analysis of (A); (C) the expression level of p‐TAK1 expression in cardiomyocytes after sham or H/R treated (n = 6 per time point). *Indicates P < 0.05 compared with the control group; (D) the quantitative analysis of (C); (E) representative immunofluorescent image of p‐TAK1 (green) and F‐ACTIN (red) in the border zone of reperfused myocardium 4 hours after reperfusion (n = 6/group), all nuclei were stained with DAPI (blue), magnification, ×400. Data are shown as means ± SEM

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  FIGURE 2. The effects of TAK1 silencing on MI/R‐induced myocardial dysfunction and myocardial injury. A, Schematic illustration of the 5Z‐7‐oxozeaenol (5Z‐7‐ox) pretreatment protocol; (B) the time course of AAV9‐mediated silence of TAK1; (C) representative M‐mode echocardiogram image after 4 hours reperfusion in mice allocated to sham, 5Z‐7‐ox, MI/R, MI/R+5Z‐7‐ox groups; (D) the quantitative analysis of left ventricular ejection fraction (LVEF) based on (C) (n = 6/group); (E) the quantitative analysis of left ventricular fractional shortening (LVFS) based on (C) (n = 6‐8/group); (F) representative M‐mode echocardiogram image (n = 6/group); (G) the quantitative analysis of LVEF (n = 6/group) and (H) LVFS (n = 6/group) based on (F); (I) representative photographs of Evans blue/TTC‐stained sections in mice allocated to sham, 5Z‐7‐ox, MI/R, MI/R+5Z‐7‐ox groups (n = 6/group), infarct area (INF: white), area at risk (AAR: red and white), and normal area (blue); (J) ratio of infarct size to area at risk (n = 6/group) based on (I); (K) ratio of area at risk to left ventricular (n = 6/group) based on (I); (L) representative Evans blue/TTC double‐stained LV transverse slices after 4 hours…

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  FIGURE 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

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  FIGURE 4. TAK1 silencing mitigated oxidative stress and dissipated ER stress in MI/R‐injured myocardium. A through L, 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 the myocardium (n = 6/group); (K) the concentration of malondialdehyde (MDA) and (L) the activity of superoxide dismutase (SOD) in myocardium (n = 6/group); (M) DHE staining and evaluate (N) the amount of ROS in cardiac tissue (n = 6/group), magnification, ×400; Data are shown as means ± SEM; *P < 0.05, between the indicated groups

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  FIGURE 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

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  FIGURE 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

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  FIGURE 7. Inhibition of ROS attenuates TAK1‐mediated increase of myocardial apoptosis in the neonatal cardiomyocytes (CMs). A, Schematic illustration of the protocol used for hypoxia/reoxygenation Injury in CMs; (B) Western blot analysing caspase 3 activity in CMs (n = 6/group); (C) the quantitative analysis of (B); (D) and (E) annexin V‐PE and 7‐AAD stained CMs were detected by flow cytometry, followed by quantitative analysis of the total apoptotic cells (annexin V+) (n = 6/group); (F through H) The protein expression levels and optical density analysis of CHOP and cleaved caspase 12 in the CMs (n = 6/group); (I) Western blot analysis of p‐TAK1 expression in CMs (n = 6/group); (J) the quantitative analysis of (I). Data are shown as means ± SEM; *P < 0.05, between the indicated groups