KLF9 Aggravates Doxorubicin-Induced Cardiotoxicity by Regulating the ROS/p53 Signalling Pathway

Fig. 1. Increased KLF9 expression in DOX-treated mice heart tissues. A Cardiac KLF9 expression in hearts from DOX-treated or Control (Ctrl) mice detected by Western blot (n = 6). B Relative KLF9 mRNA level in mice hearts with or without DOX treatment detected by qRT-PCR (n = 6). C Cardiac KLF9 expression in mice hearts with or without KLF9 overexpression (n = 6). D Immunofluorescence staining of KLF9 and α-actinin in mice hearts with or without KLF9 overexpression (n = 6). E Cardiac KLF9 expression in hearts from KLF9-deficient or Ctrl mice (n = 6). F Immunofluorescence staining of KLF9 and α-actinin in hearts from KLF9-deficient or Ctrl mice (n = 6). All results were presented as mean ± SEM. A, B, * p < 0.05 vs. Ctrl. C, D, * p < 0.05 vs. AAV9-NC. E, F, * p < 0.05 vs. AAV9-ScRNA

Fig. 2. Increased KLF9 expression in DOX-treated H9c2 cells. A KLF9 expression in H9c2 cells treated with DOX for the indicated times (0, 0.5, 1, 2, 5, and 10 µM) (n = 6). B Relative KLF9 mRNA level in H9c2 cells treated with DOX for the indicated times (0, 0.5, 1, 2, 5, and 10 µM) (n = 6). C Fluorescence intensity in H9c2 cells incubated with adenovirus carrying KLF9 (Ad-KLF9) for the indicated MOI (0, 30, 50, 100, 150, and 200) (n = 6). D Fluorescence intensity in H9c2 cells incubated with adenovirus carrying shKLF9 (Ad-shKLF9) for the indicated MOI (0, 30, 50, 100, 150, and 200) (n = 6). All results were presented as mean ± SEM. * p < 0.05 vs. Ctrl

Fig. 3. KLF9 overexpression aggravated DOX-induced cardiac injury and dysfunction in mice. A Schematic diagram of AAV9 and DOX intervention. B Cardiac KLF9 expression in mice with or without KLF9 overexpression after DOX or normal saline (NS) treatment (n = 6). C Relative KLF9 mRNA level of mice heart tissues in each group (n = 6). D The survival curves of mice (n = 20). E The average body weight gain (n = 12). F HW/TL of mice (n = 12). G Cardiac tissues HE staining and WGA staining and quantification of cardiomyocyte cross-sectional areas (scale bar = 50 μm; n = 6). H Serum levels of LDH, CK-MB and cTnT (n = 6). I Echocardiographic parameters of cardiac function (n = 8). All results were presented as mean ± SEM. * p < 0.05 vs. AAV9-NC + NS; # p < 0.05 vs. AAV9-NC + DOX. ns: not significant

Fig. 4. KLF9 deficiency alleviated DOX-induced cardiotoxicity in mice. A Cardiac KLF9 expression in mice with or without KLF9 silence after DOX or NS treatment (n = 6). B Relative KLF9 mRNA level of mice heart tissues in each group (n = 6). C The survival curves of mice (n = 20). D The average body weight gain (n = 12). E HW/TL of mice (n = 12). F Cardiac tissues HE staining and WGA staining and quantification of cardiomyocyte cross-sectional areas (scale bar = 50 μm; n = 6). G Serum levels of LDH, CK-MB and cTnT (n = 6). H Echocardiographic parameters of cardiac function (n = 8). All results were presented as mean ± SEM. * p < 0.05 vs. AAV9-ScRNA + NS; # p < 0.05 vs. AAV9-ScRNA + DOX. ns: not significant

Fig. 5. KLF9 overexpression exacerbated DOX-induced cardiomyocyte apoptosis in vivo and vitro. A TUNEL staining in mice heart tissues followed by quantification of TUNEL positive nuclei in heart samples (scale bar = 50 μm; n = 6). B The effects of KLF9 overexpression on Bax, Bcl-2 and cleaved-caspase3 expression detected by Western blot (n = 6). C KLF9 expression in H9c2 cells with or without KLF9 overexpression after DOX or phosphate buffer saline (PBS) treatment (n = 6). D Relative KLF9 mRNA level in H9c2 cells (n = 6). E The expression of Bax, Bcl-2 and cleaved-caspase3 in H9c2 cells (n = 6). F Quantification of cell apoptosis using Annexin V-APC/7-AAD followed by analysis with flow cytometry (n = 6). All results were presented as mean ± SEM. A, B, * p < 0.05 vs. AAV9-NC + NS; # p < 0.05 vs. AAV9-NC + DOX. C–F, * p < 0.05 vs. Ad-NC + PBS; # p < 0.05 vs. Ad-NC + DOX

Fig. 6. KLF9 deficiency suppressed DOX-induced cardiomyocyte apoptosis in vivo and vitro. A TUNEL staining in mice heart tissues followed by quantification of TUNEL positive nuclei in heart samples (scale bar = 50 μm; n = 6). B The effects of KLF9 deficiency on Bax, Bcl-2 and cleaved-caspase3 expression detected by Western blot (n = 6). C KLF9 expression in H9c2 cells with or without KLF9 silence after DOX or PBS treatment (n = 6). D Relative KLF9 mRNA level in H9c2 cells (n = 6). E The expression of Bax, Bcl-2 and cleaved-caspase3 in H9c2 cells (n = 6). F Quantification of cell apoptosis using Annexin V-APC/7-AAD followed by analysis with flow cytometry (n = 6). All results were presented as mean ± SEM. A, B, * p < 0.05 vs. AAV9-ScRNA + NS; # p < 0.05 vs. AAV9-ScRNA + DOX. C–F, * p < 0.05 vs. Ad-ScRNA + PBS; # p < 0.05 vs. Ad-ScRNA + DOX

Fig. 7. DOX activated p53-associated cardiomyocyte apoptosis via a ROS-dependent mechanism in vitro. A Cardiac p53 expression in H9c2 cells from DOX-treated or PBS (n = 6). B The effects of p53 deficiency on Bax, Bcl-2 and cleaved-caspase3 expression (n = 6). C Percentages of apoptotic cells for H9c2 cells incubated with or without sip53 (n = 6). D The expression of p53, Bax, Bcl-2 and cleaved-caspase3 in H9c2 cells treated with or without NAC (n = 6). E Quantitative analysis of cells apoptosis in H9c2 cells treated with or without NAC (n = 6). F ROS levels assessed by flow cytometry in H9c2 cells with or without NAC (n = 6). G ROS levels in H9c2 cells infected with or without sip53 (n = 6). All results were presented as mean ± SEM. A, * p < 0.05 vs. Ctrl. B, C, * p < 0.05 vs. siRNA + PBS; # p < 0.05 vs. siRNA + DOX. D–F, * p < 0.05 vs. PBS; # p < 0.05 vs. DOX. G, * p < 0.05 vs. siRNA + PBS; # p < 0.05 vs. siRNA + DOX. ns: not significant

Fig. 8. Txnrd2 was involved in the activation of ROS/p53 axis mediated by KLF9 in vitro. A–D The level changes of Txnrd2, ROS and p53 in H9c2 cells with KLF9 overexpression (n = 6). E–H The effects of KLF9 deficiency on Txnrd2, ROS and p53 in H9c2 cells (n = 6). I–L The changes of ROS levels and p53 expression in KLF-deficient H9c2 cells infected with siRNA or siTxnrd2 (n = 6). M Schematic illustration of PCR-amplified fragments of Txnrd2 promoter performed by chromatin immunoprecipitation (ChIP) assays. N, O Quantitative analysis of amplified fragments of Txnrd2 promoter presented by histograms (n = 3). All results were presented as mean ± SEM. A–D, * p < 0.05 vs. Ad-NC + PBS; # p < 0.05 vs. Ad-NC + DOX. E–H, * p < 0.05 vs. Ad-ScRNA + PBS; # p < 0.05 vs. Ad-ScRNA + DOX. I–L, * p < 0.05 vs. siRNA + Ad-ScRNA; # p < 0.05 vs. siRNA + Ad-shKLF9. N, * p < 0.05 vs. IgG. O, * p < 0.05 vs. PBS

Fig. 9. KLF9 affected ROS/p53 signalling pathway via inhibiting Txnrd2 in vivo. A The effects of KLF9 overexpression on Txnrd2 and p53 expression in vivo (n = 6). B The effects of KLF9 overexpression on ROS levels assessed by DHE staining in mice heart tissues (scale bar = 50 μm; n = 6). C The expression changes of Txnrd2 and p53 in mice with KLF9 silence (n = 6). D Statistical analysis of DHE staining in mice heart tissues with or without KLF9 silence (scale bar = 50 μm; n = 6). All results were presented as mean ± SEM. A, B, * p < 0.05 vs. AAV9-NC + NS; # p < 0.05 vs. AAV9-NC + DOX. C, D, * p < 0.05 vs. AAV9-ScRNA + NS; # p < 0.05 vs. AAV9-ScRNA + DOX