Tax1 banding protein 1 exacerbates heart failure in mice by activating ITCH-P73-BNIP3-mediated cardiomyocyte apoptosis

Abstract

Tax1 banding protein 1 (Tax1bp1) was originally identified as an NF-κB regulatory protein that participated in inflammatory, antiviral and innate immune processes. Tax1bp1 also functions as an autophagy receptor that plays a role in autophagy. Our previous study shows that Tax1bp1 protects against cardiomyopathy in STZ-induced diabetic mice. In this study we investigated the role of Tax1bp1 in heart failure. Pressure overload-induced heart failure model was established in mice by aortic banding (AB) surgery, and angiotensin II (Ang II)-induced heart failure model was established by infusion of Ang II through osmotic minipump for 4 weeks. We showed that the expression levels of Tax1bp1 in the heart were markedly increased 2 and 4 weeks after AB surgery. Knockdown of Tax1bp1 in mouse hearts significantly ameliorated both AB- and Ang II infusion-induced heart failure parameters. On the contrary, AB-induced heart failure was aggravated in cardiac-specific Tax1bp1 transgenic mice. Similar results were observed in neonatal rat cardiomyocytes (NRCMs) under Ang II insult. We demonstrated that the pro-heart failure effect of Tax1bp1 resulted from its interaction with the E3 ligase ITCH to promote the transcription factor P73 ubiquitination and degradation, causing enhanced BCL2 interacting protein 3 (BNIP3)-mediated cardiomyocyte apoptosis. Knockdown ITCH or BNIP3 in NRCMs significantly reduced Ang II-induced apoptosis in vitro. Similarly, BNIP3 knockdown attenuated heart failure in cardiac-specific Tax1bp1 transgenic mice. In the left ventricles of heart failure patients, Tax1bp1 expression level was significantly increased; Tax1bp1 gene expression was negatively correlated with left ventricular ejection fraction in heart failure patients. Collectively, the Tax1bp1 increase in heart failure enhances ITCH-P73-BNIP3-mediated cardiomyocyte apoptosis and induced cardiac injury. Tax1bp1 may serve as a potent therapeutic target for the treatment of heart failure.• Cardiac Tax1bp1 transgene mice were more vulnerable to cardiac dysfunction under stress.• Cardiac Tax1bp1 transgene mice were more vulnerable to cardiac dysfunction under stress.• Knockout of Tax1bp1 in mouse hearts ameliorated heart failure induced by pressure overload.• Tax1bp1 interacts with the E3 ligase Itch to promote P73 ubiquitination and degradation, causing enhanced BNIP3-mediated apoptosis.• Tax1bp1 may become a target of new therapeutic methods for treating heart failure.

Keywords: BCL2 interacting protein 3; ITCH; P73; Tax1 banding protein 1; apoptosis; heart failure.

Fig. 1. Tax1bp1 was upregulated during the pathology of cardiac remodeling.

a Tax1bp1 protein levels in mouse hearts undergoing aortic banding surgery (n = 6 for each group). b Tax1bp1 and wheat germ agglutinin (WGA) staining in mouse hearts (n = 5, Scale bars: 50 μm). c Tax1bp1 protein levels in cardiomyocytes under Ang II insult (n = 6). d Tax1bp1 staining in cardiomyocytes (n = 5, Scale bars: 50 μm). Tax1bp1 and α-actinin protein levels in cardiomyocytes (e), Tax1bp1 and α-SMA protein levels in fibroblasts (f), and Tax1bp1 and CD31 protein levels in endothelial cells (g) isolated from mouse hearts 1, 2, and 4 weeks after AB (n = 6). **P < 0.01. One-way ANOVA followed by a post hoc Tukey test was used to compare the data in Fig. 1a, e–g Student’s unpaired t test was used to compare the data in Fig. 1b–d.

Fig. 2. Tax1bp1 knockdown in vivo ameliorated cardiac remodeling in mice under AB.

a Heart weight (HW)/body weight (BW) and HW/tibia length (TL) ratios in mice subjected to AAV9-shTax1BP1 injection and AB surgery (n = 12). b H&E staining and WGA staining and cross-section area (CSA) quantification (n = 6, Scale bars: 50 μm). c PSR staining and left ventricular (LV) collagen volume (n = 6, Scale bars: 100 μm). d Echocardiography results in mice subjected to AAV9-shTax1BP1 injection and AB surgery (n = 10). LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening. e Hemodynamic results. dp/dt_max, maximal rate of pressure development; dp/dt_min, maximal rate of pressure decay. f α-actin staining in NRCMs transfected with Tax1bp1 siRNA under Ang II insult (n = 6, Scale bars: 50 μm). **P<0.01. One-way ANOVA followed by a post hoc Tukey test was used to compare the data in Fig. 2a–f.

Fig. 3. Tax1bp1 exacerbated HF in mice under AB.

a Heart weight (HW)/body weight (BW) and HW/tibia length (TL) ratios in Tax1bp1 transgenic mice 4 weeks after AB (n = 12). b H&E staining and cross-sectional area (CSA) quantification (n = 6, Scale bars: 50  μm). c PSR staining and left ventricular (LV) collagen volume (n = 6, Scale bars: 100 μm). d Echocardiography results in Tax1bp1 transgenic mice subjected to AB surgery (n = 10). LVEF, left ventricular ejection fraction, LVFS left ventricular fractional shortening. e Hemodynamic results. dp/dt_max, maximal rate of pressure development; dp/dt_min, maximal rate of pressure decay. f α-actin staining in NRCMs transfected with Ad-Tax1bp1 under Ang II insult (n = 6, Scale bars: 50 μm). **P < 0.01. One-way ANOVA followed by a post hoc Tukey test was used to compare the data in Fig. 3a–f.

Fig. 4. Tax1bp1 affected cell death.

a TUNEL and α-actin staining in mice subjected to AAV9-shTax1BP1 injection and AB surgery (n = 6, Scale bars: 50 μm). b TUNEL and α-actin staining in Tax1bp1 transgenic mice subjected to AB surgery (n = 6, Scale bars: 50 μm). c Bax, Bcl-2, and c-caspase 3 in hearts from mice subjected to AAV9-shTax1BP1 injection and AB surgery (n = 4). d Bax, Bcl-2, and c-caspase 3 in Tax1bp1 transgenic mice (n = 4). e TUNEL staining in NRCMs transfected with Ad-Tax1bp1 or Tax1bp1 siRNA (n = 6, Scale bars: 50 μm). **P < 0.01. Student’s unpaired t test was used to compare the data in Fig. 4a, b, e. One-way ANOVA followed by a post hoc Tukey test was used to compare the data in Fig. 4c, d.

Fig. 5. ITCH-P73-BNIP3 was the target of Tax1bp1.

a and b BNIP3, P73, and ITCH protein levels in NRCMs transfected with Ad-Tax1bp1 under Ang II insult (n = 6). c and d BNIP3, P73, and ITCH protein levels in NRCMs transfected with Tax1bp1 siRNA under Ang II insult (n = 6). **P < 0.01. NS, no significance. One-way ANOVA followed by a post hoc Tukey test was used to compare the data in Fig. 5a–d.

Fig. 6. Tax1bp1 interacted with ITCH, affecting P73 ubiquitination.

a Endogenous Co-IP assay in NRCMs. b Exogenous Co-IP assay in NRCMs cotransfected with psicoR-HA-Tax1bp1 and psicoR-Flag-ITCH. c Co-IP assay in NRCMs stimulated with Ang II and Co-IP assay in protein from heart tissue in mice subjected to AB surgery. d P73 expression levels in NRCMs transfected with either Ad-P73 or P73 siRNA (n = 6). e Upper: Luciferase assay in NRCMs cotransfected with Ad-P73, P73 siRNA or Ad-Tax1bp1, Tax1bp1 siRNA; lower panel: mRNA level of BNIP3 in the indicated condition (n = 6). f 293 T cells were transfected with HA-p73, Myc-ubiquitin, and Flag-Tax1bp1 or ITCH siRNA and treated with the proteasome inhibitor MG132 prior to harvesting. Ubiquitination assays and Western blotting to detect P73 ubiquitination. g 293 T cells were transfected with plasmids expressing Myc-ubiquitin, Flag-Tax1bp1, HA-P73, and Tax1bp1 mutants (Y741A and Y768A). Ubiquitination assays and Western blotting to detect P73 ubiquitination. **P < 0.01. Student’s unpaired t test was used to compare the data in Fig. 6d and e.

Fig. 7. Association between Tax1bp1 level and HF.

a Tax1bp1 protein levels in patients with heart failure (n = 8 in each group). b BNIP3 and P73 protein levels in patients with heart failure (n = 8 in each group). **P < 0.01. c Correlation model for Tax1bp1 and BNIP3 protein levels in patients with heart failure (n = 8 in each group). d Correlation model for Tax1bp1 mRNA level and LVEF and LVEDD in patients with heart failure (n = 8 in the control group, n = 8 in the HF group). Student’s unpaired t test was used to compare data in Fig. 7a, b; Spearman’s test was used in Fig. 7c, d.