TAX1BP1 overexpression attenuates cardiac dysfunction and remodeling in STZ-induced diabetic cardiomyopathy in mice by regulating autophagy

BBA - Molecular Basis of Disease 1864 (2018) 1728–1743

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TAX1BP1 overexpression attenuates cardiac dysfunction and remodeling in STZ-induced diabetic cardiomyopathy in mice by regulating autophagy

T

Yang Xiao1, Qing Qing Wu1, Ming Xia Duan, Chen Liu, Yuan Yuan, Zheng Yang, Hai Han Liao, ⁎ Di Fan, Qi Zhu Tang Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China Cardiovascular Research Institute, Wuhan University, Wuhan 430060, PR China Hubei Key Laboratory of Cardiology, Wuhan 430060, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Autophagy Diabetic cardiomyopathy Tax1 binding protein 1 Non-canonical NF-κB RelB

Diabetic cardiomyopathy is associated with suppressed autophagy and augmented inflammation in the heart. The effects of Tax1 binding protein 1 (TAX1BP1) on both autophagy and inflammation suggest that it may participate in the progression of diabetic cardiomyopathy. Mice were injected with streptozotocin (STZ) to induce experimental diabetes. An adenovirus system was used to induce heart specific TAX1BP1 overexpression 12 weeks after STZ injection. TAX1BP1 expression was significantly decreased in STZ-induced diabetic mouse hearts. TAX1BP1 overexpression in the heart alleviated cardiac hypertrophy and fibrosis, attenuated inflammation, oxidative stress, and apoptosis, and improved cardiac function in STZ-induced diabetic mice. Diabetic mice exhibited decreased autophagy. By contrast, increased autophagy was observed in diabetic mice overexpressing TAX1BP1. TAX1BP1 overexpression promoted autophagic flux, as demonstrated by increased LC3-RFP fluorescence in vitro. Furthermore, the autophagy inhibitor 3-MA abolished the protective effects of TAX1BP1 in vivo. Interestingly, we found that TAX1BP1 increased autophagy via the activation of a non-canonical NF-κB signaling pathway. Conversely, RelB knockdown disrupted the protective effects of TAX1BP1 in cardiomyocytes. TAX1BP1 thus restores the decreased autophagy level, leading to decreased inflammatory responses and oxidative stress and reduced apoptosis in cardiomyocytes.

1. Introduction Diabetes mellitus is associated with significantly higher cardiovascular morbidity and mortality than normoglycemia [1]. Cardiovascular complications are responsible for the high morbidity and mortality observed in diabetic patients, with a 2-fold greater (male) and 5-fold greater (female) relative risk for heart failure found in diabetic patients compared with age-matched controls [2,3]. This emphasizes the need to understand and manage diabetic cardiomyopathy. The prominent features of diabetic cardiomyopathy are cardiac hypertrophy and fibrosis, which are accompanied by compromised systolic and diastolic function [3–5]. Oxidative stress, pro-inflammatory responses, metabolic alterations and cell death form a complex interaction, playing an important role in the progression of diabetic cardiomyopathy [6–8]. Various cardiac diseases, including ischemia–reperfusion, chronic ischemia, cardiac hypertrophy and heart failure are associated with alterations in the autophagy level [5,9,10]. Accumulated findings have ⁎

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demonstrated that autophagy is also centrally involved in diabetic cardiomyopathy [8,11,12]. The detrimental effects of both “too little” and “too much” autophagy are becoming apparent. Well-established type 1 diabetic animal models, namely, STZ-induced diabetic mice and OVE26 mice, exhibit cardiac autophagy inhibition along with cardiomyocyte apoptosis and cardiac dysfunction [5,13]. Many molecules protect against type 1 diabetes-induced cardiac dysfunction by activating autophagy [14] [15] [16]. Upregulation of autophagy in rodents with type 2 diabetes has also been described [17].Autophagy inhibition has a beneficial effect on type 2 diabetes-induced cardiomyopathy [18]. These findings suggest that autophagy is altered diversely in different types of diabetes-induced cardiac pathologies. Targeting autophagy regulation may be a potential therapeutic strategy for diabetic cardiomyopathy. NF-κB is a crucial transcription factor that regulates inflammatory responses, immune responses, cell survival, and cell growth [19]. The canonical NF-κB pathway of NF-κB has been identified in cardiac

Corresponding author at: Department of Cardiology, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan 430060, PR China. E-mail address: [email protected] (Q.Z. Tang). Yang Xiao and Qing Qing Wu contribute equally.

https://doi.org/10.1016/j.bbadis.2018.02.012 Received 22 November 2017; Received in revised form 3 February 2018; Accepted 19 February 2018

Available online 21 February 2018 0925-4439/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. TAX1BP1 was downregulated in the heart in STZ-induced diabetic mice and in HG-stimulated cardiomyocytes A. Representative western blot and analysis of TAX1BP1 in STZ-induced diabetic hearts (n = 6). B. The protein level of TAX1BP1 in cardiomyocytes (n = 6 sample). *P < 0.05 vs the sham/PBS group. C. Immunohistochemistry of TAX1BP1 in STZ-induced diabetic hearts (n = 5). D. Immunofluorescence of TAX1BP1 (red) and α-actinin (green) in high glucose (HG)stimulated cardiomyocytes (n = 5 samples).

myocytes as a central mediator of various cardiac pathologies. Inhibition of the canonical NF-κB pathway is beneficial in the treatment of heart disease [20]. The non-canonical NF-κB RelB/P52 pathway has

also been identified in cardiomyocytes. In contrast to canonical NF-κB signaling, very few studies have investigated the role of non-canonical signaling in the heart [21]. In other cell systems, this non-canonical NF1729

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Fig. 2. TAX1BP1 overexpression alleviates cardiac hypertrophy, fibrosis and dysfunction in STZ induced-diabetic mice. A. The protein level of TAX1BP1 in mouse hearts at 0, 1, 2, 3, and 4 weeks after Ad-TAX1BP1 injection (n = 6). B and C. Effect of TAX1BP1 on echocardiographic (B) and hemodynamic measurements (C) (n = 10). The ratio of heart weight (HW) to body weight (BW) and to tibia length (TL) (n = 10). E. Representative image of the heart with H&E staining and PicroSirius red (PSR) staining. F. The cell surface area of cardiomyocytes (n = 100+ cells per group). G. Quantification of the total collagen volume in the indicated group. H and I. PCR analysis of hypertrophic markers (ANP, β-MHC) and fibrotic markers (collagen I, collagen III, TGFβ, CTGF). *P < 0.05 vs the corresponding Sham; #P < 0.05 vs Ad-NC-STZ. NC, negative control.

κB signaling was reported to act as a central and major regulator of protein aggregate clearance by modulating autophagic activity [22]. Tax1-binding protein 1 (TAX1BP1) was initially identified as an interacting protein of the human T-cell leukemia virus 1 (HTLV-1) Tax protein [23]. TAX1BP1 has been shown to serve as an NF-κB inhibitor, regulating pro-inflammatory cytokine production [24], innate immunity [25] and inflammatory responses [26]. The N-terminus of TAX1BP1 shows homology with the autophagy receptor nuclear dot protein 52 (NDP52), and the C-terminus contains two zinc fingers, which function as novel ubiquitin-binding domains [27]. Recent studies have suggested that TAX1BP1 is associated with autophagy [28,29]. The LC3-interacting region (LIR) of TAX1BP1 serves as an autophagy

receptor region that recruits proteins for degradation; the ubiquitinbinding domain of TAX1BP1 serves as a myosin VI cargo adaptor [28,29]. Since autophagy is centrally associated with the metabolic disorders and cardiac dysfunction observed in diabetic cardiomyopathy [12], we aimed to investigate the potential effect of TAX1BP1 in the pathogenesis of STZ-induced diabetic cardiomyopathy.

2. Methods 2.1. Materials STZ, the autophagy inhibitors 3-MA, chloroquine, and bafilomycin,

Fig. 3. TAX1BP1 overexpression decreased STZ-induced inflammation, oxidative stress and myocyte death. A–C. Immunohistochemistry analysis of CD45, CD68, and TNFα in diabetic hearts. Representative images and quantification (n = 6, 10+ fields per heart) are shown. D and E. PCR analysis of inflammation markers (IL-1, IL6, MCP-1, D) and P67phox and gp91phox (E) in diabetic hearts (n = 6). F. Total SOD activity and NADPH oxidase activity in diabetic hearts in the indicated group (n = 6). G and H. Representative western blot (G) and analysis (H) of SOD and P67 phox in diabetic hearts (n = 6). I. Immunohistochemistry staining of 4-HNE in diabetic hearts. J and K. TUNEL and α-actin staining (J) and analysis (K) in diabetic hearts (n = 6, 10+ fields per heart). L and M. Representative western blot (L) and analysis (M) of Bcl2, Bax and c-caspase3 in diabetic hearts (n = 6). *P < 0.05 vs the corresponding Sham group; #P < 0.05 vs Ad-NC-STZ.

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Fig. 3. (continued)

Chinese Academy of Medical Sciences (Beijing, China). The diabetic model was established by intraperitoneal streptozotocin (STZ) injection (dissolved in 0.1 mol/l citrate buffer, pH 4.5) at a dose of 50 mg/kg for 5 consecutive days. Control mice were injected with an equal volume of citrate buffer. One week after the final STZ injection, fasting blood glucose (FBG) was detected. Diabetes was defined as FBG ≥ 16.6 mmol/L in three independent measurements independently. A myocardial injection of either adenoviral (Ad)TAX1BP1 (n = 12) or Ad-LacZ (as a negative control, also called as AdNC, n = 12) was administered 12 weeks after the induction of diabetes [1 × 1010 vp (viral particles) per animal]. The mice were also given intraperitoneal injections of 3-MA (100 mg kg−1 on alternate days) from 12 weeks to 16 weeks after the final STZ injection [30].

and the autophagy inducer rapamycin were purchased from Sigma (St Louis, MO, USA). P65 siRNA, Atg5 siRNA and the scrambled si RNA were obtained from Santa Cruz. Secondary antibodies were obtained from LI-COR Biosciences (Lincoln, NE, USA). 2, 7-Dichlorofluorescin diacetate (DCFH-DA) was ordered from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals were of analytical grade. 2.2. Animals and animal model All animal experimental procedures followed National Institutes of Health guidelines and the guidelines of Renmin Hospital of Wuhan University. Eight-week-old male C57/B6 mice were purchased from the

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Fig. 4. TAX1BP1 overexpression inhibited HG-induced inflammation A. Representative western blot (Top) and analysis (Bottom) of TAX1BP1 in cardiomyocytes infected with Ad-TAX1BP1 (n = 6 sample, *P < 0.05 vs Ad-NC). B. Immunofluorescence staining (Right) and analysis (Left) of P-P65 nuclear translocation in cardiomyocytes exposed to HG and infected with Ad-TAX1BP1 (n = 5 sample, 10+ fields per coverslip). C. PCR analysis of inflammation markers (TNFα, IL-1, IL-6, MCP-1) in cardiomyocytes (n = 6 sample). *P < 0.05 vs Ad-NC-NG; #P < 0.05 vs Ad-NC-HG. D–H. Cardiomyocytes were infected with Ad-TAX1BP1 and treated with P65 siRNA. D. P65 expression after cells was treated with P65 siRNA. *P < 0.05 vs scrambled RNA. E. Cell viability was detected by an MMT assay in the indicated group (n = 6 sample). F. PCR analysis of inflammation markers in the indicated group. G. ROS level in the indicated group (n = 6 sample). H. Total SOD activity and NADPH oxidase activity in cardiomyocytes in the indicated group (n = 6 sample). *P < 0.05 vs the corresponding NG group; #P < 0.05 vs the Ad-NC-HG group. NC, negative control; NG, normal glucose; HG, high glucose.

glucose concentration (NG; 5.5 mM glucose) and 27.5 mM mannitol to control for osmolarity. To explore the mechanism by which TAX1BP1 inhibits NF-κB P65, cardiomyocytes were incubated with P65 siRNA or the scrambled RNA. To explore the mechanism by which TAX1BP1 activates autophagy, cardiomyocytes were infected with Ad-Atg5 or treated with Atg5 siRNA, chloroquine (100 μM), or bafilomycin A1 (100 nM). To knock down RelB, small interfering RNA (siRelB) was used. Samples in one experiment represent an independent replicate, and each experiment was repeated three times.

2.3. Echocardiographic and hemodynamic measurements The echocardiographic and hemodynamic measurements have been described in our previous studies [31,32]. Briefly, echocardiography was performed on anesthetized (1.5% isoflurane) mice using a MyLab 30CV ultrasound system (Biosound Esaote, Genoa, Italy) with a 10MHz linear array ultrasound transducer. LVEDd, LVESd, LVEF, and LVFS were analyzed for > 10 beats per heart. Hemodynamics were measured with a pressure-load loop in anesthetized (1.5% isoflurane) mice using cardiac catheterization. A microtip catheter transducer (SPR-839; Millar Instruments, Houston, TX, United States) was inserted into the right carotid artery and advanced into the LV, and dp/dt max and dp/dt min were analyzed.

2.7. Real-time PCR and western blot Real-time PCR and western blot were performed according to our previous study [31,32]. We performed 20 μl reactions according to the manufacturer's protocol with the following cycling parameters: 95 °C for 5 min; 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s; 95 °C for 5 s; 60 °C for 1 min; 97 °C for 0.11 s; and 40 °C for 10 min. The results were analyzed with the 2 − ΔΔCq method and normalized to GAPDH gene expression. The primers used are listed in Table 1 in the Supplemental materials, and the antibodies used are listed in Table 2 in the Supplemental materials.

2.4. Histological analysis Hematoxylin–eosin and Picrosirius Red staining, immunohistochemical analysis and TUNEL staining were performed as previously described [31,32]. The following antibodies were used for immunohistochemical staining: TAX1BP1, 4-hydroxynonenal, CD45, CD68, and TNF-α.

2.8. Detection of total superoxide dismutase activity and NADPH oxidase activity

2.5. Adenoviral vector construction 2.5.1. Construction of recombinant adenoviruses Recombinant adenoviruses expressing mouse TAX1BP1 (AdTAX1BP1) and Ad-LacZ purchased from Vigene Bioscience Company (Jinan, China) were prepared using the pAdEasy vector system (Qbiogene, Santa Ana, CA, USA). In brief, TAX1BP1 and the LacZ sequence were cloned into pShuttle-CMV (Qbiogene), and homologously recombined in bacteria BJ5183 with pAdeasy-1. The recombinant plasmids were propagated separately in HEK 293 cells. The titers of stocks measured by plaque assays were 2 × 1011 pfu/ml for Ad-LacZ and 1.6 × 1011 pfu/ml for Ad-TAX1BP1.

Freshly isolated mouse hearts (80–120 mg) were homogenized, and the cardiomyocytes were lysed. The supernatant fractions were collected to detect the activity of SOD and NADPH oxidase with commercial kits. 2.9. Detection of ROS and cell viability Cardiomyocytes cultured in six-well plates were infected with AdTAX1BP1 and stimulated with HG. DCFH-DA was used to detect ROS with a fluorescence microplate reader (excitation wavelength/emission wavelength: 485/525 mm) to quantify the result. Cell viability was determined using an MTT assay following the manufacturer's protocol.

2.5.2. Viral delivery protocol Twelve weeks after the final STZ injection, the mice were randomly chosen to receive a heart injection of either Ad-TAX1BP1 (n = 12) or Ad-NC (n = 12) at 1 × 1010 vp (viral particles) per animal. Briefly, after anesthetization with 3% sodium pentobarbital (80 mg·kg−1, intraperitoneal injection), the mouse heart was exposed, followed by removal of the pericardium. Three areas were injected with a 29-gauge syringe, including the left ventricular apex, anterior wall, and lateral wall. A total of 50 μl of adenovirus vector (1 × 1010 vp) was injected to each heart, with a single injection (10 μl) in the apex and two injections each in the anterior and lateral walls. The injections were spaced approximately 5 mm apart. Echocardiographic measurements and invasive hemodynamics measurements were performed at 16 weeks after STZ injection, and the animals were then killed.

2.10. Immunofluorescence, TUNEL staining and autophagic flux analysis Cardiomyocytes cultured in six-well plates were infected with AdTAX1BP1 and/or siRelB and then exposed to HG for 24 h. To detect PP65 or RelB nuclear translocation, the cardiomyocytes were incubated with a rabbit anti-P-P65 (1:100) or anti-RelB (1:100) antibody. To detect cell apoptosis, the cardiomyocytes were incubated with anti-ccaspase3 (1:100). TUNEL staining was also performed using a commercial kit (Millipore, Billerica, MA, USA), following the manufacturer's instructions, and the cells were co-stained with an α-actin antibody (1:100). To detect autophagic flux, mRFP-GFP-LC3 adenovirus (Ad-tf-LC3, MOI = 50) was used to infect cardiomyocytes, and the cells were then exposed to HG. Next, cells were scanned with a fluorescence microscope.

2.6. Cell culture and treatment NRCMs were isolated and cultured according to our previous study [31]. To investigate the effect of TAX1BP1, cardiomyocytes were infected with Ad-TAX1BP1 (MOI = 50) or Ad-NC for 4 h. The cardiomyocytes were also exposed to a high glucose concentration (HG; 33 mM glucose). Cells in the control group were exposed to normal

2.11. Statistical analyses SPSS22.0 was used for analysis. The data are presented as mean ± SD. One-way ANOVA was used to evaluate differences 1734

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Fig. 5. TAX1BP1 increased autophagy in STZ-induced diabetic hearts and HG-stimulated cardiomyocytes. A and B. PCR analysis of autophagy-related-genes (Atg7, Atg7, Beclin1) in diabetic hearts (A) and HG-stimulated cardiomyocytes (B) (n = 6). C–G. Representative western blot (C and F) and analysis (D and G) of autophagy-related-protein expression (Beclin1, Atg7, Atg12, P62, LC3) in diabetic hearts (C and D) and HG-stimulated cardiomyocytes (F and G) (n = 6 sample). E and H. LC3II/I ratio in the indicated group in diabetic hearts (E) and HG-stimulated cardiomyocytes (H) (n = 6). I. Representative images (Left) of RFP-LC3 puncta (red) and GFP-LC3 puncta (green) and quantified red puncta number (Right) in cardiomyocytes after infection with mRFP-GFP-LC3 adenovirus (n = 5, left, representative image; right, quantitative analysis of the number of red puncta). *P < 0.05 vs the corresponding sham/NG group; #P < 0.05 vs Ad-NC-STZ/HG.

Fig. 6. The protective effects of TAX1BP1 on HG-stimulated cardiomyocytes depend on autophagy. A. Expression level of Atg5 after cells were infected with Ad-Atg5 or treated with Atg5 siRNA. *P < 0.05 vs siRNA/Ad-NC. B. Representative images (Middle) of RFP-LC3 puncta (red) and GFP-LC3 puncta (green) and quantified red puncta number (Bottom) in cardiomyocytes infected with mRFP-GFP-LC3 adenovirus and cultured with HG, infected with Ad-TAX1BP1 and/or Ad-Atg5, or treated with Atg5 siRNA or autophagy inhibitors (chloroquine, bafilomycin A1) (n = 5). C. Cell viability in the indicated group (n = 6). D. DCF-DA fluorescence in cardiomyocytes in the indicated group (n = 6). E and G. PCR analysis of P67phox, gp91phox (E) and inflammation markers (G) in cardiomyocytes (n = 6 sample). F. Total SOD activity and NADPH oxidase activity in cardiomyocytes in the indicated group (n = 6 sample). *P < 0.05 vs Ad-NC-HG; #P < 0.05 vs Ad-TAX1BP1-HG.

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Fig. 6. (continued)

3. Results

diabetic mice as well as in cardiomyocytes isolated from neonatal rats. TAX1BP1 expression was downregulated in the heart in STZ-induced diabetic mice (Fig. 1A). The level of TAX1BP1 protein in HG-treated cardiomyocytes was approximately 3 times lower than that in control cardiomyocytes (Fig. 1B). Immunostaining analyses revealed that TAX1BP1 protein levels were dramatically reduced in the heart in STZinduced diabetic mice (Fig. 1C) and in cardiomyocytes stimulated with HG (Fig. 1D). These results indicate a role for TAX1BP1 in diabetic cardiomyopathy.

3.1. TAX1BP1 was downregulated in the heart in STZ-induced diabetic mice and in HG-stimulated cardiomyocytes

3.2. TAX1BP1 overexpression alleviates cardiac hypertrophy, fibrosis and dysfunction in STZ-induced diabetic mice

between multiple groups with a single intervention, followed by a post hoc Tukey test. Two way ANOVA followed by a post hoc Tukey test was used to analyze differences between multiple groups with two interventions (STZ-induced diabetes/HG stimulation and TAX1BP1 overexpression). Differences between two groups were analyzed by an unpaired, two-sided Student's t-test [33]. A P value < 0.05 was defined as statistically significant.

Next, we investigated whether the increased TAX1BP1 level affected

TAX1BP1 expression was assessed in the heart in STZ-induced 1737

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Fig. 7. Autophagy inhibition counteracted the protective effects of TAX1BP1 in vivo. A and B. Representative western blot (A) and analysis (B) of LC3 and P62 in diabetic hearts treated with 3-MA (n = 6). C and D. Effect of 3-MA on echocardiographic (C) and hemodynamic measurements (D) in diabetic hearts (n = 9). E. TUNEL and α-actin co-staining(E). F–H. Immunohistochemistry staining of 4-HNE (F), CD45 (G), and TNFα (H) in diabetic hearts treated with 3-MA (n = 5). I. Analysis of TUNEL and CD45-positive cells (n = 5, 10+ fields per heart). J and L. PCR analysis of P67phox, gp91phox (J) and inflammation markers (L) in diabetic hearts treated with 3-MA (n = 6). K. Total SOD activity and NADPH oxidase activity in diabetic hearts in the indicated group (n = 6). *P < 0.05 vs STZ-Ad-TAX1BP1Vehicle.

hypertrophy and fibrosis process in STZ-induced diabetic mouse hearts. After being injected with STZ, mice exhibited increased blood glucose (Fig. S1A) and reduced body weight (Fig. S1B). The blood glucose (Fig. S1C) and body weight (Fig. S1D) in mice overexpressing TAX1BP1 were not significantly different from those in control group. Both invasive (pressure-volume loops) and noninvasive (echocardiograph) cardiac function and contractility parameters were used in our study. Left ventricular (LV) ejection fraction (LVEF) and fractional shortening (LVFS) detected by echocardiography are afterload-dependent, while dp/dt max and dp/dt min detected by pressure-volume loops are preload dependent [34]. Thus, both pressure-volume loops and echocardiography were used in our study to support our results. Sixteen weeks after STZ injection, the mice developed deteriorated cardiac function, as assessed by increased LV end diastolic diameter (LVEDd) and LV end systolic diameter (LVESd) and reduced LVEF and LVFS. STZ-induced diabetic mice also displayed systolic dysfunction (decreased dP/dt max) and diastolic dysfunction (decreased dP/dt min). TAX1BP1 overexpression (Fig. 2A) in the heart improved the decreased cardiac function (Fig. 2B–C). STZ-induced diabetic mice exhibited severe cardiac hypertrophy and fibrosis, with increases in the heart weight/body weight ratio, the heart weight/tibia ratio (Fig. 2D), cell surface area, interstitial collagen deposition (Fig. 2E–G), and hypertrophic and fibrotic gene transcription (Fig. 2H and I).

ROS production decreased SOD activation, and upregulated NADPH expression and activation (Fig. S2A–D). Ad-TAX1BP1 infection inhibited these responses (Fig. S2A–D). Ad-TAX1BP1 infection also suppressed HG-induced Bax upregulation and Bcl-2 downregulation (Fig. S2E, F), thus inhibiting the release of c-caspase3 (Fig. S2G, H) and blocking cardiomyocyte apoptosis (Fig. S2I–K). P65 siRNA (to knockdown P65, Fig. 4D) was used to explore whether the protective effects of TAX1BP1 are dependent on P65 inhibition. P65 knockdown partially mimicked the anti-inflammatory, anti-oxidative stress and anti-apoptotic effects of TAX1BP1, as TAX1BP1 overexpression further augmented these protective effects (Fig. 4E–H). Thus, TAX1BP1 overexpression protected against HG-induced cardiomyocyte inflammation, oxidative stress, and apoptosis. These effects were partially dependent on the inhibition of NF-κB P65. 3.5. TAX1BP1 increased autophagy in STZ-induced diabetic hearts and HG-stimulated cardiomyocytes We first investigated the autophagy level in mouse hearts at 0, 4, 8, 12 and 16 weeks after the initiation of STZ-induced diabetes. As shown in Fig. S3, no significant difference was observed in the autophagy level in the heart in mice at 0, 4, and 8 weeks after STZ treatment, but the autophagy level decreased sharply at 12 weeks and continued to decline at 16 weeks in diabetic mouse hearts. Thus, intervening in diabetic cardiomyopathy progression at 3 months after diabetes development by overexpressing TAX1BP1 seems reasonable. There was a significant decrease in autophagy-related gene expression (Atg5, Atg7, Atg12, beclin1) and a decreased LC3II/I ratio in both diabetic hearts (16 weeks) and HG-stimulated cardiomyocytes (Fig. 5A–H). In mouse hearts or cardiomyocytes infected with Ad-TAX1BP1, the expression of these autophagy-related genes increased significantly, with an increased LC3II/I ratio and decreased accumulation of P62 (Fig. 5A–H). In cells infected with mRFP-GFP-LC3 adenovirus, an acidic lysosomal environment quenches acid-sensitive GFP fluorescence, while mRFP is not affected, and the autophagic lysosome thus exhibits red fluorescence. Therefore, red fluorescence can indicate the formation of autophagosomes, while green fluorescence can indicate the degree of autophagy flux in the presence of bafilomycin A1 or chloroquine. In our study, the number of red puncta was decreased in HG-stimulated cells compared with that in the NG group. TAX1BP1 overexpression increased the number of red puncta without affecting the accumulation of green puncta (Fig. 5I). This result indicated that TAX1BP1 increased autophagosome formation without affecting autophagosome degradation.

3.3. TAX1BP1 overexpression decreased STZ-induced inflammation, oxidative stress and myocyte death As inflammation, oxidative stress, and cell death are the key features of diabetic cardiomyopathy, the effect of TAX1BP1 was evaluated. There was a significant increase in CD68-labeled macrophage and CD45-labeled leukocyte infiltration and in inflammatory cytokine expression in diabetic mouse hearts. TAX1BP1 overexpression decreased the inflammatory response in STZ-induced diabetic mouse hearts (Fig. 3A–D). Oxidative stress is a key feature of diabetic cardiomyopathy. The STZ-induced upregulation of P67phox and Gp91phox mRNA expression was reduced in diabetic mice overexpressing TAX1BP1 (Fig. 3E). TAX1BP1 overexpression in STZ-induced diabetic mice also increased total SOD expression and activity and reduced the abnormal expression and activity of NADPH oxidase (Fig. 3F–H). TAX1BP1 overexpression also reduced myocardial lipid peroxidation in diabetic mice, as assessed by 4-HNE staining (Fig. 3I). STZ-induced diabetic mouse hearts showed an increased proportion of apoptotic cells, which was reduced in mice overexpressing TAX1BP1 (Fig. 3J and K). The expression of the apoptosis-associated proteins B-cell lymphoma 2 (Bcl2) and Bax was altered in diabetic hearts, with a decrease in Bcl-2 and an increase in Bax expression, while TAX1BP1 overexpression limited these alterations and reduced the release of the apoptosis activator ccaspase3 (Fig. 3L and M).

3.6. The protective effects of TAX1BP1 on HG-stimulated cardiomyocytes depend on autophagy We further assessed whether autophagy was involved in TAX1BP1mediated protection. Cardiomyocytes were infected with Ad-Atg5 or treated with Atg5 siRNA (Fig. 6A), chloroquine (which inhibits lysosome degradation) or bafilomycin A1 (which inhibits the fusion of autophagosomes and lysosomes). TAX1BP1 overexpression increased autophagic flux, as demonstrated by an increased number of red puncta and green puncta in the presence of chloroquine and bafilomycin A1 (Fig. 6B). Atg5 knockdown decreased the number of red puncta in cardiomyocytes overexpressing TAX1BP1. Conversely, Atg5 overexpression could not further increase red puncta number in cardiomyocytes with TAX1BP1 overexpression (Fig. 6B). These results

3.4. TAX1BP1 overexpression inhibited HG-induced inflammation, ROS generation and apoptosis Next, we determined whether TAX1BP1 protected against HG-induced myocyte injury. Cardiomyocytes were infected with AdTAX1BP1 to overexpress TAX1BP1 (Fig. 4A) and then exposed to HG for 24 h. HG increased the inflammatory response in cardiomyocytes, as evidenced by increased p-P65 nuclear translation and increased proinflammatory cytokine expression (Fig. 4B–C). Ad-TAX1BP1 infection decreased these inflammatory responses (Fig. 4B–C). HG also increased 1739

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Fig. 8. The effect of TAX1BP1 on NF-κB signaling. A–F. Representative western blot (A and D) and analysis (B, C, E, F) of NF-κB signaling proteins in diabetic hearts (A–C) and HG-stimulated cardiomyocytes (D–F) (n = 6). G and H. Immunofluorescence staining and analysis of RelB nuclear translocation in HG-stimulated cardiomyocytes (n = 5 sample, 10+ fields per coverslip). *P < 0.05 vs the corresponding NG group; #P < 0.05 vs Ad-NC-HG.

suggested that TAX1BP1 increased autophagy flux, which overlaps with the effect of Atg5 overexpression on autophagy. Consistent with this, autophagy inhibition by Atg5 siRNA, chloroquine and bafilomycin A1 abolished the protective effects of TAX1BP1 overexpression on HG-induced excessive oxidative stress, inflammatory responses and cell death (Fig. 6C–G). Nevertheless, Atg5 overexpression could not further augment the protective effect of TAX1BP1 (Fig. 6C–G). To explore whether inflammation is a downstream effect of autophagy regulation, cells were treated with P65 siRNA to inhibit the inflammatory response. We found that, consistent with TAX1BP1 overexpression, P65 knockdown had no protective effects in the presence of bafilomycin A1 (Fig. 6C–G). These data indicated that by activating autophagy, TAX1BP1 acted as a protector, subsequently suppressing downstream inflammation, oxidative stress and apoptosis. These protective effects of TAX1BP1 could be abrogated by autophagy inhibitors.

diabetic cardiomyopathy. Furthermore, TAX1BP1 overexpression inhibited remodeling processes of diabetic cardiomyopathy, including cardiac hypertrophy, fibrosis, inflammation, oxidative stress and apoptosis. These data indicated that a TAX1BP1 -mediated physiological process may constitute a protective mechanism against STZ-induced diabetic cardiomyopathy. The canonical NF-κB pathway (NF-κB P65) is a central mediator of various cardiac pathologies [22]. NF-κB P65 signaling is initiated via the stimulus-induced phosphorylation of IκB [20]. IκB phosphorylation facilitates its polyubiquitination and subsequent degradation, which unmasks a nuclear localization sequence within p65/p50 dimers, promoting their translocation to the nucleus, where they bind specific NFκB–responsive sequences within promoters or enhancer regions of specific target genes, including inflammatory, proliferative, angiogenic and MMP genes as well as a subset of fetal cardiac genes [36]. As a negative regulator of NF-κB P65, TAX1BP1 has been reported to participate in many physiological functions, including pro-inflammatory cytokine production [24], innate immunity [25] and inflammatory responses, via the inhibition of NF-κB P65 [26]. Consistent with this role, we found that TAX1BP1 inhibited the activation of P65 in STZ-induced diabetic hearts and HG-stimulated cardiomyocytes. The anti-inflammatory, anti-oxidative and anti-apoptotic effects of TAX1BP1 were partially dependent on the suppression of P65, since P65 knockdown could only partly mimic the protective effects of TAX1BP1, suggesting that other mechanisms underlie the protective effects of TAX1BP1 on the pathology of STZ-induced cardiomyopathy. Autophagy is a conserved process in which organelles and longlived proteins are degraded via vesicle and lysosomal system. Alterations in the autophagy level may be either protective or detrimental depending on the extent of autophagy and the cellular environment [37]. Consistent in type 1 diabetes, Studies have reported that a decreased autophagy level was observed in many type 1 diabetic mouse hearts [13,38]. Cardiac autophagy in type 2 diabetes has been reported to be decreased, increased or unchanged. Studies have indicated that cardiac autophagy is inhibited in high-fat-diet-induced obesity and metabolic syndrome [39,40] but activated in fructose- and fat-based-diet-induced induced insulin resistance and hyperglycemia [41,42]. These inconsistent results suggest the complexity of autophagy regulation in the progression of diabetic cardiomyopathy. Of note, not all studies have determined the autophagic flux in the heart, which may contribute to the variation observed among different reports. In our hands, STZ-induced diabetic mice exhibited decreased autophagy. By contrast, increased autophagy was observed in diabetic mouse hearts with TAX1BP1 overexpression. Most notably, autophagy flux was inhibited in HG-stimulated cardiomyocytes but significantly increased by TAX1BP1 overexpression, with increased RFP-LC3 puncta in the absence and presence of bafilomycin A1 and chloroquine. The autophagy inhibitor 3-MA counteracted the protective effects of TAX1BP1 overexpression. These results indicate that autophagy is inhibited in STZinduced diabetic cardiomyopathy. TAX1BP1 improves the phenotype of STZ-induced cardiomyopathy by activating autophagy. Very few studies have investigated the role of non-canonical NF-κB RelB/P52 signaling in cardiomyocytes [21]. In other cell systems, this non-canonical NF-κB signaling pathway has been reported to act as a central and major regulator of protein aggregate clearance by modulating autophagic activity [22]. Most studies have focused on the inhibitory effect of TAX1BP1 on canonical NF-κB signaling [26,27,43–45]. Most recently, TAX1BP1 was reported to bind to TANKbinding kinase 1 (TBK1) and to mediate the activation of non- canonical NF-κB signaling in lung cancer cells [46]. Interestingly, we found that

3.7. Autophagy inhibition abolished the protective role of TAX1BP1 in vivo The role of increased autophagy mediated by TAX1BP1 overexpression was investigated by autophagy inhibitor in vivo. Intraperitoneal injection of 3-MA resulted in an increased protein aggregation, as evidenced by a decreased LC3II/I ratio and increased P62 accumulation (Fig. 7A and B). Mice in the STZ + Ad-TAX1BP1+ 3-MA group exhibited increased LVEDd and LVESd, reduced LVEF, LVFS and augmented hemodynamic alteration (Fig. 7C and D). 3-MA injection also abolished the protective effects of TAX1BP1 on excessive oxidative stress, inflammatory responses and cell death in STZ-induced diabetic hearts (Fig. 7E–L). 3.8. The effect of TAX1BP1 on NF-κB signaling A previous study demonstrated that TAX1BP1 promotes non-canonical NF-κB signaling in lung cancer cells [35]. This non-canonical NFκB signaling contributes to activation of autophagy [22]. We found that TAX1BP1 decreased IκB phosphorylation and subsequent degradation, and decreased canonical NF-κB P65 activation and nuclear translocation in both diabetic hearts (Fig. 8A, B) and HG-stimulated cardiomyocytes (Fig. 8 D, E and Fig. 4B). Interestingly, P100 phosphorylation and RelB nuclear translocation were markedly reduced in diabetic hearts (Fig. 8A, C) and HG-stimulated cardiomyocytes (Fig. 8 D, F, G, H), while TAX1BP1 overexpression restored these changes (Fig. 8A–H), suggesting that TAX1BP1 inhibited canonical NF-κB P65 activation, while promoting the activation of non-canonical NF-κB P52/RelB signaling in STZ-induced diabetic hearts. 3.9. The precise mechanism by which TAX1BP1 activates autophagy Knockdown of RelB by siRNA (Fig. S4A) abolished the AdTAX1BP1-mediated induction of autophagy in HG-stimulated cardiomyocytes (Fig. S4B). Knockdown of RelB also resulted in an aggravated phenotype in cardiomyocytes as evidenced by reduced cell viability (Fig. S4C) and increased oxidative stress (Fig. S4D–F) and inflammatory responses (Fig. S4G) in Ad-TAX1BP1 + siRelB cardiomyocytes compared with the Ad-TAX1BP1 + siRNA group. 4. Discussion The major findings from our current study revealed impaired cardiac diastolic and systolic function in STZ induced-diabetic mice. TAX1BP1 overexpression improved cardiac function in STZ-induced 1741

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Conflict of interest

TAX1BP1 activated the non-canonical NF-κB RelB in cardiomyocytes, which was inhibited below baseline in STZ-induced diabetic heart and HG-stimulated cardiomyocytes. We also found that non-canonical NFκB signaling acted as an autophagy sensor in cardiomyocytes, which stimulated autophagy activation. Conversely, knockdown of RelB inhibited TAX1BP1-overexpression-mediated autophagy induction and even blocked the protective effects of TAX1BP1 overexpression. Interestingly, TAX1BP1 overexpression did not affect the activation of either canonical NF-κB P65 or non-canonical NF-κB RelB signaling under baseline in cardiomyocytes. These results indicate that TAX1BP1 may exert its' function only under pathological conditions. Since TAX1BP1 affects both canonical NF-κB P65 and non-canonical NF-κB RelB signaling, we investigated which signaling pathway dominates the main role of TAX1BP1 in diabetic cardiomyopathy. In our study, P65 silencing only partially partly mimic the effects of TAX1BP1, whereas increasing autophagy by Atg5 overexpression could almost completely mimic the protective effects of TAX1BP1. These findings suggested that autophagy activation, rather than P65 inhibition, is the key target of TAX1BP1. In addition, only autophagy inhibitors and RelB silencing could totally abrogate the protective effects of TAX1BP1. Even P65 silencing could not exert any protective effects in the presence of bafilomycin A1. These data indicated that by activating RelB induced autophagy, TAX1BP1 acted as a protector, subsequently suppressing downstream inflammation, oxidative stress and apoptosis. Several limitations should be considered. First, there a number of reports showing inconsistent autophagy levels in diabetic hearts in type 1 and type 2 diabetic models. More than one diabetes model should be used to explore the effects of TAX1BP1 in the pathology of diabetic cardiomyopathy. Second, in type 2 diabetic models that show increased autophagy, it is unclear whether TAX1BP1 would be a viable therapeutic option. Third, the 3-MA used in this study is a general PI3K inhibitor and therefore has a number of non-autophagy effects. Further studies with genetic techniques to confirm the effects of TAX1BP1 on autophagy are needed. In summary, we demonstrate here that TAX1BP1, a ubiquitin binding protein, may ameliorate the progression of STZ-induced cardiomyopathy by preserving autophagy. TAX1BP1 inhibits canonical NF-κB signaling and induces non-canonical NF-κB signaling, thereby regulating inflammation and autophagy.

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Transparency document The http://dx.doi.org/10.1016/j.bbadis.2018.02.012 with this article can be found, in online version.

associated

Acknowledgments Authors' contribution Yang Xiao, Qing-Qing Wu, Qi-Zhu Tang contributions to conception, designed experiments and takes full responsibility for the work as a whole; Qing-Qing Wu, Yang Xiao, Ming-Xia Duan, Chen Liu, and Yuan Yuan carried out experiments; Zheng Yang, Di Fan analyzed experimental results, revised the manuscript. Zheng Yang, Hai-Han Liao wrote, revised the manuscript.

Sources of funding This work was supported by grants from the National Natural Science Foundation of China (No. 81700353, 81530012), Hubei Province's Outstanding Medical Academic Leader program, and China Postdoctoral Science Foundation, Grant numbers: 2014M562068. Fundamental Research Funds of the Central Universities, Grant numbers: 2042017kf0060. 1742

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