Overexpression of IκBα in cardiomyocytes alleviates hydrogen peroxide-induced apoptosis and autophagy through inhibiting NF-κB activation

Background: Inammation and oxidative stress play a predominant role in the initiation and progression of ischemia/reperfusion (I/R) injury, of which nuclear factor kappa B (NF-κB) is a crucial mediator. Overexpression of the inhibitor of κB alpha (IκBα) gene is hypothesized to have protective effects against apoptosis and autophagy in cardiomyocytes subjected to hydrogen peroxide (H 2 O 2 ) through inhibiting the NF-κB pathway. Methods: The IκBα S32A, S36A gene was transfected via adeno-associated virus serotype 9 (AAV9) delivery into neonatal rat ventricular cardiomyocytes (NRVMs) prior to H 2 O 2 treatment. NRVMs were divided into control, H 2 O 2 , GFP +H 2 O 2 , IκBα+H 2 O 2 , and pyrrolidine dithiocarbamate (PDTC)+H 2 O 2 groups. Nuclear translocation of NF-κB p65 subunit was evaluated by immunouorescence and Western blot. Cell viability was assessed by Cell Counting Kit-8 assay. Supernatant lactate dehydrogenase (LDH) and intracellular malondialdehyde (MDA) were measured to identify H 2 O 2 -stimulated cytotoxicity. Apoptosis was determined by Annexin V-PE/7-AAD staining, and the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Western blot was used to detect apoptosis- and autophagy-related proteins. Results: IκBα transfection signicantly increased cell viability and ΔΨm, but decreased the supernatant LDH and cellular MDA levels in cardiomyocytes exposed to H 2 O 2 . Meanwhile, IκBα overexpression decreased H 2 O 2 -induced apoptosis by upregulating the Bcl-2/Bax ratio and reduced autophagy by downregulating the expression of Beclin-1 and the LC3- (cid:0) /LC3- (cid:0) ratio. These effects partly accounted for the ability of IκBα to inhibit the NF-κB signaling pathway, as evidenced by decreases in p65 phosphorylation and nuclear translocation. Indeed, the effects of inactivation of NF-κB signaling with the specic inhibitor, PDTC, resembled the cardioprotective effects of IκBα during H 2 O 2 stimulation. Conclusion: IκBα overexpression can ameliorate H 2 O 2 -induced apoptosis, autophagy, oxidative injury, and ΔΨm loss through inhibition of the NF-κB signaling pathway. These ndings suggest that IκBα transfection can successfully resist oxidative stress-induced damage through inhibiting NF-κB activation, which may provide a potential therapeutic target for prevention of myocardial I/R injury.


Introduction
Acute myocardial infarction (AMI) is the leading cause of death worldwide, and reperfusion therapy is the most effective treatment for AMI [1]. Paradoxically, the process of myocardial reperfusion also induces a series of adverse cardiac events such as in ammation, necrosis, apoptosis and autophagy, nally leading to myocardial ischemia/reperfusion (I/R) injury [2]. Recent evidence has suggested that excessive in ammation and oxidative stress play a predominant role in the initiation and progression of I/R injury [3,4].
Nuclear factor kappa B (NF-κB) is an in ammatory inducer and redox-sensitive transcription factor in most cell types [5]. The p65/50 heterodimer, the most common pattern of NF-κB dimer, normally exists as a component of inactive cytoplasmic complexes bound to the inhibitor of κB alpha (IκBα). Upon stimulation, IκBα is phosphorylated, and undergoes ubiquitylation and proteasomal degradation, subsequently leading to phosphorylation and nuclear translocation of the NF-κB p65 subunit [6].
Activated NF-κB then initiates the expression of corresponding target genes, many of which may regulate apoptosis, inflammation and autophagy [7].
However, whether NF-κB is protective or detrimental for cardiomyocyte apoptosis remains controversial [8]. Notably, our previous study indicated that p65 ribozyme could prevent cell apoptosis in H9C2 cardiomyocytes exposed to hydrogen peroxide (H 2 O 2 ) [9]. Autophagy, an evolutionarily conserved form of "self-digestion", plays dual roles in the heart [10]. Recent studies on autophagy have shown both the protective [11] and deleterious [12] roles of autophagy in cardiomyocytes against oxidative stress.
Evidence has revealed a strong correlation between the modulation of NF-κB and the autophagic response [13,14]. In addition, cross-talk between autophagy and apoptosis has been noted [15], and NF-κB is known to mediate the balance between autophagy and apoptosis [16].
Therefore, it is thought that NF-κB activation is the key point of I/R injury; thus, inhibiting NF-κB may be a targeted therapy for I/R injury. Phosphorylation of IκBα, the key inhibitor of the canonical NF-κB pathway, at Ser 32 and Ser 36 is necessary for its degradation, and any mutation of these two serine residues blocks IκBα degradation [6]. Recently, adeno-associated virus serotype 9 (AAV9) was demonstrated to be the best gene carrier due to its high e ciency in the heart [17]. H 2 O 2 , a common reactive oxygen species (ROS), is generally utilized to mimic I/R injury in vitro [12]. Thus, the IκBα S32A, S36A gene was transfected into cardiomyocytes via AAV9-medicated delivery to investigate the role of inhibition of the NF-κB pathway in H 2 O 2 -induced apoptosis and autophagy. Pyrrolidine dithiocarbamate (PDTC), a speci c inhibitor of NF-κB, was used as a positive control in this study.

Ethics statement
The experimental protocol was approved by the Ethics Committee of the First A liated Hospital of Xinjiang Medical University (No. IACUC-20180223-69). One-to three-day-old neonatal Sprague-Dawley (SD) rats were purchased from the Experimental Animal Center of Xinjiang Medical University, and handled in accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health.

Isolation and culture of rat cardiomyocytes
The protocol for the isolation and puri cation of neonatal rat ventricular cardiomyocytes (NRVMs) was reported in our previous study [19]. Brie y, the hearts of 1-to 3-day-old SD neonatal rats were dissected and digested with 0.1% trypsin and 0.08% collagenase II. Following differential adhesion twice for 50 min each time, nonadherent cells were resuspended and cultivated in high-glucose DMEM containing 10% FBS, 1% penicillin-streptomycin, and 0.1 mM BrdU for 48 h. The medium was replaced every 48 h.

AAV9 transfection of cardiomyocytes
After 48 h of culture, NRVMs were transfected with dsAAV9-GFP or dsAAV9-IκBα as previously described [12]. Briefly, cells were rst transfected with dsAAV9 (multiplicity of infection, MOI = 5×10 6 vg/cell) in serum-free medium, after which DMEM at an equal volume containing 20% FBS, 2% penicillinstreptomycin and 0.2 mМ BrdU was added to every dish 3 h later. Images showing GFP were captured using a fluorescence inverted microscope (Leica DMI4000B, Weztlar, Germany), and the green fluorescence intensities were analyzed using ImageJ software.

Experimental design and cell grouping
The experiment was designed is to explore whether AAV9-delivered IκBα S32A, S36A gene transfection could protect cardiomyocytes against H 2 O 2 -induced apoptosis and autophagy via inhibition of NF-κB activation. Cardiomyocytes were starved with serum-free DMEM for 12 h to ensure cell synchronization before H 2 O 2 stimulation. The experimental cardiomyocytes were randomly divided into 5 groups as follows: the (1) control group, the primary cardiomyocytes in which were cultivated under normal conditions; (2) H 2 O 2 control group (H 2 O 2 ): the model control group, the primary cardiomyocytes in which were subjected to 100 μM H 2 O 2 alone [12]; (3) GFP control group (GFP): the vector control group, the primary cardiomyocytes in which were transfected with dsAAV9-GFP virus for 5 days before being subjected to 100 μM H 2 O 2 ; (4) IκBα treatment group (IκBα): the treatment group, the primary cardiomyocytes in which were transfected with dsAAV9-IκBα virus for 5 days before being subjected to 100 μM H 2 O 2 ; and the (5) PDTC treatment group (PDTC): the positive control group, the primary cardiomyocytes in which were pretreated with 100 μM PDTC for 60 min before being subjected to 100 μM

Measurement of cardiomyocyte vitality and cytotoxicity
The Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay was used to assess cell viability. In brief, 2×10 4 cells were seeded into each well of a 96-well plate and transfected with GFP or IκBα for ve days. After the cells were exposed to H 2 O 2 , 10 μL of CCK-8 stock solution was added to each well and incubated at 37 °C for 2 h. The absorbance at 450 nm was measured with a GO microplate spectrophotometer (Thermo Fisher Scienti c, USA). The extent of cell death was determined by quantifying lactate dehydrogenase (LDH) released into the culture supernatant with an LDH Kit (Jiancheng Bioengineering Institute, Nanjing, China). Intracellular malondialdehyde (MDA), an indicator of oxidative injury, was also measured using an MDA assay kit (Jiancheng Bioengineering Institute).

Flow cytometry analysis
Cell apoptosis was measured using PE Annexin V Kit I (BD Biosciences, NJ, USA). Brie y, cells were collected and resuspended in 1× binding buffer. Thereafter, the solution (1×10 5 cells) supplemented with 5 μL of PE Annexin V and 7-AAD was incubated in the dark for 15 min at room temperature. The apoptotic cells were identified by flow cytometry (Beckman Coulter, CA, USA). All the experiments were performed in triplicate.

Immuno uorescence
Immuno uorescence was employed to identify H 2 O 2 -induced nuclear translocation of the NF-κB p65 subunit in cardiomyocytes. Brie y, 2×10 5 cells were seeded into confocal dishes. After H 2 O 2 treatment, cardiomyocytes were xed with 4% paraformaldehyde for 20 min and permeabilized with 0.25% Triton X-100 for 10 min. After blocking with 1% BSA for 1 h, cells were probed overnight with anti-p65 antibody (1:200) at 4 °C, and incubated with Alexa Fluor 594-labelled secondary antibody (Invitrogen, 1:200, labelled with red uorescence) for 2 h at room temperature, followed by 10 min of DAPI staining of nuclei (labelled with blue uorescence). Signals were detected using a confocal spectral microscope (Leica SP8, Germany).
Measurement of the mitochondrial membrane potential JC-1 is an ideal uorescent probe used to detect the mitochondrial membrane potential (ΔΨm) in cardiomyocytes. Brie y, a 10 nmol/L JC-1 working solution was prepared prior to use, and cardiomyocytes were stained at 37 ℃ in the dark for 15 min. Cells doubly stained with JC-1 were visible as either green or red fluorescence. Fluorescent images and intensities were obtained using a uorescence microscope and ImageJ software. Generally, ΔΨm is represented by the red to green fluorescence ratio, which decreases in proportion with the severity of cell injury.

Statistical analysis
All statistical analyses were performed with SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± SEM. Multiple comparisons were carried out using one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test. A value of P < 0.05 indicated statistical signi cance.

H 2 O 2 -induced activation of NF-κB in NRVMs
The results indicated that H 2 O 2 elicited time-dependent IκBα degradation and p65 translocation after the NRVMs were incubated with 100 μM H 2 O 2 for different durations (0, 15, 30, 60min), respectively. (Fig. 1A-C). The ratio of nuclear p65 to cytosolic p65 peaked at 60 min. Consistent with the nuclear translocation of p65, the level of p-p65/p65 increased following H 2 O 2 stimulation with the incubation time ( Fig. 1D and E), and was highest at 60 min. Thus, treatment with 100 μM H 2 O 2 for 60 min was identi ed for use in the following experiments.

Efficiency of IκBα transfection in NRVMs
As shown in Fig. 1F, the green fluorescence signal was robust and the dsAAV9-GFP transfection efficiency in NRVMs reached more than 70%. Western blot analysis showed that the GFP protein was more highly expressed in the GFP group than in the other groups, while the IκBα protein level was signi cantly elevated in the IκBα group compared with the control and GFP groups (Fig. 1G-I).

IκBα protected cardiomyocytes from H 2 O 2 -induced apoptosis
The proportion of apoptotic cells in control group was 7.0 ± 1.5%. After treatment with 100 μM H 2 O 2 , the apoptotic rate of cardiomyocytes in the H 2 O 2 group and GFP group increased to 21.20 ± 0.95% and 19.97 ± 0.97%, respectively, which were decreased by IκBα or PDTC pretreatment ( Fig. 2A). Indeed, compared with the levels in the control group, the anti-apoptotic protein Bcl-2 was downregulated, but the proapoptotic protein Bax was upregulated in NRVMs exposed to H 2 O 2 , leading to a higher Bax/Bcl-2 ratio, but this effect was completely abolished by pretreatment with IκBα or PDTC (Fig. 2B).

IκBα protected cardiomyocytes from H 2 O 2 -induced cell injury
Compared to that in the control group, ΔΨm was signi cantly decreased in the H 2 O 2 and GFP groups, but this decrease was rescued by IκBα or PDTC treatment (Fig. 3A). Additionally, H 2 O 2 treatment significantly decreased cell viability but elevated supernatant LDH and intracellular MDA levels; however, these changes were reversed by IκBα or PDTC treatment (Fig. 3B-D).

IκBα suppressed H 2 O 2 -induced NF-κB activation and autophagy in NRVMs
Compared with control group, H 2 O 2 treatment signi cantly elicited p65 translocation, and increased p-p65/p65 ratio, and these changes were successfully reversed by IκBα or PDTC pretreatment ( Fig. 4A and  B). Consistently, H 2 O 2 increased the expression of p-p65 in NRVMs, but IκBα or PDTC dramatically downregulated the H 2 O 2 -induced expression of p-p65. Meanwhile, Beclin-1 and the LC3-/LC3-ratio, the autophagy-associated markers, were markedly upregulated in NRVMs exposed to H 2 O 2 , whereas these effects were inhibited by IκBα or PDTC treatment (Fig. 4C).

Discussion
This study shows that IκBα degradation and NF-κB activation occurred in a time-dependent manner in NRVMs subjected to H 2 O 2 . Cells treated with H 2 O 2 showed reductions in cell vitality and ΔΨm but elevations in LDH and MDA levels, apoptosis and autophagy. IκBα transfection or PDTC pretreatment ameliorated H 2 O 2 -induced cell injury through inhibiting NF-κB activation. I/R injury severely attenuates the benefit of revascularization after AMI and hence has become an important focus of cardiovascular research [2]. The in ammatory response induced by AMI is essential for heart repair, but excessive generation of ROS and in ammation following reperfusion therapy exacerbate heart damage [21].
The NF-κB signaling pathway plays a key role in the in ammatory response, oxidative stress, apoptosis, and autophagy in the heart [8]. Phosphorylation and nuclear translocation of the p65 subunit are signs of NF-κB activation [20]. Previous studies [22][23][24] identi ed that H 2 O 2 treatment for different durations (30 min-24 h) elicited signi cant p65 phosphorylation and nuclear translocation in NRVMs. In line with these studies, p65 was time-dependently phosphorylated and translocated from the cytoplasm to the nucleus with IκBα degradation in NRVMs subjected to H 2 O 2 .
However, whether NF-κB activation protects or damages cardiomyocytes remains a matter of debate. An early study demonstrated that activation of NF-κB reduced cell apoptosis in hypoxic cardiomyocytes [25], whereas most recent studies [26,27] have shown that NF-κB is a pro-apoptotic transcription factor correlated with myocardial injury, and blockade of NF-κB activity prevents myocardial apoptosis. Gray et al [22] recently reported that ROS generated by ischemia-reperfusion could rapidly activate calmodulin kinase II (CaMKII), which decreased cell injury through inducing IκBα degradation and nuclear p65 accumulation in NRVMs exposed to H 2 O 2 . Importantly, knockout of the CaMKIIδ gene signi cantly attenuated myocardial infarct size by inhibiting IκBα degradation and NF-κB activation. All these ndings show that NF-κB activation deteriorates the heart in I/R injury.
Herein, we hypothesized that direct overexpression of IκBα to prevent NF-κB activation may have a good effect in protecting cardiomyocytes. Then, dsAAV9-IκBα Ser 32A,36A was designed to prevent IκBα degradation due to its phosphorylation at the Ser 32 and Ser 36 sites, and successfully transfected into cardiomyocytes. Western blot and immuno uorescence demonstrated that IκBα transfection successfully maintained cytoplasmic IκBα levels and suppressed p65 phosphorylation and translocation in NRVMs exposed to H 2 O 2. Additionally, IκBα elevated cell viability, decreased LDH and MDA levels, and attenuated apoptosis, implying the protective role of IκBα in H 2 O 2 -induced cell injury in NRVMs. The mechanisms may account for the role of NF-κB in mediating the expression of various proteins that promote or inhibit apoptosis. Notably, NF-κB regulates the expression of certain anti-apoptotic genes, such as Bcl-2 [28], and an increased ratio of Bcl-2/Bax decreases cell apoptosis. In this study, treatment with IκBα or PDTC signi cantly elevated the Bcl-2/Bax ratio in NRVMs subjected to H 2 O 2 . These data indicate that IκBα protects NRVMs against H 2 O 2 -induced apoptosis by decreasing the ratio of Bax/Bcl-2.
Opening of the mitochondrial permeability transition pore (MPTP) in the rst few minutes of reperfusion leads to ΔΨm loss and is responsible for necrotic and apoptotic cell death processes, contributing differentially to myocardial infarct size [29]. Importantly, inhibition of the opening of the MPTP attenuated I/R injury. Thus, ΔΨm loss re ects mitochondrial dysfunction, indicates early-stage apoptosis and is a critical determinant of I/R injury [30]. A previous study demonstrated that H 2 O 2 induced a signi cant decrease in ΔΨm [12]. In this study, H 2 O 2 treatment attenuated ΔΨm and enhanced Bax expression in NRVMs, and these effects were reversed by pretreatment with IκBα or PDTC. NF-κB is involved in the regulation of mitochondrial dysfunction [31], and Bax antagonizes the anti-apoptotic ability of Bcl-2 and simultaneously promotes permeability of the mitochondrial outer membrane and reduces the level of ΔΨm [32]. The results herein suggest that IκBα decreases cell injury and apoptosis by inhibiting NF-κB activation and Bax expression, ultimately elevating ΔΨm after H 2 O 2 stimulation.
Autophagy, a cellular process of lysosome-mediated degradation of cytoplasmic components or damaged organelles, is thought to be an adaptive response and protective for cell survival [10]. However, autophagy causes a redox effect in cardiomyocytes upon different stimuli. Evidence supports the bene t of autophagy to cardiomyocytes during myocardial ischemia through its improvement of myocardial energy metabolism and organelle recycling [33], but excessive autophagy causes lethal damage in cells during cardiac I/R injury [21], which is mediated in part by the upregulation of Beclin-1 expression [34].
However, the communication between autophagy and NF-κB is bidirectional. Autophagy is required for the activation of NF-κB [13]; in turn, NF-κB further increases autophagosome maturation by upregulating Beclin-1 and LC3-expression in I/R injury [35]. Importantly, PDTC attenuates Beclin-1 expression and the formation of autophagosomes by suppressing I/R injury-induced NF-κB activation [16]. In accordance with these ndings, the treatment of NRVMs with H 2 O 2 induced p65 phosphorylation and translocation, enhanced Beclin-1 expression, and increased the LC3-/LC3-I ratio, these effects were rescued by IκBα transfection or PDTC treatment. These results imply that IκBα can protect cardiomyocytes by inhibiting H 2 O 2 -induced autophagy. In addition, Bcl-2 can bind Beclin-1 to inhibit autophagy [36]. This study also demonstrates that IκBα transfection elevated the expression of Bcl-2, which may disturb the function of Beclin-1 and thus further inhibit H 2 O 2 -induced autophagy, implying cross-talk between apoptosis and autophagy.
This study has some limitations. The present ndings were obtained from neonatal cardiomyocytes in vitro, and may differ from ndings in animal experiments due to the complicated features of the in vivo environment. Further animal studies should be conducted to con rm the cardioprotective effects of IκBα against I/R injury.

Conclusions
The ndings of this study show that pretreatment with dsAAV9-IκBα or PDTC protected NRVMs from H 2 O 2 -induced apoptosis, autophagy, mitochondrial dysfunction, and oxidative damage by restraining the NF-κB signaling pathway, suggesting that IκBα transfection can protect cardiomyocytes against cardiac oxidative damage. Thus, AAV9 vectors, as the high-e ciency gene carrier to heart, may be used to carry IκBα gene to protect the heart by targeted inhibition of myocardial NF-κB in future preclinical or clinical studies, which may provide a promising gene therapy for preventing cardiac I/R injury.