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

Background: In�ammation 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 considered to be a crucial mediator. Inactivation of NF-κB could bene�t cardiomyocytes through inhibiting apoptosis. IκBα, an inhibitor of NF-κB, is hypothesized to protects cardiomyocytes from H 2 O 2 -induced apoptosis and autophagy through inhibiting the NF-κB pathway. Methods: We designed an AAV9-delivered mutated IκBα S32A, S36A and investigated its effect on neonatal rat ventricular cardiomyocytes (NRVMs) in response to hydrogen peroxide (H 2 O 2 ). NRVMs were divided into Normal (blank), Control (H 2 O 2 ), GFP +H 2 O 2 , IκBα+H 2 O 2 , and Pyrrolidine dithiocarbamate (PDTC)+H 2 O 2 groups. NF-κB p65 nuclear translocation was evaluated by immuno�uorescence and western blot. Cell viability was assessed by a cell counting kit-8 kit. 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, and the mitochondrial membrane potential ( △ Ψm) was detected by JC-1. Western bolt was used to detect apoptosis and autophagy related proteins. Results: Consequently, H 2 O 2 -treated NRVMs showed reductions in cell viability but increased IκBα degradation and NF-κB p65 nuclear translocation in a time-dependent manner. Furthermore, LDH and MDA content, LC3-(cid:0)/LC3-(cid:0) ratio, Bax and Beclin-1 expressions, and apoptotic cells were upregulated in NRVMs exposed to H 2 O 2 , whereas △ Ψm and Bcl-2 expression were downregulated. Additionally, IκBα transduction or PDTC pretreatment both attenuated the nuclear translocation of the p65 subunit and reversed the H 2 O 2 -stimulated effects in NRVMs. Conclusion: These �ndings suggest that IκBα could ameliorate H 2 O 2 -induced apoptosis and autophagy through targeted inhibition of NF-κB activation, which may guide strategies to prevent cardiac I/R jury.


Introduction
Acute myocardial infarction (AMI) is the leading cause of death in 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 necrosis, apoptosis, autophagy and in ammation, nally leading to myocardial ischemia/reperfusion (I/R) injury [2,3].Recent evidences have suggested that excessive in ammation and oxidative stress play a predominant role in the initiation and progression of I/R injury [4].
Nuclear factor kappa B (NF-κB) is an in ammatory inducer and redox-sensitive transcription factor existing in most cell types [5].The p65/50 heterodimer, the most common pattern of NF-κB dimers, normally exist as components of inactive cytoplasmic complexes bounded with members of the inhibitor of κB (IκBα).Upon stimulation, IκBα is phosphorylated, and undergone ubiquitylation and proteasomal degradation, subsequently leading to the release and nuclear translocation of p65/p50 dimer [6].
Activated NF-κB then initiates expression of corresponding target genes, many of which may regulate apoptosis, in ammation and autophagy [7].
However, whether NF-κB is protective or detrimental for prevention cardiomyocyte apoptosis remains controversial [8].Notably, our previous study indicated that p65 ribozyme could prevent cell apoptosis in H9C2 cardiomyocyte exposed to hydrogen peroxide (H 2 O 2 ) [9].Autophagy, an evolutionarily conserved "self-digestion", plays dual roles in heart [10].Recent researches on autophagy shows both protective [11] and deleterious [12] roles of autophagy in cardiomyocyte against oxidative stress.Evidences have elucidated a strong correlation exists between the modulation of NF-κB and autophagic response [13,14].In addition, it is noted that there exists a "cross-talk" between autophagy and apoptosis [15], and NF-κB is known as a mediator of the balance between them [16].
Therefore, we speculate that NF-κB activation is the key point of I/R injury, inhibiting NF-κB may be a targeting therapy.IκBα, as the key inhibitor of canonical NF-κB pathway, phosphorylation on Ser 32 and Ser 36 is necessary for its degradation and any mutation of these two serine will block IκBα degradation [6].Recently, adeno-associated virus serotype 9 (AAV9) is de ned as the best gene carrier due to their high e ciency in the heart [17].H 2 O 2 , as one common reactive oxygen species (ROS), is generally utilized to mimic I/R injury in vitro [12].Thus, we designed an AAV9-delivered mutated IκBα S32A, S36A gene to investigate the role of inhibition of NF-κB pathway in H 2 O 2 -induced apoptosis and autophagy in cardiomyocytes.Pyrrolidine dithiocarbamate (PDTC), a speci c inhibitor of NF-κB, was used as a positive control in this study.
Isolation, culture and treatment of rat cardiomyocytes 1-to 3-day-old Sprague Dawley rats (sex unlimited) were obtained from the Experimental Animal Center of Xinjiang Medical University.The protocol for the isolation and puri cation of neonatal rat ventricular cardiomyocytes (NRVMs) has been reported in our previous research [19].Brie y, the hearts of 1-to 3-dayold SD neonatal rats were dissected and digested with 0.1% trypsin and 0.08% collagenase II.Following 1.5 h differential adhesion, the suspensions of nonadherent cells were resuspended and cultivated in high-glucose DMEM containing 10% FBS, 1% penicillin-streptomycin, and 0.1 mM BrdU for 48 hours.The cardiomyocytes would be treated with H 2 O 2 (100 µM) to establish a myocardial injury model according to our previous study [12].Incubation of 100 µM PDTC for 60 min before H 2 O 2 treatment was de ned as the positive control group according to our preliminary experiments.The medium was replaced every 48 h.

AAV9 transfection of cardiomyocytes
After 48 h culture, NRVMs were transfected respectively with dsAAV9-GFP and dsAAV9-IκBα as previously described [12].Brie y, cells were rstly transfected with dsAAV9 (multiplicity of infection, MOI = 5 × 10 6 vg/cell) in serum-free medium, then DMEM at equal volume with 20% FBS, 2% penicillinstreptomycin and 0.2 mМ Brdu was added to every dish 3 h later.The images of GFP were captured using uorescence inverted microscope (LEICA-DMI4000B, Germany) and green uorescence intensities were analyzed using Image J software.

Measurement of cardiomyocyte vitality and cytotoxicity
A cell counting kit-8 (CCK-8; Dojindo, Japan) was used to assess the cell viability.In brief, 2 × 10 4 cells were seeded into a 96-well plate per well and transfected with GFP or IκBα for ve days, respectively.After cells 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 was measured at 450 nm by GO microplate spectrophotometer (Thermo Fisher Scienti c, USA).The extent of cell death was determined by quantifying LDH released into culture supernatant following the manufacturer's instructions of LDH Kit (Jiancheng Bioengineering Institute, China).Intracellular MDA, an indicator of oxidative injury, was also measured using the MDA assay kit (Jiancheng Bioengineering Institute, China).

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

Western blot analysis
We extracted nuclear and cytoplasmic proteins following the instructions of Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scienti c, USA) and applied the ratio of nuclear p65 to cytosolic p65 to re ect the degree of NF-κB translocation [20].Total proteins were extracted with RIPA buffer containing Halt™ Protease and Phosphatase Inhibitor Cocktail.Equal amounts of protein were loaded and separated on SDS-PAGE precast gels (Invitrogen Life Technologies) and transferred to Millipore PVDF membrane.

Immuno uorescence
Immuno uorescence was employed to identify the 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 by 4% paraformaldehyde for 20 min and permeabilized with 0.25% Triton X-100 for 10 min, respectively.After blocking with 1% BSA for 1 h, cells were probed overnight with anti-p65 (1:200) at 4 °C, and incubated with FITC-conjugated secondary antibody (1:200) for 2 h at room temperature, followed by 10 minutes of DAPI staining for nuclei.Signals were detected using a confocal spectral microscope (Leica Microsystems, Germany).

Mitochondrial membrane potential measurement
JC-1 (Millipore, USA) is generally used as an ideal uorescent probe to detect mitochondrial membrane potential (△Ψm) in cardiomyocytes.Brie y, we prepared 10 nmol/L JC-1 working solution prior to use, and then stained cardiomyocytes at 37 ℃ in the dark for 15 min.The double staining of cells by JC-1 was visible either as green or red uorescence.Fluorescent images and intensities were obtained using a uorescence microscope or Image J software.Generally, △Ψm was represented with the red-to-green uorescence ratio, which dropped proportionally with the severity of cell injury.

Statistical analysis
All statistical analysis was performed with SPSS 22.0.Data were presented as mean ± SEM.One-way ANOVA was used for multiple group comparisons.A value of p < 0.05 was regarded as statistically signi cant.

H 2 O 2 -induced activation of NF-κB in NRVMs
To con rm NF-κB activation induced by H 2 O 2 in cardiomyocytes, the translocation of the NF-κB p65 subunit from the cytosol to the nucleus was assessed.NRVMs were incubated with 100 µM H 2 O 2 for different durations (0, 15, 30, 60 min), respectively.The ndings indicated that H 2 O 2 elicited a dose-dependent IκBα degradation and p65 translocation (Fig. 1A and B).The ratio of nuclear p65 to cytosolic p65 peaked at 60 min.Thus, 100 µM H 2 O 2 treatment for 60 min was identi ed for the following researches.

Transduction e ciency of IκBα on NRVMs
To con rm transduction e ciency of dsAAV9-IκBα on NRVMs, cells were transfected with IκBα and GFP virus at a 5 × 10 6 MOI respectively.Compared with the normal group, the expressions of IκBα and GFP were signi cantly enhanced, and green uorescence intensities of dsAAV9-GFP achieved more than 70% (Fig. 1C and D).Our ndings veri ed the high-e ciency transduction of NRVMs using dsAAV9 vectors.

IκBα protected cardiomyocytes from H2O2-induced apoptosis
To assess the role of IκBα in H 2 O 2 -stimulated cardiomyocytes apoptosis, ow cytometry and western blot were both applied.The apoptosis rate (Fig. 2A) and Bax/Bcl-2 ratio (Fig. 2B) remarkedly increased in NRVMs exposed to H 2 O 2 , which were both attenuated by IκBα transduction or PDTC pretreatment.These results indicated that IκBα could prevent H 2 O 2 -induced cell apoptosis in NRVMs.

IκBα protected cardiomyocytes from H2O2-induced cell injury
△Ψm was assessed by JC-1 staining through calculating the red-to-green uorescence intensity ratio.
Compared to the normal group, △Ψm decreased signi cantly in the control or GFP group, but it was rescued by IκBα or PDTC treatment (Fig. 3A).Additionally, we determined the H Ischemia/reperfusion (I/R) injury severely attenuates the bene t of revascularization after acute myocardial infarction (AMI) and hence has become an important focus of cardiovascular research [2].It is currently believed that the in ammatory response induced by AMI is essential for heart repair, but the excessive generation of ROS and in ammation following reperfusion therapy will exacerbate heart damage [21].
NF-κB signaling pathway plays a key role in the in ammatory response, oxidative stress, apoptosis, and autophagy in heart [8].As is known, the nuclear translocation of p65 subunit is a sign for 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 a signi cant p65 nuclear translocation in NRVMs.In line with these studies, we found that p65 was time-dependently translocated from cytoplasm to nucleus with IκBα degradation in NRVMs subjected to H 2 O 2 .
However, whether NF-κB activation could protect or exacerbate cardiomyocyte is still a matter of debate.The early study demonstrated that activation of NF-kB reduced cell apoptosis in hypoxic cardiomyocyte [25], whereas most of recent studies have shown that NF-κB is a pro-apoptotic transcription factor correlated with myocardial injury [26] and blocking NF-κB activity will prevent myocardial apoptosis [27].Gray et al [22] recently report that ROS generated by ischemia-reperfusion can rapidly activate calmodulin kinase II (CaMKII), which deteriorates cell injury through inducing IκBα degradation and nuclear p65 accumulation in NRVMs exposed to H 2 O 2 .Importantly, knock-out CaMKIIδ gene signi cantly attenuates area of myocardial infarction by inhibiting IκBα degradation and NF-κB activation.All these ndings reveal that NF-κB activation deteriorates the prognosis of heart in I/R injury.
Herein, we hypothesized that directly overexpress IκBα to prevent NF-κB activation may play a better role in protecting cardiomyocyte.Then, we designed a dsAAV9-IκBα It is reported that opening of the mitochondrial permeability transition pore (MPTP) in the rst few minutes of reperfusion lead to △Ψm loss and is responsible for the necrotic and apoptotic cell death processes exhibiting differential contributions to infarct size [29].Thus, △Ψm loss re ects the dysfunction of mitochondrial, represents a sign of early stage apoptosis and is a critical determinant of I/R injury [30].Our previous study demonstrates that oxidative stress induces a signi cant decrease of △Ψm [12].In this study, we observe that H 2 O 2 treatment results in attenuated △Ψm and enhanced Bax expression in NRVMs, and the effect was reversed by pretreatment with IκBα or PDTC.NF-κB [31] and Bax [32] are reported to be involved in regulation of △Ψm.Herein, we suggest that IκBα decreases cell injury and apoptosis via inhibiting NF-κB activation and Bax expression, nally elevating △Ψm after H 2 O 2 stimulation.
Autophagy, a cellular process of lysosome-mediated degradation of cytoplasmic components or damaged organelles, is generally thought to be an adaptive response and protective for cell survival [10].However, autophagy shows redox effect on cardiomyocyte upon different stimuli.Evidence supports that autophagy bene ts heart cells during myocardial ischemia through improving myocardial energy metabolism and organelle recycling [33], but excessive autophagy produces lethal damage on cells in cardiac I/R injury [21], which is mediated in part by upregulation of Beclin-1 expression [34].
However, the communication between autophagy and NF-κB is bidirectional.It is reported that autophagy is required for the activation of NF-κB [13], in turn, NF-κB further increases autophagosome maturation via upregulating Beclin-1 and LC3 expression in I/R injury [35].Importantly, PDTC attenuates Beclin-

Figures Figure 1
Figures

Figure 4 IκBα
Figure 4 We found 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 △Ψm, but elevations in LDH, MDA, apoptosis and autophagy.IκBα transduction or PDTC pretreatment ameliorated H 2 O 2 -induced cell injury via inhibiting the NF-κB translocation.
2 O 2 -stimulated injury using CCK8, LDH, and MDA assays.H 2 O 2 treatment signi cantly decreased cell viability but elevated supernatant LDH and intracellular MDA levels, which were reversed by IκBα or PDTC treatment (Fig.3B-D).Our ndings demonstrated that IκBα played a protective role in H 2 O 2 -induced injury in NRVMs.transduction and PDTC pretreatment (Fig.4A and B).These results indicated that IκBα transduction effectively inhibited the NF-κB activation in H 2 O 2 -treated cardiomyocytes.Additionally, the autophagyassociated markers including Beclin-1 expression and LC3-/LC3-ratio were markedly upregulated in NRVMs exposed to H 2 O 2 , whereas these effects were inhibited by IκBα or PDTC treatment (Fig.4C).Thus, we infer that IκBα could alleviate H 2 O 2 -induced autophagy via inhibiting the NF-κB signaling pathway.Discussion ser 32A,36A mutant to prevent IκBα degradation from phosphorylation at serine 32 and 36 sites, which could be successfully transfected into cardiomyocytes.Western blot and immuno uorescence demonstrated that IκBα transfection could successfully maintained cytoplasmic IκBα level and suppressed the p65 translocation in NRVMs exposed to H 2 O 2 .Consistent with our speculation, IκBα elevated cell viability, decreased LDH and MDA level and attenuated apoptosis, implying a protective role of IκBα in H 2 O O 2 .These data indicate that IκBα protects NRVMs against H 2 O 2 -induced apoptosis by decreasing the ratio of Bax/Bcl-2.
2-induced cell injury in NRVMs.The mechanisms may be account for the role of NF-κB in mediating the expression of various proteins that promote or inhibit apoptosis.It is noted that NF-κB regulates the expression of certain anti-apoptotic genes, for example Bcl-2 [28], and increasing the ratio of Bcl-2/Bax will decrease cell apoptosis.In this study, treatment with IκBα mutant or PDTC signi cantly reduced Bax/Bcl-2 ratio in NRVMs subjecting to H 2