KPC1 alleviates hypoxia/reoxygenation‐induced apoptosis in rat cardiomyocyte cells though BAX degradation

Abstract Bax triggers cell apoptosis by permeabilizing the outer mitochondrial membrane, leading to membrane potential loss and cytochrome c release. However, it is unclear if proteasomal degradation of Bax is involved in the apoptotic process, especially in heart ischemia‐reperfusion (I/R)‐induced injury. In the present study, KPC1 expression was heightened in left ventricular cardiomyocytes of patients with coronary heart disease (CHD), in I/R‐myocardium in vivo and in hypoxia and reoxygenation (H/R)‐induced cardiomyocytes in vitro. Overexpression of KPC1 reduced infarction size and cell apoptosis in I/R rat hearts. Similarly, the forced expression of KPC1 restored mitochondrial membrane potential (MMP) and cytochrome c release driven by H/R in H9c2 cells, whereas reducing cell apoptosis, and knockdown of KPC1 by short‐hairpin RNA (shRNA) deteriorated cell apoptosis induced by H/R. Mechanistically, forced expression of KPC1 promoted Bax protein degradation, which was abolished by proteasome inhibitor MG132, suggesting that KPC1 promoted proteasomal degradation of Bax. Furthermore, KPC1 prevented basal and apoptotic stress‐induced Bax translocation to mitochondria. Bax can be a novel target for the antiapoptotic effects of KPC1 on I/R‐induced cardiomyocyte apoptosis and render mechanistic penetration into at least a subset of the mitochondrial effects of KPC1.

Therefore, enhancing proteasomal function in pathological conditions will be a hopeful strategy for patients with PFI.
Actually, data from bench to bedside have identified cardiomyocyte apoptosis as a typical event in myocardial I/R (Zhang et al., 2013).
Further evidence has suggested that the permeabilization of the mitochondrial outer membrane by Bax during apoptosis is considered a key step and a point of no return in the signaling pathway, mainly attributed to the altered traits in Bax expression and mitochondria translocation (Gross, Jockel, Wei, & Korsmeyer, 1998). Previous study found that proteasomal degradation of Bax involved in the fine tuning of apoptotic cell death (Dewson, Snowden, Almond, Dyer, & Cohen, 2003).
And, a novel mechanism which the proapoptotic activity of Bax was inhibited by ubiquitination has been reported (Cakir et al., 2017). Of interest, KPC1, a novel E3 ubiquitin ligase, could mediate ubiquitination and proteasomal processing of NF-κB1 and p27Kip1, and play a crucial role in cell growth (Kamura et al., 2004;Lu et al., 2009

| Adenovirus pretreatment and ischemia-reperfusion rat model
Male SD rats (250-300 g) provided by the Laboratory Animal Center (Hubei University of Medicine) were randomized into four groups: an Ad-green fluorescent protein (GFP) pretreatment sham group, an Ad-KPC1 pretreatment sham group, an Ad-GFP pretreatment and ischemia-reperfusion (I/R) group, and an Ad-KPC1 pretreatment and I/R group. After anesthetized with chloral hydrate (300 mg/kg) by intraperitoneal injection, Ad-GFP or Ad-KPC1 (1.0 × 10 9 pfu in 150 μl) was injected at three different points (50 μl per site) into myocardium sites in the left ventricle anterior wall. Seven days later, ischemiareperfusion was achieved by the ligature of the left coronary artery anterior descending branch (LAD) as described previously (Tang, Xie, Pan, Wang, & Wang, 2006). In brief, after anesthetized, the rats were ventilated with a rodent ventilator (HX-300S, Techman Soft) and then the LAD was ligated for 1 hr followed by a 24 hr reperfusion by thoracotomy surgery of the fourth intercostal space.

| TTC staining
After a 24 hr reperfusion, the rats were narcotized and the hearts were quickly removed. After frozen at −20°C for 30 min, the hearts were transverse section into four pieces with 2-3 mm thickness. The sections were incubated in 1% TTC (2, 3, 5-triphenyltetrazolium chloride) at 37°C for 20 min lucifugally in accordance with the instructions of manual (D025, Nanjing Jiancheng Bioengineering Institute). The arithmetic of infarcted zone volume was described as previously (Huang et al., 2011).

| Heart tissues TUNEL assay
After fixed with 4% paraformaldehyde and embedded in paraffin, the hearts were serially cut for obtaining sections. For detecting apoptosis, a Colorimetric terminal-deoxynucleotidyl transferasemediated nick end labeling (TUNEL) Apoptosis Assay Kit (C1091, Beyotime) was used. The number of apoptotic cells was counted using a high-power light microscope when reddish brown cells were observed under adjacent six fields. Apoptotic ratio was calculated by the number of positive cells/total number of cells × 100%.

| H9c2 cells TUNEL assay
Forty-eight hours after treated with Ad-KPC1, Ad-Ctrl, Ad-shKPC1, or Ad-shCtrl, the cells were exposed to specific treatment with H/R. H9c2

| Immunofluorescence staining
The cultured H9c2 cells in 24-well plates reached 70% cell density. After H/R treatment, 4% polyoxymethylene was used to fix the cells for 15 min, which were washed three times. After blocking the nonspecific antigen, the primary antibodies, such as Bax (#2772, CST), cytochrome c (sc-13156, Santa Cruz), KPC1 (ab57549, abcam), and Troponin T-C (cTnT, sc-515899, Santa Cruz) were added. The specific well with the corresponding second antibody (1:250) added was incubated 2 hr at room temperature. Five random field of each glass slide (Thermo Fisher Scientific) were photographed and total 30 images per group were obtained according to the same standard. Images were analyzed by three technicians who did not know grouping information using ImageJ (Java) software (National Institutes of Health).

| Flow cytometry assay
To quantitatively analyze the role of KPC1 overexpression in H9c2 cell apoptosis, 48 hr after transfected with Ad-KPC1 or Ad-Ctrl, the cells were exposed to specific treatment with H/R. Because Ad-KPC1 did not carry the specific GFP-tag, an Annexin V/propidium iodide (PI) Kit (Invitrogen) was used. After appropriate staining, the cells were analyzed by the flow cytometry.
To confirm if KPC1 knockdown by RNA (Ad-shKPC1) was involved in H9c2 cells apoptosis, 48 hr after transfection, the cells were exposed to specific treatment with H/R. Because Ad-shKPC1 carried the specific GFP-tag, the special Annexin V/TRITC Kit (Invitrogen) was used in flow cytometry analysis (Huang et al., 2011).

| Bax protein stability assay
Forty-eight hours after transfection, the cells were exposed to specific treatment with H/R. After treated with cycloheximide (CHX, 10 μg/ml) for 0-6 hr or MG132 (10 μM) for 0-8 hr, the cells were harvested for western blot analysis.

| Mitochondrial membrane potential detection
Following transfection and H/R treatment, the mitochondrial membrane potential (MMP) was measured using a MitoProbe JC-1 Assay Kit (M34152, Life). H9c2 cells were incubated with JC-1 (1 μM each well) for 20 min. After washing, the cells were observed and photographed.
The ratios of red-to-green fluorescence were quantified to evaluate the degree of damage to the mitochondrial membrane.
Because Ad-shKPC1 carried the specific GFP-tag, only the intensity of red fluorescence in Ad-shKPC1 or Ad-shCtrl treatment group was quantitated to estimate the degree of mitochondrial membrane damage.

| Co-immunoprecipitation (Co-IP)
To examine the interaction between Bax and KPC1, the cells were lyzed with RIPA buffer (Millipore). After centrifugation, the supernatant was incubated with anti-KPC1 antibody (sc-101122, Santa Cruz) at 4°C overnight. The subsequent co-immunoprecipitation (Co-IP) was performed with a Pierce Co-Immunoprecipitation Kit (26149, Thermo Fisher Scientific) following the manufacturer's instructions. After the final elution, the samples were collected for western blot analysis (Tang et al., 2018).

| Statistical analysis
Statistical analysis was performed with PASW Statistics 18 (SPSS). All data were evaluated with two-tailed, unpaired Student's t tests or one-way analysis of variances (ANOVAs) followed by t tests and are expressed as the mean ± SD. The p-values < .05 were considered significant.

| KPC1 expressions in CHD hearts, infarcted hearts, I/R hearts, and H/R H9c2 cells
To explore the expression of KPC1 in CHD and in infarcted hearts, immunohistochemical staining was used to detect it in CHD and in I/R hearts. As shown in Figure 1a-c, KPC1 expression was increased in the CHD hearts, in ischemic, and infarcted areas of hearts (quantitative analysis results were shown in Figure S1a-c). Subsequently, to mimic the increased KPC1 expression in I/R hearts in vivo, KPC1 mRNA and protein expressions in the H/R H9c2 cells in vitro were detected by qPCR and western blot analysis, respectively. As shown in Figure 1d,e the levels of both KPC1 mRNA and proteins were markedly increased when exposed to H/R (quantitative analysis results were shown in Figure S1d). Then, to further assess if KPC1 expression was mainly located within cardiomyocytes, double staining of KPC1 and cTnT were performed and as shown in Figure   1f, KPC1 expression was obviously increased in cardiomyocytes.
These results suggested that KPC1 expression was induced in cardiomyocytes following ischemia.

| Knockdown of KPC1 worsens H/R-driven H9c2 cell apoptosis
To observe the role of KPC1 in H/R-induced apoptosis of H9c2 cells, loss-of-function of KPC1 was achieved by knockdown of KPC1 by shRNA (Ad-shKPC1). The TUNEL staining was used to evaluate H9c2 cell apoptosis in H/R conditions. As shown in Figure 2a (Huang et al., 2011). To first assess if KPC1 is involved in the changes in Δψ m exposed to H/R, JC-1 staining was performed for the traits of either monomer in the cytoplasm with green fluorescence or aggregated in mitochondria with red fluorescence. As shown in Figure 4a, H/R notably decreased the degree of red fluorescence intensity compared with normoxic conditions, indicating loss of mitochondrial membrane potential. KPC1 knockdown remarkably reduced the mitochondrial membrane potential with lower red fluorescence intensity. In contrast, the specific effects could be abolished by forced KPC1 expression, which was characterized by a higher ratio of red/green fluorescence shown in Figure 4c. Thus, KPC1 protected H/Rinduced H9c2 cell apoptosis through restoring mitochondrial function.

| KPC1 involves in the regulation of cytochrome c release
Cytochrome c release from mitochondria to cytosol and activation of the caspase cascades after apoptotic stimuli, leading to cell death (Huang et al., 2011;Zhang et al., 2013). After finding the protective effects of KPC1 on H/R-induced H9c2 cell apoptosis through improving mitochondrial function, we further evaluated the effects of KPC1 on cytochrome c release. As shown in Figure 5, H/R triggered cytochrome c release to cytoplasm by immunofluorescence staining. The specific effects could be obviously eliminated by overexpression of KPC1. Combined with the effects of KPC1 on mitochondrial membrane potential recovery, we speculated that KPC1 could prevent cytochrome c release from mitochondria through decreasing mitochondrial aggregation during apoptosis.

| KPC1 regulates bax stability in H/R H9c2 cells through a proteasomal pathway
The loss of Δψm under stress was generally attributed to increased levels of Bax (Huang et al., 2011;Zhang et al., 2013) Figure S1f). To explore whether the above effects were related to Bax, the Bax expression was detected in I/R myocardial tissues using IHC staining. As shown in Figure 6a, I/R heart tissues, following pretreatment of Ad-GFP, revealed stronger expression of Bax compared with the sham groups (quantitative analysis results shown in Figure S1e). Of interest, heart pretreatment with Ad-KPC1 remarkably reduced Bax levels triggered by I/R injury.
To reveal the underlying mechanism, Bax expression was detected in

| The forced expression of KPC1 decreases bax mitochondria translocation in H/R H9c2 cells
Really, Bax, functioning as a regulator of mitochondrial activity, deeply depends on translocation of Bax to the mitochondria besides considering Bax protein levels (Gross et al., 1998). We further evaluated if KPC1 was

| KPC1 interacts with endogenous bax during apoptosis
To uncover if KPC1 directly mediated Bax function, Co-IP of KPC1, and Bax were performed. As shown in Figure 7b,

| Overexpressing KPC1 decreases bax levels and cell apoptosis in I/R hearts
To further confirm the role of KPC1 in Bax levels and cell apoptosis in I/R hearts, an I/R heart injury model in rats was used.
As a typical method, the TTC staining was used to evaluate I/R heart injury and infarcted size. Generally, TTC staining is brickred, which represents nonischemic myocardium, whereas white staining indicates infarcted area. As shown in Figure 8a,b, heart tissues with pretreatment with Ad-GFP or Ad-KPC1 did not show the changes in ischemic myocardium following the sham operation, indicating that Ad-GFP and especially Ad-KPC1 injection, did not cause heart damage. I/R myocardial tissues with pretreatment with Ad-GFP showed more than 60% white area in left ventricle, demonstrating I/R. In contrast, I/R myocardial tissues with pretreatment with Ad-KPC1 displayed less than 40% area in the left ventricle, illustrating that forced expression of KPC1 could contribute to cardioprotective effects.
To further affirm the beneficial effects of KPC1 in the I/R heart, cell apoptosis in heart tissues was investigated using the TUNEL staining. As shown in Figure 8c,d, I/R heart tissues with F I G U R E 5 KPC1 decreases cytochrome c release from mitochondria. Cytochrome c releases from mitochondria were analyzed by double fluorescence staining of cytochrome c and DAPI. Under normoxic conditions, KPC1 knockdown slightly increased cytochrome c release from mitochondria in H9c2 cells. When exposed to H/R, the cells showed increased cytochrome c release, KPC1 knockdown further accelerated cytochrome c release. The specific effects could obviously be reversed by the overexpression of KPC1, n = 3. DAPI, 4',6-diamidino-2-phenylindole; H/R, hypoxia and reoxygenation [Color figure can be viewed at wileyonlinelibrary.com] pretreatment with Ad-GFP showed an almost 40% apoptotic ratio. In contrast, overexpression of KPC1 in I/R myocardial tissues markedly reduced the apoptotic ratio of cells in the left ventricle, which approached 25%, indicating that the pretreatment with Ad-KPC1 decreased infarcted size by reducing cell apoptosis.
Combining in vitro and in vivo results, KPC1 regulated cell apoptosis in I/R hearts through controlling Bax function.

| DISCUSSION
The results of this study show that KPC1 was identified as a novel factor protecting cardiomyocytes from apoptosis during H/R stress in vitro and I/R injury in vivo. The role of KPC1 in protecting cells from H/R-and I/R-induced cell apoptosis is supported by several lines of evidence. First, cardiomyocytes showed increased KPC1 expression during H/R or I/R-caused injury. Second, KPC1 knockdown F I G U R E 6 KPC1 regulates Bax protein stability in H/R H9c2 cells through a proteasomal pathway. (a) Bax expression was detected on different groups by immunostaining, n = 6. Forty-eight hours after H9c2 cells were pretreated with Ad-KPC1, Ad-Ctrl, Ad-shKPC1, or Ad-shCtrl, the cells were exposed under H/R conditions, and 10 μg/ml CHX were added for the indicated time. (b), (c) Bax stability was decreased in H/R H9c2 cells pretreated with Ad-KPC1. Bax expressions were normalized to α-tubulin, * p < .05, # p < .001, n = 3. (d), (e) Bax degradation by proteasome pathway was abolished by MG132. Bax expressions were normalized to α-tubulin. (f), (g) Bax stability was increased in H/R H9c2 cells pretreated with Ad-shKPC1 and Bax intensities were normalized to α-tubulin intensities, * p < .05, # p < .001, @ p < .001, & p < .001, n = 3. CHX, cycloheximide; H/R, hypoxia and reoxygenation [Color figure can be viewed at wileyonlinelibrary.com] exacerbated H/R-induced H9c2 apoptosis, whereas KPC1 overexpression alleviated the damage. Finally, targeting Bax, KPC1 provided crucial protection against mitochondria-dependent cell apoptosis ( Figure S2).
When proteasomal degradation fails to satisfy the need to remove substrate proteins, PFI can occur at a specific location within the cell (Wang & Robbins, 2013). PFI frequently occurs in heart disease, especially in myocardial I/R injury (Li et al., 2011;Tian et al., 2012). Herein, we found that KPC1 expression was increased in cardiomyocytes following I/R injury in vivo and H/R in vitro.
Knockdown of KPC1 exacerbated H/R-driven H9c2 cell apoptosis, indicating that elevated KPC1 expression in ischemic hearts, especially in injured cardiomyocytes, could be an adaptative adjustment to the demand for enhancive turnover of structural and functional proteins. Indeed, KPC1 overexpression decreased cell apoptosis driven by H/R in vitro and I/R injury in vivo.
Forced KPC1 expression enhanced proteasomal function to degrade Bax, possibly through ubiquitination. Indeed, it was reported that Bax ubiquitination participated in apoptotic cell death (Cakir et al., 2017). However, literature has emerged that provides disputable findings about the E3 ligase responsibilities.
Two studies have shown that the E3 ligase, parkin, can ubiquitinate the Bax BH3 domain, therefore, before transferring to mitochondria, cytoplasmic Bax was degraded through proteasome. (Charan, Johnson, Zaganelli, Nardozzi, & Lavoie, 2014;Johnson, Berger, Cortese, & Lavoie, 2012). Of interest, a recent study provided controversial evidence that parkin promotes dysregulated Bax proteasomal degradation in the mitochondria, F I G U R E 7 KPC1 interacts with endogenous Bax and decreases translocation of Bax to mitochondria in H/R H9c2 cells. (a) Two days after treated with Ad-KPC1 or Ad-Ctrl, the cells were exposed under H/R conditions. Double fluorescence staining of Bax and Mitotracker were analyzed in H/R cells pretreated with or without Ad-KPC1. KPC1 decreases Bax translocation to mitochondria from cytoplasm (arrow). (b) Two days after H9c2 cells treated with Ad-KPC1 or Ad-Ctrl, the cells were exposed under H/R conditions. The cell lysate were immunoprecipitated with anti-KPC1 and immunoblotted with anti-Bax. H/R, hypoxia and reoxygenation [Color figure can be viewed at wileyonlinelibrary.com] causing essential protection against apoptotic activeness of Bax (Cakir et al., 2017;Lindenboim, Ferrandomay, Borner, & Stein, 2013). Our results showed that KPC1 functioned similarly to the parkin E3 ligase in the proteasomal degradation of Bax in cytoplasm with less translocation to mitochondria, resulting in recovery of mitochondrial function and reduction in cell apoptosis induced by I/R or H/R injury. Although a complete understanding of KPC1 function in protecting I/R and H/R-induced cell apoptosis through Bax requires extensive future study, our studies suggest that KPC1 may serve as a potential target for treating cardiovascular diseases characterized by PFI, especially I/R-injured myocardium.
NF-κB (p50/p65) and p53 have been shown to activate Bax in I/R-injured hearts (Liu, Xu, Cavalieri & Hock, 2006;Zhang et al., 2018). Interestingly, p53 is not only a direct transcriptional activator of Bax (Miyashita & Reed, 1995), but also activates KPC1 expression (Zhao et al., 2013). Recent studies showed that KPC1 ubiquitinates p105, leading to enhanced production of p50 but decreased levels of p65, which elicits suppression of tumor growth . Of note, knockdown of NF-κB p65 expression or inhibition of its activity lessens cell apoptosis through reducing Bax expression (Li et al., 2013;Qu, Zhang, & Wu, 2015). We found that KPC1 accelerated proteasomal F I G U R E 8 Overexpressing KPC1 decreases Bax expression and cell apoptosis in I/R hearts. (a), (b) Infarcted size was measured by the TTC staining. After I/R injury, ischemic area was easily observed compared with sham group. However, the infarcted sizes were reduced in Ad-KPC1 pretreatment compared with Ad-GFP pretreatment after I/R injury, # p < .001, *P < 0.05, n = 5. (c), (d) Apoptosis was detected on different groups by the TUNEL staining. The proportion of TUNEL-positive cells in Ad-KPC1 transfection group was significantly decreased after I/R injury compared with Ad-GFP pretreatment and the I/R group, # p < .001, * p < .05, n = 5. GFP, green fluorescent protein; I/R, ischemia-reperfusion; TTC, 5-triphenyltetrazolium chloride; TUNEL, terminal-deoxynucleotidyl transferase-mediated nick end labeling [Color figure can be viewed at wileyonlinelibrary.com] degradation of Bax through forming a complex of KPC1-Bax. Therefore, p53, KPC1, and NF-κB likely form an integrated system to regulate Bax activity during I/R or H/R-induced cardiomyocyte apoptosis.
Taken together results, our study demonstrated that KPC1 exerted protective effects on I/R-and H/R-induced cardiomyocyte apoptosis through accelerating proteasomal degradation of Bax and reducing Bax mitochondria translocation.