Knockdown of endogenous RNF4 exacerbates ischaemia‐induced cardiomyocyte apoptosis in mice

Abstract RNF4, a poly‐SUMO‐specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress‐induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H2O2/ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML‐NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H2O2/ATO‐induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno‐associated virus infection deteriorated post‐MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia‐induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia.

intracellular ROS induces cardiomyocyte apoptosis, 3 and the development of infarction is correlated with the number of apoptotic cardiomyocytes at peri-infarction areas. 4 Transgenic mice with overexpression of superoxide dismutase (SOD), an antioxidant protein, had significantly smaller infarction size compared to that of controls, which highlighted the vital role of ROS in the regulation of post-ischaemic cardiac injury. 5 Human ring-finger protein 4 (RNF4), an E3 ubiquitin ligase, targets poly-SUMO-modified proteins, leading to protein degradation via the ubiquitin-mediated pathway. 6 RNF4 has been implicated in the proteolytic control of the degradation of promyelocytic leukaemia protein (PML) and in the transcriptional activity of multiple transcription factors, such as poly-(ADP-ribose) polymerase 1 (PARP-1), hypoxia-inducible factors (HIFs) and PEA3. 7-10 Moreover, RNF4 plays a role in the response of mammalian cells to DNA damage. Cells with RNF4 elimination are hypersensitive to certain types of DNA damage, and RNF4 deficiency results in inefficient end resection. 11,12 Furthermore, RNF4 is linked to tumorigenesis since RNF4 has been shown to enhance cancer cell survival by regulating Wnt and Notch pathways. 13 A high level of RNF4 mRNA is related to poor survival among patients with breast cancer, and its protein expression is elevated in 30% of human colon adenocarcinomas. 14 However, the role of RNF4 in heart disease has not yet been investigated.
Here, we examined RNF4 expression following myocardial infarction (MI) or H 2 O 2 /arsenic trioxide (ATO) treatment in vitro and in vivo.
Upon oxidative stress, both in vitro and in vivo experiments revealed alterations in RNF4 expression whereby RNF4 first increased and then gradually decreased. Due to the rapid enhancement of RNF4, we presumed that reduction of RNF4 could contribute to alleviating oxidative injury. Nevertheless, knockdown of endogenous RNF4 exacerbated oxidative stress-induced cardiomyocyte apoptosis and ischaemia-induced cardiac dysfunction, which was associated with enhanced PML nuclear body (NB) accumulation and p53 recruitment and activation.

| Animals
All animal experiments complied with the regulations established by the Institutional Animal Care and Use Committee of Harbin Medical University and the NIH guidelines (Guide for the Care and Use of Laboratory Animals). All animal studies were approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards established in the 1964 Declaration of Helsinki and its later amendments. Male Kunming mice (20-25 g) were provided by the Experimental Animal Center of Harbin Medical University (Grade II). Mice were fed a standard chow diet, were provided tap water ad libitum and were housed under a controlled temperature (22°C) with a 12-h light-dark cycle.

| In vivo adeno-associated virus infection
Mice were randomized into several groups (three groups: Sham, Scramble and shRNF4 groups; or four groups: Sham, MI, +Scramble and +shRNF4 groups). In vivo adeno-associated virus infection was performed as described previously. 15 Briefly, mice were anesthetized with sodium pentobarbital (40 mg⋅kg −1 , i.p.). The heart was fully exposed via blunt dissection of subcutaneous tissue and the fourth intercostal muscle. The ascending aortic artery and main pulmonary artery were clamped. Then, 200 μL saline (Sham mice) or AVV9-Scramble (1 × 10 9 pfu at a volume of 200 μL, Scramble mice) or AVV9-shRNF4 (1 × 10 9 pfu at a volume of 200 μL, shRNF4 mice) was injected into the left ventricular cavity through the apex with a 30-gauge syringe. After injection, the arteries remained occluded for 10 seconds. The shRNF4 sequence is listed in Table 1.

| Mouse models of MI
Mice in the four groups were anaesthetized with sodium pentobarbital and then underwent left anterior descending coronary artery occlusion under sterile conditions. For Sham mice, a suture was crossed through the myocardium around the left anterior descending artery without ligation. Twenty-four hours after MI, mice were subjected to heart function assessment and were then euthanized by cervical dislocation following CO 2 inhalation. Heart tissue was harvested for subsequent experiments.

| Isolation of neonatal mouse cardiomyocytes
Neonatal Kunming mice (1-3 days old) were anaesthetized with 4% isoflurane inhalation before rapid heart excision. Hearts were dissected TA B L E 1 Sequences of the specific siRNAs and shRNA, and the plasmids and cut into small pieces, as described previously. 16 Briefly, cardiac tis-

| Real-time PCR
Total RNA was extracted from heart tissue at the per-infarction area by using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Extracted RNA was measured on a Nanodrop (Thermo Fisher Scientific, USA). Then, mRNA was quantified using SYBR Green I on an ABI 7500 fast real-time PCR system (Applied Biosystems, USA). GAPDH was used as a reference gene.
All primers used are shown in Table 2.

| Detection of intracellular ROS levels
ROS assays were carried out as previously described. 17

TA B L E 2
The primers used for real-time PCR to detect the mRNAs of targeting genes washed twice and then observed by a laser-scanning confocal microscope (Olympus, Tokyo, Japan) with an excitation wavelength of 370 nm and an emission wavelength of 594 nm. ROS in the representative fluorescent images were shown as green staining.

| Measurement of cell viability
NMCMs were seeded into a 96-well microplate at 4000 cells per

| Echocardiographic measurement and pulsewave Doppler
An ultrasound machine (Vivid 7 GE Medical, General Electric Company, Fairfield, CT) with a 10-MHz phase-array transducer was used to assess ventricular function. Two-dimensional and M-mode images were obtained in the short-axis view to assess systolic function. The left ventricular ejection fraction (EF) and fraction shortening (FS) were then calculated.

| Immunofluorescent staining
NMCMs were cultured on a sterile glass in 6-well plates. After drug treatment, samples were fixed in 4% paraformaldehyde for 15 min-

| Electron microscopy
Cell and tissue samples were fixed in 2.5% glutaraldehyde (pH 7.4) overnight at 4°C and were then immersed in 0.1 M cacodylate buffer with 1% osmium tetroxide for 1 hour. Samples were dehydrated with a concentration gradient of ethanol and then embedded in Epon medium and dissected into 60-to 70-nm sections.
After being stained with uranyl acetate and lead citrate, sections were observed with a JEOL 1200 electron microscope (JEOL Ltd., Tokyo, Japan).

| Masson staining
Cardiac tissue at the peri-infarction area was sectioned and immersed in 4% paraformaldehyde, embedded in paraffin for 24 hours at 4°C and was then stained with Masson's trichrome (Accustain HT15, Sigma-Aldrich). Image analysis software (Image-Pro Plus v4.0; Media Cybernetics, Bethesda, MD, USA) was used to assess the extent of interstitial fibrosis.

| Evans blue-TTC staining
The infarcted area and the ischaemic area were determined by Evans

| Extraction of cytoplasmic and nuclear protein
Nuclear protein extracts were obtained using a protein separation kit (Invent Biotechnology, Inc) according to the manufacturer's instructions. Heart tissue was washed with cold PBS and was then centrifuged at 5000 rpms for 5 minutes. The tissue was mixed with an appropriate volume of cytoplasmic extraction reagent, incubated on ice for 5 minutes and was then vortexed for 5 seconds at the highest speed. The above protocol was repeated five times. Samples were centrifuged for 5 minutes at 5000 rpms, and the supernatant (the cytoplasmic extract) was immediately transferred to a clean tube.
Then, the nuclear extraction reagent was added and the samples were vortexed at the highest setting for 15 seconds. The above protocol was repeated five times. After centrifugation at 13 500 rpms for 15 minutes, the nuclear extract was transferred to a clean tube.

| Statistical Analysis
Statistical analysis was performed with SigmaPlot (Systat Software, Inc, San Jose, CA, USA). Comparisons between two groups were determined by Student's t test. Multiple-group comparisons were performed by one-way ANOVA followed by Tukey's analysis for comparisons of mean values. P < .05 was considered statistically significant. All data are expressed as the mean ± standard deviation (SD).

| Altered RNF4 expression upon oxidative stress in vivo and in vitro
Mouse heart tissue from the peri-infarction areas was harvested and then subjected to Western blot analysis to examine RNF4 expression under ischaemia-induced oxidative stress. After MI, RNF4 increased dramatically and then decreased gradually at the peri-infarction area ( Figure 1A), but not at remote regions ( Figure S1). H 2 O 2 and ATO are often used as oxidants in vitro. As expected, RNF4 elevated initially and then attenuated after H 2 O 2 ( Figure 1B) or ATO ( Figure 1C) treatment, while Tempol partially reversed the oxidative stimulus-induced elevation of RNF4 in vitro ( Figure 1D and E). To determine the mechanism of altered RNF4 expression upon oxidative stress, the transcription and degradation of RNF4 were examined in MI mice. RNF4 mRNA expression increased within 24 hours, followed by a decrease thereafter ( Figure 1F). We also found that RNF4 degradation enhanced in a ubiquitin-mediated pathway ( Figure 1G).
Hence, oxidative stress-induced alteration in RNF4 expression was determined by both RNF4 transcription and degradation.

| Knockdown of endogenous RNF4 exacerbates oxidative stress-induced cardiomyocyte apoptosis in vitro
Due to the rapidly increased levels of RNF4 upon oxidative stress, we next examined whether reduction of RNF4 could attenuate oxidative stress-induced cellular injury. We transfected siRNF4 into NMCMs to knockdown endogenous RNF4 (Figure 2A). After 24-h H 2 O 2 /ATO treatment, overall cell viability decreased by 55% compared to that of the control group ( Figure 2B). Surprisingly, however,  Figure S2A), leading to further exacerbation of the oxidative injury to cardiomyocytes, while an ROS inhibitor significantly attenuated this injury ( Figure S2B). These results indicate that even though oxidative stimuli increased RNF4 expression and triggered cardiomyocyte apoptosis, the reduction of endogenous RNF4 did not attenuate oxidative stress-induced apoptosis, but rather, enhanced it.

| Oxidative stress triggers an altered pattern of PML SUMOylation, and PML is involved in oxidative stress-induced cell injury in vitro
PML is a ROS sensor. 19 Upon oxidative stress, PML SUMOylation was initially enhanced and was then degraded thereafter ( Figure 3A,B).

| Knockdown of endogenous RNF4 attenuates oxidative stress-induced PML-NB degradation and enhances p53 recruitment and activation in vitro
RNF4 can target the poly-SUMO chain-modified protein, leading to protein degradation via the ubiquitin-dependent pathway. PML has been identified as the first and best-characterized substrate of RNF4-mediated degradation. 10 Knockdown of RNF4 enhanced PML SUMOylation, which was accompanied by a twofold increase in the basal expression and activity of p53 compared with that of the NC group ( Figure 4A). Meanwhile, knockdown of endogenous RNF4 not only enhanced oxidative stress-induced PML SUMOylation and PML-NB formation, but also attenuated oxidative stimuli-induced PML and NB degradation ( Figure 4B-D). Also, the reduction of RNF4 increased p53 expression and activation, as well as promoted p53 recruitment into NBs ( Figure 4B-D). Thus, knockdown of RNF4 inhibited oxidative stimulation-triggered PML degradation, which led to NB accumulation, promotion of p53 activation and co-localization with NBs. These results suggest a potential role for RNF4 in cardiomyocyte apoptosis via regulation of PML and p53.

| Knockdown of endogenous RNF4 deteriorates ischaemia-induced cardiac dysfunction
To investigate the role of RNF4 in heart disease, GFP-labelled AAV9-shRNA targeting RNF4 was delivered into the heart to specifically  (Table 3). EF and FS of MI mice decreased by 34% and 40% compared to those of Sham mice, respectively ( Figure 5B). Nevertheless, in accordance with the in vitro results, knockdown of RNF4 exacerbated ischaemiainduced cardiac dysfunction, which was documented as reduced EF (by 24%) and FS (by 27%) in the + shRNF4 group compared with that of the + Scramble group ( Figure 5B; Table 3). Then, the infarction size was

| Knockdown of endogenous RNF4 aggravates ischaemia-induced cardiomyocyte apoptosis in vivo
After 24-h ischaemia in vivo, we detected increased PML SUMOylation and p53 expression and activity ( Figure 6A) with F I G U R E 1 Altered pattern of RNF4 expression upon oxidative stress in vivo and in vitro. A RNF4 expression at peri-infarction areas was detected by Western blot. n = 3. B RNF4 expression was examined by Western blot after H 2 O 2 (200 μM) treatment in NMCMs. n = 5. C Western blot was performed after ATO (2 μM) treatment in NMCMs. n = 5. D and E After 1-h pre-treatment with Tempol (3 mM), RNF4 expression was measured by Western blot after H 2 O 2 (D) or ATO (E) treatment in NMCMs. n = 3. F RNF4 mRNA levels at peri-infarction areas were determined by real-time PCR. n = 7. G Ubiquitin and RNF4 levels at peri-infarction areas were detected by Western blot. Representative images from three independent experiments are shown. * P < 0.05, ** P < 0.01, *** P < 0.001  (Table 3; Figure 5B).

| The PML/p53 axis is involved in the regulation of RNF4 knockdown-induced deterioration of cardiomyocyte apoptosis
To further investigate the correlation between PML and p53 in regulating cell apoptosis, we overexpressed PML via transfecting F I G U R E 3 Oxidative stress triggers an altered pattern of PML SUMOylation, and PML is involved in oxidative stress-induced cell injury in vitro.

| D ISCUSS I ON AND CON CLUS I ON S
This is the first investigation to reveal an altered pattern of RNF4 upon exposure to oxidative stress in vivo and in vitro. However, in contrast to our original hypothesis, knockdown of RNF4 did not attenuate, but instead aggravated, cardiomyocyte apoptosis and heart injury. Upon oxidative stimulation, PML SUMOylation and PML-NB formation were enhanced due to the elevated generation of ROS. Then, p53 was recruited into NBs and was stabilized by phosphorylation, which promoted its transcriptional activity and led to p53-dependent apoptosis.
However, ROS triggered a rapid increase in RNF4 expression that was followed by a gradual decrease in RNF4 expression. RNF4 targeted the SUMO chain of SUMOylated PML and modulated ubiquitin/proteasome-mediated PML degradation, resulting in the abolishment of p53-dependent apoptosis ( Figure 8). Thus, knockdown of RNF4 inhibited PML degradation, which promoted NB accumulation and p53 activation and stabilization, resulting in oxidative injury.
ROS are generated during oxidative stress and are hallmarks of the pathophysiology of many diseases, including cancer, cardiovascular disease, neurodegeneration and ageing-related disease. [25][26][27] In all cases, ROS serve as important signalling modulators. 28 During ischaemia, the large accumulation of ROS is the primary initiator of myocardial damage. ROS promote cardiomyocyte apoptosis, and the death of numerous cardiomyocytes leads to their replacement with collagen-based scars. 2 ROS can regulate p53 expression, DNA binding and transcriptional activity 29 and can also activate mitogen-activated protein kinases (MAPKs) 28 and trigger intracellular calcium overload, leading to cell death. 30 PML has been widely identified as an oxidation-sensitive protein.
A previous study showed that in vivo, ROS inducers, such as paraquat or paracetamol (acetaminophen), increase NB assembly very rapidly. In vitro, PML was also shown to dimerize through di-sul- and cross talk exist among ROS, PML and p53. 19 This was consistent with our study that high ROS levels dramatically induced PML SUMOylation and NB aggregation, which was followed by a reduction of SUMOylated PML and NB due to long-term oxidative stress-triggered PML degradation (Figure 3). In our previous study, high-molecular-weight SUMOylated PML increased within  (1) ubiquitination and degradation pathways and exhibited different, or even opposite regulatory functions. 37 In other words, PML-NBs have been associated with the regulation of several different cellular functions due to the functional diversity of NB partner proteins. RNF4 has also been reported to be implicated in proteolytic control, DNA repair and carcinogenesis; in the present study, we focused on its role in heart disease. An altered pattern of RNF4 expression was shown in the present investigation. After ischaemia, RNF4 increased significantly and was then gradually abated ( Figure 1A-C). It has been reported that RNF4 expression changes in a cell cycle-dependent manner to regulate DNA repair. RNF4 accumulates in the S-/G2-phases but decreases in the G0/G1phases during DNA double-strand break repair. 38 Additionally, we found that MI induced more nuclear RNF4, but less cytoplasmic RNF4, which indicated that MI might trigger RNF4 trans-localization ( Figure S7). Phosphorylation has been reported to be the essential post-translational modification of RNF4, which is required for RNF4-mediated degradation of target proteins during DNA repair. 39 Due to the oxidative stress-induced would attenuate cardiac injury. Nevertheless, we found more severe cardiac dysfunction and cardiomyocyte apoptosis following MI. The underlying mechanism responsible for this effect is that knockdown of RNF4 inhibits PML degradation, resulting in p53 recruitment into PML-NBs and p53-dependent apoptosis, which aggravates ischaemic injury. We previously reported that manipulating PML SUMOylation could regulate cardiac fibrosis via silencing UBC9 and RNF4. 20 In accordance with our present results, knockdown of RNF4 also aggravated cardiac fibrosis and dysfunction induced by TAC. As PML-NB is a platform for the recruitment and interaction of multiple SUMOylated nuclear proteins, it is likely that other NB partner proteins are also potential targets of RNF4. Komaravelli et al found that respiratory syncytial virus (RSV) induced NRF2 (nuclear factor erythroid 2-related factor 2) degradation in a RNF4-dependent manner. NRF2 localizes, in part, to PML-NBs, can undergo SUMOylation. Poly-SUMOylated NRF2 is polyubiquitylated by RNF4 and subsequently degraded by the proteasome in PML-NB domains. Silencing RNF4 expression rescued NRF2 nuclear levels and transcriptional activity, which alleviated RSV-induced cellular oxidative damage. 18 However, we did not detect an effect of RNF4 overexpression on myocardial apoptosis or cardiac function. And whether RNF4 overexpression could partially reverse oxidative stress-induced cardiac injury remains to be studied in future.
In summary, our results demonstrate an altered pattern of RNF4 expression upon oxidative stress in vivo and in vitro. We also reveal that RNF4 is crucial for the regulation of post-ischaemic myocardial apoptosis via modulation of the PML/p53 axis. Our findings provide novel evidence for future therapeutic approaches to treating cardiac ischaemia.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science Foundation of China (Grant nos. 81770281, 81570240 and 31701021). We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

CO N FLI C T O F I NTE R E S T
All of the contributors in this study declared no conflict of interest.

AUTH O R CO NTR I B UTI O N S
YL and WFC designed the experiments and supervised the project; FQ, YNH and XQS were the primary experimenters and were responsible for the writing of the manuscript; PP, MYZ and WYL established the animal models and conducted the in vivo experiments; NNT, SLG, YBC and HW conducted the in vitro experiments; and DZ performed the statistical analysis.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.