LncRNA ACART protects cardiomyocytes from apoptosis by activating PPAR‐γ/Bcl‐2 pathway

Abstract Cardiomyocyte apoptosis is an important process occurred during cardiac ischaemia‐reperfusion injury. Long non‐coding RNAs (lncRNA) participate in the regulation of various cardiac diseases including ischaemic reperfusion (I/R) injury. In this study, we explored the potential role of lncRNA ACART (anti‐cardiomyocyte apoptosis‐related transcript) in cardiomyocyte injury and the underlying mechanism for the first time. We found that ACART was significantly down‐regulated in cardiac tissue of mice subjected to I/R injury or cultured cardiomyocytes treated with hydrogen peroxide (H2O2). Knockdown of ACART led to significant cardiomyocyte injury as indicated by reduced cell viability and increased apoptosis. In contrast, overexpression of ACART enhanced cell viability and reduced apoptosis of cardiomyocytes treated with H2O2. Meanwhile, ACART increased the expression of the B cell lymphoma 2 (Bcl‐2) and suppressed the expression of Bcl‐2‐associated X (Bax) and cytochrome‐C (Cyt‐C). In addition, PPAR‐γ was up‐regulated by ACART and inhibition of PPAR‐γ abolished the regulatory effects of ACART on cell apoptosis and the expression of Bcl‐2, Bax and Cyt‐C under H2O2 treatment. However, the activation of PPAR‐γ reversed the effects of ACART inhibition. The results demonstrate that ACART protects cardiomyocyte injury through modulating the expression of Bcl‐2, Bax and Cyt‐C, which is mediated by PPAR‐γ activation. These findings provide a new understanding of the role of lncRNA ACART in regulation of cardiac I/R injury.

potentials. 3 LncRNAs participate in a number of biological processes and pathophysiological events including cancers and cardiovascular disease. 4 LncRNAs have been demonstrated to regulate cardiac hypertrophy, mitochondrial function, cardiac fibrosis and apoptosis of cardiomyocyte. 5 For example, the expression of Meg3 (maternally expressed gene 3) was up-regulated in mouse injured heart after MI and involved in the regulation of apoptosis via binding to RNAbinding protein FUS (fused in sarcoma). 6 However, detailed studies about lncRNAs' role in regulating myocardial apoptosis are still limited.
Among various apoptosis-related pathways, members of Bcl-2 family participate in regulating programmed cell death by mediating intracellular pro-apoptotic and anti-apoptotic signals. 7,8 The Bax protein, a member of the Bcl-2 family, is crucial in the activation of both intrinsic and extrinsic apoptotic pathways in response to diverse stimuli. 9 In addition, Cyt-C is known as a significant mediator of apoptosis and is released from the mitochondrial intermembrane space to the cytoplasm in response to apoptotic stimulations. 10,11 PPAR-γ (peroxisome proliferator-activated receptor-γ), a member of the nuclear hormone receptor superfamily, has been reported to be a protective molecule in tissue repair and ischaemic injury. 12,13 PPAR-γ overexpression prevents cardiomyocytes from apoptosis by up-regulating Bcl-2 and decreasing the level of reactive oxygen species. 14 Our previous study showed that lncRNA NONMMUT030245 was significantly up-regulated in the heart of mice with myocardial infarction, 15 and it is highly conserved between mouse and human (https ://blast.ncbi.nlm.nih.gov/Blast.cgi). In the present study, the role of lncRNA NONMMUT030245 named as anti-cardiomyocyte apoptosis-related transcript (ACART) was explored in cardiomyocyte apoptosis. The data showed that ACART alleviated cardiomyocyte apoptosis and PPAR-γ/Bcl-2 pathway is involved in this process, which provide new insight into regulation of cardiomyocyte injury.

| Animals
In this study, healthy male C57BL/6 mice (20-25 g) and neonatal mice (1-3 days old) were purchased from the Animal Center of the Second Affiliated Hospital of Harbin Medical University. Mice were kept under standard conditions for animals (temperature, 21 ± 1°C; humidity, 55%-60%) and received food and water ad libitum. All experimental procedures were in accordance with the Institutional Animal Care and Use and approved by Committee of the Harbin Medical University.

| Mouse model of ischaemia/reperfusion
The healthy adult male C57BL/6 mice were randomly divided into sham-operated and I/R groups. The animals were anesthetized with intraperitoneal injection of avertin (0.2 g/kg). The mouse heart was exposed by a left-sided thoracotomy. The left anterior descending coronary artery (LAD) was ligated for 45 minutes with a 7/0 silk thread followed by reopening of the artery for 24 hours. [16][17][18] The mice in sham group underwent a same procedure, but with no LAD ligation. After I/R procedure, the air was removed from the chest and the surgical wounds were sutured. To prevent infection, 0.1 mL penicillin (4 × 10 5 U/mL) was applied by intraperitoneal injection.
Finally, the animals were placed on a heating pad until full recovery of consciousness.

| Neonatal mouse ventricular cells isolation and treatment
Neonatal mouse ventricular cardiomyocytes (NMVCs) were isolated from 1-to 3-day-old mice with 0.25% trypsin at 37°C and cultured in Dulbecco's modified Eagle medium (DMEM, Hyclone Laboratories, Utah, USA) supplemented with 10% foetal bovine serum (Gibco, California, USA), 100 U/mL penicillin and 100 μg/ mL streptomycin. 19 Cells at 80% confluence were transfected with 100 nmol/L siRNA of ACART (Si-ACART) or a plasmid carrying ACART sequence (500 ng/mL) for 24 hours. Then, cells were treated with hydrogen peroxide (H 2 O 2 , 100 μmol/L) in serum-free medium for 24 hours or 48 hours or were incubated in an anoxic chamber with 95% N 2 and 5% CO 2 in glucose-free DMEM for

| Quantitative real-time PCR
Total RNA samples from heart tissues or cultured cells were extracted using TRIzol reagent (Invitrogen, Carlsbad, USA). The RNA was reverse

| Statistical analysis
All experimental data are expressed as mean ± SD. Statistical significance was estimated by ANOVA or Student's t test for multiple group or two group comparisons, using GraphPad Prism 7. P < .05 was considered to be statistically different.

| ACART was down-regulated during cardiomyocyte injury
We have found that ACART was significantly up-regulated in cardiac fibrotic tissue, 15 but its function in cardiomyocyte apoptosis was unclear. We firstly examined ACART expression in the hearts of mice subjected to I/R (ischaemia/reperfusion) injury. The qRT-PCR assay revealed that the level of ACART was significantly decreased in I/R hearts after 1, 4, 8, 16 and 24 hours of reperfusion compared with the sham group ( Figure 1A). However, there was no significant difference among these time-points. In our study, we employed H 2 O 2 to mimic the pathological change of reactive oxygen species overproduction, which has been widely employed in the field. [23][24][25] As speculated, the expression of ACART in cultured NMVCs (neonatal mouse ventricular cardiomyocytes) treated with 100 μmol/L hydrogen peroxide (H 2 O 2 ), an inducer of apoptosis for 24 hours was also down-regulated ( Figure 1B). Meanwhile, cardiomyocytes F I G U R E 1 ACART was down-regulated during cardiomyocyte injury. A, Mice were subjected to myocardial ischaemia for 45 min then the expression level of ACART was assayed by qRT-PCR at 1, 4, 8, 16 and 24 h after reperfusion. **P < .01 vs Sham, n = 5. B, ACART level was detected in NMVCs treated with 100 μmol/L H 2 O 2 for 24 h. C, NMVCs were treated with 12 h hypoxia, followed by reoxygenation for 24 h, then ACART level was detected. **P < .01 vs Ctl, n = 4 were treated with 12 hours hypoxia, followed by reoxygenation for 24 hours. After 24 hours reoxygenation, the expression of lncRNA ACART was decreased by 48% compared with the control group ( Figure 1C), which was consistent with the results from H 2 O 2 -treated group. Therefore, in this study, 100 μmol/L H 2 O 2 was used to simulate ischaemia/reperfusion injury in cardiomyocytes.

| Overexpression of ACART mitigated H 2 O 2induced cardiomyocyte injury
To test the effects of ACART manipulation on cardiomyocyte injury, the effects of ACART overexpression on cardiomyocyte injury were evaluated. We transfected the NMVCs with a plasmid carrying ACART sequence and the expression level of ACART was increased by about 11-fold ( Figure 2A). However, ACART over-

| Knockdown of ACART induced cardiomyocyte apoptosis
To further verify the regulatory role of ACART in cardiomyocyte apoptosis, we employed the siRNA for ACART (Si-ACART) to knockdown its expression. Transfection of Si-ACART in cultured NMVCs reduced ACART level by 60% as examined by qRT-PCR ( Figure 3A). Importantly, knockdown of ACART dramatically decreased cardiomyocyte viability as detected by MTT assay ( Figure 3B), and increased LDH release compared with negative control (NC) groups ( Figure 3C). We performed TUNEL assay to evaluate cell apoptosis and found that knockdown of ACART significantly increased cardiomyocyte apoptosis ( Figure 3D,E). These data indicated that down-regulation of ACART is detrimental to cardiomyocyte.
Next, we examined the effects of ACART knockdown on NMVCs injury induced by H 2 O 2 . As shown in Figure 3F

| ACART regulated Bcl-2-mediated apoptosis of cardiomyocytes
Bcl-2, a suppressor of apoptosis, is decreased during apoptosis. 3 Bax, a pro-apoptotic protein, is an important factor in the apoptotic signalling pathway. 7 Cyt-C is released from the mitochondria into the cytoplasm when apoptosis occurs. 26 Therefore, the influence of ACART on the expressions of these proteins was explored. We found that ACART overexpression had no effects on protein levels of Bcl-2, Bax and Cyt-C ( Figure 4A,B). However, Si-ACART induced significant Bcl-2 repression, Bax and Cyt-C enhancement in NMVCs ( Figure 4C,D). Importantly, overexpression of ACART counteracted H 2 O 2 -induced Bcl-2 reduction and increase of Bax and Cyt-C expression ( Figure 4E,F). We also observed that Cyt-C was significantly increased in the cytosol after H 2 O 2 treatment. ACART attenuated this effect, while Si-ACART enhanced this effect. ACART plasmid and negative control had no effect on the cytosolic Cyt-C level. Si-ACART elevated the cytosolic Cyt-C level in cardiomyocytes without H 2 O 2 treatment (data not shown). Studies have reported that H 2 O 2induced apoptosis was associated with activation of caspase-3. 27,28 As shown in Figure 4G

| PPAR-γ/Bcl-2 pathway participated in ACARTmediated regulation of NMVCs apoptosis
Studies have reported that PPAR-γ is a critical molecule in protecting cardiomyocytes from apoptosis by regulating the expression of Bcl-2 family proteins. 29 We therefore evaluated the potential involvement of PPAR-γ in ACART-mediated apoptosis and alteration of Bcl-2, Bax and Cyt-C expression. We found that the mRNA level of PPAR-γ was significantly increased by ACART overexpression ( Figure 5A) and reduced by Si-ACART in NMVCs ( Figure 5B). Moreover, over- Si-ACART were encountered by co-treatment with PPAR-γ agonist RGZ ( Figure 6G,H). These data indicate that PPAR-γ/Bcl-2 pathway participated in the regulation of ACART in NMVCs apoptosis.

| D ISCUSS I ON
In the present study, we characterized lncRNA ACART as a positive regulator of cardiomyocyte apoptosis through PPAR-γ/Bcl-2 pathway. We demonstrated that ACART expression is down-regulated in I/R mouse hearts and H 2 O 2 -or hypoxia/reoxygenation-treated Cell fate is determined by the balance between pro-and antiapoptotic factors/molecules, such as Bcl-2 family members. 35 Pro-apoptotic molecule Bax is considered as an inhibitory binding partner of Bcl-2, it exhibits an extensive amino acid homology with Bcl-2 and forms heterodimers with Bcl-2. 36 The pro-apoptotic function of Bax is activated in response to deleterious events, resulting in the formation of a channel or other structure in the mitochondrial outer membrane. This is widely accepted that Cyt-C exits mitochondria to trigger apoptosis through the conduit. 26,[37][38][39] Here, we demonstrated that H 2 O 2 down-regulated expression of Bcl-2 and ACART in NMVCs, while elevates the Bax and Cyt-C expression.
Overexpression of ACART increased Bcl-2 expression, inhibited Bax and Cyt-C expression. However, knockdown of ACART produced opposite effects. When apoptosis occurs, Cyt-C is released from the mitochondria into the cytosol where it participates in caspase activation. ACART attenuated cytosolic Cyt-C increase after H 2 O 2 treatment, while Si-ACART enhanced this effect. These results suggest that Bcl-2, Bax and Cyt-C were involved in the regulation of cardiomyocyte apoptosis by ACART.
It has been reported that PPAR-γ, a ligand-activated transcription factor protects cardiomyocytes from oxidative stress-induced apoptosis by up-regulating Bcl-2 expression. 14,40 Importantly, PPAR-γ F I G U R E 6 PPAR-γ/Bcl-2 pathway participated in ACART-mediated regulation of NMVCs apoptosis. A and B, T0070907 an antagonist of PPAR-γ attenuated protective effect of ACART on H 2 O 2 -induced apoptosis. Scale bar, 100 μm. C and D, T0070907 repressed the effects on Bcl-2, Bax and Cyt-C of ACART, protein levels of Bcl-2, Bax and Cyt-C were detected by Western blot. E and F, Effects of agonist of PPAR-γ on TUNEL-positive cells. Scale bar, 100 μm. G and H, RGZ an agonist of PPAR-γ restored the alterations of Bcl-2, Bax and Cyt-C induced by Si-ACART in NMVCs. **P < .01 vs control, #P < . 05  activation possesses potent protective actions and has been recently determined as potential therapeutic agents for cardiovascular disease. Our study demonstrates that ACART promoted PPAR-γ and Bcl-2 expression and reduced Bax and Cyt-C expression while T0070907 a PPAR-γ antagonist abolished the effects of ACART on Bcl-2, Bax and Cyt-C expressions. These data imply that ACART possesses the regulatory effects on PPAR-γ-mediated expression of Bcl-2, Bax and Cyt-C. More importantly, PPAR-γ antagonist blocked the protective effects of ACART on NMVCs apoptosis; however, PPAR-γ agonist abrogated cell apoptosis induced by ACART silencing.
Taken together, the present study demonstrates that PPAR-γ/Bcl-2 pathway is involved in the regulation of ACART in NMVCs apoptosis. The potential limitations of the present study are as follows: Firstly, the exact mechanism of PPAR-γ regulation by ACART has not yet been explored. Based on lncRNA biological function, ACART may regulate PPAR-γ expression at gene transcriptional or/and post-transcriptional levels. Therefore, it could not be ruled out that other mechanisms are included in regulation of cardiac I/R injury by ACART. Secondly, the effects of ACART on cardiac injury and function in vivo need to be evaluated in future study.
In summary, our findings demonstrate that ACART protects cardiomyocyte from apoptosis via at least PPAR-γ/Bcl-2 pathway and provide new understanding of the role of lncRNA in regulation of cardiomyocyte apoptosis.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

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.