- Research article
- Open Access
PIAS1 protects against myocardial ischemia-reperfusion injury by stimulating PPARγ SUMOylation
- Bo Xie†1,
- Xinyu Liu†3,
- Jie Yang4,
- Jinke Cheng2,
- Jianmin Gu1Email author and
- Song Xue1Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 29 May 2018
- Accepted: 25 October 2018
- Published: 12 November 2018
Myocardial ischemia-reperfusion injury (IRI) has become one of the most serious complications after reperfusion therapy in patients with acute myocardial infarction. Small ubiquitin-like modification (SUMOylation) is a reversible process, including SUMO E1-, E2-, and E3-mediated SUMOylation and SUMO-specific protease-mediated deSUMOylation, with the latter having been shown to play a vital role in myocardial IRI previously. However, little is known about the function and regulation of SUMO E3 ligases in myocardial IRI.
In this study, we found dramatically decreased expression of PIAS1 after ischemia/reperfusion (I/R) in mouse myocardium and H9C2 cells. PIAS1 deficiency aggravated apoptosis and inflammation of cardiomyocytes via activating the NF-κB pathway after I/R. Mechanistically, we identified PIAS1 as a specific E3 ligase for PPARγ SUMOylation. Moreover, H9C2 cells treated with hypoxia/reoxygenation (H/R) displayed reduced PPARγ SUMOylation as a result of down-regulated PIAS1, and act an anti-apoptotic and anti-inflammatory function through repressing NF-κB activity. Finally, overexpression of PIAS1 in H9C2 cells could remarkably ameliorate I/R injury.
Collectively, our findings demonstrate the crucial role of PIAS1-mediated PPARγ SUMOylation in protecting against myocardial IRI.
- Ischemia-reperfusion injury
With the tremendous rise in the standard of living, acute myocardial infarction (MI) has become a common cardiovascular emergency that causes a large number of deaths in modern society. Timely and effective myocardial reperfusion appears to be the only therapeutic approach for reducing acute myocardial ischemic injury and limiting MI size . However, as a direct result of blood flow restoration to the ischemic tissue, myocardial ischemia-reperfusion injury (IRI) can lead to cell death and additional cardiac dysfunction. The underlying molecular mechanisms of myocardial IRI involve inflammation, calcium overload, oxidative stress, cytokine release and infiltration of neutrophil . Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily of ligand-inducible transcription factors, which has been shown to play a vital role in various physiological and pathological processes, including glucose and lipid metabolism, immunity and cardiovascular disease . Activation of PPARγ can suppress the inflammatory response in cardiac tissue after ischemia/reperfusion (I/R) and thus alleviate ischemic pathological damage [4, 5]. In our previous study, we found that PPARγ mediates the protective effect of quercetin against myocardial IRI via suppressing the NF-κB pathway .
It has taken more than 20 years to identify protein modification by small ubiquitin-like modification (SUMOylation) . Protein SUMOylation is a reversible process catalyzed by the activating (E1), conjugating (E2) and ligating (E3) enzymes and can be reversed by a family of SUMO-specific proteases (SENPs) [8, 9]. Only one E1 and one E2 enzyme have been reported in mammalian cells, whereas more than eight SUMO E3 ligases have been found to catalyze the transfer of SUMO from E2 UBC9 to a substrate. The protein inhibitor of activated STAT (PIAS) family of proteins , including PIAS1, PIAS3, PIASxα, PIASxβ and PIASy, belong to the largest group of SUMO E3 ligases characterized by an SP-RING motif . The requirement of the location of a RING-finger domain in the middle of a PIAS is essential to the E3 ligase activity of PIAS proteins. Various studies have shown that PIAS-mediated SUMOylation of target proteins is involved in a wide range of cellular processes [12–16].
We have previously shown that SENP1 deficiency exacerbates IRI in cardiomyocytes via an HIF1α-dependent pathway , indicating the involvement of protein SUMOylation in myocardial IRI. However, it is unknown whether SUMO E3 ligases are regulated in myocardial IRI. In this study, we identify PIAS1 as a specific E3 ligase for PPARγ SUMOylation in the myocardium. PIAS1-mediated PPARγ SUMOylation protects against apoptotic and inflammatory injury by inhibiting NF-κB activation after ischemia/reperfusion. Our data suggest a potential clinical role of PIAS1 in IRI therapy.
Expression of PIAS1 is reduced after ischemia/reperfusion in mouse myocardium and H9C2 cells
PIAS1 deficiency aggravates injury after H/R via activation of NF-κB pathway
PIAS1 enhances PPARγ SUMOylation by its SUMO E3 ligase activity
PPARγ SUMOylation antagonizes injury after H/R by suppressing NF-κB activation
Ectopic expression of PAIS1 alleviates IRI via PPARγ SUMOylation
Protein SUMOylation has been considered as a vital regulator of cellular function in physiology and pathology. Recently, Ubc9, the E2 ligase for SUMOylation, has been found to play an essential role in isoflurane preconditioning-induced tolerance against cerebral ischemia-reperfusion injury . Moreover, SENP1 protects neurons by inhibiting apoptosis during transient brain ischemia/reperfusion , consistently with our previous finding in myocardial IRI . In this study, we analyzed the expression of SUMO E3 ligases during myocardial IRI using a mouse model. Among these E3 ligases, PIAS1 demonstrated the largest extent of reduction in myocardium after I/R. Notably, PIAS1 proteins undergo fast decline during the initial period of reperfusion, whereas the mRNA level of PIAS1 at 24 h of reperfusion becomes nearly equal to that at 1 h of reperfusion. These results imply that there may be a mechanism involved in up-regulating PIAS1 expression after long-term reperfusion. It is important and valuable to further explore this mechanism under the pathological process of myocardial IRI, as ectopic expression of PIAS1 can alleviate the injury. Recent studies have suggested that PIAS1 serves as an anti-inflammatory factor in adipose tissue and the lung [23, 24]. In agreement, our findings verify that PIAS1 antagonizes inflammation in myocardium through inhibition of the NF-κB pathway.
Our previous finding demonstrated that SENP1 protects against IRI in cardiomyocytes via a HIF1α-dependent pathway . On the other hand, here we highlight the important role of PIAS1-mediated PPARγ SUMOylation in protecting against myocardial IRI, indicating us that two opposite functional proteins may have the same contribution to a cellular process through different pathways. PPARγ has been shown to benefit cardiovascular disease therapies, such as those pertaining to ventricular hypertrophy, cardiac remodeling and acute myocardial infarction [25–27]. SUMOylation of PPARγ at lysine 77 in the transactivation domain blocks its transcriptional activity, possibly by promoting co-repressor recruitment [28–30], while PPARγ is also SUMOylated at lysine 365, which in macrophages results in its occupation of the promoters of inflammatory genes and inhibition of their expression . Our previous work demonstrated that PPARγ protects against myocardial IRI by suppressing the NF-κB pathway. In this study, we find that PIAS1-mediated SUMOylation of PPARγ is essential for the inactivation of NF-κB signaling. To evaluate the function of PPARγ SUMOylation in myocardial IRI, PPARγ-WT or K77R mutant was re-expressed into H9C2 cells lacking endogenous PPARγ. Unlike PPARγ-WT, K77R mutant failed to inhibit NF-κB activation effectively and alleviate inflammation and apoptosis sharply after H/R. However, we found that K77R mutant can partially rescue the phenotype of PPARγ deficiency, suggesting that the existing SUMOylation of PPARγ at lysine 365 may also play a role in regulating NF-κB activity. Further investigation is required to reveal the function of lysine 365 SUMOylation in myocardial IRI.
IRI in human myocardium
Twelve male patients with an average age of 64.83 ± 3.326 years were diagnosed with moderate mitral stenosis without other physical illnesses, and underwent mitral valve replacement under cardiopulmonary bypass. The patients are comparable. Mitral valve replacement surgery which requires the use of extracorporeal circulation can simulate the process of myocardial ischemia and reperfusion, refer to our previous publication . We acquired myocardial tissue at two time points. The first time was before the aorta was clamped (Sham) and the second time was 15 min after unclamping(IRI). The specimens were immediately washed in cold phosphate-buffered saline (PBS) and then preserved in 4% paraformaldehyde. All patients signed informed consent. This research conformed to the Declaration of Helsinki and was approved by the Institutional Review Board of Renji Hospital, Shanghai Jiao Tong University School of Medicine.
Animals and IRI in mouse myocardium
Male C57/BL6 mice (8 weeks old; weight, 22-24 g) were purchased at the Xipuer-Bika Experimental Animal Center (Shanghai, China). Mice were housed in the Animal Experimental Center of Shanghai Jiao Tong University School of Medicine. Mice were randomly divided into four groups:  sham-operation group (Sham, n = 6);  ischemia 30 min and reperfusion 2 h group (I/R 2 h, n = 6);  ischemia 30 min and reperfusion 4 h group (I/R 4 h, n = 6);  and ischemia 30 min and reperfusion 6 h group (I/R 6 h, n = 6). Mouse heart IRI surgery was performed as described . All animal research were performed in accordance with NIH guidelines (Guide for the Care and Use of Laboratory Animals) and the Use Committee of Shanghai Jiao Tong University.
RNA isolation and quantitative RT-PCR analysis
Sense and reverse primers
Immunohistochemical and immunofluorescence analysis
In brief, Human and mouse heart tissues were harvested and fixed with 4% paraformaldehyde, then were dehydrated, paraffin-embedded, and sectioned (5 μm) prior to staining. Heart sections were exposed to 3% hydrogen peroxide for 10 min and treated for 20 min in boiling 0.01 M citric acid (pH 6.0), then blocked with bovine serum albumin for 1 h. Sections were subsequently incubated with anti-PIAS1 antibody (1:200 diluted in PBS) overnight at 4 °C, and anti-rabbit IgG secondary antibody (1:1000 diluted in PBS) were incubated for 2 h at room temperature. The avidin–biotin complex (ABC) and 3,3′-diaminobenzidine tetrahydrochloride (DAB) were then incubated, nuclei were stained with hematoxylin. Images were acquired using Nikon microscope. For PIAS1 positive cell measurement, the number of positive cells per mm2 tissue section were counted in 10 random visual fields under high magnification. Immunofluorescence analysis of NF-κB was performed as described previously .
Immunoprecipitation (IP) and western blot analysis
Cells were lysed in IP buffer (50 mM Tris-HCl, pH 7.4, 400 mM NaCl, 0.5% sodium deoxycholate, 0.3% Triton X-100, 0.1% SDS, 10 mM N-ethylmaleimide, and protease inhibitors) for30 min on the ice, then cell lysates were sonicated and centrifuged at 20000×g for 10 min at 4 °C. The supernatants were transferred to new tubes. The appropriate antibodies and protein A/G beads were added for 6 h at 4 °C, beads were then washed with IP buffer and eluted in 1% SDS solution. Proteins were detected by western blot analysis, antibodies used in this study were PIAS1 (ab32119), inducible nitric oxide synthase (iNOS) (ab15323), GAPDH (ab70699) and IgG (ab210935) from Abcam, Anti-PPARγ (sc-7273) from Santa Cruz Biotechnology, Anti-Flag (F3165) from Sigma, Anti-HA (MMS-101P) from Covance. Anti-SUMO1 (18–2306) from Zymed, Cyclooxygenase-2 (COX-2) (12282), NF-κBp65 (8242), and phospho-IκBα (p-IκBα) (2859) from Cell Signaling Technology.
HEK-293 T cells and myocardial H9C2 cells were cultured in Dulbecco’s modified Eagle’s medium (4.5 g/L D-glucose) supplemented with 10% FBS. For normal condition (sham), all cells were maintained at 37 °C in a humidified 5% CO2 incubator. For hypoxia and reoxygenation (H/R), cells were cultured in an airtight incubation tank for 4 h with a < 1% oxygen concentration followed by hours of reoxygenation as indicated. The cultured H9C2 cells were randomly divided into different groups. siRNAs as indicated were transfected into H9C2 cells for 48 h before treatments, plasmids as indicated were transfected into H9C2 for 24 h before treatments.
Plasmids and RNAi
Sequences of siRNA
Negative control (NC)
Negative control (NC)
Terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) assay
The TUNEL assay was performed using the in situ Cell Death Detection kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Images were acquired using Zeiss 710 microscope; The percentage of TUNEL-positive cells/total cells was calculated in 10 visual fields chosen randomly from each section for statistical analysis.
All experiments were performed at least three times. All data were presented as mean ± standard deviation. Graphs and statistical comparisons were performed using GraphPad Prism Software. Parametrical data were compared using Student’s t-test or one-way ANOVA analysis to analyze significance (*P < 0.05; **P < 0.01).
This work was supported by National Natural Science Foundation of China (No.81670225).
Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.
BX and XL performed experiments, analyzed data and wrote the manuscript; JY performed experiments; JC provided research platform and guided experiments; JG designed experiments, analyzed data and wrote the manuscript; SX obtained funding, designed experiments, supervised research and wrote the manuscript. All authors read and proved the final manuscript.
Ethics approval and consent to participate
All cardiac tissue specimens were obtained with informed consent from patients undergoing cardiac surgery in Renji Hospital, Shanghai Jiao Tong University School of Medicine. This study conformed to the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of Renji Hospital, Shanghai Jiao Tong University School of Medicine. All animal procedures were performed in accordance with NIH guidelines (Guide for the Care and Use of Laboratory Animals) and the Use Committee of Shanghai Jiao Tong University.
Consent for publication
The authors declare that they have no competing interests.
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