Cardiovascular disease is the leading cause of death worldwide and is mainly driven by ischemic heart disease, which causes irreversible myocardial damage and eventually heart failure (1). Timely restoration of blood flow is the most effective way to salvage the ischemic myocardium and improve clinical outcomes. However, reperfusion can contribute to further damage, known as ischemia/reperfusion (I/R) injury (2). Although multiple studies have focused on cardiac I/R injury in recent decades, effective therapeutic strategies have yet to be identified (2).
Opioid receptors are known to be involved in cardioprotection against I/R injury (3). We have recently shown that the onset administration of opioids, which was defined as opioid postconditioning (OPC), confers robust cardioprotection against I/R injury both in animal experiments and clinical studies (4–6). Remifentanil is one of the most wildly used general opioid anesthetic for its ultra-fast-acting, potent characteristics (7). Remifentanil postconditioning (RPC) induced cardioprotection in simulated I/R injury of H9c2 cardiomyocytes by attenuating apoptosis (6). Apoptosis of cardiomyocytes is an important mechanism of I/R injury, and attenuated apoptosis has been proposed as an effective mechanism of RPC-induced cardioprotection. Accumulating evidence has also shown that apoptosis of cardiomyoblasts is suppressed by glycogen synthase kinase-3β (GSK-3β). Therefore, manipulation of GSK-3β may be a promising target for cardioprotective strategy against I/R injury (8). The GSK-3 family of serine/threonine kinases plays an important role in various pathologic processes of heart, including pressure overload and ischemic injury (9). GSK-3 is composed of two isoforms (α and β), which are encoded by distinct genes and are ubiquitously expressed. Numerous studies have shown that inhibition of GSK-3β via phosphorylation at Ser9 residue serves to integrate upstream protective signaling pathways to the end effector and results in cardioprotection of multiple ischemic/pharmacological pre- or postconditioning (8). We also have determined that GSK-3β was involved in the cardioprotection induced by sufentanil postconditioning in normal heart of rats (10, 11). However, whether GSK-3β is involved in RPC is unclear, and the underlying mechanisms remain obscure.
Histone acetyltransferases (HAT) and histone deacetylases (HDACs) have recently been investigated as potential pharmacological targets for patients suffering various cardiopathology, inducing I/R injury (1, 12). Gene deletion and overexpression studies have revealed important functions of several of these enzymes in pathological cardiac remodeling, including ventricular hypertrophy, apoptosis, necrosis, metabolism, contractility, and fibrosis. In mammalian cells, 18 HDACs have been described, grouped into four classes (12). HDAC3 is a member of the class I histone deacetylase family, which has been proposed as a regulator of cardiac growth, proliferation, differentiation, and hypertrophy (13, 14). Studies have shown that suppression of HDAC3 expression contributes to decreased cell hypertrophy and mitigates established LVHF. Therapeutic interventions targeting HDAC3 have also been demonstrated to be potential strategies for the treatment of cerebral I/R injury (14). We recently identified the involvement of HDAC3 in RPC-induced cardioprotection in simulated I/R injury of H9c2 cardiomyocytes (6). However, the precise role of HDAC3 and its associated mechanisms require further study.
HDACs have primarily been reported to function as classical transcriptional modulators in their capacity to deacetylate histone and enhance chromatin compaction (9). Accumulating evidence supports a cross talking role between HDAC and kinase signaling networks in pathological myocardium and other tissues (12, 15). However, whether HDAC3 signal transduction pathways participate in cross talk with GSK-3β in cardiac ischemia remains unknown. Herein, we hypothesized that cardioprotection by RPC may be associated with inhibition of apoptosis in myocardial I/R injury through the HDAC3/GSK-3β pathway.
MATERIALS AND METHODS
Cell culture and experimental protocol
H9c2 cardiomyocytes (ATCC, Manassas, Va) were cultured in DMEM/F12 medium (Hyclone) supplemented with 10% (v/v) fetal bovine serum (Wisent, Australia) and 1% (v/v) penicillin-streptomycin solution under 95% air and 5% CO2 at 37°C. H9c2 cells were treated with hypoxia/reoxygenation (H/R), followed by specific treatment. HR was induced as previously described (6). Briefly, the cells were rinsed twice, incubated in tyrode solution (130 mMNaCl, 5 mMKCl, 10 mM HEPES, 1 mM MgCl2, 1.8 mM CaCl2 at pH 7.4 and 37°C) and subsequently placed in a hypoxic chamber (Adelbio, Clermont-Ferrand, France) containing a mixture of 95% nitrogen and 5% CO2 for 5 h. After hypoxia, standard DMEM/F12 medium was added and the cells were incubated in normal growth condition (95% air and 5% CO2) for an additional 1 h, as reoxygenation. The specific treatments included postcondition with concentrations (1 μM) of remifentanil or SAHA (2 μM) at the onset of reoxygenation until conclusion.
Transfection of H9c2
H9C2 cells were seeded in culture 6-well or 96-well plates according to specific experiment requirements. After 24 h, cells were infected with lentiviruses (Genepharma, China) including lentiviral HDAC3 (lv-HDAC3) or lentiviral GFP (lv-GFP) at multiplicity of infection (MOI) between 10 and 100 (optimized by protein expression level of the transgene) and incubated with the culture medium containing 2% serum. Cells were incubated with the lentiviruses up to 72 h to allow for maximal transduction, as evaluated under a fluorescence microscope. Ultimately MOI of 10 and 48 h incubation were selected as optimal transfection conditions resulting in more than 80% efficiency.
Cell viability assay
Cell viability was assessed by the CCK-8 Counting Kit (Vazyme). Cells were seeded in 96-well plates at 10,000 per well. After the aforementioned treatments, cells were incubated with 0.5 mg/mL CCK8 for 4 h. Cell viability was determined by measuring absorbance at 570 nm using a microplate reader (Tecan Infinite M1000, Austria).
Cell apoptosis assay
Cell apoptosis was measured by flow cytometry with Annexin V-FITC/ Propidium iodide (PI) kit (Bestbio, China). Cells were trypsinized and then washed twice with cold phosphate-buffered saline (PBS). Cells were resuspended in binding buffer, incubated with 10 μLAnnexin V-FITC for 15 min and subsequently with 5 μL PI for 5 min in the dark at 4°C. Samples were analyzed using the FACScan flow cytometry (BD Biosciences) and the data were analyzed using FCS software.
Quantitative real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, Calif) and then reverse-transcribed with Hiscript II 1st Strand cDNA Synthesis Kit (Vazyme) to cDNA. The synthesized cDNA was prepared with SYBR green qPCR Master Mix (TaKaRa) and then run in triplicate in optically clear 96-well plates (Corning, New York, NY) on an ABI Prism 7500 sequence detector (ABI Biosystems). The cycling parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for1 min, and 72°C for 30 s. The primers used to target HDAC3 were 5′-GCCTCTGGCTTCTGCTATGT-3′ (forward) and 5′-GCTGATGACTGGCTGGAAA-3′ (reverse). GAPDH was used as a control. The fold change in expression of associated gene was calculated using the 2-△△Ct method.
Samples were harvested with RIPA buffer (Vazyme) and processed with a lysis buffer containing phenylmethanesulfonyl fluoride (PMSF; Amresco) at 4°C. BCA protein assay kit (Beyotime Institute of Biotechnology, China) was used to measure protein concentration according to the manufacturer's protocol. Proteins were separated by 12% SDS-PAGE gels, then transferred to PVDF membranes (Bio-Rad, Calif) and blocked in 5% nonfat milk for 2 h. Subsequently the membranes were botted with specific primary antibodies against Ser9phospho GSK-3β (Ser9; 1:1,000, Cell Signaling), total GSK-3β (1:1,000, Cell Signaling), cleaved caspase-3 (1:1,000, Cell Signaling), HDAC3 (1:1,000, Cell Signaling), and GAPDH (1:1,000, Abcam) overnight at 4°C. The next day, membranes were washed 3 times with PBS-T for 10 min followed by incubation with the corresponding HRP-conjugated secondary antibodies (1:8,000, Abcam) for 1 h at 37°C. Membranes were then washed with PBS-T 3 times. The bands were visualized with enhanced ECL reagent (Thermo Scientific) and analyzed using Quantity One software. GAPDH was used as a control.
Statistical analysis was performed using SPSS14.0. Data were expressed as the mean ± SD. One-way ANOVA with the Student–Newman–Keuls posttest was used for comparison between groups. A value of P < 0.05 was considered statistically significant.
RPC and inhibition of HDAC-protected H9c2 cardiomyoblasts from HR injury
HR and postconditioning model using H9c2 cardiomyoblasts was established as described in a previous work (6). CCK-8 and Annexin V-FITC/PI flow cytometry assays were performed to evaluate the potential cardioprotective capacity of RPC and SAHA (a widely used HDAC inhibitor) for H9c2 cardiomyoblasts subjected to HR. A significant reduction in cell viability and an increase in cell apoptosis relative to controls were observed after HR (P < 0.05). However, remifentanil at concentrations of 1 μM increased cell viability and decreased the rate of cell apoptosis (P < 0.05). SAHA, a widely used HDAC inhibitor and used at the onset of oxygenation at concentrations of 2 μM, exerted similar protective effects (P < 0.05) (Fig. 1, A–C). These data indicated RPC-protected H9c2 cardiomyoblasts from HR injury by increasing cell viability and attenuating cell apoptosis. In addition, inhibition of HDAC also contributed to protective effects on H9c2 cardiomyoblasts against HR injury.
RPC and inhibition of HDAC restores Ser9 phosphorylation of GSK-3β and suppresses HDAC3 expression to alleviate HR-induced apoptosis in H9c2 cardiomyoblasts
To confirm the involvement of GSK-3β and HDAC3 in HR-induced apoptosis, and the role of RPC-induced cardioprotection, we analyzed the phosphorylation status of Ser9 GSK-3β, and expression levels of HDAC3 and the apoptotic biomarker cleaved caspase-3 in H9c2 cardiomyoblasts subjected to HR. Significantly higher expression of cleaved caspase-3 protein, HDAC3 mRNA and protein (P < 0.05) (Fig. 2, A–D), and reduced phosphorylation of GSK-3β (P < 0.05) (Fig. 2, E and F) were observed after HR as compared with the control group (P < 0.05) (Fig. 2, A–D). These observations were reversed when H9c2 cells were treated with remifentanil (1 μM) or SAHA (2 μM) at the onset of oxygenation in response to HR (P < 0.05). These results suggest that RPC and inhibition of HDAC mitigate HR-induced apoptosis via restoring Ser9 phosphorylation of GSK-3β, as well as suppressing HDAC3 expression at both the mRNA and protein level.
Overexpression of HDAC3 has no significant effects on HR injury in H9c2 cardiomyoblasts
To determine the effect of overexpression of HDAC3 on HR injury in H9c2 cardiomyoblasts, HDAC3 coated in lentiviral vector was transfected into H9c2 cells to induce the overexpression of HDAC3, and the transfection efficiency (>80%) was monitored with GFP-labeled control. In cultured H9c2 cells, HR led to decreased cells viability and elevated apoptotic rate as detected by CCK-8 and flow cytometry, respectively (P < 0.05) (Fig. 3, A–C). In addition, overexpression of HDAC3 had no effects on cell viability or apoptotic rate as compared with the HR group (P > 0.05) (Fig. 3, A–C). We next examined the phosphorylation status of Ser9 GSK-3β, HDAC3, and the apoptotic biomarker cleaved caspase-3 after HDAC3 lentiviral vector transfection. Compared with the control group, cleaved caspase-3 protein, HDAC3 mRNA, and protein (P < 0.05) (Fig. 3, D–F) were increased, whereas phosphorylation of GSK-3β (P < 0.05) (Fig. 3, E and F) was reduced by HR. Furthermore, cells transfected with HDAC3 before HR did not have any effects on cleaved caspase-3, HDAC3, orphosphorylation of GSK-3β (P > 0.05) (Fig. 3, D–F). These results indicated that transfection with HDAC3 alone could not reduce HR tolerance, or inherently promote apoptosis.
Overexpression of HDAC3 eliminates the protective effect of PRC against HR injury
To verify the role of HDAC3 in RPC-induced cardioprotection against HR injury, we overexpressed HDAC3 through lentiviral vector transfection in H9c2 cells. A GFP lentiviral vector was used as negative control. As shown in Figure 4, A and B, RPC increased cell viability and reduced cell apoptosis relative to the HR group (P < 0.05) (Fig. 4, A and B). Cells transfected with HDAC3 lentiviral vector completely abolished the increase in cell viability and the decrease in cell apoptosis mediated by RPC (P < 0.05) (Fig. 4, A and B). Taken together, these data demonstrated that overexpression of HDAC3 eliminated the protective capacity of RPC against HR injury.
Overexpression of HDAC3 expression attenuates the effects on phosphorylation of GSK-3β of RPC in H9c2 cardiomyoblasts
As shown in Section 3, the overexpression of HDAC3 and the transfection efficiency (>80%) were monitored with GFP-labeled control. HDAC3-transfected cells did not exhibit any additional increase in phosphorylation of GSK-3β compared with cells subjected to HR injury alone (P > 0.05) (Fig. 5). Furthermore, after RPC treatment, the expression of cleaved caspase-3 was restored (Fig. 5, A and B) (P > 0.05), whereas phosphorylation of GSK-3β (Fig. 5, E and F) (P > 0.05) was downregulated in HDAC3-transfected cells. These results indicated that attenuation of HDAC3 activity in response to RPC involves Ser9 phosphorylation of GSK-3β.
During an ischemic insult of the myocardium, cellular homeostasis is disrupted, which leads to cell apoptosis or necrosis. Remifentanil pre- or postconditioning triggers survival mediator enzymes and suppresses apoptotic effectors, resulting in restoration of homeostasis between prosurvival and proapoptotic signaling (13). In this study, we identified the involvement of HDAC3 and Ser9 phosphorylation of GSK-3β in the protective effects by RPC against HR injury. Furthermore, we found that overexpression of HDAC3 attenuated the increase of cell viability and the decrease of cell apoptosis by RPC in H9c2 cardiomyoblasts after HR. We further identified the key role of HDAC3 for phosphorylating GSK-3β by RPC treatment against HR insults.
GSK-3 is a serine/threonine kinase that was originally identified as an enzyme that phosphorylates and downregulates glycogen synthase, the rate-limiting enzyme of glycogen metabolism (16). GSK-3β has received increasing attention because of its involvement in some serious diseases, including neurological disease, cancer, and I/R injury. In the cardiovascular system, GSK-3β plays major roles in glucose metabolism, cardiomyocyte hypertrophy (17), and ischemic diseases (18, 19). Extensive evidence has implicated GSK-3β as a critical element in the cardioprotective effects by pharmacological pre-g and post-conditioning. Consistent with previous studies, the present study also indicated the decrease of Ser9 phosphorylation of GSK-3β in response to HR injury, and restoration of its phosphorylation status was involved in the cardioprotective effects of RPC.
HDACs have been implicated as promising therapeutic targets for treating cardiovascular diseases via epigenetic and nonepigenetic mechanisms (15). Inhibition of HDACs with pharmacological or gene techniques have shown significant cardioprotective effects in preclinical settings (12). Furthermore, HDACs have been proposed as signaling hubs for cellular communication in cardiac diseases (20). It was previously reported that inhibition of HDAC3 might contribute to the neuronal survival against ischemic insults elicited by ischemic preconditioning in rats (4). In addition, studies showed that the activity of HDACs contributes to the effects of opioid in central nervous system. HDAC inhibitors serve as adjuvant analgesics to morphine for the management of neuropathic pain (21). Chronic treatment with morphine significantly reduced HDAC activity in the spinal cords of mice (22). Our study also found that suppression of HDAC3 was involved in RPC-induced cardioprotection. This study shows that the mRNA and protein levels of HDAC3 were decreased by RPC treatment, whereas restoration of HDAC3 reversed these effects. These findings identified a key role of HDAC3 in the RPC-induced cardioprotection against I/R injury.
HDACs are a class of epigenetic enzymes that control signal transduction and gene expression in all cell types (23–25). HDACs deacetylase histones and repress gene transcription and cell survival, which is impacted after several hours and days on time scales necessary for protein expression (12). The acetylation state of a given metabolic signaling factor may mediate its phosphorylation, methylation, and ubiquitination state, thereby determining its subcellular location, activation, or degradation, with immediate implications for cell survival in the seconds to minutes after the stress (23). Wang et al. (26) showed that the inhibition of HDAC6 mitigated hemisphere I/R injury via activating GSK-3β. Furthermore, Yan et al. (15) found that HDAC8 regulated cardiac hypertrophy via the Akt/GSK-3β pathway. The HDAC inhibitor, valproic acid, was proposed to increase the phosphorylation of GSK-3β in colorectal cancer cells (27). Our study showed that HDAC3 expression was significantly increased after HR, whereas RPC treatment significantly downregulated its expression. In addition, we further showed that overexpression of HDAC3 decreased GSK-3β Ser9 phosphorylation, suggesting that HDAC3 likely exerted its effects by phosphorylating GSK-3β.
Admittedly, this study has some limitations. First, this study was conducted in the H9c2 cell line; therefore, these results cannot be directly extrapolated to humans. In addition, we only focused on HR-induced apoptosis. However, necrosis is also an important mechanism underlying I/R injury, and was not addressed in this study. Our future studies will examine the effect of RPC on cell necrosis.
In summary, our results suggest a potential mechanism of the cardioprotective effect in which RPC attenuates apoptosis in H9c2 cardiomyoblasts after HR insults by downregulation of HDAC3 targeting GSK-3β. The key role of HDAC3 and its crosswalk with GSK-3β are emphasized in the cardioprotective effects of RPC against I/R injury, although the exact mechanisms require further study. Nonetheless, this study provides new insights into the mechanisms underlying the effectiveness of RPC during I/R injury.
1. Xie M, Kong Y, Tan W, May H, Battiprolu PK, Pedrozo Z, Wang ZV, Morales C, Luo X, Cho G, et al. Histone deacetylase inhibition blunts ischemia/reperfusion injury by inducing cardiomyocyte autophagy. Circulation
129: 1139–1151, 2014.
2. Xia Z, Li H, Irwin MG. Myocardial ischaemia reperfusion injury: the challenge of translating ischaemic and anaesthetic protection from animal models to humans. Br J Anaesth
117 (Suppl. 2):ii44–ii62, 2016.
3. Tanaka K, Kersten JR, Riess ML. Opioid-induced cardioprotection
. Curr Pharm Des
20: 5696–5705, 2014.
4. Chen L, Chen M, Du J, Wan L, Zhang L, Gu E. Hyperglycemia attenuates remifentanil postconditioning
against hypoxia/reoxygenation injury in H9c2 cardiomyoblasts. J Surg Res
203: 483–490, 2016.
5. Zuo Y, Cheng X, Gu E, Liu X, Zhang L, Cao Y. Effect of aortic root infusion of sufentanil on ischemia-reperfusion injury in patients undergoing mitral valve replacement. J Cardiothorac Vasc Anesth
28: 1474–1478, 2014.
6. Chen M, Liu Q, Chen L, Zhang L, Gu E. Remifentanil postconditioning
ameliorates histone H3 acetylation modification in H9c2 cardiomyoblasts after hypoxia/reoxygenation via attenuating endoplasmic reticulum stress. Apoptosis
22: 662–671, 2017.
7. Irwin MG, Wong GT. Remifentanil and opioid-induced cardioprotection
. J Cardiothorac Vasc Anesth
29 (Suppl. 1):S23–S26, 2015.
8. Juhaszova M, Zorov DB, Yaniv Y, Nuss HB, Wang S, Sollott SJ. Role of glycogen synthase kinase-3beta in cardioprotection
. Circ Res
104 11: 1240–1252, 2009.
9. Zuo Y, Wang Y, Hu H, Cui W. Atorvastatin protects myocardium against ischemia-reperfusion injury through inhibiting miR-199a-5p. Cell Physiol Biochem
39 3: 1021–1030, 2016.
10. Zhang Y, Zhang L, Gu E, Zhu B, Zhao X, Chen J. Long-term insulin treatment restores cardioprotection
induced by sufentanil postconditioning in diabetic rat heart. Exp Biol Med (Maywood)
241 6: 650–657, 2016.
11. Chen QL, Gu EW, Zhang L, Cao YY, Zhu Y, Fang WP. Diabetes mellitus abrogates the cardioprotection
of sufentanil against ischaemia/reperfusion injury by altering glycogen synthase kinase-3beta. Acta Anaesthesiol Scand
57 2: 236–242, 2013.
12. Aune SE, Herr DJ, Kutz CJ, Menick DR. Histone deacetylases exert class-specific roles in conditioning the brain and heart against acute ischemic injury. Front Neurol
13. Kee HJ, Kook H. Roles and targets of class I and IIa histone deacetylases in cardiac hypertrophy. J Biomed Biotechnol
14. Yang X, Wu Q, Zhang L, Feng L. Inhibition of histone deacetylase 3 (HDAC3
) mediates ischemic preconditioning and protects cortical neurons against ischemia in rats. Front Mol Neurosci
15. Yan M, Chen C, Gong W, Yin Z, Zhou L, Chaugai S, Wang DW. miR-21-3p regulates cardiac hypertrophic response by targeting histone deacetylase-8. Cardiovasc Res
105 3: 340–352, 2015.
16. Rylatt DB, Aitken A, Bilham T, Condon GD, Embi N, Cohen P. Glycogen synthase from rabbit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase. Eur J Biochem
107 2: 529–537, 1980.
17. Sugden PH, Fuller SJ, Weiss SC, Clerk A. Glycogen synthase kinase 3 (GSK3) in the heart: a point of integration in hypertrophic signalling and a therapeutic target? A critical analysis. Br J Pharmacol
153 (Suppl. 1):S137–S153, 2008.
18. Ma LL, Kong FJ, Guo JJ, Zhu JB, Shi HT, Li Y, Sun RH, Ge JB. Hypercholesterolemia abrogates remote ischemic preconditioning-induced cardioprotection
: role of reperfusion injury salvage kinase signals. Shock
47 3: 363–369, 2017.
19. Li J, Ruffenach G, Kararigas G, Cunningham CM, Motayagheni N, Barakai N, Umar S, Regitz-Zagrosek V, Eghbali M. Intralipid protects the heart in late pregnancy against ischemia/reperfusion injury via Caveolin2/STAT3/GSK-3beta pathway. J Mol Cell Cardiol
102: 108–116, 2017.
20. Yang XJ, Seto E. HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention. Oncogene
26 37: 5310–5318, 2007.
21. Uchida H, Matsushita Y, Araki K, Mukae T, Ueda H. Histone deacetylase inhibitors relieve morphine resistance in neuropathic pain after peripheral nerve injury. J Pharmacol Sci
128 4: 208–211, 2015.
22. Liang DY, Li X, Clark JD. Epigenetic regulation of opioid-induced hyperalgesia, dependence, and tolerance in mice. J Pain
14 1: 36–47, 2013.
23. Wu SY, Tang SE, Ko FC, Wu GC, Huang KL, Chu SJ. Valproic acid attenuates acute lung injury induced by ischemia-reperfusion in rats. Anesthesiology
122 6: 1327–1337, 2015.
24. Fan J, Alsarraf O, Chou CJ, Yates PW, Goodwin NC, Rice DS, Crosson CE. Ischemic preconditioning, retinal neuroprotection and histone deacetylase activities. Exp Eye Res
146: 269–275, 2016.
25. Shi W, Wei X, Wang Z, Han H, Fu Y, Liu J, Zhang Y, Guo J, Dong C, Zhou D, et al. HDAC9 exacerbates endothelial injury in cerebral ischaemia/reperfusion injury. J Cell Mol Med
20 6: 1139–1149, 2016.
26. Wang Z, Leng Y, Wang J, Liao HM, Bergman J, Leeds P, Kozikowski A, Chuang DM, Tubastatin A. an HDAC6 inhibitor, alleviates stroke-induced brain infarction and functional deficits: potential roles of alpha-tubulin acetylation and FGF-21 up-regulation. Sci Rep
27. Feng J, Cen J, Li J, Zhao R, Zhu C, Wang Z, Xie J, Tang W. Histone deacetylase inhibitor valproic acid (VPA) promotes the epithelial mesenchymal transition of colorectal cancer cells via up regulation of Snail. Cell Adh Migr
9 6: 495–501, 2015.