Author’s Accepted Manuscript PI3K/SGK1/GSK3β signaling pathway Is involved in inhibition of autophagy in neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation by Hydrogen sulfide Huan Jiang, Jian Xiao, Bo Kang, Xiaoyan Zhu, Ni Xin, Zhinong Wang www.elsevier.com/locate/yexcr
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S0014-4827(15)30035-5 http://dx.doi.org/10.1016/j.yexcr.2015.07.005 YEXCR9993
To appear in: Experimental Cell Research Received date: 27 January 2015 Revised date: 19 June 2015 Accepted date: 6 July 2015 Cite this article as: Huan Jiang, Jian Xiao, Bo Kang, Xiaoyan Zhu, Ni Xin and Zhinong Wang, PI3K/SGK1/GSK3β signaling pathway Is involved in inhibition of autophagy in neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation by Hydrogen sulfide, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2015.07.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PI3K/SGK1/GSK3β signaling pathway is involved in inhibition of autophagy in neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation by hydrogen sulfide ˆ ˆ ˆ Huan Jiang a , Jian Xiaoa , Bo Kanga , Xiaoyan Zhub, Xin Nib, Zhinong Wanga
Huan Jiangˆ, Jian Xiaoˆand Bo Kangˆcontribute equally to the work. a
Department of Cardiothoracic Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China b Department of Physiology, Second Military Medical University, Shanghai, China Corresponding authors: Dr. Zhinong Wang, Department of Cardiothoracic Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Fengyang Road #415, Shanghai 200003, China E-mail: [email protected] (Z. Wang) Dr. Xin Ni, Department of Physiology, Second Military Medical University, Xiangyin Road #800, Shanghai 200433, China E-mail:[email protected] (X. Ni)
Abstract Excessive autophagy aggravates myocardial ischemia/reperfusion (IR) injury. Hydrogen sulfide (H2S) has been shown to possess a strong cardioprotective effect due to its anti-necrosis, anti-apoptosis, anti-oxidant and anti-inflammatory properties. Our previous study showed that H2S could also protect the myocardium against IR injury through its anti-autophagy effect in vivo, but the underlying mechanism remains unclear. The aim of the present study was to determine whether PI3K/SGK1/GSK3β signaling pathway was involved in the anti-autophagy effect of H2S against myocardial hypoxia/reoxygenation (HR) injury in vitro. Autophagy was significantly increased in cardiomyocytes subjected to HR, but it was down-regulated by H2S (NaHS donor). Blocking PI3K by LY294002 (a PI3K inhibitor) or knocking down SGK1 by SGK1 siRNA augmented autophagy and attenuated the anti-autophagy effect of H2S. However, blocking GSK3β by tws119 (a GSK3β inhibitor) produced an opposite effect. In addition, while treatment of neonatal rat cardiomyocytes with HR reduced cell viability and augmented cell injury, H2S significantly reversed it. Blocking PI3K or knocking down SGK1 aggravated HR injury and weakened the protective effect of H2S, while blocking GSK3β 1
produced an opposite effect. In conclusion, H2S can inhibit autophagy in neonatal rat cardiomyocytes exposed to H/R and exert a cardioprotective effect at least partly by regulating PI3K/SGK1/ GSK3β signaling pathway. Keywords: H2S; autophagy; cardiomyocyte; hypoxia/reoxygenation; PI3K; SGK1; GSK3β Introduction Ischemic heart disease remains a major cause of morbidity and mortality worldwide, and the main therapeutic strategy is reperfusion, although it is known to further increase the infarct size[1]. The pathophysiologic change underlying IR injury involves necrosis and apoptosis. Autophagy is significantly increased in the myocardium after IR and plays an important role in the IR injury[2]. Autophagy is an evolutionarily conserved lysosomal degradative pathway to remove damaged intracellular constituents for the sake of facilitating cellular homeostasis and promoting cell survival under stressed conditions such as nutrient deprivation and hypoxia[3]. Induction of autophagy is protective in the ischemic heart[4-6], but it may also cause the death of cardiomyocytes in myocardial reperfusion injury through excessive self-digestion and degradation of essential cellular constituents[6-8]. Hydrogen sulfide (H2S) is an endogenously generated gaseous signaling molecule produced in the myocardium, fibroblasts and blood vessels from L-cysteine by enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the cardiovascular system. It exerts a cardioprotective effect by neutralizing reactive oxygen species (ROS), inhibiting leukocyte-endothelial cell interactions, promoting vascular smooth muscle relaxation, reducing apoptotic cell death, and modulating mitochondrial respiration[9-11]. However, little is known about its reaction on autophagy. It was found in our previous in vivo study that H2S could significantly down-regulate autophagy in the myocardium subjected to IR and exert a strong cardioprotective effect. The present study was designed to explore the signaling pathway of the anti-autophagy effect of H2S using neonatal rat cardiomyocytes in vitro. It is known that PI3K (phosphatidylinositol 3-kinase) and its downstream effector AKT (protein kinase B, PKB) are critical for cardiomyocyte survival by regulating mammalian apoptosis and autophagy [11-15]. However, Nagoshi et al demonstrated that PI3K-dependent and AKT-independent effectors were required for full cardioprotection after chronic IR injury [16], suggesting that there may be other downstream effectors of PI3K involved in the cardioprotection. Consistently, the catalytic domain of SGK1 (Serum- and glucocorticoid-responsive kinase-1) is 54% homologous to the catalytic domain of AKT and shares a variety of downstream substrates with AKT [16, 17]. In addition, SGK1 could be activated by PI3K[17], and has 2
been shown as a key factor for cardiomyocyte survival on hypoxic insult[18]. GSK3β was an important downstream target of SGK1 and AKT[18, 19]. And in the heart, it was also found to protect the myocardium against IR injury by modulating mTOR and autophagy[19, 20]. So we speculated that PI3K/SGK1/GSK3β signaling pathway may also contribute to the cardioprotective effect. The mechanism of the protective effect of H2S is focused on PI3K/AKT pathway at present [21-23]. In the present study we wanted to determine whether H2S could regulate autophagy in cardiomyocytes exposed to HR and exert cardioprotection through PI3K/SGK1 /GSK3β signaling pathway. Materials and Methods Isolation of neonatal cardiomyocytes and cell culture All the experimental protocols were approved by the Ethical Committee of Experimental Animals of the Second Military Medical University (Shanghai, China), and the work was in accordance with European Directive 2010/63/EU. Cardiomyocytes culture was prepared as described previously [24]. In brief, the heart dissociated from neonatal Sprague-Dawley (SD) rats younger than 3 days (SLAC Laboratory Animal Co., Shanghai, China) was minced in dissociation buffer (in mmol/L: 20 hydroxyethyl Piperazine ethanesulfonic acid (HEPES),116 NaCl, 0.8 Na2HPO4, 5.6glucose, 5.4 KCl, 0.8 MgSO4; pH 7.35) and then serially digested in dissociation buffer containing 0.1% trypsin (Invitrogen, Grand Island, NY) and 0.05% collagenase type II (Invitrogen) at 37ć. Cells were harvested and placed in culture dishes with Dulbecco modified Eagle medium(DMEM) containing 20% fetal calf serum (FBS) at 37ć for 1 h to allow selective attachment of nonmyocytes such as cardiac fibroblasts. The cardiomyocyte-enriched fraction was then collected and seeded in 6-well culture plates (Corning, Cambridge, MA) at a density of 5×105cells per cm2 and maintained in DMEM containing 15mmol/L HEPES, 20%FBS, 0.1 mmol/L bromodeoxyuridine and antibiotics (100U/mL penicillin and 100 mg/mL streptomycin) for 48 h in a 5% CO2-95% air humidified culture incubator. Cells were then changed to serum-free DMEM and immediatedly transfected with SGK1 siRNA synthesized by Jima Inc (Shanghai, China) using siPORT NeoFX Transfection Agent (Invitrogen) according to the manufacturer’s instruction. After 24 h, the cell culture medium was changed again with serum-free DMEM and LY294002 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-201426), or TWS119 (Santa Cruz Bio-technology, Santa Cruz, CA, sc-221694) was added to it 0.5 h before HR. Establishment of the cardiomyocyte hypoxia/reoxygenation (HR) model in vitro and experimental protocol To simulate myocardial tissue IR in vivo, isolated cardiomyocytes were placed into a 3
thermo chamber purged with 94% N2, 5%CO2 and 1%O2 for 24 h, followed by reoxygenation purged with 5%CO2 and 95% air for 6 h. Assessment of cell viability Cardiomyocytes were seeded in 96-well plates at a density of 1×105 cells/ml in 100μl medium. After HR, cell viability was evaluated by MTT assay as previously described [25]
.
Determination of lactate dehydrogenase activity The conditioned cell culture supernatant was collected after HR to determine lactate dehydrogenase (LDH) levels using a spectrophotometric kit (Roche Diagnostics, Indianapolis, IN). RNA interference The sequence-specific small interfering RNA (siRNA) targeting SGK1(GenBank accession No. NM019232) was designed as previously described [26] and was synthesized by Gene Pharma Corporation (Shanghai, China). The sequences of the siRNA oligo were as follows: SGK1 siRNA-sense: 5’-AGGAGAACAUCGAGCACA ATT-3’; SGK1 siRNA-antisense: 5’-UUGUGCU CGAUGUUCUCCUTG-3’;negative control siRNA- sense: 5’-UUCUCCGAACGUGUCACGUT T-3’;negative control siRNA-antisense: 5’-ACGUGACACGUUCGG AGAATT-3’. Transfection of siRNA was performed using siPORT NeoFx transfection agent (Invitrogen) according to the manufacturer’s instruction. Total RNA extraction and quantitative RT-PCR Total RNA from cardiomyocytes was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instruction, and then 2μg RNA was reverse transcribed to generate cDNA using superscript reverse transcriptase (Invitrogen). Quantitative RT-PCR was carried out using a 7300 Real Time PCR System (Applied Biosystems, New York, USA). The annealing temperature was set at 60ć and amplification was set at 40 cycles. The temperature range to detect the melting temperature of the PCR product was set from 60ć to 95ć, and the specificity of amplifications was confirmed by melting curve analysis. To determine the relative quantitation of gene expression for target and housekeeping genes, the comparative threshold cycle method with arithmetic formulae was used. The primer sequences were as follows: Atg5 (Gen Bank accession no. NM001014250) forward: 5'-AGTGGAGGCAACAGAACC-3';Atg5 reverse: 5'-GACACGAACTGGCACAT T-3'; Beclin1 (Gen Bank accession no.NM001034117) forward: 5'-GGCAGTGG CTCCTTT-3'; Beclin1 reverse: 5'-GGCGTGCTGTGCTCTGAAAA-3'; Atg9a (Gen 4
Bank accession no. NM001014218 ) forward: 5’-AGCGTGAGCTGACAGAG TT-3'; Atg9a reverse: 5’-AGC GTGAGCTGACAGAGTT-3'; β-actin (Gen Bank accession No. NM031144) forward: 5’-ATGGTGGGTATGGTCAGAAGG-3’; β-actin reverse: 5’- TGGCTGGGGTGTTGAAGGGTC-3’. Western blot analysis Cells were homogenized in ice-cold lysis buffer and scraped off the plate. Lysis buffer contains 50mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1% protein inhibitor cocktail. Protein concentrations were quantitated with the BCA protein assay kit (Thermo Scientific). Aliquots of proteins were separated using 10% or 12% SDS polyacrylamide gel and subsequently transferred to nitrocellulose membranes. After blockage in 5% nonfat milk in 0.1% Tris-buffered saline/Tween 20, membranes were immunostained using primary antibodies at 4 ć overnight. The following primary antibodies were used: PI3K(Santa Cruz Biotechnology, Santa Cruz, CA, sc-1637) (1:1000),p-PI3K (Tyr508, Santa Cruz Biotechnology,sc-12929) (1:1000),SGK1 (Santa Cruz Biotechnology, sc-33774) (1:1000), p-SGK (Thr508,Santa Cruz Biotechnology, sc-16744) (1:1000), GSK3β (Santa Cruz Biotechnology, sc-9166) (1:1000), p-GSK3β (Ser9,Santa Cruz Biotechnology, sc-11757)(1:1000),Beclin1(Santa Cruz Biotechnology, sc-11427) (1:1000), GAPDH(Santa Cruz Biotechnology, sc-32233) (1:2000), LC3 (SigmaAldrich, L8919) (1:1000). Then the membranes were incubated with a secondary horseradish peroxidase-conjugated antibody for 1.5 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blot detection system (Tanon, Shanghai, China). The ratio of band intensities to GAPDH was used to quantitate the relative protein levels. Statistical analysis All data were presented as mean±SD and analyzed using one-way classification ANOVA followed by the SNK-q test. P < 0.05 was considered statistically significant. Results PI3K/SGK1/GSK3β signaling pathway is involved in H2S-induced regulation of autophagy in cardiomyocytes during HR Blocking the PI3K/ SGK1/GSK3β activity disproportionately affected the effect of NaHS (30μM) on the mRNA expression of autophagy-related gene (ATG5, Beclin1, Atg9) in myocardial cells subjected to HR. When we used the PI3K inhibitor LY294002 (1μM), SGK1 siRNA and GSK3β blocker TWS119 (5μM) to inhibit the activity of PI3K, SGK1 and GSK3β and determined the mRNA expression of Atg5, Beclin1 and Atg9 in all groups by RT-PCR, we found that the mRNA 5
expression of Atg5, Beclin1 and Atg9 were significantly increased in HR group, and was down-regulated by exogenous H2S. Blocking the activity of PI3K and SGK1 further raised the mRNA expression and weakened the effect of H2S, while blocking the activity of GSK3β produced an opposite effect (Figure 1). Consistently, the same effect was observed at the protein level. As shown in figure 2, blocking the activity of PI3K and SGK1 further increased the protein expression of Beclin1 and LC3II/I, and significantly weakened the effect of H2S, which significantly down-regulated Beclin1 and LC3II/I in the cardiomyocytes after HR. However, the result was opposite when the activity of GSK3β was blocked. These results suggest that PI3K and SGK1 may inhibit autophagy and GSK3β may boost autophagy, and both may be involved in the anti-autophagy effect of H2S on the cardiomyocytes during HR. H2S regulates the expression of PI3K/SGK1/GSK3β in cardiomyocytes exposed to HR. Firstly, PI3K and its phosphorylation level were assessed in Con, HR, HR+H2S groups by Western blot. It was found that the expression of both t-PI3K and p-PI3K was significantly down-regulated after HR, which could be reversed by H2S (Figure 3A). Interestingly, SGK1 and p-SGK1 altered in the same way in different groups and they could be positively regulated by PI3K as they were both reductive in the presence of PI3K inhibitor LY294002 (Figure 3B). Although p-GSK3β could also be regulated by H2S, PI3K and SGK1, there was no significant difference in the expression of t-GSK3β between the groups (Figure 3C), suggesting that H2S could regulate PI3K and SGK1 in two ways and could only regulate GSK3β by phosphorylation. In addition, p-GSK3β was also regulated by both PI3K and SGK1 negatively as it was reductive in the presence of LY294002 and SGK1 siRNA (Figure 3C and 3D). The above results indicate that H2S could regulate PI3K/SGK1/GSK3β signaling pathway in neonatal rat cardiomyocytes exposed to HR. H2S protects cardiomyocytes against HR through PI3K/SGK1/GSK3β pathway. In order to further verify the injury of excessive autophagy in cardiomyocytes after HR and explicit the mechanism underlying the myocardial protective effect of H2S, cell viability was detected in each group by using MTT assay and LDH release assay. It was found that blocking PI3K and SGK1 reduced cell viability and increased LDH release as compared with HR group, and largely abolished the protective effect of H2S compared with HR+H2S group. However, an opposite result was observed when GSK3β was blocked (Figure 4).These results suggest that the protective effect of H2S on cardiomyocytes during HR may be at least partly attributed to the inhibition of excessive autophagy through PI3K/SGK1/GSK3β 6
signaling pathway. Discussion Ischemia reperfusion injury has always been an important concern in the cardiovascular field. As a gaseous signal molecule, H2S has been found to possess a strong cardioprotective effect, thus arousing much interest and attention. The mechanism underlying this cardioprotective effect has also become a hotspot of research in recent years. However, the molecular mechanism underlying the effect of H2S on autophagy (which is indicated to be detrimental to the myocardium during reperfusion) has rarely been studied. It was found in the present study that H2S could inhibit autophagy as evidenced by a significant decrease in mRNA level of autophagy-related genes (Atg5, Beclin1 and Atg9) and in protein level of Beclin1 and LC3II/I which are the most widely used markers of autophagy. In addition, PI3K/SGK1/GSK3β signaling pathway played an important role in the anti-autophagy effect of H2S and cardioprotection as directly evidenced by up-and-down MTT assay and LDH release assay. Beclin1 is an autophagy-regulating macromolecular complex (PI3K complex) and plays an active role in initiating autophagosome formation, while Atg5 and Atg9 play an important role in the elongation of phagophores, which derive from pre-autophagosomal structures[15]. According to Matsui et al [6], IR stimulated autophagy through a Beclin1-dependent but AMPK- independent mechanism and autophagy may be detrimental during reperfusion. Atg5 is known not to participate in pathways other than autophagy [27] and the expression of Atg9 gene regulates the frequency of autophagosome formation[28]. Otherwise, up-regulation of Atg5, Beclin1 and Atg9 mRNA levels has been found in several cells following induction of autophagy under certain stressed conditions [29]. It was found in our neonatal cardiomyocyte model that the expression of Atg5, Beclin1 and Atg9 mRNA was regulated by H2S, PI3K, SGK1 and GSK3β, indicating that all of them were involved in the regulation of autophagy. In addition, the divergence in the protein expression levels of Beclin1 and LC3II/I further demonstrated it. PI3K plays an important role in protecting the myocardium subjected to HR [16]. Activated PI3K converts the plasma membrane lipid phosphatidylinositol -4,5-bisphos-phate (PIP2) to phosphatidylinositol-3,4,5- trisph -osphate (PIP3), which in turn recruits pleckstrin homology (PH) domain proteins, such as the serine/threonine kinases phosphoino-sitide-dependent kinase 1 (PDK1) and AKT/PKB, to the plasma membrane[30]. Increases in the class I PI3K product PIP3 inhibited autophagy by inducing Akt activation [31]. In our study, we found that H2S could upregulate PI3K and p-PI3K. Autophagy was reduced when we used LY294002 to block PI3K with reduced myocardial injury. So we assert that PI3K is involved in 7
the regulation of autophagy and myocardial protection by H2S. SGK1 is a serine-threonine kinase highly expressing in the myocardium to regulate cardiomyocyte survival and hypertrophic response[18]. SGK1 is a target of the PI3-kinase- stimulated signaling pathway[17]. Consistently, we also showed that SGK1 knockdown aggravated cell damage caused by H/R and impaired the protective effect of H2S against H/R injury, confirming the protective role of SGK1 against H/R injury and its involvement in H2S-induced cardioprotection. As mentioned previously, SGK1 is regulated through phosphorylation and gene transcription [17, 18]. In our study we showed that H2S could regulate both SGK1 and p-SGK1. The effect of SGK1 against autophagy is coincident with its cardioprotection, confirming the role of SGK1 involved in H2S-induced down-regulation against autophagy and the cardioprotection of H2S. As an important downstream target of SGK1 and AKT[18], GSK3β has emerged as the integration point of many cardiac protective pathways and plays a central role in transferring protective signals downstream[32, 33]. Yao et al [34] reported that the induction of the phosphorylation of GSK3β (Ser9) by H2S is a novel protective signaling pathway that functions in preventing H/R-induced cardiomyocyte apoptosis [34] . It was found that GSK-3β inactivation during reperfusion is mediated through regulation of mTOR and autophagy by phosphorylation of the Ser9 residue[20, 38] .Consistently, we found that H2S could down-regulate autophagy and exert cardioprotection by regulating the phosphorylation of GSK3β (Ser9). H2S exerts its cardioprotective effect via various signaling pathways
[21, 37]
. The
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Figure legends: Figure 1 PI3K, SGK1, GSK3β is involved in the regulation of autophagy in cardiomyocytes subjected to HR by H2S at transcription level. Moderate amounts of SGK1 siRNA was transfected into cardiomyocytes 24.5 h before HR. Cultured cardiomyocytes were treated with NaHS (30μM), LY294002 (1μM) and TWS119 (5μM) 0.5 h before HR. Overall mRNA expression levels were assessed by RT-RCR. (A) Relative mRNA level of Atg5 in different groups. (B) Relative mRNA level of Beclin1 in different groups. (C) Relative mRNA level of Atg9 in different groups. (D) Representative bands of SGK1 in cardiomyocytes transfected with SGK1 siRNA. Data are presented as mean±SD (n≥4 per group). :difference to Con group, #:difference to HR group,
Ʒ:difference
to HR+H2S group, P
PII: DOI: Reference:
S0014-4827(15)30035-5 http://dx.doi.org/10.1016/j.yexcr.2015.07.005 YEXCR9993
To appear in: Experimental Cell Research Received date: 27 January 2015 Revised date: 19 June 2015 Accepted date: 6 July 2015 Cite this article as: Huan Jiang, Jian Xiao, Bo Kang, Xiaoyan Zhu, Ni Xin and Zhinong Wang, PI3K/SGK1/GSK3β signaling pathway Is involved in inhibition of autophagy in neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation by Hydrogen sulfide, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2015.07.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PI3K/SGK1/GSK3β signaling pathway is involved in inhibition of autophagy in neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation by hydrogen sulfide ˆ ˆ ˆ Huan Jiang a , Jian Xiaoa , Bo Kanga , Xiaoyan Zhub, Xin Nib, Zhinong Wanga
Huan Jiangˆ, Jian Xiaoˆand Bo Kangˆcontribute equally to the work. a
Department of Cardiothoracic Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China b Department of Physiology, Second Military Medical University, Shanghai, China Corresponding authors: Dr. Zhinong Wang, Department of Cardiothoracic Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Fengyang Road #415, Shanghai 200003, China E-mail: [email protected] (Z. Wang) Dr. Xin Ni, Department of Physiology, Second Military Medical University, Xiangyin Road #800, Shanghai 200433, China E-mail:[email protected] (X. Ni)
Abstract Excessive autophagy aggravates myocardial ischemia/reperfusion (IR) injury. Hydrogen sulfide (H2S) has been shown to possess a strong cardioprotective effect due to its anti-necrosis, anti-apoptosis, anti-oxidant and anti-inflammatory properties. Our previous study showed that H2S could also protect the myocardium against IR injury through its anti-autophagy effect in vivo, but the underlying mechanism remains unclear. The aim of the present study was to determine whether PI3K/SGK1/GSK3β signaling pathway was involved in the anti-autophagy effect of H2S against myocardial hypoxia/reoxygenation (HR) injury in vitro. Autophagy was significantly increased in cardiomyocytes subjected to HR, but it was down-regulated by H2S (NaHS donor). Blocking PI3K by LY294002 (a PI3K inhibitor) or knocking down SGK1 by SGK1 siRNA augmented autophagy and attenuated the anti-autophagy effect of H2S. However, blocking GSK3β by tws119 (a GSK3β inhibitor) produced an opposite effect. In addition, while treatment of neonatal rat cardiomyocytes with HR reduced cell viability and augmented cell injury, H2S significantly reversed it. Blocking PI3K or knocking down SGK1 aggravated HR injury and weakened the protective effect of H2S, while blocking GSK3β 1
produced an opposite effect. In conclusion, H2S can inhibit autophagy in neonatal rat cardiomyocytes exposed to H/R and exert a cardioprotective effect at least partly by regulating PI3K/SGK1/ GSK3β signaling pathway. Keywords: H2S; autophagy; cardiomyocyte; hypoxia/reoxygenation; PI3K; SGK1; GSK3β Introduction Ischemic heart disease remains a major cause of morbidity and mortality worldwide, and the main therapeutic strategy is reperfusion, although it is known to further increase the infarct size[1]. The pathophysiologic change underlying IR injury involves necrosis and apoptosis. Autophagy is significantly increased in the myocardium after IR and plays an important role in the IR injury[2]. Autophagy is an evolutionarily conserved lysosomal degradative pathway to remove damaged intracellular constituents for the sake of facilitating cellular homeostasis and promoting cell survival under stressed conditions such as nutrient deprivation and hypoxia[3]. Induction of autophagy is protective in the ischemic heart[4-6], but it may also cause the death of cardiomyocytes in myocardial reperfusion injury through excessive self-digestion and degradation of essential cellular constituents[6-8]. Hydrogen sulfide (H2S) is an endogenously generated gaseous signaling molecule produced in the myocardium, fibroblasts and blood vessels from L-cysteine by enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the cardiovascular system. It exerts a cardioprotective effect by neutralizing reactive oxygen species (ROS), inhibiting leukocyte-endothelial cell interactions, promoting vascular smooth muscle relaxation, reducing apoptotic cell death, and modulating mitochondrial respiration[9-11]. However, little is known about its reaction on autophagy. It was found in our previous in vivo study that H2S could significantly down-regulate autophagy in the myocardium subjected to IR and exert a strong cardioprotective effect. The present study was designed to explore the signaling pathway of the anti-autophagy effect of H2S using neonatal rat cardiomyocytes in vitro. It is known that PI3K (phosphatidylinositol 3-kinase) and its downstream effector AKT (protein kinase B, PKB) are critical for cardiomyocyte survival by regulating mammalian apoptosis and autophagy [11-15]. However, Nagoshi et al demonstrated that PI3K-dependent and AKT-independent effectors were required for full cardioprotection after chronic IR injury [16], suggesting that there may be other downstream effectors of PI3K involved in the cardioprotection. Consistently, the catalytic domain of SGK1 (Serum- and glucocorticoid-responsive kinase-1) is 54% homologous to the catalytic domain of AKT and shares a variety of downstream substrates with AKT [16, 17]. In addition, SGK1 could be activated by PI3K[17], and has 2
been shown as a key factor for cardiomyocyte survival on hypoxic insult[18]. GSK3β was an important downstream target of SGK1 and AKT[18, 19]. And in the heart, it was also found to protect the myocardium against IR injury by modulating mTOR and autophagy[19, 20]. So we speculated that PI3K/SGK1/GSK3β signaling pathway may also contribute to the cardioprotective effect. The mechanism of the protective effect of H2S is focused on PI3K/AKT pathway at present [21-23]. In the present study we wanted to determine whether H2S could regulate autophagy in cardiomyocytes exposed to HR and exert cardioprotection through PI3K/SGK1 /GSK3β signaling pathway. Materials and Methods Isolation of neonatal cardiomyocytes and cell culture All the experimental protocols were approved by the Ethical Committee of Experimental Animals of the Second Military Medical University (Shanghai, China), and the work was in accordance with European Directive 2010/63/EU. Cardiomyocytes culture was prepared as described previously [24]. In brief, the heart dissociated from neonatal Sprague-Dawley (SD) rats younger than 3 days (SLAC Laboratory Animal Co., Shanghai, China) was minced in dissociation buffer (in mmol/L: 20 hydroxyethyl Piperazine ethanesulfonic acid (HEPES),116 NaCl, 0.8 Na2HPO4, 5.6glucose, 5.4 KCl, 0.8 MgSO4; pH 7.35) and then serially digested in dissociation buffer containing 0.1% trypsin (Invitrogen, Grand Island, NY) and 0.05% collagenase type II (Invitrogen) at 37ć. Cells were harvested and placed in culture dishes with Dulbecco modified Eagle medium(DMEM) containing 20% fetal calf serum (FBS) at 37ć for 1 h to allow selective attachment of nonmyocytes such as cardiac fibroblasts. The cardiomyocyte-enriched fraction was then collected and seeded in 6-well culture plates (Corning, Cambridge, MA) at a density of 5×105cells per cm2 and maintained in DMEM containing 15mmol/L HEPES, 20%FBS, 0.1 mmol/L bromodeoxyuridine and antibiotics (100U/mL penicillin and 100 mg/mL streptomycin) for 48 h in a 5% CO2-95% air humidified culture incubator. Cells were then changed to serum-free DMEM and immediatedly transfected with SGK1 siRNA synthesized by Jima Inc (Shanghai, China) using siPORT NeoFX Transfection Agent (Invitrogen) according to the manufacturer’s instruction. After 24 h, the cell culture medium was changed again with serum-free DMEM and LY294002 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-201426), or TWS119 (Santa Cruz Bio-technology, Santa Cruz, CA, sc-221694) was added to it 0.5 h before HR. Establishment of the cardiomyocyte hypoxia/reoxygenation (HR) model in vitro and experimental protocol To simulate myocardial tissue IR in vivo, isolated cardiomyocytes were placed into a 3
thermo chamber purged with 94% N2, 5%CO2 and 1%O2 for 24 h, followed by reoxygenation purged with 5%CO2 and 95% air for 6 h. Assessment of cell viability Cardiomyocytes were seeded in 96-well plates at a density of 1×105 cells/ml in 100μl medium. After HR, cell viability was evaluated by MTT assay as previously described [25]
.
Determination of lactate dehydrogenase activity The conditioned cell culture supernatant was collected after HR to determine lactate dehydrogenase (LDH) levels using a spectrophotometric kit (Roche Diagnostics, Indianapolis, IN). RNA interference The sequence-specific small interfering RNA (siRNA) targeting SGK1(GenBank accession No. NM019232) was designed as previously described [26] and was synthesized by Gene Pharma Corporation (Shanghai, China). The sequences of the siRNA oligo were as follows: SGK1 siRNA-sense: 5’-AGGAGAACAUCGAGCACA ATT-3’; SGK1 siRNA-antisense: 5’-UUGUGCU CGAUGUUCUCCUTG-3’;negative control siRNA- sense: 5’-UUCUCCGAACGUGUCACGUT T-3’;negative control siRNA-antisense: 5’-ACGUGACACGUUCGG AGAATT-3’. Transfection of siRNA was performed using siPORT NeoFx transfection agent (Invitrogen) according to the manufacturer’s instruction. Total RNA extraction and quantitative RT-PCR Total RNA from cardiomyocytes was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instruction, and then 2μg RNA was reverse transcribed to generate cDNA using superscript reverse transcriptase (Invitrogen). Quantitative RT-PCR was carried out using a 7300 Real Time PCR System (Applied Biosystems, New York, USA). The annealing temperature was set at 60ć and amplification was set at 40 cycles. The temperature range to detect the melting temperature of the PCR product was set from 60ć to 95ć, and the specificity of amplifications was confirmed by melting curve analysis. To determine the relative quantitation of gene expression for target and housekeeping genes, the comparative threshold cycle method with arithmetic formulae was used. The primer sequences were as follows: Atg5 (Gen Bank accession no. NM001014250) forward: 5'-AGTGGAGGCAACAGAACC-3';Atg5 reverse: 5'-GACACGAACTGGCACAT T-3'; Beclin1 (Gen Bank accession no.NM001034117) forward: 5'-GGCAGTGG CTCCTTT-3'; Beclin1 reverse: 5'-GGCGTGCTGTGCTCTGAAAA-3'; Atg9a (Gen 4
Bank accession no. NM001014218 ) forward: 5’-AGCGTGAGCTGACAGAG TT-3'; Atg9a reverse: 5’-AGC GTGAGCTGACAGAGTT-3'; β-actin (Gen Bank accession No. NM031144) forward: 5’-ATGGTGGGTATGGTCAGAAGG-3’; β-actin reverse: 5’- TGGCTGGGGTGTTGAAGGGTC-3’. Western blot analysis Cells were homogenized in ice-cold lysis buffer and scraped off the plate. Lysis buffer contains 50mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1% protein inhibitor cocktail. Protein concentrations were quantitated with the BCA protein assay kit (Thermo Scientific). Aliquots of proteins were separated using 10% or 12% SDS polyacrylamide gel and subsequently transferred to nitrocellulose membranes. After blockage in 5% nonfat milk in 0.1% Tris-buffered saline/Tween 20, membranes were immunostained using primary antibodies at 4 ć overnight. The following primary antibodies were used: PI3K(Santa Cruz Biotechnology, Santa Cruz, CA, sc-1637) (1:1000),p-PI3K (Tyr508, Santa Cruz Biotechnology,sc-12929) (1:1000),SGK1 (Santa Cruz Biotechnology, sc-33774) (1:1000), p-SGK (Thr508,Santa Cruz Biotechnology, sc-16744) (1:1000), GSK3β (Santa Cruz Biotechnology, sc-9166) (1:1000), p-GSK3β (Ser9,Santa Cruz Biotechnology, sc-11757)(1:1000),Beclin1(Santa Cruz Biotechnology, sc-11427) (1:1000), GAPDH(Santa Cruz Biotechnology, sc-32233) (1:2000), LC3 (SigmaAldrich, L8919) (1:1000). Then the membranes were incubated with a secondary horseradish peroxidase-conjugated antibody for 1.5 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blot detection system (Tanon, Shanghai, China). The ratio of band intensities to GAPDH was used to quantitate the relative protein levels. Statistical analysis All data were presented as mean±SD and analyzed using one-way classification ANOVA followed by the SNK-q test. P < 0.05 was considered statistically significant. Results PI3K/SGK1/GSK3β signaling pathway is involved in H2S-induced regulation of autophagy in cardiomyocytes during HR Blocking the PI3K/ SGK1/GSK3β activity disproportionately affected the effect of NaHS (30μM) on the mRNA expression of autophagy-related gene (ATG5, Beclin1, Atg9) in myocardial cells subjected to HR. When we used the PI3K inhibitor LY294002 (1μM), SGK1 siRNA and GSK3β blocker TWS119 (5μM) to inhibit the activity of PI3K, SGK1 and GSK3β and determined the mRNA expression of Atg5, Beclin1 and Atg9 in all groups by RT-PCR, we found that the mRNA 5
expression of Atg5, Beclin1 and Atg9 were significantly increased in HR group, and was down-regulated by exogenous H2S. Blocking the activity of PI3K and SGK1 further raised the mRNA expression and weakened the effect of H2S, while blocking the activity of GSK3β produced an opposite effect (Figure 1). Consistently, the same effect was observed at the protein level. As shown in figure 2, blocking the activity of PI3K and SGK1 further increased the protein expression of Beclin1 and LC3II/I, and significantly weakened the effect of H2S, which significantly down-regulated Beclin1 and LC3II/I in the cardiomyocytes after HR. However, the result was opposite when the activity of GSK3β was blocked. These results suggest that PI3K and SGK1 may inhibit autophagy and GSK3β may boost autophagy, and both may be involved in the anti-autophagy effect of H2S on the cardiomyocytes during HR. H2S regulates the expression of PI3K/SGK1/GSK3β in cardiomyocytes exposed to HR. Firstly, PI3K and its phosphorylation level were assessed in Con, HR, HR+H2S groups by Western blot. It was found that the expression of both t-PI3K and p-PI3K was significantly down-regulated after HR, which could be reversed by H2S (Figure 3A). Interestingly, SGK1 and p-SGK1 altered in the same way in different groups and they could be positively regulated by PI3K as they were both reductive in the presence of PI3K inhibitor LY294002 (Figure 3B). Although p-GSK3β could also be regulated by H2S, PI3K and SGK1, there was no significant difference in the expression of t-GSK3β between the groups (Figure 3C), suggesting that H2S could regulate PI3K and SGK1 in two ways and could only regulate GSK3β by phosphorylation. In addition, p-GSK3β was also regulated by both PI3K and SGK1 negatively as it was reductive in the presence of LY294002 and SGK1 siRNA (Figure 3C and 3D). The above results indicate that H2S could regulate PI3K/SGK1/GSK3β signaling pathway in neonatal rat cardiomyocytes exposed to HR. H2S protects cardiomyocytes against HR through PI3K/SGK1/GSK3β pathway. In order to further verify the injury of excessive autophagy in cardiomyocytes after HR and explicit the mechanism underlying the myocardial protective effect of H2S, cell viability was detected in each group by using MTT assay and LDH release assay. It was found that blocking PI3K and SGK1 reduced cell viability and increased LDH release as compared with HR group, and largely abolished the protective effect of H2S compared with HR+H2S group. However, an opposite result was observed when GSK3β was blocked (Figure 4).These results suggest that the protective effect of H2S on cardiomyocytes during HR may be at least partly attributed to the inhibition of excessive autophagy through PI3K/SGK1/GSK3β 6
signaling pathway. Discussion Ischemia reperfusion injury has always been an important concern in the cardiovascular field. As a gaseous signal molecule, H2S has been found to possess a strong cardioprotective effect, thus arousing much interest and attention. The mechanism underlying this cardioprotective effect has also become a hotspot of research in recent years. However, the molecular mechanism underlying the effect of H2S on autophagy (which is indicated to be detrimental to the myocardium during reperfusion) has rarely been studied. It was found in the present study that H2S could inhibit autophagy as evidenced by a significant decrease in mRNA level of autophagy-related genes (Atg5, Beclin1 and Atg9) and in protein level of Beclin1 and LC3II/I which are the most widely used markers of autophagy. In addition, PI3K/SGK1/GSK3β signaling pathway played an important role in the anti-autophagy effect of H2S and cardioprotection as directly evidenced by up-and-down MTT assay and LDH release assay. Beclin1 is an autophagy-regulating macromolecular complex (PI3K complex) and plays an active role in initiating autophagosome formation, while Atg5 and Atg9 play an important role in the elongation of phagophores, which derive from pre-autophagosomal structures[15]. According to Matsui et al [6], IR stimulated autophagy through a Beclin1-dependent but AMPK- independent mechanism and autophagy may be detrimental during reperfusion. Atg5 is known not to participate in pathways other than autophagy [27] and the expression of Atg9 gene regulates the frequency of autophagosome formation[28]. Otherwise, up-regulation of Atg5, Beclin1 and Atg9 mRNA levels has been found in several cells following induction of autophagy under certain stressed conditions [29]. It was found in our neonatal cardiomyocyte model that the expression of Atg5, Beclin1 and Atg9 mRNA was regulated by H2S, PI3K, SGK1 and GSK3β, indicating that all of them were involved in the regulation of autophagy. In addition, the divergence in the protein expression levels of Beclin1 and LC3II/I further demonstrated it. PI3K plays an important role in protecting the myocardium subjected to HR [16]. Activated PI3K converts the plasma membrane lipid phosphatidylinositol -4,5-bisphos-phate (PIP2) to phosphatidylinositol-3,4,5- trisph -osphate (PIP3), which in turn recruits pleckstrin homology (PH) domain proteins, such as the serine/threonine kinases phosphoino-sitide-dependent kinase 1 (PDK1) and AKT/PKB, to the plasma membrane[30]. Increases in the class I PI3K product PIP3 inhibited autophagy by inducing Akt activation [31]. In our study, we found that H2S could upregulate PI3K and p-PI3K. Autophagy was reduced when we used LY294002 to block PI3K with reduced myocardial injury. So we assert that PI3K is involved in 7
the regulation of autophagy and myocardial protection by H2S. SGK1 is a serine-threonine kinase highly expressing in the myocardium to regulate cardiomyocyte survival and hypertrophic response[18]. SGK1 is a target of the PI3-kinase- stimulated signaling pathway[17]. Consistently, we also showed that SGK1 knockdown aggravated cell damage caused by H/R and impaired the protective effect of H2S against H/R injury, confirming the protective role of SGK1 against H/R injury and its involvement in H2S-induced cardioprotection. As mentioned previously, SGK1 is regulated through phosphorylation and gene transcription [17, 18]. In our study we showed that H2S could regulate both SGK1 and p-SGK1. The effect of SGK1 against autophagy is coincident with its cardioprotection, confirming the role of SGK1 involved in H2S-induced down-regulation against autophagy and the cardioprotection of H2S. As an important downstream target of SGK1 and AKT[18], GSK3β has emerged as the integration point of many cardiac protective pathways and plays a central role in transferring protective signals downstream[32, 33]. Yao et al [34] reported that the induction of the phosphorylation of GSK3β (Ser9) by H2S is a novel protective signaling pathway that functions in preventing H/R-induced cardiomyocyte apoptosis [34] . It was found that GSK-3β inactivation during reperfusion is mediated through regulation of mTOR and autophagy by phosphorylation of the Ser9 residue[20, 38] .Consistently, we found that H2S could down-regulate autophagy and exert cardioprotection by regulating the phosphorylation of GSK3β (Ser9). H2S exerts its cardioprotective effect via various signaling pathways
[21, 37]
. The
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Figure legends: Figure 1 PI3K, SGK1, GSK3β is involved in the regulation of autophagy in cardiomyocytes subjected to HR by H2S at transcription level. Moderate amounts of SGK1 siRNA was transfected into cardiomyocytes 24.5 h before HR. Cultured cardiomyocytes were treated with NaHS (30μM), LY294002 (1μM) and TWS119 (5μM) 0.5 h before HR. Overall mRNA expression levels were assessed by RT-RCR. (A) Relative mRNA level of Atg5 in different groups. (B) Relative mRNA level of Beclin1 in different groups. (C) Relative mRNA level of Atg9 in different groups. (D) Representative bands of SGK1 in cardiomyocytes transfected with SGK1 siRNA. Data are presented as mean±SD (n≥4 per group). :difference to Con group, #:difference to HR group,
Ʒ:difference
to HR+H2S group, P
