BBA - Molecular Basis of Disease · Henseleit buﬀer containing PA with indicated agents for 30 min. At the end of perfusion program, the hearts were harvested for Western blot test

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BBA - Molecular Basis of Disease

journal homepage: www.elsevier.com/locate/bbadis

Succinate accumulation impairs cardiac pyruvate dehydrogenase activitythrough GRP91-dependent and independent signaling pathways:Therapeutic effects of ginsenoside Rb1

Jia Lia, Yi-Lin Yanga, Lan-Zhu Lia, Lei Zhanga, Qun Liua, Kang Liub, Ping Lia, Baolin Liub,Lian-Wen Qia,⁎

a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Chinab Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University,Nanjing, China

A R T I C L E I N F O

Keywords:CardiomyocytesGinsenoside Rb1GPR91Pyruvate dehydrogenaseSuccinate

A B S T R A C T

Altered mitochondrial oxidation increases vulnerability to cardiac ischemia/reperfusion (I/R) injury in meta-bolic disorders. However, the metabolic signaling responsible for the dysfunction remains partly unknown. Wesought to test whether or not hypoxic succinate accumulation could inhibit pyruvate dehydrogenase (PDH)activity and subsequently aggravate I/R injury. Results showed that saturated fatty acid palmitate stimulationincreased fatty acid oxidation and induced hypoxia in cardiomyocytes, leading to succinate accumulation.Intracellular succinate induced hypoxia inducible factor-1α (HIF-1α) expression and impaired PDH activity viaupregulation of pyruvate dehydrogenase kinase 4 (PDK4) expression. Luciferase reporter assay showed thatsuccinate increased PDK4 expression through gene promoter induction in a HIF-1α-dependent manner.Palmitate also induced the release of succinate into extracellular space. By activating GRP91, extracellularsuccinate induced the translocation of PKCδ to mitochondria and further exacerbated PDH impairment. Theseresults demonstrated that succinate impaired PDH activity via GPR91-dependent and independent pathways.Ginsenoside Rb1 (a major compound isolated from ginseng) and trimetazidine (fatty acid β-oxidation inhibitor)prevented hypoxic succinate accumulation in cardiomyocytes and improved PDH activity by blocking succinate-associated HIF-1α activation and GPR91 signaling. Through improving PDH activity, Rb1 and trimetazidineprevented cardiac acidification, ameliorated mitochondrial dysfunction and thereby reduced apoptosis duringhypoxia/reoxygenation insult. In isolated working rat hearts perfused with palmitate and in high-fat diet-fedmice, early intervention of Rb1 and trimetazidine reduced succinate production and resultantly increased heartresistance to ischemia/reperfusion injury. Taken together, our findings demonstrated that early intervention bytargeting inhibition of succinate accumulation-induced PDH impairment is an effective strategy to alleviate I/Rinjury.

1. Introduction

Metabolic disorders are tightly associated with the risk of cardio-vascular diseases. Alteration in cardiac energy metabolism is re-cognized as an initial pathological factor, setting the heart at a stagesusceptible to ischemic injury [1,2]. In obesity and diabetes, elevatedlevels of circulating free fatty acids increase fatty acid availability in theheart, where increased fatty acid oxidation can decrease glucose oxi-dation by impairing pyruvate dehydrogenase (PDH) activity, leading toa reduction in energy efficiency [3,4]. The alternation of fatty acid and

glucose metabolism in the heart is considered the major reason for theincreased vulnerability to cardiac ischemic injury [5].

Ischemic damage in the heart is an insult from the depletion ofoxygen supply. Low oxygen tension or hypoxia induces hypoxia in-ducible factor 1α (HIF-1α) accumulation which acts as a transcriptionfactor to reprogram metabolism during anoxia [6,7]. HIF-1α activationis proposed to confer protection against ischemic injury in the heart[8,9]. In cancer cells, stabilized HIF-1α promotes anaerobic glycolysisand inhibits glucose carbon flow into the mitochondria by down-regulation of PDH activity, a process referred as the Warburg effect

http://dx.doi.org/10.1016/j.bbadis.2017.07.017Received 28 April 2017; Received in revised form 4 July 2017; Accepted 19 July 2017

⁎ Corresponding author.E-mail address: [email protected] (L.-W. Qi).

Abbreviations: CPT1, carnitine palmitoyl transterase 1; I/R, ischemia/reperfusion; HIF-1α, hypoxia inducible factor-1α; HFD, high-fat diet; H/R, hypoxia/reoxygenation; NRVMs,neonatal rat ventricular myocytes; PA, palmitate; PDH, pyruvate dehydrogenase; PDK4, pyruvate dehydrogenase kinase 4; SDH, succinate dehydrogenase

BBA - Molecular Basis of Disease 1863 (2017) 2835–2847

Available online 21 July 20170925-4439/ © 2017 Elsevier B.V. All rights reserved.

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contributing to tumorigenesis [10]. The presumed alternation in car-diac metabolism might raise the question of whether or not HIF-1αinduction prevents pyruvate entry into mitochondrial oxidation due tothe uncoupling of glycolysis and oxidation.

Oxidative stress, inflammation and mitochondrial dysfunction arecrucial mediators of cardiac damage [11,12,13]. A recent study sug-gests that succinate accumulation might act as a driver for such con-sequences [14]. Succinate is an intermediate of the mitochondrial citricacid cycle (CAC). The conventional direction of succinate dehy-drogenase (SDH) in the CAC is to catalyze succinate for fumarate for-mation, but in ischemic heart, the altered mitochondrial function in-creases succinate accumulation due to the reversal of SDH, a processproposed to be related to the malate/aspartate shuttle (MAS) [14]. Is-chemic succinate accumulation drives mitochondrial ROS production,contributing to I/R damage [14]. In macrophage, intracellular succi-nate accumulation causes IL-1β production dependent on HIF-1α [15].In tumor cells, succinate is involved in hypoxic signaling by stabilizingHIF-1α [16]. In addition, succinate is an extracellular ligand by bindingto a G-protein-coupled receptor GPR91, exerting a wide array of phy-siological and pathological effects [17]. Upholding levels of serumsuccinate could lead to pathological cardiac hypertrophy through directactivation of GPR91 [18]. Evidence showed that succinate throughGPR91, regulates global Ca2+ transient and induces cardiomyocytescell death [19]. These events demonstrate that succinate acts as a sig-naling molecule beyond its traditional roles in the CAC.

Ginseng is today one of the most widely used medicinal plants in theworld. Ginsenosides are the major active constituents in ginseng, andginsenoside Rb1 is the most abundant component. Ginsenoside Rb1demonstrates antiobesity and antihyperglycemic effects in high-fat diet(HFD) fed rats [20] and protects heart from I/R injury in diabetic rat[21,22]. In this work, we investigated the effects of Rb1 on cardio-protection and its regulatory mechanisms. We showed that in responseto alerted metabolism, succinate accumulated and impaired PDH ac-tivity via both GPR91 dependent and independent regulation, settingcardiomyocytes at a stage susceptible to I/R injury. Rb1 suppressedsuccinate-associated HIF-1α induction and attenuated GPR91-PKCδsignaling by combating fatty acid oxidation in cardiomyocytes, andthereby prevented I/R injury.

2. Methods

2.1. Materials

Ginsenoside Rb1 (purity> 98%, Nanjing Spring & AutumnBiological Engineering Co., Ltd., Nanjing, China), trimetazidine dihy-drochloride tablets (Servier Pharmaceutical Co., Ltd., Tianjin, China),palmitate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), 2-Methoxyestradiol (2-MeOE2) (Selleck, USA) were obtained from thecommercial sources. Aminooxyacetate (AOA) (C13408), dimethylmalonate (136441), dimethyl succinate (V900547), sodium succinate(S2378), rottlerin (R5648) were purchased from Sigma (St. Louis, MO,USA). Palmitate was dissolved in ethanol to prepare 200 mmol/L stocksolution and then was diluted with medium containing 10% FFA-freeBSA at the ratio of 1:19 before use. The following items were purchasedfrom the cited commercial sources: anti-CPT1A (ab176320), anti-PDH(phospho S293) (ab177461), anti-HIF-1-alpha (ab51608), anti-PKCδ(ab182126), Abcam (Cambridge, MA, USA); anti-GPR91 (BS2961),anti-PDHA1 (BS7208), anti-GAPDH (AP0063), Goat Anti-Rabbit IgG (H+ L) HRP (BS13278), Rabbit Anti-Goat IgG (H + L)-HRP (BS30503),Bioworld Technology (St. Paul, MN, USA); anti-PDK4 (sc-14492), SantaCruz Biotechnology, Inc. (Santa Cruz, CA, USA); anti-ATPA1 polyclonalantibody (14418-AP), anti-HADHA, anti-ACADVL, Proteintech(Chicago, IL, USA).

2.2. Animals

ICR male mice (6–8 weeks), Sprague-Dawley (SD, 200–250 g) ratsand 1- or 2-days old neonatal rats were purchased from the LaboratoryAnimal Center of Nanjing Qinglongshan. The animal care and experi-mental procedures were approved by Animal Ethics Committee ofChina Pharmaceutical University.

2.3. Cell culture and treatment

Primary neonatal rat ventricular myocytes (NRVMs) were preparedas described previously with minor modifications [23]. Briefly, thehearts from 1- or 2-day old SD rats were rinsed in cold phosphate buffersaline (PBS) and minced into pieces. The cells were isolated from theventricles by sequential digestions with 0.08% Trypsin in PBS withoutEDTA. To obtain almost pure cardiomyocytes, cells were preplated on100-mm culture dishes for 3 h and were then replated in Dulbecco'sMinimum Essential Medium (DMEM) containing 10% fetal bovineserum (FBS), supplemented with 0.1 mmol/L 5-Bromo-2-deoxyUridinefor 3–6 d before the treatment. For cellular experiments, cells weretreated with indicated agents and incubated with palmitate (PA), di-methyl succinate or sodium succinate for 2 h. For hypoxia-reoxygena-tion (H/R) treatment, after PA treatment for 2 h, cells were washed andsubjected to hypoxia (1% O2) for 4 h, followed by 1 h reoxygenation.

2.4. HFD feeding in mice and myocardial ischemia/reperfusion protocol

Mice were fed with high fat diet (10% Lard, 10% yolk, 1% choles-terol, 0.2% cholate and 78.8% standard diet) for 8 weeks with Rb1(50 mg/kg/day) or trimetazidine (20 mg/kg/day) by gavage (n= 12 ineach group). The oral doses of Rb1 and trimetazidine were chosenbased on previous studies [24–26]. The control mice were fed a stan-dard rat chow diet. At the end of feeding program, blood was collectedfor biochemical parameter assay and the hearts isolated from 6 mice ineach group were used for immunohistochemistry and immuno-fluorescence examination. For I/R protocol, left anterior descendingcoronary artery ligation was performed, according to the previous de-scription [27]. The rest mice (n = 6 in each group) were anesthetizedintraperitoneally with chloral hydrate (400 mg/kg). A fourth-inter-costal space thoracotomy was performed, and the pericardium wasexcised to expose the heart. The left anterior descending coronary ar-tery (LAD) was ligated 2 mm above the left auricle by a 6–0 silk sutureto induce ischemia. After 30 min ischemia, the ligature was loosened toallow reperfusion for 24 h. After reperfusion, the hearts were isolatedfollowed by triphenyltetrazolium chloride (TTC) staining. The heartsfrom HFD-fed mice were collected for immunohistochemistry ex-amination.

2.5. Isolated Langendorff heart perfusion and ischemia/reperfusion protocol

Hearts were isolated from SD rats and perfused according to theLangendorff technique [28]. For PA perfusion, hearts from SD rats(n = 4 in each group) were subjected to normoxic perfusion with Krebs-Henseleit buffer containing PA with indicated agents for 30 min. At theend of perfusion program, the hearts were harvested for Western blottest. In ischemia/reperfusion experiment, after PA perfusion with orwithout indicated agents for 30 min, the hearts from SD rats (n = 4 ineach group) then were subjected to 45 min global ischemia followed by60 min reperfusion. The infarct size of the hearts was viewed by TTCstaining.

2.6. Immunofluorescence for pimonidazole staining detection

To assay hypoxic regions, control or HFD-fed mice were injectedwith pimonidazole HCl (60 mg/kg, Hypoxyprobe, Inc., Burlington,USA) 30 min prior to sacrifice. The heart was removed and fixed in 4%

J. Li et al. BBA - Molecular Basis of Disease 1863 (2017) 2835–2847

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paraformaldehyde/PBS buffer. For hypoxia assay in cardiomyocytes,cells were cultured with DMEM containing pimonidazole HCl(200 μmol/L). After immunostaining with FITC-conjugated antibodiesagainst pimonidazole adducts, the images were visualized using con-focal microscopy (LSM700, Zeiss, Germany).

2.7. Immunofluorescence for HIF-1α translocation to nucleus

NRVMs were fixed with 4% paraformaldehyde and permeabilizedwith 0.2% Triton X-100 for 20 min and then blocked with 3% BSA for1.5 h. After incubation with primary antibody (anti-HIF-1α, 1:200,Abcam, ab51608) overnight at 4 °C, cells were incubated with fluor-escein isothiocyanate FITC-linked anti-rabbit IgG antibodies for 1 h at37 °C. Nuclei were stained with DAPI (Beyotime Institute ofBiotechnology, Shanghai, China). The immunofluorescence imageswere visualized using confocal microscopy (LSM700, Zeiss, Germany).

2.8. Immunofluorescence for PKCδ translocation to mitochondria andGPR91 internalization

After treatment, NRVMs were incubated with 200 nmol/L MitoTracker Red CMXRos (Molecular Probes) for 30 min at 37 °C to locatethe mitochondria. Immunofluorescence staining was performed usingprimary antibody to detect PKCδ (1:200, Abcam, ab182126) followedby incubation with a fluorescein isothiocyanate-conjugated secondaryantibody. Images were visualized using confocal microscopy.

Internalization of GPR91 was induced by sodium succinate(200 μmol/L) for 2 h at 37 °C. Cell surface was stained with 10 μmol/LCell Tracker CM-Dil (Beyotime Institute of Biotechnology, Shanghai,China). Labeled cells were incubated with primary antibody to detectGPR91 (1:200, Bioworld Technology, BS2961) followed by secondaryantibody. Images were obtained using confocal microscopy.

2.9. Immunohistochemisty

The hearts were isolated from control or HFD-fed mice and fixedwith 4% paraformaldehyde. After dehydration and the hearts wereembedded in paraffin and then cut into thickness and affixed onto theslides. After blocking with 3% peroxide-methanol at room temperaturefor 25 min, the slides were incubated with primary antibody (anti-CPT1, 1:50, Abcam, ab176320; anti-phospho-PDH, 1:50, Abcam,ab177461; anti-HIF-1α, 1:50, Abcam, ab51608; anti-GPR91, 1:50,Abcam, ab140795) overnight at 4 °C, followed by the incubation withHRP-conjugated secondary antibody for 1 h at room temperature.Nuclei were stained with DAPI. After dehydration and stabilization,staining was detected by NanoZoomer 2.0-RS (Hamamatsu, Japan).

2.10. Western blot assay

Cells or cardiac tissues were lysed in ice-cold radio-immunoprecipitation assay (RIPA) buffer, supplemented with 1 mmol/L PMSF to extract protein. The lysates was centrifuged at 12000 g for15 min at 4 °C, and the concentration was quantified by BicinchoninicAcid Protein Assay kit (Biosky Biotechnology Corporation, Nanjing,China). Equivalent amounts of protein were separated by SDS-PAGEand transferred onto PVDF membranes. After blocking at room tem-perature for 3 h, the membranes were immunoblotted with primaryantibody (anti-CPT1, 1:1000, Abcam, ab176320; anti-HIF-1α, Abcam,1:1000, ab51608; anti-phospho-PDH, 1:1000, Abcam, ab177461; anti-PDH, 1:800, Bioworld Technology, BS7208; anti-PDK4, 1:500, SantaCruz Biotechnology, sc-14492; anti-GPR91, 1:800, BioworldTechnology, BS2961; anti-ATPA1, Proteintech, 1:1000, 14418-AP; anti-HADHA, Proteintech, 1:1000, 10758-AP; anti-ACADVL, Proteintech,1:1000, 14527-1-AP; anti-GAPDH, 1:1500, Bioworld Technology,AP0063) respectively at 4 °C overnight, followed by incubation withHRP-conjugated secondary antibody (Goat Anti-Rabbit IgG (H + L)

HRP, 1:15,000, Bioworld Technology, BS13278, or Rabbit Anti-GoatIgG (H + L)-HRP, 1:15,000, Bioworld Technology, BS30503). Equalloading was verified by incubation with anti-GAPDH. Na+/K+-ATPaseacted as the loading control of cell membrane fractions. The blots werevisualized using ECL, and the signals were quantified by densitometry(Image-Pro Plus 6.0). Relevant band intensities were quantified afternormalization to the amount of loading control protein.

2.11. Small interfering RNA transfection and the construction ofoverexpression plasmid

Fetal cardiomyocyte-derived H9c2 cells were seeded into 6-wellplates and transfected with HIF-1α siRNA (sc-45919) (Santa Cruz, CA,USA) or pcDNA3.1-HIF-1α (GenePharma, Shanghai, China) at 80%confluence using siRNA transfection reagent (sc-29528) (Santa Cruz) orHieff Trans™ Liposomal Transfection Reagent (Yeasen, Shanghai,China), respectively, according to the manufacturer's instructions. At48 h post-transfection, Western blot was performed to determine theexpression levels of HIF-1α. At 48 h post-transfection, the cells withHIF-1α siRNA transfection were subjected to PA (100 μmol/L) or di-methyl succinate (5 mmol/L) for additional 2 h. The protein of the cellswas obtained for Western blot analysis.

For GPR91 knockdown, H9c2 cells were transfected with GPR91siRNA (sc-270636) (Santa Cruz, CA, USA). At 48 h post-transfection,Western blot was performed to determine the expression levels ofGPR91. The cells with GPR91 siRNA transfection were subjected tosodium succinate (200 μmol/L) for additional 2 h to detect PDH phos-phorylation by Western blot and PKCδ translocation to mitochondria byimmunofluorescence.

2.12. Luciferase reporter assay

The transfection was conducted in H9c2 cells using Hieff Trans™Liposomal Transfection Reagent with PDK4 promoter-luciferase vectors(GenePharma) or co-transfected with HIF-1α siRNA or scramble siRNA.SV40 Renilla luciferase reporter was co-transfected to serve as internalcontrol. Twenty four hours after transfection, the cells were treatedwith or without dimethyl succinate (5 mmol/L) for 2 h. Luciferase ac-tivities were determined using the dual-luciferase assay kit (Beyotime).

2.13. Quantitative real-time PCR

Total RNA was isolated from NRVMs using Trizol reagent (Yeasen).The purity and integrity of the RNA were calculated by absorbance at260/280 nm. RNA obtained was reversely transcribed into cDNA byusing the Hieff™ First Strand cDNA Synthesis Super Mix for RT-qPCR+ gDNA wiper system (Yeasen). The relative gene expression was re-latively quantified by Hieff™ qPCR SYBR Green Master Mix (No RoxPlus) kit (Yeasen) with CFX96TM realtime system (BIO-RAD, USA). Theresults of mRNA level were calculated with the 2−ΔΔCt method andpresented as a ratio to β-actin. Primer sequences were shown in OnlineSupple Table 1.

2.14. Measurement of oxygen consumption rate (OCR) and extracellularacidification rate (ECAR)

OCR and ECAR measurements were performed using the XF96Extracellular Flux analyzer (Seahorse Bioscience, North Billerica, MA).NRVMs were seeded (10,000 cells/well) in cell culture plates (SeahorseBioscience) and incubated overnight in DMEM supplemented with 10%(v/v) FBS. On the day of the assay, cells were incubated with XF assaymedium (Seahorse Biosciences, 100965–000) supplemented with10 mmol/L glucose and 1 mmol/L pyruvate at 37 °C in a CO2-free in-cubator for 1 h before the measurement. The OCRs were measuredunder basal condition and after the application of oligomycin (1 μmol/L), FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone,


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1 μmol/L), and rotenone (0.5 μmol/L) + antimycin A (0.5 μmol/L) (XFCell Mito Stress Test Kit; Seahorse Bioscience). ECAR values weremonitored under basal condition and measured after the injection ofglucose (10 mmol/L), oligomycin (1 μmol/L), and 2-DeoxyGlucose (2-DG) (50 mmol/L) to the wells. OCR and ECAR results were analyzedusing the Seahorse XF96 software. The results were derived from fourindividual experiments.

2.15. Succinate concentration measurement

NRVMs (1 ∗ 106) or cardiac tissue (10 mg) were rapidly homo-genized on ice in 100 μL of ice-cold succinate assay buffer. These werecentrifuged at 10000 g for 5 min to collect the supernatant. Succinatelevel in the supernatant was detected by a coupled enzyme reactionwith Succinate Colorimetric Assay Kit (Sigma, St. Louis, MO, USA).

For assay of extracellular succinate, cells were cultured in 24-wellplates in the presence of mentioned agents and then the condition

medium was collected to assay succinate concentration according tomanufacturer's instructions.

2.16. Ratio of NADH/NAD+ quantification

NRVMs were pretreated with Rb1 (10 μmol/L) or TMZ (1 μmol/L)followed by palmitate insult for 2 h. Cells were centrifuged at 2000 rpmfor 5 min, and extracted with 400 μL NAD+/NADH Extraction Buffer byhomogenization or freeze/thawing for 2 cycles. The resultant mixturewas then centrifuged at 13000 g for 10 min to collect the supernatant.The protein concentration was determined by Bicinchoninic AcidProtein Assay kit. The samples were then deproteinized before use byfiltering through a 10 kDa cut-off spin filter. NADtotal (NAD+ andNADH) and NADH were quantified by a colorimetric assay using aNAD+/NADH Quantification Kit (Sigma, St. Louis, MO, USA).

Fig. 1. Ginsenoside Rb1 inhibited palmitate (PA)-induced hypoxia and attenuated glycolysis in cardiomyocytes. Primary neonatal rat ventricular myocytes (NRVMs) were pretreated withRb1 or trimetazidine (TMZ) and then incubated with PA for 2 h. A: carnitine palmitoyl transferase1 (CPT1) protein expression in PA-treated NRVMs; B: mRNA expressions of fatty acyl-CoA dehydrogenase (FACD) and 3-ketoacyl CoA thiolase (KCT) in PA-treated NRVMs; C: oxygen consumption ratio (OCR) in PA-treated NRVMs (oligo, oligomycin; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; ROT, rotenone). Boxes visually depicted computed data. Basal respiration (Basal), coupled respiration (Coupled), spare respiration capacity(SRC), and uncoupled respiration (Uncoupled) were calculated. Data points represented as mean of four readings taken at each time-point (Left) and calculated results were presented asbar plot (Right); D: pimonidazole staining in PA-treated NRVMs (Scale bar, 200 μm); E: lactate release in PA-treated NRVMs; F: extracellular acidification rates (ECAR) in PA-treatedNRVMs (oligo, oligomycin; 2-DG, 2-deoxy glucose). Boxes visually depicted computed data. Glycolysis and glycolytic capacity were calculated. Data points represented as mean of fourreadings taken at each time-point (Left) and calculated results were presented as bar plot (Right); G: intracellular NADH/NAD+ ratio in PA-treated NRVMs. Data above were expressed asthe mean ± SD from four independent experiments. *p < 0.05: indicated treatment vs PA only treatment; #p < 0.05: PA only treatment vs untreated control.


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2.17. Detection of PDH and SDH activities

Given indicated treatment, cells were harvested and lysed in PBSbuffer by sonication method. The protein concentration was determinedby Bicinchoninic Acid Protein Assay kit. Equal amount of protein ofeach group was used to measure PDH activity (Solarbio, Beijing, China)and SDH activity (Jiancheng Bioengineering Institute), according to themanufacturer's instructions.

2.18. ROS production assay

After H/R treatment, cells were loaded with 5 μmol/L DHE(Invitrogen) for 0.5 h at 37 °C. After washing, cells were fixed in 4%paraformaldehyde (v/v) for 5 min at 4 °C, and ROS production wasviewed by confocal microscopy (LSM700, Zeiss, Germany) or measuredby a microplate reader.

2.19. Measurement of cellular lactate concentration, Ca2+ concentration,lactate dehydrogenase (LDH) release and ATP contents

After H/R treatment, conditional medium was collected for lactateand LDH assay with commercial kits (Jiancheng Bioengineering

Institute) and the cells were harvested for the assay of Ca2+ con-centration and ATP contents by Calcium Assay Kit (JianchengBioengineering Institute) or ATP Assay Kit (Beyotime Institute ofBiotechnology, Shanghai, China), respectively, according to the kitmethods.

2.20. Cell viability assay

After H/R treatment, cell viability was assessed by trypan bluestaining (Beyotime Institute of Biotechnology). Cells were harvestedand stained with trypan blue working solution. Three hundred cells ofeach sample (n= 4) were randomly counted under a light microscope.The cell viability was calculated as number of unstained cells dividedby total number of cells and expressed as a percentage.

2.21. Statistical analysis

The results were expressed as the means ± SD. The significance ofdifferences was analyzed by one-way ANOVA followed by theBonferroni correction. A value of p < 0.05 was considered statisticallysignificant.

Fig. 2. Hypoxic treatment and palmitate (PA) challenge induced succinate accumulation in cardiomyocytes. Neonatal rat ventricular myocytes (NRVMs) were pretreated with ginsenosideRb1, trimetazidine (TMZ), dimethyl malonate or aminooxyacetate (AOA) followed by palmitate (PA) stimulation or 1% O2 incubation for 2 h. A: intracellular succinate production in PA-or 1% O2-treated NRVMs; B: extracellular succinate concentration in PA- or 1% O2-treated NRVMs; C: succinate dehydrogenase (SDH) activity in PA-treated NRVMs. D: succinateproduction in NRVMs incubated upon PA insult; E: the proposed pathway for succinate production in reversal. AOA, aminooxyacetate; AST, aspartate amino transferase; OAA, ox-aloacetate; SDH, succinate dehydrogenase; CAC, citric acid cycle. Data were expressed as the mean ± SD derived from four independent experiments. *p < 0.05: indicated treatment vsPA or 1% O2 only treatment; #p < 0.05: PA or 1% O2 only treatment vs untreated control.


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3. Results

3.1. Ginsenoside Rb1 inhibited PA-induced hypoxia and attenuatedglycolysis in cardiomyocytes

Fatty acid oxidation occurs in mitochondria. Carnitine palmitoyltransferase 1 (CPT1) is a key enzyme allowing fatty acid entry into themitochondria. PA challenge enhanced CPT1 protein expression inNRVMs, but this change was blunted by Rb1 at concentrations of 1.0and 10 μmol/L (Fig. 1A). As Western blot examination is a semi-quantitative analysis, we further examined PA-induced CPT1 protein

induction using ELISA assay, and found that Rb1 effectively reducedCPT1 protein contents at concentrations ranging from 0.1 to 10 μmol/L(Online Supple Fig. 1A). Fatty acyl CoA dehydrogenase (FACD) and 3-ketoacyl CoA thiolase (KCT), the key enzymes in β-oxidation, increasedin transcriptional expression in PA-treated cardiomyocytes (Fig. 1B). Inaddition, PA challenge also enhanced FACD protein expression, butKCT protein expression was not affected (Online Supple Fig. 1 B & C).Rb1 treatment transcriptionally suppressed FACD and KCT gene ex-pression and reduced FACD protein induction, despite no effect on KCTprotein expression (Fig. 1B, Online Supple Fig. 1 B & C). Proteasomalinhibitor MG-132 diminished the inhibitory effect of Rb1 on CPT1 and

Fig. 3. Intracellular succinate impaired pyruvatedehydrogenase (PDH) activity dependent on hy-poxia inducible factor 1α (HIF-1α). A, B: hypoxiainducible factor-1α (HIF-1α) protein expression(A) and nuclear translocation (B) in NRVMstreated with dimethyl succinate for 2 h (Bar,20 μm); C, D: mRNA expression of pyruvate de-hydrogenase kinase (PDK4) (C) or PDH activity(D) in NRVMs treated with dimethyl succinate inthe presence or absence of 2-MeOE2 for 2 h; E, F:PDK4 (E) and p-PDH (F) expression in H9c2 cellstransfected with HIF-1α or control scrambledsiRNAs; G: the PDK4 luciferase reporter was co-transfected with or without HIF-1α siRNAs inH9c2 cells and followed by succinate stimulationfor 2 h. The luciferase activity was normalizedwith Renilla luciferase; H: PDK4 and p-PDH ex-pression in H9c2 cells transfected withpcDNA3.1- HIF-1α or vehicle. The results wereexpressed as the mean ± SD of four independentexperiments. *p < 0.05: untreated treatment vsdimethyl succinate only treatment or PA onlytreatment; #p < 0.05: vs indicated treatment.


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FACD protein induction (Online Supple Fig. 1D, E), suggesting that Rb1reduced protein expression via the regulation of protein degradation. Asexpected, Rb1 inhibited mitochondrial complex 2 activity (OnlineSupple Fig. 1F) and oxygen consumption ratio (OCR) in cardiomyocytes(Fig. 1C). Trimetazidine, an inhibitor of β-oxidation [29], worked as apositive control at concentration of 1 μmol/L [30] and showed a similarregulation to Rb1 (Fig. 1).

PA stimulation induced a hypoxia-like state in NRVMs, indicated byincreased pimonidazole staining of hypoxia adducts (Fig. 1D), whereas

the cellular hypoxia-like state was prevented by Rb1 and trimetazidinetreatment. PA stimulation led to an enhancement of glycolysis, in-dicated by increased lactate accumulation and cellular acidification(ECAR) (Fig. 1E, F). Rb1 and trimetazidine treatment prevented lactateaccumulation and ECAR, and thereby reversed the altered redox stateby reducing cytosolic NADH/NAD+ ratio (Fig. 1G).

Fig. 4. Extracellular succinate impaired pyruvate dehydrogenase (PDH) activity via GPR91 signaling. A: GPR91 expression on cell membrane and in cytoplasm in response to sodiumsuccinate for 2 h; B: GPR91 localization in response to sodium succinate or dimethyl succinate stimulation detected by immunofluorescence staining. Cell membrane was visualized byDil. Bar, 20 μm; C: PKCδ translocation to mitochondria in response to sodium succinate stimulation detected by immunofluorescence staining. Mitochondria were located by Mito TrackerRed CMXRos. Bar, 20 μm; D: PDH phosphorylation expression in NRVMs in response to sodium succinate with or without rottlerin for 2 h. E: PKCδ translocation to mitochondria inresponse to sodium succinate stimulation in H9c2 cells transfected with GPR91 or control scrambled siRNAs. Bar, 20 μm; F: PDH phosphorylation in H9c2 cells transfected with GPR91 orcontrol scrambled siRNAs in the presence or absence of sodium succinate stimulation. Data above were expressed as the mean ± SD from four independent experiments. *p < 0.05: vssodium succinate only treatment; #p < 0.05: vs indicated treatment.


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3.2. PA stimulation induced succinate accumulation in cardiomyocytes

Hypoxic treatment or PA stimulation induced succinate accumula-tion in cardiomyocytes (Fig. 2A), suggesting that saturated fatty acid-induced succinate generation resulted from hypoxia-like state in car-diomyocytes. Since succinate can move freely to extracellular milieuwhere it binds to its receptor GPR91 [31], we assayed extracellularsuccinate contents and found elevated succinate in the culture medium,confirming the release of succinate from cardiomyocytes (Fig. 2B). Theconventional direction of SDH in CAC is to catalyze succinate for fu-marate formation, and therefore, inhibition of SDH should lead tosuccinate accumulation. However, we observed that SDH inhibitor di-methyl malonate reduced PA or 1% O2-induced succinate accumulation(Fig. 2A, B), indicating that the increased succinate production shouldbe due to the activation of SDH in reversal. As a MAS inhibitor, ami-nooxyacetate (AOA) decreased succinate production in cardiomyocytes(Fig. 2A, B), suggesting the involvement of MAS in reversed succinateproduction. High cytosolic NADH/NAD+ ratio drives malate/aspartateshuttle to supply fumarate for succinate production due to the reversalof SDH [14]. AOA prevented PA-induced fumarate production byblocking MAS pathway (Online Supple Fig. 2), providing support forthe involvement of MAS. Consistently, dimethyl malonate and AOAreduced SDH activity against PA insult (Fig. 2C). Rb1 inhibited SDHactivity and prevented succinate accumulation in cardiomyocytes ex-posed to PA (Fig. 2C, D). Similarly, trimetazidine also effectively re-duced succinate accumulation in cardiomyocytes (Fig. 2D). These datasuggested that PA-induced succinate production was, at least in part,due to enhanced MAS pathway and reversal of SDH activation. Theproposed mechanism of succinate production in reversal was shown inFig. 2E.

3.3. Intracellular succinate impaired PDH activity via HIF-1α

To determine the impact of succinate on glucose oxidation, wetreated cardiomyocytes with cell-permeable dimethyl succinate, andobserved that cellular succinate accumulation increased HIF-1α in-duction and promoted the translocation of HIF-1α into nucleus (Fig. 3A,B).

PDK4 inhibits PDH activity via phosphorylation of PDH. Dimethylsuccinate treatment increased PDK4 gene expression (Fig. 3C) andprotein expression (Online Supple Fig. 3) at concentrations rangingfrom 0.01 to 5 mmol/L and then decreased PDH activity (Fig. 3D). 2-Methoxyestradiol (2-MeOE2) is an inhibitor of microtubule assemblythat inhibits HIF-1α translocation to nucleus [32]. 2-MeOE2 reduceddimethyl succinate-induced PDK4 expression and recovered PDH ac-tivity, indicating the role of HIF-1α in transcriptional regulation. Toconfirm the essential role of HIF-1α in succinate-mediated PDH in-activation, we treated H9c2 cells with specific siRNA to silence HIF-1α.As shown in Fig. 3E and F, HIF-1α knockdown diminished PA and di-methyl succinate-induced PDK4 expression and PDH phosphorylation.Luciferase reporter assay showed that succinate regulated PDK4 ex-pression through gene promoter induction (Fig. 3G). Knockdown ofHIF-1α with siRNA blocked PDK4 promoter activation, (Fig. 3G), fur-ther confirming that HIF-1α was required for succinate-induced PDK4gene promoter induction. In contrast, overexpression of HIF-1α withplasmid transfection enhanced PDK4 induction and PDH phosphoryla-tion, indicating that HIF-1α impaired PDH activity by transcriptionalregulation of PDK4 (Fig. 3H). Together, these results demonstrated thatintracellular succinate impaired PDH activity through the transcrip-tional regulation by HIF-1α.

3.4. Extracellular succinate impaired PDH activity via GPR91 signaling

Besides intracellular pathways, extracellular succinate could inducetransactivation signals via GPR91. To explore if extracellular succinatecould influence cellular metabolism through membrane receptor

GPR91, we treated NRVMs with sodium succinate, and observed thatsodium succinate increased GPR91 expression in the cytosolic fraction,while the expression in the membrane was reduced (Fig. 4A). Con-cordantly, immunofluorescence staining showed that sodium succinateinduced the internalization of GPR91 (Fig. 4B), indicating receptoractivation and signaling amplification [33]. Dimethyl succinate alsoinduced GPR91 internalization, indicating that intracellular succinatecould be released to the medium to activate GPR91 through autocrineregulation (Fig. 4B). In response to GPR91 activation, PKCδ translo-cated to mitochondria (Fig. 4C) and impaired PDH activity by phos-phorylation (Fig. 4D). Specific PKC inhibitor rottlerin attenuated PDHphosphorylation by succinate, indicative of the role of PKCδ in PDHinactivation (Fig. 4D). To prove the essential role of GPR91 in succi-nate-induced PDH impairment, we treated H9c2 cells with specificGPR91 siRNA to silence GPR91. As shown in Fig. 4E, GPR91 knock-down diminished sodium succinate-induced PKCδ translocation to mi-tochondria. Moreover, GPR91 knockdown reduced sodium succinate-induced PDH phosphorylation (Fig. 4F), indicating the essential role ofGPR91. These data suggested that extracellular succinate impaired PDHactivity through GPR91 activation and subsequent PKCδ translocationto mitochondria.

3.5. Ginsenoside Rb1 improved PDH and prevented hypoxia-reoxygenationinjury in cardiomyocytes

PA challenge induced succinate accumulation, leading to the im-pairment of PDH activity. To know the potential implication in I/Rinjury, we stimulated NRVMs with PA in the presence of Rb1 and tri-metazidine. After washing, the cells were recultured under hypoxicconditions (1% O2) followed by reoxygenation. PA pretreatment furtherimpaired PDH activity (Fig. 5A) and increased lactate accumulation(Fig. 5B) during H/R. These alterations were prevented by Rb1 andtrimetazidine treatment during PA challenge (Fig. 5A, B). Con-cordantly, PA-pretreatment exaggerated ROS production and calciumoverload during H/R insult and these alternations were reduced by Rb1and trimetazidine treatment in PA stimulation (Online Supple Fig. 4).PA-pretreatment further limited ATP contents (Fig. 5C), aggravatedLDH release (Fig. 5D) and resultantly decreased cell viability (Fig. 5E).Rb1 and trimetazidine treatment effectively increased ATP contents,reduced LDH release, and protected cell survival from H/R insult(Fig. 5C–E). SDH inhibitor dimethyl malonate treatment in PA stimu-lation exhibited similar effects as Rb1 and trimetazidine, indicating theinvolvement of succinate in H/R injury (Fig. 5F–H). Furthermore, bothdimethyl succinate and sodium succinate treatment demonstrated thesimilar action as PA to exaggerate H/R injury (Fig. 5F–H), furtherconfirming the role of succinate in H/R injury. These results indicatedthe role of succinate in H/R injury and suggested that protection of PDHactivity increased cardiomyocytes resistance to H/R damage.

3.6. Ginsenoside Rb1 limited ischemia/reperfusion damage in isolatedworking rat heart

It is considered that fetal hearts are more dependent on glucosemetabolism, whereas after birth, fatty acid oxidation rate increasedramatically and become the primary source in the developing heart[34]. The aforementioned results showed the alterations of PDH ac-tivity and glucose metabolism in neonatal cardiomyocytes. To in-vestigate the impact of fatty acids on adult cardiomyocytes, we in-vestigated PA perfusion in isolated working hearts of rats. Because thepotent effect of Rb1 on the protection of cardiomyocytes was observedat the concentration of 10 μmol/L, we investigated the role of Rb1 inisolated working heart at the same concentration. Rb1 and trimetazi-dine treatment reduced succinate accumulation (Fig. 6A), down-regulated HIF-1α expression (Fig. 6B) and decreased GPR91 expression(Fig. 6C) with PDH improvement by dephosphorylation (Fig. 6D).Consistent with the published report [14], PA perfusion exaggerated


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succinate accumulation during ischemia and then the produced succi-nate was rapidly reduced due to re-oxidation during reperfusion. Rb1and trimetazidine pretreatment in PA perfusion reduced succinateproduction during ischemia without influence on succinate concentra-tions during reperfusion (Online Supple Fig. 5). PA perfusion ex-aggerated heart injury during subsequent I/R, but Rb1 and trimetazi-dine pretreatment during PA perfusion reduced infarct size in I/R heart,indicating the resistance to I/R injury (Fig. 6E). Dimethyl malonatereduced infarct size in I/R heart, indicative of the potential role of

succinate in heart injury.

3.7. Oral administration of ginsenoside Rb1 alleviated heart ischemia/reperfusion injury in HFD-fed mice

In parallel, we observed their effects on the heart of HFD-fed mice.HFD-feeding elevated the levels of blood free fatty acids, triglyceridesand total cholesterol with hyperglycemia in mice, indicative of meta-bolic disorders. Oral administration of Rb1 as well as trimetazidine

Fig. 5. Ginsenoside Rb1 decreased pyruvate de-hydrogenase (PDH) impairment and then pre-vented hypoxia-reoxygenation injury in cardio-myocytes. A-E: Neonatal rat ventricular myocytes(NRVMs) were pretreated with ginsenoside Rb1(Rb1) or trimetazidine (TMZ) with PA stimula-tion for 2 h. After washing, cells were exposed to1% O2 for 4 h and reoxygenation for 1 h (H/R).A: PDH phosphorylation in NRVMs exposed to H/R insult; B: H/R-induced lactate release inNRVMs; C-F: ATP contents (C), LDH release (D),and cell viability (E) in NRVMs exposed to H/Rinsult. F-H: NRVMs were pretreated with di-methyl malonate with or without PA, dimethylsuccinate or sodium succinate stimulation for 2 h.After washing, cells were exposed to 1% O2 for4 h and reoxygenation for 1 h. F-H: ATP contents(F), LDH release (G), and cell viability (H) inNRVMs exposed to H/R insult. Data above wereexpressed as the mean ± SD from four in-dependent experiments. *p < 0.05: vs PA plusH/R treatment; #p < 0.05: vs indicated treat-ment.


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lowered blood glucose with reduced body weight gain, while no sig-nificant influences on other metabolic parameters were observed(Online Supple Fig. 6). HFD-feeding induced a hypoxia-like state in theheart indicated by increased pimonidazole staining, and increasedsuccinate accumulation, whereas these alterations were prevented byoral administration of Rb1 and trimetazidine (Fig. 7A and B). Con-sistently, immunohistochemistry examination showed that Rb1 andtrimetazidine reduced HIF-1α (Fig. 7C) and GPR91 expression (Fig. 7D)and then normalized PDH phosphorylation (Fig. 7E) in the heart.Compared to the normal diet, HFD feeding increased the infarct size inthe heart subjected to I/R via temporary ligation of the LAD coronaryartery (Fig. 7F), indicative of increase susceptibility to I/R injury. Rb1and trimetazidine reduced myocardial infarction and thereby protectedthe heart from I/R injury (Fig. 7F). Consistent with the effects ex vivo,

these results in vivo demonstrated that Rb1 and trimetazidine increasedthe resistance of heart to I/R insult.

4. Discussion

In this study, we showed that aside being an intermediate in CAC,succinate acted as a metabolic signaling to influence cardiac metabo-lism in a GPR91-dependent or independent manner. Intracellular suc-cinate accumulation impaired PDH activity through HIF-1α induction,while extracellular succinate impaired PDH activity through GPR91pathway. Both regulations worked together to disturb energy metabo-lism, setting cardiomyocytes at a stage susceptible to I/R injury.

Obesity is accompanied by the elevated levels of circulating freefatty acid and ectopic accumulation of lipid metabolites in nonadipose

Fig. 6. Ginsenoside Rb1 limited ischemia/reperfusion (I/R) damage in isolatedworking rat heart. A-E: succinate production(A), hypoxia inducible factor-1α (HIF-1α)induction (B), GPR91 (C) and pyruvate de-hydrogenase (PDH) phosphorylation (D) inthe working rat heart perfused with palmi-tate (PA); E: infarct staining with TTC in theworking heart subjected to global ischemiafor 45 min + 60 min reperfusion after PAperfusion. The results were expressed asmean ± SD (n = 4). *p < 0.05: vs PA onlyor PA plus I/R treatment; #p < 0.05: vs in-dicated treatment.


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tissues including the heart [35]. Rb1 reduced free fatty acid entry intomitochondria by inhibiting CPT1 induction and combated fatty acidoxidation by downregulation of FACD and KCT, the rate-limiting en-zymes in β-oxidation. Since it was unlikely that the reduction in CPT1and FACD protein by Rb1 treatment in 2 h was a result due to trans-lational regulation, we speculated the involvement of post-translationalregulation. As expected, proteasomal inhibitor MG-132 diminished theinhibitory effects of Rb1 (Online Supple Fig. 1D, E), suggesting that Rb1reduced protein expression via protein degradation. Consistent withthis, protein expression involved in fatty acid oxidation could be in-fluenced by protein degradation [36]. Inhibition of fatty acid oxidationby Rb1 should be cardioprotective, because it reduced oxygen con-sumption in mitochondria. It is generally accepted that increased fattyacid oxidation could decrease glucose oxidation through the RandleCycle regulation [4]. Impaired glucose oxidation led to lactate accu-mulation and induced cellular acidification owing to the uncoupling ofglycolysis and glucose oxidation. Rb1 reversed these alternations, de-monstrating its beneficial effect on cellular metabolic homeostasis.

PA challenge induced succinate accumulation in cardiomyocytes.

Succinate is an intermediate in CAC and regulated by SDH. In con-ventional direction, SDH operates to convert succinate to fumarate,therefore, inhibition of SDH activity should theoretically increase suc-cinate accumulation. However, we found that succinate production wasenhanced with increased SDH activity and inhibition of SDH could re-duce succinate formation, a result similar to succinate accumulationdue to the reversal of SDH in ischemic heart [14]. MAS is a biochemicalsystem for translocating electrons from the cytosol into mitochondria.MAS activation contributes to hypoxic succinate production, because itdrives NADH/NAD+ for the conversion of malate into fumarate, sup-plying substrate for succinate production through the reversed SDHaction [14,37]. In the present study, AOA, an inhibitor of aspartateaminotransferase in MAS, inhibited SDH activity and reduced succinateproduction, indicating the involvement of MAS in succinate production.In this context, reduced NADH/NAD+ ratio and inhibited SDH activityby Rb1 and TMZ should contribute to preventing succinate productionthrough the reversal of SDH.

Next, we sought to find out if succinate accumulation was involvedin impaired PDH activity. In the heart, PDK4 is the predominant

Fig. 7. Ginsenoside Rb1 prevented heart ischemia/reperfusion (I/R) injury in high-fat diet (HFD)-fed mice. A: pimonidazole staining in the heart of HFD-fed mice (Bar, 100 μm); B:succinate production in the heart of HFD-fed mice; C-E: immunohistochemistry examinations of hypoxia inducible factor 1α (HIF-1α) accumulation (C), GPR91 (D) and pyruvatedehydrogenase (PDH) phosphorylation (E) in the heart of HFD-fed mice; Bar, 200 μm. F: myocardial infarct size in HFD-fed mice subjected to cardiac ischemia and reperfusion. Dataexpressed as mean ± SD (n = 6). *p < 0.05: vs HFD feeding or HFD feeding plus I/R treatment; #p < 0.05: vs indicated treatment.


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isoform of PDKs that inactivates PDH by phosphorylation [38]. Hereinwe used dimethyl succinate as the intracellular available succinate tostimulate cardiomyocytes and found that succinate inactivated PDH bytranscriptional regulation of PDK4. HIF-1α works as a transcriptionalregulator to reprogram energy metabolism for enhancing glycolysisunder anoxic conditions [39]. HIF-1α homeostasis is controlled by itsrate of translation and degradation [40,41]. In cardiomyocytes, di-methyl succinate increased HIF-1α accumulation, suggesting that suc-cinate might act as a metabolic mediator linking hypoxia to HIF-1αinduction and subsequent consequences. This regulation is consistentwith what happens in tumor cells where succinate stabilizes HIF-1α byinhibiting degradation [16]. In the present study, we found that suc-cinate-induced PDH impairment was mediated through HIF-1α tran-scriptional regulation and further showed that HIF-1α regulated PDK4gene expression through direct interaction with the gene promoterupon succinate insult. So far, we have shown that intracellular succi-nate accumulated in hypoxic cardiomyocytes and worked as a meta-bolic signaling linking increased fatty acid oxidation and the un-coupling of glycolysis to glucose oxidation. It is worth noting that, HIF-1α has a dual face in regulating ischemia reperfusion injury. HIF-1α isdocumented to be protective during ischemic preconditioning throughmetabolic adaptations [8,9]. In contrast, some studies report the det-rimental role of HIF-1α in ischemic reperfusion injury by inducing thepro-apoptotic molecules and inflammatory cytokines [42,43]. The dif-ferent effects of HIF-1α might be due to the setting under differentmetabolic conditions.

Different to the action of intracellular succinate, extracellular orcirculating succinate can act as signaling molecule with a hormone-likefunction to regulate cellular response by binding to membrane receptorGPR91, a member of G-protein-coupled receptor family [17]. Weshowed that extracellular succinate increased as a consequence of hy-poxic succinate accumulation in cardiomyocytes. It is documented thatthe half-maximal response (EC50) for succinate-GPR91 activation is inthe range from 20 to 50 μmol/L, and succinate rather than other CACintermediates was the specific ligand for GPR91 [17,31,33]. We ob-served that the released succinate contents by hypoxia or PA treatmentwere elevated to about 9.26 ng/μL (equal to 78 μmol/L) or 5.26 ng/μL(equal to 44 μmol/L), respectively (Fig. 2B). Thus it is reasonable tobelieve that the contents of extracellular succinate released from car-diomyocytes were enough to active GRP91 receptor. We used sodiumsuccinate as extracellular succinate to treat cardiomyocytes and foundGPR91 internalization, an indicator of GPR91 activation [33]. PKCactivation is a branch of cellular signaling in response to G-protein-coupled receptor activation [44]. In response to GPR91 activation,PKCδ translocated to mitochondria accompanied by PDH inactivation.These results elucidated the signaling pathway through which extra-cellular succinate blocked glucose oxidation in cardiomyocytes. Con-sistently, recent studies showed that elevated succinate induced cardi-omyocytes hypertrophy and led to cardiomyocytes cell death throughGPR91 activation and subsequent intracellular pathways [18,19]. Pro-tein kinase C is a family of protein kinase enzymes that are involved incontrolling the function of other proteins through the phosphorylationof Ser/Thr residues. Upon activation, PKC can translocate to multiplesubcellular localizations, and the location in subcellular compartmentsis a key determinant of its biological effects. Conventional PKC isoforms(PKCs) were documented to contribute to ischemic preconditioning[45], but the cardioprotective role was challenged by a study whichshowed that inhibition of PKC failed to prevent ischemic pre-conditioning in swine [46]. In addition to conventional PKCs, translo-cation of novel PKCs to mitochondria is found to influence PDH activity[47,48]. Consistent with this, we showed that extracellular succinatepromoted PKCδ translocation to mitochondria and impaired PDH ac-tivity via GPR91 activation, working together with intracellular succi-nate to exacerbate PDH impairment. The different signalings andcompetitive binding to mitochondria might be the determining factorsfor the special role of PKCs in myocardial ischemic injury and

protection. In view of the regulation of HIF-1α induction by in-tracellular succinate, our work showed that succinate induced meta-bolic dysregulation in cardiomyocytes in a GPR91 dependent and in-dependent manner.

Lipid disorder impairs heart energy efficiency in the use of glucoseoxidation, and the alteration of energy metabolism should make theheart sensitive to I/R injury. Indeed, PA challenge amplified PDH im-pairment and exaggerated ROS production in cardiomyocytes duringhypoxia and reoxygenation in cardiomyocytes. Lactate and acidifica-tion impaired cellular ionic homeostasis, leading to increased calciumoverload. More ATP was shifted away from contractile purpose to re-establish membrane potential and remove cellular calcium through ion-exchange [5]. The futile ATP consumption could be the main reason forcardiomyocytes susceptibility to I/R injury. Rb1 protected cellularhomeostasis by improving PDH activity, and thereby increased re-sistance to apoptosis during I/R insult. This protective effects of Rb1were reproduced in PA-perfused working heart. HFD feeding in miceelevated levels of free fatty acids in blood with a hypoxia-like state inthe heart, providing new insight into the impact of lipid disorder oncardiac metabolism. In the heart of HFD-fed mice, succinate accumu-lation, HIF-1α induction, GPR91 activation and impaired PDH activityindicated the dysfunction of mitochondrial oxidation, setting the heartprone to ischemic injury. Rb1 exerted cardioprotection without af-fecting blood lipid levels, further indicating that its action was mainlyin the direct regulation of cardiac metabolism.

Taken together, our work in vitro, ex vivo and in vivo showed thatincreased fatty acid oxidation impaired cardiac energy efficiencythrough succinate-associated HIF-1α induction and GPR91-denpendentsignaling. Ginsenoside Rb1 prevented succinate accumulation by com-bating fatty acid oxidation in mitochondria, contributing to improvingcellular metabolic homeostasis and increasing cardiomyocytes re-sistance to ischemic injury (Fig. 8). This finding suggests that beneficialregulation of cardiac lipid and glucose metabolism could reduce therisks of cardiac ischemia by reducing succinate accumulation in meta-bolic disorders.

Fig. 8. The proposed pathway through which hypoxic succinate accumulation inducedpyruvate dehydrogenase (PDH) impairment and subsequently aggravated ischemia/re-perfusion (I/R) injury. Fatty acid oxidation increased oxygen consumption and inducedhypoxia in cardiomyocytes. In response to hypoxic stress, intracellular succinate accu-mulated and was released to extracellular milieu. On the one hand, intracellular succinateimpaired PDH via the transcriptional regulation by hypoxia inducible factor 1α (HIF-1α).On the other hand, extracellular succinate amplified PDH impairment through binding toGPR91 and then promoting PKCδ activation. PDH impairment resultantly increased heartsusceptibility to I/R injury. Ginsenoside Rb1 and trimetazidine suppressed succinate-as-sociated HIF-1α induction and attenuated GPR91-PKCδ signaling by combating fatty acidoxidation in cardiomyocytes, and thus prevented I/R injury.


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Disclosures

None.

Sources of funding

This study was supported in part by the National Natural ScienceFoundation of China (No. 91639115, 81421005, 81573642), andInnovation of Graduate Student Training Project in Jiangsu Province(KYLX16_1184).

Transparency document

The Transparency document associated with this article can befound, in online version.

Acknowledgments

We thank Xiao-Nan Ma, YingJian Hou, and Minhui Sun from theCellular and Molecular Biology Center of China PharmaceuticalUniversity for their technical help in confocal microscopy. We alsothank Dr. Raphael N. Alolga from the China Pharmaceutical Universityto edit the manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbadis.2017.07.017.

References

[1] G.D. Lopaschuk, J.R. Ussher, C.D. Folmes, J.S. Jaswal, W.C. Stanley, Myocardialfatty acid metabolism in health and disease, Physiol. Rev. 90 (2010) 207–258.

[2] W.C. Stanley, G.D. Lopaschuk, J.L. Hall, J.G. McCormack, Regulation of myocardialcarbohydrate metabolism under normal and ischaemic conditions. Potential forpharmacological interventions, Cardiovasc. Res. 33 (1997) 243–257.

[3] Q. Liu, J.C. Docherty, J.C. Rendell, A.S. Clanachan, G.D. Lopaschuk, High levels offatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation, J. Am. Coll. Cardiol. 39 (2002)718–725.

[4] P.J. Randle, P.B. Garland, C.N. Hales, E.A. Newsholme, The glucose fatty-acid cycle.Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus,Lancet 1 (1963) 785–789.

[5] N. Fillmore, J. Mori, G.D. Lopaschuk, Mitochondrial fatty acid oxidation alterationsin heart failure, ischaemic heart disease and diabetic cardiomyopathy, Br. J.Pharmacol. 171 (2014) 2080–2090.

[6] R. Bartrons, J. Caro, Hypoxia, glucose metabolism and the Warburg's effect, J.Bioenerg. Biomembr. 39 (2007) 223–229.

[7] I.F. Robey, R.M. Stephen, K.S. Brown, B.K. Baggett, R.A. Gatenby, R.J. Gillies,Regulation of the Warburg effect in early-passage breast cancer cells, Neoplasia 10(2008) 745–756.

[8] G. Loor, P.T. Schumacker, Role of hypoxia-inducible factor in cell survival duringmyocardial ischemia-reperfusion, Cell Death Differ. 15 (2008) 686–690.

[9] D. Tekin, A.D. Dursun, L. Xi, Hypoxia inducible factor 1 (HIF-1) and cardiopro-tection, Acta Pharmacol. Sin. 31 (2010) 1085–1094.

[10] Q. Ke, M. Costa, Hypoxia-inducible factor-1 (HIF-1), Mol. Pharmacol. 70 (2006)1469–1480.

[11] Y. Dong, V.V. Undyala, K. Przyklenk, Inhibition of mitochondrial fission as a mo-lecular target for cardioprotection: critical importance of the timing of treatment,Basic Res. Cardiol. 111 (2016) 59.

[12] J. Han, C. Zou, L. Mei, Y. Zhang, Y. Qian, S. You, et al., MD2 mediates angiotensinII-induced cardiac inflammation and remodeling via directly binding to Ang II andactivating TLR4/NF-κB signaling pathway, Basic Res. Cardiol. 112 (2017) 9.

[13] K. Szczepanek, A. Xu, Y. Hu, J. Thompson, J. He, A.C. Larner, et al.,Cardioprotective function of mitochondrial-targeted and transcriptionally inactiveSTAT3 against ischemia and reperfusion injury, Basic Res. Cardiol. 110 (2015) 53.

[14] E.T. Chouchani, V.R. Pell, E. Gaude, D. Aksentijević, S.Y. Sundier, E.L. Robb, et al.,Ischaemic accumulation of succinate controls reperfusion injury through mi-tochondrial ROS, Nature 515 (2014) 431–435.

[15] G.M. Tannahill, A.M. Curtis, J. Adamik, E.M. Palsson-McDermott, A.F. McGettrick,G. Goel, et al., Succinate is an inflammatory signal that induces IL-1β through HIF-1α, Nature 496 (2013) 238–242.

[16] M.A. Selak, S.M. Armour, E.D. MacKenzie, H. Boulahbel, D.G. Watson,K.D. Mansfield, et al., Succinate links TCA cycle dysfunction to oncogenesis byinhibiting HIF-alpha prolyl hydroxylase, Cancer Cell 7 (2005) 77–85.

[17] Fonseca M. de Castro, C.J. Aguiar, J.A. da Rocha Franco, R.N. Gingold, M.F. Leite,GPR91: expanding the frontiers of Krebs cycle intermediates, Cell Commun. Signal.14 (2016) 3.

[18] C.J. Aguiar, J.A. Rocha-Franco, P.A. Sousa, A.K. Santos, M. Ladeira, C. Rocha-Resende, et al., Succinate causes pathological cardiomyocyte hypertrophy throughGPR91 activation, Cell Commun. Signal. 12 (2014) 78.

[19] C.J. Aguiar, V.L. Andrade, E.R. Gomes, M.N. Alves, M.S. Ladeira, A.C. Pinheiro,et al., Succinate modulates Ca(2+) transient and cardiomyocyte viability throughPKA-dependent pathway, Cell Calcium 47 (2010) 37–46.

[20] Y. Xiong, L. Shen, K.J. Liu, P. Tso, Y. Xiong, G. Wang, et al., Antiobesity and an-tihyperglycemic effects of ginsenoside Rb1 in rats, Diabetes 59 (2010) 2505–2512.

[21] Y. Wu, Z.Y. Xia, J. Dou, L. Zhang, J.J. Xu, B. Zhao, et al., Protective effect of gin-senoside Rb1 against myocardial ischemia/reperfusion injury in streptozotocin-in-duced diabetic rats, Mol. Biol. Rep. 38 (2011) 4327–4335.

[22] R. Xia, B. Zhao, Y. Wu, J.B. Hou, L. Zhang, J.J. Xu, et al., Ginsenoside Rb1 pre-conditioning enhances eNOS expression and attenuates myocardial ischemia/re-perfusion injury in diabetic rats, J Biomed Biotechnol 2011 (2011) 767930.

[23] P. Simpson, S. Savion, Differentiation of rat myocytes in single cell cultures withand without proliferating nonmyocardial cells. Cross-striations, ultrastructure, andchronotropic response to isoproterenol, Circ. Res. 50 (1982) 101–116.

[24] N. Xiao, M.D. Lou, Y.T. Lu, L.L. Yang, Q. Liu, B. Liu, et al., Ginsenoside Rg5 at-tenuates hepatic glucagon response via suppression of succinate-associated HIF-1αinduction in HFD-fed mice, Diabetologia 60 (2017) 1084–1093.

[25] H.Y. Cha, J.H. Park, J.T. Hong, H.S. Yoo, S. Song, B.Y. Hwang, et al., Anxiolytic-likeeffects of ginsenosides on the elevated plus-maze model in mice, Biol. Pharm. Bull.28 (2005) 1621–1625.

[26] J. Chen, J. Lai, L. Yang, G. Ruan, S. Chaugai, Q. Ning, et al., Trimetazidine preventsmacrophage-mediated septic myocardial dysfunction via activation of the histonedeacetylase sirtuin 1, Br. J. Pharmacol. 173 (2016) 545–561.

[27] L.H. Michael, M.L. Entman, C.J. Hartley, K.A. Youker, J. Zhu, S.R. Hall, et al.,Myocardial ischemia and reperfusion: a murine model, Am. J. Phys. 269 (1995)H2147–54.

[28] G.D. Lopaschuk, M.A. Spafford, N.J. Davies, S.R. Wall, Glucose and palmitate oxi-dation in isolated working rat hearts reperfused after a period of transient globalischemia, Circ. Res. 66 (1990) 546–553.

[29] P.F. Kantor, A. Lucien, R. Kozak, G.D. Lopaschuk, The antianginal drug trimetazi-dine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidationby inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase, Circ. Res.86 (2000) 580–588.

[30] J. Kuzmicic, V. Parra, H.E. Verdejo, C. López-Crisosto, M. Chiong, García, et al.,Trimetazidine prevents palmitate-induced mitochondrial fission and dysfunction incultured cardiomyocytes, Biochem. Pharmacol. 91 (2014) 323–336.

[31] P.R. Correa, E.A. Kruglov, M. Thompson, M.F. Leite, J.A. Dranoff, M.H. Nathanson,Succinate is a paracrine signal for liver damage, J. Hepatol. 47 (2007) 262–269.

[32] H. Seeger, D. Diesing, B. Gückel, D. Wallwiener, A.O. Mueck, J. Huober, Effect oftamoxifen and 2-methoxyestradiol alone and in combination on human breastcancer cell proliferation, J. Steroid Biochem. Mol. Biol. 84 (2003) 255–257.

[33] W. He, F.J. Miao, D.C. Lin, R.T. Schwandner, Z. Wang, J. Gao, et al., Citric acidcycle intermediates as ligands for orphan G-protein-coupled receptors, Nature 429(2004) 188–193.

[34] R.J. Bing, Cardiac metabolism, Physiol. Rev. 45 (1965) 171–213.[35] J.M. McGavock, R.G. Victor, R.H. Unger, L.S. Szczepaniak, Adiposity of the heart,

revisited, Ann. Intern. Med. 144 (2006) 517–524.[36] S. Zhao, W. Xu, W. Jiang, W. Yu, Y. Lin, T. Zhang, J. Yao, et al., Regulation of

cellular metabolism by protein lysine acetylation, Science 327 (2010) 1000–1004.[37] E.T. Chouchani, V.R. Pell, A.M. James, L.M. Work, K. Saeb-Parsy, C. Frezza, et al., A

unifying mechanism for mitochondrial superoxide production during ischemia-re-perfusion injury, Cell Metab. 23 (2016) 254–263.

[38] P. Wu, J. Sato, Y. Zhao, J. Jaskiewicz, K.M. Popov, R.A. Harris, Starvation anddiabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in ratheart, Biochem. J. 329 (1998) 197–201.

[39] J.J. Lum, T. Bui, M. Gruber, J.D. Gordan, R.J. DeBerardinis, K.L. Covello, et al., Thetranscription factor HIF-1alpha plays a critical role in the growth factor-dependentregulation of both aerobic and anaerobic glycolysis, Genes Dev. 21 (2007)1037–1049.

[40] M. Ivan, K. Kondo, H. Yang, W. Kim, J. Valiando, M. Ohh, et al., HIFalpha targetedfor VHL-mediated destruction by proline hydroxylation: implications for O2 sen-sing, Science 292 (2001) 464–468.

[41] P. Jaakkola, D.R. Mole, Y.M. Tian, M.I. Wilson, J. Gielbert, S.J. Gaskell, et al.,Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292 (2001) 468–472.

[42] F. Jung, L.A. Palmer, N. Zhou, R.A. Johns, Hypoxic regulation of inducible nitricoxide synthase via hypoxia inducible factor-1 in cardiac myocytes, Circ. Res. 86(2000) 319–325.

[43] L.A. Kubasiak, O.M. Hernandez, N.H. Bishopric, K.A. Webster, Hypoxia and acidosisactivate cardiac myocyte death through the Bcl-2 family protein BNIP3, Proc. Natl.Acad. Sci. U. S. A. 99 (2002) 12825–12830.

[44] A.C. Ariza, P.M. Deen, J.H. Robben, The succinate receptor as a novel therapeutictarget for oxidative and metabolic stress-related conditions, Front. Endocrinol.(Lausanne) 3 (2012) 22.

[45] H. Miyawaki, M. Ashraf, Ca2+ as a mediator of ischemic preconditioning, Circ. Res.80 (1997) 790–799.

[46] C. Vahlhaus, R. Schulz, H. Post, R. Onallah, G. Heusch, No prevention of ischemicpreconditioning by the protein kinase C inhibitor staurosporine in swine, Circ. Res.79 (1996) 407–414.

[47] M. Caruso, M.A. Maitan, G. Bifulco, C. Miele, G. Vigliotta, F. Oriente, et al.,Activation and mitochondrial translocation of protein kinase Cdelta are necessaryfor insulin stimulation of pyruvate dehydrogenase complex activity in muscle andliver cells, J. Biol. Chem. 276 (2001) 45088–45097.

[48] E.N. Churchill, C.L. Murriel, C.H. Chen, D. Mochly-Rosen, L.I. Szweda, Reperfusion-induced translocation of deltaPKC to cardiac mitochondria prevents pyruvate de-hydrogenase reactivation, Circ. Res. 97 (2005) 78–85.


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Succinate accumulation impairs cardiac pyruvate dehydrogenase activity through GRP91-dependent and independent signaling pathways: Therapeutic effects of ginsenoside Rb1IntroductionMethodsMaterialsAnimalsCell culture and treatmentHFD feeding in mice and myocardial ischemia/reperfusion protocolIsolated Langendorff heart perfusion and ischemia/reperfusion protocolImmunofluorescence for pimonidazole staining detectionImmunofluorescence for HIF-1α translocation to nucleusImmunofluorescence for PKCδ translocation to mitochondria and GPR91 internalizationImmunohistochemistyWestern blot assaySmall interfering RNA transfection and the construction of overexpression plasmidLuciferase reporter assayQuantitative real-time PCRMeasurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)Succinate concentration measurementRatio of NADH/NAD+ quantificationDetection of PDH and SDH activitiesROS production assayMeasurement of cellular lactate concentration, Ca2+ concentration, lactate dehydrogenase (LDH) release and ATP contentsCell viability assayStatistical analysis

ResultsGinsenoside Rb1 inhibited PA-induced hypoxia and attenuated glycolysis in cardiomyocytesPA stimulation induced succinate accumulation in cardiomyocytesIntracellular succinate impaired PDH activity via HIF-1αExtracellular succinate impaired PDH activity via GPR91 signalingGinsenoside Rb1 improved PDH and prevented hypoxia-reoxygenation injury in cardiomyocytesGinsenoside Rb1 limited ischemia/reperfusion damage in isolated working rat heartOral administration of ginsenoside Rb1 alleviated heart ischemia/reperfusion injury in HFD-fed mice

DiscussionDisclosuresSources of fundingTransparency documentAcknowledgmentsSupplementary dataReferences

Documents

BBA - Molecular Basis of Disease · Henseleit buﬀer containing PA with indicated agents for 30 min. At the end of perfusion program, the hearts were harvested for Western blot test