Effect of Geranylgeranyl Pyrophosphate Synthase on Hypoxia/Reoxygenation-Induced Injury in Heart-Derived H9c2 Cells

Recent studies have revealed that geranylgeranyl pyrophosphate synthase (GGPPS), a key enzyme involved in protein prenylation, plays a critical role in postnatal heart growth by regulating cardiomyocyte size. However, the role of GGPPS in myocardial ischemia/reperfusion (MIR) injury is still not clear. The objective of this work was to investigate the effect of GGPPS on MIR injury in H9c2 cells subjected to hypoxia/reoxygenation (HR) to mimic MIR. Prior to HR, the cells were transfected with GGPPS, shGGPPS, or shGFP. The results showed that cell viability was reduced, and cell injury and cell apoptosis were increased as a result of overexpression of GGPPS. Knockdown of GGPPS improved cell viability, and decreased cell injury and cell apoptosis. Furthermore, overexpression of GGPPS increased Rac1 activity and ROS generation, while GGPPS silencing decreased Rac1 activity and ROS generation. Based on these findings, we propose that the alteration of GGPPS expression changed the Rac1 activity and ROS production, and finally led to the different severity of HR-induced injury in H9c2 cells. These findings indicate that GGPPS might be a potential target in preventing H9c2 cells from HR-induced injury. (Int Heart J 2018; 59: 821-828)

I schemic heart disease is the most important cause of cardiac mortality and morbidity. Timely recovery of coronary flow is necessary to resuscitate the ischemic or hypoxic myocardium. However, previous studies have suggested that upon reperfusion, exposure of ischemic tissues to molecular oxygen greatly augments organ damage, a phenomenon termed "ischemia/reperfusion (IR) injury". [1][2][3] IR results in the generation of reactive oxygen species (ROS). [3][4][5][6] ROS are produced in two stages, ischemia and reperfusion, at low and high levels, respectively. 7) The production of ROS and tissue function are related to each other. Excess ROS production in the reperfusion phase is injurious for cardiac tissue, since ROS can attack cellular components and provoke apoptosis and necrosis. 8) NADPH oxidases are known to be the only enzyme family producing ROS as their sole and primary function. 9,10) Furthermore, activation of the small GTP-binding protein Rac1 leads to NADPH oxidase activation. 11) Moreover, studies of nonphagocytic cells have demonstrated that the production of ROS appears to require participation of Rac 1. [12][13][14][15] Geranylgeranyl pyrophosphate synthase (GGPPS) is a key enzyme in the synthesis of geranylgeranyl pyrophosphate (GGPP). It catalyzes the formation of GGPP 16) from one molecule of farnesyl diphosphate (FPP) 17) and one molecule of isopentenyl diphosphate. 18) GGPP mainly participates in post-translational modification for various proteins, including the Rho/Rac, Rap, and Rab families. 19) Studies have indicated that Rac1 plays an important role in IR-induced injury. 14,15) In addition, recent studies have revealed that GGPPS participates in hepatocyte apoptosis, 20) as well as cardiomyocyte hypertrophic growth. 21) However, the effect of GGPPS on IR injury is still unknown.
In this study, heart-derived H9c2 cells were transfected with GGPPS or shGGPPS. We investigated the effect of GGPPS on HR-induced injury and the relevant mechanisms in heart-derived H9c2 cells.
H9c2 cells were seeded in 6-well plates, 24-well plates, or 96-well plates for 24 hours. The cells were transfected with plasmid DNA mixed with Opti-MEM (GIBCO, MO, USA) and lipofectamine 3000 transfection reagent (Invitrogen, CA, USA). Cells were transfected with pAV-GFP plasmid to represent the transfection efficiency of PAV-rGGPPS plasmid. After transfection, the cells were incubated in complete medium (DMEM with 10%FBS) for 48 hours. Cell hypoxia/reoxygenation: Transfection cells in plates were supplied with hypoxic solution 22) and the plates were placed in a sealed chamber with an anaerobic pouch at 37°C for 8 hours. Hypoxia was followed by 4 hours of reoxygenation using complete medium at 37°C and 5% CO2/95%O2. 23) Real-time polymerase chain reaction: Total RNA was extracted using RNAisoPlus (TaKaRa, Dalian, China) and transcribed into cDNA using a PrimeScript RT Reagent Kit (TaKaRa) according to the manufacturer's protocol. GGPPS mRNA expression was relatively quantified via real-time PCR (RT-PCR) using a 7500 Real-Time PCR system (Thermo Scientific, MA, USA). RT-PCR was performed using SYBR Premix Ex Taq (TaKaRa). The GGPPS primers were 5'-ATGGTGTTGCAGAAGACAG G-3' and 5'-TCTGGAAGAA GAGCCCAAGT-3'. GGPPS mRNA levels were normalized to GAPDH with the following primers: 5'-GGAAAGCTGTGGCGTGAT-3' and 5'-AAGGTGGAAG AATGGGAGTT-3' Western blot analysis: Western blot analysis was performed to detect GGPPS protein. Whole cell lysates were prepared in 1× RIPA lysis buffer (Millipore, CA, USA) containing a 1× protease and phosphatase inhibitor cocktail (Thermo Scientific, MA, USA). Protein concentrations were determined using a BCA protein assay kit (Thermo Scientific, IL, USA) according to the manufacturer's protocol. Equal volumes of lysate (30 μg) were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. After 1 hour of blocking with 5% BSA in TBST at room temperature, the PVDF membranes were incubated overnight with anti-GGPPS antibody (Abcam, London, UK) diluted in 1× TBST with 5% BSA at 4°C. Membranes were then washed 3 times with 1× TBST and incubated with horseradish peroxidase (HRP)conjugated secondary antibodies (EarthOx, CA, USA) for 1 hour at room temperature. Hybridizing bands were visualized using an ECL-HRP chemiluminescence kit (Biological Industries, BI, Israel). Tubulin was used as an endogenous control. Cell viability assessment: A CCK assay was used to assess cell viability. Cells were seeded into 96-well plates at a concentration of 5 × 10 3 cells/mL. After transfection and HR, 10 μL of Cell Counting Kit (CCK) (7sea biotech, Shanghai, China) mixed with 100 μL of complete medium was added into each well followed by incubation for 2 hours. Results were quantified using a microplate reader (Molecular Devices) at an absorbance of 450 nm. Measurement of LDH or SOD: Lactate dehydrogenase (LDH) or superoxide dismutase (SOD) was examined to evaluate cell injury. Cells were seeded into 6-well plates (1 × 10 4 cells/mL). Following transfection and HR, LDH release in the medium was measured using a LDH Assay Kit (Jiancheng Co, Nanjing, China). SOD generation in cell lysates was measured using a SOD Assay Kit (Jiancheng Co) according to the manufacturer's protocol. Results were quantified using a microplate reader (Molecular Devices) at an absorbance of 450 nm. Protein concentrations were determined using a BCA protein assay kit (Thermo Scientific, IL, USA) according to the manufacturer's protocol. Cell apoptosis assessment via flow cytometry: Apoptotic cell death was measured using a PE AnnexinV Apoptosis Detection Kit (BD Biosciences, NJ, USA) according to the manufacturer's protocol. Briefly, cells were seeded into 6-well plates. After transfection and HR, the cells were washed twice with cold PBS and then resuspended in 1 × binding buffer at a concentration of 1 × 10 6 cells/mL. A 100 μL aliquot of the solution was transferred to a 5 mL culture tube. Cells in each tube were stained using 5 μL of PE Annexin V mixed with 5 μL 7-AAD for 15 minutes at room temperature in the dark. To each tube 400 μL of 1 × binding buffer was added and the cells were analyzed using flow cytometry. Activation of RhoA, Rac1, Cdc42 and Ras: RhoA, Rac1, Cdc42 or Ras activity was determined by an absorbance-based G-LISA Activation Assay Biochemistry Kit (Cytoskeleton, Denver, CO, USA) according to manufacturer's protocol. Results were quantified using a microplate reader (Molecular Devices) at an absorbance of 490 nm. Protein concentrations were determined using a BCA protein assay kit (Thermo Scientific, IL, USA) according to the manufacturer's protocol. Measurement of ROS: A Cellular ROS Red Fluorescence Assay Kit (Genmed Scientific Inc., Alington, TX, USA) was used to detect ROS generation according to the manufacturer's protocol. Briefly, cells were seeded into 6well plates. After transfection and HR, the cells were collected into a 15 mL centrifuge tube and counted using a cell counter. Cells (1 × 10 6 ) were transferred into a new centrifuge tube and incubated with the Cellular ROS Red Fluorescence Assay Kit working reagent for 20 minutes at  37°C in the dark. Cells were measured using fluorescence microscopy with an excitation wavelength of 540 nm and emitter band pass of 590 nm. Measurement of NADPH oxidase activity: A proportion of the supernatant from the homogenized solutions extracted from the incubation cells were used to determine NADPH oxidase activity. Protein concentration was determined using a BCA protein assay kit (Thermo Scientific, IL, USA) according to the manufacturer's protocol. NADPH oxidase activity was evaluated spectrophotometrically using an NADPH Oxidase Activity Assay kit (GMS 50095.2 v.A; Genmed Scientific Inc.,) according to the manufacturer's protocol. Statistical analyses: Significant differences were determined using SPSS16.0 software. All data are represented as the mean ± SEM. For direct comparison of differences between two groups, Student's t-test was used. Analysis of variance (ANOVA) in the case of serial measurements, followed by the multiple-comparison Bonferroni t-test, was used to assess differences among groups. A P < 0.05 was considered significant. All experiments were repeated 3 times.

PAV-rGGPPS and shGGPPS altered GGPPS expression of H9c2 cells:
Approximately 50~55% of cultured H9c2 cells were transfected by pAV-rGGPPS, shGGPPS, or shGFP ( Figure 1A and 1B). Compared with the shGFP group, GGPPS protein was highly expressed in the pAV-rGGPPS group and was down-regulated in the shGGPPS group ( Figure 1C and 1D). RT-PCR results showed that GGPPS mRNA was highly increased in the pAV-rGGPPS group and markedly reduced in the shRNA group ( Figure  1E).

Overexpression of GGPPS attenuated cell viability and GGPPS silencing improved cell viability:
To investigate the effect of GGPPS on cell viability after HR in H9c2 cells, pAV-rGGPPS, shGGPPS, or shGFP was used to transfect H9c2 cells. Cell viability was quantified using Dai, ET AL  CCK assay after HR. We found that overexpression of GGPPS reduced the viability and GGPPS silencing increased the viability in H9c2 cells after HR (Figure 2). Overexpression of GGPPS increased cell injury and GGPPS silencing decreased cell injury: LDH and SOD levels were measured to assess cell injury after HR. The results indicated that LDH release in the medium significantly increased in the pAV-rGGPPS group and decreased in the shGGPPS group ( Figure 3A). SOD generation was significantly reduced in the pAV-rGGPPS group and elevated in the shGGPPS group ( Figure 3B). Figure 4, after HR, the level of apoptosis (both early and late-stage) of H9c2 cells in the shGFP group was 10.26 ± 0.66%. The transfection of GGPPS elevated the apoptotic cell ratio to 13.75 ± 0.64%. In the shGGPPS group, the apoptotic cell ratio was decreased to 7.8 ± 0.47%. GGPPS altered the Rac1 activation: Protein geranylgeranylation is necessary for the activation of small GTPases. In this experiment, it was shown that Rac1 activity was significantly increased in the pAV-rGGPPS group and decreased in the shGGPPS group ( Figure 5A) compared with the shGFP group. However, our findings did not show significant changes in Ras activity ( Figure 5B). Also, the RhoA and Cdc42 activity were not significantly changed ( Figure 5C and 5D). Thus, the results suggested a direct relationship between the GGPPS gene and Rac1. This implied that GGPPS was involved in HR-induced injury through Rac1.

Overexpression of GGPPS increased ROS generation and GGPPS silencing decreased ROS generation:
The production of ROS and tissue function are related to each other. Studies of nonphagocytic cells have demonstrated that the production of ROS appears to require the participation of the small GTP-binding protein Rac1. [12][13][14][15] Therefore, the effects of GGPPS on ROS generation in HR were studied. Figure 6 shows the measurement of intracellular ROS induced HR treatment in cultured H9c2 cells. As assessed by dihydroethidium bromide (DHE) fluorescence, the ROS positive cells as well as the fluorescence levels were increased as a result of overexpression of GGPPS, and decreased in the GGPPS silencing group. Overexpression of GGPPS increased NADPH oxidase activity and GGPPS silencing decreased NADPH oxidase activity: NADPH oxidase activity was measured to evaluate the generation of ROS. NADPH oxidase activity was significantly increased in the pAV-rGGPPS group and decreased in the shGGPPS group (Figure 7) compared Dai, ET AL Figure 6. ROS levels in H9c2 cells. A: After 48 hours transfection, following 8 hours of hypoxia and 4 hours of reoxygenation, the ROS levels were detected by DHE fluorescence (red) and nuclei were counterstained with DAPI (blue). B: Quantification of ROS levels. ImageJ software was employed to analyze the optical density in each cell represented in A. Data are expressed as the mean ± SEM (n = 3). ### P < 0.001 versus CON. **P < 0.01 versus shGFP. ***P < 0.001 versus shGFP. CON indicates control; and HR, hypoxia/reoxygenation. Figure 7. Levels of NADPH oxidase in H9c2 cells. After 48 hours of transfection, following 8 hours of hypoxia and 4 hours of reoxygenation. NADPH oxidase activity was detected. Data are expressed as the mean ± SME (n = 3). ### P < 0.001 versus CON. **P < 0.01 versus shGFP + HR. ***P < 0.001 versus shGFP + HR. CON indicates control; and HR, hypoxia/reoxygenation. with the shGFP group.

Discussion
Recovery of oxygen supply to hypoxic cardiomyo-cytes can cause necrotic and apoptotic cell death. 24) IR results in the generation of reactive oxygen species (ROS), [3][4][5][6] and oxidative stress arising from excessive production of reactive oxygen species has long been associated with IR injury, 25,26) and this pathological process can be mimicked when cultured cells are subjected to HR challenge. 24,27) A recent study indicated that the IL-1 signaling pathway participates in the process of ROS production through the recruitment and activation of neutrophils. 28) The MAPK signaling pathway plays an important role in the pathogenesis of IR injury. 29) Other studies have demonstrated that inhibition of ROS production or scavenging of ROS offers protection against tissue injury incurred during ischemia/reperfusion. Moreover, other studies have aimed at inhibiting the source(s) responsible for the production of ROS. 30,31) Studies have demonstrated that inhibition of Rac1 protects IR-induced injury by decreasing the production of ROS. 14,15) GGPP is necessary for the activation of small GTPases including Rac1. We designed this study to investigate the effect of GGPPS on HRinduced injury in vitro, and to explore its possible mechanism.
GGPPS is a key enzyme in the mevalonate pathway. GGPPS participates in the synthesis of GGPP which is necessary for protein geranylgeranylation and activation of small GTPases such as RhoA and Rac1. Recent studies have revealed that GGPPS participates in hepatocyte apoptosis, 20) as well as cardiomyocyte hypertrophic growth. 21) We speculate that GGPPS has an influence on MIR-induced injury in cardiomyocytes. In order to verify this hypothesis, we used heart-derived H9c2 cells which were subjected to 8 hours of hypoxia following 4 hours of reoxygenation to mimic MIR. PAV-rGGPPS or shGGPPS was transfected into H9c2 cells to achieve the objective of overexpression of GGPPS or GGPPS silencing. Cell viability, cell injury, and cell apoptosis were detected. Both cell viability and cell apoptosis can reflect the effect of the intervention on cells directly. Our results showed that GGPPS silencing improved cell viability and decreased cell injury and apoptotic cell death, while overexpression of GGPPS had an opposite effect. According to the results, we can conclude that knockdown of GGPPS has a protective effect on cell viability and increases cell apoptosis.
The Rho family of small GTP-binding proteins, including the Rho, Rac, and Cdc42 subfamilies, regulates cytoskeletal function in many aspects. 32) Studies have indicated that Rac1 plays an important role in IR-induced injury, 14,15) and conditional cardiac-specific overexpression of RhoA in mice protected the heart against IR-induced injury. 33) Another study has suggested that inhibition of Rhokinase protected the mouse heart against IR-induced injury. 34) To further characterize the effect of GGPPS on HR injury, small GTP-binding proteins related to HR injury such as Ras, RhoA, Rac1, and Cdc42 were examined. The results showed that Rac1 activity was significantly improved in the pAV-rGGPPS group and reduced in the shGGPPS group. There was no significant difference in basal Rac1 activity between the groups. The reason for this phenomenon is that HR is an inducing factor which stimulates the joining of the 20-carbon geranylgeranyl group (GGPP) to protein-cysteines (Rac1) at or near their carboxy-termini that is catalyzed by protein geranylgeranyltransferase-I (GGTase-I). 35) There was no significant difference compared with the shGFP group for other small GTP-binding proteins. This may be explained in terms of the alteration of GGPPS expression changed the Rac1 activation.
ROS produced during reperfusion can initiate a series of cellular events that eventually lead to inflammation, necrosis, and/or apoptosis. [36][37][38] Activation of the small GTPbinding protein rac1 leads to the activation of NADPH oxidases, which is known to be the only enzyme family producing reactive oxygen species (ROS) as their sole and primary function. 10) Also, it has been reported that the production of ROS appears to require the participation of the small GTP-binding protein rac1. [12][13][14][15] Thus, we detected ROS generation to explore whether it participates in HRinduced injury in H9c2 cells. Our study showed that the generation of ROS was significantly increased in the pAV-rGGPPS group during HR, and was significantly reduced in the shGGPPS group. Thus, we speculated that the effect of GGPPS on HR injury was associated with ROS generation which was influenced by Rac1 ( Figure 8).
In conclusion, we provide here important experimental evidence that overexpression of GGPPS aggravates HR-induced injury in heart-derived H9c2 cells, and GGPPS silencing protects heart-derived H9c2 cells against HR-induced injury. These are accompanied by the alteration of Rac1 activity and ROS generation. These findings indicate that GGPPS might be a potential target in preventing H9c2 cells from HR-induced injury. Further studies are required to investigate the effect of GGPPS in primary cardiomyocytes and in vivo.

Conflicts of interest:
Authors have no conflict of interest to declare.