Vol. 39, No. 2207 Biol. Pharm. Bull. 39, 207–214 (2016)
Regular Article
Gene Expression Profiling of H9c2 Cells Subjected to H2O2-Induced Apoptosis with/without AF-HF001 Guping Wang,a Chunlei Tang,a Guijun Yan,*,b and Bainian Feng*,a a School of Pharmaceutical Science, Jiangnan University; Wuxi, People’s Republic of China: and b Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School; Jiangsu, Nanjing 210008, China. Received July 31, 2015; accepted November 4, 2015; advance publication released online November 26, 2015
Heart failure represents a major health problem. The development of new drugs to treat this condition is essential. We previously discovered that AF-001 attenuates the cardiac defects caused by heart failure in zebrafish. In this paper, we report the identification of AF-HF001, an AF-001 derivative, and its effects on live cardiomyocytes subjected to oxidative damage. The in vitro results demonstrated that AF-HF001 attenuates the production of reactive oxygen species (ROS) and the myocardial cell apoptosis. A DNA microarray was performed to broadly analyze gene expression after H2O2 treatment with or without AF-HF001. Hierarchical clustering analysis revealed that AF-HF001 modifies the expression of certain genes (Ndufs2, Ndufb6, Ndufb8, Ndufa13, Ndufs3, Ndufs5, TPM1, MYH14, RyR1, and TIMP4) related to ROS production, cardiac contractility and extracellular matrix remodeling. AF-HF001 ameliorates oxidative damage, which may be related to the mitogen-activated protein kinase (MAPK) family and the intrinsic mitochondrial pathway. Altogether, this study suggests that AF-HF001 exhibits potential as a clinical drug candidate for the treatment of heart failure. Key words
apoptosis; oxidative damage; AF-HF001; H9c2 cell; DNA microarray
Heart failure is a complex clinical syndrome with high prevalence, high in-hospital rates, significant healthcare costs and high mortality. Thus, developing new drugs for the treatment of heart failure will be beneficial. Unfortunately, the advances in drug discovery for heart failure have been minimal in recent years. Our research team discovered a positive inotropic compound, AF-001, in a phenotype-based, zebrafish model of heart failure; AF-001 decreased the heart and venous congestion size and effectively increased the blood flow velocity in caudal artery during in zebrafish after 2 h of treatment.1) We further discovered a derivative of AF-001, which is referred to as AF-HF001 (the chemical structure is presented in Fig. 1). During heart failure progression, the adaptation of structure and function occurs in a step-wise fashion. The cellular compensation ultimately fails over time. Some studies reported that changes in the biological properties of myocardial cells and the progressive change of these cells cause heart failure at the cellular level. Previous research has demonstrated that patients with end-stage heart failure exhibited apoptosis leading
to cardiomyocyte loss, which may contribute to progressive myocardial dysfunction.2,3) However, previous studies have also demonstrated that the production of reactive oxygen species (ROS) and the level of oxidative stress increased both in patients and animals with heart failure. ROS (including free radicals, such as superoxide, hydroxyl radical and nitric oxide) promote cardiac remodeling after myocardial damage, leading to phenotypic changes in myocardial cells (hypertrophy and death), inducing abnormal regulation of calcium signaling during contraction, and changing the extracellular matrix and metabolism of cardiac fibroblasts cells, resulting in cardiac structural and functional abnormalities. A connection between ROS and heart failure has been hypothesized. Apparently, myocardial ischemia and/or reperfusion with increased ROS production cause progressive cell death. It is conceptually plausible that treating ROS-induced cardiac apoptosis may provide an innovative therapeutic approach to heart failure. A better understanding of the mechanisms by which ROS activate signaling pathways could lead to identify a helpful tool in identifying new drug targets for the treatment of heart failure in the future. To determine the role of AF-HF001 in ROS-induced cardiac apoptosis, we used hydrogen peroxide (H2O2) and cobalt chloride (CoCl2) induced H9c2 rat cardiomyoblast apoptosis. The study used DNA chip technology and aimed to determine gene expression profiling in H9c2 cells during oxidative stress injury with or without AF-HF001. Comparing these gene expression profiles would be helpful for understanding the mechanisms by which ROS induce heart failure and AF-HF001 ameliorates heart failure.
MATERIALS AND METHODS Fig. 1. 291.30
AF-HF001; Molecular Formula: C17H13N3O2, Molecular Weight:
Cell Culture and Treatment
H9c2 rat cardiomyocyte is
To whom correspondence should be addressed. e-mail: [email protected]; [email protected] * © 2016 The Pharmaceutical Society of Japan
208
Biol. Pharm. Bull.
subclone cell line derived from embryonic BD1X rat heart tissue. 6×106 H9c2 cells were seeded into 60 mm culture plates (Corning, NY, U.S.A.). H9c2 cells (Drum Tower Hospital of Nanjing University Medical School, Nanjing, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose (HyClone, Thermo Scientific, South Logan, UT, U.S.A.) supplemented with 10% heatinactivated fetal bovine serum (HyClone, Thermo Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco BRL/ Invitrogen, Carlsbad, CA, U.S.A.) in a humidified atmosphere at 37°C in 5% CO2. Following incubation for 24 h, the cell density reached 90%. H9c2 cells were treated with the indicated concentrations of H2O2 (100 µM; Sigma, St. Louis, MO, U.S.A.) or CoCl2 (200 µM; Sigma) as stimulus for the desired time with or without pretreatment with AF-HF001 for an additional 12 h. The Measurement of Intracellular ROS The generation of intracellular ROS in H9c2 cells was measured using the green fluorescence probe 6-carboxy-2′,7′-dichlorofluorescein diacetate (DCF-DA; Sigma). The cells were placed in 35-mm cell culture slides (Corning) and treated with H2O2 for 30 min. Then, the slides were washed twice with phosphate-buffered saline (PBS) and stained with 10 µM DCF-DA at 37°C for 30 min. The fluorescence images were obtained using Leica automated microscopes (Leica DM2500-3HF-FL, Solms, Germany). Scale bars indicate 50 µm. The Assessment of Apoptosis and Caspase 3 Assay Cell death was determined after H9c2 cells were treated with H2O2 or CoCl2 for 24 h as described above. Apoptosis was assessed using a cell death detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, Germany) for the qualitative and quantitative determination of the cytoplasmic histone-associated DNA fragments. Cleaved caspase 3 activity was measured by Western blot with an anti-caspase 3 antibody (1 : 750; Cell Signal Technology, Danvers, U.S.A.). Western Blotting Analysis H9c2 cells were scratched and were homogenized in an ice-cold cell lysis buffer (50.0 mmol/L Tris pH=7.6, 150.0 mmol/L NaCl, 0.1% sodium dodecyl sulfate (SDS), 1.0% NP-40). The lysate was centrifuged at 15000×g for 15 min at 4°C and the cellular supernatant was collected. Thirty micrograms protein samples per hole were separated with 15% SDS-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, U.S.A.). After the blockage of non-specific binding sites with 5% defatted milk solution at room temperature for 1 h, the membrane was incubated overnight 4°C with the anti-caspase 3 antibody. The anti-caspase 3 antibody from Cell Signaling Technology detects endogenous levels of full length of caspase 3 (35 kDa) and the large fragment of caspase 3 resulted from cleavage (17 kDa). RNA Preparation Total RNA was extracted using the Trizol Reagent (Invitrogen, Carlsbad, CA, U.S.A.) following H2O2 treatment for 24 h. The samples from each group consisted of an equal amount of total RNA from three replicates to normalize the individual differences. The RNA purity and quality were assessed with a UV spectrophotometer K5500 (Kaiao, Beijing, China), agarose gel electrophoresis and the Agilent 2200 Bioanalyzer (Agilent, U.S.A.). Samples with A260/A280≥1.5, A260/A230≥1, RNA integrity number ≥7 were subjected to DNA microarray.
Vol. 39, No. 2 (2016)
DNA Microarray and DNA Chip Data Analysis We have set up three groups: normal group (normal), hydrogen peroxide group (H2O2), pre-protected with AF-HF001 in hydrogen peroxide group (AF-HF001+H2O2). Each group prepared three separate batches. RNA was extracted and mixed three batches in each group after ensuring RNA quality. One microgram of total RNA was reverse transcribed into cRNA with the Amino Allyl messageAmp™ II kit (Invitrogen), and 8 µg of cRNA was labeled using Amino Allyl MessageAmp™ II cRNA with the Cy5 Kit (Invitrogen). RiboArray™ arrays (Ribo, Guangzhou, China) were hybridized containing 29659 rat genes according to the Ribo Gene Hybridization Protocol. After pre-hybridization, hybridization with the chip was performed at 40°C for 16 h, and the chip was subsequently washed with buffers (Ribo). The microarray was loaded, and the GenePix 4000B scanner and GenePix Pro7 software (Molecular Devices Inc., Sunnyvale, CA, U.S.A.) were used to scan and analyze the array. The quantile normalization method was used to preprocess the microarray data. All of the microarray analyses, including the preprocessing, normalization and statistical analysis, were performed in R-3.0.2. The fold change is the ratio of the normalized spot intensities between the test sample and the control sample. The standard selection criteria to identify the differentially expressed genes are established at |Fold Change|≥1.5. Statistical Analysis All of the experimental data represent the mean of at least three separate experiments and are expressed as the mean±standard deviation (S.D.). A p-value
Regular Article
Gene Expression Profiling of H9c2 Cells Subjected to H2O2-Induced Apoptosis with/without AF-HF001 Guping Wang,a Chunlei Tang,a Guijun Yan,*,b and Bainian Feng*,a a School of Pharmaceutical Science, Jiangnan University; Wuxi, People’s Republic of China: and b Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School; Jiangsu, Nanjing 210008, China. Received July 31, 2015; accepted November 4, 2015; advance publication released online November 26, 2015
Heart failure represents a major health problem. The development of new drugs to treat this condition is essential. We previously discovered that AF-001 attenuates the cardiac defects caused by heart failure in zebrafish. In this paper, we report the identification of AF-HF001, an AF-001 derivative, and its effects on live cardiomyocytes subjected to oxidative damage. The in vitro results demonstrated that AF-HF001 attenuates the production of reactive oxygen species (ROS) and the myocardial cell apoptosis. A DNA microarray was performed to broadly analyze gene expression after H2O2 treatment with or without AF-HF001. Hierarchical clustering analysis revealed that AF-HF001 modifies the expression of certain genes (Ndufs2, Ndufb6, Ndufb8, Ndufa13, Ndufs3, Ndufs5, TPM1, MYH14, RyR1, and TIMP4) related to ROS production, cardiac contractility and extracellular matrix remodeling. AF-HF001 ameliorates oxidative damage, which may be related to the mitogen-activated protein kinase (MAPK) family and the intrinsic mitochondrial pathway. Altogether, this study suggests that AF-HF001 exhibits potential as a clinical drug candidate for the treatment of heart failure. Key words
apoptosis; oxidative damage; AF-HF001; H9c2 cell; DNA microarray
Heart failure is a complex clinical syndrome with high prevalence, high in-hospital rates, significant healthcare costs and high mortality. Thus, developing new drugs for the treatment of heart failure will be beneficial. Unfortunately, the advances in drug discovery for heart failure have been minimal in recent years. Our research team discovered a positive inotropic compound, AF-001, in a phenotype-based, zebrafish model of heart failure; AF-001 decreased the heart and venous congestion size and effectively increased the blood flow velocity in caudal artery during in zebrafish after 2 h of treatment.1) We further discovered a derivative of AF-001, which is referred to as AF-HF001 (the chemical structure is presented in Fig. 1). During heart failure progression, the adaptation of structure and function occurs in a step-wise fashion. The cellular compensation ultimately fails over time. Some studies reported that changes in the biological properties of myocardial cells and the progressive change of these cells cause heart failure at the cellular level. Previous research has demonstrated that patients with end-stage heart failure exhibited apoptosis leading
to cardiomyocyte loss, which may contribute to progressive myocardial dysfunction.2,3) However, previous studies have also demonstrated that the production of reactive oxygen species (ROS) and the level of oxidative stress increased both in patients and animals with heart failure. ROS (including free radicals, such as superoxide, hydroxyl radical and nitric oxide) promote cardiac remodeling after myocardial damage, leading to phenotypic changes in myocardial cells (hypertrophy and death), inducing abnormal regulation of calcium signaling during contraction, and changing the extracellular matrix and metabolism of cardiac fibroblasts cells, resulting in cardiac structural and functional abnormalities. A connection between ROS and heart failure has been hypothesized. Apparently, myocardial ischemia and/or reperfusion with increased ROS production cause progressive cell death. It is conceptually plausible that treating ROS-induced cardiac apoptosis may provide an innovative therapeutic approach to heart failure. A better understanding of the mechanisms by which ROS activate signaling pathways could lead to identify a helpful tool in identifying new drug targets for the treatment of heart failure in the future. To determine the role of AF-HF001 in ROS-induced cardiac apoptosis, we used hydrogen peroxide (H2O2) and cobalt chloride (CoCl2) induced H9c2 rat cardiomyoblast apoptosis. The study used DNA chip technology and aimed to determine gene expression profiling in H9c2 cells during oxidative stress injury with or without AF-HF001. Comparing these gene expression profiles would be helpful for understanding the mechanisms by which ROS induce heart failure and AF-HF001 ameliorates heart failure.
MATERIALS AND METHODS Fig. 1. 291.30
AF-HF001; Molecular Formula: C17H13N3O2, Molecular Weight:
Cell Culture and Treatment
H9c2 rat cardiomyocyte is
To whom correspondence should be addressed. e-mail: [email protected]; [email protected] * © 2016 The Pharmaceutical Society of Japan
208
Biol. Pharm. Bull.
subclone cell line derived from embryonic BD1X rat heart tissue. 6×106 H9c2 cells were seeded into 60 mm culture plates (Corning, NY, U.S.A.). H9c2 cells (Drum Tower Hospital of Nanjing University Medical School, Nanjing, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose (HyClone, Thermo Scientific, South Logan, UT, U.S.A.) supplemented with 10% heatinactivated fetal bovine serum (HyClone, Thermo Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco BRL/ Invitrogen, Carlsbad, CA, U.S.A.) in a humidified atmosphere at 37°C in 5% CO2. Following incubation for 24 h, the cell density reached 90%. H9c2 cells were treated with the indicated concentrations of H2O2 (100 µM; Sigma, St. Louis, MO, U.S.A.) or CoCl2 (200 µM; Sigma) as stimulus for the desired time with or without pretreatment with AF-HF001 for an additional 12 h. The Measurement of Intracellular ROS The generation of intracellular ROS in H9c2 cells was measured using the green fluorescence probe 6-carboxy-2′,7′-dichlorofluorescein diacetate (DCF-DA; Sigma). The cells were placed in 35-mm cell culture slides (Corning) and treated with H2O2 for 30 min. Then, the slides were washed twice with phosphate-buffered saline (PBS) and stained with 10 µM DCF-DA at 37°C for 30 min. The fluorescence images were obtained using Leica automated microscopes (Leica DM2500-3HF-FL, Solms, Germany). Scale bars indicate 50 µm. The Assessment of Apoptosis and Caspase 3 Assay Cell death was determined after H9c2 cells were treated with H2O2 or CoCl2 for 24 h as described above. Apoptosis was assessed using a cell death detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, Germany) for the qualitative and quantitative determination of the cytoplasmic histone-associated DNA fragments. Cleaved caspase 3 activity was measured by Western blot with an anti-caspase 3 antibody (1 : 750; Cell Signal Technology, Danvers, U.S.A.). Western Blotting Analysis H9c2 cells were scratched and were homogenized in an ice-cold cell lysis buffer (50.0 mmol/L Tris pH=7.6, 150.0 mmol/L NaCl, 0.1% sodium dodecyl sulfate (SDS), 1.0% NP-40). The lysate was centrifuged at 15000×g for 15 min at 4°C and the cellular supernatant was collected. Thirty micrograms protein samples per hole were separated with 15% SDS-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, U.S.A.). After the blockage of non-specific binding sites with 5% defatted milk solution at room temperature for 1 h, the membrane was incubated overnight 4°C with the anti-caspase 3 antibody. The anti-caspase 3 antibody from Cell Signaling Technology detects endogenous levels of full length of caspase 3 (35 kDa) and the large fragment of caspase 3 resulted from cleavage (17 kDa). RNA Preparation Total RNA was extracted using the Trizol Reagent (Invitrogen, Carlsbad, CA, U.S.A.) following H2O2 treatment for 24 h. The samples from each group consisted of an equal amount of total RNA from three replicates to normalize the individual differences. The RNA purity and quality were assessed with a UV spectrophotometer K5500 (Kaiao, Beijing, China), agarose gel electrophoresis and the Agilent 2200 Bioanalyzer (Agilent, U.S.A.). Samples with A260/A280≥1.5, A260/A230≥1, RNA integrity number ≥7 were subjected to DNA microarray.
Vol. 39, No. 2 (2016)
DNA Microarray and DNA Chip Data Analysis We have set up three groups: normal group (normal), hydrogen peroxide group (H2O2), pre-protected with AF-HF001 in hydrogen peroxide group (AF-HF001+H2O2). Each group prepared three separate batches. RNA was extracted and mixed three batches in each group after ensuring RNA quality. One microgram of total RNA was reverse transcribed into cRNA with the Amino Allyl messageAmp™ II kit (Invitrogen), and 8 µg of cRNA was labeled using Amino Allyl MessageAmp™ II cRNA with the Cy5 Kit (Invitrogen). RiboArray™ arrays (Ribo, Guangzhou, China) were hybridized containing 29659 rat genes according to the Ribo Gene Hybridization Protocol. After pre-hybridization, hybridization with the chip was performed at 40°C for 16 h, and the chip was subsequently washed with buffers (Ribo). The microarray was loaded, and the GenePix 4000B scanner and GenePix Pro7 software (Molecular Devices Inc., Sunnyvale, CA, U.S.A.) were used to scan and analyze the array. The quantile normalization method was used to preprocess the microarray data. All of the microarray analyses, including the preprocessing, normalization and statistical analysis, were performed in R-3.0.2. The fold change is the ratio of the normalized spot intensities between the test sample and the control sample. The standard selection criteria to identify the differentially expressed genes are established at |Fold Change|≥1.5. Statistical Analysis All of the experimental data represent the mean of at least three separate experiments and are expressed as the mean±standard deviation (S.D.). A p-value
