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Acta Cardiologica Sinica logoLink to Acta Cardiologica Sinica
. 2014 Jan;30(1):56–66.

Dung-Shen Downregulates the Synergistic Apoptotic Effects of Angiotensin II Plus Leu 27-IGF II on Cardiomyoblasts

Ko-Shih Chang 1, Nien-Hung Lee 2, Wei-Wen Kuo 3, Wei-Syun Hu 4,5, Mu-Hsin Chang 1, Fuu-Jen Tsai 6, Kun-Hsi Tsai 7,8, Yuh-Shyong Yang 8, Tung-Sheng Chen 9*, Chih-Yang Huang 6,9,10*
PMCID: PMC4804822  PMID: 27122769

Abstract

Background

Insulin growth factor II (IGFII) is expressed after ischemic stress in pig hearts and after myocardial infarction in humans. However, its receptor (IGFIIR) cannot be found in normal adult hearts. Moreover, a mouse IGFII overexpression model showed a heart and kidney hypertrophy phenomenon similar to Beckwith-Wiedemann syndrome in humans. The previous studies from our lab showed that an increase in AngII in H9c2 cells causes an elevation in IGFII and IGFIIR through MEK and JNK activation, leading to a rise in intracellular Ca2+ ions, activation of calcineurin by PLC-β3 via Gαq, insertion into mitochondrial membranes of BAD, and apoptosis via activation of caspases 9 and 3. Codonopsis pilosula (Dung-shen) has various uses in traditional Chinese medicine, including lowering blood pressure, and increasing red and white blood cell counts.

Methods

The purpose of our study is to investigate whether the addition of C. pilosula will attenuate the AngII plus Leu27-IGFII-induced apoptosis in H9c2 cardiomyoblast cells.

Results

From MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-tetrazolium bromide] results, it was revealed that AngII plus Leu27-IGFII significantly reduced cell viability, which was reversed by C. pilosula. Additionally, C. pilosula also reversed apoptosis (TUNEL staining) increased by AngII plus Leu27-IGFII. Up-regulation of caspase 3 by AngII plus Leu27-IGFII was attenuated by C. pilosula treatment, as shown in western blotting assay and immunofluorescence microscopy results.

Conclusions

C. pilosula is able to suppress the apoptotic pathway enhanced by AngII plus Leu27-IGFII in myocardial cells.

Keywords: Angiotensin II, Apoptosis, Codonopsis pilosula, Leucine27-insulin like growth factor II, Mitochondrial outer membrane permeability

INTRODUCTION

Insulin growth factor I (IGFI) is a 70 amino acid long polypeptide hormone growth factor with a molecular weight of 7649 daltons that structurally resembles insulin. IGFI is produced in the liver under the control of pituitary growth hormone, and it binds to IGFI binding proteins (IGFBPs), to be carried to target tissues or cells by the circulatory system. 1 Once it finds its target, IGFI binds to a cell surface IGFI receptor (IGFIR) with high specificity. The IGFI gene is located on chromosome 12 in humans, and on chromosome 10 in mice.

The structure of the IGFI receptor contains 2 extracellular α-chains and 2 intracellular β-chains. 1 IGFIR is a receptor tyrosine kinase, which dimerizes once IGFI is bound and whose intracellular domain becomes autophosphorylated. 2 The phosphorylated tyrosine residues can be found in Src homology 2 (SH2) domains, which then activate insulin receptor substrate 1 (IRS-1) and Shc by phosphorylation via growth factor receptor binding protein 2 (Grb-2). Subsequently, phosphatidylinositol 3-kinase (PI3K) is activated, which then activates Akt. Finally, Akt phosphorylates at serine 136 on BAD, and then unphosphorylated BAD departs from mitochondrial membranes, stabilizing mitochondrial membrane potential and inducing cell survival. In addition, depending on the cell type, IGFI/IGFIR can lead to cell proliferation via Ras/Raf/MEK/ERK signaling pathway or cell migration via Rac.

IGFI gene expression in rat liver declines with advancing age, and is reduced to its lowest levels in young adulthood. 3 Other organs, such as the brain and heart, also display different IGFI expression patterns in different developmental stages. These may be caused by different prepro-IGFIs, which also occurs in humans. In a human placental insulin-like growth factor I re-ceptor (IGFIR) model, IGFIR autoantibodies and therefore IGFI resistance were found in certain patients with diabetes or rheumatic disorders. 4

The insulin-like growth factor II (IGFII) gene is located in chromosome 11p15, 30 kbp long, and is composed of 9 exons and 4 promoters. IGFII is an imprinted gene and associated with allele-specific CpG methylation patterns in the IGFII-H19 region. 5,6 IGFII prepro-hormone (20.1 kilodaltons) is cleaved to generate a 7.5-kilodalton, 67-amino-acid-long IGFII monomer, which is 47% identical to insulin. 5 IGFII is synthesized in the liver and binds to type I IGF receptor with higher affinity than that of type II, initiating a tyrosine kinase activity and a protein kinase cascade. Moreover, IGFII receptor (IGFIIR) functions as a clearance receptor and a possible IGFBP in the fetus. Therefore, IGFII was categorized as an embryonic gene. 7

IGFIIR, also called the cation-independent mannose-6-phosphate receptor (CI-MPR), is a protein that in humans is encoded by the IGFIIR gene. 8,9 IGFIIR is a multifunctional protein receptor that binds IGFII at the cell surface and mannose-6-phosphate (M6P)-tagged proteins in the trans-Golgi network. 9 IGFIIR is a type I transmembrane protein containing a large extracellular domain, a relatively short intracellular tail and a transmembrane domain. 10 The extracellular domain consists of a small region homologous to the collagen-binding domain of fibronectin and 15 repeats of approximately 147 amino acid residues in length. Each of these repeats is homologous to the 157-residue extracellular domain of the mannose 6-phosphate receptor. Binding to IGFII is mediated through one of the repeats, while two different repeats are responsible for binding to mannose-6-phosphate. The IGFIIR is approximately 300 kilodaltons in size, and it appears to exist and function as a dimer.

IGFII plays a role in mammalian postnatal and fetal growth functioning in an autocrine or paracrine manner. SHR rats display high levels of ventricular and heart IGFII and IGFIIR, and low levels of IGFI mRNA and protein expression during fetal, neonatal and postnatal periods, 11 , 12 whereas limb, muscle, lungs, intestine, kidneys, liver and brain vary in the degree of low IGFIIR mRNA concentration. 13 However, IGFII expression declines after birth and it goes through a transition during the neonatal stage. 12 In a porcine model of brief coronary occlusions, which resulted in prolonged contractile dysfunctions and increased tolerance of myocardium against repeated challenges, such as ischemia/reperfusion, expression of IGFII and IGFBP-5 were activated under stressful conditions. 14 In addition, Matthews et al. postulated that following myocardial infarction, high IGFII expressions were shown in cardiomyocytes in surviving and necrotic areas of post-infarct myocardium. 15 So, IGFII seemed to act as a rescuer.

The IGFII gene is imprinted and loss of its imprinting or otherwise overexpression is involved in several growth disorders and tumors, including Beckwith-Wiedemann syndrome (BWS). 16 BWS involves IGFII deregulation and is characterized by pre- and postnatal overgrowth, multiple organ overgrowth including macroglossia (large tongue), and increased risk of developing childhood tumors. A similar phenomenon was observed in a mouse IGFII overexpression model, in which many BWS syndromes were displayed, such as prenatal overgrowth, polyhydramnios, fetal and neonatal lethality, and disproportionate organ size including heart and kidneys and skeletal abnormalities. 17 Therefore, IGFII may be likely to cause damage rather than to protect.

The role of IGFII in cardiac hypertrophy has been debated for years. In 2002, IGFII was proven to induce hypertrophy in cultured adult cardiomyocytes via two alternative signaling pathways: an IGFI-dependent pathway via ERK1/2, or a lysosome-dependent pathway. 17 IGFII expression can be induced in adult animals under stressful conditions, such as brief coronary occlusions, as shown in a porcine model, 14 as described previously.

The synthesis of angiotensin II (AngII) is a result of the rennin-angiotensin aldosterone system (RAAS), which is a cascade of hormones that maintains homeostasis of arterial pressure, tissue perfusion, and extracellular volume.18 The precursor of AngII, angiotensinogen (452 amino acids long in human), is first synthesized in the liver and travel to the kidneys through the blood stream, to be converted to angiotensin I (AngI) (10 amino acids long) by removal of N-terminal of the precursor by renin, 19 which is secreted by juxtaglomerular (JG) cells that line the afferent arteriole of the renal glomerulus. 18 AngI is converted to AngII (7-9 amino acids long) by angiotensin converting enzyme in the lungs. AngII affects the cardiovascular system by causing oxidative stress20 vasoconstriction, increased blood pressure, increased cardiac contractility and vascular and cardiac hypertrophy. 18 In addition, AngII stimulates zona glomerulosa to secrete aldosterone to enhance re-absorption of Na+ ions and water in the distal tubules and collecting ducts, and to promote K+ secretion by binding to type I angiotensin receptor (AT1R) and via Gαq. 18

From previous studies of Chu et al. and other senior graduates from our lab, we know that AngII stimulated expression of IGFII and IGFIIR in H9c2 cardiomyoblasts, which are not terminally differentiated and displays properties of cardiomyocytes, via MEK and JNK. 21 Then, IGFIIR somehow activates calcineurin, which was previously known as phosphatase 2B (PP2B) and dephos-phorylates BAD at serine 136. Un-phosphorylated BAD then inserts itself into mitochondrial membranes, causing mitochondrial outer-membrane permeability (MOMP) or mitochondrial membrane potential instability and release of mitochondrial proteins, such as Apaf-1 and cytochrome c (Cyto c). Apaf-1 and cytochrome c then form an apoptosome with pro-caspase 9, which is then converted to caspase 9. Finally, caspase 9 activates caspase 3, leading to apoptosis. This pathway was confirmed by using a mouse abdominal aortic ligation (which simulated pressure overload and induced AngII), IGFII antisense RNA, IGFIIR antibody, U-0126 (ERK inhibitor), SP-600125 (JNK inhibitor) and CsA (cyclosporine A; calcineurin inhibitor). In later studies, it was proven that binding of IGFII to cell at serine 537 occurs via Gαq. 22 PLC-β3 cleaves phosphatidylinositol-4, 5-bisphosphate (PIP2) is cleaved into diacyl glycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3). DAG remains bound to the membrane, and IP3 is released as a soluble structure into the cytosol. 23 IP3 then diffuses through the cytosol to bind to IP3 receptors, which are part of the calcium channels in sarcoplasmic reticulum (SR) membrane. This causes an increase of cytosolic Ca2+ concentration, which activates calcineurin. Moreover, calcium and DAG together work to activate protein kinase Cα (PKCα), which goes on to phosphorylate Na+/Ca2+ exchanger (NCX) in the cell membrane of a cardiomyocyte, leading to a further increase in intracellular Ca2+ and calcineurin activity. 22 Calcineurin then dephosphorylates BAD for its insertion into mitochondrial membranes to cause MOMP and release of Apaf-1 and Cyto c, leading to activation of caspases 9 and 3 and apoptosis.

Therefore, it was necessary to screen traditional Chinese medicine (TCM) herbs to ascertain which one suppresses IGF-II-induced cardiomyocyte apoptosis. Dr. Chu previously cloned full-length and different truncated versions of IGFIIR promoter sequences (designated P1-P6) and ligated these DNA sequences to luciferase reporter gene in order to monitor IGFIIR promoter activity under treatment of AngII and TCMs. In this screening system, Null (negative control), CMV (positive control), P1 (full-length IGF-IIR promoter sequence) and PGL2 plasmids was used. So far, several TCMs were already tested and categorized according to their effective doses. Dung-shen (Codonopsis pilosula) was one of the TCMs that significantly reduced IGFIIR promoter activity at 1, 5, and 50 unit/μl (Figure 1), and was selected for subsequent experiments.

Figure 1.

Figure 1

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C. pilosula attenuated AngII plus Leu27-IGFII-inhibited cell viability in a dose-dependent manner. (A) 10-8 M Leu27-IGFII showed a significant reduction of cell viability. * p < 0.05 vs. C. (B) 10-7 M AngII plus 10-8 M Leu27-IGFII caused a decrease in cell viability. Co-treated with C. pilosula showed a restoring and increasing trend at 20, 40 and 60 mg/ml. * p < 0.05 vs. C (decrease), # p < 0.05 vs. 0 (increase) and ## p < 0.01 vs. 0 (increase).

Leu27-IGFII is a 7.42 kilodalton, human IGFII analog that results from tyrosine27-to-leucine27 mutation. 24 Its affinity for IGFIIR in L6 myoblasts is about 16 nM. It was found that Leu27-IGFII specifically binds to IGFIIR with a high affinity and is able to induce IGFIIR-induced H9c2 cell apoptosis via Gαq, which involves increased activity of PLC-β3, calcineurin, BAD, and caspases 9 and 3. 25,26

Finally, Codonopsis pilosula is a perennial species of flowering plant native to Northeast Asia and Korea, and usually found growing around stream banks and forest openings under the shade of trees. The roots of C. pilosula (radix) are used in traditional Chinese medicine to lower blood pressure, increase red and white blood cell numbers, cure appetite loss, strengthen the immune system, and replenish chi. 27 The roots are harvested from the plant during the third or fourth year of growth and dried prior to sale.

In this study, we investigated whether C. pilosula may attenuate synergistic apoptotic effect of AngII plus Leu27-IGFII on H9c2 cardiomyoblast cells and rat neonatal primary cells. The current findings revealed that AngII was able to increase IGFIIR promoter activity, which was reduced by C. pilosula, and that AngII plus Leu27-IGFII along induced MOMP and apoptosis, and C. pilosula reversed these situations. AngII plus Leu27-IGFII also unregulated levels of IGFIIR, Gαq, p-PLC-β3, calcineurin, BAD, cytochrome c and caspases 9 and 3, and C. pilosula downregulated their protein levels and even activities. C. pilosula also increased level of p-Bad Ser136 and Bcl-2. Therefore, C. pilosula is able to reduce IGFIIR promoter activity, IGFIIR signaling pathway, MOMP and apoptosis induced by IGFIIR signaling activation in H9c2 cells.

MATERIALS AND METHODS

Cell culture

H9c2 cardiomyoblast cells were purchased from American Type Culture Collection (ATCC; CRL-1446) (Rockville, MD, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (D5523-1L; Sigma Aldrich, St. Louis, MO, USA) with 10% cosmic calf serum (CCS) (DMK0096, HyClone, USA) in humidified air with 5% CO2 at 37 °C. The cell medium was changed 48 hours after sub-cultivation. Five ml Dulbecco’s phosphate-buffered saline (PBS) (21600-010, GIBCO, Auckland, New Zealand) was used to wash each culture plate or vessel. After the cells were deprived of serum (i.e. in serum-free DMEM) for 4 hours and treated with different drugs at different concentrations and time points.

MTT assay

H9c2 cardiomyoblasts per well were grown in a 12-well plate (1 × 105 cells/well) containing DMEM (10% CCS). 24 hours later, the cells were deprived of serum for 4 hours, and then different doses of C. pilosula (20, 40, 60, 80 and 100 μg/ml) were added to appropriate wells followed by 10 μl 10-7 M AngII 1 hour later, followed by 10-8 M Leu27-IGFII (L27IGF-II receptor Grade; JJG-T01, GroPep, Australia) after an additional 2 hours. Twenty-four hours after administration of Leu27-IGFII, 1 ml 0.5 mg/ml MTT (thiazolyl blue tetrazilium bromide; M2128, Sigma, MO, USA) per well in serum free medium was added in the absence of light. The cells were incubated for 2 to 4 hours in humidified air with 5% CO2 at 37 °C and then purple precipitation was present in the bottom of each well. Medium from each well was transferred to 1.5-ml microcentrifuge tubes and centrifuged at 12000 rpm at 4 °C for 20 minutes and supernatants were discarded. 400 ml isopropanol per well was added to dissolve the purple precipitation and the plate was rocked for 20 minutes. Isopropanol from each well was transferred to the microcentrifuge tubes to dissolve the remaining purple precipitation after centrifuge, transferred back to the appropriate wells and finally to a 96-well plate (200 ml/well) to measure light absorbance at 570 nm. The level of light absorbance is proportional to H9c2 cell survival.

TUNEL assay

H9c2 cardiomyoblasts per well were grown in a 12-well plate (1 × 105 cells/well) containing DMEM (10% CCS). Twenty-four hours later, the cells were deprived of serum for 4 hours, and then 20, 40, and 60 μg/ml C. pilosula were added to the appropriate wells. 10-7 M AngII was added 1 hour later, followed by 10-8 M Leu27-IGFII after an additional 2 hours elapsed. The cells were incubated in humidified air (5% CO2) for 24 hours at 37 °C. Twenty-four hours after administration of Leu27-IGFII, each well was washed with PBS (1 ml/well) and then fixation solution (4% paraformaldehyde in PBS, pH 7.4) was added at room temperature (1 ml/well) and the plate was rested for 1 hour. Blocking buffer (3% H2O2 in methanol) was added (1 ml/well) and the plate was rested for 10 minutes at room temperature. Permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) was added (0.5 ml/well) and the 12-well plate was placed on ice for 2 minutes without shaking. 10X diluted TUNEL reagent [Enzyme Solution (11684795910, Roche, IN, USA) and Label Solution (11684795910, Roche, IN, USA)] was added (200-250 μl /well) and the plate was placed in humidified air at 37 °C for 1 hour for the reagent to react with cell nuclei. 10000X diluted DAPI (4′, 6-Diamidino-2-phenylindole; D9564, Sigma, MO, USA) was added (200 μl /well) and the plate was covered with tin foil and rested for 25 minutes. Finally, the cells were observed under fluorescent microscope.

Western blotting of caspase 3

Protein extraction

H9c2 cells were grown in 10-cm culture dishes containing DMEM (10% CCS) to 80-90% confluency. Twenty-four hours later, the cells were deprived of serum for 4 hours, and then 20, 40, and 60 μ g/ml C. pilosula were added to appropriate dishes, followed by 10-7 M AngII added 1 hour later, followed by 10-8 M Leu27-IGFII after an additional 2 hours. The cells were incubated in humidified air (5% CO2) for 24 hours at 37 °C. Twenty-four hours after administration of Leu27-IGFII, each plate was washed with 3 ml PBS twice and the remaining fluid in each plate was sucked off. Then 100 μl cell lysis buffer [50 mM pH 7.5 Tris-base, 0.5 M NaCl, 1 mM pH 8 EDTA, 1 mM BME, 1% NP40, 1% glycerol and 2 protease inhibitor tablets (04-693-H9-001, Roche, Mannheim, Germany)] per plate was added to lyse the cells. The cells were scraped down and collected in appropriate 1.5-ml microcentrifuge tubes on ice, which were then vortexed once every 10 minutes, 3 times, and centrifuged for 20 minutes at 12000 rpm, 4 °C. The supernatants were transferred to another set of microcentrifuge tubes. These were the total protein samples.

Protein quantification - Lowry assay

Lowry assay is used to determine protein concentrations in different solutions, in which color change of sample solutions is proportional to protein concentration and can be detected by using colorimetric methods. This assay involves reactions of Cu2+ ions with peptide bonds under alkaline conditions with the oxidation of aromatic amino acid residues. Lowry assay is best used with protein concentrations of 0.01-1.0 mg/ml, and is based on the reaction of Cu+ ions, produced by the oxidation of peptide bonds, with Folin-Ciocalteu reagent. The concentration of reduced Folin-Ciocalteu reagent is measured by absorbance at 750 nm and the results are calibrated in a standard calibration curve. As a result, the total concentration of protein in the sample can be deduced from the concentration of tryptophan and tyrosine residues that reduce the Folin-Ciocalteu reagent.

Bovine serum albumin (BSA; A-7906, Sigma, MO, USA) was used to prepare standard solutions (0.1, 0.2, 0.3, 0.4 and 0.5 mg/ml). We mixed 3.968 ml 2% Na2CO3 in 0.1 M NaOH, 16 μl 2% potassium sodium tartrate and 16 μl 1% CuSO4 in a 50-ml centrifuge tube, vortexed and rested for 10 minutes at room temperature. The alkaline Cu2+ mixture was added to each standard sample (250 μ l/tube). Twenty-five μ l of Folin-Ciocalteu reagent (Folin-Ciocalteu’s phenol reagent, 2N; F9252-500ML, Sigma-Aldrich, MO, USA) was added to each Eppendorf tube. The samples were vortexed, centrifuged and rested for 30 minutes in the absence of light. Finally, the samples were loaded onto a 96-well plate (200 μl/well) and light absorbance was measured at 750 nm to generate a standard curve.

Second, 45 μl DDW was mixed with 5 μl protein sample and each 10X diluted sample was placed on ice. The previous method was followed and light absorbance values were used for interpretation with the standard curve to calculate protein concentrations.

Western blotting analysis

12% separating gels and 10% stacking gels were made 1 day before the experiment and stored at 4 °C. Thirty μg of each protein sample and protein marker were loaded to the appropriate wells in the stacking gel, in the presence of running buffer in a protein electrophoresis system. The running process took 150 minutes at 75 V, 400 amps, powered by a power supply. Then, proteins were transferred to PVDF membranes (Immobilon transfer membranes; IPVH000 10, Millipore, USA) for 3 hours at 85 V, 400 amps. Nonspecific protein binding was blocked in blocking buffer (5% milk in 1X TBS) for 1 hour at room temperature. 1X TBS was used to wash off with blocking buffer and the PVDF membranes were rocked in 1:1000 primary (1°) antibody (Ab) solutions overnight, depending on protein concentration and Ab binding affinity. The following primary antibodies were used: anti-Bad (sc-8044, Santa Cruz Biotechnology, CA, USA), anti-phospho-Bad Ser136 (#9295, Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2 (610539, BD, Pharmingen, San Jose, CA, USA), anti-calcineurin (610260, BD, Pharmingen, San Jose, CA, USA), anti-caspase 3 (sc-7148, Santa Cruz Biotechnology, CA, USA), anti-caspase 9 (sc-8166, Santa Cruz Biotechnology, CA, USA), anti-cytochrome c (sc-81752, Santa Cruz Biotechnology, CA, USA), anti-Gαq/11 (sc-393, Santa Cruz Biotechnology, CA, USA), anti-IGFIIR (sc-25462, Santa-Cruz Biotechnology, CA, USA ), anti-PLC-β3 (#2482, Cell Signaling, Danvers, MA, USA ), anti--phosoho-PLC-β3 Ser537 (#2481, Cell Signaling Danvers, MA, USA), and anti-α-Tubulin (sc-58667, Santa Cruz Biotechnology, CA, USA). Then the 1° Ab TBS solutions were recycled. The PVDF membranes were washed with 1X TBS 3 times and soaked and rocked in 1:1000 secondary (2°) Ab solutions for 1 hour at room temperature. The following secondary antibodies were used: anti-goat-HRP (sc-2354, Santa Cruz Biotechnology, CA, USA), anti-rabbit-HRP (sc-2004, Santa Cruz Biotechnology, CA, USA) and anti-mouse-HRP (sc-2005, Santa Cruz Biotechnology, CA, USA).

The PVDF membranes were washed with 1X TBS 3 times and protein expressions were detected with Western Blotting Luminol Reagent (21059, PIERCE, Rockford, IL, USA). Restore western blot stripping buffer (S-2048; Santa Cruz Biotechnology, CA, USA) was used to strip off any Ab on the PVDF membranes, and the membranes were washed with DDW 2 to 3 times to remove any residue of the buffer. Again, blocking buffer was used to prevent any nonspecific binding. New 1° and 2° Abs were used.

Immunofluorescent microscopy on caspase 3 activity

H9c2 cardiomyoblasts per well were grown in a 12-well plate (1 × 105 cells/well) containing DMEM (10% CCS). Twenty-four hours later, the cells were deprived of serum for 4 hours, and then 20, 40, and 60 μg/ml C. pilosula were added to appropriate wells. 10-7 M AngII was added 1 hour later, followed by 10-8 M Leu27-IGFII after another 2 hours. The cells were incubated in humidified air with 5% CO2 at 37 °C. Twenty-four hours after administration of Leu27-IGFII, each well was added with 0.5 ml fixation solution (4% paraformaldehyde in PBS, pH 7.4) at room temperature. The plate was rested for 1 hour. An 0.5 ml permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) was added to each well, and the 12-well plate was placed on ice for 2 minutes without shaking. CCS was diluted 10X with DDW to generate blocking buffer. 1 ml blocking buffer was added to each well and the 12-well plate was rested for 1 hour at room temperature. 1:100 1° Ab (1% anti-caspase 3) PBS solution was made. 300 μl 1:100 1° Ab solutions was added to each well and the plate was rocked for 20 seconds, sealed with plastic wrap, and then stored at 4 °C for 24 hours. 1° Ab solutions were recycled. Fluorescent 2° Ab (fluorescein conjugated secondary antibody anti-rabbit IgG) was diluted 10X with PBS and 200 μl 1:100 fluorescent 2° Ab solution was added to each well. The plate was rested for 1 hour at room temperature. 200 to 300 μl 10000X diluted DAPI reagent was added to each well and the plate was covered with tin foil and rested for 2 to 5 minutes. The H9c2 cells were observed under microscopy.

Statistical analysis

Each sample was analyzed based on results that were repeated at least three times, and SigmaPlot 10.0 software and standard t-test was used to analyze each numeric data. In all cases, differences at p < 0.05 were regarded as statistically significant, and ones at p < 0.01 or p < 0.001 were considered to have higher statistical significance.

RESULTS

Optimal doses of C. pilosula that decreased IGFIIR-induced H9c2 apoptosis

To assess doses of C. pilosula that attenuates AngII plus Leu27-IGFII, first dose of Leu27-IGFII was assessed at 10-7 M AngII. At 10-7 M AngII, only 10-8 M Leu27-IGFII significantly reduced cell viability (22.49% reduction compared to control) [Figure 1(A)] and therefore this dose of Leu27-IGFII was chosen for later experiments. Then doses of C. pilosula were assessed: 0, 2, 4, 6, 8 and 10 μl of 10 mg/ml C. pilosula solution was added to each well (which contained 1 ml serum-free medium) to give final concentrations of 0, 20, 40, 60, 80 and 100 μg/ml in a 12-well plate; only 20, 40 and 60 μg/ml displayed increased cell survival compared to that of AngII plus Leu27-IGFII (36.21%, 45.34%, 58.82% increase, respectively) [Figure 1(B)]. Although 80 and 100 μg/ml also showed higher survival than AngII plus Leu27-IGFII (35.16% and 37.45%, respectively), these doses showed less cell survival than that of 60 μg/ml and therefore, 20, 40 and 60 μg/ml were chosen for later experiments.

C. pilosula is able to decrease H9c2 apoptosis induced by AngII plus Leu27-IGFII

As mentioned previously, the insertion of unphosphorylated Bad into mitochondrial membrane MOMP, resulted in activation of caspases 9 and 3 and apoptosis. To measure levels of H9c2 cell apoptosis influenced by AngII, Leu27-IGFII and C. pilosula, TUNEL assay was conducted. In Figure 2(A), the number of TUNEL-positive cells (indicated by green spots observed in FITC) in the control well was small. DAPI marks locations of cell nuclei. 10-7 M AngII plus 10-8 M Leu27-IGFII along caused a significant increase in TUNEL-positive cells [4.84-fold increase compared to control; Figure 2(B)]. However, this was attenuated by C. pilosula in a dose dependent manner (29.71 %, 45.52 % and 70.52 % decrease by 20, 40 and 60 μg/ml C. pilosula compared to AngII plus Leu27-IGFII, respectively). These results indicated that AngII plus Leu27-IGFII causes are able to induce apoptosis in H9c2 cells and this situation was negatively regulated by C. pilosula.

Figure 2.

Figure 2

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C. pilosula attenuated AngII plus Leu27-IGFII-induced H9c2 apoptosis. Twenty-four hours after growing H9c2 cells in 12-well plates, serum was derived for 4 hours. C. pilosula was added first, followed by 10-7 M AngII after 1 hour and then 10-8 M Leu27-IGFII after another 2 hours. (A) TUNEL staining assay was conducted 24 hours after addition of Leu27-IGFII. DAPI was used to label nuclei (blue, upper panels) and TUNEL was used to label apoptotic nuclei (green, lower panels). (B) Statistical analysis of apoptosis of H9c2 cells after treating with AngII plus Leu27-IGFII and C. pilosula, AngII, concentration: 10-7 M; L27: Leu27-IGFII, concentration: 10-8 M; CP: Codonopsis pilosula. *** p < 0.001 vs. control (mean ± S.E., n = 3), # p < 0.05 vs. AngII plus Leu27-IGFII treatment (mean ± S.E., n = 3) and ## p < 0.01 vs. AngII plus Leu27-IGFII treatment (mean ± S.E., n = 3).

AngII plus Leu27-IGFII-induced caspase 3 activity is downregulated by C. pilosula in H9c2 cells

From previous results, C. pilosula downregulated apoptosis induced by AngII plus Leu27-IGFII in H9c2 cells. But, how does C. pilosula downregulate AngII plus Leu27-IGFII-induced caspase 3 activity? Western blot analysis was conducted to investigate the downregulating effect of C. pilosula on AngII plus Leu27-IGFII-induced caspase 3 activity. Figure 3 shows that caspase 3 activity was significantly induced by 10-7 M AngII plus 10-8 M Leu27-IGFII, although there wasn’t a clear trend.

Figure 3.

Figure 3

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C. pilosula inhibited the caspase-3 activation induced by AngII plus Leu27-IGFII of H9c2 cardiomyoblast cells. Twenty-four hours after growing H9c2 cells in 10-cm plates, serum was derived for 4 hours. C. pilosula was added first, followed by 10-7 M AngII after 1 hour and then 10-8 M Leu27-IGFII after another 2 hours. Western blotting assay was conducted 24 hours after addition of Leu27-IGFII.

To determine this attenuation of caspase 3 activity, immunofluorescence microscopy was conducted. In Figure 4, there is an absence of caspase 3 activity in the control, indicated by the absence of FITC (green) signal. DAPI marks locations of cell nuclei. AngII plus Leu27-IGFII along significantly increased caspase 3 activity (second column from the left). C. pilosula dose-dependently downregulated this situation at 20, 40 and 60 μg/ml.

Figure 4.

Figure 4

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C. pilosula attenuated AngII plus Leu27-IGFII-induced caspase 3 activity in H9c2 cells. Twenty-four hours after growing H9c2 cells in 24-well plates, serum was derived for 4 hours. 20, 40 and 60 mg/ml C. pilosula doses were added first, followed by 10-7 M AngII after 1 hour and then 10-8 M Leu27-IGFII after another 2 hours. Immunofluorescence microscopy was conducted 24 hours after addition of Leu27-IGFII. DAPI was used to label nuclei (blue, upper panels). A: AngII, concentration: 10-7 M; L27: Leu27-IGFII, concentration: 10-8 M; CP: Codonopsis pilosula.

In summary, the above results indicated an attenuation effect of C. pilosula on AngII plus Leu27-IGFII-induced caspase 3 activity.

DISCUSSION

Chu et al. from our lab previously discovered that AngII or abdominal aortic ligation was able to induce expression of IGFII and IGFIIR and that IGFII is capable of binding to IGFIR and IGFIIR in order to cause cardiac hypertrophy. 25 In earlier findings, binding of IGFI to IGFIR induces pathological cardiac hypertrophy. 28 In addition, binding of IGFI to IGFIR or IGFIIR both induces physiological hypertrophy, in which when demand for higher blood flow is removed, cardiomyocytes return to non-hypertrophied size. 29 However, the binding of IGFII to IGFIIR induces pathological hypertrophy, in which there is an accumulation of intracellular Ca2+ ions and an increased activity of calcineurin. 21 However, the elevated Ca2+ concentration and calcineurin activity lead to mitochondrial membrane potential instability and apoptosis of cardiomyocytes. 21,25 Besides, Leu27-IGFII, an analog of IGFII, 24 strongly binds to IGFIIR and induces cardiomyocytes apoptosis via Gαq. 26 Therefore, it was used here to enhance the apoptotic effect of AngII.

In the present study, AngII plus Leu27-IGFII was able to increase the level of H9c2 cell apoptosis, as indicated by the increase in TUNEL-positive cells (Figure 2). However, this situation was reversed dose-dependently by C. pilosula, leading to an apoptotic level that was similar to control. Protein level of activated caspase 3 was increased by AngII plus Leu27-IGFII (Figure 3) although there wasn’t a distinct trend.

In Figure 4, again, AngII plus Leu27-IGFII induced a significant rise in caspase 3 activity, as indicated by a sharp increase in FITC fluorescence signal intensity. However, this increase in FITC fluorescence was reversed by C. pilosula in a dose-dependent manner (20, 40 and 60 μg/ml), indicating a dose-dependent decrease in caspase 3 activity by C. pilosula. Moreover, these results mirrored the fact that AngII plus Leu27-IGFII induced a significant apoptosis of H9c2 cells which was reverted by C. pilosula, as shown in our lab’s previous report by Tsai KH, et al. 30

Again, from studies of Chu et al., AngII is able to induce expression of IGFII and IGFIIR, leading to apoptosis via elevated Ca2+ concentration and MOMP induction in H9c2 cells. 21,25 It was also pointed out in his studies that AngII also caused an increase in release of mitochondrial components (such as cytochrome c) and activation of caspases 9 and 3 and that Leu27-IGFII also induced the same apoptotic pathway. In the present study, AngII plus Leu27-IGFII induced a synergistic increase in apoptosis and caspase 3 activity. Chu, et al. and Tsai KH, et al. also observed the same phenomena by treating H9c2 cells with AngII and/or Leu27-IGFII. 21,25,30 C. pilosula was able to reduce such effect dose-dependently. These means that C. pilosula must be capable of reducing AngII plus Leu27-IGFII-induced Ca2+ influx, MOMP, caspase 3 activity and apoptosis in H9c2 cardiomyoblasts, as observed in a previous study from our lab. 30 However, the upstream molecular marker in the pathway C. pilosula mediates through still remains to be determined.

Finally, certain inherited heart diseases with increased intracellular Ca2+ levels and deregulated heart functions often associates with elevated AngII and IGFII. 21,22,25,31,32 In this study, C. pilosula was shown to inhibit AngII plus Leu27-IGFII-induced apoptosis. This implies that clinically, C. pilosula can be used as a pre-ventive treatment or as a solution to the above problems.

CONCLUSION

In conclusion, in this study, it was found that AngII plus Leu27-IGFII induced a significant increase in caspase 3 activity and apoptosis in H9c2 cells. However, C. pilosula was able to reduce these events in a dose--dependent manner. Therefore, C. pilosula must be able to directly inhibit AngII plus Leu27-IGFII-induced apoptotic pathway (Figure 5).

Figure 5.

Figure 5

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C. pilosula suppresses the synergistic apoptotic effects induced by AngII plus Leu27-IGFII H9c2 cardiomyoblasts.

Acknowledgments

This study is supported by CMU98-S-42, CMU98-P-01 and M and in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TB-B-111-004).

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