CHAPTER I Introduction……………………………………….…………………1-13
2. Cardiac hypertrophy
Hypertrophy is used to describe cardiac response to stress under one or a few specific pathophysiological conditions, in which cardiac enlargement was largely assumed to be the result of increased cardiomyocyte size (Dorn et al, 2003). It is often associated with disease, including ischemic heart disease, hypertension and heart failure.
At the cellular level, cardiomyocyte hypertrophy is characterizedby an increment in cell size, increased protein synthesis, andchanges in the organization of sarcomeres. In contrast to developmental growth of heart through hyperplasia, growth of the postnatal heart occurs primarily through hypertrophy due to significant reduction in cardiac myoctes proliferation capacity soon after birth.
2.2 Classification
Cardiac hypertrophy is not all detrimental, as extensive aerobic conditioning through exercise induces a state of physiological growth regarded as adaption in the long term. Hypertrophy can be classified into two types. One is ‘physiological’, which is the normal response to healthy exercise or pregnancy, and the other is ‘pathological’, which is the response to stress or disease (Figure II) (Barry et al, 2008). Physiological hypertrophy is reversible and occurs without morbid effect on cardiac function. This normal growth of heart or in conditioned athletes enhances cardiac output to meet increased metabolic demands. Pathological hypertrophy occurs in response to pathological stress signals, such as neurohormonal activation, inflammation or cardiac injury. It has traditionally been considered to be adaptive initially, allowing the heart to increase cardiac output and compensate for adverse hemodynamics. In animal models,
inhibition of cardiac hypertrophy with cyclosporine A was demonstrated to result in increased mortality because of heart failure (Meguro et al, 1999). Prolonged hypertrophy is, however, associated with a significant increase in the risk for sudden death or progression to heart failure. At the molecular level, pathological hypertrophy is characterized by the activation of fetal genes, for example ANP, BNP, and β-MHC, which are considered and employed as hypertrophic markers (Cameron & Ellmers, 2003;
Rohini et al, 2010). Classically, hypertrophic growth develops in two ways: (1) concentric hypertrophy due to chronic pressure overload leading to reduced left ventricular volume and increased wall thickness, and (2) eccentric hypertrophy due to volumeoverload or prior infarction causing dilation and thinning of the heart wall.
Eccentric hypertrophy occurs by addition of the contractile sarcomeres in series causing cell elongation whereas concentric hypertrophy is caused by parallel addition of sarcomeres and lateral growth of individual cardiomyocytes (Heineke & Molkentin, 2006). Physiological hypertrophy is referred to as eccentric hypertrophy, and pathological hypertrophy can produce concentric hypertrophy.
Figure II. Pathological and physiological hypertrophic response to stimuli (Barry et al, 2008).
3. Endoplasmic reticulum (ER) stress
example, GRP78, which is the ER-located member of the family of heat shock protein 70 molecular chaperones, promotes the folding of hydrophobic regions in polypeptides to the interior in a Ca2+-dependent manner (Gething, 1999). If the influx of nascent, unfolded polypeptides exceeds the folding and/or processing capacity of the ER, the ER homeostasis is compromised. Perturbation of ER-associated functions leads to ER stress via the activation of complex cytoplasmic and nuclear signaling pathways, collectively termed the unfolded protein response (UPR), also known as misfolded protein response, resulting in upregulation of expression of ER resident chaperones, facilitating the degradation of misfolded proteins and inhibiting protein synthesis to return the ER to its normal physiological state. When these adaptive responses are not sufficient to relieve ER stress, cells subjected to sustained and irreversible stress undergo programmed cell death.3.2 UPR signaling
Three steps are involved in UPR activation. First, translation is attenuated to avoid further accumulation of misfolded proteins in the ER; second, chaperone and protein folding genes are activated transcriptionally; and the last, ER-associated degradation is activated in an attempt to rectify the accumulation of misfolded protein (Schröder &
Kaufman, 2005b). The UPR to ER stress can be initiated by activating three ER membrane receptors to initiate adaptive responses, including protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring protein-1 (IRE1), and the transcriptional factor activating transcription factor-6 (ATF6) (Kaufman, 2002; Kim et al, 2008; Ron & Walter, 2007). These receptors are located with their N-terminus inside the lumen of the ER and their C-terminus in the cytosol, thereby connecting the ER with the cytosol. All three receptors are maintained in an inactive state through the interaction of their N-terminus with GRP78. When unfolded proteins accumulate in the ER, GRP78 releases these receptors to allow their activation (Figure III). PERK is activated first, rapidly followed by ATF6, whereas IRE1 is the last. Activated PERK phosphorylates eukaryotic translation initiation factor 2 (eIF2α) and consequently blocks its translation. However, eIF2α phosphorylation enables translation of ATF4, which occurs through an eIF2α-independent translation pathway. ATF4 translocates to the nucleus and induces the UPR-related genes. ATF6 is activated by limited proteolysis after its translocation from the ER to the Golgi apparatus. ATF6 is also a transcription factor and activates a subset of the UPR-related genes, including X-box binding protein 1 (XBP1). To achieve its active form, XBP1 must undergo mRNA splicing, which is carried out by IRE1. The three arms of the UPR, including ATF4, XBP1 and ATF6, all of which can induce transcription of CHOP, coordinately regulate the transcription of UPR-related genes encoding ER chaperones and protein folding enzymes to reduce the accumulation of unfolded proteins.
Figure III. The unfolded protein response (Szegezdi et al, 2006).
3.3 The relationship of DEPs and ER stress
A recent study on the molecular basis of PM2.5-induced intracellular events has highlighted the activation of a pathophysiological ER stress response upon PM2.5 exposure (Laing et al, 2010). It suggested that PM2.5 exposure differentially activates the UPR branches in C57BL/6 male mice, leading to ER stress-induced apoptosis through the PERK-eIF2α-CHOP UPR branch. PM2.5 also caused the upregulation and activation of caspase-12, and also induced ER stress in human bronchial epithelial cells (Watterson et al, 2009; Watterson et al, 2007). Similarly, the human bronchial epithelial cells treated with organic DEP extracts were showed an induction in a proinflammatory response accompanied by the UPR, such as Hsp70, ATF4, IL-6, and IL-8 (Jung et al, 2007). In addition, 1-nitropyrene (1-NP) is one of the most abundant nitro-PAHs in diesel exhaust and a major contributor to the mutagenicity of diesel exhaust particulate matter. And previous study has demonstrated that low levels of 1-NP (≦10 μM) induced DNA damage, increased intracellular ROS levels and increased protein
expression of the ER stress chaperone GRP78 in human umbilical vein endothelial cells (Andersson et al, 2009).
3.4 ER in the heart
As other cells, cardiac cells require ER membrane for housekeeping functions like protein turnover, modification and folding. ER in cardiomyocytes is involved in continuous turnover and synthesis of many membrane proteins including ion channels gap junction components and cell surface receptors (Mesaeli et al, 2001). The ER in non-muscle cells and SR in cardiac and skeletal muscle cells are also considered one of the most important and metabolically relevant sources of cellular Ca2+ for variety of functions including secretion, contraction-relaxation, cell motility, cytoplasmic and mitochondrial metabolism, protein synthesis, modification and folding, gene expression, cell cycle progression and apoptosis (Clapham, 1995; Pozzan et al, 1994). In the myocardium, the ER involves in maintenance of cellular Ca2+ homeostasis and synthesis of secretory proteins such as ANP, BNP (Forssmann et al, 1989), and vascular cardiovascular community. The UPR and/or ER-initiated apoptosis have been implicated in the pathophysiology of various cardiovascular diseases such as cardiac hypertrophy, heart failure, atherosclerosis, and ischemic heart disease (Table I). Heat, hypoxia, ischemia, disease, glucose and metabolic starvation are potent inducers of the
ER stress. The response to ER stress occurs in two phases (Groenendyk et al, 2010).
First, the pathway is turned on in an attempt to address one consequence of the insult, the accumulation of protein in the ER. The fetal gene program is also activated in attempt to remodel the diseased tissue. If the heart continues being stressed, the UPR triggers autophagy and apoptosis to deal with the problem. This may ultimately result in heart failure and death. Therefore, the response to insult or injury can be classified into a pathologically relevant ER stress response and physiologically relevant ER stress response. XBP1 and ATF6 mediate induction of ER chaperones, protecting the heart from ischemia/reperfusion injury (Martindale et al, 2006), whereas activation of the PERK/ATF4/CHOP branch of the UPR triggers the pro-apoptotic signals. Recent observations indicate that in the heart, the UPR is activated during acute stresses, including ischemia/reperfusion, as well as upon longer term stresses that lead to cardiac hypertrophy and heart failure. For example, GRP78, CHOP, caspase-12, and phospho-JNK were significantly increased in rats with abdominal aortic coarctation (Guan et al, 2011). ATF6 was activated by ischemia but inactivated upon reperfusion in a cultured cardiomyocyte model system of simulated ischemia/reperfusion, suggesting that it may play a role in the induction of ER stress response genes during ischemia that could have a preconditioning effect on cell survival during reperfusion (Doroudgar et al, 2009). Mice subjected to transverse aortic constriction had cardiac hypertrophy and failure and showed an induction in prolonged ER stress, which might contribute to cardiomyocyte apoptosis during progression from cardiac hypertrophy to failure (Kitakaze & Tsukamoto, 2010; Okada et al, 2004). Besides, ER Ca2+ depletion occurs in the presence of ER stress inducer thapsigargin. Increases in cytosolic Ca2+ through changes in excitation-contraction coupling in cardiomyocytes such as that demonstrated for angiotensin II (Gusev et al, 2009) may drive the hypertrophic pathway and lead to
ER stress induction because of ER Ca2+ depletion. On the other hand, CHOP has shown to be responsible for ER stress-induced Puma, p53-upregulated modulator of apoptosis, activation during neonatal cardiomyocyte apoptosis (Nickson et al, 2007).
Cardiomyocytes treated with imatinib showed activation of the ER stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death (Kerkelä et al, 2006). In cardiomyocytes, activation of AMP-activated protein kinase (AMPK) contributes to protection of the heart against hypoxic injury through down regulation of ER stress (Terai et al, 2005). And inhibition of protein synthesis via eukaryotic elongation factor 2 inactivation may be the mechanism of cardioprotection by AMPK. Overexpression of ATF6, GRP78 and Derlin3 protects cardiomyocytes from ischemia/reperfusion damage (Belmont et al, 2009; Fu et al, 2008; Martindale et al, 2006)
Table I. ER stress and cardiovascular disease (Minamino & Kitakaze, 2010).
4. Specific aims
Numerous Epidemiological Studies have reported consistent associations between exposures to particulate air pollution and cardiovascular mortality and morbidity. In particulate air pollution, DEPs with a mean size of about 0.2 μm are a major component of PM2.5. Previous study demonstrated that traffic exposure is associated with higher left ventricular mass in adults. In cellular level, it was reported that DEPEs decreased cardiomyocytes viability and it might be due mainly to reactive oxygen species formation. However, the mechanisms by which DEPs produce adverse cardiovascular effects at the cellular level are not fully understood. The present study is designed to examine the actions and possible mechanisms of DEPs on growth and function of cardiomyocytes.
CHAPTER II
Materials and Methods
1. Reagents and Antibodies
Standard diesel particulate matter (DPM, SRM 2975) was purchased from National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA). Anti-phospho (P)-p38, anti-P-JNK, anti-GRP78, anti-CHOP, secondary horseradish peroxidase (HRP)-conjugated antibodies, and polyclonal rabbit anti-desmin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-PKC was from Cell Signaling Technology (Danvers, MA, USA). FITC-goat anti-rabbit IgG secondary antibodies were purchased from Zymed Laboratories (South San Francisco, CA, USA). Hoechst 33258 nuclear dye and SP600125 (JNK inhibitor) were obtained from Sigma–Aldrich Corporation (St. Louis, MO, USA)
2. Preparation of DEPEs
This method was carried out using a protocol modified from Tzeng et al.
(2003). Certified values for concentratio ns are provided for 11 PAHs in Table 1 (National Institute of Standards and Technology, 2009). Reference values for concentrations are provided for 28 additional PAHs in Table 2 and for 17 nitro-substituted PAHs in Table 3 (National Institute of Standards and Technology, 2009). In brief, DEPs were immersed in solvent (dichloromethane : n-hexane [1:1, v/v]) in the dark for 18 hours (Escobal et al, 1997).
And the DEPs were then sonicated for 1 hour and centrifuged at 6,000 rpm for 15 min at
4℃. The supernatant was filtered by HPLC filter to obtain the DEPEs solution (Figure IV). The mixture was resuspended in solvent, sonicated and centrifuged at 6,000 rpm for 15 min at 4℃. The three steps were repeated for 4 times. The DEPEs solution was concentrated by a vacuum evaporator to get DEPEs (Figure V). Vacuum evaporation is the process of causing the pressure in a liquid-filled container to be reduced below the vapor pressure of the liquid, causing the liquid to evaporate at a lower temperature than normal. The crude DEPEs was dried by air flow, weighed, dissolved in dimethyl sulfoxide (DMSO) at a desired concentration, and stored at -20℃. After DEPEs were diluted by a certain medium, the final concentration of DMSO was 0.05 % (v/v).
Figure IV. Filtration system. Figure V. Vacuum evaporator.
3. Preperation of Neonatal Rat Cardiomyocytes
This method was carried out using a protocol modified from Cheng et al.
(1999). Primary cultures of neonatal rat cardiomyocytes were prepared from ventricles
of 1- to 3-day-old neonatal Wistar rats. Briefly, hearts were cut into chunks of approximately 1 mm3 using scissors and treated with pancrease (Sigma–Aldrich Corporation) at 37 ℃ for 15 min. Pancrease digested cells were collected by centrifugation at 1,600 rpm for 3 min. The cell pellets were resuspended in F10 medium containing 20% (v/v) fetal bovine serum (FBS) and plated into a Petri dish for 2 hours.
The nonattached myocytes in the F10 medium (Sigma–Aldrich Corporation) were collected and centrifuged at 1,600 rpm for 3 min. The cell pellets were resuspended in Dulbecco’s modified Eagle medium (DMEM; Gibco) containing 0.1 mM 5-bromo-2'-deoxyuridine (BrdU; Sigma–Aldrich Corporation) and 10% (v/v) FBS. The cells were then seeded on culture dishes at an appropriate cell density and cultured at 37°C in 95% air-5% CO2. After 2 days, cardiomyocytes obtained were 80% pure as revealed by their contractile characteristics under light microscopy. The culture medium was changed to fresh DMEM containing BrdU and 0.2% (v/v) FBS for 12 hours before exposure to DEPEs.
4. H9c2 Cell Cultures
H9c2 cells derived from embryonic rat heart myocardium were obtained from American Type Tissue Collection (ATCC, Rockville, MD, USA). The cells were grown in DMEM supplemented with 10% FBS at 37°C in 95% air-5% CO2. When they reached 70–85% confluency, the cells were washed with phosphate-buffered saline (PBS) (pH 7.4), trypsinized, and centrifuged at 1,000 rpm for 5 min. the cell pellets were resuspended in DMEM containing 10% (v/v) FBS and further passaged or appropriately treated. The culture medium was changed to fresh DMEM 1% (v/v) FBS for 12 hours before exposure to DEPEs.
5. Preparation of Total Cell Lysates
Cells were washed with ice-cold PBS and lysed with RIPA buffer (20 mM Tris-HCl (pH7.4), 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 0.1%
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium fluoride (NaF), 1 μg/ml leupeptin, and 1 μg/ml aprotinin). PMSF and NaF in the buffer were the serine protease inhibitor and phosphatase inhibitor, respectively. The lysates were left on ice for 10 min, and centrifuged at 10,000 rpm for 10 min at 4℃. The supernatants were normalized for protein concentration by BCATM Protein Assay Kit (PIERCE) with bovine serum albumin as standard.
6. Western Blotting
Equal amounts of proteins (50 μg per lane) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electrotransfer to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked with 5% nonfat powder milk in 0.1% Tris-buffered saline + Tween 20 (TBST) for 1 hour and probed with various primary antibodies at 4℃. Subsequently, membranes were washed three times with 0.1% TBST and incubated with secondary HRP-conjugated antibodies at room temperature for 1 hour. After three washes, the signals were visualized by an enhanced chemiluminescence reagent detection system according to the manufacturer’s protocol (Millipore Corporation).
7. Measurement of Cell Viability
The yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT;
Sigma-Aldrich Corporation) is reduced by mitochondrial succinate dehydrogenase in
viable cells to form insoluble purple formazan crystals, which are solubilized by DMSO.
In brief, cells seeded in 96-well plates were treated with DEPEs for 24 hours and then stained with MTT (0.5 mg/ml) for 4 hours. The media were removed, and formazan crystals produced were dissolved in 100 μL DMSO (Sigma–Aldrich Corporation). The absorbance was measured at 570 nm.
8. Lactate Dehydrogenase (LDH) Release Assay
After exposure to DEPEs for 24 hours, the amount of LDH leaked from the cytosol of damaged cells into the medium was detected. The released LDH was quantified by Cyto 96® Non-Radioactive Cytotoxicity Assay (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. The absorbance was measured at 492 nm.
9. Analysis of Intracelluler ATP Levels
The intracellular ATP content was measured by Adenosine 5’-triphosphate bioluminescent assay kit (FL-AA, Sigma-Aldrich Corporation). After exposure to different concentrations of DEPEs for 24 hours, cells were washed two times with PBS and lysed with RIPA buffer. The lysates were centrifuged at 10,000 rpm for 10 min at 4℃. Subsequently, 100 μL supernatant and 100 μL ATP Assay Mix solution were mixed and immediately measured the amount of light produced with Orion L Microplate Luminometer (Berthold Detection Systems, Bad Wildbad, Germany).
10. Annexin V-FITC apoptosis detection
H9c2 cells were cultured in 6-well plates. Cells were treated with DEPEs for 24
hours, and then apoptosis was assessed by using an annexin V-FITC apoptosis detection kit (Becton Dickinson, Franklin Lakes, NJ, USA). In brief, cells were dissociated by 0.05% trypsin/EDTA for 3 min, and then centrifuged at 1,000 rpm for 5 min and re-suspended in 100 μL 1X binding buffer, and transferred into a 5 mL FACS tube, and added 5 μL annexin V-FITC (conjugated with fluorescein isothiocyanate) and 10 μL propidium iodide (PI). After incubation for 30 min at room temperature in dark, 400 μL of 1X binding buffer was added to each tube and the samples were immediately analyzed using a FACS flow cytometer.
11. Total RNA Extraction
Total RNA was isolated from cells with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) (1 mL to 1 × 106 cells), followed by chloroform extraction and isopropanol precipitation. The RNA was washed with 75% ethanol, resuspended in RNase-free water and, incubated at 65℃ for 5 min.
12. Real-time Reverse Transcription-Polymerase Chain Reaction
1 μg RNA was reverse transcribed using SuperScriptTM III First-Strand Synthesis System (Invitrogen). The RT reaction products were diluted to the volumes of 256 μL, and 1-μL aliquots were used as template. The mRNA expression levels were quantified by StepOne Real-Time PCR Detection Systems (Applied Biosystems, Warrington, UK) using SYBR® GreenERTM qPCR SuperMix (Invitrogen). Thermal cycler conditions were initiated at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15
s, and annealing/extension at 60 °C for 1 min. Polymerase chain reaction products were confirmed by melting curve analysis to ascertain the specificity of the primers and the purity of the final PCR product. The sequences of the primers used are shown in table 4.
The mRNA expression was normalized to expression of the housekeeping gene GAPDH amplified in a separate reaction.
13. Measurement of Protein Content Per Cell
After treatment with DEPEs for 24 hours, cells were trypsinized and washed twice with PBS. Cells were then collected via centrifugation at 1,000 rpm for 5 min, stained with trypan blue, and the viable cells were counted by microscopic examination. Cells were lysed with RIPA buffer as described above, and the lysates was centrifuged at 10,000 rpm for 10 min at 4℃. Protein concentration in the lysates was measured using BCATM Protein Assay Kit (PIERCE). Protein content per cell was determined by dividing the total amount of protein by the cell number.
14. Immunofluorescence
This method was carried out using a protocol modified from Liu et al.
(2009). To measure the cell size of cardiomyocytes, cells cultured in 12-well plates were washed with PBS followed by fixation with 4% paraformaldehyde in PBS at room temperature for 10 min. After three washes, the cells were permeabilized with 0.2%
Triton-100 for 5 min at room temperature. Non-specific binding of the fixed cells was blocked with PBS containing 2% bovine serum albumin for 1 hour, and cells were incubated overnight at 4℃ with polyclonal rabbit anti-desmin antibody diluted with blocking solution to 1:200. After three washes with PBS, cells were incubated for 1
hour at room temperature in the dark with FITC-goat anti rabbit IgG secondary antibodies diluted in blocking solution to 1:1,000. Finally, Hoechst 33258 nuclear dye was added with secondary antibodies at a final concentration of 1 μg/mL for 15 min, and cells were washed three times in PBS. Fluorescent images were captured by a Leica DMIL inverted microscope equipped with a charge-coupled device camera and SPOT
hour at room temperature in the dark with FITC-goat anti rabbit IgG secondary antibodies diluted in blocking solution to 1:1,000. Finally, Hoechst 33258 nuclear dye was added with secondary antibodies at a final concentration of 1 μg/mL for 15 min, and cells were washed three times in PBS. Fluorescent images were captured by a Leica DMIL inverted microscope equipped with a charge-coupled device camera and SPOT