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ARTICLE Involvement of cardiomyocyte apoptosis in myocardial injury of hereditary epileptic rats Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV GUELPH on 11/18/14 For personal use only.
Fan Chen, Yong-gang Cao, Han-ping Qi, Lei Li, Wei Huang, Ye Wang, and Hong-li Sun
Abstract: Many clinical cases have been reported where epilepsy profoundly influenced the pathophysiological function of the heart; however, the underlying mechanisms were not elucidated. We use the tremor (TRM) rat as an animal model of epilepsy to investigate the potential mechanisms of myocardial injury. Cardiac functions were assessed by arrhythmia score, heart rate, heart:body mass ratio, and hemodynamic parameters including left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), and maximum rate of left ventricular pressure rise and fall (+dp/dtmax and –dp/dtmax). Catecholamine level was detected by HPLC. Apoptotic index was estimated by TUNEL assay. The expressions of Bcl-2, Bax, caspase-3, extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinases (JNK), and p38 were evaluated by Western blot. The results indicated that there existed cardiac dysfunction and cardiomyocyte apoptosis, accompanied by increasing catecholamine levels in TRM rats. Further investigation revealed that apoptosis was mediated by reducing Bcl-2, upregulating Bax, and activating caspase-3. Additional experiments demonstrated that P-ERK1/2 was decreased, whereas P-JNK and P-p38 were upregulated. Our results suggest that the sympathetic nervous system activation and cardiomyocyte apoptosis are involved in the myocardial injury of TRM rats. The mechanisms of apoptosis might be associated with the activation of the mitochondriainitiated and the mitogen-activated protein kinase pathways. Key words: epilepsy, tremor rat, myocardial injury, cardiomyocyte, apoptosis. Résumé : Plusieurs cas cliniques où l'épilepsie influençait profondément les fonctions pathophysiologiques du cœur ont été rapportés ; toutefois, les mécanismes sous-jacents n'ont pas été élucidés. Nous avons utilisé le rat « tremor » (TRM) comme modèle animal d'épilepsie afin d'examiner les mécanismes potentiellement responsables du dommage myocardique. Les fonctions cardiaques ont été évaluées par le score d'arythmie, le rythme cardiaque, le rapport du poids du cœur sur le poids corporel, ainsi que par les paramètres hémodynamiques, dont le taux maximum d'élévation et de chute de la pression ventriculaire gauche (+dp/dtmax et –dp/dtmax). Le niveau de catécholamines a été détecté par HPLC. L'indice d'apoptose a été estimé par un dosage TUNEL. L'expression de Bcl-2, de Bax, de la capspase-3, des kinases ERK (extracellular signal-regulated kinase), JNK (c-Jun NH2-terminal protein kinase) et p38 a été évaluée par buvardage Western. Les résultats ont révélé une dysfonction cardiaque et la présence d'apoptose chez les cardiomyocytes, accompagnées par un accroissement des niveaux de catécholamines chez les rats TRM. Une recherche plus poussée a révélé que la réduction de Bcl-2, l'augmentation de Bax et l'activation de la caspase-3 étaient les médiateurs de l'apoptose. Des expériences additionnelles ont démontré que le niveau de P-ERK1/2 était diminué alors que ceux de P-JNK et de P-p38 étaient accrus. Nos résultats suggèrent que l'activation du système nerveux sympathique et l'apoptose des cardiomyocytes sont impliqués dans le dommage myocardique chez les rats TRM. Les mécanismes d'apoptose pourraient être associés a` l'activation des voies signalétiques issues des mitochondries et des protéines kinases activées par les mitogènes. [Traduit par la Rédaction] Mots-clés : épilepsie, rat « tremor », dommage myocardique, cardiomyocyte, apoptose.
Introduction Epilepsy is one of the most common neurological disorders. Characterized by recurrent seizures, it affects up to 50 million people worldwide (Carpio and Hauser 2009). Patients with epilepsy have consistently been found to have a higher mortality rate than the general population (Cockerell et al. 1994; Nashef et al. 1995), with a higher frequency in the younger patients (Nilsson et al. 1997; Annegers and Coan 1999; Ding et al. 2006). The increased mortality in people with epilepsy is due to a wide range of causes (Nilsson et al. 1997), including heart disease and pneumonia (Nashef et al. 2007; Hitiris et al. 2007; Sevcencu and Struijk 2010; Jansen and Lagae 2010). Sudden unexpected death in epilepsy (SUPEP) is the most common cause of seizure-related mortality in people with refractory
epilepsy. In 1906, Finny first documented cardiac asystole during a seizure. Since then, a wide range of cardiac arrhythmias have been reported, ranging from atrial fibrillation, supraventricular tachycardia, prolonged QT intervals, and torsades de pointes (Nilsson et al. 1997; Nei et al. 2004; Schuele 2009; Sevcencu and Struijk 2010). Also, a number of studies have indicated that seizure activity produces cardiac dysfunction and myocardial damage in human patients (Legriel et al. 2008; Shimizu et al. 2008) as well as animals experiencing seizures that were induced by lethal nerve agents (Tryphonas et al. 1996). Several studies have shown that seizures could result in myocardial injury by a variety of mechanisms. Available evidence has demonstrated that excessive stimulation of the sympathetic nervous system of the heart during seizures can result in cardiac contractile dysfunction, characterized by myofilament damage of the cardiomyocytes, myocardial
Received 3 January 2013. Accepted 16 May 2013. F. Chen,* Y.-g. Cao,* H.-p. Qi, W. Huang, Y. Wang, and H.-l. Sun. Department of Pharmacology, Harbin Medical University – Daqing, Daqing, Heilongjiang 163319, China. L. Li. Department of Surgery, The Fifth Clinical College of Harbin Medical University, Daqing, Heilongjiang 163316, China. Corresponding author: Hong-li Sun (e-mail: [email protected]). *Fan Chen and Yong-gang Cao contributed equally to this study.
Can. J. Physiol. Pharmacol. 91: 804–811 (2013) dx.doi.org/10.1139/cjpp-2013-0005
Published at www.nrcresearchpress.com/cjpp on 30 May 2013.
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fibrosis, arrhythmogenic alterations in cardiac electrical activity, and subsequently increased susceptibility to cardiac arrhythmias or ischemia (Ishiguro and Morgan 2001; Shimizu et al. 2008). So far, although the myocardial injury in epileptic patients has been reported, most of the data were acquired from a drug-induced model of epilepsy (Metcalf et al. 2009), and there has been no ideal animal model, comparable with epileptic patients, used in the experiments to evaluate the mechanisms of myocardial damage during seizures. The tremor rat (TRM; tm/tm), a genetic mutant that is found in a Kyoto–Wistar colony, exhibits both absence-like seizures and tonic convulsions without any external stimuli (Seki et al. 2004). Previous study has indicated that the absence-like seizures in the TRM rat are characterized by the paroxysmal occurrence of 5–7 Hz spike-wave-like complexes in hippocampal electroencephalograms (EEGs) at 8 weeks (Serikawa et al. 1987). However, in the intervening periods, normal EEG recordings free of absence-like seizures were found in the TRM rat (Serikawa et al. 1987). Thus, this TRM rat is most likely to be a useful genetic animal model of epilepsy. In this study, the TRM rat was selected as an animal model of epilepsy to determine the cardiac effects of seizures, and significant cardiac dysfunction has been observed in this animal. Therefore, these experiments were designed to investigate the potential mechanisms of myocardial injury in the TRM rat.
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Table 1. Alteration of heart rate and arrhythmia score in TRM rats. Group
n
Heart rate (beats/min)
Arrhythmia score
Control TRM
6 6
367±17 435±6**
0 56±4**
Note: Control, wild-type Wistar rats; TRM, tremor rats. Values are the mean ± SE; n = number of rats used; **, p < 0.01 compared with the control group.
Fig. 1. Alteration of heart:body mass ratio in tremor (TRM) rats. Statistical analysis of heart:body mass ratio. Data are the mean ± SE (n = 6 rats); **, p < 0.01 compared with the wild-type Wistar control group.
Materials and methods Experimental animals and reagents All experimental protocols were pre-approved by the Experimental Animal Ethic Committee of Harbin Medical University, China (Animal Experimental Ethical Inspection Protocol No 2009104). Wild-type Wistar rats (control) and TRM rats of either sex (250–300 g) at the age of 24 weeks were used in this study. Animals had free access to food and water and were housed under a controlled environment (12 h (light): 12 h (dark) cycle; 50%–70% humidity; 24 °C). Primary antibodies anti-Bcl-2, anti-Bax, anti-caspase-3, antiERK1/2, anti-JNK, and anti-p38 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). Anti-GAPDH was from Kangcheng, Inc. (Shanghai, China). The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) kit was from Roche (Mannheim, Germany). Arrhythmia scores Arrhythmia was quantitatively estimated according to the method described by Mest and Förster (1979). The degree of severity was assessed according to the following scale: 0 = sinus rhythm; 20 = first degree atrial–ventricular block, supraventricular arrhythmias; 40 = ventricular premature bigeminy, trigeminy, second degree atrial–ventricular block; 60 = polyphyleticism ventricular premature, paroxysmal ventricular tachycardia; 80 = ventricular fibrillation; 100 = death. Hemodynamic measurements The methods and procedures were exactly according to the methodology described in Liu et al. (2012). Briefly, rats were anesthetized with sodium pentobarbitone (40 mg/kg body mass, intraperitoneal (i.p.)), and a heparin-filled cannula was inserted into the right carotid artery and then advanced into the left ventricle. The hemodynamic parameters reflecting cardiac performances, including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and maximum rate of left ventricular pressure rise and fall (+dp/dtmax and –dp/dtmax) were measured at the same time by a pressure transducer interfaced with a BL-420E organism function experiment system (Cheng Du Tai Meng, Cheng Du, China).
Plasma sample preparation Blood samples were taken from all the groups, and an equal volume of sterile heparinized physiological saline was added. Each sample was centrifuged at 4000g for 15 min for preparing the plasma, and 200 L HClO4 (0.1 mol/L, Sigma), 50 L DHBA (2 g/mL), and 250 L plasma were added to a test tube. The mixture was kept at 4 °C for 5 min and centrifuged at 14 000g for 10 min. The supernatant was filtered (0.5 m) and made ready for catecholamine analysis. Catecholamine analysis by HPLC Adrenaline (AD) and noradrenaline (NA) are 2 main types of catecholamine in plasma. The levels of AD and NA in rat plasma were detected using HPLC. DHBA (3,4-dihydroxybenzylamine) was used as an internal standard in each sample. To the mobile phase was added 0.144 g sodium dodecyl sulfate (SDS; Sigma), 0.5 mL triethylamine (TEA; Merck), 10.5 g citric acid (CA; Merck), 4.1 g sodium acetate (SA, Sigma), 120 mL methanol (ME, Merck), 0.186 g ethylenediaminetetraacetic acid disodium salt (EDTA–2Na, Sigma), and 880 mL triple-distilled water. The flow rate was 0.8 mL/min. The fluorescence detector (RF-10Axl, Shimadzu; 330 nm for emission, 280 nm for excitation) was used to measure the levels of AD and NA. TUNEL assay The apoptotic ratio of cardiomyocytes was measured by TUNEL assay in accordance with the protocol of apoptosis detection kit. As seen under a light microscope, the normal nucleolus of cardiomyocytes stained blue and the apoptotic nucleolus stained brown. Apoptotic cells in each group were carefully evaluated under double-blind conditions. Ten different high-power fields (×400) were randomly selected, and the percentage of apoptotic cells was calculated as the ratio of the numbers of positivestaining nuclei to the numbers of total nuclei of the cells. Published by NRC Research Press
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Fig. 2. Alteration of hemodynamic parameters in tremor (TRM) rats. The hemodynamic parameters reflecting cardiac function were measured by left ventricle intubation via a pressure transducer interfaced with BL-420E organism function experiment system. (A) Left ventricular systolic pressure (LVSP). (B) Left ventricular end-diastolic pressure (LVEDP). (C) Maximum rate of left ventricular pressure rise and fall (+dp/dtmax and –dp/dtmax). Data are the mean ± SE (n = 6 rats per group); *, p < 0.05 compared with the wild-type Wistar control group.
Western blot analysis The left ventricular tissue was homogenized in PBS containing 1% triton X-100 and protease inhibitor cocktail. The homogenate was centrifuged at 14 000g for 30 min at 4 °C and the supernatant was collected, which was then mixed with sample loading buffer [2.5% sodium dodecyl sulphate (SDS), 10% glycerol, 50 mmol/L Tris–HCl, pH 6.8, 0.5 mol/L -mercaptoethanol, and 0.01% bromophenol blue] and boiled for 5 min. Samples of 50 g of protein from different experimental groups were separated by 10% SDS– PAGE and transferred to nitrocellulose membranes by electroblotting (200 mA for 1.5 h). The membranes were blocked with 5% non-fat milk in Tris-buffered saline – Tween 20 (TBS-T) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with the primary antibodies anti-Bax (1:200), antiBcl-2 (1:200), anti-caspase-3 (1:200), anti-ERK1/2 (1:200), anti-p38 (1:200), anti-JNK (1:200), and anti-GAPDH (1:5000), respectively. After rinsing 3 times with TBS-T at 10 min intervals, the membranes were then incubated with the secondary antibody (1:5000) for 1 h at room temperature in the dark. Immunolabeled protein bands were detected by an enhanced chemiluminescence kit (Pierce, Calif.). The volumes of the protein bands were quantified using a Bio-Rad Chemi Doc EQ densitometer and Bio-Rad Quantity One software (Bio-Rad Laboratories). GAPDH was used as an internal control for the relative quantification assay. Statistical analysis Data are the mean ± SE, and analyzed using SPSS 15.0 software. Student's t test was used to evaluate the differences between 2 groups. Values for p < 0.05 were considered to be statistically significant.
Results Alteration of cardiac function in TRM rats As shown in Table 1, there were no arrhythmias in the control group of rats. However, in TRM rats, premature ventricular contraction (PVC) and ventricular tachycardia (VT) were observed. The results from the statistical analyses showed that the arrhythmia score and heart rate were significantly increased (p < 0.01, compared with the control rats). Moreover, the heart:body mass ratio was also altered (3.16 ± 0.20 for the control compared with 4.68 ± 0.25 for TRM, p < 0.01) (Fig. 1). Other important indexes of cardiac function, such as LVSP, LVEDP, and ±dp/dtmax, were measured in both the control and TRM rats (Fig. 2). Compared with the control rats, TRM rats demonstrated a significant increase in LVEDP (7 ± 1 mm Hg for the control compared with 13 ± 1 mm Hg for TRM, p < 0.05; 1 mm Hg = 133.322 Pa) and decreases in LVSP (164 ± 6 mm Hg for the control compared with 92 ± 8 mm Hg for TRM, p < 0.05), +dp/dtmax (7196 ± 1000 mm Hg/s for the control compared with 2927 ± 1200 mm Hg/s for TRM, p < 0.05) and –dp/dtmax (–5447 ± 1050 mm Hg/s for the control compared with –2943 ± 1090 mm Hg/s for TRM, p < 0.05). These results indicated that cardiac dysfunction was developed in TRM rats. Detection of catecholamine level in rat plasma using HPLC It has been shown that epilepsy is associated with higher levels of catecholamines, which are known to cause myocardial injury (Sakamoto et al. 2008; Shimizu et al. 2008). To evaluate whether the plasma contents of catecholamine were changed in TRM rats, the levels of AD and NA were measured using Published by NRC Research Press
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Fig. 3. Detection of catecholamine level in rat plasma using HPLC. Adrenaline (AD) and noradrenaline (NA) are 2 main types of catecholamine in plasma. The levels of AD and NA in rat plasma were detected using HPLC. (A) Bar graph of AD. (B) Bar graph of NA. Data are the mean ± SE (n = 6 rats); **, p < 0.01 for tremor (TRM) rats compared with the wild-type Wistar control group.
Fig. 4. Cardiomyocyte apoptosis in tremor (TRM) rats was determined by TUNEL staining. (A) Representative examples of TUNEL staining. Nuclei with staining were TUNEL-positive cells (brown, on the Web site only), which were apoptotic. Magnification 400×. (B) Mean data of TUNEL-positive stained cells (n = 6 rats); **, p < 0.01 compared with the wild-type Wistar control group.
HPLC. As indicated in Fig. 3, the plasma AD and NA values were both higher in TRM rats than in the control rats. The level of AD was increased from 0.57 ± 0.03 g/mL to 0.72 ± 0.05 g/mL (p < 0.01), and NA was elevated from 0.13 ± 0.01 g/mL to 0.21 ± 0.03 g/mL (p < 0.01). Cardiomyocyte apoptosis in TRM rats was determined using TUNEL staining To elucidate the causes of cardiac dysfunction observed in TRM rats, cardiomyocyte apoptosis was assessed using TUNEL staining. The histological sections of the hearts were selected for locating apoptotic nuclei. As shown in Fig. 4, the apoptotic ratio of cardiomyocytes in TRM rats was increased significantly compared with
the control rats (3.4 ± 0.9 for the control compared with 18.5 ± 1.6 for the TRM, p < 0.01). Detection of Bcl-2 and Bax protein expression in rat hearts using Western blot To explore the potential mechanisms of epilepsy-induced myocardial damage, the cytoplasmic level of the anti-apoptotic signaling molecule Bcl-2 and pro-apoptotic molecule Bax were detected using Western blot. Averaged data (Fig. 5) revealed that in TRM rats, the expression of intracellular Bcl-2 was diminished (1.13 ± 0.06 for the control compared with 0.96 ± 0.07 for TRM, p < 0.05), and Bax expression was upregulated (0.34 ± 0.09 for the control compared with 1.16 ± 0.10 for TRM, p < 0.01), respectively. Published by NRC Research Press
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Fig. 5. Detection of Bcl-2, Bax, and caspase-3 protein expression in rat hearts by Western blot. Shown are Western blot results with protein samples extracted from tremor (TRM) rat hearts. (A) Top, examples of Western blot bands; bottom, expression level of Bcl-2. (B) Top, examples of Western blot bands; bottom, expression level of Bax. (C) Top, examples of Western blot bands; bottom, expression level of caspase-3. Data are the mean ± SE (n = 6 rats); *, p < 0.05; **, p < 0.01 compared with the wild-type Wistar control group.
Detection of caspase-3 protein expression in rat hearts by Western blot Activation of the caspase cascade is crucial in the initiation of apoptosis in diverse biological processes. Caspase-3 mediates the proteolytic cleavage of a range of proteins responsible for the rearrangement of the cytoskeleton, plasma membrane, and nucleus. To address whether caspase-3 was involved in the myocardial injury of TRM rats, the expression of caspase-3 was tested. The present data showed that caspase-3 protein expression was significantly increased in TRM rats compared with the control animals (0.96 ± 0.06 for the control compared with 1.10 ± 0.06 for TRM, p < 0.05) (Fig. 5), which indicated that activation of caspase-3 was likely involved in the cardiomyocyte apoptosis of myocardial dysfunction in TRM rats. Detection of ERK1/2, JNK, and p38 protein expression in rat hearts using Western blot To determine whether epilepsy induced apoptosis through the mitogen-activated protein kinase (MAPK) signal pathway, extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 protein expression were analyzed by Western blot. p38 and JNK are known to regulate apoptotic or death signal, whereas ERK1/2 mediates cell growth and differentiation signal in the cells. The results showed that the total ERK1/2,
JNK, and p38 were equivalent in the 2 groups; however, phosphorylated ERK1/2, JNK, and p38 were changed in the TRM group. P-ERK1/2 expression was decreased (1.06 ± 0.03 for the control compared with 0.95 ± 0.04 for TRM, p < 0.05) in TRM rats. By comparison, P-JNK (0.54 ± 0.05 for the control compared with 1.19 ± 0.04 for TRM, p < 0.01) and P-p38 (0.92 ± 0.05 for the control compared with 1.11 ± 0.06 for TRM, p < 0.05) were upregulated in TRM rats (Fig. 6).
Discussion The results of this study provide novel experimental evidence that epilepsy-like tremor deteriorates cardiac functions in TRM rats. The TRM rat would be a useful animal model to study the potential mechanisms of epileptic-mediated cardiomyocyte dysfunction. Using this animal model, we reported for the first time that the activation of the sympathetic nervous system and cardiomyocyte apoptosis are involved in the myocardial injury of TRM rats. The mechanisms of apoptosis might be associated with the activation of the mitochondria-initiated and the mitogen-activated protein kinase pathways. The animal models that have been employed to study the pathogenesis of epilepsy are mainly the inbred Wistar Albino Glaxo Rats from Rijswijk (WAG/Rij) and the Genetic Absence Epilepsy Rats Published by NRC Research Press
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Fig. 6. Detection of ERK1/2, p38, and JNK protein expression in rat hearts by Western blot. Shown are Western blot results with protein samples extracted from tremor (TRM) rat cardiac tissue. (A) Top, representative examples of Western blot bands; bottom, quantification of ERK1/2. (B) Top, representative examples of Western blot bands; bottom, quantification of JNK. (C) Top, representative examples of Western blot bands; bottom, quantification of p38. All the data are the mean ± SE (n = 6 rats); *, p < 0.05; **, p < 0.01 compared with the wild-type Wistar control group.
from Strasbourg (GAERS) (Vergnes et al. 1982; Kovács et al. 2011). Spike-wave-like complexes of 7–10 Hz and 7–11 Hz followed by behavioral absence-like seizures have been found in WAG/Rij and GAERS stains, respectively (Vergnes et al. 1982; Drinkenburg et al. 1993). Besides, genetic studies have recently reported that loci on chromosomes 5 and 9 in the WAG/Rij strain, and on chromosomes 4, 7, and 8 in GAERS, control the features of spike-wave discharges (SWDs) (Gauguier et al. 2004; Rudolf et al. 2004). Compared with these rats, the TRM rat selected for this study was characterized by the paroxysmal occurrence of 5–7 Hz spike-wave-like complexes associated with behavioral absence seizures, and without other abnormal EEG recordings. The TRM seizure linked with the tm mutant, which was mapped to rat chromosome 10 (Kuramoto et al. 1994). These EEG and behavioral characteristics in TRM rats are similar to the symptoms featured with a 3 Hz spike-wave-like complex and transient absence in pykno-epilepsy in humans. Thus, this TRM rat is very likely to be a very useful genetic model for the research of human absence epilepsy. Myocardial injury in patients with epilepsy has been reported in recent years, such as changes in heart rhythm, conduction, and
even subtle signs of ischemia (Tigaran et al. 2003). This study found that there existed cardiac arrhythmias including PVC, VT, and increased heart rate in TRM rats. Meanwhile, abnormal heart: body mass ratio and hemodynamic parameters were detected, indicating that cardiac function had deteriorated in the TRM rats. These data are consistent with the reported studies in other druginduced animal models of epilepsy (Walton et al. 1995; Sakamoto et al. 2008). A number of studies have demonstrated that epilepsy produces activation of the sympathetic nervous system in both human patients and in animal models (Sakamoto et al. 2008; Shimizu et al. 2008), resulting in cardiac contractile dysfunction characterized by myocyte damage and ventricular arrhythmias (Ishiguro and Morgan 2001). Therefore, we measured the catecholamine level, and found AD and NA in TRM rat plasma were increased significantly. The data suggested that excessive cardiac stimulation of sympathetic nervous system might partly participate in the detrimental effects of epilepsy on hearts. Apoptosis plays an important role in cell death in cultured cardiomyocytes and isolated hearts (Gottlieb et al. 1994; Maulik et al. Published by NRC Research Press
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1998; Bialik et al. 1999; Toufektsian et al. 2000; Stephanou et al. 2002). The process of apoptosis is regulated by a complex interaction of pro-apoptotic (Bax group of proteins) and anti-apoptotic (Bcl-2 family of proteins) mitochondrial membrane proteins and the activation of effector caspase (Bishopric et al. 2001). It is known that the anti-apoptotic protein Bcl-2 resides in the surface of mitochondrial membrane and inhibits the release of cytochrome c; however, the pro-apoptotic protein Bax promotes the release of cytochrome c. Caspase-3 is the apoptotic executor. In this study, we used TUNEL staining to confirm that the apoptotic ratio of cardiomyocytes was increased significantly in TRM rats. At the same time, we observed the down-regulation of Bcl-2, the up-regulation of Bax, and the activation of caspase-3 in TRM rats. We hypothesized that the activation of the mitochondriainitiated pathway may partly account for the induction of cardiomyocyte apoptosis by epilepsy. In addition, the MAPKs such as ERK, p38, and JNK are essential for the regulation of proliferation, differentiation, and apoptosis. The ERK pathway plays a critical role in regulating cell growth and differentiation by inhibiting components of the cell death machinery and enhancing the transcription of pro-survival genes. Phosphorylated JNK has been demonstrated to be associated with induction of apoptosis (Gabai et al. 2000). Activation of the JNK pathway is important for the release of cytochrome c from the mitochondria, followed by activation of the caspase cascade. The participation of activated p38 MAPK is still controversial (Armstrong 2004). In the studies of ischemic preconditioning, the increased activation of p38 MAPK seemed to be the main contributor of cardioprotection, and the inhibition of p38 MAPK phosphorylation could abolish the cardioprotective effects (Weinbrenner et al. 1997). However, it was also reported that activation of p38 promoted myocardial necrosis/apoptosis during sustained ischemia–reperfusion, and the inhibition of p38 MAPK reduced the apoptosis and improved post-ischemic cardiac function (Ma et al. 1999; Mackay and Mochly-Rosen 1999). To confirm whether epilepsy induces apoptosis through the MAPKs, we analyzed the ERK1/2, JNK, and p38 by Western blot. Our results showed that the levels of P-JNK and P-p38 were increased in TRM rats, and P-ERK1/2 was decreased. The results demonstrated that the epilepsy induced apoptosis may partly through the MAPKs pathway in TRM rats. In summary, the results of this study show, for the first time, that excessive stimulation of cardiac sympathetic nervous system receptors and cardiomyocyte apoptosis are involved in myocardial injury of TRM rats. The apoptotic mechanisms were very likely due, at least in part, to the activation of the mitochondriainitiated pathway, as well as the mitogen-activated protein kinase pathway. These findings could provide good therapeutic targets for myocardial injury in genetic epilepsy, but further investigation is needed.
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Epilepsia, 48(5): 859–871. doi: 10.1111/j.1528-1167.2007.01082.x. PMID:17433051. Nei, M., Ho, R.T., Abou-Khalil, B.W., Drislane, F.W., Liporace, J., Romeo, A., et al. 2004. EEG and ECG in sudden unexplained death in epilepsy. Epilepsia, 45(4): 338–345. doi:10.1111/j.0013-9580.2004.05503.x. PMID:15030496. Nilsson, L., Tomson, T., Farahmand, B.Y., Diwan, V., and Persson, P.G. 1997. Cause-specific mortality in epilepsy: a cohort study of more than 9,000 patients once hospitalized for epilepsy. Epilepsia, 38(10): 1062–1068. doi:10.1111/ j.1528-1157.1997.tb01194.x. PMID:9579951. Rudolf, G., Bihoreau, M.T., Godfrey, R.F., Wilder, S.P., Cox, R.D., Lathrop, M., et al. 2004. Polygenic control of idiopathic generalized epilepsy phenotypes Published by NRC Research Press
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in the genetic absence rats from Strasbourg (GAERS). Epilepsia, 45(4): 301– 308. doi:10.1111/j.0013-9580.2004.50303.x. PMID:15030491. Sakamoto, K., Saito, T., Orman, R., Koizumi, K., Lazar, J., Salciccioli, L., et al. 2008. Autonomic consequences of kainic acid-induced limbic cortical seizures in rats: peripheral autonomic nerve activity, acute cardiovascular changes, and death. Epilepsia, 49(6): 982–996. doi:10.1111/j.1528-1167.2008. 01545.x. PMID:18325014. Schuele, S.U. 2009. Effects of seizures on cardiac function. J. Clin. Neurophysiol. 26(5): 302–308. doi:10.1097/WNP.0b013e3181b7f13b. PMID:19752744. Seki, T., Matsubayashi, H., Amano, T., Kitada, K., Serikawa, T., Sasa, M., et al. 2004. Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats. Neurochem. Int. 45(1): 171–178. doi:10. 1016/j.neuint.2003.10.009. PMID:15082234. Serikawa, T., Ohno, Y., Sasa, M., Yamada, J., and Takaori, S. 1987. A new model of petit mal epilepsy: spontaneous spike and wave discharges in tremor rats. Lab. Anim. 21(1): 68–71. doi:10.1258/002367787780740635. PMID:3104669. Sevcencu, C., and Struijk, J.J. 2010. Autonomic alterations and cardiac changes in epilepsy. Epilepsia, 51(5): 725–737. doi:10.1111/j.1528-1167.2009.02479.x. PMID: 20067509. Shimizu, M., Kagawa, A., Takano, T., Masai, H., and Miwa, Y. 2008. Neurogenic stunned myocardium associated with status epileptics and postictal catecholamine surge. Intern. Med. 47(4): 269–273. doi:10.2169/internalmedicine. 47.0499. PMID:18277028. Stephanou, A., Scarabelli, T.M., Townsend, P.A., Bell, R., Yellon, D., Knight, R.A., et al. 2002. The carboxyl-terminal activation domain of the STAT-1 transcrip-
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tion factor enhances ischemia/reperfusion-induced apoptosis in cardiac myocytes. FASEB J. 16(13): 1841–1843. doi:10.1096/fj.02-0150fje. PMID:12223448. Tigaran, S., Mølgaard, H., McClelland, R., Dam, M., and Jaffe, A.S. 2003. Evidence of cardiac ischemia during seizures in drug refractory epilepsy patients. Neurology, 60(3): 492–495. doi:10.1212/01.WNL.0000042090.13247.48. PMID: 12578934. Toufektsian, M.C., Boucher, F., Pucheu, S., Tanguy, S., Ribuot, C., Sanou, D., et al. 2000. Effects of selenium deficiency on the response of cardiac tissue to ischemia and reperfusion. Toxicology, 148(2–3): 125–132. doi:10.1016/S0300483X(00)00203-1. PMID:10962131. Tryphonas, L., Veinot, J.P., and Clement, J.G. 1996. Early histopathologic and ultrastructural changes in the heart of Sprague–Dawley rats following administration of soman. Toxicol. Pathol. 24(2):190–198. doi:10.1177/ 019262339602400207. PMID:8992609. Vergnes, M., Marescaux, C., Micheletti, G., Reis, J., Depaulis, A., Rumbach, L., et al. 1982. Spontaneous paroxysmal electroclinical patterns in rat: a model of generalized non-convulsive epilepsy. Neurosci. Lett. 33(1): 97–101. doi:10. 1016/0304-3940(82)90136-7. PMID:6818498. Walton, N.Y., Rubinstein, B.K., and Treiman, D.M. 1995. Cardiac hypertrophy secondary to status epilepticus in the rat. Epilepsy Res. 20(2): 121–124. doi:10. 1016/0920-1211(94)00074-7. PMID:7750508. Weinbrenner, C., Liu, G.S., Cohen, M.V., and Downey, J.M. 1997. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J. Mol. Cell. Cardiol. 29(9): 2383–2391. doi:10.1006/jmcc.1997.0473. PMID:9299362.
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ARTICLE Involvement of cardiomyocyte apoptosis in myocardial injury of hereditary epileptic rats Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV GUELPH on 11/18/14 For personal use only.
Fan Chen, Yong-gang Cao, Han-ping Qi, Lei Li, Wei Huang, Ye Wang, and Hong-li Sun
Abstract: Many clinical cases have been reported where epilepsy profoundly influenced the pathophysiological function of the heart; however, the underlying mechanisms were not elucidated. We use the tremor (TRM) rat as an animal model of epilepsy to investigate the potential mechanisms of myocardial injury. Cardiac functions were assessed by arrhythmia score, heart rate, heart:body mass ratio, and hemodynamic parameters including left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), and maximum rate of left ventricular pressure rise and fall (+dp/dtmax and –dp/dtmax). Catecholamine level was detected by HPLC. Apoptotic index was estimated by TUNEL assay. The expressions of Bcl-2, Bax, caspase-3, extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinases (JNK), and p38 were evaluated by Western blot. The results indicated that there existed cardiac dysfunction and cardiomyocyte apoptosis, accompanied by increasing catecholamine levels in TRM rats. Further investigation revealed that apoptosis was mediated by reducing Bcl-2, upregulating Bax, and activating caspase-3. Additional experiments demonstrated that P-ERK1/2 was decreased, whereas P-JNK and P-p38 were upregulated. Our results suggest that the sympathetic nervous system activation and cardiomyocyte apoptosis are involved in the myocardial injury of TRM rats. The mechanisms of apoptosis might be associated with the activation of the mitochondriainitiated and the mitogen-activated protein kinase pathways. Key words: epilepsy, tremor rat, myocardial injury, cardiomyocyte, apoptosis. Résumé : Plusieurs cas cliniques où l'épilepsie influençait profondément les fonctions pathophysiologiques du cœur ont été rapportés ; toutefois, les mécanismes sous-jacents n'ont pas été élucidés. Nous avons utilisé le rat « tremor » (TRM) comme modèle animal d'épilepsie afin d'examiner les mécanismes potentiellement responsables du dommage myocardique. Les fonctions cardiaques ont été évaluées par le score d'arythmie, le rythme cardiaque, le rapport du poids du cœur sur le poids corporel, ainsi que par les paramètres hémodynamiques, dont le taux maximum d'élévation et de chute de la pression ventriculaire gauche (+dp/dtmax et –dp/dtmax). Le niveau de catécholamines a été détecté par HPLC. L'indice d'apoptose a été estimé par un dosage TUNEL. L'expression de Bcl-2, de Bax, de la capspase-3, des kinases ERK (extracellular signal-regulated kinase), JNK (c-Jun NH2-terminal protein kinase) et p38 a été évaluée par buvardage Western. Les résultats ont révélé une dysfonction cardiaque et la présence d'apoptose chez les cardiomyocytes, accompagnées par un accroissement des niveaux de catécholamines chez les rats TRM. Une recherche plus poussée a révélé que la réduction de Bcl-2, l'augmentation de Bax et l'activation de la caspase-3 étaient les médiateurs de l'apoptose. Des expériences additionnelles ont démontré que le niveau de P-ERK1/2 était diminué alors que ceux de P-JNK et de P-p38 étaient accrus. Nos résultats suggèrent que l'activation du système nerveux sympathique et l'apoptose des cardiomyocytes sont impliqués dans le dommage myocardique chez les rats TRM. Les mécanismes d'apoptose pourraient être associés a` l'activation des voies signalétiques issues des mitochondries et des protéines kinases activées par les mitogènes. [Traduit par la Rédaction] Mots-clés : épilepsie, rat « tremor », dommage myocardique, cardiomyocyte, apoptose.
Introduction Epilepsy is one of the most common neurological disorders. Characterized by recurrent seizures, it affects up to 50 million people worldwide (Carpio and Hauser 2009). Patients with epilepsy have consistently been found to have a higher mortality rate than the general population (Cockerell et al. 1994; Nashef et al. 1995), with a higher frequency in the younger patients (Nilsson et al. 1997; Annegers and Coan 1999; Ding et al. 2006). The increased mortality in people with epilepsy is due to a wide range of causes (Nilsson et al. 1997), including heart disease and pneumonia (Nashef et al. 2007; Hitiris et al. 2007; Sevcencu and Struijk 2010; Jansen and Lagae 2010). Sudden unexpected death in epilepsy (SUPEP) is the most common cause of seizure-related mortality in people with refractory
epilepsy. In 1906, Finny first documented cardiac asystole during a seizure. Since then, a wide range of cardiac arrhythmias have been reported, ranging from atrial fibrillation, supraventricular tachycardia, prolonged QT intervals, and torsades de pointes (Nilsson et al. 1997; Nei et al. 2004; Schuele 2009; Sevcencu and Struijk 2010). Also, a number of studies have indicated that seizure activity produces cardiac dysfunction and myocardial damage in human patients (Legriel et al. 2008; Shimizu et al. 2008) as well as animals experiencing seizures that were induced by lethal nerve agents (Tryphonas et al. 1996). Several studies have shown that seizures could result in myocardial injury by a variety of mechanisms. Available evidence has demonstrated that excessive stimulation of the sympathetic nervous system of the heart during seizures can result in cardiac contractile dysfunction, characterized by myofilament damage of the cardiomyocytes, myocardial
Received 3 January 2013. Accepted 16 May 2013. F. Chen,* Y.-g. Cao,* H.-p. Qi, W. Huang, Y. Wang, and H.-l. Sun. Department of Pharmacology, Harbin Medical University – Daqing, Daqing, Heilongjiang 163319, China. L. Li. Department of Surgery, The Fifth Clinical College of Harbin Medical University, Daqing, Heilongjiang 163316, China. Corresponding author: Hong-li Sun (e-mail: [email protected]). *Fan Chen and Yong-gang Cao contributed equally to this study.
Can. J. Physiol. Pharmacol. 91: 804–811 (2013) dx.doi.org/10.1139/cjpp-2013-0005
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fibrosis, arrhythmogenic alterations in cardiac electrical activity, and subsequently increased susceptibility to cardiac arrhythmias or ischemia (Ishiguro and Morgan 2001; Shimizu et al. 2008). So far, although the myocardial injury in epileptic patients has been reported, most of the data were acquired from a drug-induced model of epilepsy (Metcalf et al. 2009), and there has been no ideal animal model, comparable with epileptic patients, used in the experiments to evaluate the mechanisms of myocardial damage during seizures. The tremor rat (TRM; tm/tm), a genetic mutant that is found in a Kyoto–Wistar colony, exhibits both absence-like seizures and tonic convulsions without any external stimuli (Seki et al. 2004). Previous study has indicated that the absence-like seizures in the TRM rat are characterized by the paroxysmal occurrence of 5–7 Hz spike-wave-like complexes in hippocampal electroencephalograms (EEGs) at 8 weeks (Serikawa et al. 1987). However, in the intervening periods, normal EEG recordings free of absence-like seizures were found in the TRM rat (Serikawa et al. 1987). Thus, this TRM rat is most likely to be a useful genetic animal model of epilepsy. In this study, the TRM rat was selected as an animal model of epilepsy to determine the cardiac effects of seizures, and significant cardiac dysfunction has been observed in this animal. Therefore, these experiments were designed to investigate the potential mechanisms of myocardial injury in the TRM rat.
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Table 1. Alteration of heart rate and arrhythmia score in TRM rats. Group
n
Heart rate (beats/min)
Arrhythmia score
Control TRM
6 6
367±17 435±6**
0 56±4**
Note: Control, wild-type Wistar rats; TRM, tremor rats. Values are the mean ± SE; n = number of rats used; **, p < 0.01 compared with the control group.
Fig. 1. Alteration of heart:body mass ratio in tremor (TRM) rats. Statistical analysis of heart:body mass ratio. Data are the mean ± SE (n = 6 rats); **, p < 0.01 compared with the wild-type Wistar control group.
Materials and methods Experimental animals and reagents All experimental protocols were pre-approved by the Experimental Animal Ethic Committee of Harbin Medical University, China (Animal Experimental Ethical Inspection Protocol No 2009104). Wild-type Wistar rats (control) and TRM rats of either sex (250–300 g) at the age of 24 weeks were used in this study. Animals had free access to food and water and were housed under a controlled environment (12 h (light): 12 h (dark) cycle; 50%–70% humidity; 24 °C). Primary antibodies anti-Bcl-2, anti-Bax, anti-caspase-3, antiERK1/2, anti-JNK, and anti-p38 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). Anti-GAPDH was from Kangcheng, Inc. (Shanghai, China). The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) kit was from Roche (Mannheim, Germany). Arrhythmia scores Arrhythmia was quantitatively estimated according to the method described by Mest and Förster (1979). The degree of severity was assessed according to the following scale: 0 = sinus rhythm; 20 = first degree atrial–ventricular block, supraventricular arrhythmias; 40 = ventricular premature bigeminy, trigeminy, second degree atrial–ventricular block; 60 = polyphyleticism ventricular premature, paroxysmal ventricular tachycardia; 80 = ventricular fibrillation; 100 = death. Hemodynamic measurements The methods and procedures were exactly according to the methodology described in Liu et al. (2012). Briefly, rats were anesthetized with sodium pentobarbitone (40 mg/kg body mass, intraperitoneal (i.p.)), and a heparin-filled cannula was inserted into the right carotid artery and then advanced into the left ventricle. The hemodynamic parameters reflecting cardiac performances, including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and maximum rate of left ventricular pressure rise and fall (+dp/dtmax and –dp/dtmax) were measured at the same time by a pressure transducer interfaced with a BL-420E organism function experiment system (Cheng Du Tai Meng, Cheng Du, China).
Plasma sample preparation Blood samples were taken from all the groups, and an equal volume of sterile heparinized physiological saline was added. Each sample was centrifuged at 4000g for 15 min for preparing the plasma, and 200 L HClO4 (0.1 mol/L, Sigma), 50 L DHBA (2 g/mL), and 250 L plasma were added to a test tube. The mixture was kept at 4 °C for 5 min and centrifuged at 14 000g for 10 min. The supernatant was filtered (0.5 m) and made ready for catecholamine analysis. Catecholamine analysis by HPLC Adrenaline (AD) and noradrenaline (NA) are 2 main types of catecholamine in plasma. The levels of AD and NA in rat plasma were detected using HPLC. DHBA (3,4-dihydroxybenzylamine) was used as an internal standard in each sample. To the mobile phase was added 0.144 g sodium dodecyl sulfate (SDS; Sigma), 0.5 mL triethylamine (TEA; Merck), 10.5 g citric acid (CA; Merck), 4.1 g sodium acetate (SA, Sigma), 120 mL methanol (ME, Merck), 0.186 g ethylenediaminetetraacetic acid disodium salt (EDTA–2Na, Sigma), and 880 mL triple-distilled water. The flow rate was 0.8 mL/min. The fluorescence detector (RF-10Axl, Shimadzu; 330 nm for emission, 280 nm for excitation) was used to measure the levels of AD and NA. TUNEL assay The apoptotic ratio of cardiomyocytes was measured by TUNEL assay in accordance with the protocol of apoptosis detection kit. As seen under a light microscope, the normal nucleolus of cardiomyocytes stained blue and the apoptotic nucleolus stained brown. Apoptotic cells in each group were carefully evaluated under double-blind conditions. Ten different high-power fields (×400) were randomly selected, and the percentage of apoptotic cells was calculated as the ratio of the numbers of positivestaining nuclei to the numbers of total nuclei of the cells. Published by NRC Research Press
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Fig. 2. Alteration of hemodynamic parameters in tremor (TRM) rats. The hemodynamic parameters reflecting cardiac function were measured by left ventricle intubation via a pressure transducer interfaced with BL-420E organism function experiment system. (A) Left ventricular systolic pressure (LVSP). (B) Left ventricular end-diastolic pressure (LVEDP). (C) Maximum rate of left ventricular pressure rise and fall (+dp/dtmax and –dp/dtmax). Data are the mean ± SE (n = 6 rats per group); *, p < 0.05 compared with the wild-type Wistar control group.
Western blot analysis The left ventricular tissue was homogenized in PBS containing 1% triton X-100 and protease inhibitor cocktail. The homogenate was centrifuged at 14 000g for 30 min at 4 °C and the supernatant was collected, which was then mixed with sample loading buffer [2.5% sodium dodecyl sulphate (SDS), 10% glycerol, 50 mmol/L Tris–HCl, pH 6.8, 0.5 mol/L -mercaptoethanol, and 0.01% bromophenol blue] and boiled for 5 min. Samples of 50 g of protein from different experimental groups were separated by 10% SDS– PAGE and transferred to nitrocellulose membranes by electroblotting (200 mA for 1.5 h). The membranes were blocked with 5% non-fat milk in Tris-buffered saline – Tween 20 (TBS-T) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with the primary antibodies anti-Bax (1:200), antiBcl-2 (1:200), anti-caspase-3 (1:200), anti-ERK1/2 (1:200), anti-p38 (1:200), anti-JNK (1:200), and anti-GAPDH (1:5000), respectively. After rinsing 3 times with TBS-T at 10 min intervals, the membranes were then incubated with the secondary antibody (1:5000) for 1 h at room temperature in the dark. Immunolabeled protein bands were detected by an enhanced chemiluminescence kit (Pierce, Calif.). The volumes of the protein bands were quantified using a Bio-Rad Chemi Doc EQ densitometer and Bio-Rad Quantity One software (Bio-Rad Laboratories). GAPDH was used as an internal control for the relative quantification assay. Statistical analysis Data are the mean ± SE, and analyzed using SPSS 15.0 software. Student's t test was used to evaluate the differences between 2 groups. Values for p < 0.05 were considered to be statistically significant.
Results Alteration of cardiac function in TRM rats As shown in Table 1, there were no arrhythmias in the control group of rats. However, in TRM rats, premature ventricular contraction (PVC) and ventricular tachycardia (VT) were observed. The results from the statistical analyses showed that the arrhythmia score and heart rate were significantly increased (p < 0.01, compared with the control rats). Moreover, the heart:body mass ratio was also altered (3.16 ± 0.20 for the control compared with 4.68 ± 0.25 for TRM, p < 0.01) (Fig. 1). Other important indexes of cardiac function, such as LVSP, LVEDP, and ±dp/dtmax, were measured in both the control and TRM rats (Fig. 2). Compared with the control rats, TRM rats demonstrated a significant increase in LVEDP (7 ± 1 mm Hg for the control compared with 13 ± 1 mm Hg for TRM, p < 0.05; 1 mm Hg = 133.322 Pa) and decreases in LVSP (164 ± 6 mm Hg for the control compared with 92 ± 8 mm Hg for TRM, p < 0.05), +dp/dtmax (7196 ± 1000 mm Hg/s for the control compared with 2927 ± 1200 mm Hg/s for TRM, p < 0.05) and –dp/dtmax (–5447 ± 1050 mm Hg/s for the control compared with –2943 ± 1090 mm Hg/s for TRM, p < 0.05). These results indicated that cardiac dysfunction was developed in TRM rats. Detection of catecholamine level in rat plasma using HPLC It has been shown that epilepsy is associated with higher levels of catecholamines, which are known to cause myocardial injury (Sakamoto et al. 2008; Shimizu et al. 2008). To evaluate whether the plasma contents of catecholamine were changed in TRM rats, the levels of AD and NA were measured using Published by NRC Research Press
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Fig. 3. Detection of catecholamine level in rat plasma using HPLC. Adrenaline (AD) and noradrenaline (NA) are 2 main types of catecholamine in plasma. The levels of AD and NA in rat plasma were detected using HPLC. (A) Bar graph of AD. (B) Bar graph of NA. Data are the mean ± SE (n = 6 rats); **, p < 0.01 for tremor (TRM) rats compared with the wild-type Wistar control group.
Fig. 4. Cardiomyocyte apoptosis in tremor (TRM) rats was determined by TUNEL staining. (A) Representative examples of TUNEL staining. Nuclei with staining were TUNEL-positive cells (brown, on the Web site only), which were apoptotic. Magnification 400×. (B) Mean data of TUNEL-positive stained cells (n = 6 rats); **, p < 0.01 compared with the wild-type Wistar control group.
HPLC. As indicated in Fig. 3, the plasma AD and NA values were both higher in TRM rats than in the control rats. The level of AD was increased from 0.57 ± 0.03 g/mL to 0.72 ± 0.05 g/mL (p < 0.01), and NA was elevated from 0.13 ± 0.01 g/mL to 0.21 ± 0.03 g/mL (p < 0.01). Cardiomyocyte apoptosis in TRM rats was determined using TUNEL staining To elucidate the causes of cardiac dysfunction observed in TRM rats, cardiomyocyte apoptosis was assessed using TUNEL staining. The histological sections of the hearts were selected for locating apoptotic nuclei. As shown in Fig. 4, the apoptotic ratio of cardiomyocytes in TRM rats was increased significantly compared with
the control rats (3.4 ± 0.9 for the control compared with 18.5 ± 1.6 for the TRM, p < 0.01). Detection of Bcl-2 and Bax protein expression in rat hearts using Western blot To explore the potential mechanisms of epilepsy-induced myocardial damage, the cytoplasmic level of the anti-apoptotic signaling molecule Bcl-2 and pro-apoptotic molecule Bax were detected using Western blot. Averaged data (Fig. 5) revealed that in TRM rats, the expression of intracellular Bcl-2 was diminished (1.13 ± 0.06 for the control compared with 0.96 ± 0.07 for TRM, p < 0.05), and Bax expression was upregulated (0.34 ± 0.09 for the control compared with 1.16 ± 0.10 for TRM, p < 0.01), respectively. Published by NRC Research Press
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Fig. 5. Detection of Bcl-2, Bax, and caspase-3 protein expression in rat hearts by Western blot. Shown are Western blot results with protein samples extracted from tremor (TRM) rat hearts. (A) Top, examples of Western blot bands; bottom, expression level of Bcl-2. (B) Top, examples of Western blot bands; bottom, expression level of Bax. (C) Top, examples of Western blot bands; bottom, expression level of caspase-3. Data are the mean ± SE (n = 6 rats); *, p < 0.05; **, p < 0.01 compared with the wild-type Wistar control group.
Detection of caspase-3 protein expression in rat hearts by Western blot Activation of the caspase cascade is crucial in the initiation of apoptosis in diverse biological processes. Caspase-3 mediates the proteolytic cleavage of a range of proteins responsible for the rearrangement of the cytoskeleton, plasma membrane, and nucleus. To address whether caspase-3 was involved in the myocardial injury of TRM rats, the expression of caspase-3 was tested. The present data showed that caspase-3 protein expression was significantly increased in TRM rats compared with the control animals (0.96 ± 0.06 for the control compared with 1.10 ± 0.06 for TRM, p < 0.05) (Fig. 5), which indicated that activation of caspase-3 was likely involved in the cardiomyocyte apoptosis of myocardial dysfunction in TRM rats. Detection of ERK1/2, JNK, and p38 protein expression in rat hearts using Western blot To determine whether epilepsy induced apoptosis through the mitogen-activated protein kinase (MAPK) signal pathway, extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 protein expression were analyzed by Western blot. p38 and JNK are known to regulate apoptotic or death signal, whereas ERK1/2 mediates cell growth and differentiation signal in the cells. The results showed that the total ERK1/2,
JNK, and p38 were equivalent in the 2 groups; however, phosphorylated ERK1/2, JNK, and p38 were changed in the TRM group. P-ERK1/2 expression was decreased (1.06 ± 0.03 for the control compared with 0.95 ± 0.04 for TRM, p < 0.05) in TRM rats. By comparison, P-JNK (0.54 ± 0.05 for the control compared with 1.19 ± 0.04 for TRM, p < 0.01) and P-p38 (0.92 ± 0.05 for the control compared with 1.11 ± 0.06 for TRM, p < 0.05) were upregulated in TRM rats (Fig. 6).
Discussion The results of this study provide novel experimental evidence that epilepsy-like tremor deteriorates cardiac functions in TRM rats. The TRM rat would be a useful animal model to study the potential mechanisms of epileptic-mediated cardiomyocyte dysfunction. Using this animal model, we reported for the first time that the activation of the sympathetic nervous system and cardiomyocyte apoptosis are involved in the myocardial injury of TRM rats. The mechanisms of apoptosis might be associated with the activation of the mitochondria-initiated and the mitogen-activated protein kinase pathways. The animal models that have been employed to study the pathogenesis of epilepsy are mainly the inbred Wistar Albino Glaxo Rats from Rijswijk (WAG/Rij) and the Genetic Absence Epilepsy Rats Published by NRC Research Press
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Fig. 6. Detection of ERK1/2, p38, and JNK protein expression in rat hearts by Western blot. Shown are Western blot results with protein samples extracted from tremor (TRM) rat cardiac tissue. (A) Top, representative examples of Western blot bands; bottom, quantification of ERK1/2. (B) Top, representative examples of Western blot bands; bottom, quantification of JNK. (C) Top, representative examples of Western blot bands; bottom, quantification of p38. All the data are the mean ± SE (n = 6 rats); *, p < 0.05; **, p < 0.01 compared with the wild-type Wistar control group.
from Strasbourg (GAERS) (Vergnes et al. 1982; Kovács et al. 2011). Spike-wave-like complexes of 7–10 Hz and 7–11 Hz followed by behavioral absence-like seizures have been found in WAG/Rij and GAERS stains, respectively (Vergnes et al. 1982; Drinkenburg et al. 1993). Besides, genetic studies have recently reported that loci on chromosomes 5 and 9 in the WAG/Rij strain, and on chromosomes 4, 7, and 8 in GAERS, control the features of spike-wave discharges (SWDs) (Gauguier et al. 2004; Rudolf et al. 2004). Compared with these rats, the TRM rat selected for this study was characterized by the paroxysmal occurrence of 5–7 Hz spike-wave-like complexes associated with behavioral absence seizures, and without other abnormal EEG recordings. The TRM seizure linked with the tm mutant, which was mapped to rat chromosome 10 (Kuramoto et al. 1994). These EEG and behavioral characteristics in TRM rats are similar to the symptoms featured with a 3 Hz spike-wave-like complex and transient absence in pykno-epilepsy in humans. Thus, this TRM rat is very likely to be a very useful genetic model for the research of human absence epilepsy. Myocardial injury in patients with epilepsy has been reported in recent years, such as changes in heart rhythm, conduction, and
even subtle signs of ischemia (Tigaran et al. 2003). This study found that there existed cardiac arrhythmias including PVC, VT, and increased heart rate in TRM rats. Meanwhile, abnormal heart: body mass ratio and hemodynamic parameters were detected, indicating that cardiac function had deteriorated in the TRM rats. These data are consistent with the reported studies in other druginduced animal models of epilepsy (Walton et al. 1995; Sakamoto et al. 2008). A number of studies have demonstrated that epilepsy produces activation of the sympathetic nervous system in both human patients and in animal models (Sakamoto et al. 2008; Shimizu et al. 2008), resulting in cardiac contractile dysfunction characterized by myocyte damage and ventricular arrhythmias (Ishiguro and Morgan 2001). Therefore, we measured the catecholamine level, and found AD and NA in TRM rat plasma were increased significantly. The data suggested that excessive cardiac stimulation of sympathetic nervous system might partly participate in the detrimental effects of epilepsy on hearts. Apoptosis plays an important role in cell death in cultured cardiomyocytes and isolated hearts (Gottlieb et al. 1994; Maulik et al. Published by NRC Research Press
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1998; Bialik et al. 1999; Toufektsian et al. 2000; Stephanou et al. 2002). The process of apoptosis is regulated by a complex interaction of pro-apoptotic (Bax group of proteins) and anti-apoptotic (Bcl-2 family of proteins) mitochondrial membrane proteins and the activation of effector caspase (Bishopric et al. 2001). It is known that the anti-apoptotic protein Bcl-2 resides in the surface of mitochondrial membrane and inhibits the release of cytochrome c; however, the pro-apoptotic protein Bax promotes the release of cytochrome c. Caspase-3 is the apoptotic executor. In this study, we used TUNEL staining to confirm that the apoptotic ratio of cardiomyocytes was increased significantly in TRM rats. At the same time, we observed the down-regulation of Bcl-2, the up-regulation of Bax, and the activation of caspase-3 in TRM rats. We hypothesized that the activation of the mitochondriainitiated pathway may partly account for the induction of cardiomyocyte apoptosis by epilepsy. In addition, the MAPKs such as ERK, p38, and JNK are essential for the regulation of proliferation, differentiation, and apoptosis. The ERK pathway plays a critical role in regulating cell growth and differentiation by inhibiting components of the cell death machinery and enhancing the transcription of pro-survival genes. Phosphorylated JNK has been demonstrated to be associated with induction of apoptosis (Gabai et al. 2000). Activation of the JNK pathway is important for the release of cytochrome c from the mitochondria, followed by activation of the caspase cascade. The participation of activated p38 MAPK is still controversial (Armstrong 2004). In the studies of ischemic preconditioning, the increased activation of p38 MAPK seemed to be the main contributor of cardioprotection, and the inhibition of p38 MAPK phosphorylation could abolish the cardioprotective effects (Weinbrenner et al. 1997). However, it was also reported that activation of p38 promoted myocardial necrosis/apoptosis during sustained ischemia–reperfusion, and the inhibition of p38 MAPK reduced the apoptosis and improved post-ischemic cardiac function (Ma et al. 1999; Mackay and Mochly-Rosen 1999). To confirm whether epilepsy induces apoptosis through the MAPKs, we analyzed the ERK1/2, JNK, and p38 by Western blot. Our results showed that the levels of P-JNK and P-p38 were increased in TRM rats, and P-ERK1/2 was decreased. The results demonstrated that the epilepsy induced apoptosis may partly through the MAPKs pathway in TRM rats. In summary, the results of this study show, for the first time, that excessive stimulation of cardiac sympathetic nervous system receptors and cardiomyocyte apoptosis are involved in myocardial injury of TRM rats. The apoptotic mechanisms were very likely due, at least in part, to the activation of the mitochondriainitiated pathway, as well as the mitogen-activated protein kinase pathway. These findings could provide good therapeutic targets for myocardial injury in genetic epilepsy, but further investigation is needed.
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