Skip Navigation

Cardiovascular Research 2006 72(1):163-174; doi:10.1016/j.cardiores.2006.06.028
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Xu, Y.-J.
Right arrow Articles by Dhalla, N. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, Y.-J.
Right arrow Articles by Dhalla, N. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2006, European Society of Cardiology

MAPK activation and apoptotic alterations in hearts subjected to calcium paradox are attenuated by taurine

Yan-Jun Xu, Harjot K. Saini, Ming Zhang, Vijayan Elimban and Naranjan S. Dhalla*

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

* Corresponding author. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue Winnipeg, Manitoba, Canada R2H 2A6. Tel.: +1 204 235 3421; fax: +1 204 233 6723. Email address: nsdhalla{at}sbrc.ca

Received 22 March 2006; revised 27 June 2006; accepted 28 June 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The Ca2+-paradox is an important phenomenon to study cell injury induced by Ca2+-overload in myocardium. Although intracellular Ca2+-overload acts as a trigger and modulator of cell death due to apoptosis under various pathophysiological conditions, the presence of apoptosis in hearts subjected to Ca2+-paradox has not been demonstrated. Since taurine attenuates the changes in cardiac function due to Ca2+-paradox, this study investigated the occurrence and mechanisms of apoptosis in Ca2+-paradoxic hearts treated in the absence and presence of taurine.

Methods: Ca2+-paradox was induced by perfusing the isolated rat heart with Ca2+-free medium for 5 min followed by reperfusion with Ca2+-containing medium for 30 min. Apoptosis related signal transduction mechanisms were determined in Ca2+-paradoxic hearts perfused with or without 10 mM taurine.

Results: Marked alterations in cardiac function and the presence of apoptosis were seen in Ca2+-paradoxic hearts reperfused for 30 min. Unlike the total protein contents in hearts subjected to Ca2+-paradox, the contents of phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK)1, ERK2 and c-jun amino-terminal kinase were increased by 125±8.6%, 296±14.3%, 213±8.5% and 133±4.2%, respectively vs. control. Caspase-3 and phosphorylated Bcl-2 contents were also increased by 193±10.2% and 134±5.0% vs. control whereas phosphorylated Bad and the ratio of Bcl-2/Bad were depressed by 32±10.8% and 0.23±0.5% vs. control in Ca2+-paradoxic hearts. The apoptosis as seen in Ca2+-paradoxic hearts reperfused for 30 min was not evident in hearts at 10 min Ca2+-repletion but was similar to hearts subjected to 60 min Ca2+-repletion. These changes in the apoptotic pathway in cardiomyocytes subjected to Ca2+-paradox were prevented by taurine. Furthermore, taurine attenuated the KCl- or ATP-induced increase in intracellular concentration of Ca2+ in cardiomyocytes.

Conclusions: This study suggests that cardiac dysfunction due to Ca2+-paradox may be associated with apoptosis. In addition, the beneficial effects of taurine on cardiac function may be related to the attenuation of changes in MAPK and apoptotic signal transduction mechanisms in Ca2+-paradoxic hearts.

KEYWORDS Apoptosis; Calcium (cellular); Contractile function; MAPK


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Reperfusion of the isolated heart with a medium containing Ca2+ after a brief period of Ca2+-free perfusion was observed to produce dramatic changes in cardiac structure and function and this phenomenon was termed as Ca2+-paradox [1,2]. The Ca2+-paradoxic heart has been considered to form an excellent model for studying the mechanisms of cell injury due to intracellular Ca2+-overload at cellular level in myocardium by reoxygenation after anoxia or ischemia [3]. In fact, cardiac dysfunction due to Ca2+-paradox has been associated with abnormalities in energy production and utilization, electrophysiological and mechanical derangements, development of sustained contracture, membrane disruption as well as degradation of myofilaments and cytoskeleton [3]. Various investigators have revealed the release of intracellular proteins, such as lactate dehydrogenase, creatine kinase and myoglobin (which are the indices of necrotic cell death) as well as depression in activities of different enzymes including β-hydroxybutylate dehydrogenase, succinate dehydrogenase and glycogen phosphorylase in hearts undergoing Ca2+-paradox [4,5]. However, the occurrence of apoptosis, a distinct mode of programmed cell death, which has been observed in ischemic heart disease [6], was not investigated in Ca2+-paradoxic hearts. Because intracellular Ca2+ overload can modulate various pathways involved in the process of apoptotic cell death [7], the present study was designed to examine the presence of apoptosis in hearts subjected to 5 min Ca2+-depletion followed by 30 min of Ca2+-repletion. It should be noted that hearts subjected to 30 min ischemia followed by 60 min reperfusion [8] have also shown to produce apoptotic cell death [9]. Since the process of apoptosis is considered to be regulated by a complex interplay of proapoptotic (Bax group of proteins) and antiapoptotic (Bcl-2 family of proteins) mitochondrial membrane proteins as well as the activation of effector caspases [6], the status of these targets was also investigated in Ca2+-paradoxic hearts. In addition, alterations in mitogen-activated protein kinases (MAPKs) such as extracellular signal regulated kinase (ERK), p38 MAPK and c-jun amino-terminal kinase (JNK), the known mediators of apoptotic cell death [10], were determined in hearts undergoing Ca2+-paradox. Because taurine (2-aminoethane sulfonic acid), a sulphur-containing free amino acid, has been shown to protect the heart from Ca2+-paradox-mediated injury [11,12], the effects of taurine on alterations in signal transduction mechanisms for apoptosis were examined in Ca2+-paradoxic hearts. The rationale for using taurine was based on the observation that taurine caused a significant depression in apoptosis due to ischemia in neonatal cardiomyocytes [13].


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
All experimental protocols were approved by the University of Manitoba Animal Care Committee according to the guidelines of the Canadian Council on Animal Care and the guidelines of National Institute of Health.

2.1. Perfusion of isolated rat heart and experimental protocol
Male Sprague–Dawley rats (250–300 g) were anaesthetized with a mixture of ketamine (90 mg/kg) and xylazine (9 mg/kg). The hearts were quickly excised and perfused with Krebs–Henseleit (K–H) buffer gassed with 95% O2–5% CO2, 37 °C, pH 7.4 at a constant flow of 10 ml/min [14]. The composition of K–H solution was (mM): 120 NaCl; 4.7 KCl; 1.2 KH2PO4; 1.2 MgSO4; 25 NaHCO3; 1.25 CaCl2 and 11 glucose. The hearts were electrically stimulated at 300 beats/min via a square wave current of 1.5 ms by using Phipps and Bird stimulator (Richmond, VA, USA). The left ventricular systolic pressure (LVSP), the rate of change of pressure development (+dP/dt) and rate of change of pressure decay (–dP/dt) were measured via a transducer (Model 1050 BP-Biopac System Inc., Goleta, CA, USA), which was connected with a water-filled latex balloon inserted into the left ventricle. At beginning of the experiment, left ventricular end diastolic pressure (LVEDP) was adjusted to approximately 10 mm Hg by inflating the balloon. All data were recorded online and stored in a computer program (Acqknowledge 3.5.3) by using a Biopac Data Acquisition System (Biopac Systems Inc., Goleta, CA, USA) as described previously [14]. Left ventricular developed pressure (LVDP) was taken as the difference between the LVSP and LVEDP.

After 20 min of stabilization, the hearts were divided into four experimental groups. The control hearts were perfused with oxygenated K–H medium for 35 min whereas the Ca2+-paradoxic hearts were perfused with Ca2+-free medium for 5 min followed by 30 min perfusion with normal K–H buffer containing 1.25 mM Ca2+ [2,15]. For the taurine treatment group, taurine (10 mM) (Sigma-Aldrich, Oakville, ON, Canada) infusion was started 10 min before inducing Ca2+-paradox and was carried out throughout the Ca2+-depletion and Ca2+-repletion periods. In another set, control hearts were treated with 10 mM taurine for 35 min. The selection of this concentration of taurine was based on our observations showing maximal improvement of cardiac function in this experimental model. After measurement of the left ventricular function, the hearts were frozen in liquid N2 and stored at –70 °C for biochemical analysis. These frozen hearts were randomly divided in two groups; one group of hearts were used for in situ nick end labelling and the other group of hearts were used for Western blot analysis. It is pointed out that the concentration of Ca2+ in the K–H buffer was calculated as described previously [2,15] and no attempt was made to directly measure the Ca2+-levels in Ca2+-paradoxic hearts in the absence or presence of taurine.

2.2. In situ nick end labelling
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed by using APO-DIRECTTM kit (BD Biosciences Pharmingen, Mississauga, ON, Canada) according to the manufacturer's instructions. In brief, the ventricular tissue from the experimental groups was immersed in OCT compound (Miles Inc., Elkhart, IN, USA), serial cryostat sections (7 µM) were mounted on ProbeONTM/MC Plus slides (Fisher Scientific, Ottawa, Ontario, Canada) and a minimum of 6 sections from different regions of hearts in each group were processed. The tissue sections were fixed with 1% paraformaldehyde for 15 min at room temperature. After rinsing with phosphate buffer saline (PBS), the sections were treated overnight in a moisture chamber at room temperature with staining solution containing reaction buffer, terminal deoxynucleotidyl transferase enzyme and FITC labeled dUTP nucleotides; Hoechst 33342 (5 µg/ml) (Sigma-Aldrich, Oakville, ON, Canada) was used to stain the nuclei. The tissue sections were examined under a Nikon ECLIPSE 800 microscope equipped with epifluorescence optics and appropriate filters. In each group, approximately 200 cells obtained from minimum of 6 sections from different regions of hearts were counted. Percentage apoptotic index was calculated as the ratio of TUNEL positive cell nuclei (green fluorescence) to the total number of nuclei (blue fluorescence) multiplied by hundred as described previously [13].

2.3. 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 100,000xg for 10 min at 4 °C and the supernatant was collected. The supernatant was mixed with sample loading buffer [2.5% sodium dodecyl sulphate (SDS), 10% glycerol, 50 mM Tris–HCl, pH 6.8, 0.5 M β-mercaptoethanol, and 0.01% bromophenol blue] and boiled for 3 min. Protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The membranes were probed with phosphospecific and total primary antibodies against ERK1/2, p38 MAPK, JNK, Bcl and Bad (Cell Signaling Technology Inc, Beverly, MA, USA). In some of the experiments, membranes were incubated with caspase-3 primary antibody (Cell Signaling Technology Inc, Beverly, MA, USA). The protein bands were visualized by using the ECL Plus (Amersham; Arlington Heights, IL, USA) according to the manufacturer's instructions and quantified by densitometric analysis [16]. The scan value for control in each group was taken as 100% and accordingly others were expressed as % of control. Equal loading of protein samples was determined by staining membranes subjected to Western blotting with Coomassie brilliant blue.

2.4. Measurement of intracellular Ca2+ concentration ([Ca2+]i)
Ventricular cardiomyocytes were isolated and loaded with fura-2 AM (Molecular Probes, Eugene, OR, USA) as described previously [17]. For [Ca2+]i measurements, alterations in fluorescence intensity were monitored by a SLM DMX-1100 dual-wavelength spectrofluorometer (SLM Instruments, Urbana, IL, USA) adjusted to excitation wavelength of 340/380 nm, emission wavelength of 510 nm, integration time of 0.95 s, and resolution time of 1.0 s [14,17]. Treatment with different concentrations of taurine (2, 10 or 30 mM) was performed by incubating the fura-2 AM-loaded cells in a buffer containing taurine for 60 min at room temperature before the addition of KCl (30 mM), a known depolarizing agent or ATP (50 µM), a known purinergic receptor agonist [14,17]. It is pointed out that both KCl and ATP are known to cause increase in [Ca2+]i in isolated cardiomyocytes [14,17] whereas the selection of different concentrations of taurine was based on the previous observations [18]. Treatments with different pharmacological agents, which are known to modulate [Ca2+]i, were performed by incubating the fura-2 loaded cells in the buffer containing the desired concentration of pharmacological agents for 10 min before the measurement of fluorescence. The concentrations of different pharmacological interventions for the present study were selected on the basis of previous studies [19,20]. The increase in [Ca2+]i at peak [Ca2+]i was calculated as the net increase above the basal value in each experiment. To eliminate the possibility of any Ca2+-chelating effect of taurine, the alteration in fluorescence of fura-2 pentapotassium salt (Molecular Probes, OR, USA) was determined in the presence of different concentrations of taurine with 1 mM Ca2+.

2.5. Induction of Ca2+-paradox and enzyme-linked immunoassay (ELISA) for quantification of apoptosis in cardiomyocytes
The freshly isolated adult rat cardiomyocytes were subjected to Ca2+-paradox according to the method given by Takahashi et al. [21]. In brief, cardiomyocytes were incubated for 1 h in Ca2+-free medium-199 containing 1 mM EDTA followed by 24 h incubation with 1 mM Ca2+-containing medium. In taurine treated group, different concentrations of taurine (2, 10 or 30 mM) were included before starting the Ca2+-paradox protocol. The cell viability was determined by trypan blue exclusion method [14]. The cardiomyocyte apoptosis and necrosis were quantitatively estimated by using the Cell Death Detection ELISA PLUS kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's instructions. In brief, 1 ml of cardiomyocyte suspension (1x105 cells) was centrifuged at 300xg for 5 min. The resultant supernatant was used for the assessment of necrosis. The cell pellet was incubated in 200 µl lysis buffer for 30 min at room temperature and the supernatant containing cytoplasmic histone associated DNA fragments was used for measuring cardiomyocyte apoptosis. Twenty µl each of these supernatant fractions were added to a streptavidin-coated microtitre plate with 80 µl of the immunoreagent containing anti-histone biotin against histone and anti DNA-peroxidase against DNA to each well. The plate was gently shaken (300 rpm) for 2 h at 15–25 °C and the unbound antibodies removed by washing the plate three times with 250 µl of incubation buffer. One hundred microliters of ABTS [2,2'-azino-di (3-ethylbenzthiazolin-sulfonate)] reagent was added as a substrate and the fluorescence was measured, using a microtitre plate reader, at emission wavelength 450 nm with 490 nm as reference wavelength. Apoptosis was taken as the difference of absorbance (A405 nm–A490 nm) in the lysed cell supernatant, whereas necrosis was taken as the difference of absorbance (A405 nm–A490 nm) in the cell supernatant obtained from the first step. The fluorescence was measured, using a microtitre plate reader, at an emission wavelength 450 nm with 490 nm as a reference wavelength [22].

2.6. Statistical analysis
Data are expressed as means±S.E.M. Statistical analysis was performed with Microcal Origin version 6 (Microcal Software, Northampton, MA, USA). The differences between two groups were evaluated by Student's t-test. The data from more than two groups were evaluated by one-way ANOVA followed by the Student–Newman–Keuls test. Values showing P<0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Cardiac dysfunction due to Ca2+-paradox mediated injury
Five minutes of Ca2+-free perfusion (depletion phase) followed by 30 min of perfusion with Ca2+-containing solution (repletion phase) caused a dramatic impairment in cardiac performance (Fig. 1). These changes in LV function were reflected by an increase in LVEDP and a marked decrease in LVDP, +dP/dt and –dP/dt. Treatment of Ca2+-paradoxic hearts with taurine (10 mM) throughout the Ca2+-depletion and Ca2+-repletion period produced a marked improvement in all these parameters (Fig. 1). On the other hand, no recovery in cardiac function was observed when taurine was absent during the 30 min Ca2+-repletion period. In addition, taurine treatment did not affect different parameters of LV function vs. control perfusion (LVDP 108±8.4 vs. 112±10.7; LVEDP 7.2±2.1 vs. 8.9±3.2; +dP/dt 4539±560 vs. 4502±760; –dP/dt 3530±415 vs. 3750±680).


Figure 1
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Cardiac performance of isolated rat hearts subjected to Ca2+-paradox in the absence and presence of taurine (10 mM). Panel A: left ventricular developed pressure (LVDP), panel B: left ventricular end diastolic pressure (LVEDP), panel C: left ventricular rate of change of pressure development (+dP/dt), panel D: left ventricular rate of change of pressure decay (–dP/dt). The exposure times for taurine treatment are indicated by the arrows. Each data point represents the mean±S.E.M. of 6 experiments in each group. *P<0.05 vs. Ca2+-paradox group.

 
3.2. Effect of Ca2+-paradox on apoptotic index
To examine the effect of Ca2+-paradox on cell death due to apoptosis, apoptotic index was calculated by using TUNEL assay. Fig. 2A and C show representative photomicrographs of the TUNEL assay from control and Ca2+-paradoxic hearts (5 min Ca2+-depletion and 30 min Ca2+-repletion), respectively. A significant increase in apoptotic nuclei number (% of total) was observed in hearts subjected to Ca2+-paradox as compared to control group (Fig. 2E). Treatment with taurine significantly decreased the Ca2+-paradox-induced apoptotic cell death (Fig. 2D). On the other hand, taurine treated hearts were not different from control (Fig. 2B and E). In order to determine the optimal duration for the induction of apoptosis, 5 min Ca2+-depleted hearts were reperfused with Ca2+-containing K–H buffer for 10 or 60 min. No changes were evident when the Ca2+-repletion period was 10 min whereas apoptotic alterations in 60 min Ca2+-repleted hearts were similar to those seen in hearts subjected to 30 min Ca2+-repletion. These experiments indicate that 5 min Ca2+-depletion followed by 30 min Ca2+-repletion produced maximal apoptotic alterations in the heart.


Figure 2
View larger version (84K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 TUNEL staining of heart sections subjected to Ca2+-paradox in the absence (Ca2+-P) and presence of taurine (Ca2+-P+T). Panel A: representative photomicrographs of TUNEL staining of sections obtained from control hearts, panel B: representative photomicrographs of TUNEL staining of sections obtained from control hearts treated with taurine (10 mM), panel C: representative photomicrographs of TUNEL staining of sections obtained from Ca2+-paradoxic hearts, panel D: representative photomicrographs of TUNEL staining of sections obtained from Ca2+-paradoxic hearts treated with taurine (10 mM), panel E: the apoptotic index in all experimental groups calculated as the ratio of TUNEL positive cell nuclei to the total number of nuclei. Hoeschst 33342 (5 µg/ml) was used to stain the nuclei. In each group approximately 200 cells obtained from minimum of 6 sections from different regions of hearts of each group were counted. *P<0.05 vs. control; #P<0.05 vs. Ca2+-paradox group.

 
3.3. Ca2+-paradox mediated alterations in MAPK protein content
Since MAPKs are the known mediators of cell death due to apoptosis under various pathophysiological conditions [10], we determined the protein contents of different MAPKs in Ca2+-paradoxic hearts. An increase in protein contents of phosphorylated forms of p38 MAPK, ERK1/2 as well as JNK was observed in hearts subjected to Ca2+-paradox (Fig. 3A, B and C). In contrast, no change in total protein content of these MAPKs was observed under Ca2+-paradoxic conditions (Fig. 3D, E and F). From the data shown in Fig. 3, it can be seen that the increase in protein contents of phosphorylated MAPKs was significantly attenuated by taurine treatment without any change in total protein contents of these kinases.


Figure 3
View larger version (60K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Immunoreactive bands and Western blot analysis for protein content of different mitogen activated protein kinases (MAPKs) in Ca2+-paradoxic hearts in the absence (Ca2+-P) and presence of taurine (Ca2+-P+T). Panel A: phosphorylated p38 MAPK (phospho-p38 MAPK), panel B: phosphorylated ERK 1/2 (phospho-ERK 1/2), panel C: phosphorylated JNK (phospho-JNK), panel D: total p38 MAPK, panel E: total ERK 1/2 and panel F: total JNK. Data represent mean±S.E.M. of 6 separate experiments in each group. *P<0.05 vs. control; #P<0.05 vs. Ca2+-paradox group.

 
3.4. Effect of Ca2+-paradox on different regulators of apoptosis
In order to investigate the effect of Ca2+-paradox on signal transduction mechanisms for apoptosis, we investigated changes in phosphorylated and total Bcl-2 and Bad as well as caspase-3 in hearts subjected to Ca2+-paradox. The protein content of phosphorylated Bcl-2 was increased whereas a decrease in phosphorylated Bad was observed in Ca2+-paradoxic hearts (Fig. 4A and B). From Fig. 4D and E, it can be seen that Bcl-2 content was attenuated in Ca2+-paradoxic hearts without any change in Bad protein content under similar conditions. It should be noted that the ratio of Bcl-2/Bad was significantly depressed in hearts subjected to Ca2+-paradox (Fig. 4F). In addition, an increase in caspase-3 protein content was observed in Ca2+-paradoxic hearts (Fig. 4C). All these alterations in phosphorylated Bcl-2, phosphorylated Bad and ratio of Bcl-2/Bad as well as caspase-3 protein content under conditions of Ca2+-paradox were attenuated by treatment with taurine (Fig. 4). From Fig. 4D, it should be noted that Bcl-2 protein content was markedly increased in taurine treated Ca2+-paradoxic hearts, which may be due to either increased synthesis or less degradation of protein in the presence of taurine. In view of the short time period of Ca2+-repletion, it is possible that the apparent increase in Bcl-2 protein content may be due to increased reactivity of this protein to its antibody. However, no effort was made to determine the cause of such a change.


Figure 4
View larger version (55K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Immunoreactive bands and Western blot analysis for different apoptotic signal transduction proteins Ca2+-paradoxic hearts in the absence (Ca2+-P) and presence of taurine (Ca2+-P+T). Panel A: phosphorylated Bcl-2, panel B: phosphorylated Bad, panel C: caspase-3, panel D: Bcl-2, panel E: Bad and panel F: ratio of Bcl-2/Bad. Data represent mean±S.E.M. of 6 separate experiments in each group. *P<0.05 vs. control; #P<0.05 vs. Ca2+-paradox group.

 
3.5. Effect of Ca2+-paradox on apoptosis and necrosis in cardiomyocytes
To verify the occurrence of apoptosis due to Ca2+-paradox, we examined the effect of Ca2+-depletion and repletion in isolated cardiomyocytes. Both apoptosis as well as necrosis were increased in cardiomyocytes subjected to Ca2+-paradox under in vitro conditions (Table 1). Taurine at higher concentrations (10 and 30 mM) caused a significant decrease in cell death due to apoptosis as well necrosis (Table 1). On the other hand, no reduction in cell death was observed at lower (2 mM) concentrations of taurine. It is important to note that extent of necrosis in cardiomyocytes undergoing Ca2+-paradox seems to be less than apoptosis (Table 1). Removal of dead cells in the supernatant during isolation and after Ca2+ repletion may the reason for such an effect. However, the viability of cardiomyocytes as determined by trypan blue exclusion method was significantly less in Ca2+-paradoxic cardiomyocytes as compared to control and treatment with different concentrations of taurine (2, 10 or 30 mM) prevented the reduction in cell viability (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1 Effect of different concentrations of taurine on viability, apoptosis and necrosis of isolated cardiomyocytes

 
3.6. Effect of taurine on KCl- and ATP-induced increase in [Ca2+]i
In order to understand if taurine was capable of modifying the [Ca2+]i, isolated cardiomyocytes in the absence or presence of taurine were exposed to KCl or ATP. From the data given in Fig. 5, it can be seen that taurine caused a significant attenuation of KCl-mediated augmentation of [Ca2+]i. Similarly, ATP-mediated increase in [Ca2+]i was significantly attenuated by taurine in a concentration dependent manner (Fig. 5). It is pointed out that the fluorescence of fura-2 pentapotasssium salt with 1 mM Ca2+ did not change in the presence of different concentrations of taurine (340/380 nm fluorescence ratio: control 103±7.3; taurine 10 mM 102±7.5 and taurine 30 mM 115±5.2) ruling out the possibility of any Ca2+-chelating effect of taurine.


Figure 5
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Representative tracings showing the effect of different concentrations of taurine (10 and 30 mM) on intracellular Ca2+ concentration ([Ca2+]i) in isolated cardiomyocytes. Panel A: effect of taurine on KCl (30 mM)-induced increase in [Ca2+]i in cardiomyocytes, panel B: effect of taurine on ATP (50 µM)-induced increase in [Ca2+]i in cardiomyocytes. Data represent mean±S.E.M. of 4 separate experiments in each group. *P<0.05 vs. control; #P<0.05 vs. Ca2+-paradox group.

 
3.7. Mechanisms of taurine-mediated alterations in [Ca2+]i
To understand the mechanisms of taurine-mediated reduction in [Ca2+]i, isolated cardiomyocytes were treated with different pharmacological agents, which are known to affect sarcolemmal (SL) and sarcoplasmic reticulum (SR) Ca2+-regulating sites. It can be seen from Table 2 that taurine treatment caused a significant decrease in KCl-mediated increase in [Ca2+]i. Pretreatment with tetrodotoxin (TTX) (10 and 25 µM), a known SL Na+-channel blocker [20], and verapamil (1 and 5 µM), SL Ca2+-channel antagonist [19], caused a decrease in taurine-mediated attenuation of KCl response. Similarly, pretreatment with caffeine (10 and 20 mM), which keeps SR Ca2+-release channels in an open state [16] attenuated the taurine-induced response. On the other hand, KB-R7943 (5 and 10 µM), a known Na+–Ca2+ exchange inhibitor [19], did not cause any alterations in taurine response. Furthermore, basal [Ca2+]i remained unaltered in the presence of different concentrations of pharmacological agents (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2 Effect of different concentrations of pharmacological agents on KCl-induced increase in [Ca2+]i in absence or presence of taurine in isolated cardiomyocytes

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present study, we have shown that Ca2+-paradox caused a dramatic depression in mechanical function as reflected by an increase in LVEDP as well as a decrease in LVDP, +dP/dt and –dP/dt. These changes due to Ca2+-paradox are in agreement with our previous observations under similar experimental conditions [8,15]. Ultrastructural damage, depletion of ATP content, abnormalities in electrical activity and increase in cytosolic Ca2+-content are the major alterations associated with irreversible cell injury in Ca2+-paradoxic hearts [3]. Various investigators have shown that the uncontrolled entry of Ca2+ during Ca2+-paradox through voltage-dependent L-type Ca2+-channels, reverse mode of Na+–Ca2+ exchanger and passive diffusion of Ca2+ is the main reason for cell death due to necrosis in Ca2+-paradoxic hearts [3,23]. Since cell death due to apoptosis may be associated with increase in [Ca2+]i under different pathological conditions [7], it is possible that the loss of cardiomyocytes in Ca2+-paradoxic hearts may in part be related to cell death due to apoptosis. This view was supported by the occurrence of apoptosis as determined by TUNEL assay and ELISA, in Ca2+-paradoxic hearts and cardiomyocytes undergoing Ca2+-paradox, respectively. Since both TUNEL and ELISA are more sensitive techniques for the determination of apoptosis as compared to DNA laddering [24], no attempt was made to check the status of DNA laddering under different experimental conditions.

In order to understand the mechanisms of apoptosis, we examined the status of different signal transduction pathways involved in this process. Previous studies in ischemic and failing hearts have implicated the role of MAPKs in the signal transduction mechanisms of apoptosis [10]. In the present study, an increase in protein content of phosphorylated MAPKs such as p38 MAPK, ERK1/2 and JNK without any change in total protein content of these kinases was seen in Ca2+-paradoxic hearts. Phosphorylated JNK has been shown to be associated with induction of apoptosis [25] as well as cell survival pathways [26]. Similarly, the participation of activated p38 MAPK and ERK1/2 is still controversial [27]. Some studies have shown that inhibition of p38 MAPK phosphorylation is protective in ischemic–reperfused (I/R) hearts [28], while others have shown the protective effect of phosphorylated p38 MAPK [29]. Activation of ERK1/2 has also been implicated in cell protection during I/R injury [30] whereas the reactive oxygen species (ROS)-induced Ca2+-overload has been shown to be mediated by the activation of ERK1/2 [31]. Variations in physiological responses of different MAPKs in diverse cell types and the differences in their responsiveness to pathophysiological stimuli under in vivo and in vitro conditions are the major reasons for these controversial findings [10].

Since mitochondria associated Bcl-2 family of proteins and caspases are the key regulators of apoptotic cell death [32], we determined the changes in Bcl-2, Bad and caspase-3 in Ca2+-paradoxic hearts. A decrease in antiapoptotic protein, Bcl-2, with no change in proapoptotic protein, Bad, was observed in hearts undergoing Ca2+-paradox; however, the ratio of Bcl-2/Bad, a parameter of apoptotic cell death, was decreased in Ca2+-paradoxic hearts. Ca2+-paradox was found to increase the phosphorylation of Bcl-2 with a significant depression in Bad-phosphorylation. It is important to point out that hyperphosphorylation of Bcl-2 through cAMP-dependent protein kinase has been shown to be linked with apoptosis in MCF-7 and MDA-MB-231 cells [33]. In addition, phosphorylation of Bcl-2 was found to inhibit the ability of Bcl-2 to lower endoplasmic reticulum Ca2+ and protect against Ca2+-dependent death stimuli [34]. Depression in phosphorylation of Bad has also been linked with the translocation of Bad into mitochondria and thus cause the down-regulation of Bcl-2 expression, opening of permeability transition pore (PTP) and leading to apoptosis [35]. Furthermore, we have shown an increase in the protein content of caspase-3, an effector protease in the apoptotic signal transduction mechanism [32], in Ca2+-paradoxic hearts. Nonetheless, the relation between cardiac contractile dysfunction in Ca2+-paradoxic hearts and caspase-3 activation cannot be defined on the basis of results obtained in the present study. No evidence of apoptosis was detected in Ca2+-paradoxic hearts subjected to 5 min Ca2+-depletion followed by 10 min Ca2+-repletion period while cardiac function was markedly altered in these hearts. Some investigators have reported a positive correlation between caspase-3 activation and contractile dysfunction [36,37], while others have denied this fact [38].

Treatment of Ca2+-paradoxic hearts with taurine was observed to cause a marked improvement in cardiac function. Previous studies have also shown that taurine treatment attenuated the morphological changes in cardiomyocytes due to Ca2+-paradox [11]. In addition, Kramer et al. [39] have demonstrated that taurine treatment caused an improvement in mechanical function of the heart undergoing Ca2+-paradox. Although no improvement in cardiac function by taurine was observed in hearts undergoing low-flow ischemia followed by reperfusion [39], taurine supplementation attenuated the ischemia-induced apoptosis and necrosis [40]. Alterations in MAPKs, the known mediators of apoptosis [10], due to Ca2+-paradox were also attenuated by treatment with taurine. In addition, changes in phosphorylated Bcl-2 and Bad in Ca2+-paradoxic hearts were depressed by taurine treatment. In this regard, Takahasi et al. [40] have also shown an elevation of Bcl-2 protein content by taurine as this was considered to render the cell resistant to ischemia-mediated injury. Our previous study has shown that mitochondrial respiration and oxidative phosphorylation rate was significantly depressed in mitochondria isolated from Ca2+-paradoxic hearts [41]. Although in the present study we have not examined the effect of taurine on the status of mitochondrial membrane potential and cytochrome c release, no effect of taurine on these parameters was observed in a model of simulated ischemia [13]. Taurine has also been demonstrated to decrease the protein content of caspase-3. In this regard, it should be noted that inhibition of the formation of Apaf-1/caspase 9 leading to caspase-3 activation was suggested to be the mechanism in ischemia-induced apoptosis [13].

To understand the protective effects of taurine at cellular level, isolated cardiomyocytes were subjected to Ca2+-paradox. Although Piper et al. [42] have shown that that Ca2+-paradox does not occur in cardiomyocytes unlike isolated heart, others have established the existence of Ca2+-paradox in cardiomyoctes [43]. The occurrence of Ca2+-paradox in the present study was evident from the observation that a significant reduction in the number of rod shape cardiomyocytes and their ability to exclude trypan blue after Ca2+-repletion was observed in accordance with the previous studies [44]. While, the potential importance of cell death due to apoptosis by increase in [Ca2+]i is not fully understood, an increase in [Ca2+]i as a stimulus for apoptotic cell death has been demonstrated in lymphoid cells, leukemic T-cells and cytotoxic T-cells [7]. On the other hand, a transient elevation of intracellular [Ca2+]i was reported to elicit signalling events leading to prolonged inhibition of apoptosis in neutrophils [45]. Differences in responsiveness to intracellular Ca2+ overload under various experimental conditions may be the reason for such a controversy. In our experimental conditions, development of intracellular Ca2+-overload due to Ca2+-paradox [2] seems to be the trigger for the induction of contractile dysfunction and apoptosis. Excessive amount of Ca2+-sequestration by mitochondria followed by PTP opening subsequent to the release of cytochrome c into the cytoplasm, which in turn activates the effector caspases and apoptotic pathways, may be one of the mechanisms of apoptosis [46]. The participation of activated cysteine proteases, calpains, which transduce apoptotic signals together with caspases [7], cannot be ruled out on the basis of data in the present study. However, the participation of Ca2+ in initiating apoptosis is supported by our observation that taurine, which prevented apoptosis due to Ca2+-paradox, caused a significant reduction in KCl- or ATP-mediated increase in [Ca2+]i in a concentration dependent manner. In addition, Failli et al. [47] have shown that taurine antagonises the increase in [Ca2+]i by {alpha}-adrenergic stimulation in isolated guinea pig cardiomyocytes. The inhibition of [Ca2+]i by taurine appears to be mediated by L-type Ca2+ channels, SL Na+ channels and SR without the involvement of SL Na+–Ca2+ exchange. Previous studies have also shown the involvement of various SL sites in taurine-mediated inhibition of increase in [Ca2+]i [48]. Since different investigators have demonstrated that ROS is a major determinant for causing apoptosis and cardiac dysfunction in cardiomyocytes [17,49], it can be argued that the protective effects of taurine in Ca2+-paradoxic hearts may be related to its antioxidant effect [50]. However, this may not be the case as no improvement in cardiac function was observed in Ca2+-paradoxic hearts upon treatment with different antioxidants as well as an antioxidant mixture containing superoxide dismutase and catalase (unpublished observations). Since the relative changes in apoptosis in hearts subjected to Ca2+-paradox with or without taurine are substantially less than those seen for cardiac function, it appears that there is no direct relationship between apoptosis and cardiac contractile dysfunction. This view is supported by the observations made by other investigators in different models of cardiac injury [51,52]. Nonetheless, our findings suggest that cardiomyocyte loss due to apoptosis in Ca2+-paradoxic hearts may be associated with dramatic depression in cardiac function.


   Acknowledgements
 
The work reported in this article was supported by a grant from the Canadian Institutes of Health Research. H.K. Saini is a predoctoral fellow of the Heart and Stroke Foundation of Canada.


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Zimmerman A.N., Hulsmann W.C. Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart. Nature (1966) 211:646–647.[CrossRef][Medline]
  2. Alto L.E., Dhalla N.S. Myocardial cation contents during induction of calcium paradox. Am J Physiol Heart Circ Physiol (1979) 237:H713–H719.[Abstract/Free Full Text]
  3. Piper H.M. The calcium paradox revisited: an artefact of great heuristic value. Cardiovasc Res (2000) 45:123–127.[Free Full Text]
  4. Ziegelhoffer A., Ravingerova T., Tribulova N., Slezak J., Okolicany J., Tregerova V., et al. Partial prevention of calcium paradox in isolated perfused rat hearts by diltiazem. Biomed Biochim Acta (1989) 48:S96–S101.[ISI][Medline]
  5. Hunt W.G., Willis R.J. Calcium exposure required for full expression of injury in the calcium paradox. Biochem Biophys Res Commun (1985) 126:901–904.[CrossRef][ISI][Medline]
  6. Bishopric N.H., Andreka P., Slepak T., Webster K.A. Molecular mechanisms of apoptosis in the cardiac myocyte. Curr Opin Pharmacol (2001) 1:141–150.[CrossRef][Medline]
  7. Orrenius S., Zhivotovsky B., Nicotera P. Regulation of cell death: the calcium–apoptosis link. Nat Rev Mol Cell Biol (2003) 4:552–565.[CrossRef][ISI][Medline]
  8. Temsah R.M., Kawabata K., Chapman D., Dhalla N.S. Preconditioning prevents alterations in cardiac SR gene expression due to ischemia–reperfusion. Am J Physiol Heart Circ Physiol (2002) 282:H1461–H1466.[Abstract/Free Full Text]
  9. Maulik N., Yoshida T., Engelman R.M., Deaton D., Flack J.E. III, Rousou J.A., et al. Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion. Mol Cell Biochem (1998) 186:139–145.[CrossRef][ISI][Medline]
  10. Feuerstein G.Z., Young P.R. Apoptosis in cardiac diseases: stress- and mitogen-activated signaling pathways. Cardiovasc Res (2000) 45:560–569.[Abstract/Free Full Text]
  11. Takahashi K., Harada H., Schaffer S.W., Azuma J. Effect of taurine on intracellular calcium dynamics of cultured myocardial cells during the calcium paradox. Adv Exp Med Biol (1992) 315:153–161.[Medline]
  12. Yamauchi-Takihara K., Azuma J., Kishimoto S., Onishi S., Sperelakis N. Taurine prevention of calcium paradox-related damage in cardiac muscle. Its regulatory action on intracellular cation contents. Biochem Pharmacol (1988) 37:2651–2658.[CrossRef][ISI][Medline]
  13. Takatani T., Takahashi K., Uozumi Y., Shikata E., Yamamoto Y., Ito T., et al. Taurine inhibits apoptosis by preventing formation of the Apaf-1/caspase-9 apoptosome. Am J Physiol Cell Physiol (2004) 287:C949–C953.[Abstract/Free Full Text]
  14. Saini H.K., Elimban V., Dhalla N.S. Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia–reperfusion. Am J Physiol Heart Circ Physiol (2005) 289:H614–H623.[Abstract/Free Full Text]
  15. Zhang M., Xu Y.J., Saini H.K., Turan B., Liu P.P., Dhalla N.S. TNF-alpha as a potential mediator of cardiac dysfunction due to intracellular Ca2+-overload. Biochem Biophys Res Commun (2005) 327:57–63.[CrossRef][ISI][Medline]
  16. Xu Y.J., Rathi S.S., Chapman D.C., Arneja A.S., Dhalla N.S. Mechanisms of lysophosphatidic acid-induced DNA synthesis in vascular smooth muscle cells. J Cardiovasc Pharmacol (2003) 41:381–387.[CrossRef][ISI][Medline]
  17. Saini H.K., Dhalla N.S. Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia–reperfusion. Am J Physiol Heart Circ Physiol (2005) 288:H2260–H2270.[Abstract/Free Full Text]
  18. Bkaily G., Haddad G., Jaalouk D., Gros-Louis N., Benchekroun M.T., Naik R., et al. Modulation of Ca2+ and Na+ transport by taurine in heart and vascular smooth muscle. Adv Exp Med Biol (1996) 403:263–273.[Medline]
  19. Saini H.K., Tripathi O.N., Zhang S., Elimban V., Dhalla N.S. Involvement of Na+/Ca2+ exchanger in catecholamine-induced increase in intracellular calcium in cardiomyocytes. Am J Physiol Heart Circ Physiol (2006) 290:H373–H380.[Abstract/Free Full Text]
  20. Bokenes J., Sjaastad I., Sejersted O.M. Artifactual contractions triggered by field stimulation of cardiomyocytes. J Appl Physiol (2005) 98:1712–1719.[Abstract/Free Full Text]
  21. Takahashi K., Takahashi T., Suzuki T., Onishi M., Tanaka Y., Hamano-Takahashi A., et al. Protective effects of SEA0400, a novel and selective inhibitor of the Na+/Ca2+ exchanger, on myocardial ischemia–reperfusion injuries. Eur J Pharmacol (2003) 458:155–162.[CrossRef][ISI][Medline]
  22. Cheema K.K., Dent M.R., Saini H.K., Aroutiounova N., Tappia P.S. Prenatal exposure to maternal undernutrition induces adult cardiac dysfunction. Br J Nutr (2005) 93:471–477.[CrossRef][ISI][Medline]
  23. Ruigrok T.J. Is an increase of intracellular Na+ during Ca2+ depletion essential for the occurrence of the calcium paradox? J Mol Cell Cardiol (1990) 22:499–501.[CrossRef][ISI][Medline]
  24. Stadelmann C., Lassmann H. Detection of apoptosis in tissue sections. Cell Tissue Res (2000) 301:19–31.[CrossRef][ISI][Medline]
  25. Gabai V.L., Yaglom J.A., Volloch V., Meriin A.B., Force T., Koutroumanis M., et al. Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death. Mol Cell Biol (2000) 20:6826–6836.[Abstract/Free Full Text]
  26. Dougherty C.J., Kubasiak L.A., Prentice H., Andreka P., Bishopric N.H., Webster K.A. Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress. Biochem J (2002) 362:561–571.[CrossRef][ISI][Medline]
  27. Armstrong S.C. Protein kinase activation and myocardial ischemia/reperfusion injury. Cardiovasc Res (2004) 61:427–436.[Abstract/Free Full Text]
  28. Weinbrenner C., Liu G.S., Cohen M.V., Downey J.M. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J Mol Cell Cardiol (1997) 29:2383–2391.[CrossRef][ISI][Medline]
  29. Schulz R., Belosjorow S., Gres P., Jansen J., Michel M.C., Heusch G. p38 MAP kinase is a mediator of ischemic preconditioning in pigs. Cardiovasc Res (2002) 55:690–700.[Abstract/Free Full Text]
  30. Yue T.L., Wang C., Gu J.L., Ma X.L., Kumar S., Lee J.C., et al. Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res (2000) 86:692–699.[Abstract/Free Full Text]
  31. Rothstein E.C., Byron K.L., Reed R.E., Fliegel L., Lucchesi P.A. H2O2-induced Ca2+ overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway. Am J Physiol Heart Circ Physiol (2002) 283:H598–H605.[Abstract/Free Full Text]
  32. Reeve J.L., Duffy A.M., O'Brien T., Samali A. Don't lose heart–therapeutic value of apoptosis prevention in the treatment of cardiovascular disease. J Cell Mol Med (2005) 9:609–622.[CrossRef][ISI][Medline]
  33. Srivastava R.K., Srivastava A.R., Korsmeyer S.J., Nesterova M., Cho-Chung Y.S., Longo D.L. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol (1998) 18:3509–3517.[Abstract/Free Full Text]
  34. Bassik M.C., Scorrano L., Oakes S.A., Pozzan T., Korsmeyer S.J. Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis. EMBO J (2004) 23:1207–1216.[CrossRef][ISI][Medline]
  35. Rajesh K.G., Suzuki R., Maeda H., Yamamoto M., Yutong X., Sasaguri S. Hydrophilic bile salt ursodeoxycholic acid protects myocardium against reperfusion injury in a PI3K/Akt dependent pathway. J Mol Cell Cardiol (2005) 39:766–776.[CrossRef][ISI][Medline]
  36. Communal C., Sumandea M., de Tombe P., Narula J., Solaro R.J., Hajjar R.J. Functional consequences of caspase activation in cardiac myocytes. Proc Natl Acad Sci U S A (2002) 99:6252–6256.[Abstract/Free Full Text]
  37. Laugwitz K.L., Moretti A., Weig H.J., Gillitzer A., Pinkernell K., Ott T., et al. Blocking caspase-activated apoptosis improves contractility in failing myocardium. Hum Gene Ther (2001) 12:2051–2063.[CrossRef][ISI][Medline]
  38. Ruetten H., Badorff C., Ihling C., Zeiher A.M., Dimmeler S. Inhibition of caspase-3 improves contractile recovery of stunned myocardium, independent of apoptosis-inhibitory effects. J Am Coll Cardiol (2001) 38:2063–2070.[Abstract/Free Full Text]
  39. Kramer J.H., Chovan J.P., Schaffer S.W. Effect of taurine on calcium paradox and ischemic heart failure. Am J Physiol Heart Circ Physiol (1981) 240:H238–H246.[Abstract/Free Full Text]
  40. Takahashi K., Ohyabu Y., Takahashi K., Solodushko V., Takatani T., Itoh T., et al. Taurine renders the cell resistant to ischemia-induced injury in cultured neonatal rat cardiomyocytes. J Cardiovasc Pharmacol (2003) 41:726–733.[CrossRef][ISI][Medline]
  41. Dhalla N.S., Singh J.N., McNamara D.B., Bernatsky A., Singh A., Harrow J.A.C. Experimental medical biology. Spitzer J.J., ed. (1983) New York: Plenum Publishing Corporation. 305–316.
  42. Piper H.M., Spahr R., Hutter J.F., Spieckermann P.G. The calcium and the oxygen paradox: non-existent on the cellular level. Basic Res Cardiol (1985) 80:159–163.[ISI][Medline]
  43. Chapman R.A., Suleiman M.S., Rodrigo G.C., Tunstall J. The calcium paradox: a role for [Na]i, a cellular or tissue basis, a property unique to the Langendorff perfused heart? A bundle of contradictions! J Mol Cell Cardiol (1991) 23:773–777.[CrossRef][ISI][Medline]
  44. Suleiman M.S., Edmond J.J., Bulstrode G.K. Calcium paradox in guinea-pig ventricular myocytes. Exp Physiol (1997) 82:657–664.[Abstract]
  45. Whyte M.K., Hardwick S.J., Meagher L.C., Savill J.S., Haslett C. Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro. J Clin Invest (1993) 92:446–455.[ISI][Medline]
  46. Halestrap A.P., Kerr P.M., Javadov S., Woodfield K.Y. Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart. Biochim Biophys Acta (1998) 1366:79–94.[Medline]
  47. Failli P., Fazzini A., Franconi F., Stendardi I., Giotti A. Taurine antagonizes the increase in intracellular calcium concentration induced by alpha-adrenergic stimulation in freshly isolated guinea-pig cardiomyocytes. J Mol Cell Cardiol (1992) 24:1253–1265.[CrossRef][ISI][Medline]
  48. Satoh H., Sperelakis N. Review of some actions of taurine on ion channels of cardiac muscle cells and others. Gen Pharmacol (1998) 30:451–463.[CrossRef][ISI][Medline]
  49. von Harsdorf R., Li P.F., Dietz R. Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation (1999) 99:2934–2941.[Abstract/Free Full Text]
  50. Oriyanhan W., Yamazaki K., Miwa S., Takaba K., Ikeda T., Komeda M. Taurine prevents myocardial ischemia/reperfusion-induced oxidative stress and apoptosis in prolonged hypothermic rat heart preservation. Heart Vessels (2005) 20:278–285.[CrossRef][ISI][Medline]
  51. Perrin C., Ecarnot-Laubriet A., Vergely C., Rochette L. Calpain and caspase-3 inhibitors reduce infarct size and post-ischemic apoptosis in rat heart without modifying contractile recovery. Cell Mol Biol (Noisy-le-grand) (2003) 49:OL497–OL505. Online Pub.
  52. Borutaite V., Jekabsone A., Morkuniene R., Brown G.C. Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia. J Mol Cell Cardiol (2003) 35:357–366.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Xu, Y.-J.
Right arrow Articles by Dhalla, N. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, Y.-J.
Right arrow Articles by Dhalla, N. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?