HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 91/3/591    most recent expphysiol.2005.032615v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Meng, D. Articles by Zhang, J.-N. Search for Related Content PubMed PubMed Citation Articles by Meng, D. Articles by Zhang, J.-N. Related Collections Cardiovascular control

¹ Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China² Research Institute of Cardiovascular Disease, First Affiliated Hospital and Human Functional Genetics Laboratory of Jiangsu province of Nanjing Medical University, Nanjing, 210029, China

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Dysregulation of intracellular Ca²⁺ homeostasis plays an important role in mediating myocardial injury. We tested the hypothesis that treatment with trimetazidine (TMZ) would improve intracellular Ca²⁺ handling in myocardial injury of rats. The control group received saline only (10 ml kg^–1 day^–1, I.P.) for 7 days. In a second group, isoprenaline (ISO; 5 mg kg^–1 day^–1, S.C.) was administered to rats for 2 days to induce an acute injury of the myocardium. In a third group, treatment with TMZ (10 mg kg^–1 day^–1, I.P.) was initiated 1 day before ISO administration and continued for 7 days (n= 7 rats in each group). Histopathological evaluation showed that TMZ prevented ISO-induced myocardial damage. TMZ preserved the ATP levels and decreased the maleic dialdehyde (MDA) content in the hearts compared with ISO-treated rats. The diastolic [Ca²⁺]_i measured by loading with fura-2 AM in isolated cardiomyocytes was increased significantly in ISO-treated rats compared to the control animals. TMZ prevented the rise of diastolic [Ca²⁺]_i and the depression of caffeine-induced Ca²⁺ transients caused by ISO administration. The reduction in sarcoplasmic reticulum (SR) Ca²⁺ content in the heart cells and in cardiac SR Ca²⁺-ATPase activity in ISO-treated rats was abolished by TMZ, although there were no differences in SR Ca²⁺-ATPase protein levels between the control, ISO and ISO + 7 mz-treated rats. In addition, TMZ prevented the reduction in sarcolemmal L-type Ca²⁺ current density in the heart cells induced by ISO treatment. These results demonstrate that the treatment of rats with TMZ inhibited the increase of diastolic [Ca²⁺]_i and prevented the decrease of SR Ca²⁺ content, SR Ca²⁺-ATPase activity and L-type Ca²⁺ current density in cardiomyocytes in ISO-mediated myocardial injury of rats. These changes in Ca²⁺ handling could help to explain the favourable action of TMZ in myocardial injury.

(Received 17 October 2005; accepted after revision 3 February 2006; first published online 9 February 2006)
Corresponding author D. Meng: Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China. Email: dmeng{at}sibs.ac.cn

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Dysregulation of intracellular Ca²⁺ homeostasis plays an important role in mediating myocardial injury (Piper et al. 2003; Valen, 2003). A marked increase in cytosolic free calcium ([Ca²⁺]_i) has been reported in ischaemic myocardial injury, and the occurrence of intracellular Ca²⁺ overload has been suggested to lead to arrhythmias, contractile failure and ultimately cell death (Orrenius et al. 2003), while pretreatment with Ca²⁺ antagonists completely prevented the occurrence of myocardial injury in rats (Ferrari & Visioli, 1991). In the normal heart, intracellular Ca²⁺ movements critically regulate subsequent mechanical contractions. In cardiac excitation–contraction coupling, a small amount of Ca²⁺ first enters through the L-type Ca²⁺ channel during membrane depolarization. This Ca²⁺ influx triggers a large-scale Ca²⁺ release through the Ca²⁺ release channel of the sarcoplasmic reticulum (SR). The released Ca²⁺ then binds to troponin C within the myofilaments, which induces activation of the myofilaments and a consequent muscle contraction. Relaxation is initiated by dissociation of Ca²⁺ from troponin C, followed by its reuptake into the SR through SR Ca²⁺-ATPase and subsequent trans-sarcolemmal Ca²⁺ removal through the Na⁺–Ca²⁺ exchanger (Yano et al. 2005). Impairment of contractility during myocardial injury is primarily associated with changes in intracellular Ca²⁺ handling. These changes are predominantly decreases in Ca²⁺ influx through the L-type Ca²⁺ channel, reduced Ca²⁺ release from the SR and impaired SR Ca²⁺ reuptake (Tani, 1990; Sayer, 2002).

Trimetazidine (TMZ) is a clinically effective anti-anginal agent that protects energy metabolism against ischaemia and reperfusion injury, termed as a cellular anti-ischaemic agent (Marzilli, 2003). Trimetazidine acts by inhibiting long-chain 3-ketoacyl coenzyme A (CoA) thiolase in the heart, resulting in a reduction in fatty acid oxidation and an increase in glucose oxidation, directly improving myocardial energy metabolism (Kantor et al. 2000). Trimetazidine has been shown to limit myocardial necrosis after transient coronary occlusion (Noble et al. 1995). The effects may be due to protection of myocardial cell function during ischaemia by prevention of the fall in ATP concentration (Stanley, 2004), limitation of the free radicals (Monteiro et al. 2004) and prevention of the accumulation of Ca²⁺ in the heart cells (Renaud, 1988; D'hahan et al. 1997). Recent studies demonstrated that TMZ reduced basal cytosolic Ca²⁺ concentration during hypoxia in single skeletal myocytes (Stary et al. 2003) and corrected disturbances of transmembrane ion exchange leading to Ca²⁺ overload in ischaemic cardiomyopathy (Belardinelli, 2000). It suggests that TMZ might have an effect on intracellular Ca²⁺ regulation under pathological conditions. However, the effects of TMZ on intracellular Ca²⁺ handling during myocardial injury remain largely unknown.

In this study, isoprenaline (ISO)-induced acute injury of the myocardium in rats was used as an experimental model (Rona et al. 1959; Todd et al. 1985). We examined the effects of TMZ on ISO-mediated myocardial injury. Histopathological evaluation in sections of hearts, and maleic dialdehyde (MDA) and ATP levels in the left myocardium of rats were measured. We attempted to investigate the hypothesis that treatment with TMZ would improve intracellular Ca²⁺ handling in ISO-mediated myocardial injury of rats. To test this hypothesis, diastolic [Ca²⁺]_i, caffeine-induced Ca²⁺ transients and L-type Ca²⁺ current density in isolated heart cells, and cardiac SR Ca²⁺-ATPase protein level and activity were measured.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals

Male Sprague–Dawley rats, weighing 230–270 g, were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. Rats were housed in environmentally controlled conditions with a 12 h–12 h light–dark cycle and on standard laboratory diet. Animal care was in accordance with the guidelines of the Chinese Committee for Experiments on Animals.

Drugs and chemicals

Isoprenaline, ATP, fura-2 AM and caffeine were purchased from Sigma (St Louis, MO, USA). Crude collagenase (type II) was purchased from Worthington Biochemical Corp. (Lakewood, NJ, USA). Trimetazidine was a gift from IRIS (Institut de Recherches Internationales Servier, France).

Experimental protocols

Rats were divided randomly into the following three treatment groups (n= 7 rats in each group): (i) control, saline only (10 ml kg^–1 day^–1, I.P.) for 7 days; (ii) ISO, isoprenaline (5 mg kg^–1 day^–1, S.C.) was given for 2 days to produce myocardial injury in the rats; and (iii) ISO + TMZ, ISO administration was as for the ISO group, and trimetazidine (10 mg kg^–1 day^–1, I.P.) was initiated 1 day before ISO administration and continued for 7 days. At the end of the experiment, rats were killed with intraperitoneal injection of pentobarbitone (50 mg kg^–1), and the hearts were rapidly excised. The apexes of the hearts were placed for 24 h in 10% formaldehyde solution, and parts of the left ventriculum were frozen in liquid nitrogen and stored at –70°C for further determination of ATP levels, MDA content and SR Ca²⁺-ATPase protein levels and activity. Single heart cells were prepared from the remaining tissues. All procedures were in accordance with the guidelines of the Chinese Committee for Experiments on Animals.

Histopathological evaluation

The apex of the heart from each rat was embedded in paraffin, and sections 4 µm thick were cut serially from base to apex. Four sections were mounted on slides from 10–20 slices at 1 mm intervals, and then stained with Haematoxylin and Eosin (HE) for histopathological evaluation. The 4x objective of a Nikon Optiphot microscope equipped with a CCD colour video camera was used to display the images. Four sections from each rat were analysed according to the pathology grade (modified from Rona et al. 1959) as follows: Grade 0, no lesions; Grade I, focal lesions of the subendocardial portion of the apex; Grade II, sheets of lesions without confluence throughout the wall of the apex; Grade III, confluent lesions throughout the wall of the apex; and Grade IV, confluent lesions throughout the heart, including infarct-like massive necrosis, occasionally with acute aneurysm or mural thrombi. Scoring was done on coded samples by an experienced pathologist in a blinded manner.

Determination of ATP levels

Adenosine triphosphate levels in left ventricular tissues were determined by high-performance liquid chromatography (HPLC) as previously described (Botker et al. 1994). In brief, mechanical homogenization was performed with 100 mg of left ventricular tissue from each rat in 0.42 mM perchloric acid, homogenate was centrifuged at 12,000 g for 10 min, and the supernatant was neutralized by 2 mM potassium hydrogen carbonate (pH adjusted to 7.0 with Tris) for 10 min and centrifuged at 12,000 g for 5 min. Ten microlitres of neutralized supernatant was injected for analysis.

Maleic dialdehyde content assay

Approximately 100 mg of left ventricular tissue from each rat was homogenized in an ice-bath for 30 s. Supernatants were collected after centrifugation at 12,000 g for 10 min, and protein concentrations in supernatants were measured using Coomassie Plus Protein Assay Reagent Kit (PIERCE Biotechnology, IL, USA). The MDA contents in supernatants were determined by assay reagent kits (Jiancheng Bioengineering Institute, Nanjing, China), and the results were obtained in micromoles per gram tissue weight.

Preparation of single cardiac myocytes

Myocytes were enzymatically dissociated from the heart as previously described (Kawamura & Wahler, 1994). Briefly, the heart was rapidly cannulated via the aorta, and perfused through the coronary artery with Ca²⁺-free Tyrode solution for 6 min. The composition of Ca²⁺-free Tyrode solution was (mM): NaCl, 126; KCl, 5.4; NaH₂PO₄, 0.9; MgCl₂, 1; glucose, 10; and Hepes, 5; pH adjusted to 7.35 with NaOH at room temperature. The heart was then perfused with low-Ca²⁺ Tyrode solution containing CaCl₂, 50 µM; BSA, 0.6 g l^–1; and collagenase, 0.4 g l^–1 for 20–30 min. All perfusates were gassed with 95% O₂–5% CO₂, and the temperature was maintained at 37°C. After perfusion, the left ventricle was cut into small chunks and agitated gently in Krebs buffer (KB) solution composed of (mM): KOH, 70; glutamic acid, 70; KCl, 40; taurine, 20; KH₂PO₄, 20; MgCl₂, 3; Hepes, 10; glucose, 10; and EGTA, 0.5; pH adjusted to 7.4 with KOH. Then the cells were filtered, centrifuged at 500 g and resuspended in Tyrode solution. The living cell yield was 50–80%; cells were used within 6 h of isolation.

Measurement of diastolic [Ca²⁺]_i and caffeine-induced Ca²⁺ transient

The diastolic [Ca²⁺]_i in resting heart cells was measured as previously described (Nicolas et al. 1998). In brief, isolated heart cells were loaded with fura-2 AM (2.5 µM) at 37°C for 30 min in a physiological saline solution containing (mM): NaCl, 140; KCl, 6; CaCl₂, 1; MgCl₂, 1; glucose, 10; and Hepes, 10, pH adjusted to 7.4 with NaOH. Then the cells were washed with physiological saline solution for at least 30 min, plated onto glass coverslips and placed on the stage of an inverted microscope. An epifluorescence 40x oil immersion objective lens (Olympus, Japan) was used. The fluorescence measurement was performed by an image system (TILL Imaging System, Grafelfing, Germany), which used a fast CCD sensor and a 41 Msample/sA/D converter to acquire full-frame 12 bit per pixel digitized images with a time resolution of 30 ms. The fluorescence image collected by the CCD camera was fed to a computer analysing system (TILLvisIONs, Grafelfing, Germany). Excitation wavelengths were 340 and 380 nm, and emitted fluorescence was measured at 510 nm. The fluorescence signal was acquired by the system at a sampling rate of 500 Hz. At each of these sampling points, a calculated fluorescence ratio was determined. The data from 10 sequential runs were averaged to increase signal-to-noise ratio. Fluorescence ratios were obtained from whole cells by dividing the 340 nm image after background subtraction by the 380 nm image after background subtraction. The ratio images of cells were visualized with an intensity-defined pseudocolour gradient as previously described (Scheuerlein et al. 1991). The ratios were calibrated into calcium concentration by the method of Sipido & Callewaert (1995). To evaluate the sarcoplasmic reticulum Ca²⁺ loading state, caffeine-induced Ca²⁺ transients were measured. Caffeine (10 mM) was perfused directly onto the cell for 20 s using a puffer pipette. Images were obtained at 30 ms time resolution throughout the time course of caffeine action. Fluorescence values at each time point were expressed as a ratio, and this ratio was calibrated in terms of [Ca²⁺]_i. The amplitudes of caffeine-induced Ca²⁺ transients were expressed as changes of [Ca²⁺]_i ([Ca²⁺]_i/[Ca²⁺]_i). Time-to-peak Ca²⁺ of the transients was measured, and time constants of [Ca²⁺]_i decay were obtained by fitting of the decay fluorescence trace to a single exponential function as previously described (Yao et al. 1998).

Immunoblot assay

The protein content of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) in the left ventricular tissue was determined as previously described (Temsah et al. 1999). Briefly, protein samples were separated by electrophoresis through 10% mini-SDS-PAGE gels. Samples were transferred to nitrocellulose membranes. The membranes were probed with monoclonal anti-SERCA2a antibody (Santa Cruz Biotechnology, CA, USA). A peroxidase-linked antimouse IgG was used as a secondary antibody. Antibody–antigen complexes in all membranes were detected by the ECL kit (Amersham Life Science). The immunoreactive bands were quantified using a GS800 calibrated densitometer and Quantitive One version 4 software (Bio-Rad, Hercules, CA, USA).

Measurement of cardiac SR Ca²⁺-ATPase activity

The cardiac Ca²⁺-ATPase activity of SR can be selectively inhibited at high (21 mM) Ca²⁺ concentrations, which saturates the low-affinity inhibitory binding site on the enzyme. This feedback inhibition phenomenon was exploited to determine SR Ca²⁺-ATPase activity according to the procedure developed by Simonides & van Hardeveld (1990). Cardiac SR was prepared according to the method of Kodavanti et al. (1990). Briefly, the ventricular tissues were homogenized with nine volumes of solution containing (mM): NaHCO₃, 0.01; sodium azide, 0.05; and EGTA, 0.001. The homogenate was centrifuged at 12 000g twice, and the supernatant was ultracentrifuged at 120 000g twice for 45 min. The precipitate obtained was designated the SR fraction and suspended in cold sucrose–histidine buffer containing (mM): sucrose, 200; L-histidine, 40; EDTA, 1; and sodium azide, 10; pH adjusted to 7.8 with NaOH. The standard assay medium additionally contained 1 or 21 mM CaCl₂, 10 mM sodium azide, 0.005% Triton X-100, and an adequate amount of cardiac SR. The absorbance decrease was recorded 10 min after the addition of ATP. The difference between the rate measured in the presence of 1 mM CaCl₂ and the rate in the presence of 21 mM CaCl₂ was taken as the SR Ca²⁺-ATPase activity.

Electrophysiological studies

Whole-cell recordings were carried out by the standard gigaseal patch clamp technique (Hamill et al. 1981). Isolated cells were placed in a recording chamber mounted on the stage of an inverted microscope (IX70, Olympus, Japan). Patch pipettes were fabricated from borosilicate glass (TW150F-4, World Precision Instruments, USA) by using a pipette puller (PIP5, HEKA, Freiburg, Germany). The pipettes had resistances of 3–6 M, when filled with an internal solution of the following composition (mM): CsCl, 133; Mg-ATP, 5; phosphocreatine disodium, 5; Na-GTP, 0.5; EGTA, 10; CaCl₂, 0.062; and Hepes, 15; pH adjusted to 7.3 with CsOH. The pipette was connected through a Ag–AgCl wire to a patch-clamp amplifier (EPC 9, HEKA). The extracellular solution was composed of (mM): choline chlorine, 126; CsCl, 21; MgCl₂, 1; CaCl₂, 1.8; Hepes, 15; and glucose, 10; pH adjusted to 7.4 with 2 mM Tris. After gigaohm seal formation and cell membrane rupture, a period of 5–10 min was allowed for intracellular dialysis. L-type Ca²⁺ current was evoked by depolarization of myocytes from a holding potential of –50 mV to +40 mV at a frequency of 2 kHz. The sequence of clamped pulses and holding potential, collection of signals, and analysis of results were established by Pulse+PulseFit 8.0 software (HEKA). The magnitudes of L-type Ca²⁺ currents were normalized to the membrane capacitance of each cell (in pF) and expressed as current density (in pA pF^–1). The steady-state inactivation curves were fitted with a Boltzmann equation by IGOR Pro 4.0+ software. The time constants for the inactivation process were obtained by fitting a double exponential function to individual current traces (Li et al. 2001).

Statistical analysis

The pathology grades were evaluated by rank sum test. Data are expressed as means ±S.E.M. The significance of differences between individual groups was determined by using a one-way analysis of variance (ANOVA). A probability value less than 0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Pathology grade and changes in ATP levels and MDA contents

The heart sections from the control rats showed well-arranged muscle fibres without lesions, and the pathology grade of these sections was 0 (n= 7 rats). Isoprenaline produced subendocardial necrosis in the hearts; the grade for five rats was II, one rat was I, and another was III (n= 7 rats; P < 0.01 compared with the control). The treatment with TMZ completely prevented the myocardial damage induced by ISO; the pathology grade of the sections was 0 (n= 7 rats; P < 0.01 compared with ISO-treated rats), showing that TMZ had a protective effect on ISO-mediated myocardial injury. To test whether TMZ protects energy metabolism and prevents lipid peroxidation, ATP levels and MDA contents in the left myocardium of rats were measured. As shown in Table 1, the ATP levels were decreased in ISO-treated rats compared with the controls (P < 0.01), while TMZ prevented the decrease of ATP levels induced by ISO administration (P < 0.05). It should be noticed that ATP levels in the control hearts in this study are lower than the previously published values (Botker et al. 1994). The lower values may be due to the fact that we used frozen tissues compared with the fresh tissues reported by others; it is possible that storing tissues at –70°C led to a decrease in ATP contents. In addition, ISO intervention resulted in an increase in the MDA contents of left myocardium compared with the control values (P < 0.05). The treatment with TMZ markedly reduced the ISO-induced increase of MDA contents (P < 0.01). These results suggest that TMZ preserves the ATP pool and reduces lipid peroxidation in ISO-mediated myocardial injury of rats.

The images of Ca²⁺ fluorescence ratios recorded from cardiomyocytes loaded with fura-2 AM are shown in Fig. 1A. The fluorescence ratios (F₃₄₀/F₃₈₀) were visualized with a colour gradient. The diastolic [Ca²⁺]_i in living heart cells at rest was determined without electrical stimulation. The mean diastolic [Ca²⁺]_i was significantly increased in the myocytes from ISO-treated rats (164.8 ± 7.1 nM; n= 104 cells from 5 rats) compared to the controls (110.4 ± 4.5 nM; n= 90 cells from 5 rats, P < 0.01), while it was markedly decreased in ISO + TMZ-treated rats (123.9 ± 1.6 nM; n= 96 cells from 5 rats, Fig. 1B) compared with ISO-treated rats (P < 0.01). This indicates that TMZ prevents the rise of diastolic [Ca²⁺]_i in the heart cells caused by ISO administration.

View larger version (32K): [in this window] [in a new window]   Figure 1.  The diastolic [Ca2+]i in isolated ventricular myocytes of rats A, representative images of fluorescence ratios (F340/F380) visualized with an intensity-defined pseudocolour gradient. The images were recorded from cardiomyocytes preloaded with fura-2 AM. The fluorescence intensity scale is shown to the left of the images. Scale bar, 20 µm. B, pooled data of diastolic [Ca2+]i. Data from 5 rats are represented as means ±S.E.M. Control, n= 90 cells; ISO, n= 104 cells; and ISO + TMZ, n= 96 cells. **P < 0.01 versus control; P < 0.01 versus ISO.

View larger version (13K): [in this window] [in a new window]   Figure 2.  Caffeine-induced Ca2+ transients in isolated ventricular myocytes of rats A, typical recordings of caffeine-induced Ca2+ transients in cells from 3 groups of rats. Top, control rats; middle, ISO-treated rats; and bottom, ISO + TMZ treated rats. B, the average amplitudes of caffeine-induced Ca2+ transients ([Ca2+]i/[Ca2+]i) in cardiomyocytes from control rats (n= 12 cells), ISO-treated rats (n= 10 cells) and ISO + TMZ-treated rats (n= 16 cells). C, the average time-to-peak Ca2+ of the transients in cells from the control, ISO and ISO + TMZ-treated rats. D, the average time constants for decay in caffeine-induced Ca2+ transients, which were obtained by fitting of the decay fluorescence trace to a single exponential function in cells from the control, ISO and ISO + TMZ-treated rats. The data in Figure 2B, 2C and 2D represent means ±S.E.M. from 5 rats. **P < 0.01 versus control; P < 0.05, P < 0.01 versus ISO.

Protein levels of SERCA2a, one of key proteins involved in Ca²⁺ handling, were unchanged in the three groups of rats when examined by immunoblot analysis (Fig. 3A and B). Sarcoplasmic reticulum Ca²⁺-ATPase activity in ventricular myocardium, however, was significantly lower in ISO-treated rats (108.3 ± 8.3 µmol g^–1 min^–1; n= 4 rats) than the control value (147.8 ± 8.6 µmol g^–1 min^–1; n= 4 rats, P < 0.01), while it was increased in ISO + TMZ-treated rats (135.8 ± 4.6 µmol g^–1 min^–1; n= 4 rats) compared with ISO-treated rats (P < 0.05, Fig. 3C). This suggests that treatment with TMZ prevents the decrease of cardiac SR Ca²⁺-ATPase activity caused by ISO administration.

View larger version (21K): [in this window] [in a new window]   Figure 3.  SR Ca2+-ATPase protein expression and activity in the rat heart Immunoblots of SERCA2a (A) in samples obtained from the control (lane 1), ISO-treated rats (lane 2) and ISO + TMZ-treated rats (lane 3) and analysis of contents of SERCA2a (B). C, cardiac SR Ca2+-ATPase activities from the 3 groups of rats. The Ca2+-dependent ATPase activity of SR was determined from cardiac SR (see Methods). The results are means ±S.E.M. of data from 4 rats. **P < 0.01 versus control; P < 0.05 versus ISO.

To determine whether Ca²⁺ influx in heart cells was altered in the three groups of rats, we recorded the L-type Ca²⁺ currents (I_Ca,L) in the isolated ventricular myocytes. There were no significant changes in myocyte size as estimated by the cell membrane capacitance between the three groups (n.s., Table 2). Figure 4A shows current tracings obtained in a similar manner. There was a difference in current shapes between the control and ISO-treated rats, and the peak of I_Ca,L was significantly depressed in ISO-treated rats compared with the controls (P < 0.01, Table 2). Cardiomyocytes from ISO + TMZ-treated rats showed similar current shapes to control cells, and the peak of I_Ca,L was slightly but not significantly increased compared with ISO-treated rats (n.s., Table 2). Figure 4B shows the peak current density–voltage (I–V) relationships of I_Ca,L. Calcium currents were detected at –40 mV and peaked at 0 mV in the control and ISO + TMZ-treated rats, while the I–V curve in cells from ISO-treated rats shifted to a more negative potential and peaked at –10 mV. L-type Ca²⁺ current densities in cells of ISO-treated rats were decreased over all the test potentials between –40 and 40 mV compared with the control rats, while the maximal peak of I_Ca,L densities was significantly increased in ISO + TMZ-treated rats compared with ISO-treated rats (P < 0.01, Table 2). These results suggest that TMZ prevents the reduction of I_Ca,L density in ISO-treated rats.

View larger version (11K): [in this window] [in a new window]   Figure 4.  L-type Ca2+ currents (ICa,L)recorded from isolated ventricular myocytes of rats A, examples of ICa,L recorded from the control, ISO-treated and ISO + TMZ-treated rats. Currents were elicited from a holding potential of –50 mV to 0 mV in a step. B, current density–voltage relationships of ICa,L. Peak ICa,L was normalized to the cell capacitance to give current density (in pA pF–1). Data points represent means ±S.E.M. from 5 rats.

View larger version (19K): [in this window] [in a new window]   Figure 5.  Time constants of inactivation for L-type Ca2+ currents The data represent means ±S.E.M. (n= 7 cells from 5 rats). fast, fast time constant of inactivation; slow, slow time constant of inactivation. **P < 0.01 versus control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Excessive levels of circulating catecholamines have been shown to cause myocardial hypertrophy, myocyte damage and cardiomypathy (Rona, 1985). ISO acts through ß-adrenergic receptors, augmenting the consumption of oxygen and depletion of ATP. It causes severe stress in the myocardial tissue and produces acute myocardial injury (Todd et al. 1985; Grimm et al. 1998). In the present study, ISO-induced myocardial injury in rats was used as an experimental model. We observed that ISO produced sheets or confluent lesions in the hearts, which was in agreement with previous reports (Rona, 1985; Todd et al. 1985). We examined the effects of TMZ on ISO-mediated myocardial injury in rats. Pathological evaluation indicated that pretreatment with TMZ prevented the histological damage of the hearts induced by ISO, indicating that TMZ exerts a pronounced preventative effect on myocardial injury. We observed that TMZ prevented the decrease of ATP levels and the increase of MDA content induced by ISO treatment. These results are consistent with previous findings, which demonstrated that TMZ reduced MDA formation and improved myocardial energy metabolism in the ischaemic rat heart (Guarnieri & Muscari, 1993; Opie & Sack, 2002). Thus, our data suggest that the beneficial effects of TMZ on myocardial injury are associated with the preservation of ATP levels and the limitation of lipid peroxidation in the hearts.

We next attempted to investigate whether intracellular Ca²⁺ handling was altered by TMZ treatment in ISO-mediated myocardial injury of rats. Heart cells were isolated after in vivo studies, and they revealed intrinsic functional changes that could not be ascribed to the extracellular matrix. Firstly, the diastolic [Ca²⁺]_i in resting heart cells was measured by loading with fura-2 AM. The results showed that diastolic [Ca²⁺]_i in heart cells from ISO-treated rats increased significantly compared with the control rats; TMZ prevented the rise of diastolic [Ca²⁺]_i caused by ISO administration (Fig. 1). A similar result was obtained in a previous study, which demonstrated that TMZ inhibited the increase of diastolic [Ca²⁺]_i in cultured cardiac cells under acid-load conditions (Renaud, 1988). The favourable action of TMZ on Ca²⁺ homeostasis was also supported by the prior finding that TMZ prevented high myocardial Ca²⁺ contents with long-term therapeutic procedures (D'hahan et al. 1997). The rise in diastolic [Ca²⁺]_i tends to block many vital enzymatic functions and leads to cell necrosis (Gilloteaux et al. 1990). The prevention of the increase in diastolic [Ca²⁺]_i by TMZ may, at least in part, account for the beneficial effects of TMZ on myocardial injury.

The mechanisms underlying the inhibition of the increase in diastolic [Ca²⁺]_i by TMZ during myocardial injury remain poorly understood. The rise in diastolic [Ca²⁺]_i has been shown to be related to abnormal Ca²⁺ handling by the impaired SR (Meissner & Morgan, 1995; Pei et al. 2003). Therefore, we determined whether caffeine-induced SR Ca²⁺ release and SR Ca²⁺-ATPase protein levels and activity were altered with TMZ treatment in the rats. The results showed that caffeine-induced Ca²⁺ transients were depressed in ISO-treated rats compared with the control values. However, TMZ improved the depression of caffeine-induced Ca²⁺ transients caused by ISO, including prevention of the decease of peak Ca²⁺ and prolongation of the time constant for decay (Fig. 2A and D). It is interesting to speculate that the improvement of the depressed Ca²⁺ transients by TMZ may contribute to improved cardiac contraction during myocardial injury. Indeed, it has been shown that TMZ can improve ischaemic regional myocardial function in patients with chronic coronary artery disease without affecting the haemodynamic determinants (Belardinelli, 2000; Kantor et al. 2000). It was reported that the amplitude of caffeine-induced Ca²⁺ transients can be used as an index of the SR Ca²⁺ content (Ziolo et al. 2001). In the present study, the steady-state SR Ca²⁺ contents in resting cells from ISO-treated rats were decreased by 49% compared with the control values, and this decrease was abolished by TMZ treatment (Fig. 2B). The steady-state SR Ca²⁺ contents reflect a balance between SR Ca²⁺ uptake (via the SR Ca²⁺-ATPase) and efflux (via Ca²⁺ leak pathways; Yang & Steele, 2001). This indicates that TMZ either preserved the activity of SR Ca²⁺-ATPase or reduced the Ca²⁺ efflux associated with the ryanodine receptors. Our results showed that TMZ prevented the decrease of cardiac SR Ca²⁺-ATPase activity caused by ISO administration (Fig. 3C), although the protein levels of SR Ca²⁺-ATPase (SERCA2a) were unchanged (Fig. 3A and B). It was reported that SR Ca²⁺-ATPase activity can be regulated by phosphorylated phospholamban (Kim et al. 2001), but whether TMZ had an effect on the phosphorylation of phospholamban remains to be determined. Thus, our results support the concept that TMZ preserves the activity of SR Ca²⁺-ATPase during myocardial injury, which could increase the reuptake and refilling of Ca²⁺ stores in SR and so lead to a decrease of the diastolic [Ca²⁺]_i compared with ISO-treated rats. This finding is consistent with a recent study in skeletal myoctyes, which showed that TMZ might preserve the SR Ca²⁺-ATPase reuptake activity under conditions of hypoxia (Stary et al. 2003). Thus, our data suggest that the preservation of cardiac SR Ca²⁺-ATPase activity by TMZ might be a possible mechanism for the prevention of the increase of diastolic [Ca²⁺]_i in ISO-mediated myocardial injury of rats.

In addition, we found that TMZ prevented the reduction of I_Ca,L density caused by ISO treatment (Table 2 and Fig. 4), which might favour the improvement of the Ca²⁺ transients and contraction function of cardiomyocytes, although TMZ had little effect on the time constant of I_Ca,L inactivation (Fig. 5). These results seem paradoxical compared with the observation that TMZ prevented the increase of diastolic [Ca²⁺]_i. It should be noted that the diastolic [Ca²⁺]_i reflects a balance between the Ca²⁺ influx (including L-type Ca²⁺ channels and ryanodine receptors) and the Ca²⁺ efflux (mainly including SR Ca²⁺-ATPase and the Na⁺–Ca²⁺ exchanger). Compared with ISO-treated rats, although the Ca²⁺ influx through L-type Ca²⁺ channels increased with TMZ treatment, the Ca²⁺ efflux via SR Ca²⁺ reuptake was enhanced by TMZ as well. In fact, in rat cardiac cells the SR Ca²⁺-ATPase pump removes 92% of the activator Ca²⁺ during diastole and plays an crucial role in regulating the diastolic [Ca²⁺]_i (Bers, 2002). Thus, the summation of these two opposing effects induced by TMZ resulted in the decrease of diastolic [Ca²⁺]_i compared with ISO-treated rats.

In summary, this study shows that TMZ had beneficial effects in preventing histological damage, preserving the ATP levels and limiting lipid peroxidation in rat myocardial injury. The treatment of rats with TMZ inhibited the increase of diastolic [Ca²⁺]_i and prevented the decrease of SR Ca²⁺ contents, SR Ca²⁺-ATPase activity and I_Ca,L density in the heart cells in ISO-mediated myocardial injury. These changes in Ca²⁺ handling could help to explain the favourable action of TMZ on myocardial injury.

	References

Top Abstract Introduction Methods Results Discussion References

 
Belardinelli R (2000). Trimetazidine and the contractile response of dysfunctional myocardium in ischaemic cardiomyopathy. Rev Port Cardiol 19 (Suppl. 5), V35–V39.

Bers DM (2002). Cardiac excitation-contraction coupling. Nature 415, 198–205.[CrossRef][Medline]

Botker HE, Kimose HH, Helligso P & Nielsen TT (1994). Analytical evaluation of high energy phosphate determination by high performance liquid chromatography in myocardial tissue. J Mol Cell Cardiol 26, 41–48.[CrossRef][Medline]

D'hahan N, Taouil K, Dassouli A & Morel JE (1997). Long-term therapy with trimetazidine in cardiomyopathic Syrian hamster BIO 14:6. Eur J Pharmacol 328, 163–174.[CrossRef][Medline]

Ferrari R & Visioli O (1991). Protective effects of calcium antagonists against ischaemia and reperfusion damage. Drugs 42 (Suppl. 1), 14–26.

Gilloteaux J, Bissler JJ, Kondolios P & Jarjoura D (1990). Cardiomyocyte aging and hypertrophy: atrial and ventricular changes in normal and myopathic Syrian hamsters. J Submicrosc Cytol Pathol 22, 249–264.[Medline]

Grimm D, Elsner D, Schunkert H, Pfeifer M, Griese D, Bruckschlegel G, Muders F, Riegger GA & Kromer EP (1998). Development of heart failure following isoproterenol administration in the rat: role of the renin-angiotensin system. Cardiovasc Res 37, 91–100.[Abstract/Free Full Text]

Guarnieri C & Muscari C (1993). Effect of trimetazidine on mitochondrial function and oxidative damage during reperfusion of ischemic hypertrophied rat myocardium. Pharmacology 46, 324–331.[Medline]

Hamill OP, Marty A, Neher E, Sakmann B & Sigworth FJ (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391, 85–100.[CrossRef][Medline]

Kantor PF, Lucien A, Kozak R & Lopaschuk GD (2000). The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase. Circ Res 86, 580–588.[Abstract/Free Full Text]

Kawamura A & Wahler GM (1994). Perforated-patch recording does not enhance effect of 3-isobutyl-1-methylxanthine on cardiac calcium current. Am J Physiol 266, C1619–C1627.

Kim SJ, Kudej RK, Yatani A, Kim YK, Takagi G, Honda R, Colantonio DA, Van Eyk JE, Vatner DE, Rasmusson RL & Vatner SF (2001). A novel mechanism for myocardial stunning involving impaired Ca²⁺ handling. Circ Res 89, 831–837.[Abstract/Free Full Text]

Kodavanti PR, Cameron JA, Yallapragada PR & Desaiah D (1990). Effect of chlordecone (Kepone) on calcium transport mechanisms in rat heart sarcoplasmic reticulum. Pharmacol Toxicol 67, 227–234.[Medline]

Li S, Blaschke M, Heubach JF, Wettwer E & Ravens U (2001). Effects of azelastine on contractility, action potentials and L-type Ca²⁺ current in guinea pig cardiac preparations. Eur J Pharmacol 418, 7–14.[CrossRef][Medline]

Marzilli M (2003). Cardioprotective effects of trimetazidine: a review. Curr Med Res Opin 19, 661–672.[CrossRef][Medline]

Meissner A & Morgan JP (1995). Contractile dysfunction and abnormal Ca²⁺ modulation during postischemic reperfusion in rat heart. Am J Physiol 268, H100–H111.

Monteiro P, Duarte AI, Goncalves LM, Moreno A & Providencia LA (2004). Protective effect of trimetazidine on myocardial mitochondrial function in an ex-vivo model of global myocardial ischemia. Eur J Pharmacol 503, 123–128.[CrossRef][Medline]

Nicolas JM, Renard-Rooney DC & Thomas AP (1998). Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes. J Mol Cell Cardiol 30, 2591–2604.[CrossRef][Medline]

Noble MI, Belcher PR & Drake-Holland AJ (1995). Limitation of infarct size by trimetazidine in the rabbit. Am J Cardiol 76, 41B–44B.[CrossRef][Medline]

Opie LH & Sack MN (2002). Metabolic plasticity and the promotion of cardiac protection in ischemia and ischemic preconditioning. J Mol Cell Cardiol 34, 1077–1089.[CrossRef][Medline]

Orrenius S, Zhivotovsky B & Nicotera P (2003). Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4, 552–565.[CrossRef][Medline]

Pei JM, Kravtsov GM, Wu S, Das R, Fung ML & Wong TM (2003). Calcium homeostasis in rat cardiomyocytes during chronic hypoxia: a time course study. Am J Physiol Cell Physiol 285, C1420–C1428.[Abstract/Free Full Text]

Piper HM, Meuter K & Schafer C (2003). Cellular mechanisms of ischemia-reperfusion injury. Ann Thorac Surg 75, S644–S648.[Abstract/Free Full Text]

Renaud JF (1988). Internal pH, Na⁺, and Ca²⁺ regulation by trimetazidine during cardiac cell acidosis. Cardiovasc Drugs Ther 1, 677–686.[CrossRef][Medline]

Rona G (1985). Catecholamine cardiotoxicity. J Mol Cell Cardiol 17, 291–306.[CrossRef][Medline]

Rona G, Chappel CI, Balazs T & Gaudry R (1959). An infarct-like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. AMA Arch Pathol 67, 443–455.[Medline]

Sayer RJ (2002). Intracellular Ca²⁺ handling. Adv Exp Med Biol 513, 183–196.[Medline]

Scheuerlein R, Schmidt K, Poenie M & Roux SJ (1991). Determination of cytoplasmic calcium concentration in Dryopteris spores: a developmentally non-disruptive technique for loading of the calcium indicator fura-2. Planta 184, 166–174.[CrossRef][Medline]

Simonides WS & van Hardeveld C (1990). An assay for sarcoplasmic reticulum Ca²⁺-ATPase activity in muscle homogenates. Anal Biochem 191, 321–331.[CrossRef][Medline]

Sipido KR & Callewaert G (1995). How to measure intracellular [Ca²⁺] in single cardiac cells with fura-2 or indo-1. Cardiovasc Res 29, 717–726.[CrossRef][Medline]

Stanley WC (2004). Myocardial energy metabolism during ischemia and the mechanisms of metabolic therapies. J Cardiovasc Pharmacol Ther 9 (Suppl. 1), S31–S45.[Abstract/Free Full Text]

Stary CM, Kohin S, Samaja M, Howlett RA & Hogan MC (2003). Trimetazidine reduces basal cytosolic Ca²⁺ concentration during hypoxia in single Xenopus skeletal myocytes. Exp Physiol 88, 415–421.[Abstract]

Tani M (1990). Mechanisms of Ca²⁺ overload in reperfused ischemic myocardium. Annu Rev Physiol 52, 543–559.[Medline]

Temsah RM, Netticadan T, Chapman D, Takeda S, Mochizuki S & Dhalla NS (1999). Alterations in sarcoplasmic reticulum function and gene expression in ischemic-reperfused rat heart. Am J Physiol 277, H584–H594.

Todd GL, Baroldi G, Pieper GM, Clayton FC & Eliot RS (1985). Experimental catecholamine-induced myocardial necrosis. I. Morphology, quantification and regional distribution of acute contraction band lesions. J Mol Cell Cardiol 17, 317–338.[CrossRef][Medline]

Valen G (2003). Cellular signalling mechanisms in adaptation to ischemia-induced myocardial damage. Ann Med 35, 300–307.[CrossRef][Medline]

Yang Z & Steele DS (2000). Effects of cytosolic ATP on spontaneous and triggered Ca²⁺-induced Ca²⁺ release in permeabilised rat ventricular myocytes. J Physiol 523, 29–44.[Abstract/Free Full Text]

Yang Z & Steele DS (2001). Effects of cytosolic ATP on Ca²⁺ sparks and SR Ca²⁺ content in permeabilized cardiac myocytes. Circ Res 89, 526–533.[Abstract/Free Full Text]

Yano M, Ikeda Y & Matsuzaki M (2005). Altered intracellular Ca²⁺ handling in heart failure. J Clin Invest 115, 556–564.[CrossRef][Medline]

Yao A, Su Z, Nonaka A, Zubair I, Lu L, Philipson KD, Bridge JH & Barry WH (1998). Effects of overexpression of the Na⁺–Ca²⁺ exchanger on [Ca²⁺]_i transients in murine ventricular myocytes. Circ Res 82, 657–665.[Abstract/Free Full Text]

Ziolo MT, Katoh H & Bers DM (2001). Expression of inducible nitric oxide synthase depresses ß-adrenergic-stimulated calcium release from the sarcoplasmic reticulum in intact ventricular myocytes. Circulation 104, 2961–2966.[Abstract/Free Full Text]

	Acknowledgements

 
This study is supported by National Natural Science Foundation of China (no. 30200374) and Foundation of Human Functional Genetics Laboratory of Jiangsu province and Jiangsu province 135 Key Laboratory in China.

This article has been cited by other articles:

				Corrigendum. Exp Physiol, July 1, 2006; 91(4): 799 - 799. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 91/3/591    most recent expphysiol.2005.032615v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Meng, D. Articles by Zhang, J.-N. Search for Related Content PubMed PubMed Citation Articles by Meng, D. Articles by Zhang, J.-N. Related Collections Cardiovascular control