Li XQ et al / Acta Pharmacol Sin 2003 Dec; 24 (12): 1224-1230

Effects of tumor necrosis factor-alpha on calcium movement in rat ventricular myocytes1

LI Xiao-Qiang, ZHAO Ming-Gao, MEI Qi-Bing2, ZHANG Yan-Feng, CAO Wei, WANG Hai-Fang, CHEN Dan3, CUI Yi

Department of Pharmacology, 3Electron Microscopy Center, The Fourth Military Medical University, Xi-an 710032, China

1 Project supported by the National Natural Science Foundation of China, No 39500177.

2 Correspondence to Prof MEI Qi-Bing. Phn 86-29-337-4555. Fax 86-29-337-4552. E-mail qbmei@hotmail.com

Received 2002-11-26 Accepted 2003-10-20

KEY WORDS tumor necrosis factor; myocardium; calcium; patch-clamp techniques; confocal microscopy

ABSTRACT

AIM: To study the effects of tumor necrosis factor-alpha (TNF-) on calcium movement in rat ventricular myocytes. METHODS: Intracellular free Ca2+ concentration was measured with calcium fluorescent probe Fluo-3/AM and laser confocal microscope. L-type calcium current (ICa,L) was recorded with the whole-cell configuration of the patch-clamp techniques. RESULTS: At 2, 20 and 200 µg/L, TNF- was found to increase intracellular free Ca2+ concentration in a dose-dependent manner illustrated by the increment of calcium fluorescence density with laser confocal microscope. Nicardipine 0.5 µmol/L slightly attenuated TNF--induced response. When the cardiac myocytes were exposed to caffeine (100 mmol/L) for 30 min, TNF- failed to induce any change of intracellular free calcium. However, it was found that TNF- inhibited ICa,L in whole-cell patch-clamp experiments. At 2, 20, and 200 µg/L, TNF- decreased peak ICa,L by 3.9 % (-5.1 pA/pF±0.3 pA/pF vs -4.9 pA/pF±0.2 pA/pF, n=9, P>0.05), 15.7 % (-5.1 pA/pF±0.3 pA/pF vs -4.3 pA/pF±0.3 pA/pF, n=9, P<0.05) and 19.6 % (-5.1 pA/pF±0.3 pA/pF vs -4.1 pA/pF±0.4 pA/pF, n=9, P<0.01), respectively. It shifted the steady-state inactivation curve of ICa,L to the left (V1/2 shifted from -28.7 mV±0.3 mV to -37.8 mV±1.4 mV, n=7, P<0.05), while it took no effects on steady-state activation and recovery from inactivation. CONCLUSION: TNF- inhibited ICa,L in rat ventricular myocytes, while increasing the intercellular free Ca2+ level due to the release of Ca2+ from intracellular stores.

INTRODUCTION

TNF- is a potentially powerful anti-neoplastic substance. In the heart it is produced by both cardiac myocytes and resident macrophages under conditions of cardiac stress, and is thought to be responsible for many of the untoward manifestations of cardiac diseases. When expressed in large amounts, it can produce widespread deleterious effects, such as cytostatic and cytotoxic activity[1]. Clinical evidence demonstrated that TNF- induced left ventricular dysfunction, acute pulmonary edema, and congestive cardiomyopathy[2]. Calcium ion plays important physiological roles, including mediation of cell contraction, secretion, protein phosphorylation, and gene transcription. In the cardiac myocyte, how does TNF- affect calcium movement? In this aspect, there is not a categorical and uniform conclusion at present. To answer this question, the present experiments were designed to investigate the effects of TNF- on calcium movement in rat ventricular myocytes.

MATERIALS AND METHODS

Detection of intracellular free Ca2+ concentration ([Ca2+]i) by laser confocal microscopy Reproducible single cell cultures were obtained by the procedure described previously[3,4]. Sprague-Dawley rats aging 1-3 d (provided by the Experimental Animal Center of Fourth Military Medical University, Grade II, Certificate No C98008) were used. Cardiomyocytes were cultured in MEM (Eagle's) culture medium containing 10 % fetal bovine serum in 5-mm culture plates with cell density of 1×104 L-1. Cells were incubated at 37 ºC in humidified air with 5 % CO2 for 2 d. On the d 3, cardiomyocytes were rinsed with D-Hanks' solution for three times and then incubated in D-Hanks' solution containing Fluo 3-acetoxymethyl ester (Fluo-3/AM) 10 µmol/L at 37 ºC for 30 min. The cells were then washed with D-Hanks' solution to remove the extracellular Fluo- 3/AM. The fluorescence was detected with a MRC-1024 laser scanning confocal microscope (BIO-RAD Int, USA). An argon laser was used to excite Fluo-3 at 488 nm and emit at 526 nm. Systolic [Ca2+]i changes were shown by fluorescence intensity (FI). TNF- (Sigma) was a kindly gift from Dr WANG Hui in Biotechnology Center, Administration of Science Research, Fourth Military Medical University, and was dissolved in the modified Tyrode's solution.

Whole-cell patch-clamp techniques Five-week-old male Sprague-Dawley rats (provided by the Experimental Animal Center of Fourth Military Medical University, Grade II, Certificate No C98008 ) weighing 160-180 g were used.

The single myocyte was isolated from left ventricle of adult rats as described previously[3,5]. In brief, the rats were anesthetized with pentobarbital sodium (30 mg/kg, ip) and anticoagulated with heparin sodium (300 U/kg, iv). The heart was rapidly excised and mounted on a Langendorff apparatus. It was perfused conversely via the aorta for 5 min with a modified Tyrode's solution (in mmol/L: NaCl 144; KCl 4, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.33, glucose 5.5, and HEPES 5.5, adjust pH to 7.4 with NaOH) equilibrated with 100 % O2 at 37 ºC at a rate of 5 to 10 mL/min, followed for 5 min with Ca2+-free Tyrode's solution (omitting CaCl2 from Tyrode's solution). The heart was then perfused with 0.1 % collagenase (type I, Sigma) dissolved in Ca2+-free Tyrode's solution until the solution flowed freely (15 to 25 min). Then left ventricular free wall was dissected and gently blowed in a Kraftbruhe (KB) solution (in mmol/L: KOH 70, KCl 40, L-glutamic acid 50, taurine 20, KH2PO4 10, MgCl2 0.5, glucose 11, egtazic acid 0.5, and HEPES 10, adjusted pH to 7.4 with KOH). The isolated myocytes were stored in the KB solution at 4 ºC and studied within 12 h after isolation.

Rod-shaped noncontracting cells with clear striations and resting potential of at least -75 mV were used. At room temperature (18-22 ºC), currents were recorded by the gigaohm seal patch-clamp technique in whole-cell configuration with a CEZ 2300 amplifier (Nihon Kohden). The resistance of patch pipette ranged from 4 to 6 M¸ when filled with the pipette solution. Pipette capacitance and series resistance were compensated to minimize the duration of capacitive currents. The run-down of ICa,L averaged 18 % over 15 min. All the recordings of ICa,L were, therefore, performed at 15 min after the establishment of a gigaohm seal. The current signal was sampled directly into a computer and analyzed by using pClamp software (version 7.0, AXON instruments Inc). For the recording of ICa,L, the external solution was composed of (in mmol/L) NaCl 137 or choline chloride 137 (for activation and inactivation curves), MgCl2 0.5, CaCl2 1.8, HEPES 5, glucose 10, and CsCl 4.6 (pH 7.3 with NaOH). The pipette solution was composed of (in mmol/L) CsCl 140, MgCl2 0.5, Na2ATP 4, egtazic acid 1, HEPES 5, and glucose 5.5 (pH 7.2 with CsOH). TNF- (Sigma) was dissolved in the modified Tyrode's solution. Solutions were gased with 95 % O2 and 5 % CO2.

To obtain current-voltage (I/V) curves, ICa,L was recorded by applying 300 ms depolarizing pulses at a test potential ranging from -40 mV to +50 mV in 10-mV steps from a holding potential of -40 mV at an interval of 5 s (0.2 Hz). To study steady-state activation of ICa,L, cells were maintained at a holding potential of -80 mV. ICa,L was elicited by depolarizing the cells to test potentials from -40 to +10 mV in 10 mV increments for 300 ms. A double-pulse protocol was used to determine the steady-state inactivation curves. Cells were clamped at a holding potential of -80 mV for 300 ms to a range of potentials from -80 mV to +10 mV (10-mV steps), then stepped to +10 mV for 200 ms at 1-s intervals. The time dependence of ICa,L recovery from inactivation was determined by using a double-pulse protocol: two depolarizing pulses to +10 mV with varying interpulse intervals (times of 20 ms) were applied from a holding potential of -40 mV every 5 s. The extent of recovery at each interpulse interval was obtained by expressing the amplitude of ICa,L.

Data were expressed as mean±SD and the statistical significance of differences was estimated according to t-test for grouped observations.

RESULTS

Effect of TNF- on intracellular free Ca2+ of myocardium Myocytes loaded with Fluo-3/AM beat synchronously in Hanks' solution. TNF- 20 µg/L increased fluorescence intensity of [Ca2+]i at least for 1 min (Fig 1) and increased it in a dose-dependent manner (Fig 2A). This increment was slightly attenuated by adding dihydropyridine calcium antagonist, nicardipine 0.5 µmol/L (Fig 2 B), or replacing the extracellular Hanks' solution with D-Hanks' solution, showing that L-type calcium channels took the minor effects of TNF- on [Ca2+]i metabolism. When the cardiac myocytes were exposed to caffeine (100 mmol/L) for 30 min, TNF- 2, 20 µg/L failed to induce any changes of intracellular free Ca2+ (Fig 2C).

Fig 1. Fluorescence intensity dynamic process of intracellular free Ca2+ (Microscopic analysis of the intracellular calcium increase) in neonatal rat ventricular myocytes in the presence of TNF- 20 µg/L. The first two lattices of the last line display fluorescence intensity of the myocyte in the absence of TNF- . TNF- 20 µg/L was added at the third lattice of the last line. Fluo-3-loaded neonate rat ventricular myocyte was recorded every 1 s from left to right and from down to up using laser confocal microscope system. Fluorescence intensity was seen to increase with the time at least for 60 s.

Fig 2. The dynamic process of fluorescence intensity in neonatal rat ventricular myocytes. The lowest curve in each graph stands for the basal fluorescence intensity used as control background. The other ones showed the dynamic process of fluorescence intensity changing with the drugs addition. A: arrow 1, 2, and 3 show the time of adding TNF- 2, 20, and 200 µg/L respectively. B: arrow 1 and 2 show the time of adding TNF- 2 µg/L and nicardipine (0.5 µmol/L). C: when the myocytes were exposed to caffeine 100 mmol/L for 30 min, adding TNF- 2 µg/L (arrow 1), 20 µg/L (arrow 2) failed to induce any changes of fluorescence intensity (two curves show the responses of two cells respectively).

Effect of TNF- on L-type calcium current (ICa,L) in rat ventricular myocytes The cell membrane capacitance (Cm) was 150 pF±25 pF (n=27 from 16 rats). The threshold for the activation of ICa,L and the potential of peak current were -40 mV and 0 mV respectively at the holding potential of -40 mV. TNF- 2, 20, and 200 µg/L decreased peak ICa,L density (the amplitude normalize to cell membrane capacitance) at the test potential of 0 mV by 3.9 % (-5.1 pA/pF±0.3 pA/pF vs -4.9 pA/pF±0.2 pA/pF), 15.7 % (-5.1 pA/pF±0.3 pA/pF vs -4.3 pA/pF±0.3 pA/pF) and 19.6 % (-5.1 pA/pF±0.3 pA/pF vs -4.1 pA/pF±0.4 pA/pF) respectively (Fig 3). At the same time, TNF- did not change the threshold (-40 mV) of the activation of ICa,L and the potential (0 mV) of peak current.

Fig 3. Effects of TNF- on ICa,L in rat ventricular myocytes. A: protocol and original current recording. B: rundown of ICa,L with the time and the effects TNF- 2, 20, 200 µg/L on ICa,L. C: the I-V relationship of ICa,L in ventricular myocytes in the absence and presence of TNF- . TNF- 2 µg/L, n=9 cells from 9 hearts. TNF- 20 µg/L, n=9 cells from 9 hearts. TNF- 200 µg/L, n=8 cells from 9 hearts. Mean±SD. bP<0.05, cP<0.01 vs control.

Effect of TNF- on steady-state activation and inactivation kinetics of ICa,L Steady-state activation or inactivation were obtained by conventional protocols, and the corresponding curves of ICa,L were fitted with Boltzman equation of the following form: I/Imax= 1/{1+EXP[(V-V1/2)/]}. I is the calcium current, Imax is the maximal amplitude of calcium current, V is the voltage of conditioning pulse, V1/2 is the potential of half activation or inactivation and is the slope factor. For each individual cell, data were fitted to the Boltzmann distribution of the form: V1/2 and slope were compared and used to generate a continuous curve that fitted the average normalized data. TNF- 200 µg/L did not markedly influence activation properties. Half activation potential (V1/2) and slope factor () were (-24.1±0.6) mV and (2.0±0.3) under control conditions, and at (-24.2±0.5) mV and (2.2±0.3) in the presence of TNF- 200 µg/L (Fig 4A, P>0.05). Steady-state inactivation was determined by a double-pulse protocol. TNF- 200 µg/L shifted half inactivation potential (V1/2) from (-28.7±0.3) mV to (-37.8±1.4) mV, and slope factor () was not affected (5.5±0.3 vs 5.7±1.2, Fig 4B, P<0.05).

Fig 4. Steady-state activation and inactivation and time-dependent recovery of ICa,L in the absence and presence of TNF- 200 µg/L. Protocols are given in the insets. A: Half activation potential (V1/2) and slope factor (k) were (-24.1± 0.6) mV and (2.0±0.3) in control (open symbols), and (-24.2± 0.5) mV and (2.2±0.3) in the presence of TNF- 200 µg/L (closed symbols) (n=6 cells from 5 hearts, aP>0.05 ). B: Steady-state inactivation was determined by a double-pulse protocol. n=7 cells from 6 hearts.

The time dependence of ICa,L recovery from inactivation In the course of ICa,L recovery from inactivation, the normalized data from 6 myocytes were fitted by a biexponential function according to the equation: y= y0+A1[1-exp(-x /t1)]+A2[1-exp(-x /t2)], where x is the time, A1 and A2 represent the proportion of recovery accounted for by the time constants t1 and t2 respectively. TNF- 200 µg/L did not affect the half recovery time of ICa,L from inactivation (46.7 ms vs 47.9 ms, Fig 5, P>0.05).

Fig 5. Recovery time curves from inactivation of ICa,L. The cells were depolarized from -40 mV to +10mV with a duration of 200 ms and various interpulse durations (times of 20 ms) were applied. Half recovery time of ICa,L shifted from 46.7 ms under control conditions (open symbols) to 47.9 ms in the presence of TNF- 200 µg/L (closed symbols), (n=5 cells from 5 hearts, aP>0.05).

DISCUSSION

At the end of 19th century William Coley, a New York surgeon, was the first to describe necrosis of the tumor induced by bacterial toxins[6]. In 1975, a protein responsible for the induction of this process was identified and called TNF-. Since its discovery, the understanding of the roles of TNF- in human biology and diseases has grown. In the heart, both myocardial macrophages and cardiac myocytes themselves can synthesize TNF-. Accumulating evidence indicates that myocardial TNF- is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in a variety of experimental and clinical conditions, including ischemia-reperfusion injury, sepsis, chronic heart failure, viral myocarditis, and cardiac allograft rejec-tion[7]. So, the spectrum of biological activities for TNF- is not limited to cytotoxic effects but rather TNF- exerts pleiotropic effects[8].

Although the precise role of TNF- in the heart is not known, the elaboration of TNF- in cardiac pathophysiological contexts suggests that TNF- may play a pathogenetic role in above-mentioned diseases. TNF- is involved in the regulation of normal tissue homeostasis affecting cell proliferation, differentiation, and death[9]. Patients with HF have been shown increased levels of TNF- in the myocardium[10]. However, the mechanisms by which this pleiotropic cytokine alters cardiac mechanical function remain unclear. It is reported that Ca2+ is also involved in TNF--mediated cell damage through the activation of proteases[11].

As a ubiquitous intracellular second messenger in the signal transduction pathways, Ca2+ plays a pivotal role in many biological processes, including muscle contraction, gene regulation, enzymatic reaction, cell injury and apoptosis[12]. Therefore, the effects of TNF- on calcium movement probably mediate some cellular functions. But, there is not a categorical and uniform conclusion of TNF-'s effect on calcium movement in the cardiac myocyte at present. It has been reported that TNF- inhibited cardiac L-type Ca2+ channel current (ICa,L)[13] and decreased peak systolic [Ca2+]i[14]. Whereas Amadou et al reported that at a low concentration TNF- produced a 40 % increase and at a high concentration TNF- evoked a biphasic effect comprising an initial positive effect peaking at 5 min, followed by a sustained negative effect[15].

In this study, we found that TNF- 2, 20, and 200 µg/L, increased intracellular free Ca2+ concentration significantly in a dose-dependent manner in the cardiac myocytes. When sarcoplasmic reticulum (SR) calcium store was exhausted by caffeine, TNF- failed to induce any changes of intracellular free calcium. Moreover, The whole-cell configuration of the patch-clamp indicated TNF- inhibited cardiac L-type calcium channel current (ICa,L). It suggests that TNF--induced increment of Ca2+ concentration be due to calcium releasing from the sarcoplasmic reticulum (SR). This process is independent of voltage-dependent L-type Ca2+ channels. In addition, inactivation curves showed TNF- 200 µg/L shifted half inactivation potential to the left. So, TNF- accelerated steady-state inactivation of ICa,L. It suggests that the effect of TNF- on inactivation state be stronger than on activation state of L-type calcium channel.

These phenomena indicate TNF- must participate in other signal transduction gateways to induce intracellular free Ca2+ release from the sarcoplasmic reticulum (SR) and inhibit Ca2+ influx feedback in ventricular myocytes of adult rats. Now growing evidence suggests that most of the biologic effects of TNF- are mediated by the p55 receptor or tumor necrosis factor receptor 1 (TNFR1)[16]. The effects of TNF- on calcium movement appear to involve a modulatory effect on G-protein-mediated signal transduction via its TNFR1 receptor. The specific target could be at the level of either G-protein or phospholipase C.

ACKNOWLEDGEMENTS The authors thank the valuable technical assistance from Dr ZHANG Xiao-Dong (Department of Physiology, The Fourth Millitary Medical University).

REFERENCES