Cardiovascular Research

Cardiovascular Research 2002 53(2):334-340; doi:10.1016/S0008-6363(01)00501-6
© 2002 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

Cytosolic free magnesium modulates Na/Ca exchange currents in pig myocytes

Shao-kui Wei^a, John F Quigley^a, Stephen U Hanlon^a, Brian O'Rourke^b and Mark C.P Haigney^a,*

^aDivision of Cardiology, A-3060, Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
^bInstitute of Molecular Cardiobiology, Johns Hopkins University, Baltimore, MD, USA

^* Corresponding author. Tel.: +1-301-295-3826; fax: +1-301-295-3557 mcph{at}aol.com

Received 22 June 2001; accepted 5 October 2001

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: Cardiac Na/Ca exchanger (NCX) protein is up-regulated and intracellular free magnesium ([Mg²⁺]_i) is significantly reduced in experimental heart failure. We asked whether changes in [Mg²⁺]_i in a physiologically relevant range could alter the I_NCX. Methods: The nickel-sensitive current was measured in voltage-clamped myocytes (Yorkshire pig; left ventricular) exposed to ramp pulses at 37°C in Tyrode's solution containing ouabain, nifedipine and ±Ni²⁺ (5 mmol/l). The intracellular free [Ca²⁺] and [Mg²⁺] concentrations were set at 50 nmol/l and 1.25 mmol/l (HiMg) or 0.13 mmol/l (LoMg), respectively, through pipette dialysis. Results: Reducing [Mg²⁺]_i resulted in a significant increase in both outward and inward Ni-sensitive current without a shift in the reversal potential. This effect was not due to the inadvertent reduction of intracellular free [ATP] secondary to binding of ATP to Mg²⁺; reducing intracellular [ATP] in LoMg cells from 1.35 mmol/l to 0.18 mmol/l did not affect I_NCX. The intracellular free [Ca²⁺] was raised from 50 to 200 nmol/l, resulting in augmented inward and outward current due to calcium activation. HiMg attenuated both inward and outward currents significantly compared to LoMg, suggesting that [Mg²⁺]_i competes with [Ca²⁺]_i at the allosteric regulatory site. Conclusion: Cytosolic free magnesium modulates the I_NCX over a physiologic range independent of [ATP]_i. Reduced [Mg²⁺]_i in heart failure could contribute to altered calcium regulation of the NCX, contributing to the altered heart failure phenotype through enhanced NCX activity.

KEYWORDS Intra/extracellular ions; Ion channels; Myocytes; Na/Ca-exchanger

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This article is referred to in the Editorial by T. Kiyosue (pages 290–291) in this issue.

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The Na/Ca exchanger (NCX) has an important role in regulating cytosolic calcium ([Ca²⁺]) that is incompletely understood. Early in the action potential, low cytosolic [Ca²⁺] and membrane depolarization favor outward movement of Na⁺ and inward movement of Ca²⁺ in a 3:1 ratio, resulting in a net hyperpolarizing effect (‘outward current’ or ‘reverse mode exchange’). Following the action potential plateau, the membrane potential falls and cytosolic [Ca²⁺] rises, causing the exchanger to reverse direction, resulting in Ca²⁺ efflux and a depolarizing current (‘forward mode exchange’) [1,2]. Little is known about the factors (either intracellular or extracellular) which act to modify the activity of the exchanger. Evidence is accumulating that increased [Ca²⁺]_i serves to rapidly activate both inward and outward currents despite the fact that the concentration gradient favors only inward current [3]. Understanding the cytosolic modulators of the exchanger is crucial to exploring the role of the exchanger in normal and pathologic states.

Cytosolic free magnesium ([Mg²⁺]_i) appears to alter cellular calcium handling through interactions with a wide variety of proteins. Free magnesium acts as a co-factor for SERCA 2 and the sarcolemmal Ca-ATPase, enhancing calcium uptake at low concentrations but competing with calcium at higher levels [4]. Free magnesium appears to regulate adaptation and the open probability of the ryanodine receptor, so that increasing [Mg²⁺]_i results in fewer spontaneous openings and a reduction in calcium release [5]. The peak L-type calcium current is inhibited by free magnesium [6]. The effect of changing [Mg²⁺]_i in a physiologically relevant range on Na/Ca exchange, however, is unknown.

We have recently reported that [Mg²⁺]_i is reduced by 50% in a canine pacing heart failure model. Furthermore, manipulation of pipette magnesium between 0.5 and 1.0 mmol/l significantly altered the duration and variability of the action potential in both control and failing myocytes [7]. Because the NCX is significantly upregulated in heart failure, it is crucial to test whether a reduction in [Mg²⁺]_i would alter the magnitude of the NCX current (I_NCX). We chose to study the effect of altering [Mg²⁺]_i in isolated myocytes from Yorkshire pigs, which have an action potential contour similar to that of humans. Extrapolating from the described effects of magnesium on other calcium-handling proteins, we hypothesized that a reduction in [Mg²⁺]_i would result in an increase in I_NCX, since there would be less competition for binding to the exchanger between calcium and magnesium. We found that [Mg²⁺]_i does modulate the I_NCX, and that this effect is relatively independent of the ATP concentration. Reducing [Mg²⁺]_i appears to enhance the I_NCX at both low and high diastolic calcium concentrations. Finally, the data suggests that cytosolic Mg²⁺ does not simply compete with Ca²⁺ for binding to the exchanger, but acts at a site that regulates both forward and reverse mode NCX activities.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 Myocyte isolation and culture
The methods for cell isolation and culture have been described previously [8]. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Yorkshire domestic pigs (10 weeks old) were euthanized with pentobarbital sodium. The hearts were harvested by left lateral thoracotomy, and immersed in ice-cold saline. The region of ventricle perfused by the left anterior descending coronary artery was excised, cannulated, and perfused at 15 ml/min for 10 min with nominally Ca²⁺-free modified Tyrode's solution (in mmol/l, NaCl 138, KCl 4, MgCl₂ 1, NaH₂PO₄ 0.33, glucose 10, and HEPES 10 (pH 7.3 with NaOH) at 37°C and oxygenated with 100% O₂). Perfusion was continued with the same solution but containing 0.24% (w/v) collagenase type I (Sigma), and 0.028% protease XIV (Sigma) for 12 min, and then for 10 min with washout modified Tyrode's solution with 0.1 mmol/l CaCl₂ and 0.02% albumin. Sections of well-digested ventricular tissue from the mid-myocardial layer of the ventricle were dissected out, and cells were mechanically dissociated and resuspended in buffers of gradually increasing [Ca²⁺]. To remove dead myocytes and residual contaminating cell types, the myocytes suspension was centrifuged through a discontinuous Percoll gradient, usually resulting in over 90% rod-shape cells. To allow the myocytes to recover from enzymatic digestion, the cells were cultured overnight at 37°C in serum-free medium 199 supplemented with 5 mmol/l carnitine, 5 mmol/l creatine, 5 mmol/l taurine, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin. We have found that this approach increases the rate of successful giga-seal formation without significantly altering their phenotype with regard to NCX function (data not shown).

2.2 Electrophysiology
Whole-cell recordings were obtained at 37°C using standard patch-clamp techniques. Membrane current was assessed by use of an Axopatch-100A amplifier and a 1/100 CV-3 headstage (Axon instruments, USA). Experimental control, data acquisition and data analysis were accomplished by use of the software package Pclamp 8.0 with the Digidata 1200 acquisition system (Axon instruments). Patch-pipettes were pulled from thin-walled glass capillary tubes and heat-polished. The electrode resistance ranged between 1 and 2 M. The external solution contained (mmol/l): NaCl 145, MgCl₂ 1, HEPES 5, CaCl₂ 2, CsCl 5 and glucose 10 (pH 7.4, adjusted with NaOH). Ouabain (0.02 mmol/l) and nifedipine (0.01 mmol/l) were added to the solution. The internal solution contained (mmol/l): CsCl 65, NaCl 20, Na₂ATP 5, CaCl₂ 6 or 13, MgCl₂ 4.0 or 8.0, HEPES 10, tetraethyl ammonium chloride (TEA) 20, EGTA 21 and ryanodine 0.05 (pH 7.2 adjusted with CsOH). The concentrations of free Ca²⁺, Mg²⁺, ATP and MgATP in different internal solution are shown in Table 1, which were calculated using ‘Maxchelator for Windows’ software (Dr. C. Patton, Johns Hopkins University). Membrane currents were elicited by using a standard voltage ramp protocol. From a holding potential of –40 mV, a 100 ms step depolarization to +75 mV was followed by a descending voltage ramp (from +75 mV to –115 mV at 100 mV/s). The protocol was applied every 10 s. I_NCX was measured as the Ni-sensitive current. Ni²⁺ (5 mmol/l) is added to define the fraction of current that derives from NCX (total current remaining after subtraction of post-Ni²⁺ trace). Membrane capacitance was directly recorded from the membrane test function of Pclamp8.0 before compensating series resistance and membrane capacitance. The mean capacity of pig ventricular cells was 81.2±2.2 pF; there was no difference between high and low intracellular [Mg²⁺] groups. The calcium-activated chloride current was not blocked. In a separate group of experiments using an identical protocol in 11 cells, Ni-sensitive currents were measured before and after the addition of 100 µmol/l Niflumic acid (Sigma). We found no evidence of calcium-activated chloride current in this species, similar to reports in Guinea pig [9].

View this table: [in this window] [in a new window]   Table 1 The concentration of free Ca2+, Mg2+, ATP and MgATP in internal solutions (mM)a

 
2.3 Data statistical analysis
Data are presented as the mean±S.E.M. Significant differences were evaluated by unpaired t-test. A P-value of <0.05 was regarded as a statistically significant finding.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Effect of intracellular [Mg²⁺] on I_NCX
Fig. 1A shows the basic protocol used for measuring I_NCX, where [Ca²⁺]_i was buffered to 50 nmol/l. [Mg²⁺]_i was set at 0.13 mmol/l (LoMg) or at 1.25 mmol/l (HiMg, see Table 1). L-type calcium, potassium, and Na–K pump currents were blocked using specific inhibitors. The holding potential was set at –40 mV to inactivate Na current, then the voltage was stepped to +75 mV and ramped down to –115 mV at a rate of 100 mV/s to elicit outward and inward current. The protocol was repeated in the presence of 5 mmol/l Ni²⁺ to obtain Ni-sensitive I_NCX. Fig. 1B shows representative I_NCX normalized to cell capacitance for myocytes with low [Mg²⁺]_i or high [Mg²⁺]_i. Both outward and inward I_NCX in a myocyte with low [Mg²⁺]_i were enhanced compared to a myocyte with high [Mg²⁺]_i. Fig. 1C shows mean data of peak outward and inward I_NCX in myocytes with low and high [Mg²⁺]_i. Fig. 1D shows the relationship of I_NCX to voltage. It is clear that reduced intracellular [Mg²⁺] enhanced both outward and inward I_NCX. The apparent reversal potential was unchanged in the two groups (–25.9±2.8 in LoMg cells vs. –22.6±5.0 in HiMg cells; P=0.82); the value was similar to that reported by Convery and Hancox when using a descending ramp protocol [10].

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 The effect of altering intracellular [Mg2+] on INCX in pig ventricular myocytes. (A) Voltage protocol used for measuring INCX, the typical membrane currents obtained in a myocyte in the presence or absence of 5 mmol/l Ni2+, and the Ni-sensitive INCX. (B) The representative INCX normalized to cell capacitance for myocytes with low (upper tracing) or high (lower tracing) intracellular [Mg2+], demonstrating the reduction in INCX due to increased pipette [Mg2+]. (C) Mean data of peak outward (at 70 mV) and inward (at –110 mV) INCX density from myocytes with low (LoMg) or high (HiMg) intracellular [Mg2+]. *P=0.010/0.002 (outward/inward current) LoMg versus HiMg. (D) Current–voltage relationship for INCX from the same cells as in (C). *P<0.05 LoMg versus HiMg.

 
3.2 Effect of free ATP on I_NCX
Because Mg²⁺ avidly binds ATP in intact myocytes, changing intracellular [Mg²⁺] significantly alters intracellular free [ATP] and [MgATP]. In the above experiment, reducing intracellular free [Mg²⁺] from 1.25 mmol/l to 0.13 mmol/l resulted in an increase in cytosolic free [ATP] of nearly an order of magnitude; [MgATP]_i was slightly decreased as well (see Table 1). Several studies have reported that cytosolic ATP regulates NCX activity [11,12] and that increasing intracellular ATP significantly enhances I_NCX. Consequently it was important to determine whether the increase of I_NCX seen in low intracellular [Mg²⁺] was due to a change in either cytosolic free [ATP] or [MgATP]. To test this possibility, we decreased intracellular free [ATP] through pipette dialysis to the level seen in the high [Mg²⁺]_i group, while keeping intracellular [Mg²⁺] equal to that in the low [Mg²⁺]_i group (solution 3 in Table 1). Fig. 2A demonstrates representative I_NCX from myocytes with high and low free [ATP], showing that decreased free [ATP] does not significantly change I_NCX. Mean data of peak outward and inward current for myocytes with high and low free [ATP] are given in Fig. 2B. The results showed that decreasing [ATP]_i and [MgATP]_i in this range did not significantly alter the I_NCX, supporting the notion that the increase in I_NCX seen in low intracellular [Mg²⁺] is attributable to actions of the free [Mg²⁺]_i.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 The effect of intracellular [ATP] on INCX. (A) Representative INCX normalized to cell capacitance for myocytes with high (upper tracing) or low (lower tracing) intracellular [ATP]. Note the absence of a significant effect on the INCX due to reducing [ATP]. (B) Mean data of peak outward (at 70 mV) and inward (at –110 mV) INCX density from myocytes with high (HiATP) or low (LoATP) intracellular [ATP], P=0.78/0.52 (outward/inward current) HiATP versus LoATP.

 
3.3 Effect of intracellular [Ca²⁺] on NCX activity
The previous experiments were performed at low cytosolic calcium concentrations. Since the exchanger is activated by increased [Ca²⁺] in most species, we asked whether [Mg²⁺]_i can still modulate I_NCX in higher intracellular [Ca²⁺]. To test this hypothesis, we increased intracellular [Ca²⁺] to 200 nmol/l, while the intracellular [Mg²⁺] was again held at either 0.13 or 1.25 mmol/l. Fig. 3A shows representative I_NCX normalized to cell capacitance from myocytes with low [Mg²⁺]_i (upper tracing) or high [Mg²⁺]_i (lower tracing) when intracellular [Ca²⁺] was 200 nmol/l. Both outward and inward I_NCX in a myocyte with low [Mg²⁺]_i were still significantly enhanced compared to a myocyte with high [Mg²⁺]_i. The mean peak current data (Fig. 3B) confirm these trends. Fig. 3C shows the relationship of mean I_NCX to voltage. These results suggest that [Mg²⁺]_i still modulates the I_NCX even at higher intracellular Ca²⁺ concentrations. Fig. 4A shows the effect of increasing cytosolic Ca²⁺ from 50 nmol/l to 200 nmol/l on the current–voltage relationship for myocytes with low intracellular [Mg²⁺] (0.13 mmol/l). Increased intracellular [Ca²⁺] enhanced both outward and inward I_NCX in low intracellular [Mg²⁺] cells, even though the electrochemical gradient only favors inward current. This phenomenon is consistent with the report by Levitsky et al. that increasing [Ca²⁺]_i enhances I_NCX via Ca²⁺-dependent NCX activation in canine myocytes [13]. But while this observed increase in outward current confirms the presence of a Ca-dependent NCX activation in porcine myocytes at low free [Mg²⁺]_i, we were only able to demonstrate a trend towards an increase in outward current in the high [Mg²⁺]_i cells (P=0.18; Fig. 4B). Finally, to see whether increasing intracellular [Mg²⁺] had a greater effect on inward versus outward currents (consistent with competition for binding of calcium to the exchanger), we compared the ratio of peak inward to outward currents at low and high intracellular [Mg²⁺] at both calcium concentrations. The ratios of peak inward to outward currents were unchanged by raising intracellular [Mg²⁺] (Fig. 4C), indicating a balanced reduction in NCX activity. This finding suggests that [Mg²⁺] is not primarily competing with calcium at the exchanger (which would only affect inward current), but at the allosteric activation site.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 The effect of altering intracellular [Mg2+] on INCX in myocytes with high intracellular [Ca2+] (200 nmol/l). (A) Representative INCX normalized to cell capacitance for myocytes with low (upper tracing) or high (lower tracing) intracellular [Mg2+]. Note that the INCX are increased at the lower value of [Mg2+]i. (B) Mean data of peak outward (at 70 mV) and inward (at –110 mV) INCX density from myocytes with low (LoMg) or high (HiMg) intracellular [Mg2+], *P=0.011/0.005 (outward/inward current) LoMg versus HiMg. (C) Current–voltage relationship from myocytes with low (LoMg) or high (HiMg) intracellular [Mg2+] when intracellular [Ca2+] was 200 nmol/l. *P<0.05 LoMg versus HiMg.

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 The effect of altering intracellular [Ca2+] on INCX. (A) Current–voltage relationship from myocytes with high (HiCa) or low (LoCa) intracellular [Ca2+] when intracellular [Mg2+] was 0.13 mmol/l, *P<0.05 HiCa versus LoCa. (B) Current–voltage relationship from myocytes with high (HiCa) or low (LoCa) intracellular [Ca2+] when intracellular [Mg2+] was 1.25 mmol/l, #P=0.178 (outward current)/*P=0.020 (inward current) HiCa versus LoCa. Note that while increasing intracellular [Ca2+] from 50 nmol/l to 200 nmol/l resulted in a significant increase in outward current in the low intracellular [Mg2+] cells, the increase seen in the high intracellular [Mg2+] cells failed to reach significance. (C) The effect of altering intracellular [Mg2+] on the ratio of inward to outward INCX in both calcium concentrations. Note that increasing [Mg2+]i has no effect on the ratio of inward to outward INCX in either calcium concentration. P=0.52 (LoCa) or 0.77 (HiCa) LoMg versus HiMg.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The major finding from this study is that cytosolic free magnesium modulates the I_NCX over a physiologically relevant range in Yorkshire pig myocytes. This effect is not due to binding of free ATP by magnesium, and it appears that reducing cytosolic free magnesium increases both the inward and outward currents. These findings are significant in light of increasing interest in the regulation of the NCX in heart failure and the recent finding that intracellular free magnesium is significantly reduced in that condition.

4.1 Regulation of the NCX
Previous studies revealed that intracellular ATP stimulates NCX activity. Dipolo and Bauge reported that the exchanger has a high affinity for ATP with half-maximal activation at about 200 µmol/l in squid axons [14]. The mechanism of modulation of the NCX by ATP is unclear, and may involve exchanger phosphorylation, activation of phospholipid translocases, ATP-dependent cytoskeletal processes, phosphatidylinositol-4,5-biphosphate modulation, and a direct effect of ATP on the exchanger [15]. Recently, DiPolo et al. found that NCX activity was greatly enhanced as [ATP] was increased from 0.2 to 1.0 mmol/l in squid axons [16]. By comparison, altering intracellular free [ATP] (from 1.35 mmol/l to 0.177 mmol/l) and [MgATP] (from 3.65 mmol/l to 0.48 mmol/l) via pipette dialysis did not significantly change NCX activity in our mammalian model. Our results suggest that in the pig the effect of intracellular [Mg²⁺] on NCX activity is due to the change in [Mg²⁺] itself as opposed to [ATP]_i or [MgATP]_i, at least over this range of values.

On a beat-to-beat basis, perhaps the most important regulatory mechanism of NCX is Ca²⁺-dependent activation, which involves the enhancement of transport activity through the binding of cytosolic Ca²⁺ to regulatory sites in the central hydrophilic domain of NCX [12,17,18]. Philipson et al. have identified the Ca²⁺-binding regions at amino acids 371 to 508 in exchanger [19]. In our experiments on myocytes with low intracellular [Mg²⁺], increasing intracellular [Ca²⁺] from 50 to 200 nmol/l significantly enhanced NCX activity as represented by increased outward I_NCX.

Other investigators have suggested a possible role for magnesium in modulating NCX activity. Howarth and Levi have reported that increasing pipette [Mg²⁺] to 7.1 mmol/l eliminated the calcium transient during depolarizations to very positive potentials in the presence of I_Ca, blockers in rabbit cardiac myocytes. When dialyzed at nominal magnesium levels (2.9 µmol/l), however, a significant contraction and calcium transient was elicited by depolarization, consistent with magnesium-sensitive calcium entry through the NCX [20]. In this study, we examined I_NCX and demonstrated significant modulation over a physiologically relevant range, thus directly confirming their previous findings. The mechanism of inhibition is unclear. Our finding that both the inward and outward currents were attenuated almost equally by magnesium argues against competition for the internal calcium binding site on the exchanger as the predominate mechanism. Although further experiments are needed, these results are consistent with competition between magnesium and calcium for binding to an allosteric regulatory site. Indeed, such an effect was predicted by Levitsky et al., who demonstrated inhibition of ⁴⁵Ca²⁺ binding to a fusion protein containing amino acids 240–532 of the canine NCX by magnesium with a K_i,Mg of 0.3 mM [13].

4.2 Implications for heart failure
Several groups have reported increased expression of sarcolemmal NCX protein in experimental and human heart failure [21–24], but controversy persists regarding the measurement of I_NCX. The present study suggests that close attention to pipette free Mg²⁺ concentrations may be required to accurately assess the contribution of upregulated-NCX to excitation–contraction coupling in heart failure. An increase in NCX activity might alter the shape of the action potential in heart failure, contributing to arrhythmogenesis.

Time for primary review 33 days

	Acknowledgements

 
This work was supported in part by grants 9807745 from the American Heart Association Mid-Atlantic Consortium, Baltimore MD (MH) and USUHS CO83ME (SKW) and CO83MD (SUH) and NIH grant R01HL61711.

	Notes

 
Disclaimer: the views expressed in this paper reflect the opinions of the authors only and not the official policy of the Uniformed Services University or the Department of Defense.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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				S.-k. Wei, A. Ruknudin, S. U. Hanlon, J. M. McCurley, D. H. Schulze, and M. C.P. Haigney Protein Kinase A Hyperphosphorylation Increases Basal Current but Decreases {beta}-Adrenergic Responsiveness of the Sarcolemmal Na+-Ca2+ Exchanger in Failing Pig Myocytes Circ. Res., May 2, 2003; 92(8): 897 - 903. [Abstract] [Full Text] [PDF]

				T. Kiyosue Removal of intracellular Mg2+ activates cardiac Na+/Ca2+ exchanger Cardiovasc Res, February 1, 2002; 53(2): 290 - 291. [Full Text] [PDF]

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