Cardiovascular Research

Cardiovascular Research 2001 49(1):38-47; doi:10.1016/S0008-6363(00)00205-4
© 2001 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Overexpression of the Na⁺/Ca²⁺ exchanger and inhibition of the sarcoplasmic reticulum Ca²⁺-ATPase in ventricular myocytes from transgenic mice

Cesare M.N. Terracciano^a,*, Kenneth D. Philipson^b and Kenneth T. MacLeod^a

^aImperial College School of Medicine at NHLI, Cardiac Medicine, Dovehouse Street, London SW3 6LY, UK
^bDepartments of Physiology and Medicine, UCLA, Los Angeles, CA, USA

^* Corresponding author. Tel.: +44-171-352-8121; fax: +44-171-351-8145 c.terracciano{at}ic.ac.uk

Received 21 March 2000; accepted 21 July 2000

	Abstract

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

 
Background: Myocytes from failing hearts produce slower and smaller Ca²⁺ transients associated with reduction in expression of sarcoplasmic reticulum (SR) Ca²⁺ ATPase and an overexpression of Na⁺/Ca²⁺ exchanger. Since the physiological role of both these proteins is competing for, and removing, Ca²⁺ from the cytoplasm, overexpression of the exchanger may compensate for less effective SR Ca²⁺ uptake. This study demonstrates this compensatory effect and provides a quantitative description of the results. Methods: Ventricular myocytes from transgenic mice overexpressing the Na⁺/Ca²⁺ exchanger (TR) and nontransgenic littermates (NON) were used. Cell shortening, cytoplasmic [Ca] (using indo-1 AM) and electrophysiological parameters were monitored. Results: TR myocytes displayed faster Ca²⁺ transients and twitches compared with NON myocytes. Superfusion with thapsigargin prolonged the time-course of Ca²⁺ transients of TR myocytes until these were equal to the ones measured in NON myocytes. The amount of SR Ca²⁺-ATPase (SERCA) inhibition needed to obtain such transients was calculated as a function of V_max for the Ca²⁺ flux via SERCA and found to be 28%. In TR myocytes V_max for the Ca²⁺ flux via Na⁺/Ca²⁺exchange was 240% of NON myocytes. When Ca²⁺ transients in TR myocytes were slowed by thapsigargin to similar values to the ones recorded in NON myocytes, SR Ca²⁺ content was also correspondingly reduced. Conclusions: The results suggest that in pathophysiological conditions where there is a reduction in SERCA function, overexpression of Na⁺/Ca²⁺ exchanger can compensate and allow normal Ca²⁺ homeostasis to be maintained. In mouse ventricular myocytes a 2.4-fold increase in Na⁺/Ca²⁺ exchange activity compensates for a reduction in SERCA function by 28% so maintaining the duration of the Ca²⁺ transient.

KEYWORDS Na/Ca-exchanger; SR (function); Myocytes; Calcium (cellular); e–c coupling

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This article is referred to in the Editorial by G. Isenberg (pages 1–6) in this issue.

	1 Introduction

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

 
In heart muscle current evidence suggests that the process of contraction is mainly initiated by the action potential activating L-type Ca²⁺ channels to cause an influx of Ca²⁺ into the cells. The voltage-dependent Ca²⁺ influx across the sarcolemma promotes further release of stored Ca²⁺ from the sarcoplasmic reticulum (SR) by a process known as Ca-induced Ca²⁺ release [1]. Together both sources of Ca²⁺ initiate contraction [2–4]. Relaxation takes place as a result of two main systems decreasing the cytoplasmic Ca²⁺ concentration. The SERCA pumps Ca²⁺ back into the SR ready for release at the next beat and the sarcolemmal Na⁺/Ca²⁺ exchanger extrudes the Ca²⁺ that entered the cell via the Ca²⁺ channels. Although other mechanisms (the sarcolemmal Ca²⁺ ATPase and the mitochondria) are involved in the maintenance of a low cytoplasmic Ca²⁺ concentration, the proteins that perform the major roles on a beat-to-beat basis are SERCA and the Na⁺/Ca²⁺ exchanger [2].

In heart failure intracellular Ca²⁺ regulation has been shown to be altered (e.g. [5,6]) manifesting as a slower and smaller cytoplasmic Ca²⁺ transient. Associated with the change to the profile of the Ca²⁺ transient are alterations in the expression of the proteins involved in Ca²⁺ regulation and in the mRNA encoding their production [6,7]. In failing human hearts it has been shown that there is a reduction in the expression of SERCA sometimes accompanied by an overexpression of the Na⁺/Ca²⁺ exchanger [8,9]. Since both SERCA and the Na⁺/Ca²⁺ exchanger compete for, and remove, Ca²⁺ from the cytoplasm during the Ca²⁺ transient, overexpression of the Na⁺/Ca²⁺ exchanger in heart failure may result in more effective Ca²⁺ removal by this route to compensate for less effective SR Ca²⁺ uptake.

Quantification of the relative contributions of the Ca²⁺ regulation mechanisms to relaxation in different species has been rigorously examined [2] in normal physiological circumstances but is poorly understood in the pathophysiological setting of heart failure. Although some groups have recently tried to examine the contributions of the two main mechanisms in failing animal and human hearts (e.g. [10–12]), the pathophysiological condition itself confounds the interpretation and so the relationship between changes in protein expression and their functional consequences remain poorly understood.

The supposition that overexpression of the Na⁺/Ca²⁺ exchanger should compensate for less effective SERCA function remains untested and there has been no quantification of the degree of compensation that could be achieved with a two- to three-fold overexpression of the exchanger — roughly the increase in protein levels measured in human heart failure [8]. Accordingly, the aims of this work were (1) to quantify the amount of reduction in SERCA activity that could be compensated by the overexpression of the Na⁺/Ca²⁺ exchanger in an otherwise unchanged system and (2) to study the possible mechanisms involved in such compensation. Using transgenic mice overexpressing the Na⁺/Ca²⁺ exchanger and by gradually and partially inhibiting SERCA with thapsigargin, this study shows for the first time that functional compensation for reduced SERCA activity is possible and quantifies the degree of compensation that can be achieved for a two-to three-fold overexpression of the Na⁺/Ca²⁺ exchanger.

	2 Methods

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

 
2.1 Transgenic mice overexpressing the Na⁺/Ca²⁺ exchanger
Transgenic (TR) mice overexpressing the Na⁺/Ca²⁺ exchanger have been produced and characterised as described previously [13–15]. A transgene construct consisting of the open reading frame of the canine cardiac Na⁺/Ca²⁺ exchanger [16] under the control of the -myosin heavy chain (-MHC) promoter was used. The SV40 transcriptional terminator was inserted downstream of -MHC to provide a polyadenylation signal. The transgene was purified and microinjected into the nuclei of C57Bl/6xC3HF1 mice. Southern blot analysis on genomic DNA extracted from tail clippings was used to demonstrate the presence of the construct (TR mice). Littermates which did not show the transgene construct were used as controls (NON mice).

2.2 Cell isolation
Mice were killed by cervical dislocation. Isolation of mice ventricular myocytes has been performed following a method previously described [15]. Myocytes were stored in enzyme solution (for composition see Solutions) at room temperature and used within 7–8 h of isolation.

2.3 Cell shortening and calcium measurements
Cell shortening was measured using a video-based motion detector described by Steadman et al. [17]. Cell shortening is expressed as an absolute value.

A Ca²⁺-sensitive, single excitation, dual emission fluorescent dye, indo-1, was used as described previously [18]. The cells were loaded with indo-1 by incubation with the acetoxymethyl ester form (10 µM) of the indicator (indo-1 AM, Molecular Probes, Eugene, OR, USA) for 15 min at room temperature. Supernatant was then discarded and substituted with Dulbecco's Modified Eagle's Medium (Gibco BRL, Life technologies, Paisley, UK). The cells were used after at least 30 min (to allow de-esterification of the intracellular indicator). Calibration of indo-1 fluorescence was performed. R_min, R_max and b (ratio of free to bound indo-1 fluorescence at 485 nm) were obtained in vivo using cells loaded with indo-1 AM. Intracellular [Ca²⁺] was varied by using the non-fluorescent Ca²⁺ ionophore 4BrA-23187 (Molecular Probes) in solutions of varying [Ca²⁺]. The cells were also incubated with 3 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma, Poole, UK). This, together with the substitution of glucose with 10 mM 2-deoxy-D-glucose (Sigma), was used to obtain metabolic inhibition to avoid hypercontracture during the application of solution containing high [Ca²⁺] [19]. The experiments were performed at 37°C and a K_d value of 395 nM was used [20].

2.4 Electrophysiology
The electrophysiological experiments were performed using an Axoclamp-2B system (Axon Instruments). To avoid dialysis of the cells and to minimise the effects of changing the intracellular environment, high resistance (20–30 M) microelectrodes pulled from borosilicate glass (Clark Electromedical) were used.

The microelectrode filling solution contained: KCl, 2 M; EGTA; 0.1 mM; HEPES, 5 mM, pH 7.2. The cells were stimulated in current clamp mode at 0.5 Hz with a 1.0 nA pulse of depolarising current of 10 ms duration. Voltage-clamp experiments were performed in switch-clamp mode (switching rate: 5–8 kHz). When the cells were not impaled with microelectrodes, they were field-stimulated using a pair of platinum wires in the superfusing chamber (volume 60 µl).

When integration of caffeine-induced transient inward currents was performed, the baseline was taken at the end of the caffeine pulse, as reported in previous papers [18,21].

Current clamp and voltage clamp protocols were controlled with PCLAMP 6.0.3 software (Axon Instruments).

2.5 Calculation of Ca²⁺ fluxes
A method described by Bassani et al. [19] was used. Briefly, indo-1 fluorescence was converted to [Ca²⁺] using the calibration parameters described in Terracciano et al. [15]. The caffeine-induced changes in [free Ca²⁺] were converted to [total Ca²⁺] using empirically derived intracellular buffering parameters calculated for rabbit ventricular myocytes [22]. Cytoplasmic [indo-1] was assumed to be 50 µM. This value is not known but it has previously been used in several studies in rat and rabbit ventricular myocytes [19,23,24] since the fluorescence produced is similar to the one obtained when [indo-1] was calculated using patch pipette loading [25]. To calculate V_max, K_m and the Hill coefficient, the traces representative of the decline in [total Ca²⁺] were smoothed by fitting with a LOWESS method using GraphPad prism 3.0 software. The time derivative from these traces was calculated. The pCa–velocity relationship subsequently obtained was fitted with the equation:

where J is the flux, V_max is the maximum velocity, K_m is the Michaelis–Menten constant and H is the Hill coefficient.

2.6 Acquisition systems and statistical analysis
The data were recorded on computer using PCLAMP 6.0.3 (rate of sampling: 0.5–3 kHz). Time-to-peak (TTP) was measured between the point before the initial increase of the trace and the peak of the signal. Time-to-50% relaxation (T50) was measured between the peak of the signal and the point on the declining phase corresponding to half of the total size of the signal. To assess statistical differences between means, the Student's t test was performed. Unless otherwise specified, the results are expressed as mean±standard error of the mean (SEM). The number of myocytes that have undergone investigation is represented by n which were isolated from a minimum of three hearts per n number.

2.7 Solutions
Composition (in mM):

• Normal tyrode (NT) solution: NaCl, 140; KCl, 6; MgCl₂, 1; CaCl₂, 1; glucose, 10; HEPES, 10; pH to 7.4 with 2 M NaOH.
  
• Enzyme solution (ES): NaCl, 120; KCl, 5.4; MgSO₄, 5; CaCl₂, 0.2; pyruvate, 5; glucose, 20; taurine, 20; HEPES, 10; bubbled with 100% O₂; pH 7.4
  
• Na⁺-free/Ca²⁺-free solution: LiCl, 140; KOH, 6; MgCl₂, 1; glucose, 10; HEPES, 10; EGTA, 0.75; pH to 7.4 with 1 M LiOH.
  

Chemicals were generally purchased from BDH (Poole, UK). Thapsigargin was purchased from Calbiochem (La Jolla, CA, USA).

The temperature of the superfusing solution was 37°C throughout the experiments. The rate of superfusion was 2–3 ml/min except during fast application of caffeine when it was 12–15 ml/min.

	3 Results

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

 
3.1 Overexpression of the Na⁺/Ca²⁺ exchanger and duration of the Ca²⁺ transient
In TR myocytes field-stimulated at 1 Hz at 37°C the time course of the Ca²⁺ transient was faster compared with NON myocytes (TTP in TR myocytes: 110±4 ms (n = 14); in NON myocytes: 146±7 (n = 24); P<0.01; T50 in TR myocytes: 124±4 ms (n = 14); in NON myocytes: 185±7 (n = 24); P<0.01; TTP+T50 in TR myocytes: 234±6 ms (n = 14); in NON myocytes: 332±11 (n = 24); P<0.01) (Fig. 1, left panel). We reported this phenomenon previously in similar experiments performed at room temperature [15]. The overexpression of the Na⁺/Ca²⁺ exchanger in mouse ventricular myocytes is therefore responsible for a faster Ca²⁺ transient. Partial inhibition of SERCA can balance this effect in TR myocytes producing Ca²⁺ transients of similar duration to the one recorded in NON myocytes. This was shown by the following experiments. TR myocytes were superfused with 200 nM thapsigargin. At this concentration, thapsigargin produces a slow, partial and irreversible inhibition of SERCA [21]. After varying periods of time thapsigargin gradually slowed the time course of the Ca²⁺ transient in TR myocytes until this was equal (Fig. 1, right panel) or slower than the one measured in NON myocytes. Thus, if the time-course of the Ca²⁺ transient is considered, overexpression of the Na⁺/Ca²⁺ exchanger compensates for a reduction in SERCA function.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Ca2+ transients recorded in a TR myocyte and in a NON myocyte were scaled and superimposed (left). Application of 200 nM thapsigargin (TG) gradually slowed the time course of the Ca2+ transient in the TR myocyte until the rate was equal (right) or slower than that measured in NON myocytes.

 
No significant changes in the amplitude of the Ca²⁺ transients were detected in the two groups [Ca²⁺ amplitude in TR: 125.6±18 nM (n = 14); in NON: 133±25 (n = 22)] as previously reported [15]. Ca²⁺ transient amplitude increased slightly at the start of application of thapsigargin in TR myocytes but at the point shown in Fig. 1 was not significantly different from control (198±38 nM, n = 10). The traces were therefore normalised and emphasis has been placed on the time course of the transients in all the experiments described.

3.2 Quantification of Ca²⁺ fluxes via SERCA and Na⁺/Ca²⁺ exchanger
We then quantified the amount of inhibition of SERCA required to slow the Ca²⁺ transients of TR myocytes until they were of the same duration as the NON myocytes (Fig. 2). TR and NON myocytes were field-stimulated at 1 Hz until Ca²⁺ transients of steady-state amplitude were recorded. Stimulation was stopped and the superfusing solution changed to a Na⁺-free/Ca²⁺-free solution for 2 s. Caffeine (10 mM) was added to this solution for 1 s in order to obtain Ca²⁺ release from the SR (caff 1). Caffeine was then removed from the superfusing solution. Na⁺-free/Ca²⁺-free solution was maintained for a further 12 s and cytoplasmic Ca²⁺ returned to its baseline value. Under these conditions, Ca²⁺ removal from the cytoplasm is mainly due to SERCA and this Ca²⁺ decline was used to calculate the net Ca²⁺ flux into the SR mediated by this protein (J_SR). A second application of 10 mM caffeine was then performed but after 1 s the superfusing solution was changed to NT+10 mM caffeine and maintained for a further 12-s period. Under these conditions Ca²⁺ extrusion from the cytoplasm is due almost entirely to the Na⁺/Ca²⁺ exchanger. Net Ca²⁺ flux mediated by the Na⁺/Ca²⁺ exchanger was calculated using the declining phase of the Ca²⁺ transient elicited by this second application of caffeine (caff 2). We correlated the level of activity of the two Ca²⁺ regulation mechanisms with the duration of the steady-state Ca²⁺ transients recorded before each protocol.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 This protocol was performed in control conditions and at 2-min intervals after starting superfusion with thapsigargin. An example recorded from a NON myocyte is shown. Two consecutive applications of caffeine (in Na+-free/Ca2+-free solution) were performed to release Ca2+ from the SR. Ca2+ decline after the first application of caffeine in Na+-free/Ca2+-free solution was used to calculate Ca2+ flux via SERCA (JSR). Ca2+ decline after the second application in the continuous presence of caffeine was employed to measure Ca2+ flux via the Na+/Ca2+ exchanger (JNa/Ca).

 
The values for the caffeine-induced Ca transient (in Na⁺-free/Ca²⁺-free solution) amplitude were: TR myocytes: 1556±227 nM (n = 11); NON myocytes: 880±147 nM (n = 13); P = 0.01. These values support the findings previously reported [15] that the SR Ca²⁺ content is increased in TR myocytes. A further quantification of the SR Ca²⁺ content has been carried out using integration of the caffeine-induced transient inward current and described below.

In both TR and NON myocytes the amplitude of Ca²⁺ transients elicited by the two consecutive applications of caffeine was not different (caff 2/caff 1 in TR myocytes: 0.98±0.16 (n = 9); caff 2/caff 1 in NON myocytes: 1.14±0.12 (n = 12); P = 0.438). This suggests that during the 12-s period in Na⁺-free/Ca²⁺-free solution after the first application of caffeine, much of the cytoplasmic Ca²⁺ had been taken up by the SR with no significant efflux of Ca²⁺ from the cytoplasm mediated by the sarcolemmal Ca²⁺ ATPase or uptake by mitochondria. This, together with previous findings [15], supports the assumption that these mechanisms play a minimal role in Ca²⁺ removal from mouse ventricular myocytes and, for the purpose of this study, can be ignored.

At the end of the protocol described above, stimulation was restored and the superfusing solution was changed to one with similar composition but containing 200 nM thapsigargin. The protocol described in Fig. 2 was repeated every 2 min after onset of thapsigargin application. After this time interval and before applying the next paired caffeine application protocol, a stimulation train elicited twitches and Ca²⁺ transients of constant amplitude suggesting that steady-state was achieved and reloading of the SR with Ca²⁺ was complete.

In order to quantify SERCA and Na⁺/Ca²⁺ exchange function, V_max of the Ca²⁺ fluxes following each of the two applications of caffeine were calculated. Fig. 3, upper panel shows traces of the caffeine-induced Ca²⁺ transient and the decline of Ca²⁺ in Na⁺-free/Ca²⁺-free solution (caff 1) in control conditions and after 3 min of thapsigargin exposure. The lower panel in Fig. 3 shows values of the corresponding Ca²⁺ fluxes plotted against pCa and the sigmoidal fits obtained as described in Methods in control conditions and after 3 min superfusion with thapsigargin. Thus we could measure the V_max for net flux of Ca²⁺ into the SR (J_SR) produced by TR myocytes when the Ca²⁺ transient was of similar duration to NON myocytes in control conditions. We could also calculate V_max for J_Na/Ca (obtained from the declining phase of caff 2) allowing concurrent assessment of the degree of overexpression of the Na⁺/Ca²⁺ exchanger activity.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effects of superfusion with 200 nM thapsigargin in TR and NON myocytes on JSR are shown. Ca2+ traces for the first application of caffeine (Ca2+ decline in Na+-free/Ca2+-free solution) in control conditions and after 3 min in TG in the same TR myocyte were scaled and superimposed in the upper part of the figure. Ca/t against pCa was plotted to show how Vmax is reduced after 3 min in TG (lower graph). (Goodness-of-fit: control R2: 0.812; Sy.x=10.2; TG R2=0.76; Sy.x=8.08).

 
Table 1 shows mean values of V_max, K_m and the Hill coefficient for J_SR and J_Na/Ca in TR and NON myocytes before thapsigargin addition. J_SR was not different in the two groups. This result correlates well with the observation that the expression of SR Ca²⁺ proteins (SERCA, phospholamban and calsequestrin) was unchanged in TR myocytes compared with NON myocytes [15].

View this table: [in this window] [in a new window]   Table 1 Ca2+ fluxes via the SR Ca2+ uptake and the Na+/Ca2+ exchanger in TR and NON myocytes

 
Since the protocol allowing the flux calculation was repeated every 2 min we could determine the relationship between J_SR and the duration of the Ca²⁺ transient (TTP+T50) during application of thapsigargin in TR myocytes. This is shown in Fig. 4. Firstly, for each experiment with TR myocytes a monoexponential relationship was derived between the V_max of J_SR and the duration of the Ca²⁺ transient. Then V_max of J_SR at 332 ms (mean TTP+T50 of the Ca²⁺ transient measured in NON myocytes; the vertical rectangular area on x axis represents the mean±SEM of TTP+T50 of the Ca²⁺ transient in 24 NON myocytes) was calculated for each TR myocyte. The horizontal rectangular area on the y axis represents the mean±SEM of V_max of J_SR in nine TR myocytes calculated at this point. This was 72±5% (n = 9) of the control value suggesting that the SR Ca²⁺ uptake can be reduced by approximately 28% of its original value before the duration of the Ca²⁺ transient becomes larger than the average transient in NON myocytes. In other words, the overexpression of the Na⁺/Ca²⁺ exchanger in these myocytes allows a 28% reduction in SERCA activity.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Quantification of JSR and JNa/Ca in TR myocytes when Ca2+ transients are prolonged to NON myocyte values by application of thapsigargin is shown. The upper graph shows data for Vmax of JSR in TR myocytes after applications of TG plotted against the TTP+T50 value of the Ca2+ transient measured immediately before the corresponding caffeine application. Application of TG produced a gradual reduction in JSR and prolongation of duration of the Ca2+ transient. The vertical rectangular area represents the mean±SEM of the Ca2+ transient duration in NON myocytes in control conditions. For each experiment in TR myocytes a monoexponential curve was fitted on the JSRVmax–duration of Ca2+ transient relationship. The value at 332 ms (this being average TTP+T50 of Ca2+ transients in NON myocytes) was derived. The horizontal rectangular area is the projection of the mean value±SEM of JSRVmax calculated in this fashion. This represents the level of SERCA inhibition which produces the same Ca2+ transient duration in TR myocytes. The lower graph compares this result with the calculation of Vmax for JNa/Ca.

 
In order to quantify the overexpression of the Na⁺/Ca²⁺ exchanger in functional terms, V_max of J_NaCa in TR myocytes was measured as described above and found to be 240±15% (n = 13) of the value measured in NON myocytes. That is, an approximately 2.4-fold increase of Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger at maximal Ca²⁺ was found in TR myocytes supporting previous molecular biological and functional observations [13,15]. The lower panel in Fig. 4 summarises the results of the determinations of V_max for both fluxes when the Ca²⁺ transients had the same duration in the two groups of myocytes (Fig. 1B). The results suggest that a 2.4-fold increase in Na⁺/Ca²⁺ exchanger activity can compensate for an approximately 28% reduction in SERCA function.

3.3 Role of the SR Ca²⁺ content
The amount of releasable Ca²⁺ from the SR (SR Ca²⁺ content) is an important determinant of the size of contraction [2]. TR myocytes show an increased SR Ca²⁺ content [15]. This could, at least partially, be the cause of the faster relaxation observed in TR myocytes particularly when the Na⁺/Ca²⁺ exchanger is inhibited. In order to investigate the importance of differences in the SR Ca²⁺ content in the compensatory effect described above, experiments were performed to measure SR Ca²⁺ content in control conditions and during application of 200 nM thapsigargin. In these experiments, no indo-1 was used to avoid the buffering effect of the indicator and cell shortening was monitored instead. Cells were voltage-clamped at –75 mV and stimulated at 1 Hz using voltage-clamp steps to 0 mV (step duration: 300 ms). Stimulation was stopped, membrane potential was continued to be held at –75 mV and 10 mM caffeine rapidly applied as described before [18,21,26] and maintained for 10 s. SR Ca²⁺ content was calculated using the integral of the caffeine-induced transient inward current [15]. Caffeine was then washed out and stimulation restored. Thapsigargin (200 nM) was then applied and the SR Ca²⁺ content measured again at 2-min intervals.

Once again, the time-course of the twitches was initially faster in TR myocytes compared with NON myocytes (TTP+T50 in TR myocytes: 128±7 ms (n = 15); in NON myocytes: 164±4 ms (n = 14); P<0.01). The SR Ca²⁺ content was measured at the point at which thapsigargin slowed the duration of the twitch in TR myocytes to a similar value compared with that in NON myocytes. Fig. 5A shows examples of the caffeine-induced transient inward current recorded after stimulation train in NT solution in a TR myocyte under control conditions (left), NON myocyte (centre) and in a TR myocyte after 3 min in thapsigargin (TR (TG), right). Using the assumptions reported in previous studies [27,18,28], the SR Ca²⁺ content was calculated by integrating this caffeine-induced current. The SR Ca²⁺ content was significantly larger in TR myocytes compared with NON myocytes in NT solution but application of thapsigargin reduced this difference (Fig. 5C). Fig. 5B shows the relationship between the duration of the twitch and the SR Ca²⁺ content in TR myocytes. The rectangular area running parallel with the x axis represents the SR Ca²⁺ content calculated at the mean TTP+T50 of the NON myocyte twitch (164 ms). This was 79±4% (n = 10) of the value measured in TR myocytes in control conditions (TR myocyte SR Ca²⁺ content in control: 121±9 µM non-mitochondrial volume (accessible volume; a.v.) (n = 15); TR myocyte SR content at twitch of 164 ms duration: 98±10 µM a.v. (n = 10); paired t-test, P = 0.003). When thapsigargin prolonged the TTP+T50 of twitches of TR myocytes until they had a comparable time-course to the twitches in NON myocytes (164 ms duration), the SR Ca²⁺ contents were not significantly different (NON myocytes in control conditions: 91.4±6 µM a.v. (n = 12); t-test, P = 0.54). These data are summarised in Fig. 5C) and suggest that a reduction in SERCA function of TR myocytes, so that they produce a twitch of similar duration to that in NON myocytes, is accompanied by a reduced SR Ca²⁺ content to NON myocyte values.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effects of application of thapsigargin on the SR Ca2+ content in TR myocytes. Traces from a TR myocyte in control, after application of thapsigargin and from a NON myocyte are shown in (A). Integration of these currents allowed calculation of the SR Ca2+ content. In 15 TR myocytes in control conditions the integral of the caffeine-induced inward current was 251±23 pC; in the same myocytes during TG application, when the duration of the twitches was comparable to the one measured in NON myocytes, the integral of the current was reduced to 187±24 pC. In 12 NON myocytes the integral of the current was 188±16 pC. SR Ca2+ content values calculated as described in the text after different intervals in TG were plotted against the duration of the twitch (B). Rectangular areas represent the SR Ca2+ content for twitches of similar duration to those recorded in NON myocytes with an approach similar to the one described for Fig. 4A. (C) SR Ca2+ content is significantly larger in TR compared to NON myocytes. When twitch duration becomes similar between the two groups (in TR with thapsigargin), the SR Ca2+ content in TR myocytes significantly reduces to a value similar to NON myocyte values.

 

	4 Discussion

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

 
Na⁺/Ca²⁺ exchanger activity during the cardiac cycle has been investigated in several studies in different species (for review see [2]). In rabbit and guinea-pig myocardium Ca²⁺ extrusion has been shown to be the predominant mode of action of this mechanism whereas in rat and mouse myocardium the role of the Na⁺/Ca²⁺ exchanger during the cardiac cycle is still unclear. We reported in a previous work that, when the Na⁺/Ca²⁺ exchanger function was inhibited in mouse ventricular myocytes, a faster Ca²⁺ decline was observed, particularly at cytoplasmic [Ca²⁺] close to diastolic values. To explain this phenomenon, we suggested that the Na⁺/Ca²⁺ exchanger in this species might be taking Ca²⁺ into the cell rather then extruding Ca²⁺ during rest and during the latter part of the Ca²⁺ transient [15]. This idea is not new; the mechanism has been proposed to exist in the rat myocardium [29]. We have also previously shown that in TR myocytes the decline of cytoplasmic Ca²⁺ was faster than in NON myocytes even when Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger was abolished. We speculated that this could be due to a larger SR Ca²⁺ content, a larger Ca²⁺ release and its effect on the SR Ca²⁺ uptake [15]. It could be suggested that in mouse ventricular myocytes the Na⁺/Ca²⁺ exchanger controls the speed of relaxation by modifying the SR Ca²⁺ content and by competing directly for cytoplasmic Ca²⁺. The effect of Na⁺/Ca²⁺ exchanger function on SR Ca²⁺ content and its consequence on relaxation may be present to different degrees in other species [30] in physiological and pathophysiological conditions but this still needs to be tested. If, in addition, SERCA activity is inhibited it is very difficult to predict the effects of the overexpression of the Na⁺/Ca²⁺ exchanger on cytoplasmic Ca²⁺ handling. We studied this relationship from a quantitative point of view in mouse ventricular myocytes aware that this quantification maybe different in other species and in pathophysiological conditions.

4.1 TR myocytes: a suitable model to investigate the overexpression of the Na⁺/Ca²⁺ exchanger
Several functional and molecular biological aspects of Ca²⁺ regulation have been previously investigated in cardiac myocytes isolated from the transgenic model used in this paper [13–15]. Protein levels of Ca²⁺ regulatory mechanisms such as SERCA, phospholamban and calsequestrin appear unchanged compared with NON myocytes [15] and no difference in the size of the L-type Ca²⁺ current was detected [14]. However, the speed of contraction and relaxation in TR myocytes was faster, the SR Ca²⁺ content was increased [15] and the action potential duration prolonged [31] compared with NON myocytes and these appear to occur as a result of the overexpression of the Na⁺/Ca²⁺ exchanger itself [15].

4.2 Comparative parameter: time-course of Ca²⁺ transients and twitches
Functional assessments of one Ca²⁺ regulation mechanism can be limited by a compensatory functional adjustment of others. This is why the investigation of the activity of such cellular components is performed more correctly in the presence of complete inhibition of the other known mechanisms [19,32]. Nevertheless, this approach makes it difficult to understand the relationship between the different mechanisms when they are working together, e.g. during a twitch. In order to evaluate the relationship between overexpression of the Na⁺/Ca²⁺ exchanger and reduction in SERCA activity, a comparative parameter was needed. We choose to use the time-course of the Ca²⁺ transients and the twitches for two reasons: (a) Ca²⁺ transients and twitches are the result of the combined action of all Ca²⁺ regulatory mechanisms working together; (b) as already mentioned, Ca²⁺ transient and twitch duration are clearly affected in pathophysiological conditions. The development of heart failure appears to involve alterations to them.

It should be noted that during the Ca²⁺ transient, Ca²⁺ release and Ca²⁺ extrusion overlap and this makes it difficult to analyse the mechanisms involved in contraction and relaxation separately using the rate of increase or decay of the transient, respectively. Moreover, if for example SR Ca²⁺ content was increased, a larger and faster Ca²⁺ release from the SR would be expected [33,15], cytoplasmic [Ca²⁺] would be higher and there would be a different activation of Ca²⁺ extrusion mechanisms. These effects can all be consequences of changes in Na⁺/Ca²⁺ exchanger and SERCA function since they influence both TTP and T50 and they need to be considered as a whole. For this reasons the parameter TTP+T50 of twitches and Ca²⁺ transients has been preferred in this paper rather than the single rate of increase or decay.

4.3 Relationship between the function of the Na⁺/Ca²⁺ exchanger and SERCA
For the functional assessment of SERCA and the Na⁺/Ca²⁺ exchanger a technique to measure Ca²⁺ fluxes described by Bassani et al. [19] was used. The rate of Ca²⁺ decline after Ca²⁺ release from the SR was not used as a measure of the Ca²⁺ transport processes since there may be differences in cytoplasmic [Ca²⁺] in TR and NON myocytes and in the presence of thapsigargin that may affect the rate of Ca²⁺ decline [23]. V_max, a [Ca²⁺] independent parameter, was measured and this provided an index of the level of activity of the mechanism under investigation. V_max was calculated for the net Ca²⁺ fluxes mediated by SERCA and the Na⁺/Ca²⁺ exchanger in the same myocyte and compared with the time-course of the Ca²⁺ transient or the twitch.

A reduction in SERCA function was obtained using 200 nM thapsigargin, previously shown to produce a gradual reduction in SR Ca²⁺ uptake [21]. Na⁺/Ca²⁺ exchanger function was modified by increasing the level of expression of this protein. This amount of overexpression coincides with values previously reported in human heart failure by Studer et al. [8]. Similar values have also been reported by Owen et al. [34].

This study shows that reducing SERCA function could reverse the effect of the Na⁺/Ca²⁺ exchanger overexpression on the time-course of the Ca²⁺ transient. Not surprisingly, a correlation between reduction in function of SERCA and prolongation of the Ca²⁺ transient was found in both TR and NON myocytes. This can be ascribed to the combined effect of two different actions of reduced SERCA activity on the time-course of the Ca²⁺ transient: a slower Ca²⁺ removal from the cytoplasm (to affect Ca²⁺ decline) and a reduced [33] and slower SR Ca²⁺ release (due to a reduction in SR Ca²⁺ content [21], to affect Ca²⁺ release). In TR myocytes, increased function of the Na⁺/Ca²⁺ exchanger has been shown to have the opposite effect: increased rate of Ca²⁺ decline and increased SR Ca²⁺ content [15]. This study confirms that a reduction in function of SERCA can be compensated by the overexpression of the Na⁺/Ca²⁺ exchanger. The relationship between overexpression of the Na⁺/Ca²⁺ exchanger and reduction in SERCA function was quantified. It was shown that for a 2.4-fold increase in the function of the Na⁺/Ca²⁺ exchanger, SERCA function could be reduced by 28% before a prolongation of the Ca²⁺ transient is produced. Values of similar magnitude have been reported in failing human hearts [8,34].

This quantitative relationship may help to explain the absence of a prolongation or even a faster Ca²⁺ transient in some models of cardiac hypertrophy where alterations in Ca²⁺ regulation mechanisms may be present [35]. It can be speculated that, when alterations to Ca²⁺ regulatory proteins are found, the time-course of the Ca²⁺ transient is affected in a manner depending on how much SERCA function is reduced and how much Na⁺/Ca²⁺ exchanger function is increased. However, it should be emphasised that these conclusions cannot necessarily be applied to the situation in human heart failure because of the diverse nature of the human disease and the possible different roles of the Ca²⁺ regulatory mechanisms in the human myocardium.

Another important caveat is that the quantitative assessments presented in this study are only valid if calibration of indo-1 fluorescence faithfully allows calculation of cytoplasmic [Ca²⁺]. We used a well-characterised calibration technique in this study, and derived calibrated Ca²⁺ transients of similar size and time-course to those shown by others in mouse myocytes [36,37]. Nevertheless, the Ca²⁺ transients were somewhat smaller and slower than expected from previous studies performed in other species and in different conditions. These effects could be due to species-dependent compartmentalization of indo-1 or Ca²⁺ buffering, limitations associated with many Ca²⁺ sensitive fluorescence indicators. Although the measured systolic level of Ca² was low, myofilament Ca²⁺ affinity in intact adult mouse cardiac myocytes is unknown and it is possible that the changes in cytoplasmic [Ca²⁺] reported are sufficient to produce activation of contraction in this species. The Ca²⁺ transients were slow suggesting that if they did not shorten at higher stimulation frequencies (e.g. in vivo), then appropriate changes in cytoplasmic Ca²⁺ could not be sustained.

We tried to determine whether the compensatory mechanism due to the overexpression of the Na⁺/Ca²⁺ exchanger was the result of an effect on the SR Ca²⁺ content. An increased SR Ca²⁺ content in TR myocytes was observed and this appears to influence the time-course of the twitch. When thapsigargin prolonged twitches in TR myocytes to similar values as those measured in NON myocytes, the difference in SR Ca²⁺ content could no longer be detected. This suggests that an increased SR Ca²⁺ content can account for a faster twitch (we have previously shown that this was the case even during inhibition of the Na⁺/Ca²⁺ exchanger [15]), and that relaxation is the effect of competition between SERCA and Na⁺/Ca²⁺ exchanger for cytoplasmic Ca²⁺ when SR Ca²⁺ content is similar. In conclusion, both an effect on SR Ca²⁺ content and on cytoplasmic Ca²⁺ removal appear responsible for the compensatory effect of the overexpression of the Na⁺/Ca²⁺ exchanger in the presence of reduced function of SERCA.

Time for primary review 28 days.

	Acknowledgments

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

 
We thank the British Heart Foundation, the Wellcome Trust and NIH (HL48509) for financial support.

	References

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

 

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