Effects of Overexpression of the Na+-Ca2+ Exchanger on [Ca2+]i Transients in Murine Ventricular Myocytes
Abstract—We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.
In cardiac ventricular myocytes, an increase in [Ca2+]i is considered the mechanism of excitation-contraction coupling. It has been generally accepted that Ca2+ influx through the SL L-type Ca2+ channel induces Ca2+ release (CICR) from the SR, which leads to contraction.1 2 Subsequently, Ca2+ is taken up by the SR Ca2+-ATPase and extruded from the myocyte by the SL Na+-Ca2+ exchanger operating in the “forward” mode. Therefore, the Na+-Ca2+ exchanger competes with the SR Ca2+-ATPase to lower [Ca2+]i and cause relaxation. The Na+-Ca2+ exchanger can also operate in a “reverse” mode, causing the influx of 1 Ca2+ in exchange for 3 Na+ early during excitation.2 The reverse function of the Na+-Ca2+ exchanger has been proposed to increase the content of Ca2+ in SR,3 4 5 directly activate the contractile elements,5 and/or trigger CICR, contributing to contraction.6 7 8 9 However, it remains unclear the extent to which these various components of Na+-Ca2+ exchanger function are important in physiological excitation-contraction coupling and relaxation.
TG mice overexpressing the Na+-Ca2+ exchanger in ventricular myocardium have been produced by K.D. Philipson and associates, and initial studies of voltage-clamped isolated myocytes from these animals have demonstrated that forward function of the exchanger is enhanced 2.5-fold.10 These myocytes provide a model to examine further the effects of increased function of the Na+-Ca2+ exchanger in intact myocytes. We therefore compared values of diastolic and peak systolic [Ca2+]i and the time course of rise and decline of [Ca2+]i transients in TG and WT myocytes. We also examined the effects of overexpression of the Na+-Ca2+ exchanger on the action potential, ICa, [Na+]i, and SR Ca2+ content and the extent to which the Na+-Ca2+ exchanger contributes to Ca2+ influx.
Materials and Methods
Production of TG Mice
TG mice were produced as described by Adachi-Akahane et al.10 Briefly, the transgene construct consisted of the open reading frame of the canine cardiac Na+-Ca2+ exchanger connected downstream to the α-myosin heavy chain promoter. This promoter contains 4.5 kb of 5′ upstream sequence and 1 kb of the α-myosin heavy chain gene encompassing exons 1 through 3 of the untranslated region. An SV40 transcriptional terminator was used to provide a polyadenylation signal. TG mice lines were heterozygous so that non-TG littermates could be used as control WT mice in the present study.
Dissociation of Adult Mouse Ventricular Myocytes
Adult mouse myocyte isolations were performed as previously described.11 Briefly, hearts were removed from anesthetized mice and immediately attached to an aortic cannula. After perfusion with Ca2+-free modified Tyrode’s solution for 5 minutes, hearts were digested with 0.90 mg/mL collagenase D (Boehringer-Mannheim Biochemicals) in 25 μmol/L CaCl2–containing modified Tyrode’s solution for 7 to 12 minutes. These two solutions consisted of (mmol/L) NaCl 126, KCl 4.4, MgCl2 1.0, NaHCO3 18, glucose 11, HEPES 4, and BDM 30, along with 0.13 U/mL insulin, and were gassed with 5% CO2/95% O2, which maintained the pH at 7.4. The digested hearts were removed from the cannula, and the left ventricles were cut into small pieces in 100 μmol/L Ca2+–containing modified Tyrode’s solution. These pieces were gently agitated and then incubated in the same solution containing 2% albumin at 30°C for 20 minutes. The cell suspension was then centrifuged at 300 rpm for 3 minutes, and the pellet of the cells was resuspended in 200 μmol/L Ca2+ and 2% albumin Tyrode’s solution and allowed to settle for 20 minutes at 30°C. The cells were then resuspended in culture medium composed of 5% heat-inactivated fetal bovine serum (Hyclone), 47.5% MEM (GIBCO Laboratories), 47.5% modified Tyrode’s solution, 10 mmol/L pyruvic acid, 4.0 mmol/L HEPES, and an additional 6.1 mmol/L glucose at 30°C in a 5% CO2 atmosphere until use. Isolated cells were all used for experiments within 6 hours after isolation.
Measurement of [Ca2+]i Transients
The [Ca2+]i in isolated myocytes was measured by a previously described method.12 Myocytes were attached to laminin-coated glass coverslips and then incubated in a 1 μmol/L fluo 3-AM (Molecular Probes)–containing HEPES solution (loading solution) at 30°C in the dark for 30 minutes. The loading solution was prepared by diluting a 100 μmol/L fluo 3 stock solution, which contained 0.45% pluronic F-127 (Molecular Probes), 10% dimethyl sulfoxide, and 90% heat-inactivated fetal calf serum (GIBCO). HEPES solution consisted of (mmol/L) NaCl 126, KCl 4.4, MgCl2 1.0, CaCl2 1.08, HEPES 24, NaOH 13, glucose 11, and probenecid 0.5, along with 5 mg/L insulin (pH 7.4). Fluo 3–loaded myocytes on the coverslip were then washed twice with dye-free HEPES solution and placed in a flow-through chamber on the microscope. Fluo 3–loaded myocytes were excited by a mercury-arc lamp system at a 485-nm wavelength through an epifluorescence attachment (505-nm dichroic mirror, Omega) and a ×40 Fluor oil objective lens (Nikon). Fluorescence (530 nm, DF30, Omega) was detected with a photomultiplier tube (Hitachi). The intensity of the fluorescence at 530 nm increases with an increase in [Ca2+]i.
Myocytes were field-stimulated with platinum electrodes with 7-ms pulses of alternating polarity, and [Ca2+]i transients and pacing signals were simultaneously recorded on tape for the further analysis. In most of the experiments, calibration of the [Ca2+]i transients was performed with a modification of the method of Kao et al.13 After the fluorescence transients were recorded, the myocyte was superfused with 10 μmol/L ionomycin and 30 mmol/L BDM–containing HEPES solution. In 5 minutes, the intensity of the fluorescence increased and then the myocyte was perfused with 10 mmol/L MnCl2, 10 μmol/L ionomycin, and 30 mmol/L BDM–containing HEPES solution. Mn2+ quenched fluo 3 in the cytosol, yielding an intensity of fluorescence of FMn2+, which is 1/5 Fmax. After FMn2+ was recorded, the intensity of the fluorescence from the field (Fbkg) was measured by blowing the myocyte away from the field with a pipette. The mean value of the autofluorescence of 10 unloaded myocytes in the same day was used as an autofluorescence (Fauto) for each myocyte. The variance of Fauto was very small. Fmax was calculated with the following formula: Fmax=(FMn2+−Fbkg−Fauto)×5. Fmin is 1/40 of Fmax. Therefore, [Ca2+]i could be calculated with the following formula: [Ca2+]i= Kd×[(F−Fbkg−Fauto)−Fmin]/[Fmax−(F−Fbkg−Fauto)]. In this formula, F is the measured fluorescence intensity of the myocyte, and Kd is the dissociation constant. All experiments were performed at 25°C. The Kd of fluo 3 is known to be temperature dependent and is reported to be 400 and 864 nmol/L at 22°C and 37°C, respectively.13 Therefore, we used 493 nmol/L as the Kd at 25°C, assuming a linear relationship between Kd and temperature. In some experiments, we reported fluorescence intensity in arbitrary units, normalized to the peak amplitude of the electrically stimulated transient.
Caffeine-Induced Ca2+ Transients
In some experiments, we abruptly exposed a myocyte to 10 mmol/L caffeine with an SW.14 With this device, the bulk solution bathing a myocyte can be changed within 4 ms. Myocytes were exposed to caffeine for 4 s (long caffeine pulse) or 100 ms (short caffeine pulse). The long caffeine pulse protocol permitted assessment of the rate of decline of [Ca2+]i when the SR was disabled by the continued presence of caffeine. In the short caffeine pulse protocol, the Ca2+ released from the SR by the initial exposure to caffeine could be resequestered by the SR as the caffeine effects were rapidly washed out and the function of Na+-Ca2+ exchanger was blocked in 0 Na+/0 Ca2+ solution.
Measurement of SR Ca2+ Content
SR Ca2+ content was determined by measuring the integral of the caffeine-induced inward INa/Ca.15 In brief, myocytes were voltage-clamped at −80 mV with a single suction pipette filled with a solution composed of (mmol/L) NaCl 15, CsCl 100, tetraethylammonium chloride 30, MgATP 5, HEPES 10, and dextrose 5.5 (pH 7.1 adjusted with CsOH). Then the voltage-clamped cell was superfused in a microstream containing (mmol/L) NaCl 138, KCl 4.4, MgCl2 1.0, CaCl2 1.08, CsCl 2, BaCl2 0.1, dextrose 11, and HEPES 24 (pH 7.4 was adjusted with NaOH to give a final [Na+]o of 145 mmol/L). After a train of steady-state conditioning pulses (eight 200-ms pulses to 0 mV, 0.25 Hz), the cell was abruptly immersed for 6 seconds in an adjacent switcher microstream of solution in which 10 mmol/L caffeine was added to release SR Ca2+. On the basis of the stoichiometry of the electrogenic Na+-Ca2+ exchange (3:1), the integral (nA · ms=pC) of the resulting inward INa/Ca was converted to the amount of Ca2+ (pmol) extruded by Na+-Ca2+ exchange during the sustained exposure of cells to caffeine normalized by cell capacitance (pF). The decline of the current curve was well fitted with a single exponential, with a τ value of ≈500 ms in WT myocytes under our experimental conditions. Two current values at >5×τ (≈2500 ms) from the peak of the current were used to define a baseline for current transient integration.
Measurement of [Na+]i
[Na+]i was measured with a modified method of Harootunian et al16 and Levi et al17 using two Na+-sensitive fluorescent dyes, Sodium Green and SBFI (Molecular Probes). Myocytes on laminin-coated coverslips were incubated at 25°C for 30 minutes in 5 μmol/L Sodium Green tetraacetate–containing HEPES solution or for 120 minutes in 10 μmol/L SBFI acetoxymethyl ester–containing HEPES solution. The loading solution for each dye was prepared with the same method as described for fluo 3-AM. The loaded myocytes were then washed and incubated in dye-free HEPES solution for 15 minutes. Then the myocytes on the coverslips were placed in a flow-through chamber and perfused with HEPES solution at 25°C. For Sodium Green–loaded myocytes, the fluorescence was collected with exactly the same method as that for fluo 3, because the excitation and the emission wavelengths are both the same as those of fluo 3. As described, the fluorescence intensity at 530 nm was used as F, an indicator for [Na+]. SBFI has two different excitation (340- and 380-nm) and one emission (510-nm) wavelength. Myocytes were illuminated sequentially at 60 Hz by 340- and 380-nm excitation light passing through band-pass filters (P10–340, and P10–380, Corion) with an optical switcher (DX-1000, Solamere Technology Group), and the fluorescence at 510 nm (P10–510, Corion) was continuously recorded. The ratio of the fluorescence intensities during excitation with 340-nm light to that with 380-nm light was used as R, an indictor for [Na+]. After recording the emission intensities, an in vivo calibration was performed for each dye. For calibration, the myocyte was sequentially exposed to three calibration solutions of 5, 10, and 15 mmol/L Na+ containing (μmol/L) gramicidin D 2, monensin 40, and strophanthidin 100. In each solution, [Na+]i was equilibrated to [Na+]o, and the stable fluorescence at each [Na+]i was then obtained. Calibration solutions were made from appropriate mixtures of high-Na+ solution and high-K+ solution. The former consisted of (mmol/L) NaCl 30, sodium gluconic acid 110, EGTA 2, and HEPES 10, and the latter was identical except for complete replacement of Na+ by K+. The pH of both solutions were adjusted to 7.2 with NaOH and KOH, respectively. Data were all digitized and directly acquired by a computer. The relationships between F or R and [Na+]i were fitted with mathematical software, Origin (Microcal), to the following formula: [Na+]i=Kd×(T−Tmin)/(Tmax−T), where T indicates F for Sodium Green and R for SBFI. By using this curve, the fluorescence intensity from the myocyte was then converted to [Na+]i.
ICa was measured as described by Chin et al.18 A myocyte was superfused with HEPES solution, which contained the same components as described above except for the elimination of KCl, insulin, and probenecid, and voltage-clamped (Axopatch 200A, Axon Instruments) with a suction pipette (2 to 3 MΩ) filled with solution containing (mmol/L)NaCl 20, MgCl2 0.3, EGTA 14, MgATP 3.0, glucose 5.5, and HEPES 10. The pH was adjusted to 7.1 with CsOH, and the concentration of Cs+ in the solution was brought to 140 mmol/L by adding CsCl. The myocyte was held at a potential of −40 mV, and ICas were then activated by depolarizing the myocyte membrane to more positive values for 500 ms. Between measurements, the myocyte was maintained at a holding potential of −80 to −90 mV in order to prevent the rundown phenomenon. Data were all digitized, acquired, and analyzed by a computer with PCLAMP6 software (Axon Instruments Inc). The cell capacitance was also measured for the determination of the current density.
Measurement of Action Potentials
Action potentials were recorded in whole-cell current-clamp mode using an AxoClamp 2B (Axon Instruments Inc). The “physiological” pipette filling solution used for recording action potentials contained (mmol/L) KCl 113, NaCl 15, MgCl2 0.5, ATP (potassium salt) 5, HEPES 10, and dextrose 5.5, with pH adjusted to 7.1 with KOH. Myocytes were superfused in HEPES solution (same composition as used for the measurement of [Ca2+]i transients) and stimulated at 0.25 Hz with 3-ms current pulses (1 nA). In a few experiments, simultaneous measurements of action potentials and [Ca2+]i transients were performed in myocytes loaded with fluo 3 as described.
For analysis, all records of [Ca2+]i transients and pacing signals were simultaneously digitized and acquired at the sampling rate of 1 kHz (Axo Scope, Axon Instruments Inc). Digitized [Ca2+]i transients were analyzed with Origin. Results were expressed as mean±SEM. An unpaired t test was performed for the comparison between WT and TG myocytes. Significance was also tested by ANOVA if multiple comparisons were made. Values of P<.05 were considered significant.
[Ca2+]i Transients of Electrically Stimulated Beats in TG and WT Myocytes
Fig 1⇓ shows representative recordings of [Ca2+]i transients in a TG and a WT myocyte. The mean values of the diastolic and peak systolic [Ca2+]i and the amplitudes of the [Ca2+]i transients from the myocytes are given in Table 1⇓. Peak systolic [Ca2+]i and the [Ca2+]i transient amplitude were somewhat higher in TG myocytes than in WT myocytes, but this difference did not reach statistical significance. However, the time to peak systolic [Ca2+]i was significantly prolonged in TG myocytes. The diastolic [Ca2+]i was similar in the two cell groups.
We also examined characteristics of decline of [Ca2+]i in TG and WT myocytes. The time constant (τ) was calculated with a modified method of Bers and Berlin.19 Briefly, the digitized data from 90% to 10% of the peak in the decline phase of [Ca2+]i transients were extracted for curve fitting. The decay in a [Ca2+]i transient was well fitted with the following formula: [Ca2+]i=C+Co×e−(t−t0)/τ. We defined C as the end-diastolic [Ca2+]i level and CO as the initial [Ca2+]i at the beginning of the decline. Values of t90–50% and t50–10% were also calculated. The τ value was significantly shorter in TG myocytes (128±6 ms) compared with WT myocytes (166±12 ms) (P=.004, n=27 and 20). The most prominent difference was in the terminal phase of the decline of the [Ca2+]i transients (t50–10%, 205±12 ms [TG] and 284±22 ms [WT]; P<.001). These data clearly show that in myocytes overexpressing the Na+-Ca2+ exchanger, the decline of [Ca2+]i transients is accelerated, suggesting an enhanced effectiveness of Ca2+ extrusion by Na+-Ca2+ exchange and thus a more effective competition with Ca2+ uptake by the SR Ca2+-ATPase.
Characteristics of Caffeine-Induced [Ca2+]i Transients
To confirm that the overexpression of the Na+-Ca2+ exchanger induces enhanced Ca2+ extrusion, we exposed the myocytes to a long pulse of 10 mmol/L caffeine with the SW. In this protocol, a myocyte was paced at 0.25 Hz in HEPES solution in one stream of the SW for 2 minutes and then abruptly exposed for 4 s to a 10 mmol/L caffeine–containing HEPES solution stream with SW 4 s after the last pacing stimulus (see Fig 2⇓). The myocyte was subsequently paced again for 2 minutes in control solution. After the solutions perfusing SW were changed to include 5 mmol/L Ni2+ (Ni2+ solution and Ni2+-caffeine solution), the myocyte was exposed to Ni2+ solution streaming from the first barrel of SW. When the myocyte failed to respond to the electrical stimulus, it was abruptly exposed to the Ni2+-caffeine solution with the SW for 4 s. The rationale for this experiment is that the decline in the caffeine-induced [Ca2+]i transient under these conditions is thought to be primarily dependent on the Na+-Ca2+ exchanger, with the SL Ca2+-ATPase, mitochondria, and the intracellular buffering system playing a less important part.20 21 On the other hand, 5 mmol/L Ni2+ is known to inhibit the function of the Na+-Ca2+ exchanger.22 In the Ni2+-caffeine solution, the decline in the caffeine-induced [Ca2+]i transient is, therefore, mediated by systems other than the Na+-Ca2+ exchanger and the SR Ca2+-ATPase. As shown in examples in Fig 2a⇓, the speed of the decline of the [Ca2+]i signal in caffeine was markedly faster in the TG myocyte, whereas the amplitudes of both fluorescence signals relative to the amplitude of the last electrically stimulated beats were identical in the two myocytes. In the presence of 5 mmol/L Ni2+, the [Ca2+]i decline was prolonged in both types of myocytes, but there was no apparent difference in the rate of decline between the myocytes. Fig 2b⇓ shows average results. In the absence of Ni2+, the average decline of the long-pulse caffeine-induced [Ca2+]i transient was significantly more rapid in TG myocytes. Time from the peak signal to 50% of the peak, which was measured with an interpolation method, was 464 ms in TG myocytes and 1750 ms in WT myocytes. This enhanced Ca2+ extrusion by Na+-Ca2+ exchange probably accounts for the more rapid decline in [Ca2+] in electrically stimulated transients in TG myocytes. The rate of decline did not differ in the presence of Ni2+, suggesting that there is no significant difference in the combined [Ca2+]i reducing function of the SL Ca2+-ATPase, mitochondria, and Ca2+ buffering between WT and TG myocytes.
The amplitude of the caffeine-induced [Ca2+]i transient in the presence of Ni2+ is caused only by the Ca2+ release from SR and is an indicator of SR Ca2+ content. In the presence of Ni2+, the amplitude of the caffeine-induced [Ca2+]i transient relative to that of the last electrically stimulated transient was 196±33% for WT myocytes (n=6) and 213±37% for TG myocytes (n=7, P=NS). We also measured the inward exchange current (INa/Ca), induced by a rapid exposure to caffeine, under voltage-clamp conditions. The peak current was higher in TG than WT myocytes (5.2±1.3 versus 1.8±0.2 pA/pF). However, the amounts of Ca2+ released estimated from the integrals of the INa/Ca were similar (7.96±0.69 pmol/μF for WT and 8.61±0.56 pmol/μF for TG, P=.54, n=9 and 5). Thus, these results suggest that SR Ca2+ content is similar in WT and TG myocytes under these experimental conditions.
[Na+]i in TG and WT Myocytes
[Na+]i is recognized as one of the factors affecting the function of the Na+-Ca2+ exchanger.2 23 For comparison of the function of the Na+-Ca2+ exchanger in TG and WT myocytes, it is therefore necessary to measure [Na+]i in both types of the myocytes. The resting [Na+]i in WT myocytes was 16.4±0.7 and 15.6±0.7 mmol/L when measured with Sodium Green and SBFI, respectively, and was similar in TG myocytes (16.9±0.6 and 14.1±0.7 mmol/L, n=21 and 20 for Sodium Green and n=11 and 7 for SBFI). Using identical calibration protocols, we measured an [Na+]i of 5 to 6 mmol/L in rabbit ventricular myocytes, a result similar to that reported by Levi et al.17 Thus, the [Na+]i in murine ventricular myocytes is quite high compared with that in rabbit (4 to 7 mmol/L17 23 ) and guinea pig (≈8.0 mmol/L24 ) ventricular myocytes and comparable to that in rat myocytes (≈16 mmol/L23 ). [Na+]i, although high in murine myocytes, is not affected by the overexpression of the Na+-Ca2+ exchanger; therefore, a difference in [Na+]i seems unlikely to account for the difference in the function of the Na+-Ca2+ exchanger in TG and WT myocytes noted above.
SR Ca2+-ATPase Function in TG and WT Myocytes
To rule out the possibility that the acceleration of the decline of the electrically stimulated [Ca2+]i transients in TG myocytes was in part mediated by modified function of SR Ca2+-ATPase, we directly examined the function of the SR in TG and WT myocytes. For this purpose, we superfused a myocyte with 0 Ca2+ solution for 1 minute, and then the solution was exchanged to 0 Na+/0 Ca2+ solution, with the complete replacement of Na+ by Li+. (If the bathing solution was simultaneously changed to 0 Na/0 Ca2+ solution, myocytes developed [Ca2+]i oscillations.) The myocyte was then captured in one stream of 0 Na+/0 Ca2+ solution from the SW and subsequently abruptly exposed to 10 mmol/L caffeine–containing 0 Na+/0 Ca2+ solution for 100 ms. In this experiment, the time from the switching point to the peak of the caffeine-induced [Ca2+]i was always >200 ms, so that during the decline of the [Ca2+]i transient, the extracellular caffeine concentration was decreased to less than an effective concentration for releasing Ca2+ from the SR. Hence, this decline reflects sequestering of Ca2+ by a normally functioning SR Ca2+-ATPase, because the Na+-Ca2+ exchanger is disabled in 0 Na+/0 Ca2+ solution. Fig 3⇓ shows a representative recording of the short caffeine pulse–induced [Ca2+]i transients obtained in both types of myocytes. Both myocytes had a similar resting and peak [Ca2+]i and showed an identical rate of decline of the [Ca2+]i transient. The mean values of τ, t90–50%, and t50–10% were similar in TG and WT myocytes. In addition, the mean diastolic and peak systolic [Ca2+]i values did not differ significantly (Table 2⇓). These data indicate that the SR Ca2+-ATPase function is not altered in TG myocytes and further support the conclusion that the overexpressed Na+-Ca2+ exchanger is most likely responsible for the more rapid decline of electrically stimulated [Ca2+]i transients noted in Table 1⇑.
Function of the L-type Ca2+ Channel
There is normally a balance between Ca2+ influx via the L-type Ca2+ channel and Ca2+ extrusion by Na+-Ca2+ exchange.25 Therefore, increased forward function of the Na+-Ca2+ exchanger might be expected to result in increased Ca2+ influx via the L-type Ca2+ channel to maintain Ca2+ homeostasis. To examine whether the L-type Ca2+ channel is upregulated in TG myocytes, we measured ICa in both types of myocytes. ICa normalized to membrane capacitance, as a function of membrane potential, was very similar in the two groups of myocytes (Fig 4⇓). This finding is consistent with that reported by Adachi-Akahane et al10 and suggests that the Ca2+ influx through the L-type Ca2+ channel is similar in TG and WT myocytes.
Excitation-Contraction Coupling in the Presence of Ca2+ Channel Blockade
There has been disagreement as to whether the Na+-Ca2+ exchanger operating in the reverse mode can trigger or contribute to CICR.2 6 7 8 9 26 27 28 To examine whether overexpression of the Na+-Ca2+ exchanger results in a difference in the magnitude of reverse Na+-Ca2+ exchange–mediated Ca2+ influx, we exposed TG and WT myocytes to 10 μmol/L nifedipine for 5 minutes. The [Ca2+]i transient (Fig 5a⇓) was almost completely eliminated by nifedipine in the WT myocyte within 1 minute but was clearly maintained in the TG myocyte for 5 minutes in nifedipine-containing HEPES solution. Furthermore, in the presence of 1 μmol/L ryanodine and 0.5 μmol/L thapsigargin in addition to nifedipine, the [Ca2+]i transient became almost undetectable in the TG myocyte within 5 minutes (Fig 5a⇓, lower trace). The mean values of the amplitudes of the [Ca2+]i transients in the nifedipine-containing HEPES solutions are shown in Fig 5b⇓. The mean amplitude of the [Ca2+]i transients in the nifedipine solution without ryanodine and thapsigargin was significantly larger in TG myocytes than in WT myocytes. Thus, overexpression of the Na+-Ca2+ exchanger seems to result in a relative maintenance of the [Ca2+]i transients in nifedipine. In the presence of ryanodine and thapsigargin, the amplitude of [Ca2+]i transients became significantly smaller (Fig 5b⇓), indicating that SR Ca2+ content is necessary for this response of TG myocytes. Hence, it seems possible that Ca2+ influx via the Na+-Ca2+ exchanger is contributing to CICR rather than directly activating the contractile elements, and this maintains the electrically stimulated [Ca2+]i transient magnitude in TG myocytes under these experimental conditions.
To further assess this possibility, we abruptly blocked the L-type Ca2+ channel without changing SR Ca2+ content. Briefly, after pacing at 0.1 Hz, a myocyte was exposed to 20 μmol/L nifedipine with the SW for 9 s before the next electrical stimulation. A prominent [Ca2+]i transient was observed in the TG myocyte, although only a small transient could be seen in the WT myocyte (Fig 6⇓). The myocyte was subsequently paced in control solution until the Ca2+ signal again stabilized and then was exposed to 5 mmol/L Ni2+–containing solution with the same protocol. In the Ni2+ solution, the [Ca2+]i transient was completely eliminated in both myocytes. These experiments clearly indicate that nifedipine-insensitive and Ni2+-sensitive Ca2+ influx, which is most likely caused by the reverse function of the Na+-Ca2+ exchanger, is able to induce a [Ca2+]i transient in TG myocytes. In Fig 6b⇓ are shown superimposed high-speed recordings of the control [Ca2+]i transient and the [Ca2+]i transient after abrupt exposure to nifedipine. The rise in [Ca2+]i is clearly prolonged in the nifedipine-treated transient compared with the control transient. On the other hand, the decline of the two transients was similar. This result suggests that the rate at which Ca2+ influx via reverse Na+-Ca2+ exchange contributes to CICR is slower than CICR caused by L-type Ca2+ channel Ca2+ influx. This is consistent with a previous report.27
Effects of Overexpression of the Na+-Ca2+ Exchanger on Action Potentials
Examples of action potentials recorded from TG and WT myocytes are shown in Fig 7⇓, and average parameter values are given in Table 3⇓. The resting membrane potentials were similar. However, the TG myocyte action potentials had a lower peak amplitude (APA), a more rapid initial repolarization (APD50), and a delayed terminal repolarization (APD90). An increase in APD90 in TG myocytes was also noted in a preliminary report by Silverman et al.29 These action potential characteristics are consistent with but do not prove the hypothesis that the TG myocytes have a more marked reverse function of the Na+-Ca2+ exchanger early during depolarization, with an increase in outward current causing a decrease in APA and APD50, and a more marked forward function during the [Ca2+]i transient, causing an increase in inward current and an increase in APD90.
Degree of Functional Overexpression of the Na+-Ca2+ Exchanger in TG Myocytes
The Na+-Ca2+ exchanger is functionally overexpressed in TG myocytes, as shown by the accelerated decline of [Ca2+]i in the long caffeine pulse–induced [Ca2+]i transients. This result was not due to an alteration in [Na+]i and is consistent with the findings of Adachi-Akahane et al10 in voltage-clamped myocytes. The difference in the rates of the decline of the long caffeine pulse–induced [Ca2+]i transients between TG (t1/2=464 ms) and WT (t1/2=1750 ms) myocytes and in the magnitudes of the peak INa/Ca measured during abrupt exposure to caffeine is consistent with the 2.5- to 3-fold increase in intracellular Na+–dependent 45Ca uptake in sarcolemmal vesicles obtained from TG and WT myocytes.10
Alteration in [Ca2+]i Transients and Action Potential Morphology Associated With Overexpression of the Na+-Ca2+ Exchanger
In electrically stimulated [Ca2+]i transients, the decline of [Ca2+]i was significantly accelerated in TG myocytes, particularly during the terminal phase of decline. This was associated with a prolonged APD90. Our recent findings in rabbit ventricular myocytes12 indicate that Na+-Ca2+ exchanger function is most apparent during the terminal phase of Ca2+ decline. On the basis of the short-pulse caffeine results, SR Ca2+-ATPase function appears similar in both types of myocytes. Taken together, these results indicate that the acceleration of decline of the Ca2+ transient is due to the functional overexpression of the Na+-Ca2+ exchanger and the enhanced forward exchange in TG myocytes. Since ICa, a major source of Ca2+ influx, was similar in the two groups of myocytes (Fig 4⇑), one might predict that enhanced forward function of the Na+-Ca2+ exchanger would result in Ca2+ depletion and a reduced [Ca2+]i transient magnitude. However, in electrically stimulated cells, the magnitude of the Ca2+ transient was somewhat greater in TG than in WT myocytes, although this difference did not reach statistical significance. In addition, as shown in Table 1⇑, diastolic levels did not differ significantly in TG and WT myocytes. The amplitudes of both the long- and short-duration caffeine pulse–induced Ca2+ transients were similar, as was the integrated inward current activated by abrupt caffeine exposure, suggesting that SR Ca2+ loading was similar in TG and WT myocytes. The presence of a normal resting [Ca2+]i, SR Ca2+ content, and a normal [Ca2+]i transient amplitude in TG myocytes therefore indicates that Ca2+ influx in these myocytes is enhanced, probably by augmented reverse Na+-Ca2+ exchange. The changes in the early action potential morphology (decreased APA and decreased APD50) are consistent with this hypothesis.
Ca2+ Influx by Reverse Na+-Ca2+ Exchange in TG Myocytes
To assess further the possible enhanced contribution of reverse Na+-Ca2+ exchange to Ca2+ influx, we examined the effects of nifedipine. We found less sensitivity of the [Ca2+]i transient to nifedipine in TG myocytes. Furthermore, the maintenance of [Ca2+]i transients in nifedipine required SR Ca2+ content, suggesting that the Na+-Ca2+ exchanger–mediated Ca2+ influx can contribute to CICR in TG myocytes. On the basis of our results, it is not clear whether the Ca2+ influx due to reverse Na+-Ca2+ exchange actually “triggers” SR Ca2+ release itself or potentiates the effects of the very small residual Ca2+ influx possibly occurring via unblocked L-type Ca2+ channels. It should be noted that Adachi-Akahane et al10 reported only inconsistent triggering of CICR in TG ventricular myocytes by Ca2+ influx through the Na+-Ca2+ exchanger during large (+80-mV) voltage clamps and failed to observe sufficient influx of Ca2+ via the exchanger to cause CICR in the physiological range of membrane potentials (−10 to +20 mV). This different result may have been obtained because Adachi-Akahane et al used 10 mmol/L Na+ in the voltage-clamp pipette solution, although the measured [Na+]i in our present study was ≈16 mmol/L. Using two values of [Na+]i (7 and 10 mmol/L), Bers4 simulated a [Ca2+]i transient and an action potential in a rabbit cardiac myocyte. He predicted that with [Na+]i of 10 mmol/L, Ca2+ influx by Na+-Ca2+ exchange occurred during most of the action potential but that at [Na+]i of 7 mmol/L, this occurred only in the initial phase of AP. This simulation implies that just a 3-mol/L difference in [Na+]i could lead to a significant difference in Na+-Ca2+ exchanger–mediated Ca2+ influx. Kohmoto et al8 also found that the magnitude of shortening in ventricular myocytes sensitive to the Na+-Ca2+ exchange inhibitor XIP (indicating exchanger inhibitory peptide) was increased with increasing [Na+]i. A difference in [Na+]i may thus account for our detection of apparent Na+-Ca2+ exchanger contribution to Ca2+ release in intact non–voltage-clamped TG myocytes under relatively physiological conditions. This possibility is also supported by work of Bers et al,30 who showed a decreased sensitivity to nifedipine in rabbit myocardium treated with acetylstrophanthidin to increase [Na+]i.
We found that the rate of Ca2+ release induced by the Na+-Ca2+ exchanger is somewhat slow (Fig 6⇑). It remains to be determined how much reverse Na+-Ca2+ exchange contributes to the [Ca2+]i transient in the presence of a functioning L-type Ca2+ channel, since Ca2+ influx via the L-channel could enhance Na+-Ca2+ exchange via a catalytic effect of Ca2+ on the exchanger.31 In addition, the triggering by INa/Ca and ICa may not add up in a simple linear fashion, because the relationship between open probability and [Ca2+] for the ryanodine receptor is sigmoid.32 The activity of Na+-Ca2+ exchange alone may, for example, bring Ca2+ in the vicinity of the ryanodine receptors close to the foot of this relationship, whereas the presence of both ICa and INa/Ca could bring pCa levels onto the steep region of the relationship. Under this circumstance, the effect of both triggers would not be a simple linear combination of each trigger acting independently.33 However, the prolongation of time to peak systolic [Ca2+]i in TG myocytes (Table 1⇑) may reflect an increased and delayed contribution to Ca2+ release by the Na+-Ca2+ exchanger operating in the reverse mode, although further analysis will be necessary to examine this possibility.
In conclusion, the overexpressed Na+-Ca2+ exchanger is shown to function in vivo in TG myocytes. A significant increase in the rate of decline of electrically stimulated and long caffeine pulse–induced [Ca2+]i transients indicates enhanced forward Na+-Ca2+ exchange function. In both TG and WT mouse ventricular myocytes, the [Na+]i was high compared with that in other species. This seems to favor an enhanced reverse Na+-Ca2+ exchange, with increased influx of Ca2+, which maintains SR Ca2+ loading and contributes to triggering of CICR in TG myocytes. The L-type Ca2+ channel and SR Ca2+-ATPase function appear to be unaltered by overexpression of the Na+-Ca2+ exchanger in murine ventricular myocytes.
Selected Abbreviations and Acronyms
|APA||=||action potential amplitude|
|APD||=||action potential duration|
|APD50, APD90||=||APD at 50% and 90% repolarization|
|CICR||=||Ca2+-induced Ca2+ release|
|F (with subscript)||=||fluorescence|
|I Ca||=||L-type Ca2+ channel current|
|I Na/Ca||=||Na+-Ca2+ exchange current|
|SW||=||solenoid-based rapid solution-switching device|
|t50–10%||=||time required for [Ca2+]i to decline from 50% to 10% of peak|
|t90–50%||=||time required for [Ca2+]i to decline from 90% to 50% of peak|
This study was supported by National Institutes of Health grants HL-42357, HL-52338, HL-53773, and HL-52335 and awards from the Nora Eccles Treadwell Foundation and the Richard A. and Nora Eccles Harrison Fund for Cardiovascular Research. We are indebted to Pam Larson for assistance in preparing the manuscript.
- Received June 11, 1997.
- Accepted January 27, 1998.
- © 1998 American Heart Association, Inc.
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