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Am J Physiol Heart Circ Physiol 275: H1441-H1448, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 4, H1441-H1448, October 1998

Abnormal myocyte Ca2+ homeostasis in rabbits with pacing-induced heart failure

Atsushi Yao1, Zhi Su1, Akihiko Nonaka1, Iram Zubair1, Kenneth W. Spitzer2, John H. B. Bridge2, Gerhard Muelheims1, John Ross Jr.3, and William H. Barry1

1 Division of Cardiology and 2 Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah Health Sciences Center, Salt Lake City, Utah 84132; and 3 Cardiology Division, University of California, San Diego, California 92093

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

To determine whether there are abnormalities in myocyte excitation-contraction coupling and intracellular Ca2+ concentration ([Ca2+]i) homeostasis in pacing-induced heart failure (PF), we measured L-type Ca2+ current (ICa,L) and Na+/Ca2+ exchanger current (INa/Ca) with voltage clamp and measured intracellular Na+ concentration ([Na+]i) and [Ca2+]i with the use of sodium-binding benzofuran isophthalate (SBFI) and fluo 3 in ventricular myocytes isolated from control and paced rabbits. The peak systolic and diastolic levels and the amplitude of electrically stimulated [Ca2+]i transients (0.25 Hz, extracellular Ca2+ concentration = 1.08 mM) were significantly less in PF myocytes. Also, there was prolongation of the times to peak and decline of [Ca2+]i transients. ICa,L density was markedly decreased in PF myocytes. INa/Ca at -40 mV elicited by rapid exposure to 0 Na+ solution with a rapid solution switcher was significantly reduced in PF myocytes, suggesting that the function of the Na+/Ca2+ exchanger is impaired in these myocytes. In PF myocytes the decline of the [Ca2+]i transient when the Na+/Ca2+ exchanger was abruptly disabled was markedly prolonged compared with the decline in control myocytes, consistent with depressed sarcoplasmic reticulum (SR) Ca2+-ATPase function. RNase protection assay showed decreased levels of Na+/Ca2+ exchanger and SR Ca2+-ATPase mRNA in PF hearts, consistent with the function studies. We conclude that the functions of L-type Ca2+ channels, Na+/Ca2+ exchanger, and SR Ca2+-ATPase are impaired in myocytes from rabbit hearts with failure induced by rapid pacing. These abnormalities result in reduced [Ca2+]i transients and systolic and diastolic dysfunction and appear to account for the abnormal ventricular function observed.

isolated myocyte; calcium transient; calcium channel; sodium/calcium exchanger; sarcoplasmic reticulum calcium adenosine 5'-triphosphatase

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ABNORMAL intracellular Ca2+ concentration ([Ca2+]i) homeostasis with a slow decline in [Ca2+]i (6, 14) and a reduced peak [Ca2+]i may be present in myocytes from hypertrophied and failing hearts and could contribute to impaired systolic and diastolic ventricular function (2). Mechanisms involved may include a decrease in the expression and/or function of the sarcoplasmic reticulum (SR) Ca2+-ATPase and a reduced SR Ca2+ content (2). However, a decrease in SR Ca2+-ATPase expression in failing myocardium has not been a uniform finding (15), and the decline of [Ca2+]i is mediated by both the SR Ca2+-ATPase and the Na+/Ca2+ exchanger during relaxation (3). Because Na+/Ca2+ exchanger expression may be altered in hypertrophy and failure (11, 13, 30), it is necessary to consider the function of both the SR Ca2+-ATPase and the Na+/Ca2+ exchanger in analyzing [Ca2+]i transients in myocytes from failing myocardium.

Recently we have developed techniques using a rapid solution switcher (SW) and voltage clamp to assess function of the SR Ca2+-ATPase (34) and to quantitate Na+/Ca2+ exchanger function (8, 9) in single rabbit ventricular myocytes. In this study we have examined the changes in [Ca2+]i transients and the function of the SR Ca2+-ATPase and the Na+/Ca2+ exchanger in ventricular myocytes from rabbits in which failure was caused by rapid pacing. We have also investigated associated changes in the L-type Ca2+ current and intracellular Na+ concentration ([Na+]i), which can influence [Ca2+]i homeostasis.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animal model. New Zealand White adult rabbits were used in this study. Rapid pacing was performed as described previously (26), and the investigation conformed with the Guide for the Care and Use of Laboratory Animals (Washington, DC: Natl. Acad. Press, 1996). Two pacemaker leads for ventricular pacing were attached to the left ventricle, and two pairs of leads were also implanted for monitoring the electrocardiogram. After recovering from surgery, the rabbits were paced at 380-400 beats/min with a stimulator (J & MO Technologies, San Diego, CA) until clinical symptoms of dyspnea, appetite loss, and body weight loss were observed (3-5 wk). After the fractional shortening (FS) in sinus rhythm was confirmed as being <22% with echocardiography (mean FS 16.9 ± 1.1%, n = 13 rabbits), dissociation of ventricular myocytes was performed.

Dissociation of ventricular myocytes. Adult rabbit myocyte isolation was performed by a modification of a previously reported method (34). Hearts were removed from rabbits anesthetized with pentobarbital sodium (65 mg/kg iv). The heart was immediately attached to an aortic cannula, and continuous retrograde coronary arterial perfusion at 37°C by pump (Masterflex, Cole-Parmer, Chicago, IL) was initiated at a coronary perfusion pressure of 60 mmHg. The heart was first perfused with nominally Ca2+-free modified Krebs-Ringer bicarbonate buffer solution for 5 min, immediately followed by 15 min of recirculating perfusion with the same solution containing 0.3 mg/ml collagenase (type 1, Worthington Biochemicals, Freehold, NJ), 0.15 mg/ml hyaluronidase (type I-S, Sigma Chemical, St. Louis, MO), and 50 µM CaCl2. Both cell isolation solutions contained (in mM) 91.7 NaCl, 30 KCl, 1.2 MgSO4, 19 NaHCO3, 1.2 NaHPO4, 15 glucose, 20 taurine, and 0.5 adenosine and were gassed with 5% CO2-95% O2 (pH 7.40). The heart was then detached from the cannula. After left ventricular wet weight was measured, the left ventricle was cut into small pieces, which were agitated in the same solution without hyaluronidase. These pieces were then incubated with an equal volume of solution containing 0.0025% trypsin inhibitor (Sigma) and 1% BSA and centrifuged at 250 revolutions/min for 5 min. The supernatant was discarded, and the cells were resuspended in minimum essential medium (GIBCO). The isolated myocytes were used for physiology studies within 6 h after dissociation.

Measurement of [Ca2+]i and [Na+]i. The [Ca2+]i in isolated myocytes was measured with a previously described method (34). Myocytes were attached to laminin-coated glass coverslips and then incubated in a HEPES (loading) solution containing 1 µM fluo 3-acetoxymethyl ester (fluo 3-AM; Molecular Probes) at 30°C in the dark for 30 min. The loading solution was prepared by diluting a 100 µM fluo 3 stock solution, which contained 0.45% Pluronic F127 (Molecular Probes), 10% dimethyl sulfoxide, and 90% heat-inactivated fetal calf serum (Gibson). HEPES solution consisted of (in mM) 126 NaCl, 4.4 KCl, 1.0 MgCl2, 1.08 CaCl2, 24 HEPES, 13 NaOH, 11 glucose, and 0.5 probenecid (pH 7.4). Fluo 3-loaded myocytes on a coverslip were then washed twice with dye-free HEPES solution and placed in a flow-through chamber on the microscope. Myocytes were excited with 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 increased with an increase in [Ca2+]i. More than 90% of the dye within myocytes was rapidly released with 50 µM digitonin, consistent with accumulation primarily in the cytosol.

Myocytes were electrically stimulated with the use of platinum electrodes with 7-ms pulses of alternating polarity, and [Ca2+]i transients and pacing signals were simultaneously recorded on tape for further analysis. In most of the experiments, the calibration of the [Ca2+]i transients was performed with a modification of the method of Yao et al. (34). After fluorescence transients were recorded, the myocyte was superfused with HEPES solution containing 10 µM ionomycin and 30 mM 2,3,-butanedione monoxime (BDM). In 5 min, the intensity of the fluorescence increased, and then the myocyte was perfused with HEPES solution containing 10 mM MnCl2, 10 µM ionomycin, and 30 mM BDM. Mn2+ quenched fluo 3 in the cytosol of the myocyte, providing fluorescence intensity at saturating Mn2+ (FMn2+). After FMn2+ was recorded, the intensity of the fluorescence from the background 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 isolated in the same day was used as an autofluorescence (Fauto) for each myocyte. The variance of the autofluorescence was very small. Maximum fluorescence intensity (Fmax) was calculated with the following formula: Fmax = (FMn2+ - Fbkg - Fauto) × 5. Minimum fluorescence intensity (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)]}, where F is the measured fluorescence intensity of the myocyte and Kd is the dissociation constant. The [Ca2+]i calibration was performed at 37°C using a Kd of fluo 3 at 37°C of 864 nM.

[Na+]i was measured with a modified method of Levi et al. (18) using the Na+-sensitive fluorescent dye benzofuran isophthalate (SBFI; Molecular Probes). Myocytes on laminin-coated coverslips were incubated at room temperature for 120 min in HEPES solution containing 10 µM SBFI-AM. The loading solution for each dye was prepared with the same method described for fluo 3-AM. The loaded myocytes were then washed and incubated in dye-free HEPES solution for 15 min. The myocytes on the coverslips were then placed in a flow-through chamber and perfused with HEPES solution at 37°C. SBFI has two different excitation wavelengths (340 and 380 nm) and one emission wavelength (510 nm). 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 R of the fluorescence intensity during excitation with 340-nm light to that with 380-nm light was used as an indicator of [Na+]. After the emission intensities were recorded, an in vivo calibration was performed. For calibration, the myocyte was sequentially exposed to three calibration solutions of 5, 10, and 15 mM Na+ containing (in mM) 2 gramicidin D, 40 monensin, and 100 strophantidin. In each solution, [Na+]i was equilibrated to the extracellular Na+ concentration, 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 solution consisted of (in mM) 30 NaCl, 110 sodium gluconic acid, 2 EGTA, and 10 HEPES, and the latter solution was identical except for complete replacement of Na+ by K+. The pH of each solution was adjusted to 7.2 with NaOH and KOH, respectively. Data were all digitized and directly acquired by a computer. The relationship between R and [Na+]i was fitted with the mathematical software Origin (Microcal) to the following formula: [Na+]i = C × (R - Rmin)/(Rmax - R), where C = Kd × beta . With the use of this curve, the fluorescence intensity from the myocyte was then converted to [Na+]i.

Solution switching. A solenoid-driven device (SW) was used to make fast changes of the extracellular solution surrounding a myocyte (34). Two adjacent microstreams flowed simultaneously from neighboring square glass tubes (each 200 µm wide) separated by a 70-µm glass septum. A myocyte was positioned in the control stream so that activation of the solenoid moved the two streams laterally and suddenly exposed the cell to the test solution stream in ~4 ms.

Measurement of L-type Ca2+ current and Na+/Ca2+ exchanger current. L-type Ca2+ current (ICa,L) and the outward current induced by the Na+/Ca2+ exchanger (INa/Ca) were measured sequentially in the same myocyte at room temperature with a voltage-clamp technique described previously (9). A myocyte was voltage-clamped with a suction pipette and a voltage-clamp circuit (Axopatch 200A, Axon Instruments, Foster City, CA). The suction pipettes were made from borosilicate capillary tubing (1.65-mm OD, 1.2-mm ID; Corning 7052, A-M Systems, Everett, WA) and had resistances of 1.5-3 MOmega . The pipette solution contained (in mM) 10 NaCl, 0.2 MgCl2, 14 EGTA, 3.0 MgATP, 5.5 glucose, and 10 HEPES. Ca2+ (3.9 mM) was added as H2Ca-EGTA. The solution pH was adjusted to 7.1 with CsOH. CsCl was added to give a final Cs+ concentration of 130 mM. The free Ca2+ was estimated to be 100 nM. The myocyte was held at a potential of -40 mV and initially superfused in a microstream from the SW solution, which contained (in mM) 126 NaCl, 1.0 MgCl2, 2.7 CaCl2, 11 glucose, and 24 HEPES. The pH was adjusted to 7.4 with NaOH to give a final Na+ concentration of 140 mM.

For the measurement of ICa,L, L-type Ca2+ currents were activated by depolarizing the myocyte membrane to more positive values at increments of 10 mV from -40 mV for 500 ms.

After the measurement of ICa,L, an outward Na+/Ca2+ exchange current was activated when the myocyte was abruptly immersed in an adjacent microstream from the SW solution, in which Li+ replaced Na+. With this procedure, the elimination of extracellular Na+ elicits the reverse function of the Na+/Ca2+ exchanger (3 Na+ out, 1 Ca2+ in), resulting in an outward current (INa/Ca). The magnitude of the Na+/Ca2+ exchange current was measured after 20 min of dialysis of a myocyte with the suction pipette, with the Na+ pump inhibited by zero extracellular K+. The elicited outward current has been shown to be inhibited by Ni2+ and XIP, a specific exchange inhibitory peptide for the Na+/Ca2+ exchanger (9), and has been shown to be correlated with mRNA levels of the Na+/Ca2+ exchanger in neonatal and adult rabbit myocytes examined by RNase protection assay (8). The cell capacitance was measured for the determination of the current density.

Data were all digitized, acquired, and analyzed by a computer with pCLAMP 6 software (Axon Instruments).

Assessment of SR Ca2+-ATPase function. We have recently reported a method to assess the SR Ca2+-ATPase function in single ventricular myocytes (34). Briefly, we abruptly exposed a myocyte to 140 mM K+ (0 Na+-0 Ca2+ EGTA) solution with SW solution at 4 s after the last of a series of electrical stimulations (ES) at 0.25 Hz. This 140 mM K+ (0 Na+-0 Ca2+ EGTA) solution contained (in mM) 126 KCl, 1.0 MgCl2, 13 KOH, 24 HEPES, 2 EGTA, and 0.5 probenecid, the pH of which was titrated with additional KOH and adjusted to 7.4. In the SW solution the membrane is depolarized by high K+ and maintained at ~0 mV with the initial phase identical to the action potential, and this induced a [Ca2+]i transient similar in magnitude to that evoked by the ES. The Na+/Ca2+ exchanger is unable to function in either forward or reverse modes during the decline of the [Ca2+]i transient because of the elimination of extracellular Na+ and Ca2+. The function of the SR Ca2+-ATPase and the contribution of the Na+/Ca2+ exchanger to the decline of [Ca2+]i transients can be assessed by comparing the time courses of decline of the ES and SW [Ca2+]i transients. In some experiments, control myocytes were superfused with a 0.6 mM Ca2+ solution to reduce the magnitude of the [Ca2+]i transient before elimination of Na+/Ca2+ exchange with the SW.

RNase protection assay. The expression of Na+/Ca2+ exchanger and SR Ca2+-ATPase was examined with RNase protection assay as described by Chin et al. (8). Total RNA was extracted from isolated myocytes or apex muscle homogenated in a Trizol reagent (Life Technologies, Gaithersburg, MD) as described by the manufacturer. Riboprobes for the canine heart Na+/Ca2+ exchanger (380 bp) and the rabbit sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a; 300 bp) as well as human U1 gene were used. Fifteen micrograms of each sample of total RNA were probed with the Na+/Ca2+ exchanger and the sarcoplasmic reticulum Ca2+-ATPase riboprobes together with the antisense U1 snRNA riboprobe as an internal control for RNA loading. After digestion with RNase A and RNase T1, the protected bands for each mRNA were separated over 2 h on a 6% sequencing gel at 1,500 V. The protected species for Na+/Ca2+ exchanger appeared as two distinct bands, and the SR Ca2+-ATPase protected species ran as a doublet. The relative amounts of Na+/Ca2+ exchanger and SR Ca2+-ATPase were quantified with PhosphorImager analysis and normalized to the amount of U1 snRNA.

Data analysis. [Ca2+]i data were digitized and analyzed using a Digidata 1200 A analog-to-digital converter and Axoscope 1.0 (Axon Instruments) and Micro Origin 4.0 (Microcal Software) software running on a personal computer (Innovation Computer, West Valley City, UT). Data were expressed as means ± SE, and the statistical significance of differences was assessed by ANOVA and t-test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cardiac enlargement induced by pacing. The left ventricular wet weight (LVW) and body weight (BW) of control and pacing-induced heart failure (PF) animals are shown in Table 1. In PF rabbits, LVW and the ratio of LVW to BW were significantly increased compared with the same values in control rabbits, whereas BW did not show statistically significant changes. The purpose of our experiments was to examine the Ca2+ cycling system in PF rabbits, and therefore it was necessary to measure the LVW after perfusion with enzyme to get a good yield of isolated myocytes. Nonetheless, our data on LVW and the ratio of LVW to BW are consistent with a previous report (19).

                              
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  		Table 1.   Heart and body weight

[Ca2+]i transients in myocytes from failing hearts. [Ca2+]i transient data are summarized in Table 2. Systolic and diastolic [Ca2+]i and the amplitude of [Ca2+]i transients were significantly decreased in PF myocytes. The time courses of the [Ca2+]i transients are shown in Table 3. Time to peak [Ca2+]i was significantly prolonged in PF myocytes compared with that in control myocytes. The terminal decline of [Ca2+]i transients [time from 50 to 10% of peak (t50-10)] was significantly prolonged in PF myocytes, whereas the difference in the initial rate of decline of [Ca2+]i transients [time from 90 to 50% of peak (t90-50)] did not reach statistical significance. In physiological excitation-contraction (EC) coupling, Ca2+ influx through L-type Ca2+ channel and via Na+/Ca2+ exchanger operating in reverse mode provide influx of Ca2+ from the extracellular space, and SR Ca2+ release is the main internal source of cytosolic Ca2+ for contraction. The SR Ca2+-ATPase and the Na+/Ca2+ exchanger mediate the decline of [Ca2+]i during relaxation (3). Therefore, we examined the functions of L-type Ca2+ channel, Na+/Ca2+ exchanger, and SR Ca2+-ATPase to determine whether alteration of these Ca2+ transport systems might account for the observed changes in the [Ca2+]i transient.

                              
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  		Table 2.   [Ca2+]i transients

                              
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  		Table 3.   Time courses of [Ca2+]i transients

ICa,L density. Figure 1 shows ICa,L densities in control and PF myocytes. Figure 1, inset, shows examples of raw current traces. ICa,L density from -10 to +60 mV was significantly decreased in PF myocytes. It seems likely that the reduced influx of Ca2+ via the L-type Ca2+ channel is at least in part responsible for the observed reduction of peak systolic [Ca2+]i.


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  		Fig. 1.   Peak current-voltage relationship of L-type Ca2+ channel current (ICa,L). Peak ICa,L density at -10 to 60 mV was significantly decreased in pacing-induced heart failure (PF) myocytes (bullet , n = 25) as compared with control myocytes (, n = 17). * P < 0.01, by ANOVA and unpaired t-test. Inset: representative ICa,L. ICa,L was elicited by depolarizing the membrane for 500 ms from a holding potential of -40 to +60 mV in 10-mV increments in a control and a PF myocyte. ICa,L was markedly decreased in PF myocyte.

INa/Ca density. As described above, the Na+/Ca2+ exchanger functions in a forward mode to extrude cytosolic Ca2+ and in a reverse mode to produce influx of Ca2+. We directly examined the Na+/Ca2+ exchanger function in control and PF myocytes. As shown in Fig. 2, outward currents were observed in myocytes abruptly exposed to 0 Na+ solution with the SW. Current magnitudes normalized to the membrane capacitance indicate that the INa/Ca density was significantly reduced in PF myocytes (0.701 ± 0.026 pA/pF, n = 21, P < 0.01) compared with that in control myocytes (0.919 ± 0.039 pA/pF, n = 26). Measured capacitance was somewhat greater in PF myocytes (170 ± 9 pF, n = 29) than in control myocytes (155 ± 7 pF, n = 28), but this difference was not statistically significant. The function of Na+/Ca2+ exchanger in vivo is very dependent on the [Na+]i (4, 27). The [Na+]i was 5.2 ± 1.5 and 4.4 ± 1.8 mM in control (n = 30) and PF (n = 20, P < 0.05) non-voltage-clamped myocytes, respectively. This small decrease (0.8 mM) in the [Na+]i in PF myocytes increases Na+ equilibrium potential by 4 mV and could theoretically contribute to some extent to a lower diastolic [Ca2+] and help maintain relaxation by Na+/Ca2+ exchange. However, this effect is probably not marked in view of the slight decrease in exchanger density in PF myocytes estimated from INa/Ca measurements.


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  		Fig. 2.   Examples of outward Na+/Ca2+ exchanger current (INa/Ca). INa/Ca was elicited by abruptly exposing a myocyte to 0 Na+ solution for 5 s with a holding membrane potential at -40 mV (see MATERIALS AND METHODS). In 0 Na+ solution, the Na+/Ca2+ exchanger functions reversely to extrude 3 Na+ in exchange for 1 Ca2+, inducing an outward current. INa/Ca was decreased in PF myocyte.

SR Ca2+-ATPase function in congestive heart failure myocytes. In congestive heart failure (CHF) myocytes, it has been reported that the decline of [Ca2+]i transients is prolonged and the SR Ca2+-ATPase function is impaired (6, 14). In the present study, we observed a slower decline of the ES [Ca2+]i transients in PF myocytes as shown in Table 3. However, in ES [Ca2+]i transients the decline is mediated by both the SR Ca2+-ATPase and the Na+/Ca2+ exchanger (3). Therefore, the prolonged decline may be due in part to the decreased function of the Na+/Ca2+ exchanger. Furthermore, Bers and Berlin (5) have recently shown that the time constant of [Ca2+]i decline is accelerated by a higher peak level of [Ca2+]i. The prolongation of the decline in ES [Ca2+]i transients may therefore also be the result of the lower peak of [Ca2+]i observed in our PF myocytes and in the previous reports (6, 14). Furthermore, as we recently showed (34), an increase in Ca2+ buffering might prolong the decline of the [Ca2+]i transients. To rule out these possibilities, we examined the contribution of the SR Ca2+-ATPase to the decline of ES [Ca2+]i transients using a rapid SW.

After warm-up pacing of myocytes at 0.25 Hz, we abruptly exposed a myocyte to 140 K+ (0 Na+-0 Ca2+ EGTA) solution, which induced the identical peak of [Ca2+]i transient (SW [Ca2+]i transient) while disabling Na+/Ca2+ exchange (34). We observed no difference in the initial decline of [Ca2+]i between ES and SW [Ca2+]i transients in control myocytes (Fig. 3), similar to our previous report (34). In contrast, the initial decline of the SW [Ca2+]i transient was dramatically prolonged compared with the ES [Ca2+]i transient in PF myocytes. The mean values of t90-50 of ES and SW [Ca2+]i transients are summarized in Fig. 4. In control myocytes in normal extracellular Ca2+ concentration (Fig. 4A), there was no significant difference in t90-50 between ES and SW [Ca2+]i transients, suggesting that the initial decline is mainly mediated by SR Ca2+-ATPase in normal EC coupling. On the other hand, in PF myocytes, t90-50 of SW [Ca2+]i transients was significantly prolonged compared with that of ES [Ca2+]i transients. This result suggests that, in PF myocytes, the function of the SR Ca2+-ATPase is decreased and the contribution of the Na+/Ca2+ exchanger to the initial decline in ES [Ca2+]i is relatively increased despite the reduced density of the Na+/Ca2+ exchanger. The reduced [Ca2+]i transient magnitude could affect the relative dependence of the [Ca2+]i decline on Na+/Ca2+ exchanger and SR function. We therefore performed similar experiments in control myocytes in which peak [Ca2+]i was reduced to levels comparable to those in PF myocytes (246.2 ± 18.2 nM estimated by pseudocalibration; Ref. 20) by lowering extracellular Ca2+. The results are shown in Fig. 4B. Reducing peak [Ca2+]i in control myocytes produced a greater relative dependence of Ca2+ decline on Na+/Ca2+ exchange, manifest by the change in the t90-50 when Na+/Ca2+ exchange was abruptly inhibited with the SW. However, the PF myocytes showed a greater prolongation in t90-50 than control myocytes at comparable peak [Ca2+]i values.


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  		Fig. 3.   Examples of effects of abrupt elimination of Na+/Ca2+ exchange on decline of intracellular Ca2+ concentration ([Ca2+]i). Myocytes were electrically stimulated (ES) at 0.25 Hz for >2 min and then abruptly exposed to 140 mM K+ (0 Na+-0 Ca2+ EGTA) solution with a rapid switcher (SW) 4 s after the last pacing stimulus. In this solution, myocytes were depolarized by high extracellular K+ to ~0 mV. Na+/Ca2+ exchanger was disabled by elimination of extracellular Na+ and Ca2+. Therefore, decline of SW [Ca2+]i transients was mediated only by SR Ca2+-ATPase. There was no apparent difference in initial decline of ES and SW [Ca2+]i transients in a control myocyte (top tracing). In a PF myocyte (bottom tracing), initial decline in SW [Ca2+]i transient was prolonged compared with that of ES [Ca2+]i transient, consistent with impaired sarcoplasmic reticulum (SR) function.


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  		Fig. 4.   A: comparison of time from 90 to 50% of peak (t90-50) in ES and SW [Ca2+]i transients in 1.08 mM extracellular Ca2+. Averaged t90-50 in ES and SW [Ca2+]i transients were measured in control (, n = 13) and PF (bullet , n = 20) myocytes. In control myocytes, t90-50 in SW transients (150 ± 11 ms) was similar to that in ES transients (170 ± 15 ms), suggesting little contribution of Na+/Ca2+ exchanger to initial [Ca2+]i decline. In contrast, in PF myocytes, t90-50 in SW transients (535 ± 108 ms) was significantly prolonged compared with that in ES transients (212 ± 19 ms), implying a significant contribution of Na+/Ca2+ exchanger to initial decline of [Ca2+]i and, thus, impaired SR function. * P < 0.01, by paired t-test. B: comparison of t90-50 in ES and SW [Ca2+]i transients at comparable peak [Ca2+]i. Peak [Ca2+]i was reduced in control myocytes (n = 18) to a value similar to that in PF myocytes (n = 20) by lowering extracellular Ca2+ concentration to 0.6 mM. In low-Ca2+ control myocytes, prolongation of t90-50 (Delta t90-50) in SW beats was greater than in normal-Ca2+ control myocytes but significantly less than in PF myocytes (P < 0.05).

Expression of SR Ca2+-ATPase and Na+/Ca2+ exchanger. We also examined the expression of the SR Ca2+-ATPase (SERCA2a) and the Na+/Ca2+ exchanger in control and PF rabbit hearts. As shown in Fig. 5, the message levels for the SR Ca2+-ATPase and the Na+/Ca2+ exchanger were markedly reduced, whereas the loading conditions in each lane defined by the message levels of U1 gene were similar. Mean values of normalized expression of the SR Ca2+-ATPase and the Na+/Ca2+ exchanger were 64.0 ± 11.4% (n = 10) and 66.4 ± 8.0% (n = 6) in PF hearts, respectively (P < 0.05 for both). Although mRNA levels may not accurately reflect protein levels, these data are consistent with the changes we have observed in the functional activity of the SR Ca2+-ATPase and the Na+/Ca2+ exchanger.


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  		Fig. 5.   Expression of Na+/Ca2+ exchanger and SR Ca2+-ATPase in 3 control and 3 PF hearts. Representative RNase protection assay shows that expressions of Na+/Ca2+ exchanger and SR Ca2+-ATPase (SERCA2a) were decreased in PF rabbits, whereas the loading of RNA was similar in each lane as shown by expression of U1 snRNA.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Alteration in [Ca2+]i in pacing-induced failure. It is well established that contractile function is depressed in hearts with PF (19, 29, 32), but the mechanisms involved have not been well established and may vary with the species studied. We have investigated changes in myocytes from rabbits with PF. This is the smallest animal in which PF has been reported, and the electrophysiological and hemodynamic characteristics in rabbits are similar to those in larger mammals. Thus myocytes from rabbits provide a convenient and appropriate model for this type of failure.

In rabbits, a marked decrease in the [Ca2+]i transient is present in myocytes isolated from ventricles with PF. This finding differs from the report of Perreault et al. (24), who found a similar peak [Ca2+]i and diastolic [Ca2+]i measured with aequorin in trabeculae from dogs with PF. These investigators suggested that a decrease in Ca2+ availability was not responsible for the decreased contraction. However, an alteration in Ca2+ homeostasis rather than an alteration in myofilament responsiveness to Ca2+ appears to be the principal cause of myocyte dysfunction in PF in rabbits. We have identified several factors that may contribute to the observed decrease in the [Ca2+]i transient in these cells.

Reduction of L-type Ca2+ channel function. L-type Ca2+ channel function has been studied in myocardium of several pacing-induced failure models (10, 16, 22, 23, 25) and human heart failure [reviewed by Hasenfuss et al. (15)]. However, the results have not been consistent. Vatner et al. (32) reported that neither dihydropyridine binding nor affinity for [3H]PN200-110 was changed in dog PF hearts. Recently Kääb et al. (16) also reported no change in the function of the L-type Ca2+ channel assessed with voltage clamp in ventricular myocytes isolated from dogs with PF. In contrast to the dog model, Mukherjee et al. (22, 23) reported a decrease in L-type Ca2+ current density in PF in pigs. Colston et al. (10) reported a 50% reduction in Ca2+ channel antagonist binding sites in ventricular sarcolemmal membranes in the rabbit model of PF. Rozanski et al. (25) reported that basal L-type Ca2+ current was not altered in the rabbit model, although the current in the presence of isoproterenol was reduced. These differences in changes in ICa,L in the rabbit appear to reflect the degree of failure (25).

Consistent with this possibility, we have found a 43% reduction in basal ICa,L density at +10 mV in rabbit ventricular myocytes isolated from rabbits with severe PF. In the study of Rozanski et al. (25), rabbits were paced for only 2-3 wk at a slower pacing rate (360-380 beats/min). Also, the degree of ventricular dysfunction induced was less severe than in our study and the study of Ryu et al. (26). The reason for the difference noted in L-type Ca2+ channel function with PF in different species is not clear. Nonetheless, it seems likely that the reduced Ca2+ current accounts in part for the decreased [Ca2+]i transient we have observed.

Alteration of Na+/Ca2+ exchanger function. In the present study, we have examined the in vivo activity of the Na+/Ca2+ exchanger in PF myocytes, as well as its expression. Studer et al. (30) reported that, in myocardium from humans with CHF due to dilated and ischemic cardiomyopathy, the expression and protein level of the Na+/Ca2+ exchanger were increased compared with normal hearts. However, Dhalla et al. (11) reported that expression and protein levels of the Na+/Ca2+ exchanger were decreased in myocardium from rats with CHF induced by infarction. To our knowledge there have been no reports of alteration in Na+/Ca2+ exchanger function in myocytes from animals with PF. In the PF model, we detected decreased activity of the Na+/Ca2+ exchanger as well as downregulation of expression of Na+/Ca2+ exchanger message, presumably reflecting changes in the protein level. Previous work has suggested that the Na+-K+-ATPase activity and glycoside receptor density are depressed in pig myocardium with PF (28). However, we found only a very small decrease in [Na+]i in myocytes from rabbits with PF. This indicates that Na+-K+-ATPase activity is probably not markedly altered in these myocytes and that the decreased activity of the Na+/Ca2+ exchanger should result in both decreased forward and reverse exchange. It is not clear from the present study what effect, if any, decreased expression of the Na+/Ca2+ exchanger might have on the Ca2+ transient, although the slowing of the terminal phase of relaxation we have observed might be anticipated (34).

Reduced SR function in CHF myocytes. The SR Ca2+-ATPase sequesters cytosolic Ca2+ during relaxation, competing with Ca2+ extrusion via the Na+/Ca2+ exchanger to maintain SR Ca2+ content for subsequent Ca2+-induced Ca2+ release during EC coupling (2, 3). In various types of human CHF (1, 2, 21, 30, 31) and CHF animal models (2, 12, 17), the expression and/or the protein level of SR Ca2+-ATPase has been reported to be decreased, suggesting that the impairment in relaxation is due to reduced uptake of Ca by the SR. Williams et al. (33) have reported that mRNA levels for the SR Ca2+-ATPase were unchanged in the dog heart with PF, although Perreault et al. (24) noted a slowed decline of the aequorin Ca2+ transient in myocardium from dogs with PF. However, there has not been any report in which SR function or [Ca2+]i transients were directly examined in intact ventricular myocytes isolated from PF hearts. In the present study, we have shown a markedly slower decline in [Ca2+]i in PF myocytes when the Na+/Ca2+ exchanger is abruptly disabled. This finding, as well as the reduced SERCA2a mRNA levels, supports the hypothesis that SR Ca2+-ATPase function is decreased. This likely contributes to slow relaxation and to decreased Ca2+ release from the SR, which could combine with the decreased Ca2+ current to reduce the [Ca2+]i transient magnitude. This could also lead to the observed prolongation of the time to peak systolic [Ca2+]i, as suggested by Cannell et al. (7).

In summary, in myocytes isolated from rabbit hearts with abnormal and severe pacing-induced CHF, there is a marked decrease in the magnitude of the Ca2+ transient that is associated with a highly significant decrease in the L-type Ca2+ current. The Na+/Ca2+ exchanger function as well as SR Ca2+-ATPase function is also impaired in these cells. The mechanisms by which rapid pacing causes these alterations are not established. However, the changes noted in rabbit myocytes with PF appear to resemble more closely those reported in the pig, whereas findings in the dog seem quite different. These results emphasize possible important species differences in responses to rapid pacing and suggest that different mechanisms causing myocardial dysfunction may be involved.

    ACKNOWLEDGEMENTS

We are indebted to J. Thorburn for assistance in the RNase protection assays and to Pam Larson for preparation of the manuscript.

    FOOTNOTES

This work was supported in part by Specialized Center of Research on Heart Failure Grant HL-53773. The contributions of the first two authors are equal.

Address for reprint requests: W. H. Barry, Div. of Cardiology, Univ. of Utah Health Sciences Center, 50 N. Medical Drive, Salt Lake City, UT 84132.

Received 10 December 1997; accepted in final form 19 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(4):H1441-H1448
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


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