Abstract Cellular Ca2+ regulation is abnormal in diseased hearts. We designed this study to assess the role of the Na+-Ca2+ exchanger in excitation-contraction coupling in surviving myocardium of the infarcted heart. We measured cellular contractions and whole-cell currents in single left ventricular myocytes isolated from the hearts of rabbits with healed myocardial infarction (MI). Eight weeks after MI, rabbits had left ventricular dysfunction without overt heart failure. Myocytes isolated from regions adjacent to the infarcted zone were significantly longer than cells from control hearts. At low stimulation rates (0.5 Hz), the amplitude of field-stimulated contractions was increased (11.6±0.5% versus 10.2±0.6% resting cell length), whereas the time to peak shortening and action potential duration were prolonged in the MI cells. When stimulation frequency was increased to 2.0 Hz, cellular shortening did not change or decreased in myocytes from infarcted hearts, whereas control cells had a positive shortening-interval relationship. Cells from infarcted hearts had a significantly decreased (31%) L-type Ca2+ current (ICa) density but no change in the current-voltage relationship or the kinetics of ICa inactivation. Maximal Na+-Ca2+ exchange current density was significantly increased (32%) in the cells from infarcted hearts. Sarcoplasmic reticulum (SR) Ca2+ content during a stable train of contractions, as estimated from caffeine-induced inward currents, was slightly increased (P=NS) in the MI myocytes. To determine whether Na+-Ca2+ exchange influenced SR Ca2+ content, cells were clamped at potentials between −70 and +90 mV for 400 ms. The amplitude of the contraction during a subsequent clamp step to +10 mV was then measured as an index of SR loading that occurred during the preceding clamp step. Steps to positive potentials produced greater augmentation of the subsequent contraction in MI than in control myocytes. In myocytes from the infarcted heart, increased activity of the Na+-Ca2+ exchanger may promote Ca2+ entry or decrease Ca2+ extrusion. This relative augmentation of inward Ca2+ flux by the exchanger may enhance SR Ca2+ loading and thus support contractility that would otherwise be impaired as a result of decreased Ca2+ current. However, Ca2+ influx by the exchanger may contribute to the prolongation of contractions in myocytes from infarcted hearts.
The development of heart failure after MI is a gradual process resulting from geometric and biochemical changes in both the infarcted and noninfarcted portions of the heart.1 Surviving myocytes, particularly those adjacent to the zone of infarcted tissue, elongate in response to hemodynamic stresses and/or neurohumoral influences.2,3 Although this hypertrophic response may partially compensate for the loss of contractile tissue in the infarcted zone, there is mounting evidence that abnormalities of excitation-contraction coupling develop in the surviving myocytes of the infarcted heart. For example, several groups have observed prolongation of intracellular Ca2+ transients and contractions in papillary muscles or isolated cells from noninfarcted regions of the rat heart.4,5
A number of systems involved in cellular Ca2+ homeostasis have been considered as potential causes of the prolongation of the intracellular Ca2+ transients in myocytes from diseased hearts. Some investigators have focused on the significance of changes in SR function. In this regard, decreased expression of the SR Ca2+-ATPase (mRNA and protein) has been reported in several forms of cardiac overload.6,7 Although these changes are clearly important, they may not explain all of the functional abnormalities found in cells from hypertrophied or failing hearts. If impaired sequestration of Ca2+ by the SR were the only abnormality, then SR Ca2+ content should be depleted during repetitive stimulation, since the SR Ca2+-ATPase and the sarcolemmal Na+-Ca2+ exchanger compete for Ca2+ removal from the cytoplasm.8 Significantly decreased cellular contractions or Ca2+ transients should, therefore, be consistently observed in failing or nonfailing hypertrophied myocardium. Contrary to this prediction, many investigators have found only modest reductions or no differences in the amplitude of contractions or Ca2+ transients in tissue or cells from diseased hearts.5,9–12 Hence, other Ca2+ regulatory systems that may contribute to the observed changes in Ca2+ transients of dysfunctional hearts have been examined.
Several groups have reported that the sarcolemmal Na+-Ca2+ exchanger is upregulated in myocardium from humans and experimental animals with myocardial hypertrophy or heart failure.13–16 Based on these findings, it has been hypothesized that increased Ca2+ extrusion by the Na+-Ca2+ exchanger is an adaptive mechanism that may “compensate” for decreased Ca2+ uptake by the SR and may help to maintain normal diastolic Ca2+ levels.15 There is some evidence to support this view11; however, the Na+-Ca2+ exchanger can transport Ca2+ in either direction and, hence, could promote Ca2+ entry as well as Ca2+ extrusion.17 There are many factors that affect Na+-Ca2+ exchange activity, including membrane potential and the transsarcolemmal Na+ and Ca2+ gradients. Since we have very limited information regarding these variables in pathological states, it is difficult to predict the function of the exchanger in diseased hearts. Therefore, we measured contractility, sarcolemmal Ca2+ transport, and SR Ca2+ content in surviving myocytes from infarcted hearts. We found evidence suggesting that Ca2+ influx by the Na+-Ca2+ exchanger might support contractility that would otherwise be impaired as a result of reduced Ca2+ current; however, Ca2+ entry by the exchanger may prolong cellular contractions or delay the onset of relaxation.
Materials and Methods
Male New Zealand White rabbits weighing 2.0 to 3.0 kg were used for all experiments. Animals were cared for according to the guidelines of the American Physiological Society. The studies were approved by the institutional animal care committee.
Production of MI
MIs were induced using a modification of a published protocol.18 Briefly, rabbits were injected with acepromazine (16 mg/kg) and xylazine (3 mg/kg) intramuscularly. Approximately 10 to 15 minutes later, they were anesthetized with 2% isoflurane delivered by face mask. They were orotracheally intubated and ventilated at 40 breaths/min with 1% isoflurane supplemented with oxygen (2 L/min). A thoracotomy was made in the left fourth intercostal space. The large branch of the circumflex artery that runs on the lateral aspect of the heart was identified and ligated 1 to 2 mm below the atrioventricular groove using 6–0 silk suture. Lidocaine HCl (1 mg/kg IV) was administered at the time of coronary occlusion, and a second dose (0.5 mg/kg) was given 10 minutes later. Transthoracic defibrillation with 10 to 50 J of energy delivered via pediatric paddles was used if required (≈20% of infarcted rabbits). Procaine penicillin G (300 000 U) was administered intramuscularly just before the operation.
In ≈10% to 20% of the rabbits undergoing surgery, the artery was very small or could not be visualized. These rabbits sustained minimal or no infarctions. In a subgroup of animals, the chest was opened, but the coronary artery was intentionally not ligated (sham surgery). To minimize the number of survival surgeries, we compared characteristics (cell size, action potentials, Ca2+ current density, and current-voltage relationships) of cells from rabbits with minimal or no infarcts and from sham-operated animals with cells from unoperated rabbits. Since we did not see significant differences (data not shown), we included animals from all three groups (minimal infarct, sham surgery, and unoperated) as controls. It seems unlikely that residual effects of the surgical procedure would still be present 8 weeks after surgery.
Rabbits were killed 8±0.7 weeks after MI. This duration of time was chosen because scar healing is complete and compensatory processes are likely to be activated, but direct effects of the surgical procedure were anticipated to be largely dissipated. Left ventricular myocytes from the peri-infarct border zone were isolated by using a variation of standard methods. We chose to use only cells from this specified region to reduce variability in data due to regional differences in the infarcted ventricle. In addition, we anticipated that the differences in cellular physiology would be greatest in this region, since previous investigators have shown that the infarct border zone has more contractile dysfunction and a greater amount of myocyte hypertrophy.2,19 Rabbits were euthanized with Beuthanasia (Schering-Plow Animal Health) administered through an ear vein. The heart was quickly excised and then retrogradely perfused via the aorta (50 mm Hg pressure) with a Ca2+-free HEPES-buffered saline solution that was bubbled with 100% O2. After 5 minutes, the solution was changed to one containing 0.08% collagenase and 0.02% protease, and perfusion was continued for an additional 15 to 25 minutes. When the heart was soft, the right ventricle and atria were removed. The scar tissue was carefully cut away from the rest of the left ventricle. A rim of 2 to 3 mm of surviving myocardium was left intact. The rim of surviving tissue was then dissected from the scar, and this tissue was minced. In some rabbits, cells from the remote portion of the infarcted left ventricle were also studied. The minced tissue was gently shaken in a low-Ca2+ saline solution free of digestive enzymes and strained, and the dissociated myocytes were allowed to settle in a storage solution containing 1.0 mmol/L Ca2+. In a subgroup of rabbits, the atria, right ventricle, left ventricle, and the scar were weighed after perfusion with collagenase, but before mincing. Large numbers of Ca2+-tolerant rod-shaped cells with clear striations were consistently obtained in both control and infarcted hearts.
In a subgroup of animals, intracardiac pressures were measured before they were killed. Ketamine (50 mg/kg IM) and xylazine (5 mg/kg IM) were administered. The right carotid artery was isolated, and a 2F micromanometer-tipped catheter (Millar Instruments) was inserted and passed retrogradely into the left ventricle.
General Features of All Studies
Studies were performed within 8 hours after cell dissociation. The cells were affixed with natural mouse laminin (Collaborative Biomedical Products) to a glass coverslip, which formed the bottom of a bath. The bath was continuously perfused with a HEPES-buffered modified Tyrode’s solution (mmol/L: NaCl 138, MgCl2 1.0, KCl 4.4, dextrose 11.0, CaCl2 2.7, and HEPES 24.0, pH adjusted to 7.4 with NaOH) maintained at 30°C. Cells were viewed with an inverted phase-contrast microscope (Nikon or Olympus). Cell motion was monitored with a video edge-detection system (Crescent Electronics). Solutions superfusing cells were rapidly changed using a modification of a previously described solution switcher.20 The modified switching device directs the solutions through two square glass tubes (200 μm) separated by a 70-μm glass septum. The bulk solution over the cell may be changed in 7 ms using this device.20 Command voltage pulses were generated by pClamp software, which controlled an Axopatch 200A or an Axoclamp 2B voltage-clamp circuit (Axon Instruments Inc). Pipettes were fashioned from 7052 borosilicate glass (1.2-mm inner diameter, 1.65-mm outer diameter) using a horizontal micropipette puller (P97, Sutter Instruments Co). When the pipettes were filled with solution, tip resistance was typically 1 to 2 MΩ. Stray capacitance was compensated as fully as possible. Current or membrane potential and cell motion were digitized at 1 to 2 kHz with an analogue-to-digital converter (TL-1 or Digidata 1200, Axon Instruments) and stored on a personal computer for later analysis using pClamp software (Axon Instruments).
In all studies, quiescent rod-shaped myocytes with clear striations and lack of granulation were selected. In voltage-clamp experiments, very long, thin, or irregularly shaped cells were avoided, since voltage control may be suboptimal. In experiments in which cell shortening was measured, myocytes with more square ends were preferentially selected, since tracking cell motion is more reliable. In all cases, an effort was made to avoid the use of cell size as a selection criteria.
Measurements of Cell Size
Cell size (length and width) was measured in randomly selected cells from control and infarcted hearts. To avoid bias, 50 to 100 cells from each heart (4 or 5 in each group) were measured by randomly moving the microscope stage (×20 objective) and measuring all the quiescent rod-shaped cells in each field. In two hearts, cells remote from the region of infarction were also measured.
Field Stimulation Studies
Cellular contractions were recorded at pacing intervals of 0.5, 1.0, and 2.0 Hz during field stimulation. The advantage of this approach is that the intracellular milieu (ion concentrations, pH, etc) is unperturbed. Constant current pulses of 5-ms duration (≈10% above threshold) were delivered by a pair of parallel platinum wires positioned near the cell. Cells were stimulated to contract at 0.5 Hz for at least 2 minutes to achieve stable SR Ca2+ loading. Steady-state contractions were recorded ≈30 s after each change in stimulation frequency. Maximal amplitude of shortening (percent resting cell length), peak rate of shortening, time to peak shortening, and peak rate of cellular relengthening were determined for contractions at each stimulation frequency. In every cell, measurements were made on four or five contractions at each stimulation frequency.
Measurement of Action Potentials
Action potentials were measured using a discontinuous voltage-clamp circuit in bridge mode (Axoclamp 2B, Axon Instruments Inc). For these experiments, the pipette solution contained (mmol/L) KCl 113, dextrose 5.5, K2ATP 5.0, HEPES 10, EGTA 0.02, MgCl 0.5, and NaCl 10, with pH adjusted to 7.1 using KOH. Five-ms current injections were adjusted to a level ≈10% above threshold. Cells were stimulated at 0.5 Hz for at least 2 minutes before making recordings. Action potentials were measured at stimulation frequencies of 0.5, 1.0, 2.0, and 3.0 Hz. Resting membrane potential, peak and plateau potentials, and APD90 were measured on at least three action potentials at each frequency.
Measurement of Ca2+ Current Density and Inactivation Kinetics
For measurements of ICa density and inactivation, the pipette filling solution contained (mmol/L) CsCl 130, dextrose 5.5, K2ATP 5.0, EGTA 14, MgCl 0.5, CaCl2 3.92, HEPES 10, and tetraethylammonium chloride 10, with pH adjusted to 7.1 using KOH. Free Ca2+ concentration in the pipette solution was calculated to be 0.1 μmol/L. Cells were held at a potential of −80 mV. A 75-ms prepulse to −40 mV was applied to inactivate Na+ current. Voltage-clamp steps (400 ms) to test potentials between −40 and +60 mV were then applied at 5-s intervals. Cells were held at −80 mV between episodes to minimize rundown of Ca2+ current. To calculate current density, membrane capacitance was measured at the beginning of each study. This was performed by recording capacitative transients induced by a 15-ms hyperpolarizing voltage-clamp step (5 mV). The area under the capacitative transient was integrated and divided by the voltage difference. Peak Ca2+ current was taken as the difference of the peak current and the current 350 ms after the start of the voltage-clamp pulse. Each current was then expressed relative to membrane capacitance (pA/pF).
Steady-state inactivation of Ca2+ current was determined using a double-pulse protocol.21 Cells were held at −40 mV to inactivate Na+ current. Voltage-clamp pulses to potentials between −50 and +40 mV were applied for 400 ms. These prepulses were of sufficient duration to induce maximal inactivation. The cell was then briefly clamped back to −40 mV (10 ms), followed by a 300-ms clamp step to +10 mV. The amplitude of the Ca2+ current during each test pulse (ICa) to +10 mV was normalized to the maximum current (ICamax) and plotted against the voltage of the preceding clamp step. The normalized current-voltage relationship (ICa/ICamax) was fitted to a Boltzmann function: ICa/ICamax=1/[1+exp(V1/2−Vt)/k], where Vt is the test pulse. The half-point (V1/2) and slope factor (k) were determined from this fit.
The time course of Ca2+ current inactivation, ICa(t), was assessed by fitting the declining phase of individual current records to an exponential relationship, ICa(t)=A0+A1e(−t/τ), with a Chebyshev noniterative fitting technique (pClamp, Axon Instruments).
Maximal INaCa density was recorded using previously described methods.22 First, cells were patch-clamped with a pipette containing (mmol/L) CsCl 85, dextrose 5.5, MgATP 3.0, EGTA 14, MgCl 0.5, CaCl2 3.92, HEPES 10, and NaCl 15, with pH adjusted to 7.1 using CsOH (total Cs, 130 mmol/L). Free Ca2+ concentration in the pipette solution was calculated to be 0.1 μmol/L. After the membrane was ruptured, the cell was superfused with a solution similar to the normal Tyrode’s solution, except that no K+ was added. Extracellular K+ was eliminated to inhibit the Na+ pump and thus allow more rapid accumulation of intracellular Na+. In addition, this approach should also eliminate overlapping currents due to activation of the Na+ pump during changes in sarcolemmal Na+ gradients. In some experiments, 3 mmol/L ouabain was also added to the superfusing solution. Results were comparable whether ouabain was present or not, suggesting that the Na+ pump was effectively inhibited by elimination of extracellular K+ (data not shown). The cell was held at −40 mV, and the superfusing solution was rapidly changed to a solution in which LiCl was substituted for NaCl. Exposure to zero extracellular Na+ was continued for 3 s. The sudden decrease in extracellular Na+ caused rapid extrusion of Na+ by the Na+-Ca2+ exchanger. This produced an outward current. After patch rupture, intracellular Na+ concentration gradually increased (as a result of dialysis of the cell by the pipette filling solution), and the amplitude of the outward current increased. Rapid solution switches were performed every 2 to 3 minutes until current amplitude reached steady state. Cells were held at −80 mV between rapid solution switches. INaCa amplitude reached a plateau 2 to 15 minutes after rupture of the patch. The largest value of the exchange current (difference between holding current and the maximal outward current) was recorded for each cell and expressed relative to membrane capacitance.
In these experiments, intracellular Ca2+ concentration was buffered to 0.1 μmol/L. Therefore, INaCa are probably not truly maximal, since Ca2+ binding to the regulatory site on the exchanger may enhance exchanger activity up to Ca2+ concentrations of ≈1 μmol/L. However, the measurements should provide physiologically relevant data, since they are obtained near normal diastolic Ca2+ levels.
Voltage Dependence of Cellular Contractions
The voltage-dependence of cellular shortening rate was determined by applying 400-ms voltage-clamp steps from a holding potential of −40 mV to potentials between −30 and +60 mV. The pipette solution contained (mmol/L) CsCl 130, dextrose 5.5, K2ATP 5.0, EGTA 0.02, MgCl 0.5, HEPES 10, and NaCl 10, with pH adjusted to 7.1 using KOH. This solution was used to minimize outward K+ currents but included enough K+ in the pipette solution to support normal SR Ca2+ release. Stable SR Ca2+ loading was maintained by a series of five conditioning pulses (400-ms pulses at 3-s intervals) to +10 mV between each test pulse. Peak Ca2+ current was taken as the difference between the maximal inward current and the current measured near the end of the 400-ms voltage-clamp step. The peak rate of cellular shortening was analyzed as described above.
Measurements of SR Ca2+ Content
SR Ca2+ content was estimated by applying a train of six conditioning pulses (400-ms clamp steps at 2-s intervals) from −40 to +10 mV to establish uniform SR loading conditions. The cell was then rapidly superfused with normal Tyrode’s solution containing 20 mmol/L caffeine. The caffeine was applied for ≈5 s. The application of caffeine induced a large contraction accompanied by an inward current. The inward current induced by caffeine has previously been shown to be caused by Na+-Ca2+ exchange, which extrudes the Ca2+ released from the SR.23 Integration of the inward current gives an estimate of the total amount of Ca2+ released from the SR.23 Since cell size differs between control and infarcted hearts, we expressed the caffeine current integral relative to cell capacitance. Currents were integrated for 5 s. Longer applications of caffeine (10 s) were associated with a small inward current that did not decay and a tonic component of contraction that did not relax until the caffeine was removed. This tonic component of the contraction may be due to increased myofilament sensitivity to Ca2+ induced by caffeine.
Influence of Na+-Ca2+ Exchange on SR Ca2+ Content
To determine the effect of Na+-Ca2+ exchange on SR Ca2+ content, we held cells at −40 mV and applied five conditioning pulses to +10 mV (300-ms pulses applied every 3 s) to achieve steady-state SR content. We then applied a 400-ms voltage-clamp step to a potential between −70 and +90 mV (“loading pulses”). Three seconds later, another clamp step to +10 mV was applied (“test pulse”). We assumed that the cellular contraction during the test pulse would reflect any changes in SR content that occurred during the loading pulse. Since ICa has a bell-shaped relationship with voltage and Na+-Ca2+ exchange has an exponential current-voltage relationship, pulses to positive potentials should produce a small ICa and a relatively larger INaCa. Thus, after loading pulses to positive potentials, changes in SR content should predominantly reflect the effects of reverse Na+-Ca2+ exchange. Therefore, we expected that steps to positive potentials (loading pulse) might cause more augmentation of the subsequent contraction (test pulse) in the MI than the control myocytes.
The peak rate of cell shortening during the test pulse was used as an index of changes in SR Ca2+ content induced by the prior loading pulse. The peak cellular shortening rate of each contraction was expressed relative to that of the first postloading contraction for the same cell. In these experiments, the pipette solution contained (mmol/L) CsCl 130, dextrose 5.5, K2ATP 5.0, EGTA 0.02, MgCl 0.5, HEPES 10, and NaCl 10, with pH adjusted to 7.1 using KOH. Cells were held at −80 mV between each episode to minimize rundown of Ca2+ current. To determine if augmentation of contractions was due to changes in SR content, the protocol was performed in cells (n=3) that were pretreated with ryanodine (1 μmol/L) and thapsigargin (1 μmol/L). This treatment abolished the augmentation seen with loading pulses to positive potentials.
All data are shown as mean±SEM. Comparisons of cellular contractions, action potentials, or whole-cell currents in cells from control and infarcted hearts were performed using a two-tailed unpaired Student’s t test. A value of P<05 was considered to be significant.
Data from a total of 991 (control, 441; MI, 550) cells isolated from 59 (control, 28; MI, 31) rabbits were included in this report. Two rabbits that survived the initial surgery died before the anticipated study date. At autopsy, both of these rabbits had large pleural effusions and ascites. There was no gross evidence of fluid retention in any of the other rabbits. Hearts from the infarcted rabbits showed transmural scarring of the posterolateral wall and apex (Fig 1⇓). Infarct size was variable, with the largest infarctions involving ≈25% of the left ventricle. During the early portions of this study, infarct size was estimated as minimal, small, moderate, or large on the basis of visual assessment. To better characterize this animal model and to aid in comparison of our findings with those seen in other animal models of left ventricular dysfunction, we measured chamber weights and intracardiac pressures in all rabbits during the later portions of the study. These data are shown in the Table⇓. Since infarct sizes could not be measured histologically in hearts perfused with collagenase, we stratified them into small, moderate, and large infarcts on the basis of scar weight (small, <1.0 g; moderate, 1.0 to 2.0 g; and large, >2.0 g). Small, moderate, and large infarctions corresponded to ≈>10%, 10% to 20%, and >20% of total left ventricular mass, respectively. Measurements of infarct size based on weight probably underestimate the true percentage of the left ventricle that is infarcted, because the viable portions of the heart become quite edematous after collagenase perfusion, whereas the scar does not. During the portion of the study when we routinely measured scar weights, 7 rabbits had small infarcts, 7 rabbits had moderate infarcts, and 20 rabbits had large infarcts (on the basis of the above criteria). Data from hearts with moderate and large infarcts were included in the MI group of the present study. Cell length and width were measured in randomly selected cells from control and infarcted hearts. Cells from the infarct border zone (n=300) were significantly increased in length, with a minimal increase in width (P=NS) compared with those from control cells (n=300, Fig 2B⇓).
To investigate cellular mechanical function under relatively physiological intracellular conditions, we measured changes in cell length during field stimulation (control, n=28; MI, n=39; Fig 3⇓). With this approach, cytoplasmic ion concentrations (eg, Na+) are determined largely by the actions of cellular ion pumps or transporters. At 0.5- and 1.0-Hz stimulation frequencies, contractions were larger in cells from the infarcted hearts (Fig 3B⇓). However, the time to peak shortening was consistently prolonged in the MI cells (Fig 3D⇓). The shortening-interval relationship was shifted upward at slower stimulation rates but showed more of a decline at 2.0 Hz in the myocytes from infarcted hearts compared with control myocytes. These findings were similar whether contractile amplitude or rate of shortening was measured (Fig 3B⇓ and 3C⇓).Cellular relengthening rate also failed to increase at the 2.0-Hz stimulation rate in the MI cells (Fig 3E⇓). The time to 50% cellular relengthening was significantly prolonged in the post-MI myocytes at all stimulation frequencies. However, the degree of prolongation was less pronounced at 2.0 Hz.
We also recorded action potentials in control (n=28) and MI (n=17) myocytes at different stimulation frequencies (Fig 4⇓). Resting membrane potential was slightly, but consistently, more negative (P<.05) in the MI myocytes (Fig 4C⇓). The peaks of the action potentials were not different at any of the stimulation frequencies (Fig 4B⇓). The action potential overshoots (peak minus plateau potential) were similar at 0.5, 1.0, and 2.0 Hz but modestly increased at 3.0 Hz in the MI myocytes (Fig 4D⇓). APD90 was significantly prolonged in cells from infarcted hearts (Fig 4E⇓). APD90 shortened as stimulation frequency was increased in both cell types. However, it decreased more in the MI myocytes, so that APD90 was not different in the control and MI myocytes at the 3.0-Hz stimulation rate.
Transsarcolemmal Ca2+ influx is a major determinant of contractile function in cardiac myocytes. Therefore, we measured ICa in cells from infarcted hearts (n=32) and cells from control hearts (n=34). We found that there was a significant decrease in peak ICa density in the cells from infarcted hearts (Fig 5B⇓). However, there was no change in the current-voltage relationship (Fig 5B⇓). Voltage-dependent and steady-state inactivation kinetics of ICa were similar in cells from infarcted and control hearts (Fig 5C⇓ and 5D⇓).
We next measured INaCa density in myocytes from infarcted (n=13) and control (n=10) hearts. Exchange currents were activated by rapidly reducing extracellular Na+ concentration in voltage-clamped cells (see “Materials and Methods”). These currents generally increased in amplitude with time after patch rupture (Fig 6A⇓). Current amplitude reached a plateau 2 to 15 minutes after rupture of the membrane. Maximal INaCa density (measured after steady state was achieved) was increased by 32% in the infarcted hearts (Fig 6B⇓).
To determine the functional significance of the increased activity of the exchanger in the infarcted hearts, we performed several experiments. First, we tested the hypothesis that Ca2+ entry by Na+-Ca2+ exchange might affect the voltage dependence of contractions in myocytes from the infarcted heart. We found an increased peak cellular shortening rate during square voltage-clamp steps in the MI cells; however, there was no difference in the voltage dependence of peak shortening rate (Fig 7⇓). This finding suggests that any component of SR Ca2+ release triggered by reverse Na+-Ca2+ exchange is comparable in the control and MI myocytes.
Next, we examined SR Ca2+ content and the ability of Na+-Ca2+ exchange to modulate SR Ca2+ content. SR Ca2+ content was estimated by integrating the inward current induced by rapid application of caffeine.23 We found that releasable SR Ca2+ stores (normalized to membrane capacitance) during a stable train of conditioning pulses were slightly increased (11%, P=NS) in myocytes from the infarcted hearts (Fig 8⇓). We then tested the hypothesis that enhanced reverse Na+-Ca2+ exchange might augment SR Ca2+ content to a greater extent in MI than control myocytes. This was determined by applying conditioning pulses to various potentials and then measuring the rate of cellular shortening during a standard voltage-clamp step from −40 to +10 mV applied 3 s later (see “Materials and Methods”). We found that voltage-clamp steps to positive potentials (>+30 mV) produced significantly greater augmentation of the subsequent cellular contraction in the myocytes from the infarcted hearts than in those from control hearts (Fig 9⇓). No augmentation of the contraction after clamp steps to positive potentials was seen in cells pretreated with ryanodine and thapsigargin (Fig 9B⇓). Thus, the augmentation must be due to changes in SR Ca2+ content. These data imply that reverse Na+-Ca2+ exchange is more effective in producing Ca2+ influx in the cells from the infarcted hearts than in control cells. Moreover, this Ca2+ influx may help to load SR stores.
Our goal was to begin defining the role of the sarcolemmal Na+-Ca2+ exchanger in excitation-contraction coupling in surviving myocytes of the infarcted heart. Although infarctions involved as much as 25% of the left ventricle, animals had relatively compensated left ventricular dysfunction at the time of study. Myocytes from the infarct border zone in the rabbit were significantly elongated, with little increase in cell width. These myocytes had contractions of normal or increased amplitude but prolonged duration. Peak Ca2+ current density was significantly decreased, whereas Na+-Ca2+ exchange density was increased. SR Ca2+ content after a standard train of conditioning pulses was slightly increased, and voltage-clamp steps to positive potentials produced greater augmentation of a subsequent contraction in the myocytes from infarcted hearts compared with control hearts. These data provide evidence that in surviving myocytes from chronically infarcted hearts, Ca2+ influx by the Na+-Ca2+ exchanger may support contractility that would otherwise be reduced.
ICa in Myocytes From the Infarcted Heart
We found a significant reduction in peak ICa density in myocytes from infarcted hearts without significant changes in the current-voltage relationship or inactivation kinetics. These data suggest that the number of Ca2+ channels is reduced relative to cell surface area. Reductions in dihydropyridine receptor density or ICa density have been observed in myocardium from patients with heart failure and in several experimental animal models.14,24–27 However, it should be recognized that not all forms of left ventricular hypertrophy or failure show decreased ICa density.9,28,29 Furthermore, there may be complex changes in Ca2+ channel expression during the progression of disease in some models.30 Hence, findings in one experimental model may not be generalizable to all forms of left ventricular dysfunction. ICa is commonly thought to serve as the primary trigger for SR Ca2+ release.31,32 As such, the size of the Ca2+ current generally has a very significant effect on the amplitude and rate of cellular contraction. If Ca2+ entry via L-type Ca2+ channels is reduced in our model, why is cellular contractility not more impaired? A likely explanation is that there is another source of cellular Ca2+ influx.
Na+-Ca2+ Exchanger in Myocytes From the Infarcted Heart
Several investigators have found evidence of increased expression of the Na+-Ca2+ exchanger (mRNA and/or protein) in tissue from failing or hypertrophied hearts.13–16 It has been hypothesized that increased activity of the Na+-Ca2+ exchanger may “compensate” for impairment of SR function by enhancing the ability of the cells to restore diastolic Ca2+ to normal levels.15 This view may be incomplete, since the exchanger can transport Ca2+ into or remove Ca2+ from the cell. Although the Na+-Ca2+ exchanger clearly is a critical mechanism for cellular Ca2+ extrusion, the Ca2+ influx mode may be more important than previously appreciated. For example, recent work suggests that Ca2+ entering a cell via the exchanger may contribute to the trigger for SR Ca2+ release.33–35 It has also been proposed that SR Ca2+ loading may be mediated through Na+-Ca2+ exchange.36
Our finding that contractions were of normal or increased amplitude in myocytes from infarcted hearts despite a decrease in peak ICa density suggests that there might be another source of Ca2+ influx in these cells. Since there is an increase in INaCa density, this might be an alternative mechanism of Ca2+ entry. Cellular Ca2+ entry by the reverse mode of Na+-Ca2+ exchange could enhance contractility by triggering release of Ca2+ from the SR, enhancing SR Ca2+ content, or directly activating the myofilaments. We tested each of these hypotheses. If Ca2+ entry by reverse exchange had a greater role in triggering SR Ca2+ release in cells from infarcted hearts, we would have expected to see a rightward and upward shift in the relationship between cellular contraction and membrane potential. However, we did not observe a change in the voltage dependence of contraction in the MI myocytes (Fig 7⇑). This observation is similar to one previously reported in a model of pressure-overload left ventricular hypertrophy.27 Therefore, it seems unlikely that Ca2+ influx by the Na+-Ca2+ exchanger has a greater role in inducing SR Ca2+ release in MI than in control myocytes. The shape of the shortening-voltage relationships suggests that under the conditions of these experiments, triggering of SR Ca2+ release by reverse Na+-Ca2+ exchange is relatively small in both control and post-MI myocytes. In contrast, we found evidence that SR Ca2+ content tended to be enhanced in the MI cells and that this may be mediated by Na+-Ca2+ exchange. Since the fractional release of Ca2+ tends to increase with SR Ca2+ content, a relatively normal SR release might occur despite a smaller trigger for SR release (ie, decreased Ca2+ current amplitude) in the post-MI myocytes.37 Together, these data suggest that any additional Na+-Ca2+ exchangers that may be present in the remodeled myocytes of the infarcted heart may not be directly apposed to the Ca2+ release channels of the SR. Direct activation of the myofilaments by Ca2+ entering via the exchanger could also contribute to the normal or enhanced contractility in the MI myocytes despite a reduced stimulus for Ca2+-induced Ca2+ release from the SR (decreased ICa). Direct myofilament activation by reversed Na+-Ca2+ exchange could partially explain why the time to peak shortening was significantly prolonged.
During the cardiac action potential, there are likely to be extremely complex interactions between ICa and INaCa. Both of these currents will simultaneously affect membrane potential and intracellular Ca2+ concentration. In turn, these variables will affect the electrochemical forces that drive the ionic currents. The situation is further complicated by the fact that Ca2+ channels exhibit Ca2+-dependent inactivation, whereas the Na+-Ca2+ exchanger has a regulatory site that tends to stimulate the exchanger when Ca2+ is bound.17 Thus, the size and shape of the two currents during an action potential are very difficult to predict.
Although the amplitude or peak rate of cellular contraction was normal or enhanced in the MI myocytes, the contractions were prolonged. The main prolongation seemed to be in the time to peak shortening, whereas the rates of cellular shortening and relengthening were relatively preserved (at least at slower stimulation frequencies). This finding suggests that there might be continued transsarcolemmal Ca2+ influx, prolonged SR Ca2+ release, or a delay in the Ca2+ reuptake (SR) or extrusion (Na+-Ca2+ exchange) processes. We hypothesize that continued Ca2+ entry via the Na+-Ca2+ exchanger during the prolonged plateau of the action potential in the MI cells might explain the prolongation of the mechanical contraction. Additional Ca2+ influx might also occur through L-type Ca2+ channels during the prolonged plateau of the action potential. Since the peak ICa density appears to be reduced, prolongation of the action potential may be an important mechanism for increasing Ca2+ influx by either mechanism. Furthermore, shortening of the action potential at more rapid stimulation frequencies might partially explain the flat slope of the shortening-interval relationship at higher stimulation rates in the MI myocytes.
Overall, our data support the notion that the Na+-Ca2+ exchanger may have a more important role in beat-to-beat regulation of excitation-contraction coupling in surviving myocytes from the infarcted left ventricle than in normal myocytes. We have focused on the potential importance of Ca2+ influx by the exchanger. However, enhanced Ca2+ efflux by Na+-Ca2+ exchange probably assumes greater significance as well. Because of these changes, the Na+-Ca2+ exchanger could represent an important new target for therapeutic interventions in the damaged heart.
Selected Abbreviations and Acronyms
|APD90||=||action potential duration at 90% repolarization|
|I Ca||=||L-type Ca2+ current(s)|
|I NaCa||=||Na+-Ca2+ exchange current(s)|
This study was supported by grants from the Department of Veterans Affairs, the National Heart, Lung, and Blood Institute (HL-42357), and the University of Utah. Dr Litwin was the recipient of a Clinician Scientist Award from the American Heart Association. The authors wish to acknowledge the excellent technical assistance provided by Phyllis Roberge and Gary Webster.
- Received April 23, 1997.
- Accepted September 5, 1997.
- © 1997 American Heart Association, Inc.
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