INTRODUCTION

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HIGH-VOLTAGE DEFIBRILLATION shock remains the only reliable therapy available to salvage the life of a patient experiencing ventricular fibrillation. Unfortunately, the efficacy of defibrillation shocks is inconsistent both between individuals and even within the same individual between bouts of fibrillation (11). Although some mechanisms contributing to this variation are understood, such as poor electrode contact with high electrode impedance and improper electrode placement, other factors leading to variation and prolonged delay to return of sinus rhythm and protracted poor or slow recovery of hemodynamic function postshock are not readily explained. Return to sinus rhythm has been reported to occur <50% in out-of-hospital cardiac defibrillation (32). Clinically there are several instances of documented poor hemodynamics and depressed contractile function after defibrillation shocks. Various mechanisms have been suggested to contribute to this depression, including electrolyte shifts, ischemia, electro poration of the sarcolemma, postischemic myocardial stunning, as well as contributions from a variety of dysfunctions already present before the fibrillation bout.

In experimental models, electroporation has clearly been shown to occur in the sarcolemma (11); however, recovery of membrane integrity is rapid (seconds) and cannot readily explain protracted depression lasting 10 min (15). Also, protracted alterations are seen in action potential characteristics (16) that are not modified by agents that affect sarcolemmal Ca²⁺ handling, such as propranolol and verapamil (15). In the mammalian heart, 65-80% of the beat-to-beat Ca²⁺ transient occurs via the sarcoplasmic reticulum (SR) (2). However, no studies have specifically examined the effects of defibrillation shocks on Ca²⁺ handling by the SR. The objective of the present study was to test the hypothesis that defibrillation shocks interfere with Ca²⁺ transport function of cardiac SR.

	METHODS

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Animals. Male Wistar rats (Charles River, Mississauga, Canada) weighing 250-300 g were housed in the animal quarters on a 12:12-h light-dark cycle and an ad libitum food and water regime. All animals were cared for in accordance with the guidelines of the Canadian Council on Animal Care, and the animal use protocol was approved by the University of Western Ontario Animal Care Committee. On the day of death, the rats were anesthetized with pentobarbital sodium (Somnitol, 60 mg/kg; Canada Packers, Hamilton, Canada) and 1-cm incisions were made on the left and right chest for subcutaneous insertion of two 0.8-cm diameter gold-plated circular electrodes (Grass Instruments, Quincy, MA) just beneath the skin on the chest wall.

Shock delivery. Two rats were connected in series to a trapezoidal defibrillator (model 2376 or 2394, Medtronic, Mississauga, Canada). Pairs were randomized to receive 10 shocks separated by 10 s at stored voltages of 0, 50, 100, 150, or 250 stored volts. Shocks were trapezoidal monophasic pulses of 5-ms duration from the 50-µF capacitor bank. Stored voltages and paired animals were selected to provide peak current per gram of heart for the rat heart, which approximated those of subthreshold and successful defibrillation shocks in patients and large animal studies (10, 11) and previous studies with shocks delivered to papillary muscle (17).

Isolation of SR-enriched membrane vesicles. Immediately after the last shock, or ~1 min after electrode placement for control rats receiving no shocks, the hearts were rapidly removed, cleaned of major vessels and atria, and then washed in ice-cold 10 mM NaHCO₃ (pH 6.8) buffer to remove blood, and the ventricular tissue was used for the isolation of SR membranes as described previously (28). Briefly, the tissue was minced and homogenized in 6 vols (based on ventricular weight) of ice-cold buffer (10 mM NaHCO₃, pH 6.8) using a Brinkman Instruments Polytron homogenizer (3 bursts of 15-s duration with 30-s intervals, speed setting 8). The homogenate was centrifuged at 1,000 g for 10 min at 4°C. The supernatant was decanted and kept in an ice slurry. The pellet was resuspended in four times the ventricular weight of ice-cold buffer and then centrifuged at 1,000 g for 10 min at 4°C. The supernatant was decanted and combined with the first supernatant, and the pellet was discarded. The combined supernatant was centrifuged at 8,000 g for 20 min at 4°C. The supernatant was decanted, and the pellet was discarded. Solid KCl (44 mg/ml) was added to the supernatant (final concn 0.6 M), swirled to dissolve, and left on ice for 25 min and then centrifuged at 40,000 g for 60 min at 4°C. The supernatant was discarded, and the pellet containing SR-enriched membrane vesicles was resuspended in a 10 mM tris(hydroxymethyl)aminomethane (Tris)-maleate-100 mM KCl buffer (pH 6.8) to give a protein concentration of ~3 mg/ml. Protein was determined using the method of Lowry et al. (18) using bovine serum albumin as a standard.

Preparation of heart homogenates. In addition to SR, homogenates of ventricular myocardium from control and shocked rats were also used in some of the experiments. For this, the homogenates were prepared by homogenizing the ventricles in 10 vols (based on ventricular weight) of 10 mM Tris-maleate-100 mM KCl buffer (pH 6.8) using a Polytron PT-10 homogenizer (3 bursts of 15-s duration with 30-s interval, speed setting 8), returning the beaker to the ice between bursts. In the second experiment, in which the blood pressure was recorded, hearts were removed 15 min after the last shock, or 20 min after surgery for nonshocked control rats, for preparation of homogenates.

Determination of Ca²⁺ uptake and Ca²⁺-ATPase activities. ATP-dependent, oxalate-facilitated Ca²⁺ uptake by SR and cardiac homogenates was determined using a Millipore filtration technique as detailed elsewhere (20). The standard incubation medium (total volume 1 ml) contained (in mM) 50 Tris-maleate (pH 6.8), 5 MgCl₂, 2.5 ATP, 120 KCl, 5 potassium oxalate, 5 NaN₃, and 0.1 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), membrane or homogenate fraction (30 µg protein in SR, 80 µg protein in homogenate), and varying concentrations of ⁴⁵CaCl₂ (8,000-10,000 counts · min¹ · nmol¹). All assays were performed at 37°C. The Ca²⁺ uptake reaction was initiated by the addition of membrane fraction after preincubation of the rest of the assay components for 3 min. The free Ca²⁺ concentration in the assay medium was determined according to the computer program of Fabiato (4). The data on Ca²⁺ concentration dependence on Ca²⁺ uptake were analyzed using nonlinear regression curve fitting using the SigmaPlot program (Jandel Scientific). The data were fit to the equation where v is the measured mean Ca²⁺ uptake activity at a given Ca²⁺ concentration ([Ca²⁺]); V_max is the maximum activity achieved; K_0.5 is the [Ca²⁺] giving one-half V_max, and n_H is the Hill coefficient.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein composition of cardiac SR isolated from control and shocked rats was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as previously described (20).

Data analysis. The results are presented as means ± SE. Statistical significance was evaluated by Student's t-test. Regression lines were determined with nonlinear regression curve fitting using the SigmaPlot program. Blood pressure, contractility, and half-relaxation time were analyzed with analysis of variance (ANOVA) (treatment × subject design) and paired Student's t-test. A probability of <5% (P < 0.05) was taken as the level of significance.

	RESULTS

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Figure 1 shows the effect of varying shock intensities on Ca²⁺ uptake by SR measured at a saturating free Ca²⁺ (8.2 µM) in the assay. Increasing shock voltage depressed the Ca²⁺ uptake activity of SR membranes in a dose-dependent manner. A plateau appeared to occur between 100 and 250 V, and the largest change occurred between 50 and 100 V. Even at the lowest voltage of 50 V, the decrease (~10%) was statistically significant.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Shock voltage-dependent reduction in Ca2+ uptake by cardiac sarcoplasmic reticulum (SR) vesicles. Initial free Ca2+ concentration in the assay medium was 8.2 µM. Each value is significantly below that of control (0 V). Values at 100, 150, and 250 V were not statistically different from each other. Symbols are means; bars are SE. Number of animals is given in parentheses.

Figure 2 shows the time course of Ca²⁺ uptake by SR derived from control rats and rats receiving 150-V shocks. The rate of Ca²⁺ uptake, measured in the presence of 8.2 µM free Ca²⁺ in the assay medium, was significantly lower (~33%) in SR from the shocked rats compared with that from the control rats (Fig. 2A). The diminished rates of Ca²⁺ uptake by SR from shocked rats persisted when the Ca²⁺ uptake assay medium was supplemented with ruthenium red (50 µM), an SR Ca²⁺-release channel blocker (Refs. 3, 19; Fig. 2B). Therefore, the observed depression in Ca²⁺ uptake activity of SR from the shocked rats does not appear to be caused by an increase in the rate of Ca²⁺ release from the SR. Ruthenium red augmented the rates of Ca²⁺ uptake by SR from both control and shocked rats to a similar extent (~40%). Thus the drug was equally effective in blocking Ca²⁺ release from SR derived from control and shocked rats.

View larger version (15K): [in this window] [in a new window]   View larger version (23K): [in this window] [in a new window]   Fig. 2.   A: time course of Ca2+ uptake by SR from control and shocked (150 V) rats. Rate of Ca2+ uptake is lower in shocked compared with control SR. Values of shocked rats were significantly (P < 0.01) lower than corresponding values of control rats. B: comparison of rates of Ca2+ uptake by cardiac SR from control and shocked (150 V) rats in absence and presence of Ca2+-release channel blocker ruthenium red (50 µM). Initial free Ca2+ concentration in assay medium was 8.2 µM; n = 8 animals for each group.

Figure 3 shows the effects of varying Ca²⁺ concentrations on the Ca²⁺ uptake activities of SR from control rats and rats receiving 150-V shocks. When the free Ca²⁺ concentration in the assay medium was varied from 0.237 to 8.241 µM, SR from shocked rats showed significantly lower rates (56 to 61%; P < 0.05) of Ca²⁺ uptake than the membranes from control rats at all Ca²⁺ concentrations tested. The kinetic parameters of Ca²⁺ uptake derived from these data indicated that the V_max of Ca²⁺ uptake by SR, but not the K_0.5 for Ca²⁺, was altered in the shocked rats [V_max (nmol Ca²⁺ · mg¹ · min¹): control, 66.3 ± 1.3; shocked, 40.1 ± 1.4; K_0.5 for Ca²⁺ (µM): control, 1.14 ± 0.05; shocked, 1.04 ± 0.07; n_H: control, 2.0 ± 0.1; shocked, 2.0 ± 0.2].

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of varying Ca2+ concentration on Ca2+ uptake by cardiac SR from control and shocked (150 V) rats. Values are significantly lower in SR from shocked rats at all concentrations (P < 0.01). Curves fit by nonlinear regression yielded r2 = 0.998 for control and r2 = 0.991 for shocked (150 V) rats; n = 8 animals for each group.

To exclude the possibility that differences in the relative purity of SR membranes isolated from control and shocked rats contributed to the observed difference in the Ca²⁺ uptake activities of these membranes, additional experiments were performed using unfractionated cardiac muscle homogenates from control and shocked (150 V) rats. In these experiments, Ca²⁺ uptake activities were measured at a saturating free Ca²⁺ concentration (8.2 µM) using cardiac tissue homogenates as well as SR isolated from the same hearts (control and shocked). The results showed significantly reduced Ca²⁺ uptake activity in both homogenates and SR from shocked compared with control rats (Fig. 4). The magnitude of shock-induced depression of Ca²⁺ uptake activity was similar in SR (31%) and homogenate (36%) fractions from shocked compared with control rats (Fig. 4). The data from these experiments also showed that the relative enrichment in the Ca²⁺ uptake activity of SR compared with homogenate was similar (~9-fold) in the case of control and shocked rats. Thus it is unlikely that differences in the relative purity of SR preparations from control and shocked rats contributed to the observed differences in Ca²⁺ uptake activity. In further support of this, the polypeptide composition of SR (determined by SDS-polyacrylamide gel electrophoresis) derived from the hearts of control and shocked (150 V) rats was found to be essentially similar (Fig. 5). Scanning and quantification of the electropherograms did not reveal any significant difference in the amount of protein in individual peptide bands including the ~105-kDa band representing Ca²⁺-ATPase. The 97.4-kDa peptide band, which presumably represents phosphorylase, was, however, diminished in the SR from shocked rats.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Comparison of Ca2+ uptake by cardiac homogenates and SR from control and shocked (150 V) rats. Depression of Ca2+ uptake by homogenate was 36% and SR was 31%. Initial free Ca2+ concentration in assay medium was 8.2 µM; n = 6 animals for each group.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Comparison of polypeptide profiles of cardiac SR isolated from control and shocked (150 V) rats. Polypeptide composition and relative intensity of individual peptide bands are similar in control and shocked SR except for diminished content of phosphorylase (97.4-kDa band) in shocked SR. Protein profiles similar to those shown were obtained in 3 additional experiments using separate SR preparations. Numbers on left indicate molecular mass.

In additional experiments, we examined whether the observed shock-induced depression in the Ca²⁺ uptake activity of SR was also accompanied by concomitant impairment in the energy transduction function of the Ca²⁺-pumping ATPase. In these experiments, ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATP hydrolysis were determined using the same SR preparations isolated from control and shocked (150 V) rats. The results showed that the Ca²⁺-uptake activity but not the Ca²⁺-stimulated ATP hydrolysis was depressed in SR from shocked rats (Fig. 6). The shock-induced depression in Ca²⁺ uptake appeared to parallel cardiac function abnormalities as demonstrated by the transient reduction in blood pressure monitored during shock delivery and postshock. Systolic, mean, and diastolic pressures were all depressed significantly at 30 and 60 s postshock. There was a rebound at 5-10 min postshock (P = not significant), with eight of nine rats having pressures above baseline values at 5 min. All values had returned to baseline preshock values ~15 min postshock (Table 1). Uptake of Ca²⁺ by homogenates derived from the ventricular myocardium of rats killed 15 min after shock delivery averaged 13.3 ± 2.6 nmol · µg protein¹ · min¹ (n = 6), which did not differ from uptake values of similarly operated, nonshocked control rats (15.6 ± 3.1 nmol · µg protein¹ · min¹, n = 5).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Comparison of ATP-dependent Ca2+ uptake (left) and Ca2+-stimulated ATP hydrolysis (right) by cardiac SR from control and shocked (150 V) hearts. Initial free Ca2+ concentration in assay medium was 8.2 µM. Decrease in Ca2+ uptake activity of SR from shocked (150 V) compared with control rats is significant, whereas there was no difference in Ca2+-stimulated ATP hydrolysis.

To determine the effects of shocks on contractile function in the absence of hormonal and/or autonomic influence, shocks were also delivered to hearts supported on the Langendorff apparatus, with contractility/relaxation measured with ultrasound crystals (Fig. 7). There was a significant reduction in shortening of the distance between the two chambers (short axis, P < 0.01) and a similar trend in the shortening in the long axis, which did not reach significance (P < 0.27). The half-relaxation time increased in all six hearts, on average approximately doubled at 1 min postshock (211.5 ± 58.4 ms) from baseline (112 ± 26.3 ms, P < 0.05). However, there was considerable variability between hearts in the subsequent responses at 5, 10, and 15 min postshock, with all six hearts returning towards baseline by 15 min (Fig. 7C).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effects of 150-V shocks on contractility and relaxation of Langendorff isolated hearts. A: average percentage long-axis shortening after shocks. There was a trend that approached but was not statistically significant (F = 1.424, P < 0.27). B: average percentage short-axis shortening after shocks (F = 4.477, P < 0.01). C: sets of individual half-relaxation times after shocks (P < 0.05 for control and 1st minute only). 0, Preshock recording; 1, 5, 10, and 15, times from last shock delivery.

	DISCUSSION

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The present results demonstrate that the ATP-energized Ca²⁺ uptake activity of cardiac SR isolated from rats receiving defibrillation shocks is significantly depressed (35-40%) compared with controls. The shock-induced depression in Ca²⁺ uptake activity of SR was voltage dependent but was not accompanied by alterations in the Ca²⁺-ATPase content of the SR, Ca²⁺-stimulated ATP hydrolysis, or the apparent affinity of the Ca²⁺-ATPase for Ca²⁺. Thus the shock-induced depression in Ca²⁺ uptake activity of the SR is apparently caused by impaired efficiency of coupling ATP hydrolysis to Ca²⁺ transport. The mechanistic basis of this shock-induced uncoupling of ATP hydrolysis and Ca²⁺ transport is unclear at present. Altered Ca²⁺ transport activities without parallel alterations in ATPase activities have been encountered in a number of instances, indicating that the intrinsic ATP-hydrolyzing activity of the transport system is not the sole determinant of the ion transport function. For example, 1) binding of anti-ATPase antibodies to SR membranes has been found to cause marked inhibition of Ca²⁺ transport without altering ATPase activity and passive permeability to Ca²⁺ of the SR membranes (29). 2) Diminished Ca²⁺ transport rates and enhanced ATPase activities have been observed in reconstituted "Ca²⁺ pump" vesicles of SR compared with original membranes. The decline in Ca²⁺ transport rates could not be accounted for by altered Ca²⁺ permeability of the reconstituted vesicles (23, 24). 3) Tryptic cleavage at a specific region of the ATPase (26) or a single amino acid substitution (Tyr⁷⁶³  Gly) in the primary structure of the ATPase (1) was found to abolish Ca²⁺ transport function while ATP hydrolysis still remained functional. 4) Aging was found to be accompanied by a decline in the Ca²⁺ uptake but not the Ca²⁺-stimulated ATPase activity of SR from rat cardiac (20, 21) and slow-twitch skeletal muscles (22).

In the present study, effective blockade of the ryanodine receptor/Ca²⁺-release channel by ruthenium red (3, 19) did not overcome the shock-induced depression in Ca²⁺ uptake activity of SR. Therefore, it is unlikely that a shock-induced enhancement in Ca²⁺ release through the Ca²⁺-release channel contributes to the observed depression in the Ca²⁺ uptake activity of SR. Injury to the cardiac sarcolemmal membrane (electroporation) after defibrillation shocks has been reported (13). Whether defibrillation shocks induce any electroporation of the SR is not known. Myocardial functional deficits that may occur as a result of electroporation of the sarcolemma are short lived, and recovery rapidly occurs within seconds (12, 13). The defibrillation-induced impairment in SR function reported here, apparently, persists for minutes.

It is possible that shock-induced depression in the Ca²⁺-sequestering activity of SR may contribute to the depression in cardiac function observed in some patients after defibrillation shocks (10). The uptake and release of Ca²⁺ by the SR is central to the maintenance of a normal excitation-contraction-relaxation cycle of the heart (2, 5, 14). The ATP-energized Ca²⁺ uptake activity of isolated SR vesicles is widely accepted as the manifestation of the Ca²⁺-sequestering (Ca²⁺ pump) function of these membranes in vivo (5, 9). Thus the depressed Ca²⁺ sequestration by SR membranes would be expected to reflect a similar depression in vivo. Such diminution in SR function in vivo would lead to prolongation of cardiac muscle relaxation and thus impaired mechanical restitution. Furthermore, with depressed Ca²⁺ uptake into the SR compartment, there would be diminished release of Ca²⁺ for subsequent contractions and this would compromise cardiac contractile force.

When 150-V shocks were delivered to rats that had indwelling femoral arterial cannulas, all blood pressure measures were reduced immediately postshock (P < 0.01). These transient blood pressure responses remained below baseline for all animals beyond 60 s, had rebounded by 5-10 min, and subsequently returned to baseline by 15 min. This rebound is consistent with the previously demonstrated autonomic activation postshock (30), which would also alter sarcolemmal Ca²⁺ transients postshock (2, 10, 14, 30). The return to baseline corresponded to a time when the Ca²⁺ uptake activity of heart homogenates also did not differ from those of similarly operated control nonshocked rats. This time course of 10-15 min for recovery is similar to our previous finding of depressant electrophysiological effects after similar intensity shocks delivered to isolated papillary muscles (16, 17).

When shocks were delivered directly to the hearts on the Langendorff apparatus, they resulted in an immediate prolongation of relaxation. There was a disparity between the magnitude and duration of effects on contractility and relaxation and, indeed, between contractility measured in the two different axes. Some of these differences could be due to differences in effects of the shocks on the SR and sarcolemma, as well as the current density profiles through the different regions of the myocardium. There were also differences between the time course of effects seen on the Langendorff heart and the whole animal or isolated papillary muscle. One possible factor contributing to these differences may be the relative distribution of current delivered directly to the heart in the Langendorff hearts versus that which was shunted either through the tissue bath containing the papillary muscle or through the rest of the body in the whole animal transthoracic shock study. In the Langendorff heart, it was not possible to match resistance, peak current, peak voltage, and total energy delivery to that of the whole animal, and these differences could explain some of the differences in results. Determining the relative contributions of these factors requires consideration of current distribution, heart geometry, and electrode configurations (30), which is beyond the scope of the present investigation. On the other hand, it is noteworthy that the postshock restitution response of the Langendorff hearts demonstrated considerably more variability than the contractility responses as a group and even in the same heart. Variability in postshock cardiac function is one characteristic also clearly evident in patients after defibrillation shock (10, 11).

It is possible that this depression in Ca²⁺ uptake contributes to the postshock myocardial dysfunction observed in some patients after a defibrillation shock (10). Why this might differ from patient to patient is unclear. Age-associated diminution in general cardiac performance is well known, and there is an age-associated decline in SR Ca²⁺ pump activity (6, 20), which might contribute to poor cardiac performance postshock and inability to pace the heart (16). However, there does not appear to be a good correlation between patient characteristics and defibrillation success or postshock recovery (10, 11).

Additional factors that may contribute to poor cardiac performance postshock include the magnitude and characteristics of the delivered defibrillation energy. A relationship between energy delivery and myocardial damage has been demonstrated previously (7, 25, 30), which is consistent with the shock intensity-related depression of SR Ca²⁺ pump function observed in the present study. Another factor that may contribute to depressed function is alteration in the phosphorylation status of Ca²⁺ cycling proteins at the level of the SR (5, 8, 14, 33) and/or myofilaments (31). At present, we have not investigated this possibility. Also, the observed decrease in SR-associated phosphorylase suggests that there may be other subcellular alterations not yet determined that may contribute to postshock cardiac dysfunction. These remain to be determined.

In conclusion, we have demonstrated that trapezoidal direct current shocks, with parameters similar to defibrillation shocks, elicit protracted and dose-dependent depression of Ca²⁺-sequestering activity of cardiac SR. The impairment of SR Ca²⁺ pump function was caused by an uncoupling of ATP hydrolysis from Ca²⁺ transport. This shock-induced impairment of Ca²⁺ pump function is suggested to contribute to postshock myocardial dysfunction, consistent with the shock-induced reduction in contractility and relaxation.