DAVID JOHNSON. Department of Physiology and Department of Medical Biochemistry,. The Ohio State University, Columbus,. Ohio 43210, USA. ABSTRACT.
Elevated
myocardial
cardiac GEORGE
calcium
and
E. BILLMAN,’
BRIAN
McILROY,
and Department
ANDJ.
of Medical
ABSTRACT The contribution of intracellular calcium to ventricular fibrillation (VF) was investigated using chronically instrumented dogs with healed myocardial infarctions. A 2-minute coronary occlusion was initiated during the last minute of exercise. Fourteen animals developed ventricular fibrillation (susceptible) whereas the remaining 12 did not (resistant) during this exercise plus ischemia test. The test was then repeated for the susceptible animals after pretreatment with the intracellular calcium chelator BAPTA-AM (1.0 mg/kg). BAPTA-AM significantly reduced left ventricular dp/dt max and prevented VF in 8 of 12 susceptible animals. Conversely, myocardial cytosolic calcium levels were increased in resistant animals using the calcium channel agonist Bay K 8644 (30 jig/kg) or phenylephrine (10 jig#{149} kg min’ 3-5 mm before occlusion). Bay K 8644 induced VF in all 5 resistant animals tested whereas phenylephrine induced VF in 8 of 12 resistant animals. BAPTA-AM pretreatment attenuated the hemodynamic effects of Bay K 8644 or phenylephrine and prevented VF in five of five Bay K 8644- and four of seven phenylephrine-treated animals. Finally, the endogenous level of calcium/calmodulin (Ca-CaM)-dependent phosphorylation of 170- and 55-kDa substrate proteins was measured (as an index of intracellular free calcium concentration). In the susceptible dog heart, the endogenous level of Ca-CaM-dependent phosphorylation was estimated to be two- to threefold higher than that observed in resistant dog heart. Treatment of resistant dog tissue with the calcium ionophore A23187 increased the level of Ca-CaM-dependent phosphorylation of these two proteins to the level observed in susceptible dog heart. These data suggest that elevated cytosolic calcium facilitates development of malignant arrhythmias and that elevated cytosolic calcium levels may be present in animals particularly susceptible to ventricular fibrillation. Billman, G. E.; Mcllroy, B.; Johnson, J. D. Elevated myocardial calcium and its role in sudden cardiac death. FASEBJ. 5: 2586-2592; 1991.
-
cardiac arrhythrnias ventricular and fi-adrenergic receptors
fibrillation
cytosolic
A GROWING BODY OF EVIDENCE suggests that disturbances in the autonomic control of the heart play a critical role in the development of ventricular fibrillation (VF)2 In general, activation of the sympathetic nervous system tends to reduce cardiac electrical stability whereas parasympathetic activation may protect against malignant arrhythmias (1). Recent clinical (2, 3) and experimental studies (4-6) demonstrated that individuals with the greatest increases in sympathetic tone and/or reductions in parasympathetic tone after myocardial infarction also have the greatest propensity for sudden cardiac death.
2586
in sudden
death
Department of Physiology Ohio 43210, USA
Key Words: calcium a
its role DAVID JOHNSON Biochemistry,
The Ohio
State University,
Columbus,
The mechanism by which these alterations in autonomic tone provoke disturbances in cardiac electrical stability, particularly at the cellular level, remains largely unknown. Ultimately, transmitter substances released from nerve terminals must bind with postsynaptic receptors on the cardiac cells to alter the concentration of second messengers (calcium, cyclic AMP, cyclic GMP, diacylglyceral, or ITP), protein phosphorylation, and myocardial contractility (7-10). It is probable that these alterations in second-messenger levels elicited by autonomic neural activity contribute significantly to the formation of malignant arrhythmias. In particular, large excesses in intracellular calcium can provoke oscillatory after-depolarization of cardiac membrane, and if sufficient to reach threshold, can provoke spontaneous action potential generation (10). This triggered activity is believed to be a potential mechanism for initiation of ventricular fibrillation (11). In fact, recent studies (32) using isolated rabbit hearts demonstrated that a slow inward calcium current was required for initiation and maintenance of ventricular fibrillation. In intact preparations, Billman (12) demonstrated that both organic (verapamil, nifedipine) and inorganic (magnesium) calcium channel antagonists could prevent VF elicited by the combination of exercise and ischemia whereas the calcium channel agonist Bay K 8644 provoked VF in animals resistant to malignant arrhythmias. These findins suggest that calcium entry and subsequent elevation in cytosolic calcium contribute significantly to the development of VF. Thus, this series of experiments investigates the effects of the intracellular calcium chelator, BAPTA-MA, on susceptibility to ventricular fibrillation. Preliminary results of some aspects of this work have been reported elsewhere (13).
METHODS Surgical
preparation
Forty-two mongrel dogs weighing 11.6-20.7 kg were used in this study. The animals were anesthetized and instrumented to measure left circumflex coronary blood flow, left ventricular pressure, and ventricular electrogram as previously described (4-6, 12). The left anterior descending artery was ligated and an anterior wall myocardial infarction was then produced (4-6, 12).
‘To whom correspondence should be addressed, at: Department of Physiology, Ohio State University, 4196 Graves Hall, 333 W. 10th Ave., Columbus, Ohio 43210, USA. 2Abbreviations: VF, ventricular fibrillation; BAPTA-AM, the acetoxymethyl ester of 1,2 bis-(O-aminophenoxy ethane) N,N,N ,N’tetraacetic acid; LVP, left ventricular pressure; LVSP, left ventricular diastolic pressure; LVDP, left ventricular systolic pressure; CaM, calmodulin; DMSO, dimethyl sulfoxide.
092-663811005-2586101.50.
©
FASEB
All leads to the cardiovascular instrumentation were tunneled under the skin to exit on the back of the animal’s neck. Pentazocine lactate (Talwin, Winthrop-Breon Lab., New York, N.Y., 30 mg., i.m.) was given to minimize postoperative pain. In addition, the long-acting local anesthetic bupivacaine HCI (Marcaine, Winthrop-Breon Lab.) was used to block the intercostal nerves (i.e., pain fibers) in the area of the incision to minimize discomfort to the animal. Each animal was placed on antibiotic therapy (penicillin G x 106 units, i.m., Burns Veterinary Supply, Oakland, Calif.) twice daily for 7 days. The animals were placed in an intensive care setting for the first 24 h and given antiarrhythmic drugs for the next 4 days, as previously described (4-7, 12). Twelve dogs died suddenly during surgery or within the first 72 h. Four additional animals could not be classified because of rupture of the coronary occluder (see below) and were also eliminated from the study. Of the original 42 animals, 26 entered the studies described below. The principles governing the care and treatment of animals as expressed by The American Physiological Society were followed throughout this study. Classification
of animals:
exercise
plus
ischemia
test
Three to four weeks after production of the myocardial infarction the studies began. The animals were walked on a motor-driven treadmill and adapted to the laboratory environment during this period. The susceptibility to ventricular fibrillation was tested as previously described (4-6, 12). Briefly, the animals ran on a motor-driven treadmill while work load increased every 3 mm. During the last minute of exercise, the left circumflex coronary artery was occluded, the treadmill was then stopped, and the occlusion was maintained for an additional minute. The occlusion lasted a total of 2 mm. Large metal plates (11-cm diameter) were placed across the animal’s chest so that electrical defibrillation could be achieved with a minimal delay but only after the animal was unconscious (10-20 s after VF began). This model has been shown to induce ventricular fibrillation reliably and reproducibly (4-6, 12) in susceptible animals. The control (untreated) exercise plus ischemia test was repeated as many as four times in each animal. The time to ventricular fibrillation was averaged for each animal.
reductions in heart rate. Atropine did not induce malignant arrhythmias in these animals. One week later the Bay K 8644 or phenylephrine studies were repeated after pretreatment with BAPTA-AM as described previously. A final control (DMSO) exercise plus ischemia test was repeated 1 wk after the BAPTA-AM studies. Biochemical
Five to seven days after the last exercise plus ischemia test animals were euthanized with an overdose of sodium pentobarbitol followed by saturated KC1. The heart was rapidly removed (within 5-10 mm) and tissue samples were frozen in a - 70#{176}C freezer for future biochemical analysis. Thirty-milligram samples of posterior papillary muscle obtained from susceptible (n = 5) and resistant (n = 5) animals were incubated for 10 mm in 1 ml of a physiological saline solution either in the presence or absence of 10 jiM A23187. After incubation, 100 mM NaPO4 and 10 mM NaF were added to inhibit protein phosphatases; the tissue was homogenized with ground glass homogenizer and the protein concentration was determined by the Bradford method (15). A portion of the homogenized tissue (250 jig total protein) was incubated in 0.2 ml of buffer containing 50 mM NaPO4, 5 mM NaF’, 3 mM MgC12, pH 7.0, with either no calcium (+1 mM EGTA), calcium (2 mM CaC12), or calcium plus 5 jiM calmodulin. In each case the phosphorylation reaction was started by the addition of 1 jiM [32P]ATP (20,000 cpm/nmol) for 10 mm and stopped by addition of 40 jil of stop solution (1.2% DTE, 10% SDS, 40% glycerol). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was then performed on each reaction mixture using 7-15% gradient gels that were stained with Coomassie blue and exposed to Kodak X-AR film for determination of phosphate incorporation into substrate proteins. These autoradiographs were scanned and relative peak areas were determined using an LKB Ultrascan XL. The relative peak area for the 170- and 55-kDa protein in the resistant animals was divided by the corresponding areas obtained from susceptible animals and the increase in phosphorylation of these proteins was calculated. These data were averaged for the five susceptible and five resistant animals. Data
Experimental
protocol
The exercise plus ischemia test described previously was then performed after the following treatments. The susceptible (n = 12) dogs were treated with the intracellular calciumspecific chelator BAPTA-AM [the acetoxymethyl ester of 1,2 bis(O-aminophenoxy ethane)-N,N, -N’ ,N’ tetraacetic acid 1 mg/kg, Molecular Probes, Eugene, Oreg.]. The drug was dissolved in I ml of dimethyl sulfoxide (DMSO) and injected into a cephalic vein. Because the drug slowly incorporates into the cell, the exercise plus ischemia test was performed 45-60 mm after the BAPTA-AM injection. One week later the exercise plus ischemia test was repeated with vehicle alone (DMSO). The resistant animals (n = 12) were given either the calcium channel agonist Bay K 8644 (30 jig/kg dissolved in 1 ml 95% ethanol, n = 5)or phenylephrine HCI(10 jig.kg #{149}min’, Sigma Chemical Co., St. Louis, Mo., n = 12) to increase intracellular calcium levels. The drugs were injected via a catheter placed in a cephalic vein 3-5 mm before the occlusion, i.e., while the animal was running. Atropine sulfate (50 jigS kg, iv., Lyphomed, Rosemont, Ill.) was given to the animal receiving the phenylephrine infusion to prevent reflex
ELEVATED CELL CALCIUM
AND
VENTRICULAR
FIBRILLATION
assays
analysis
All data were recorded on a Gould Model 2800S 8 channel chart recorder and a Teac Model MR-30 cassette tape recorder as previously described (12). The hemodynamic data were averaged over 5-s periods during the last minute of each exercise level, immediately before the coronary occlusion and at the 60-s time point (or immediately before VF) during the occlusion. The data were analyzed using analysis of variance for repeated measures. When the F ratio was found to exceed a critical value (P < 0.05), Scheffe’s test was used to compare the means (16). The effects of the various drug treatments on ventricular fibrillation were determined using a Chi-square test with Yate’s correction for continuity (17). All data are reported as the mean ± SEM. Cardiac arrhythmias were analyzed at a paper speed of 25 mm/s while P-R interval was determined at paper speed of 100 mm/s.
RESULTS The dogs were divided to the exercise plus malignant arrhythmias
in two groups based on the response ischemia test: 14 animals exhibited (ventricular flutter that deteriorated
2587
S.
into ventricular fibrillation) and were classified as susceptible to sudden death whereas 12 animals did not exhibit arrhythmias and were designated resistant. The time to onset for malignant arrhythmias in the susceptible animals was 45.4 ± 4.2 s (range 37.0-81.0 s); four animals developed VF shortly after the treadmill was stopped whereas the remaining 10 had VF while running. Two susceptible animals were not successfully resuscitated and were eliminated from the study. In agreement with previous studies, no animals developed VF in response to exercise alone (i.e., the coronary occlusion was necessary to induce the malignant arrhythmias). The arrhythmias were reproducibly induced during each presentation of control exercise plus ischemia tests in the susceptible animals. Conversely, malignant arrhythmias were never noted during control exercise plus ischemia tests for the resistant animals. Effects of the calcium ventricular fibrillation:
chelator, BAPTA-AM, susceptible animals
4000 ‘C
Cu 3000
>E 0 0-
2000
-o ‘
BAPTA-AM Control
r 0/0
4.8/0
6.4/0
6.4/4
EXERCISE
6.4/8
6.4/12
6.4/16
LEVEL
Speed (kph)/Grade (%) Figure
1. Effect of BAPTA-AM
S*P < 0.01 control
on
left ventricular
on inotropic
vs. BAPTA-AM
pressure,
d(LVP)/dt
response
to exercise.
for a given exercise level. LVP, served as an index of inotropic
state. The intracellular calcium chelator, BAPTA-AM, was given to 12 susceptible animals. This drug significantly (x2 = 9.2, P < 0.005) reduced the incidence of malignant arrhythmias preventing VF in 8 of 12 animals. The onset of VF was delayed in each of the remaining four animals (control 36.1 ± 5.2 s, BAPTA-AM 70.7 ± 17.5 s), with onset delayed to after the occlusion release in one animal. The vehicle, DMSO, failed to prevent VF in any susceptible animal. The hemodynamic effects of BAP1’A-AM before and during the coronary occlusion are shown in Table 1. BAPTA-AM significantly reduced the preocclusion values of d(LVP)/dt as well as the change in d(LVP)/dt elicited by the coronary occlusion. In a similar manner, BAPTA-AM significantly reduced the d(LVP)/dt max response to exercise (Fig. 1) and significantly increased preexercise P-R interval 22.0 ± 6.7% (control 84.5 ± 6.9 ms BAPTA-AM 99.8 ± 4.8 ms). Previous studies demonstrated that these variables reflect myocardial free calcium levels (18, 19). The coronary occlusion elicited significant increases in heart rate and left ventricular diastolic pressure (LVDP) where left ventricular systolic pressure (LVSP) and d(LVP)/dt maximum decreased significantly. The hemodynamic reTABLE
1. Hetnodynamic ejects of 1/ic intraerllular
calcium
chelator,
sponse to coronary BAPTA-AM.
207.6 194.6
BAPTA-AM,
on susceptible
Control BAPTA-AM
153.2 146.3
252.0 249.8
± 8.3
Control BAPTA-AM
“P < 0.01 or last 5 s
2588
diastolic
pressure,
6.5 ± 1.7 8.2 ± 2.4
Control BAPTA-AM
4858 3635 occlusion
vs. preocclusion
values.
27.9 24.3 maximum,
K 8644
by
on
animals
Change
2708 2404
± 364.0k control
± 10.3 ± 13.3
44.6 54.1
14.8 ± 15.7
±
± 1l.0 ± 7.8
-46.7 -45.5
± 10.8 ± 6.1
mmHg ± 5.2 ± 3.8
21.4 ± 6.1 18.3 ± 6.2
mmHg/s
± 403.0
5P < 0.01,
affected
mmHg
106.6 103.7
d(LVP)/dt
VF
systolic pressure,
± 4.7 ± 4.9 Left ventricular
Bay
not
rate (beats/mm)
± 9.0
Left ventricular
was
The calcium channel agonist Bay K 8644 was used to increase calcium entry and thereby elevate cytosolic calcium in five resistant animals. Bay K 8644 elicited VF in all five resistant animals (x2 = 6.4 P < 0.025 time to onset 30.7 ± 3.4 s) and provoked several significant hemodynamic changes as shown in Table 2. In contrast, pretreatment with BAP’TA-AM prevented the VF induced by Bay K 8644 (x2 = 6.4, P < 0.025) and significantly attenuated the hemodynamic effects of this drug (Table 2). For example, this intracellular calcium chelator prevented Bay K 8644-induced increases in heart rate and d(LVP)/dt max and attenuated the increase in LVSP. As in susceptible animals, BAPTA-AM significantly reduced predrug (i.e., before Bay K 8644 injection) values of d(LVP)/dt max.
Occlusion Heart
occlusion
Effect of the calcium channel agonist susceptibility to ventricular fibrillation
Preocclusion
Control BAPTA-AM
artery
vs. BAPTA-AM;
± 524.4
± 420.9a LVP,
left
ventricular
-2346 ± 319.7 1407 ± l90.4 pressure, occlusion last seconds before
before treadmill stopped.
Vol. 5
August 1991
The FASEB Journal
BILLMAN
ET AL.
TABLE
2. Hemodynamic
response to Bay K 8644 before and after BAPTA-AM”
Pre-Bay K
Ba y K
Heart Control Bay K 8644 BAPTA-AM
223.6 201.6 191.4
plus Bay K 8644
rate (beats/mm)
± 16.0 ± 8.4 ± 6.4
133.0 135.3 137.0
plus Bay K 8644
systolic pressure,
d(LVP)/dt Control Bay K 8644 BAPTA-AM “Bay and
drug
3658 3761 2692
plus Bay K 8644
K 8644 was injected 3-5 mm for other groups) vs. occlusion.
Effect of the a-adrenergic susceptibility to ventricular
diastolic
3. Hemodynamic
pressure,
maximum,
plus PE
143.8 134.4 143.8
plus PE
+
-
19.4
± 996
13.6
± 4.3
19.2
± 6.9
4924 3148
±
Control PE PE
phenylephrine
was infused 6p
< 0.01
beginning preocclusion
vs. phenylephrine
ELEVATED CELL CALCIUM
AND
3-5 (i.e.,
5.8
±
3043 2318.6 2436
225.5’ ± 754.8’
Bay
± 362.4 ± 3562k ± 561.5
< 0.01 preocclusion (i.e., predrug for control ‘1P < 0.01 pre-Bay K vs. Bay K 8644.
bp
K 8644.
PE
193.0
±
12.4
±
7.1
254.1
±
776
±
50’.
216.0
±
9.O
466
-
235.3 190.9 systolic
pressure,
mmHg 115.7
±
230.0
±
12.3’
188.5
±
14.36
158.7
±
756/
126.8
±
8.9
-
22.0
±
396
6.3’ ± 3.0’
31.0 26.0
±
786
-
diastolic
pressure,
maximum,
mmHg 21.1 15.1
±
± ±
±
369.1 ± 451.6
6995
4202
±
3760
3143
±
3546b
553.4’
5162
±
5037k
384.9w
3236
±
-
438.O
before the occlusion, predrug for control group
± 6.6k
mmHg/s
4368 4846 mm
Occlusion
rate (beats/mm)
11.1 ± 2.5 6.2 ± 1.8 7.6 ± 1.6
plus PE
“Phenylephrine
± 8.9k ± ± 6.0
given during the exercise plus ischemia test did not induce VF in these animals. Phenylephrine elicited significant increases in LVSP, d(LVP)/dt max, and LVDP (Table 3). The intracellular calcium chelator BAPTA-AM was then given to seven resistant animals that had VF in response to phenylephrine, and the exercise plus ischemia test was repeated. BAPTA-AM prevented phenylephrine-induced VF in four animals and delayed the onset of VF in the remaining
4.2 ± 6.4 ± 4.3
d(LVP)/dt
phenylephrine.
116.1 130.1 130.5
±
±
Left ventricular
plus
±
ventricular pressure.
198 ± 7.8 231.6 ± 6.9 214.6 ± 8.6
Control PE
BAPTA-AM
lO.5 6.5’
receptor agonist phenylephrine”
Left ventricular
Control PE BAPTA-AM
34.0’
mmHg/min
BAPTA-AM
on
Heart
BAPTA-AM
169b
±
-
left
23.2
15.9 18.0
Pre-PE
Control PE BAPTA-AM
±
±
mmHg
384.7 ± 210.0 ± 657.9’
before coronary occlusion. LVP, ‘P < 0.01 Bay K 8644 vs.
effects of the a-adrenergic
±
±
agonist phenylephrine fibrillation
216.4 249.2 196.2
-
The a-adrenergic receptor agonist phenylephrine was used to increase intracellular calcium levels in resistant animals. Phenylephrine infusion induced ventricular arrhythmias in 10 of 12 resistant animals; 8 dogs had VF (x2 = 9.2, P < 0.005, time to VF onset 27.4 ± 6.6 s). Atropine was used to prevent reflex changes in heart rate. Atropine alone
TABLE
±
184.6 153.6
8.2 ± 1.6 8.5 ± 1.0 12.8 ± 3.6
plus Bay K 8644
±
mmHg
± 8.6 ± 3.3 ± 10.9
Left ventricular Control Bay K 8644 BAPTA-AM
19.3 0.3
-
183.6 189.0
Left ventricular Control Bay K 8644 BAPTA-AM
Occlusion
continuing and drug
throughout the for other groups).
occlusion. ‘P
LVP, < 0.01
left ventricular pre-PE vs. PE.
pressure; dp
