131
Calcium Depletion in Rabbit Myocardium
Calcium Paradox Protection by Hypothermia and Cation Substitution
Terrell L. Rich and Glenn A. Langer
From the Departments of Medicine and Physiology and the American Heart Association, Greater Los Angeles Affiliate
Cardiovascular Research Laboratory, University of California, Los Angeles, Center for the Health Sciences, Los Angeles, California
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SUMMARY. The purpose of this study was to define further the basis of control of myocardial
membrane permeability by further examination of the "calcium paradox." To this end, the protective
effect of hypothermia and addition of micromolar amounts of divalent cations during the Ca-free
perfusion period were studied. Damage during Ca ++ repletion to the isolated arterially perfused,
interventricular rabbit septum was assessed by contracture development, loss of developed tension,
and loss of 42K and creatine kinase. Progressive hypothermia prolongs the time of Ca-free perfusion
needed to cause similar 42K, creatine kinase and developed tension losses upon Ca ++ repletion.
Complete protection against the Ca-paradox after 30-60 minutes Ca-free perfusion is seen at 18°C.
The inclusion of 50 JIIM Ca ++ during 30 minutes "Ca-free" perfusion also provides complete
protection during Ca ++ repletion, i.e., there was full mechanical recovery with no 42K or creatine
kinase loss. Other divalent cations perfused in 50 JUM concentrations during the Ca-free period
exhibited variable ability to protect when Ca ++ was reperfused. The order of effectiveness (Ca ++ >
Cd + + > Mn + + > Co + + > Mg ++ ) was related to the crystal ionic radius, with those cations whose radii
are closest to that of Ca ++ (0.99 A) exerting the greatest protective effect. The cation sequence for
effectiveness in Ca-paradox protection is the same sequence for potency of excitation-contraction
uncoupling. The mechanism of hypothermic protection is likely a phase transition in the membrane
lipids (from a more liquid to a less liquid state) which stabilizes membrane structure and preserves
Ca ++ permeability characteristics during the Ca-free period. The mechanism of protection via cation
addition is perhaps a cation's ability to substitute for Ca ++ (dependent on unhydrated crystal ionic
radius) at critical sarcolemmal binding sites to preserve control of Ca ++ permability during the Cafree period. (Ore Res 51:131-141, 1982)
IF isolated hearts are perfused with calcium-free solutions, contractile activity quickly declines (Crevey
et al., 1978) when only minor changes in electrical
activity have occurred (Locke and Rosenheim, 1907).
Upon calcium (Ca) repletion, there is loss of tissue
high-energy phosphates (Boink et al., 1976; Bulkley
et al., 1978), development of contracture (Hearse et
al., 1978; Ruigrok et al., 1975), influx of Ca (Holland
and Olson, 1975; Dhalla et al., 1976; Alto and Dhalla,
1979), and massive tissue disruption evidenced by
ultrastructural damage (Zimmerman et al., 1967;
Muir, 1968; Yates and Dhalla, 1975; Ashraf, 1979) and
release of intracellular enzymes (DeLeiris and Feuvray, 1973; Hearse et al., 1976). This phenomenon is
called the Ca-paradox (Zimmerman and Hiilsmann,
1966; Zimmerman et al., 1967). Likewise, oxygen paradox is the term used to describe similar tissue damage which results from reoxygenation of anoxic tissue.
A number of factors attenuate the extent of damage
occurring during the Ca-paradox including lowered
perfusate pH (Bielecki, 1969), hypothermia (Boink et
al., 1980; Holland and Olson, 1975; Diggerness et al.,
1980), and lowered perfusate Na + during Ca-free
exposure. The mechanism(s) of protection, however,
remains unclear.
It has been shown that small concentrations of
cations whose unhydrated, crystal radii are close to
that of Ca ++ are effective in uncoupling excitation
from contraction (Bers and Langer, 1979). The more
closely the crystal radius of a cation matches that of
Ca ++ , the more effective it is in the uncoupling process. It has also been found that cations have the effect
of increasing the phase transition temperature of
membrane lipids, i.e., addition of cations changes the
membrane lipids from a more fluid to a less fluid
state (Trauble and Eibl, 1974; Jacobson and Papahadjopoulos, 1975; Gordon el al., 1978). It is our intent in
this study to test several divalent cations as substitutes
for Ca ++ during Ca-free perfusions to determine
whether they serve as protective agents against the
Ca-paradox and to compare this possible protective
effect to that seen during hypothermia. We have
found that inclusion of as little as 50 ;U.M concentrations
of divalent cations during Ca-free perfusions does
have a protective effect against the Ca-paradox, the
order of efficacy being Ca ++ > Cd + + > Mn + + > Co + +
> Mg t + . This cation sequence was very similar to the
cation sequence seen for potency of excitation-contraction uncoupling (Bers and Langer, 1979).
This paper will analyze and correlate functional
and flux responses to Ca depletion as affected by
temperature and cationic substitution. The goal is to
define further the mechanisms for control of sarcolemmal permeability in the heart.
Circulation Research/Vo/. 51, No. 2, August 1982
132
Methods
Experimental Preparation
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The experimental preparation used in these experiments
was the arterially perfused, isolated, interventricular septum
of male, albino rabbits (2-3 kg) (Rau et al., 1977). The
animals were killed by an overdose intravenous injection of
heparinized sodium pentabarbitol. The hearts were rapidly
removed from the animals through a midsternal thoracotomy, rinsed with oxygenated Krebs bicarbonate solution,
and placed on a dissecting block where the atria and right
ventricular wall were trimmed away. With the aid of a
dissecting microscope, the septal branch of the left coronary
artery was cannulated with a small polyethylene cannula
and perfused. Total ischemic time for the septum from
death of the animal to onset of perfusion was 2-2.5 minutes.
The perfused area of the septum was isolated from the rest
of the heart, secured by two clamps at its base, and tied
with suture to a Statham force transducer for the vector
sum recordings of isometric tension development and dT/
dt.
The perfusate was pumped through the cannulated septal
artery by a variable speed peristaltic pump at a rate of 2.2
ml/g wet wt per min. Average septal weight was 1.20 ±
0.03 g (SEM). The septa were paced at 42 beats/min in the
control state by attaching electrodes from a Grass model
SD9 stimulator to the clamps at the base of the muscle.
Stimulation rates were varied from 6 to 120 beats/min as
required by the experimental protocol. The temperature of
the perfusate was controlled by means of a Peltier unit on
each side of a stainless steel chamber (< 0.1 ml volume)
immediately preceding the cannula. A manifold assembly
preceding the Peltier unit allowed rapid switching between
perfusates of different composition. Developed isometric
tension, rest tension, maximal rate of tension development,
and relaxation were routinely recorded. A dome surrounding the apparatus allowed control of ambient temperature
and air composition.
Solutions
The control solution for perfusion was a modified Tyrode's solution containing (in millimoles per liter): NaCl,
130; KC1, 6; MgCl2, 1; NaH2PO4, 0.43; dextrose, 5.6; CaCl2,
1.5; and NaHCO.3, 9-11. All solutions were equilibrated at
room temperature with 98% O2-2% CO2 with a resultant pH
of 7.35-7.40.
Zero Ca (0-Ca) solutions were meticulously prepared by
first rinsing all containers and spatulas with 0.1 N HNO3
and then with distilled deionized water. Composition of the
0-Ca perfusate was identical to control solutions except that
Ca++ had not been included. Total Ca in "0-Ca" solutions
was less than 7 /JM measured by atomic absorption spectrophotometry. Reagent grade chemicals were used to minimize Ca contamination. For those experiments in which
various cations were to be used to test for protection against
the Ca-paradox, appropriate aliquots from stock solutions
of the desired cation-chloride salt prepared in water were
added to the Ca-free solutions.
Enzyme Assay and Calculations
Timed collections of droplets during an experiment were
made as effluent perfusate drained from the surface of the
septum and fell from a common area near the cannula.
These samples were weighed to determine flow rate and
then analyzed for enzyme activity and/or specific activity
if 42K experiments were being done. Enzyme activity was
first calculated as international units (IU)/L, normalized for
flow rate and muscle weights and expressed as IU/g dry wt
per min. The resultant enzyme activity for each sampling
period was entered into a computer which plotted the data
and calculated the total amount released during any sampling period. Creatine kinase (CK,EC2.7.3.2) activity was
assayed according to a modification of the Rosolki method
(1967) using a CPK Max-Pack assay kit from Cal Biochem.
Lactic dehydrogenase (LDH,ECl.1.1.27) and malate dehydrogenase (MDH,ECl.l.l.37) activity were measured at 340
nm in thermostated Beckman Spectrophotometers, model
35 or DU-8, as the oxidation of NADH according to the
methods of Wroblewski and LaDue (1955) and Siegel and
Bing (1956), respectively. Glutamic oxaloacetic transaminase
(GOT,EC2.6.1.1) activity was assayed by the colorimetric
method of Reitman and Frankel (1957), using Sigma kit
no.505. Net absorbance of the effluent perfusate samples
was measured at 260 and 280 /im to indicate approximate
concentrations of neucleosides/nucleotides and protein
content, respectively.
Protocol
The septum was cannulated, isolated, and perfused at
28°C, 42 beats/min and 2.2 ml/g wet wt per min for an
equilibration time of 90 minutes during which time enzyme
release, tissue ionic changes, and water content of the
septum reached a steady state. The temperature of the
septum was controlled (±0.2°) by passing current through
the Peltier unit by means of a rheostat. In those experiments
in which Ca-free exposure and Ca++ repletion were to be
carried out at temperatures other than 28°C, the control
perfusate and muscle temperature were altered to the desired level within 2 minutes and maintained at that new
temperature for 15 minutes prior to any Ca-free exposure.
The stimulation rate to the septum was adjusted at the new
temperature to allow controlled pacing of the septum. At
higher temperatures, the rate generally had to be increased
in order to override the increased temperature-dependent
intrinsic rate. Lowered pacing rates at cooler temperatures
were necessary to allow for complete relaxation.
After the muscle function had stabilized at 90 minutes,
the appropriate perfusion lines were rinsed with 0.1 N
HNO3, and distilled, deionized H2O and Ca-free solutions
or Ca-free perfusates plus variable concentrations of desired
cations were perfused. Following Ca-free perfusion periods
of up to 60 minutes, solutions with control levels of 1.5 min
Ca were again perfused for 30-60 minutes. Active developed tension, passive resting tension, and rates of tension
development and relaxation were monitored continuously
and recorded throughout.
Results
Functional Characteristics of the Ca-free
Experimental System
The rate at which active isometric tension development declined in response to 0-Ca perfusions was
dependent on and directly related to the pacing rate,
flow rate, and temperature of the muscle (Fig. 1).
Figure 1A demonstrates the rate of tension decline in
a septum during repeated, short-term 0-Ca exposures
as the flow rate during successive 0-Ca perfusions
was lowered from 4.0 to 0.8 ml/min with pacing rate
and temperature held constant at 42 beats/min and
28°C, respectively. The half-time (to.s) of tension decline during 0-Ca perfusions increased, monophasically, from 18 to 35 seconds as the flow rate was
Rich and Langer/Hypothermia and Cation Addition Protect Ca-Paradox
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of 2.6 ml/min and 28°C, respectively. The to.5 of
tension decline during 0-Ca increased, monophasically, from 19.5 seconds to 29 seconds as the stimulation rate was lowered 10-fold from 120 to 12 beats/
min with a correlation coefficient r = —0.975. By
contrast to the two preceding manipulations, the rate
of tension decline during 0-Ca showed a biphasic
response as muscle temperature was decreased. The
transition point occurred at approximately 28°C (Fig.
1C). The rate of tension fall in 0-Ca decreased much
more rapidly as the septal temperature was lowered
from 28 to 16°C (to.s increased from 20.1 seconds to
47.1 seconds) than it did when the muscle temperature
was lowered from 37 to 28°C (to.5 increased from 17.8
to 20.1 seconds). The correlation coefficient r relating
temperature and t05 was -0.985 for temperatures
below 28°C.
The flow, stimulation, and temperature relationships were defined such that each could be isolated in
the analysis of the response to 0-Ca perfusion. The
flow rate and stimulation rate for hypothermia experiments were selected so that clearance of Ca++ from
the tissue was not a limiting factor in the Ca-paradox
phenomenon, i.e., the [Ca++]o level was reduced to a
value capable of production of the paradox. For cation
substitution experiments, the flow rate, stimulation
rate, and temperature were kept constant at levels
(arrows in Fig. 1) where tissue Ca++ clearance was not
limiting in the Ca-paradox protocol.
Protective Effect of Hypothermia
co
r = -0.985
20-
10
20
24
28
32
36
TEMPERATURE (°C)
FIGURE 1. The effect of flow rate, stimulation rate, and muscle
temperature on the rate of tension decline of a rabbit septum
during short-term, repetitive, Ca-free perfusions. A: with the septa/
temperature and stimulation rate held constant at 28°C and 42
beats/min, respectively, the flow rate during Ca-free perfusions
was altered over the range 4.0 to 0.8 ml/min. The half-time ft or,) of
tension decline was directly correlated with the flow rate (r =
-0.994), i.e., faster flow rates resulted in lower half-times. B:
Stimulation rate was varied over the range 120 to 12 beats/min
during successive Ca-free perfusions with temperature and flow
rate held constant at 28 °C and 2.6 ml/min, respectively. The rate
of tension decline in 0-Ca was linearly correlated with beating rate
(r = —0.975), i.e., faster beating rates resulted in lower half-times.
C: Summary of the rate of tension decline in several muscles during
0-Ca perfusions at different temperatures. The flow rate was constant at 2.6 ml/min. Stimulation rates varied from 15 beats/min at
16°C to 72 beats/min at 37°C.
16
decreased from 4.0 to 0.8 ml/min, with a correlation
coefficient r = —0.994 over this range. Figure IB
shows the rate of decline during successive, shortterm 0-Ca exposures in a septum as the pacing rate
was increased at a constant flow rate and temperature
Damage to the heart upon reperfusion with Ca++
following Ca-free exposure was assessed by recovery
of developed tension, development of contracture
tension, and release of intracellular enzymes and K+.
Temperature was important in the determination of
the length of time the heart required to be perfused
with 0-Ca such that the paradox occurred. Perfusion
with Ca-free solutions for 2 minutes at 37°C allowed
full recovery of developed tension upon return to
control Ca++ (Fig. 2). Five minutes of 0-Ca perfusion
at 37°C allowed only 50% recovery of tension upon
Ca++ repletion and 10 minutes 0-Ca exposure at 37°C
allowed only 26% tension recovery in the septum.
When hearts were perfused with 0-Ca for variable
times at lower temperatures, longer exposures were
required to achieve similar losses of tension recovery
upon Ca++ repletion (see Fig. 2). Whereas 50% developed tension could be recovered upon Ca++ repletion
after 5 minutes 0-Ca perfusion at 37°C, muscles
needed to be perfused with 0-Ca for 17.5 and 33
minutes at 28°C and 22°C, respectively, to demonstrate 50% developed tension recovery. Even after 60
minutes of 0-Ca perfusion at 18°C, tension recovered
to nearly 70% of control when Ca was restored. These
data confirm, via functional recovery, the marked
protective effect of hypothermia on the Ca-paradox.
Creatine kinase release patterns from the above
muscles corresponded inversely to the degree of tension recovery. Figure 3 shows that at 37°C, 2 minutes
of 0-Ca perfusion followed by Ca++ repletion caused
Circulation Research/Vo/. 51, No. 2, August 1982
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20
30
40
50
1000
60
TIME(MIN) OF Ca-FREE EXPOSURE
FIGURE 2. Active tension recovery upon Ca ++ repletion following
variable times of Ca-free perfusion at different muscle temperatures. A septum was equilibrated at the particular temperature for
at least 15 minutes before Ca-free perfusion. Ca + + depletion was at
the same temperature as the Ca-free perfusion.
<
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very little additional CK release. Five- and 10-minute
0-Ca perfusions at 37°C caused progressively larger
amounts of CK release upon Ca++ repletion. The
largest release of CK corresponded to the least recovery of developed tension with the CK release vs.
developed tension recovery showing a correlation
coefficient r = -0.993 at 37°C. CK release upon Ca++
repletion at 28°C increased progressively as the length
of 0-Ca exposure increased, and reached similar levels
caused by 5 minutes 0-Ca at 37°C only after 26
minutes 0-Ca perfusion. CK release vs. developed
tension recovery at 28°C showed a correlation coefficient r = -0.911. Lowering the muscle temperature
to 22°C showed no additional CK release upon Ca
repletion even after 20 minutes of 0-Ca perfusion. As
perfusion time of 0-Ca was lengthened at 22°C, CK
release upon Ca++ repletion was progressively increased, but even after 60 minutes of 0-Ca perfusion
the CK release during the Ca-paradox was only 1/3
of the amount released at 28°C following 30 minutes
0-Ca exposure, although active tension recovery was
similar in both cases (see Fig. 2). CK release vs. active
tension recovery at 22°C had a correlation coefficient
r = -0.969. CK release upon Ca++ repletion at 18°C
following 30 and 60 minutes 0-Ca perfusion increased
only slightly over control levels.
The enzyme release patterns of LDH, MDH, and
GOT (data not shown) were very similar to the patterns of CK release. These data confirm via intracellular enzyme release patterns the marked protective
effect of hypothermia on the Ca-paradox.
K+ Flux during Ca-Paradox
The results of Ca + + repletion on 42K exchange and
mechanical function following 60 minutes of 0-Ca
10
20
30
40
50
60
TIME(MIN) OF Ca-FREE EXPOSURE
FIGURE 3. Total creatine kinase release during the first 30 minutes
of Ca + + repletion following variable times of Ca-free perfusion at
different muscle temperatures. Data were obtained from the same
muscles and experimental conditions seen in Figure 2.
perfusion at 28°C are shown in Figure 4A. After 115
minutes of labeling the septum to near asymptote
with 42K (1 juCi/ml), the septum was perfused for 60
minutes with 0-Ca solution of matched 42K specific
activity. The level of 42K uptake did not change appreciably during the 0-Ca perfusion. When Ca was
reperfused, there was then evidenced a massive loss
of K+ (-50% of labeled 42K). The 42K uptake then
stabilized at the new level. The net loss of K+ was
similar during Ca++ repletion after 30 minutes of 0Ca perfusion at 28°C
Developed tension fell rapidly to zero when perfused with 0-Ca in Figure 4A. Rest tension rose from
a stable control level of 5.8 to 9.6 g at 60 minutes of
0-Ca perfusion. Within 2 minutes of Ca++ repletion,
contracture tension was 99% of total control tension
before 0-Ca perfusion. This contracture tension gradually declined but was still 219% (12.7 g) of the control
rest tension after 30 minutes of Ca++ repletion. Developed tension recovered to only 6.3% of pre-O-Ca
control levels.
Figure 4B demonstrates the effect of hypothermia
on 42K uptake during Ca repletion after 60 minutes 0Ca perfusion. The pacing rate was slowed from 42 to
20 and then to 15 beats/min as the muscle was cooled
to 18°C to allow for more complete relaxation. 42K
uptake was constant during 30 minutes at 18°C. The
muscle function was stable at 15 beats/min and 18°C.
135
Rich and Langer/Hypothermia and Cation Addition Protect Ca-Paradox
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FIGURE 4. 42K uptake (dots) and isometric tension response to Ca-paradox at different temperatures. A: Ca-free perfusion (O-Ca) for 60
minutes was followed by Ca ++ repletion at 28 °C,. 2.6 ml/min, 42 beats/min stimulation rate throughout. B: After 80 minutes of 42K Jabeiing
at 28 °C, 42 beats/min, the muscle temperature was decreased to 18°C and the pacing rate was decreased to 20 and then to 15 beats/min.
After 30 minutes of total equilibration at 18°C, the septum was perfused Ca-free at 18°C for 60 minutes followed by 35 minutes Ca**
repletion at 18 "C. The muscle then was warmed to 28°C and paced at 42 beats/min for an additional 30 minutes. Note the marked protective
effect of hypothermia (18°C) against 42 K + and tension loss during the Ca-paradox.
During the subsequent 60 minutes O-Ca perfusion at
18°C, the rate of 42K uptake decreased slightly, probably due to a temperature-induced inhibition of the
Na + -K + -pump which would increase K+ loss from the
heart. Rest tension actually decreased from 6.6 g to
5.0 g after 60 minutes O-Ca at 18°C Upon Ca ++
repletion at 18°C, there was not only a stabilization
but a slight increase in IC1" uptake for 35 minutes.
Peak contracture tension was 29.0 g at 3-4 minutes
after Ca ++ repletion at 18°C (63.9% of pre-O-Ca total
tension). Developed tension continued to increase and
was 46.4% of pre-O-Ca control after 30 minutes. When
the septum was warmed to 28°C for an additional 35
minutes, the K+ uptake increased further to approximately the same level recorded before cooling. Muscle
function increased much further at 28°C. Rest tension
fell quickly when the muscle was warmed and continued to decline to 6.0 g after 35 minutes of perfusion
at 28°C (150% of control at 28°C). Developed tension
also increased further to 74.0% of control after 35
minutes at 28°C. This recovery of mechanical function and preservation of 42K exchange at 18°C is in
marked contrast to the loss of function and 42K seen
during the Ca-paradox at 28°C. The effect of the Caparadox protocol was also investigated during 42K
washout, and these experiments confirmed the 42K
uptake results.
Cation Substitution—Protection against Tension
and K+ Loss
The effect on tension development and 42K uptake
of including 50 JUM concentrations of various divalent
Circulation Research/Voi. 51, No. 2, August 1982
136
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cations during 30 minutes "Ca-free" perfusion at
28°C followed by 1.5 miu Ca ++ repletion is seen in
Figure 5. Figure 5A demonstrates that the inclusion of
50 fiM Ca + + in this septum during the 30 minute "Cafree" period allowed nearly complete developed tension recovery, no contracture tension, and no change
in the 42K uptake pattern during normal Ca ++ repletion. In another septum (Fig. 5B), inclusion^of 50 ;UM
Cd ++ , whose crystal ionic radius of 0.97 A is very
close to that of Ca + + (0.99 A), during the 30 minutes
of 0-Ca perfusion also prevented 42K loss and all but
minimal contracture upon Ca + + repletion. Tension
development after 30 minutes of normal Ca ++ reperfusion was only 34% of control, probably due to
residual, but diminishing, uncoupling effects of Cd + +
(Bers and Langer, 1979). 50 JUM Mn + + (crystal ionic
radius of 0.80 A) had less protective effect on the Caparadox when included during the 0-Ca period (see
Fig. 5C). On Ca ++ repletion, the septum lost 11.4% of
its 42K and developed considerable contracture tension which eventually relaxed to approximately twice
the pre-paradox rest tension level. Developed tension
recovered to about 35% of control. If only 50 /IM Mg + +
(crystal ionic radius of 0.65 A) was included during
the 0-Ca period, Ca + + repletion produced 43% 42K
loss, high contracture tension development and small
(7%) active tension development recovery (see Fig.
5D). Sr ++ (crystal ionic radius of 1.13 A) also demonstrated poor protection if included at 50 /tin concentrations during the 0-Ca perfusions (data not shown).
CK Release—Protective Effect of Substituted
Cations
The time course of CK release caused by 1.5 ITIM
Ca ++ reperfusion following 30 minutes of perfusion
with Ca-free + 50 JUM concentrations of four divalent
cations is seen in Figure 6. These four cations have
crystal radii similar than that of Ca ++ (Cd ++ > Mn + +
> Co ++ > Mg + + ). It can be seen that Cd ++ , whose
crystal ionic radius (0.97 A) is nearly that of Ca ++
(0.99 A), shows nearly complete protection against
CK release upon Ca ++ repletion. Ca ++ (not shown)
also prevented the Ca-paradox and shows identical,
near complete protection against CK release. As cations whose unhydrated radii are further and further
removed from that of Ca ++ are included during the 0Ca exposure, the protection they provide against CK
release during Ca ++ repletion is progressively less.
Hence, muscles that had 50 JIM Mn + + (0.80 A) included in the Ca-free perfusates showed CK losses of
1060 IU/gdw with the time course shown in Figured
during 30 minutes of Ca ++ repletion. Co + + (0.72 A)
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FIGURE +5. "K counts (dots) and+ isometric
tension responses to Ca + + repletion following the inclusion of 50 concentration of (A)
(B) Cd* , (Q Mn**, or (D) M g + during 30-m/nufe Ca-free perfusions. Experimental conditions were 28"C, 2.6 ml/min, 42 beats/min.
Rich and Langer/Hypothermia and Cation Addition Protect Ca-Paradox
25Or Co**- FREE
sion recovery with little additional CK release when
Ca ++ was reperfused (data not shown). Sr ++ also
demonstrated the ability to provide nearly complete
protection against CK release upon Ca ++ repletion if
1.5 mvi Sr ++ was included during the 0-Ca perfusion
(data not shown). It is important to note, however,
that it requires 200 and 30 times higher concentrations
of Mg + + and Sr ++ , respectively, to provide the same
CK release protection that 50 /XM Cd + + does, which
has a crystal ioniuc radius nearly equal that of Ca ++ .
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50
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FIGURE 6. Creatine kinase (CK) release during the period of Ca "
repletion after 30-minute Ca-free + 50 JIM cation addition. Each
point is the mean of CK in the effluent at the particular time point.
Standard error values are shown at 5, 10, and 20 minutes of Ca **
repletion. Perfusions were performed at 28 °C, 2.6 ml/min, 42
beats/min.
and Mg + + (0.65 A) showed progressively less protection against CK release upon Ca ++ repletion.
The relative protective effects of 50 JUM concentrations of divalent cations on total CK release from the
heart during the Ca-paradox can be seen in Figure 7.
The area (during 30 minutes of Ca ++ repletion) under
the CK release curve for each cation tested was integrated and plotted as a function of its crystal ionic
radius. It is evident that 50 /XM Ca ++ provides nearly
complete protection against the Ca-paradox, as does
Cd ++ . As the crystal ionic radius of a cation differs
increasingly from that of Ca ++ , either less or greater,
the ability of the cation to protect against the CK
release seen during the Ca-paradox is progressively
diminished. Thus, 50 JUM Mg ++ , whose unhydrated
ionic radius is furthest removed from that of Ca ++ , is
the least protective against the Ca-paradox.
Any divalent cation of those tested was capable of
providing nearly complete protection against the Caparadox if included in high enough concentrations
during the Ca-free period. It has been shown above
that Mg + + at 50 JUM concentration during 30 minutes
0-Ca was not effective in preventing CK release or K+
loss upon Ca ++ repletion. The inclusion of 1.0 mM
Mg ++ during the Ca-free perfusion period allowed
half as much CK release. Increasing [Mg ++ ] to 10 mM
during 0-Ca perfusions allowed nearly complete ten-
The time course of contracture tension caused by
1.5 mM Ca reperfusion following 30 minutes of perfusion with Ca-free + 50 JUM concentrations of five
divalent cations is seen in Figure 8. The inclusion of
50 JUM Ca ++ during the "Ca-free" period results in
very little and only transitory additional rest tension
development upon Ca ++ repletion. Cd ++ , with unhydrated ionic radius very close to that of Ca ++ , shows
greater contracture development which is largely sustained. This would indicate some damage has occurred, although there is no indication of that from
the enzyme release data (see Figures 6 and 7). As the
crystal ionic radius of the other tested cations is
progressively more different from that of Ca ++ , the
contracture tension during Ca ++ repletion following
Ca-free + 50 JUM cation perfusions is progressively
greater, indicating progressively greater damage.
Figure 9 depicts, in summary form, the relative
relationship of the tested cations and their crystal
ionic radius with their ability to protect against the
Ca-paradox as measured by K+ loss, amount of con-
3000-
2 2000LU
CC
1000UJ
0.6
CRYSTAL
0.8
IONIC RADIUS
1.0
(X)
1.2
FIGURE 7. Total creatine kinase (CK) release during the 30-minute
period of Ca ** repletion following 30 minutes of Ca-free + 50 JIM
cation perfusion as a function of the added cation's crystal ionic
radius. Total CK release was calculated by plotting each CK release
pattern and integrating the area under the curve. Each point is the
mean ± sc of these areas.
Circulation Research/Vo/. 51, No. 2, August 1982
138
0-Ca
++
Ca or Cd"1"1" were included during 0-Ca perfusions.
Increasing amounts of K+ were lost as the cation
radius deviated increasingly from that of Ca + + :Mn + + ,
2.6 ± 2.9 SEM%; Co ++ , 33.3 ± 1.6%; Mg ++ , 40.2 ±
6.1%, Sr ++ , 41.7%. Contracture tension (expressed as
total sustained rest tension) at 5 minutes after Ca ++
repletion compared to total control tension (active +
rest tension) also followed nearly the same cation
sequence: Ca ++ , 18.0%; Cd ++ , 35.8 ± 7.1 SEM%; Mn ++ ,
42.1 ± 6.8%; Co ++ , 59.7 ± 4.6%; Mg + + , 59.0 ± 6.0%;
and Sr ++ , 42.3 ± 5.2%. Total CK release, expressed as
IU/g dry wt, followed a similar cation sequence: Ca ++ ,
58.6; Cd ++ , 46.2 ± 19 SEM; Mn + + , 1063 ± 356; Co ++ ,
1780 ± 478; Mg ++ , 2614 ± 318; and Sr ++ , 2103 ± 202.
450 fj.M Cation
30r
20
g
CO
CO
10
a:
Cation Addition during Ca-reperfusion
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0L
10
20
30
TIME (min)
FIGURE 8. Total rest tension during the 30-minute period of Ca ++
repletion following 30 minutes ofCa-free + 50 JUM cat/on perfusion.
Experimental conditions and muscles were the same as for Figure
tracture tension after 5 minutes of Ca ++ reperfusion,
and total CK released. The protocol for each experiment was the same: 30 minutes 0-Ca ++ /50 JJLM cation
perfusion at 28°C and 2.2 ml/g wet wt per min
followed by 1.5 mM Ca ++ reperfusion for 30 minutes.
There was no K+ loss upon Ca + + reperfusion when
It is possible that the added cations provide protection by preventing Ca ++ reentry and overload during
the Ca ++ repletion. The results of experiments to test
this possibility are seen in Figure 10. The inclusion of
50 jitM Cd ++ during 0-Ca perfusion periods prevents
the damage indicated by CK release during subsequent Ca ++ reperfusion (closed circles). If septa are
perfused Ca-free for 30 minutes followed by Ca ++
repletion, there is massive CK release, contracture
development, and K+ loss (data described above). The
inclusion of 50 and 500 JUM Cd + + during the Ca ++
repletion period following Ca-free perfusion protects
only partially against the damage as evidenced by CK
release. Maximum contracture tension (total rest tension) during 1.5 mM Ca ++ + A/XM Cd + + perfusion after
30 minutes of 0-Ca exposure was 71.0 ± 3.8% and
l00r
%
K* LOST
%
5 min CONTRACTURE TENSION
TOTAL CONTROL TENSION
CK RELEASE
80
3000
(5)
(6)
(6) _
(5)
60
o
o
Q
110)
(5)
(2) (6)
2000 lo
UJ
(10)
: 40-
(4)
UJ
or
o
<r
UJ
oo
UJ
Q.
(2)
20
UJ
(2)
(2)
0
CATION
IONIC
Caz
RADIUS (A) 0.99
UJ
CE
O
(2)
Cdz
Mnz
Co*
Mg
Sr 2
0.97
0.80
0.72
0.65
1.13
FIGURE 9. Relationship of K + loss, contracture tension development, and CK release during Ca ++ repletion to crystal ionic radius. Septa were
perfused for 30 minutes with Ca-free + 50 JUM substituted cation followed by 30 minutes of Ca *+ repletion. K * loss and total CK loss were
determined for 30 minutes. Total rest tension (contracture tension) as percent of total control tension was determined at 5 minutes of the
Ca ++ repletion period.
Rich and Ldnger/Hypothermia and Cation Addition Protect Ca-Paradox
400
0-Co
300
LJJ
Cd 2
<
UJ
200
cr
UJ
UJ
100
or
o
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15
30
50
40
60
TIME (min)
FIGURE 10. The effect on creatine kinase release of including
variable concentrations of Cd ++ during the Ca ++ repietion period
following 30 minutes of 0-Ca perfusion (open symbols). For comparison, the CK release pattern during normal Ca ++ repletion is
shown following 30 minutes 0-Ca + 50 JIM Cd** perfusion (closed
circles). Maximum protection (lack of CK release) is seen oniy if
Cd*+ is included during the Ca-free perfusion period.
90.7 ± 6.0% of total control tension for 50 and 100
JUM Cd ++ , respectively. Thus, Cd ++ addition during
the Ca ++ reperfusion period was also not functionally
protective.
Discussion
The progressive, protective effect of hypothermia
against the Ca-paradox is evidenced by data shown in
Figures 2, 3, and 4. As hearts are cooled from 37°C,
it is necessary to perfuse with 0-Ca for longer periods
of time to cause the same amount of tension loss (Fig.
2) or CK release (Fig. 3) upon Ca ++ repletion. K+ loss
was also prevented during Ca ++ repletion if the 0-Ca
perfusion was at 18°C (Fig. 4). It has been shown that
membranes undergo phase transitions from a more
liquid to a less liquid state as the temperature is
lowered (McMurchie et al., 1973; Gordon et al., 1978;
Charnock et al., 1980). In mammalian heart sarcolemma membranes, the phase transition seems to
occur in the 21-32°C range (Gordon et al., 1978). It
has been hypothesized that this phase transition with
decreased temperature might result in the protection
against the Ca-paradox shown by hypothermia (Holland and Olson, 1975). The results seen here would
support this proposal. Thus hypothermia, leading to
a less fluid membrane state, may prevent the changes
in membrane permeability associated with Ca-free
perfusion.
Recent studies have proposed that the energy state
of the muscle may be determinative in the demonstration of the Ca-paradox. Ruigrok et al. (1978) found
that an energy-depleted state is protective. Boink et
139
al. (1980) interpret hypothermia as protective via an
"energy-dependent" mechanism and not via a lipid
phase transition—implying that lower temperature
leading to energy conservation protects against the
paradox. This is, then, opposed to the view of Ruigrok
et al. (1978). The present study using cation substitution suggests that the paradox can be demonstrated
or not at the same energy level. The metabolism of
the muscle would be expected to be little different
under conditions of "zero" Ca ++ perfusion at 28°C
(Fig. 4), 50 JUM Ca ++ perfusion at 28°C (Fig. 5) and 50
juM Mg + + perfusion at 28°C (Fig. 5). In all three
protocols, the levels of ATP and CP would be expected to be similarly high. Ca ++ repletion, however,
induces markedly different results: large contracture
tension, failure to redevelop tension, loss of 42K and
intracellular enzymes in the "zero" Ca ++ and 0-Ca +
50 JUM Mg ++ -perfused septa, but nearly complete recovery of developed tension, little contracture tension,
and no loss of 42K or intracellular enzymes in the 50
jiiM Ca ++ -perfused muscles.
It is clear that the cations that were substituted
during Ca-free perfusions were effective in protection
according to their crystal ionic radius and not their
hydrated ionic radius. The cation sequence for prevention of the Ca-paradox was: Ca + + > Cd + + > Mn + +
> Co + + > Mg ++ . The crystal ionic radius (less than
Ca ++ ) is exactly the same: Ca ++ > Cd + + > Mn + + >
Co + + > Mg ++ . A recent study (Bers and Langer, 1979)
also demonstrated that the cation sequence for potency of EC uncoupling and Ca ++ displacement is of
the same order found to be protective against the Caparadox in the present study. The hydrated ionic
radius sequence, however, is much different: Mn + + ,
4.38 A > Mg ++ , 4.28 A > Cd ++ , 4.26 A > Co + + , 4.23
A > Ca ++ , 4.12 A (Nightingale, 1959). The fact that
the order of effectiveness for cation protection against
the paradox is identical to the order of effectiveness
for EC uncoupling and Ca ++ displacement by the
cation may indicate a membrane-binding site common to the control of Ca ++ permeability or lipid
orientation in the membrane and for binding that
Ca ++ which is important in EC coupling.
The results of these experiments do not rule out
the possibility that a membrane phase transition induced by cation addition may contribute to the protection against the Ca-paradox. It has been shown that
divalent cations have the effect of raising the transition temperature of membranes (Trauble and Eibl,
1974; Gordon et al., 1978). That is, at a given temperature, the addition of divalent cations and/or H + have
the effect of making the membrane less liquid, or
more gel-like. The protection effects of acidosis (Bieleki, 1969) and lowered perfusate Na + (Alto and
Dhalla, 1979) on the Ca-paradox—both of which
increase the phase transition temperature of the membrane—are consistent with this mechanism. It is unlikely, however, that the degree of protection via this
mechanism provided by cation addition in micromolar concentrations as done in these experiments would
be nearly as great as that seen in hypothermia.
Circulation Research/Vo/. 51, No. 2, August 1982
140
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It is also possible that the protective effects of
hypothermia and cation substitution are based not in
the lipid bilayer but in the glycocalyx. The addition
of 50 JUM Ca++ during "Ca-free" perfusions prevents
peeling of the external lamina and the Ca-paradox
(Crevey et al., 1978). Protective levels of hypothermia
also prevent the glycocalyx peeling. In fact, all interventions that we have examined which will protect
against the development of the Ca-paradox also prevent glycocalyx peeling. An ultrastructural examination of the paradox and the effects of hypothermia
and cation substitution is in this issue (Frank et al.,
1982).
An additional possible mechanism for prevention
of the Ca-paradox by cation addition is the prevention
of Ca++ reentry during the Ca++ repletion period.
Those cations which are the most effective in Ca++
displacement from the membrane and EC uncoupling
should be most effective in preventing Ca++ reentry
during Ca++ repletion and thus most effective in
preventing the damage caused by cellular Ca++ overload during Ca++ repletion. Figure 10 presents evidence that Cd++ prevents the changes in Ca++ permeability that occur during Ca-free perfusions. If the
changes in the membrane are allowed to occur during
Ca-free perfusions [see Frank et al., (1982)], the addition of Cd++ during the Ca++ repletion period can
only partially prevent the damage caused by Ca++
overload.
We are very appreciative of the excellent technical assistance of
Bradley Williams.
Supported by U.S. Public Health Service Grant HL 11351-14,
American Heart Association, Greater Los Angeles Affiliate Research Award #694 Gl-1, and the Castera Foundation.
Address for reprints: Terrell L. Rich, Ph.D., Cardiovascular
Research Laboratory, A3-381 CHS, UCLA School of Medicine, Los
Angeles, California 90024.
Received November 2, 1981; accepted for publication May 14,
1982.
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INDEX TERMS: Calcium paradox • Hypothermia • Phase transition • Crystal ionic radius • lnterventricular septum • Creatine
kinase
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Calcium depletion in rabbit myocardium. Calcium paradox protection by hypothermia and
cation substitution.
T L Rich and G A Langer
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Circ Res. 1982;51:131-141
doi: 10.1161/01.RES.51.2.131
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