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The possible role of endogenous amphiphiles in the membrane abnormalities of ischemic and reperfused myocardium
The Possible Role of Endogenous Amphiphiles in the Membrane Abnormalities of Ischemic and Reperfused Myocardium Frank C. Messineo,
MD
Calcium entry into cardiac cells is believed to be controlled by transmembrane-voltage dependent, protein regulated “channels.” The sarcoplasmic reticulum participates in the regulation of cytosolic calcium by ATP dependent Ca2+ sequestration during diastole, and by action potential stimulated calcium release. Massive calcium overloading occurs during reperfusion
following myocardial ischemia. Calcium overloading activates phospholipases, which may activate another mechanism involved in lethal cellular injury, that is, the accumulation of long chain fatty acids and their derivatives. These compounds are soluble amphiphiles, and once liberated, they may insert into biological membranes and change membrane composition, physiology, and response to ions and drugs. Sarcoplasmic reticulum vesicles were used as an in vitro model to study the effects of palmitic acid, oleic acid, and pahnitylcarnitine on the ability of this membrane system to sequester calcium within the vesicles. In the absence of phosphate, pahnitic acid enhanced the ability of the vesicles to sequester calcium. Oleic acid and palmitylcarnitine inhibited calcium sequestration. In the presence of phosphate, palmitic acid also inhibited the sequestration of calium by sarcoplasmic reticulum, although not as severely as oleic acid and palmitycarnitine. These results suggest that the disturbances in cellular calcium homeostasis following ischemia may be due, in part, to the incorporation of accumulated long chain fatty acids into membranes. From the Division sity of Connecticut
of Cardiology, Department Health Center, Farmington,
of Medicine, Connecticut.
Univer-
Dr. Messineo is the recipient of an NIH Clinical Investigator Award, HL-00911. Research cited in this article has been supported by Grants HL-21812, HL-22165, and HL-26903. Address reprint requests to Frank C. Messineo, MD, Division of Cardiology, Dept of Medicine, University of Connecticut Health Center, Farmington, CT 06032. Key Words: Membrane structure, calcium; long chain fatty acids; ischemia; sarcoplasmic reticulum.
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Introduction Mechanisms of Normal Calcium Homeostasis Excitation-contraction coupling in heart muscle depends on the structural and functional integrity of a number of membrane systems that act as regulated barriers for the movement of calcium ions. During the cardiac cycle, the cytosolic calcium concentration, aproximately 10.’ M during diastole, increases to aproximately 10e5 M and serves to activate the contractile proteins and initiate cardiac systole. ’ Relaxation occurs, in part, by mechanisms that sequester cytosolic calcium to delimited membrane sites unavailable to the contractile proteins. The cardiac sarcolemma and the sarcoplasmic reticulum are two membrane systems instrumental to this cyclic regulation of the cytosolic calcium concentration.2J3 The phospholipid portions of these membranes act as the permeability barrier for ions while intramembraneous proteins are the structures through which controlled ion movement occurs. 4 In the sarcolemma, under physiologic conditions, calcium entry occurs primarily through specific membrane channels that are regulated; that is, opened and closed as a function of membrane potential and time. During the plateau phase of the cardiac action potential these channels open and allow calcium to move down its electrochemical gradient into the myocardial cytosol.‘,” The sarcolemma also contains a Na-Ca counter-transport mechanism that can move these two ions across the membrane as a function of the respective ion concentrations and their relative binding affinities at each membrane surface. This mechanism may contribute to a Ca2+ entry or serve to prevent cellular calcium overload by extruding a portion of the calcium that enters the cell through calcium channels. This movement of calcium outside the cell against its electro-chemical gradient is coupled to sodium entry down an electro-chemical gradient; established by operation of the sarcolemmal ATP-consuming Naf, K’ pump that actively extrudes sodium.’ The sarcoplasmic reticulum (SRI is an intracellular membrane delimited space whose primary function is to control cytosolic calcium by effecting calcium movements in both directions across the membrane. The calcium-ATPase protein of this system uses the chemical energy of ATP to remove calcium from the cytosol and sequester it within the membranedelimited SR network. The sarcoplasmic reticulummediated removal of calcium from the cytosolic matrix results in myocardial relaxation; in addition, this se-
American lournal of Emergency Medicine 1983J: 162- 167
CEREBRAL RESUSCITATION
questration of calcium results in the accumulation of a pool of calcium that is available for subsequent release down a concentration gradient into the cytosol.* The release of calcium from this intracellular store is triggered by an event occurring during the action potential. The most likely signal is the entry of a small amount of calcium through the sarcolemmal calcium channel triggering a regenerative release of activator calcium from the intracellular SK pool (calciumtriggered calcium release).‘19 The orderly regulation of these transmembrane calcium movements determine the changes in cytosolic calcium concentration that result in the generation and dissipation of myocardial tension.
Ischemic
Disturbance
of Calcium
Homeostasis
The mechanisms responsible for the functional and structural aberrations in cardiac muscle that occur upon interruption of myocardial blood flo~,~‘-~~ or after the reperfusion of muscle rendered hypoxic or ischemic,13J14 remains obscure. Possible mechanisms include the depletion of high energy phosphate compounds, the accumulation of phosphate and its subsequent precipitation with calcium in a number of cellular organelles, and acidosis.” One possible common pathway of these mechanisms may be alterations in the function of the membranes that govern cellular calcium movement. Nayler and colleaguesl’ suggest that during hypoxia and ischemia the initial depression in myocardial contractility and the subsequent increase in resting tension may result from changes in both sarcolemmal calcium entry and sarcoplasmic reticulum calcium sequestration, respectively. Furthermore, irreversible myocardial damage is associated with a net gain in cellular calcium; such a gain may reflect more extensive derangements in the processes governing the maintenance of cytosolic calicum concentrations. 15-17 The calcium paradox18 is an intriguing phenomenon first described in cardiac muscle but also observed in vascular smooth muscle and kidney, which occurs when after a period of exposure to low calcium perfusate, tissue is re-exposed to a physiologic calcium concentration. Upon re-exposure, there is an immediate massive gain in cellular calcium that in cardiac muscle is associated with consumption of high energy phosphate compounds, sustained contracture with loss of active tension development, ultrastructural damage, enzyme and protein release, and finally cell death.” Similar changes are observed after reperfusion of ische-
l
MESSINEO . AMPHIPHILES IN ISCHEMIC MYOCARDIUM
mic muscle or the re-exposure to oxygen of hypoxic muscle.13-17~20 The loss of the functional integrity of sarcolemmal and sarcoplasmic reticulum calcium transport regulation may be important in the massive entry of calcium into the cytosol under these conditions. The initial uncontrolled elevation in the cytosolit calcium concentration would have dramatic stimulatory effects on both calcium-dependent energy consuming processes including contractile protein activation and mitochondrial and sarcoplasmic reticulum ATPases, as well as potentially destructive catabolic enzyme systems such as calcium-dependent proteases and phospholipases. l9 An additional mechanism that could contribute to a derangement in membrane function during myocardial ischemia may be the accumulation of both long-chain fatty acids and a number of derivatives of these fatty acids in the cytosol of the myocardial ce11.12 Three metabolic abnormalities that obtain during hypoxia or ischemia result in myocardial lipid accumulation. They include 1) catecholamine-induced increases in circulating free fatty acids; 2) hypoxia-induced inhibition of mitochondrial fatty acid beta-oxidation resulting in long-chain acylcarnitine accumulation2’; 31 activation of phospholipases that generate lysophosphatides and free fatty acids from membrane phospholipids.12f22 Studies have documented the accumulation of neutral lipids during myocardial infarction and ischemia in animals,23 and an increase in free fatty acids as well as long-chain acylcarnitine and longchain acyl-coenzyme A in ischemic cardiac muscle W1 Because long chain fatty acids and their derivatives are soluble amphiphiles, compounds with both hydrophilic and hydrophobic portions these compounds may insert into biological membranes and, at low concentrations, change the physical state and composition of the membrane bilayer, or displace calicum from phospholipid binding sites, and thereby could alter the function of’ membrane-bound proteins (Figure 1).12124 At high concentrations, these compounds could act as detergents and disrupt the structural integrity of the membrane barrier.25 A large number of studies examining the effects of endogenous amphiphiles have shown that these compounds can alter the drug binding characteristics,26 the ion transport properties, and the enzyme function of a variety of isolated cardiac and skeletal muscle membrane systems in vitro. I2 In order to characterize further amphiphile effects on muscle membrane calcium transport we have ex-
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Figure 1. Schematic representation of possible mechanisms by which the incorporation of amphiphihc molecules into membrane phospholipids could modify membrane function. (Reproduced with permission from Katz AM, Messineo FC. Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ Res 1981;48: 1.)
amined the calcium transport properties of sarcoplasmic reticulum membranes exposed to low concentrations of two fatty acids, palmitic a Cl, saturated fatty fatty acid, acid, oleic a Cl, cis-9 mono-unsaturated and the fatty acid derivative palmityl carnitine.
Methods Sarcoplasmic reticulum vesicles were prepared from rabbit “fast” skeletal muscle or canine cardiac muscle as described previously.” Calcium sequestration was measured at 25°C with 6 or 12 &ml sarcoplasmic reticulum protein in 0.12 M KCl, 40 mM histidine buffer (pH 6.81, and either 5 or 1 mM MgATP. Reactions were initiated by the addition of sarcoplasmic reticulum to reaction mixtures containing 10 to 11.1 PM 45CaC1, and terminated by Routine methods of the laboratory Millipore filtration. were used to calculate calcium sequestration.27 Under these conditions, calcium sequestration was determined at various times after initiation of the reaction. The calcium sequestration reached a maximal and stable amount at two minutes so two-minute samples were taken in later experiments. The effects of palmitic acid, oleic acid, and palmitycarnitine were examined by addition of small volumes of these amphiphiles in ethanol before initiation of the sequestration reaction or at a time when the amount of calicum sequestered was maximal and
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stable. The amount of ethanol added (0.5% by volume) had no effect. The effect of two amphiphiles together, palmitic acid and palmitylcarnitine was examined by measuring calcium sequestration in reaction mixtures containing a fixed concentration of palmitic acid and increasing concentrations of palmitylcarnitine. Additional studies to determine the effects of the fatty acids on phosphate-supported sarcoplasmic reticulum calcium uptake were also performed. (Phosphate, by forming a precipitate with calcium within the sarcoplasmic reticulum vesicles, allows calcium transport to persist and calcium uptake velocities to be determined.) These studies were carried out in the presence of 50 mM phosphate and 48 PM 45CaC12.28
Results In the absence of a calcium-precipitating anion, palmitic acid in micromolar concentrations 16 to 24 PM) enhanced calcium sequestration in both skeletal (Figure 21 and cardiac (data not shown) sarcoplasmic reticulum vesicles in a concentration-dependent manner. The extent of this effect was similar whether palmitic acid was present from the onset of the reaction or added to vesicles that had sequestered a maximal amount of calcium. At concentrations similar to palmitic acid, both oleic acid and palmitylcarnitine inhibited calcium sequestration by skeletal SR and, when added to calcium-filled vesicles, caused a rapid release of a portion of the sequestered calcium (Figures 2 through 41; at higher concentrations (: 24 FM), these compounds caused the release of all the calcium that had been accumulated by the sarcoplasmic reticulum vesicles. Palmitic acid could partially overcome the inhibitory effect of palmitylcarnitine and also decrease the sensitivity of the skeletal sarcoplasmic reticulum to palmitylcarnitine (Figure 41. Thus, in the absence of palmitic acid, a 50% inhibition of calcium sequestration by sarcoplasmic reticulum occurs at a palmitylcarnitine concentration of aproximately 6 PM, whereas in the presence of 12 PM palmitic acid, half-maximal inhibition requires 14 PM palmitylcarnitine (Figure 4, insert). In the presence of phosphate, both oleic and palmitic acids inhibit the initial calcium uptake velocity of skeletal SR vesicles; at 8 FM, oleic acid inhibited calcium uptake velocity by approximately 90% whereas 8 PM palmitic acid inhibited uptake by only 25% (Table 11.
CEREBRAL
RESUSCITATION
.
MESSINEO
.
AMPHIPHILES
IN ISCHEMIC
Cat+ CONTENT
Ca++CONTENT CALCIUM
CONTENT
III PA
0 CONTROL
6uM
.5moks/mg
1 181
1.5unmoles/mg
MYOCARDIUM
l
/r
CONTROL
0 12uM
OLEIC
OA
w
UM
0
2
4
6
8
2.0 flmoles/mg
2. Concentration dependence of the effects of palmitic and oleic acid added to skeletal sarcoplasmic reticulum vesicles before the start of a calcium sequestration reaction. Values am mean +SEM of at least three experiments. The upper abscissa expresses the fatty acid concentration and the lower abscissa the number of moles of fatty acid per milligram of sarcoplasmic reticulum protein. (For experimental details see Methods and Messineo FC, Pinto PB, Katz AM. Palmitic acid enhances calcium sequestration by isolated sarcoplasmic reticulum. J Mol Cell Cardiol 1980; 12:725.J Figure
10
1
0
2
4
6
8
IO
TIME
Figure 3. Effect of the addition of 12 PM palmitic and 12 uM oleic acid two minutes after the start of a calcium sequestration reaction. Values are mean -tSEM for three experiments for each fatty acid. (For experimental details see Methods.1
Discussion The findings presented indicate that palmitic acid, oleic acid, and palmitylcarnitine, naturally occurring, amphiphilic compounds, have complex effects on the calcium transport properties of sarcoplasmic reticulum membranes. In the absence of phosphate, fatty acids can both increase sarcoplasmic reticulum sequestration or promote calcium release. The nature of the SR response to these compounds appears to depend partially on the structure of both the aliphatic chain and the hydrophilic headgroup; so that the presence of a double bond in the aliphatic chain of oleic acid, imparting a 30” angle to its structure, results in an inhibitory effect on calcium sequestration whereas palmitic acid, a straight saturated fatty acid of similar molecular length, enhances calcium sequestration. The addition of the bulky hydrophilic moiety, carnitine, to palmitic acid results in an amphiphile with potent inhibitory effect on sarcoplasmic reticulum calcium sequestration. Our results also indicate (Figure 4) that the effect of one amphiphile can be modified by the presence of a second amphiphile and the final effect cannot bc predicted solely by the effects of each amphiphile alone. When the characteristics of the SR calcium transport mechanism are changed by the addition of the calcium precipitating anion, phosphate, the effect of pal-
0
IO [Pahnitylcarnitine]
20 x
10e8
30
M
Concentration dependence of the effect of Figure 4. palmitylcarnitine on calcium sequestration in the absence (open circles) and presence (closed circles] of 12 PM palmitic acid. Reaction conditions were as described in Figure 2. Values are mean +-SD of four experiments in the absence of palmitic and three experiments in the presence of palmitic acid. The inset displays the data normalized such that 100% equals the control values obtained in the absence and presence of 12 PM palmitic acid. (Reprinted with permission from Plenum Publishing, Advanced Myocardiology, Vol 3, 1982. In press.1
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Table I. Calcium uptake velocity phate and 48 PM 4”CaCl,. Amphiphile Control 8 PM Palmitic 8 PM Oleic
in the presence
MEDICZNE
.
VOL
of 50 mM phos-
CaZf Uptake Velocity l~mol/mg* mini 1.65 f
SEPTEMBER
I.
Katz AM. Physiology
2.
Fabiato A, Fabiato F. Calcium lum. CIIC Res 1977;40:119-129.
However, the disturbances in cellular calcium economy that occur during tissue hypoxia and more dramatically after reperfusion of hypoxic tissue may depend in part upon disturbances in cellular calcium transport secondary to the incorporation of accumulated endogenous amphiphiles into membranes. If such a mechanism is operative in the membrane failure of post-ischemic cell death, be it in heart or brain, potential therapeutic approaches could be directed at the removal of amphiphiles by stimulating their metabolism32 or the addition of exogenous amphiphiles that could interfere with the detrimental effects of endogenous lipids until their normal metabolism resumes. It is intriguing then to speculate on the mechanism of cerebral protection reported for the highly lipid soluble calcium channel blocking agents33 or barbituratesM The author wishes to thank Dr. AM Katz for his invaluable discuwons and support, and P. Pinto, C. Favreau and M. Kathier for their cxcellcnt technical assistance.
of the Heart.
New York: Raven Press, IY77.
release
from the sarcoplasmic
rettcu-
3. Langer GA. The role of calcium tractility:
0.14 f 0.03 (51
mitic acid is also qualitatively changed and under these conditions this amphiphile inhibits calcium transport. These results suggest that the effect of any amphiphile on membrane calcium transport is influenced by the size and charge of its hydrophilic portion, the functional state of the membrane transport system3’ and the length and saturation of the hydrocarbon chain, which as suggested by Klausner et a129may determine preferential incorporation into different membrane lipid domains.
1983
References
0.04 (61
1.15 f 0.12 (4)
Initial calcium uptake velocity by 6 pg/ml SR protein was determined in the presence of 50 mM potassium phosphate and 48 PM 45CaCl Reactions were initiated by addition of sarcoplasmic reticulum vehicles to a reaction mixture containing 0.12 M KCl, 40 mM histidine buffer (ph 6.8) and 5 mM MgATP at 25°C. Reactions were terminated at 30.set intervals for 2.5 min by Millipore filtration. The values are mean f SEM. The numbers of individual determinations are in parentheses. (Reprinted with permission from Academic Press, \ Mol Cell Cardiol 1982: 14(suppl). In press.)
166
1 .
An update.
I
III the control of myocardlal Mol Cell Cardiol 1980;12:231-239.
con-
4.
Singer SJ. The molecular Biochem 1974;43:805-833.
5.
Reuter H. Properties of two Inward Physiol 1979;41:413-424.
6.
Katz AM, Messmeo FC, Herbette Clrc Res lYH2;65(suppl 11%10.
7.
Reuter H. Exchange of calcium tons m mammalian myocardmm. Mechanisms and physiologic signtficance. Clrc Res 1974;34:599605.
x.
Tada M, Yamamoto T, Tonomura Y. Molecular mecharnsm of active calcium transport by sarcoplasmic reticulum. Physiol Rev 1978;58:1-79
9.
Endo M. Calcium 1977;57:71-108
release
organization
of
membranes
currents
m the heart.
L Ion channels
from sarcoplasmic
Ann
Rev
Ann Rev
m membranes.
reticulum.
Physlol
Rev
10.
Kubler W, Katz AM. Mechanism of early “pump” failure of tbc tschemic heart: Possible role of adenosine triphosphate depletion and inorganic phosphate accumulation. Am I Cardiol 1977;40:467-471,
11.
Nayler WC, Poole-Wilson P, Williams Mel Cell Cardiol 1979; I I :683-706.
12.
Katz AM, Messineo FC. Lipid-membrane interacuons and the pathogenesls of ischemic damage m the myocardium. Circ Rcs 1981;48:1-16.
13.
Hearse Cardiol
14.
Hearse DI, Gorlick PB, Humphrey myocardium: Mechanisms and 1977;39:986-993.
DJ Reperfusion 1978;9:605-616.
of the
A
lschemic
Hypoxia
myocardium.
SM. lschenuc prevention.
I
Mol Cell
contractwe Am
I
15.
Jennings RB, Ganote CE, Rclmer Path”1 1975;81:179-198.
16.
Henry PD, Schuleib R, Doris I et al. Myocardial contractore cumulation of calcium in ischemlc rabbit heart. Am 1977,233:H677-H6R4.
KA. lschemic
f
and calcium.
of the Cardiol
ttssue miury
j
Am
I
and acPhysiol
17.
Harding DP, Poole-Wilson P. Calcium exchange in rabbtt myocardlurn during and after hypoxta: Effect of temperature and substrate Cardiovasc Res 198O;XIV:435-445.
IR.
Zunmerman AN, Hulsmann WC. Paradoxical influence of calcium ions on the permeability of the cell membrane of the isolated rat heart. Nature IY66;211:646-647.
19.
Crtnwald PM, Naylcr WC. Calcium 1Mel Cell Cardiol 1981;13:867-880.
20.
Hearse Dl, Humphrey SM. Bullock CR. The oxygen paradox and the calcium paradox: Two facets of the same problem? I Mel Cell Cardiol 1978;10:605-616.
21.
Idell-Wenger IA, Neely IR. Regulation of uptake and metabolism of fatty acids by muscle. In Dietschy IM, Gott AM, Ontko IA (edsl. Disturbances in Lipld and Lipoprotein Metabolism. Bethesda: Am Physiol Sot, 1978:269-284.
entry
in the calcwm
paradox
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22.
Chien KR, Abrams I, Serroni A et al. Accelerated phospholipid gradation and associated membrane dysfunction in irreversible, chemic liver injury. I Biol Chem 1978;253:4809-4817.
23.
Belheimer DW, Buja LM, Parker RW et al. Fatty acid accumulation and abnormal lipid deposition in peripheral and border zones of experimental myocardial infarction. 1 Nucl Med 1978; 19:276-283.
24.
Seeman P. The membrane actions Pharmacol Rev 1972;24:583-655.
of anesthetics
deis-
. MESSINEO . AMPHIPHILES IN ISCHEMIC MYOCARDIUM
29.
Klausner RD, Kleinfeld AM, Hoover RL et al. Lipid domains in membranes. Evidence derived from structural perturbations induced by free fatty acids and lifetime heterogeneity analysis. I Biol Chem 1980;255:1286-1294.
30.
Katz AM, Nash-Adler P, Watras I et al. Fatty acid effects on calcium influx and efflux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Biochim Biophys Acta 1982,687: 17-26.
31.
Swoboda G, Fritzche I, Hasselbach and albumin on calcium-dependent tion of sarcoplasmic reticulum. Eur
32.
Liedtke Al, Nellis SH. Effects of carnitine in ischemic and fatty acid supplemented swine hearts. J Clin Invest 1978;64:440-447.
33.
White BC, Winegar C, lackson RE et al. Cerebral cortical perfusion following resuscitafrom cardiac arrest in dogs. Am I Emerg Med 1983;2:128-138.
34.
Blaustein MP, Ector AC. Barbiturate inhibition of calcium uptake in depolarized nerve terminals in vitro. Mel Pharmacol 1975,22:369378.
and tranquilizers.
25.
Helenius A, Simons K. Solubilization Biochim Biophys Acta 1975;415:29-79.
of membranes
by detergents.
26.
Adams RI, Pitts BI, Woods IM et al. Effect of palmitylcarnitine ouabain binding to Na, K-ATPase. I Mel Cell Cardiol 1979;l 959.
on I :941-
27
Messineo FC, Pinto PB, Katz AM. Palmitic acid enhances calcium sequestration by isolated sarcoplasmic reticulum. I Mol Cell Cardiol 1980;12:725-732.
28.
Katz AM, Repke DI, Hasselbach W. Dependence of tonophore- and caffeine-induced calcium release from sarcoplasmic reticulum on external calcium ion concentrations. I Biol Chem 1977,252: 193X-1949.
W. Effects of phospholipase A2 ATPase and the lipid composiI Biochem 1979;99:77-88.
167
MD
Calcium entry into cardiac cells is believed to be controlled by transmembrane-voltage dependent, protein regulated “channels.” The sarcoplasmic reticulum participates in the regulation of cytosolic calcium by ATP dependent Ca2+ sequestration during diastole, and by action potential stimulated calcium release. Massive calcium overloading occurs during reperfusion
following myocardial ischemia. Calcium overloading activates phospholipases, which may activate another mechanism involved in lethal cellular injury, that is, the accumulation of long chain fatty acids and their derivatives. These compounds are soluble amphiphiles, and once liberated, they may insert into biological membranes and change membrane composition, physiology, and response to ions and drugs. Sarcoplasmic reticulum vesicles were used as an in vitro model to study the effects of palmitic acid, oleic acid, and pahnitylcarnitine on the ability of this membrane system to sequester calcium within the vesicles. In the absence of phosphate, pahnitic acid enhanced the ability of the vesicles to sequester calcium. Oleic acid and palmitylcarnitine inhibited calcium sequestration. In the presence of phosphate, palmitic acid also inhibited the sequestration of calium by sarcoplasmic reticulum, although not as severely as oleic acid and palmitycarnitine. These results suggest that the disturbances in cellular calcium homeostasis following ischemia may be due, in part, to the incorporation of accumulated long chain fatty acids into membranes. From the Division sity of Connecticut
of Cardiology, Department Health Center, Farmington,
of Medicine, Connecticut.
Univer-
Dr. Messineo is the recipient of an NIH Clinical Investigator Award, HL-00911. Research cited in this article has been supported by Grants HL-21812, HL-22165, and HL-26903. Address reprint requests to Frank C. Messineo, MD, Division of Cardiology, Dept of Medicine, University of Connecticut Health Center, Farmington, CT 06032. Key Words: Membrane structure, calcium; long chain fatty acids; ischemia; sarcoplasmic reticulum.
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Introduction Mechanisms of Normal Calcium Homeostasis Excitation-contraction coupling in heart muscle depends on the structural and functional integrity of a number of membrane systems that act as regulated barriers for the movement of calcium ions. During the cardiac cycle, the cytosolic calcium concentration, aproximately 10.’ M during diastole, increases to aproximately 10e5 M and serves to activate the contractile proteins and initiate cardiac systole. ’ Relaxation occurs, in part, by mechanisms that sequester cytosolic calcium to delimited membrane sites unavailable to the contractile proteins. The cardiac sarcolemma and the sarcoplasmic reticulum are two membrane systems instrumental to this cyclic regulation of the cytosolic calcium concentration.2J3 The phospholipid portions of these membranes act as the permeability barrier for ions while intramembraneous proteins are the structures through which controlled ion movement occurs. 4 In the sarcolemma, under physiologic conditions, calcium entry occurs primarily through specific membrane channels that are regulated; that is, opened and closed as a function of membrane potential and time. During the plateau phase of the cardiac action potential these channels open and allow calcium to move down its electrochemical gradient into the myocardial cytosol.‘,” The sarcolemma also contains a Na-Ca counter-transport mechanism that can move these two ions across the membrane as a function of the respective ion concentrations and their relative binding affinities at each membrane surface. This mechanism may contribute to a Ca2+ entry or serve to prevent cellular calcium overload by extruding a portion of the calcium that enters the cell through calcium channels. This movement of calcium outside the cell against its electro-chemical gradient is coupled to sodium entry down an electro-chemical gradient; established by operation of the sarcolemmal ATP-consuming Naf, K’ pump that actively extrudes sodium.’ The sarcoplasmic reticulum (SRI is an intracellular membrane delimited space whose primary function is to control cytosolic calcium by effecting calcium movements in both directions across the membrane. The calcium-ATPase protein of this system uses the chemical energy of ATP to remove calcium from the cytosol and sequester it within the membranedelimited SR network. The sarcoplasmic reticulummediated removal of calcium from the cytosolic matrix results in myocardial relaxation; in addition, this se-
American lournal of Emergency Medicine 1983J: 162- 167
CEREBRAL RESUSCITATION
questration of calcium results in the accumulation of a pool of calcium that is available for subsequent release down a concentration gradient into the cytosol.* The release of calcium from this intracellular store is triggered by an event occurring during the action potential. The most likely signal is the entry of a small amount of calcium through the sarcolemmal calcium channel triggering a regenerative release of activator calcium from the intracellular SK pool (calciumtriggered calcium release).‘19 The orderly regulation of these transmembrane calcium movements determine the changes in cytosolic calcium concentration that result in the generation and dissipation of myocardial tension.
Ischemic
Disturbance
of Calcium
Homeostasis
The mechanisms responsible for the functional and structural aberrations in cardiac muscle that occur upon interruption of myocardial blood flo~,~‘-~~ or after the reperfusion of muscle rendered hypoxic or ischemic,13J14 remains obscure. Possible mechanisms include the depletion of high energy phosphate compounds, the accumulation of phosphate and its subsequent precipitation with calcium in a number of cellular organelles, and acidosis.” One possible common pathway of these mechanisms may be alterations in the function of the membranes that govern cellular calcium movement. Nayler and colleaguesl’ suggest that during hypoxia and ischemia the initial depression in myocardial contractility and the subsequent increase in resting tension may result from changes in both sarcolemmal calcium entry and sarcoplasmic reticulum calcium sequestration, respectively. Furthermore, irreversible myocardial damage is associated with a net gain in cellular calcium; such a gain may reflect more extensive derangements in the processes governing the maintenance of cytosolic calicum concentrations. 15-17 The calcium paradox18 is an intriguing phenomenon first described in cardiac muscle but also observed in vascular smooth muscle and kidney, which occurs when after a period of exposure to low calcium perfusate, tissue is re-exposed to a physiologic calcium concentration. Upon re-exposure, there is an immediate massive gain in cellular calcium that in cardiac muscle is associated with consumption of high energy phosphate compounds, sustained contracture with loss of active tension development, ultrastructural damage, enzyme and protein release, and finally cell death.” Similar changes are observed after reperfusion of ische-
l
MESSINEO . AMPHIPHILES IN ISCHEMIC MYOCARDIUM
mic muscle or the re-exposure to oxygen of hypoxic muscle.13-17~20 The loss of the functional integrity of sarcolemmal and sarcoplasmic reticulum calcium transport regulation may be important in the massive entry of calcium into the cytosol under these conditions. The initial uncontrolled elevation in the cytosolit calcium concentration would have dramatic stimulatory effects on both calcium-dependent energy consuming processes including contractile protein activation and mitochondrial and sarcoplasmic reticulum ATPases, as well as potentially destructive catabolic enzyme systems such as calcium-dependent proteases and phospholipases. l9 An additional mechanism that could contribute to a derangement in membrane function during myocardial ischemia may be the accumulation of both long-chain fatty acids and a number of derivatives of these fatty acids in the cytosol of the myocardial ce11.12 Three metabolic abnormalities that obtain during hypoxia or ischemia result in myocardial lipid accumulation. They include 1) catecholamine-induced increases in circulating free fatty acids; 2) hypoxia-induced inhibition of mitochondrial fatty acid beta-oxidation resulting in long-chain acylcarnitine accumulation2’; 31 activation of phospholipases that generate lysophosphatides and free fatty acids from membrane phospholipids.12f22 Studies have documented the accumulation of neutral lipids during myocardial infarction and ischemia in animals,23 and an increase in free fatty acids as well as long-chain acylcarnitine and longchain acyl-coenzyme A in ischemic cardiac muscle W1 Because long chain fatty acids and their derivatives are soluble amphiphiles, compounds with both hydrophilic and hydrophobic portions these compounds may insert into biological membranes and, at low concentrations, change the physical state and composition of the membrane bilayer, or displace calicum from phospholipid binding sites, and thereby could alter the function of’ membrane-bound proteins (Figure 1).12124 At high concentrations, these compounds could act as detergents and disrupt the structural integrity of the membrane barrier.25 A large number of studies examining the effects of endogenous amphiphiles have shown that these compounds can alter the drug binding characteristics,26 the ion transport properties, and the enzyme function of a variety of isolated cardiac and skeletal muscle membrane systems in vitro. I2 In order to characterize further amphiphile effects on muscle membrane calcium transport we have ex-
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Figure 1. Schematic representation of possible mechanisms by which the incorporation of amphiphihc molecules into membrane phospholipids could modify membrane function. (Reproduced with permission from Katz AM, Messineo FC. Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ Res 1981;48: 1.)
amined the calcium transport properties of sarcoplasmic reticulum membranes exposed to low concentrations of two fatty acids, palmitic a Cl, saturated fatty fatty acid, acid, oleic a Cl, cis-9 mono-unsaturated and the fatty acid derivative palmityl carnitine.
Methods Sarcoplasmic reticulum vesicles were prepared from rabbit “fast” skeletal muscle or canine cardiac muscle as described previously.” Calcium sequestration was measured at 25°C with 6 or 12 &ml sarcoplasmic reticulum protein in 0.12 M KCl, 40 mM histidine buffer (pH 6.81, and either 5 or 1 mM MgATP. Reactions were initiated by the addition of sarcoplasmic reticulum to reaction mixtures containing 10 to 11.1 PM 45CaC1, and terminated by Routine methods of the laboratory Millipore filtration. were used to calculate calcium sequestration.27 Under these conditions, calcium sequestration was determined at various times after initiation of the reaction. The calcium sequestration reached a maximal and stable amount at two minutes so two-minute samples were taken in later experiments. The effects of palmitic acid, oleic acid, and palmitycarnitine were examined by addition of small volumes of these amphiphiles in ethanol before initiation of the sequestration reaction or at a time when the amount of calicum sequestered was maximal and
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1983
stable. The amount of ethanol added (0.5% by volume) had no effect. The effect of two amphiphiles together, palmitic acid and palmitylcarnitine was examined by measuring calcium sequestration in reaction mixtures containing a fixed concentration of palmitic acid and increasing concentrations of palmitylcarnitine. Additional studies to determine the effects of the fatty acids on phosphate-supported sarcoplasmic reticulum calcium uptake were also performed. (Phosphate, by forming a precipitate with calcium within the sarcoplasmic reticulum vesicles, allows calcium transport to persist and calcium uptake velocities to be determined.) These studies were carried out in the presence of 50 mM phosphate and 48 PM 45CaC12.28
Results In the absence of a calcium-precipitating anion, palmitic acid in micromolar concentrations 16 to 24 PM) enhanced calcium sequestration in both skeletal (Figure 21 and cardiac (data not shown) sarcoplasmic reticulum vesicles in a concentration-dependent manner. The extent of this effect was similar whether palmitic acid was present from the onset of the reaction or added to vesicles that had sequestered a maximal amount of calcium. At concentrations similar to palmitic acid, both oleic acid and palmitylcarnitine inhibited calcium sequestration by skeletal SR and, when added to calcium-filled vesicles, caused a rapid release of a portion of the sequestered calcium (Figures 2 through 41; at higher concentrations (: 24 FM), these compounds caused the release of all the calcium that had been accumulated by the sarcoplasmic reticulum vesicles. Palmitic acid could partially overcome the inhibitory effect of palmitylcarnitine and also decrease the sensitivity of the skeletal sarcoplasmic reticulum to palmitylcarnitine (Figure 41. Thus, in the absence of palmitic acid, a 50% inhibition of calcium sequestration by sarcoplasmic reticulum occurs at a palmitylcarnitine concentration of aproximately 6 PM, whereas in the presence of 12 PM palmitic acid, half-maximal inhibition requires 14 PM palmitylcarnitine (Figure 4, insert). In the presence of phosphate, both oleic and palmitic acids inhibit the initial calcium uptake velocity of skeletal SR vesicles; at 8 FM, oleic acid inhibited calcium uptake velocity by approximately 90% whereas 8 PM palmitic acid inhibited uptake by only 25% (Table 11.
CEREBRAL
RESUSCITATION
.
MESSINEO
.
AMPHIPHILES
IN ISCHEMIC
Cat+ CONTENT
Ca++CONTENT CALCIUM
CONTENT
III PA
0 CONTROL
6uM
.5moks/mg
1 181
1.5unmoles/mg
MYOCARDIUM
l
/r
CONTROL
0 12uM
OLEIC
OA
w
UM
0
2
4
6
8
2.0 flmoles/mg
2. Concentration dependence of the effects of palmitic and oleic acid added to skeletal sarcoplasmic reticulum vesicles before the start of a calcium sequestration reaction. Values am mean +SEM of at least three experiments. The upper abscissa expresses the fatty acid concentration and the lower abscissa the number of moles of fatty acid per milligram of sarcoplasmic reticulum protein. (For experimental details see Methods and Messineo FC, Pinto PB, Katz AM. Palmitic acid enhances calcium sequestration by isolated sarcoplasmic reticulum. J Mol Cell Cardiol 1980; 12:725.J Figure
10
1
0
2
4
6
8
IO
TIME
Figure 3. Effect of the addition of 12 PM palmitic and 12 uM oleic acid two minutes after the start of a calcium sequestration reaction. Values are mean -tSEM for three experiments for each fatty acid. (For experimental details see Methods.1
Discussion The findings presented indicate that palmitic acid, oleic acid, and palmitylcarnitine, naturally occurring, amphiphilic compounds, have complex effects on the calcium transport properties of sarcoplasmic reticulum membranes. In the absence of phosphate, fatty acids can both increase sarcoplasmic reticulum sequestration or promote calcium release. The nature of the SR response to these compounds appears to depend partially on the structure of both the aliphatic chain and the hydrophilic headgroup; so that the presence of a double bond in the aliphatic chain of oleic acid, imparting a 30” angle to its structure, results in an inhibitory effect on calcium sequestration whereas palmitic acid, a straight saturated fatty acid of similar molecular length, enhances calcium sequestration. The addition of the bulky hydrophilic moiety, carnitine, to palmitic acid results in an amphiphile with potent inhibitory effect on sarcoplasmic reticulum calcium sequestration. Our results also indicate (Figure 4) that the effect of one amphiphile can be modified by the presence of a second amphiphile and the final effect cannot bc predicted solely by the effects of each amphiphile alone. When the characteristics of the SR calcium transport mechanism are changed by the addition of the calcium precipitating anion, phosphate, the effect of pal-
0
IO [Pahnitylcarnitine]
20 x
10e8
30
M
Concentration dependence of the effect of Figure 4. palmitylcarnitine on calcium sequestration in the absence (open circles) and presence (closed circles] of 12 PM palmitic acid. Reaction conditions were as described in Figure 2. Values are mean +-SD of four experiments in the absence of palmitic and three experiments in the presence of palmitic acid. The inset displays the data normalized such that 100% equals the control values obtained in the absence and presence of 12 PM palmitic acid. (Reprinted with permission from Plenum Publishing, Advanced Myocardiology, Vol 3, 1982. In press.1
165
AMERZCAN
IOURNAL
OF EMERGENCY
Table I. Calcium uptake velocity phate and 48 PM 4”CaCl,. Amphiphile Control 8 PM Palmitic 8 PM Oleic
in the presence
MEDICZNE
.
VOL
of 50 mM phos-
CaZf Uptake Velocity l~mol/mg* mini 1.65 f
SEPTEMBER
I.
Katz AM. Physiology
2.
Fabiato A, Fabiato F. Calcium lum. CIIC Res 1977;40:119-129.
However, the disturbances in cellular calcium economy that occur during tissue hypoxia and more dramatically after reperfusion of hypoxic tissue may depend in part upon disturbances in cellular calcium transport secondary to the incorporation of accumulated endogenous amphiphiles into membranes. If such a mechanism is operative in the membrane failure of post-ischemic cell death, be it in heart or brain, potential therapeutic approaches could be directed at the removal of amphiphiles by stimulating their metabolism32 or the addition of exogenous amphiphiles that could interfere with the detrimental effects of endogenous lipids until their normal metabolism resumes. It is intriguing then to speculate on the mechanism of cerebral protection reported for the highly lipid soluble calcium channel blocking agents33 or barbituratesM The author wishes to thank Dr. AM Katz for his invaluable discuwons and support, and P. Pinto, C. Favreau and M. Kathier for their cxcellcnt technical assistance.
of the Heart.
New York: Raven Press, IY77.
release
from the sarcoplasmic
rettcu-
3. Langer GA. The role of calcium tractility:
0.14 f 0.03 (51
mitic acid is also qualitatively changed and under these conditions this amphiphile inhibits calcium transport. These results suggest that the effect of any amphiphile on membrane calcium transport is influenced by the size and charge of its hydrophilic portion, the functional state of the membrane transport system3’ and the length and saturation of the hydrocarbon chain, which as suggested by Klausner et a129may determine preferential incorporation into different membrane lipid domains.
1983
References
0.04 (61
1.15 f 0.12 (4)
Initial calcium uptake velocity by 6 pg/ml SR protein was determined in the presence of 50 mM potassium phosphate and 48 PM 45CaCl Reactions were initiated by addition of sarcoplasmic reticulum vehicles to a reaction mixture containing 0.12 M KCl, 40 mM histidine buffer (ph 6.8) and 5 mM MgATP at 25°C. Reactions were terminated at 30.set intervals for 2.5 min by Millipore filtration. The values are mean f SEM. The numbers of individual determinations are in parentheses. (Reprinted with permission from Academic Press, \ Mol Cell Cardiol 1982: 14(suppl). In press.)
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Idell-Wenger IA, Neely IR. Regulation of uptake and metabolism of fatty acids by muscle. In Dietschy IM, Gott AM, Ontko IA (edsl. Disturbances in Lipld and Lipoprotein Metabolism. Bethesda: Am Physiol Sot, 1978:269-284.
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Katz AM, Nash-Adler P, Watras I et al. Fatty acid effects on calcium influx and efflux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Biochim Biophys Acta 1982,687: 17-26.
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167