Alterations of M embrane Potential and Ca2+ Flux of Sarcoplasmic Reticulum Vesicles in Ischemic Myocardium*f

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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 13, No. 6 Copyright 1983, Institute for Clinical Science, Inc. Alterations of M embrane Potential and Ca2+ Flux of Sarcoplasmic Reticulum Vesicles in Ischemic Myocardium*f C. F. PENG, Ph.D., K. D. STRAUB, M.D., Ph.D., and M. L. MURPHY, M.D. Medical Research Service, Veterans Administration Medical Center and Depts. of Biochemistry and Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, ABSTRACT The sequence of contraction-relaxation for myocardial cells is believed to be linked to Ca2+ flux across the sarcoplasmic reticulum. Alterations of sarcoplasmic reticulum function during ischemia may result in depressing the myocardial contraction-relaxation sequence. This study examines the relationship between the membrane potential and Ca2+ flux across sarcoplasm ic reticulum vesicles isolated from non-ischem ic and ischem ic myocardium. Ischemic myocardium was produced by ligating the coronary artery of swine hearts for 15 and 30 minutes. M em brane potential was determ ined by use of the fluorescence-sensitive dye, 3,3'-diethylthiadicarbocyanine, and Ca2+ uptake was studied spectrophotometrically with the use of murexide. Results are as follows: (1) m em brane potential and Ca2+ uptake by sarcoplasmic reticulum from ischemic myocardium progressively decreased with the length of ischemia; and (2) preincubation of sarcoplasmic reticulum from non-ischemic myocardium with deoxycholate (0.01 ~ 0.09 percent) resulted in progressively decreasing membrane potential and Ca2+ uptake. Apparently a correlation exists between membrane potential and the rate of Ca2+ uptake. These results suggest that m em brane characteristics of sarcoplasmic reticulum are altered within as early as 15 m inutes of the onset of ischemia. Alteration of m em brane perm eability in sarcoplasmic reticulum from ischemic myocardium may be responsible for the observed decrease in m em brane potential and Ca2+ uptake. * This work was supported by the Veterans Administration and the American Heart Association, Arkansas Chapter. t Please send reprint requests to Chun Fu Peng, Ph.D., VA Medical Center (111B), 300 E. Roosevelt Road, Little Rock, AR Introduction The use of fluorescent probes to study m em brane potentials has been reported in intact cell preparations such as squid /83/11C $01.50 Institute for Clinical Science, Inc.

2 5 12 P E N G, STRAUB, A N D M U R PH Y giant axon,7' ,29 leech seg m en tal ganglion,25 and red blood cells.13,28 Generally, w hen the m em brane potential of intact cells becomes negative inside, cyanine dyes move from the external m e dium to the inner m em brane vesicular space. T here they aggregate and becom e dim ers owing to an increased dye concentration and, as a result, the fluorescence intensity decreases. On the other hand, when the membrane potential inside the cells becom e positive, the dyes are discharged from the inside to the external m edium and the fluorescence intensity increases owing to an increase in the m onom er dye concentration in the medium. Thus, changes in the fluorescence in te n sity of cyanine dyes are a linear function of changes in m em brane potential. Based on this property, these dyes have been widely used as optical probes for m em brane potentials in isola te d s u b c e llu la r fra c tio n s s u c h as synaptosom e p re p a ra tio n,11 m ito ch o n d ria,16,30 sarcoplasm ic reticu lu m,3,4,24,35 and sarcolemmal vesicles.2 These studies suggest that nondestructive fluorescent probes may become useful tools for monitoring m em brane potentials in subcellular vesicle preparations and subcellular organelles as well as in intact cells. It is generally believed that depolarization of the transverse tubular system causes C a2+ release from th e sarcoplasm ic reticu lu m (SR). T he released C a 2+ a c tiv a te s th e c o n tr a c tile m a ch in e ry.10 D u rin g relaxation, C a2+ is reaccum ulated by SR through the adenosine-triphosphate (ATP)-dependent Ca2+pum p. In this process, it is thought that a change in m em brane potential of SR associated w ith th e C a2+ m ovem ent across the SR m em brane may play an important physiological ro le.14,19,22 Recently, m easurem ent of Ca2+ flux and the m em brane potential of SR vesicles from skeletal muscle and reconstituted liposomal SR Ca2 + -ATP ase vesicles has been carried out by several investigators using several d y es.1,3,4,6,24,35 D epending upon the experim ental conditions and vesicles used, either no correlation betw een the m ag n itu d e of th e artificially im posed m em brane potential and the rate of Ca2+ uptake or release by isolated SR vesicles was reported,4 or inhibition and stimulation of Ca2+ uptake was found to be a function of the imposed mem brane pote n tia l on th e re c o n stitu te d v esicular m em brane.36 Since a decrease in the rate of C a2+ u p tak e by SR iso lated from th e ischem ic m yocardium has b een found,12,21,27 it thus would seem pertinent to investigate the relationship betw een th e m agnitude of m em brane potential and C a2+ u p tak e by SR vesicles from ischemic myocardium. In this study, the relation of m em brane potential and Ca2+ flux across the m em brane and the effect of brief ischemia on the membrane pote n tia l and C a2+ u p tak e by SR was studied. M aterials and Methods E x p e r im e n t a l P r o t o c o l The farm pig, weighing 40 to 50 kg, was used as the experim ental model for this study because its major coronary distribution, collateral circulation, and conduction system s blood supply are virtually id en tical to those of m a n.15,18 Sixteen animals w ere anesthetized with six percent phenobarbital adm inistered intravenously following an 18-hr fast. A tracheostomy was perform ed and respiration m aintained with a Harvard pum p respirator using room air supplem ented by oxygen and adjusted to maintain an arterial P a 0 2 above 100 m m H g and a ph of 7.4. A midline sternal splitting thoracotomy was performed and the heart suspended in a pericardial cradle exposing the distribution of the left anterior descending coronary artery. Aortic p ressure, left ventricular pressure, and lead II of the electrocardiogram were continuously monitored.

3 M E M B R A N E PO T E N T IA L A N D C a2+ F L U X O F SA R C O PLA SM IC R E T IC U L U M V E S IC L E S 5 13 The left anterior descending coronary artery was occluded just distal to the first or second diagonal branch using a nontraum atic rubber ligature. Thus, an area of ischemia consisting of approximately 25 to 30 percent of the total left ventricu lar m ass was p ro d u ced. T he lig atu re was applied for 15 m inutes in half the animals and for 30 m inutes in the other half. Ventricular arrhythmia during the occlusion period was treated with minimal effective doses of lidocaine or, in the case of ventricular fibrillation, electrical countershock. At the end of the experiment, 2.5 cc of 10 percent alphazurine 2-G blue dye was injected in a systemic vein in order to identify areas of the myocardium not well perfused. This dye stains p erfused and p resu m ab ly viable tissu e a vivid blue but does not stain nonperfused ischemic tissue23 and has no effect on sarcoplasm ic reticu lu m C a2+ u p tak e or membrane potential measurement. Im m ediately after injection of the dye, the heart was rem oved and taken to a cold room w here samples w ere excised from th e ischem ic area of th e left v en tricle using surface anatomy and dye staining as guidelines. The dye-stained non-ischemic left ventricle was used as a control experim ent. S a r c o p l a s m ic R e t ic u l u m V e s ic l e s Sarcoplasmic reticulum vesicles w ere p rep ared from the sw ine left ventricle with a slight modification of the procedure previously described by Harigaya and Schwartz.12 Essentially, the centrifugation force for ventricular hom ogenate was increased from 8,700 x g to 12,000 x g for the first two spins (20 m inutes each spin) to elim in ate m ito ch o n d rial fragments as com pletely as possible. The p o st-m ito ch o n d rial fraction was th e n centrifuged at 35,000 x g for 30 minutes. T he p elle ts from th e 35,000 x g spin w ere suspended in buffered 0.6 M KC1 and recentrifuged at 35,000 x g for 30 m inutes. The pellets w ere then washed and suspended in 20 mm Tris-m aleate (ph 7.0) and 160 mm KC1 or 160 mm NaCl with a final protein concentration of 4 mg p er ml. Protein concentration of SR vesicles was determ ined by Lowry s m ethod.17 C a 2+ U p t a k e M e a s u r e m e n t s The rate of Ca2+ uptake by isolated SR vesicles was m easured in a spectrophotom eter* using m urexide as a Ca2+ ind icato r T he in cu b atio n m edium contained 160 mm NaCl, 20 mm Trism aleate buffer (ph 7.0), 10 mm M gs04, mm CaCl2, 0.33 mm oxalate, 0.3 mm murexide. Sarcoplasmic reticulum vesicles, Ca2+ and ATP w ere added to the reaction m ixture (final volume three ml) to in itia te C a2+ accum ulation at 23 C. D ual w avelength m easurem ents w ere perform ed at a wavelength pair of 542 and 507 nm. M e a s u r e m e n t o f M e m b r a n e P o t e n t ia l w it h F l u o r e s c e n c e o f C y a n in e D y e For absorption and fluorescence measurements, a spectrophotometer equipped with a locally m ade spectrofluorom eter accessory! was used. 3,3'-D iethylthiadicarbocyanine iodide dye (dis-c2(5)) was used in this study. T he excitation and em ission w avelengths for fluorescence m easurem ents with the dye w ere 622 nm and 670 nm, respectively.28 The incubation m edium for m em b ran e p o ten tial studies is indicated in the legend of the figures. Results In figure 1 is shown an example of m u rexide indicated Ca2+ uptake by SR ves- * Aminco DW2 spectrophotometer, t Cary II spectrophotometer.

4 5 14 P E N G, STRAUB, A N D M U R PH Y F i g u r e 1. An example of Ca2+ uptake by (SR) vesicles preparation from pig h eart left ventricle with the use of murexide indicator. Incubation system contained 160 mm Buffer NaCl, 20 mm Tris-maleate Oxalate (ph 7.0), 10 mm MgS04, Murexide 0.33 mm oxalate, 0.3 mm SR murexide and oligomycin (2 Ag per ml). Sarcoplasmic reticulum vesicles Ca (60 (J.g-a, M-g-c, 240 pog-d and 360 jjig-e) were added in the reaction system, respectively. Ca2 + (400 nmoles O.D=0.005 or a final concentration of 13.3 x 10 5 M) and ATP (3 mm) were added to initiate reaction at 23 C with a final volum e of 3 ml. Inset shows the rate of Ca2 + uptake vs the concentration of Ca2+ added in the reaction system Tim e( m inutes ) Ca (10"5M) icle preparations isolated from pig left ventricle. As noted in figure 1, the slope of SR Ca2+ accumulation increased with the increase of SR vesicle concentration in th e reaction m ixture. However, the rate of Ca2+ uptake by SR vesicles, expressed as nmoles per mg per min (numeral adjacent to each tracing) was not significantly different and reached the maximal rate w hen 100 to 200 fxg protein w ere used. It is im portant to note that oligomycin (2 jxg per ml) was added to each reaction m ixture to eliminate possible C a2+ uptake from m itochondrial contam in atio n. T he in set in figure 1 show s th a t th e concen tratio n of C a2 + used in this study was that which initiated the maximal rate of Ca2+ uptake by SR vesicles under experim ental conditions. It is shown in table I that the rate of Ca2+ uptake by SR vesicle preparations decreased from 263 nmoles per mg per min in the non-ischemic preparation to 190 and 148 nmoles per mg per min in 15- and 30-m inute ischem ic preparations, respectively. A ddition of oligomycin did not significantly inhibit Ca2+ uptake by SR vesicle preparations in all three groups. These results suggest that e ith e r m ito ch o n d rial contam ination of the preparations was minimal or the conta m in a te d m ito c h o n d ria w e re fra g m ented and thus incapable of taking up Ca2+. The Ca2+ uptake dem onstrated in table I is thus primarily attributed to the SR vesicles. TABLE I Rate of Ca2+ Uptake by Isolated Sarcoplasmic Reticulum Vesicles from Pig Left Ventricle* n moles Ca^+ per mg per minute Control 15-Minute 30-Minute Nonischemic Ischemic Ischemic Additions Myocardiurn Myocardiurn Myocardiurn ATP 263 ± 66(n=14) 190 ± 69 (n=7) f 148 ± 73(n=7)$ ATP ± 60(n=14) 188±± 68<n=7)t 140 ± 54(n=7)î Oligomycin Data expressed as mean ± standard error. Incubation conditions were as described in the Methods Section. Oligomycin (2 ug per ml), if used was added to the reaction system after Sarcoplasmic reticulum vesicles were added. fcompared to controls, p < ^Compared to controls, p < with the Student's t-test for paired observations.

5 M E M B R A N E P O T E N T IA L A N D C a2+ F LU X O F SA R C O PLA SM IC R E T IC U L U M V E S IC L E S 5 15 In figure 2 is shown a relationship between the fluorescence intensity of dis- C2-(5) and the m em brane potential of SR vesicles. Fluorescence intensity d e creased with the addition of valinomycin (hyperpolarization) to K+-loaded SR vesicles which w ere incubated in 160 mm N ac l or L ic l m ed iu m (2a). H ow ever, valinom ycin addition did not decrease fluorescence in te n sity of Na + -loaded vesicles which w ere incubated in KC1 or LiCl m edium (2b). Instead, an increase in fluorescence intensity was observed. This result is similar to a previous finding in K +-depleted, N a+-loaded red blood cell ghosts.28 Since dis-c2-(5) is a positively charged molecule, the results in figure 2 suggest th at this fluorescence sensitive dye is accum ulated inside the SR vesicles in an aggregated form when electrical potential inside is negative rel- F i g u r e 3. The rate of K + -gradient dissipation in SR vesicles. One hundred-sixty mm KCl-loaded SR vesicles (40 jxg per 10 jjli) were diluted to 3 ml with medium containing 160 mm NaCl, 10 mm Tris-HCl, ph 7.0 and 5 xm dis-c2-(5). Valinomycin (2 J.M) was added one (I), two (II), three (III), four (IV) and ten (V) minutes after SR vesicle addition as indicated. S.R. TIM E (m inute ) F i g u r e 2. Effect of types of salt-loaded vesicles on membrane potential development, (a) One hundred sixty mm KCl-loaded SR vesicles (40 (xg per 10 (xl) were diluted to 3 ml of medium containing 160 mm NaCl (or LiCl), 10 mm Tris-HCl, ph 7.0 and 5 (jlm dis-c2-(5), (b) 160 mm NaCl-loaded vesicles (40 (xg/ 10 were diluted to 3 ml of medium containing 160 mm KC1 (or LiCl), 10 mm Tris-HCl, ph 7.0 and 5 pal dis-c2-(5). Valinomycin (2 xm) was added to the reaction system to initiate membrane potential. ative to th e outsid e. Thus, vesicles loaded with K + and then incubated in a K + -free m edium develop a m em brane potential due to a diffusion potential of K + out of the vesicles. In figure 3 is shown the rate of K + - gradient dissipation in SR vesicles. Addition of valinomycin at increasing intervals after addition of KCl-loaded vesicles to a K +-free medium produced fluorescence intensity alterations which indicate the m agnitude of the K +-gradient that is present at the tim e of valinomycin addition. These results suggest that the K + - gradient in KCl-loaded SR vesicles gradually dissipates into the K + -free external medium. In order to obtain comparable and maximal m em brane potential m easurem ents, valinomycin was added one m inute after SR vesicle addition in the following studies. In figure 4 is show n a d ecrease in m e m b ra n e p o te n tia l c a u s e d by i n creasing K + concentration in the external m edium (depolarization) resulted in a depression in fluorescence decreases

6 516 P E N G, STRAUB, A N D M U R PH Y S.R. F i gu re 4. Correlation between membrane potential and fluorescence alterations: Incubation medium contained 10 mm Tris-HCl, ph 7.0 and 110 mm NaCl plus 50 mm KC1 (a); 150 mm NaCl plus 10 mm KC1 (b); 155 mm NaCl plus 5 mm KC1 (c); 159 mm NaCl plus 1 mm KC1 (d); and 160 mm NaCl (e). dis- C2-(5) (5 (jlm) and SR vesicles (40 (jlg per 10 jxl) were added to the incubation system as indicated. Valinomycin (2 am) was added one minute after SR vesicles were added. tions of SR vesicles. The result is shown in figure 5. As noted in figure 5, valinomycin-induced fluorescence changes, i.e., magnitude of m em brane potential, increased w hen SR protein concentration increased from 3 xg to approximately 30 (xg and reached a plateau betw een 40 to 50 (xg. U nder such conditions, the m em brane p o te n tia l in SR vesicles isolated from ischemic myocardium decreased in comparison to th e control group. This d e crease was more significant in 30-minute than in 15-minute ischemic myocardium and is in agreem ent with the decrease of Ca2+ uptake by SR vesicles from ischemic tissue. In figure 6 is shown a positive correlation was observed betw een the maximal m em brane potential and the rate of Ca2+ uptake by SR vesicles. In figure 7 is shown a preincubation of SR vesicles from nonischemic myocardium with various concentrations of deof K +-loaded SR vesicles when valinom ycin was ad d ed. T he h ig h er the K + concentration used, the less the fluorescence intensity changed. W hen d e creases in fluorescence intensity reach a minimum, an increase in fluorescence intensity after valinomycin addition is believed to be caused by a loss of the m em brane potential as a result of diffusion of K + from vesicles to the external m e dium. The greater the membrane potential SR vesicles possess, the longer the time required for m em brane potential to collapse. The results of this fluorescence study in relation to m em brane potential are in agreem ent with previous reports on SR vesicles from skeletal muscles, red blood cells and sarcolemmal m em brane v esicles.2 3,4 24,28 T he valinom ycin-induced fluorescence decrease which represents the m agnitude of the m em brane potential in the SR preparation was determ ined at various protein concentra- k Sf 5 3 I 1 <* k. ISi SR PROTEIN CONCENTRATION (u «) F i g u r e 5. Effect of ischemia on SR membrane potential: Incubation medium contained 160 mm NaCl, 10 mm Tris-HCl, ph 7.0 and 5 xm dis-c2(5). SR vesicles (160 mm KCl-loaded) at various concentrations were used and are indicated in the horizontal axis. Valinomycin (2 jjlm) was added one minute after SR vesicles were added. Percent fluorescence decrease is shown in the vertical axis. Normal ( ), 15-minute ischemia ( A), 30-minute ischemia ( -- ).

7 _ 50 M E M B R A N E P O T E N T IA L A N D C a2+ F L U X O F SA R C O PLA SM IC R E T IC U L U M V E S IC L E S 5 17 Ischemic Ischemic Ischemic F i g u r e 6. Correlation between the magnitude of the membrane potential and the rate of Ca2+ uptake by isolated SR vesicle preparations. Experimental conditions were the same as described in table I and figure 5. F i g u r e 7. Effects of deoxycholate preincubation of SR vesicles on Ca2+ uptake and membrane potentials. Aliquots of 0.1 ml of SR vesicles (4 mg per ml) were treated with ml of varying concentrations of deoxycholate (producing final concentrations from 0.01 to 0.9% deoxycholate) at 4 C for two minutes. At the end of two minutes, ten microliters of treated SR vesicles (approximately 36 xg protein) were then used in the reaction systems for both membrane potential measurements and Ca2 + uptake studies. This treatm ent resulted in d e creased m em brane potentials and Ca2+ uptake oxycholate caused a collapse of m em brane potential and a decrease of Ca2 + uptake. B oth m em brane potential and C a2+ uptake d ecre a sed pro p o rtio n ally with the increase of deoxycholate concentration. The results suggest that the rate of Ca2+ uptake and the m agnitude of m em b ran e p o te n tia l of vesicles are closely related. Discussion This study demonstrates that SR vesicles isolated from ischemic myocardium have decreased rates of Ca2+ uptake and decreased m em brane potential generation. The alteration of these m em brane characteristics o ccu rred in as early as 15-m inute ischem ic m yocardium. The decrease of Ca2+ uptake was apparently related to the decrease of m em brane potential developed in the ischemic SR vesicles. This result supports the finding of Zimniak and Racker36 that inhibition and stimulation of Ca2+ uptake is a function of the m em brane potential on the reconstituted vesicles. Beeler also found that the rate of Ca2+ uptake by SR was stimulated by inside-negative membrane po- DEOXYCHOLATE CONCENTRATION Ott added. Non-treated SR vesicles were used for a control by placing them into the reaction systems first and then adding the same amounts of deoxycholate as used above. Deoxycholate did not affect membrane potentials or Ca2+ uptake when added in this manner.

8 518 PEN G, STRAUB, AND M URPHY tential.3 However, the mechanism to explain the parallel m anner in which the d ep ressio n of b o th C a2 + uptake and m em brane potential generation occur is not understood. Beeler suggests that the rate of Ca2+ uptake is activated by the generation of inside-negative potential and that the generation of K + -diffusion potentials was influenced by the anions present in the m edium.3 It is thus conceivable that alteration of the m em brane perm eability to anions, such as C l-, may occur in the ischemic SR vesicles and, as a result, may decrease the rate of Ca2+ uptake by dissipating the imposed membrane p o ten tial. This explanation is supported by the study of the deoxycholate -p retreated SR vesicles from nonischem ic m yocardium. P re tre a tm e n ts re su lte d in p ro g ressiv ely decreasing m em brane potential and Ca2+ uptake. Loss of Ca2+ uptake and membrane poten tial d e v elo p m en t by deoxycholate tre a tm e n t suggest th a t p h ospholipid com ponents of m em brane vesicles are im portant in m aintaining these m em brane functions. Phospholipid com ponents could have a direct effect on the conformation of the m em brane proteins and the perm eability of the SR m em brane. This was evidenced by the study of Ca2 + -ATPase reco n stitu ted vesicles using different phospholipids as previously reported.36 The disappearance of fluorescence intensity w hen non-ischem ic SR vesicles w ere pretreated with deoxycholate is not artifact b u t was caused by the change of m em brane potential due to an alteration of m em brane permeability. A similar result has been rep o rted for SR vesicles isolated from rabbit skeletal muscle.35 Although fluorescence changes of cyanine dyes w ere reportedly not due to direct interaction of the dye with lipids, but to the m em brane potential and SR Ca2+- ATPase,35 the integrity of certain phospholipids on the m em brane is believed to be essential to maintain the membrane potential of SR vesicles.36 Alteration of phospholipid com ponents in SR vesicles from ischemic myocardium may thus be responsible for the observed decrease in m em brane potential and C a2+ uptake. Fatty acid accumulation and abnormal lipid deposition and metabolism in ischemic m yocardium have been re p o rte d.26,33,34 Recently, an alteration of phospholipids which correlated with m em brane dysfunction in ischem ic m yocardium has been found.5 It is thus conceivable that activation of phospholipases may occur in the early stage of ischemic insult and result in the alteration of membrane permeability of SR vesicles. This, in turn, depresses mem brane potential developm ent and Ca2+ uptake and thus interferes with the contraction-relaxation sequence in the ischemic myocardium. It is generally believed that depolarization of the transverse tubular system causes Ca2+ release from the SR during excitation.10 The released Ca2+activates the contractile machinery. The formation of an inside-negative membrane potential in the SR during contraction has been show n in m uscle fib er p re p a ra tio n s.32 However, it is not known whether fluorescence alterations observed in muscle fibers are due to changes in m em brane potential generation by Ca2+ release or other unknown causes such as binding of Ca2+ to contractile proteins, and uptake or release of Ca2+ by mitochondria or by SR. A correlation of the rate of Ca2+ uptake and th e m ag n itu d e of th e insidenegative potential dem onstrated in the isolated SR vesicles may suggest that the regulation of the rate of Ca2+ transport during relaxation is m ediated through the magnitude of negative membrane potential generated by Ca2+-release during excitation. Acknowledgments The skillful surgical assistance of Mr. Wasson Snow is acknowledged as well as the excellent technical assistance of Ms. Shawn Pruitt and Ms. Karen

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