BJoch~,nica et Biophyslca Acta. 1073(1991)394-401 1991 ElsevierScience Publishersn.Y. 0304-4165/01/$03.50 A D O N I S 0?34416591O0]074

Release of fatty acid-binding protein from ischemic-reperfused rat heart and its prevention by mepacrine D i p a k K . D a s 1, P a n k a j K . B a r u a 2 a n d R a n d a l l M . J o n e s 1 ' Vnh,ers~ty o/Canneca~t School n/Med, eine, F~mington. C T (U.S.A.) and 2 Veterans Administration Medical Center. Newingtan. CTeU.S.A.)

(Received 13 June, 1990)

In an attempt to resolve the issue of whether there is a loss of fatty acid binding pretein (H-FABP) from ~ during ischemia and repedusion, and to further examine the role of this protein in isehemle-repeduslon injury, the amount of H-FABP of heart was monitored during isehenda and reperloslon. Excellent con'elatiou was obtained between the loss of H-FABP from heart and its appearance in the pedusate bof|er when examined by Western blot using the specific antibody to H-FABP. Further quantitation was achieved by densltometric scanning of the Western blot and roc.ket electrophoresis. Maximum release of H-FABP was obsereed within 20 rain of repedusion, the t~tal release being 10% of the H-FABP content of the heart. Mepacrine, a membrane stabilizer and a phosplmllpaso inhibitor, reduced the release of H.FABP from the heart and prevented the accumulation of nonesterified fatty acids in the tissue during ischemia and repedusion. In view of the established role of H-FABP in the preservation of membrane plmsphollpids by either scavenging free radicals daring isehenda and re~rfosinn or by modulating the enzymes of phosl~ltoll#d synthesis, it seems likely that the loss of H-FABP may have some eontribmlon towards the ischemle-repedaslon injury.

A growing body of evidence suppnrts a role for abnormal lipid metabolism in the pathogenesis of myocardial ischemic-mperfusion injury. Prolonged ischemia [1 4] or reperfusion following an iscbemic insult [5,6] have been shown to be associated with the accumulation of long chain nonesterified fatty acids (FA) and their thioesters. These amphiphilic substances can induce major changes in membrane function either by inserting free amphiphile molecules into the lipid bilayer or by their detergent-like effects that deplete the membrane lipids [7]. A number of interrelated factors may function in concert to potentiate FA release and their st,~sequent accumulation in ischemic-reperfused myoeardium. Isehemia-indueed inhibition of #-oxidation of fatty acids [8,9] and degradation of membrane phospholipids [1012] may be the two most important factors which can play significant roles in the accumulation of FA and Abbreviations: FABP, fatty acid bindingprolein; FA. fatty acid. Correspondence: D.K. Das, CardiovascularDivision, Department of Surgery, Urfiversayof Connecticut School of Medicine, Farmington, CT O6032U.S.A.

their long-chain acyl CoA esters in the ischemic-reperfused heart. Previous studies have demonstrated that preservation of membrane phospholipids with a corresponding inhibition of FA accumulation may provide a means of protecting an ischemic heart from repcrfusion injury [13-15], During recent years, a specific fatty acid binding protein (FABP) has been described which can bind with FA and their thiol esters [16-18]. The presence of a relatively high concentration of this protein, designated as H-FABP, in mammalian heart cytosol suggests its role in storage of FA and their derivatives [16]. Recent studies demonstrated release of H-FABP from the isolated rat heart subjected to ischemia and reperfusion [19,20]. However, controversies remain as to the extent of H-FABP release from the ischemic-reperfused heart. While the study by Knowlton et al. [20] claimed a reduction of over 50% H-FABP in heart after 60 rain of iscbemia and 60 min of reperfusion, the study by Glatz et al. [19] demonstrated lower amounts of H-FABP release. The present study was undertaken to resolve this problem and to further examine the significance of such H-FABP release from the ischermc-reperhised heart. In the present study, the release of H-FABP was followed continuously as a function of duration of

395 ischemia and reperfusion. The H-FABP ia heart and released H-FABP in perfusate were identified by Western blot analysis using a specific antibody to H-FABP. and quantified using a densitomelric scanner. In addition, rocket electrophoresis was also performed to measure the concentration of released H-FABP from ischemic-reperfused heart. Our s~udies have demonstrated that a specific pbospholipase inhibitor and membrane stabilizer, mepacrine, was able to inhibit the release of H-FABP from the heart. Materials and Methods

Isolation and purification of FABP Rat heart cyfosolic FABP was essentially prepared according to the procedure described by Ockner et al. [21] with slight modification, in brief, the cytosol (100000 x g supernatant containing 5 mg p r o te in /m l) was dialyzed against 10 rnM Tris-HCI (pH 7.5) (buffer A), and the dialyzate was loaded to a Sephadex G-75 (1 × 100 cm) gel filtration column previously equilibrated with buffer A. The second peak eluting in the low molecular mass region ( 2 5 - t 0 kDa) from gel filtration column was loaded onto a DEAE-cellulose (2 × 10 cm) column previously equilibrated with buffer A. Elution was performed with a linear gradient of 0-0.4 M KCI in buffer A. The fractions eluting in 0.1 M KC| concentration were pooled and assayed for FABP activity. The fractions containing FABP activity from DEAE-cellulose column were dialyzed against 10 m M sodium acetate buffer (pH 5.0) and applied to a CMcellulose cation exchange column (1 x 10 cm) previously equilibrated with l 0 m M sodium acetate (pH 5.0) (buffer B). The column was eluted by a linear gradient of sodium acetate (10-200 raM). Fractions containing FABP activity were pooled and the p H was adjusted to 7.4. The purity of the resulting FABP was monitored by sodium dodecyl sulfate (SDS) polyacrylamide gel dectrophoresis under reducing conditions with a 15% separation gel and 3% a stacking gel, using a discontinuous buffer system by the method of Laemmli 122]. Antibody to H-FABP Rabbit polyclonal antiserum raised against purified rat H-FABP was kindly provided by Drs. M.M. Vork and J.F.C. Glatz [23]. Isolated perfused rat heart preparation Sprague-Dawley male rats of about 300 g body wt. were anesthetized with intrapcritoneal pcntobarbital (8 rag/100 g body wt.). Isolated and perfused rat heart was prepared according to the Langendorff te,:hnique as described previously [24]. Hearts were perfused with Krebs-Henseleit bicarbonate (KHB) buffer (pH 7A) equilibrated with a gas mixture of 95~ O 2 and 5~ C O 2 in a nonrecirculating mode for 10 rain for washout, and

then for a further period of 15 rain in the presence or absence of 5O ~M mepacrine. Ischemia was then induced at normothermia for 6O rain, which was followed by 6O min reperfusion with fresh recirculating K H B buffer (50 ml). Perfusates were withdrawn at regular intervals. At the end of reperfusion, heart was freezeclan,fled at liquid nitrogen temperature.

Sodium dode~yl sulfate polyacrylamide gel electrophoresis and Western blot assay Extracled tissue components were separated by SDS- PAG E in presence of reducing agent according to the method of Laemmli [22] using a 15% resolving gel. The molecular mass markers and the tissue components were solubilized at 1 0 0 ° C in SDS- PAG E sample buffer. Ttie molecular mass markers (Bio-Rad) were: phosphorylase B, 97 kDa: bovine serum albumin, 66 kDa; ovalalbumin, 43 kDa; carbonic anhydrase, 31 kDa; soybean-trypsin inhibitor 21.5 kDa; and lysozyme 14,4



e o , c 'B a

Fig. 1. SDS-PAGE of myocardial cytosollc protein~. From right to left: (A) slandard proteins: phosphorylase B. 97 kOa; bovine serum albumin, b6 kDa; ovahilbumin.43 kDa, carbonic anhydrase, 31 kDa; soybean-trypsin inhibitor 21.5 kDa: and lysc~yme 14.4 kDa. (U) Control heart; (C) i~.hemic heart (30 rain): (D) ischemic heart (60 min); (E) 30 rain ischemia followedby 60 rain ~eperfusion; and (F) 60 rain ischen~a followed by 6O rain reperfusion. Lanes B to F contain 200/~g of protein. Arrow indicale$location of FABP band.

396 kDa. Proteins were detected by slaining with Coomassie brilliant blue. For immunohlotting, proteins were transferred electrophoretically from SDS-polyacrylamide gels to nitrocellulose paper as described by Towbin et al. [25] using a Bio-Rad electroblotting apparatus. After nonspecific binding sites ",,,'ere blocked with 3% bovine serum albumin in 0.1 M Tris-HCI buffer (pH 7.4), the nitrocellulose paper was incubated for 2 h with 1 : 5 0 H-FABP antiserum. The nitro~,elhilose papers were then washed two times to remove the excess antibody by Tris buffer (0.02 M Tris-HCI (pH 7.4), containing 0.2% Tween-20). The filters were incubated for 1 h with 1 : 1000 dilution of peroxidase-conjugated goat anti-rabbit immunoglobulin (Bio-Rad). After removing the excess antigens by washing, the immunoreoetive bands were visualized by using 0.05% 4-chloro-l-naphthol and 0.015% H202 as substrate. The reaction was stopped by washing the filters with distilled water.

Assay for FA Myocardial lipids were extracted with c h l o r o f o r m/ methanol mixture according to the method of Foleh et al. [27]. Heptadecenoic acid (17:0) was used as the internal standard. Lipids were subjected to silica gel G, thin-layer chromatography (TLC) using b e x a n e / e t b e r / acetic acid ( 8 0 : 2 0 : 1 , v/v) containing 25 nag butylated hydroxy tohine (BHT) per 100 ml solvent system. The spot corresponding to FA was scraped off, converted to methyl esters, and subjected to gas chro:~mtographic analysis using Model HP5890A Hewlett Packard gas chromatography as described previously [28].

Rocket electrophoresis Rocket immunoelectrophoresis [26] in 1% agarose gels containing (1 : 25) H-FABP antisera was performed m 10 V / c m for 4' h. After electrophoresis, the plates were washed extensively with 0.9% NaCI, dried, and stained with 0.1% Coomassie brilliant blue R-250.


Statistical analysis Student's t-test was used for statistical analysis for comparison between two groups. For the comparison of several independent groups, analysis of variance followed by Scheffe's test also were also performed. Results were considered significant when P < 0.05.

Release of lt-FABP from heart When isolated and iscbemic rat heart was reperfused after 60 min of ischemia, H-FABP leaked out of the heart as a function of the duration of reperfusion.

v io.v}


i! ...... _





Fig. ~ Analysisof cytosolic proteins of heart. From left to fight: SDS-PAGE, Western blot using antibody to FABP, ana ~canalngdensitometl3,. Heart was subjected to 30 rain of ischemlc insult, followedby reperfustorl (A) 15 rain repcrtusion; (B) 30 rain reperfusion; (C} 45 mln repertusiom (D) 60 min reperfusion. Arrow indicates location of FABP band.

~ ~- ~ ~



...L ,

_% , _



ii A







Fig, 3. Analysis of cylOSO]icproleins released from heart, From lefl to r.ght: SDS-PAGF~Western blot using FABF, and scanning densitOmelt% Heart was subjected Io 30 rain of ischemia followed by reper fusion. (A) control (preischemic); (El) 15 rain repeffusion: (C) 30 mill repeffusion: (D) 6(I rain r¢'t~rfusion. ArrOwindicates I~alion of EABP band.

Cytosolic p r o t e i n o b t a i n e d from contro!, ischemic, a n d reperfused heart subjected to S D S - P A G E d e m o n s t r a t e d progressive d e c r e a s e of H - F A B P (14.5 k D a ) in heart w!th the reperfusion time (Fig. l). Ischemic heart re-

rained H - F A B P prior to reperfusion. T h e rate of HF A B P loss was significant u p to 20 rain of reperfusion: thereafter, the rate of loss was r e d u c e d (Fig. 2). T h e a p p e a r a n c e of H ~ F A B P in the perfusatc was


Di~appca~ncc ef H.FABP from Iwart during reperfmion and its appearance in the perJ~ate Isolated rat he,~rt was made isehemi¢ for 60 min, followed by (~0 rain repaffuslon, H-FABP in heart and in perfusate were measured during reperfusion of i~hemie my~ardium. Seven experiments wcrc performed at each time poinl. R~ults are exptes,~edas mean±~E. Minutes after repotfusion O"

Hear t cytosolic H-FABP ( pg protein) Perfusate H-FABP Otg protein)


1387+23 0




1278±19 *

~ 5 1 ±30 *

~45:[: 15 *

T'252:[: 17 *

97 ± 5 *

119± 12 *

125 ± 9 *

121 i 14 *

• P • 0.05 compared to 0 mill (aft~ ischentia at the onset of repaffuslon).

II ~0,l~J




Fig. 4. Effectsof mopacrine on etflux of FABPduring teperrusi0n 0f ischenu¢ heart. From !eft to right: $D$-PAGE,Western btol using antibody to FABP, and scanning densitometl3,. Heart was preperfused in the pl'e~enceof rll~aerlne and ti,en subjected to 30 rain ischemlaas described in Materialsand Methods. Heart wm then reperfused for indicated lime intervals. (A} prei~hemic; (B) IS rain reperfusion; (C) 60 rain repertusion. Arrow indicates location of FABPhand.

increased in concert with the disappearance of the protein from heart (Fig. 3). Analysis of densitometrie scanning of immunoblot demonstrated excellent correlation between the disappearance of H-FABP (14.5 kDa) from heart and its corresponding reappearance in the perfusate (Table 1).

nificant amount of H-FABP loss during reperfusion (Fig. 4). These results compared favorably with the results of the appearance of H-FABP into the perfusate. Western blot analysis failed to identify the 14,5 kDa band, suggesting that mepacrine completely inhibited the loss of H-FABP from heart.

Inhibition of H-FABP release by mepacrine

Quantification of H-¥ABP by rocket electrophoresis

When isolated heart was preperfused with mepacrine prior to ischemia, the heart appeared to suffer an insig-

We further quantified tho amounts of the loss of H-FABP from heart and its appearance in the per[usat¢

399 by performing rocket electrophoresis. As shown in Fig. 5, control h~art shows no loss of H-FABP and the perfusare demonstrates complete absence of this protein. The heart subjected to 60 rain of repcrfusion following ischemia indicated a significant loss of HFABP with a corresponding appearance in the perfusate. Once again, mepacrine-treated hearts showed complete retention of H-FABP, further confirming the results shown in Fig. 4.

Appearance of FA in heart and inhibition by mepacrine The FA content was analyzed in both the treated and untreated hearts. As shown in Table 11, the FA concentration increased significantly in the ischemic heart. It further increased with the duration of reperfusien. Preperfusion of the heart with ,uepaerine almost completely prevented the appearance of FA in the heart. Disotssian Accumulation of FA and their thioesters appears to be one of the major causes for myocardial dysfunction during ischemia and reperfusiou [1-7]. During the past years, a number of interventions have been developed in order to prevent such accumulation of amphiphilic metabolites [1334,29,301. However, enough FA are still accumulated in an isehemic-reperfased heart to cause a number of biochemical and physiological disorders [7]. The intracellniar accumulation of FA and their acyl CoA esters is now believed 1o be regulated by a low molecular weight intracenular protein, FABP [31-34]. These FABPs are present in various organs, including heart 135,36]. Under normal conditions, H-FABP presumably protects the heart from the harmful effects of



17 E

Fig. 5. Rocket electrophoresis of H-FABPoblained from untreated and mepacrine-tre=ted heart, Each lane contained an equal amo~t o[ protein. (A) Control: (el 6O win rcpcrfusion {heart ¢yto~ol); IC) 6O

lain re[effusion mepacfiae-ue.aled(hecLrtcyto~ol);(D) 60 win leperfusion (perftmat¢); and rE) 60 rain reperfuslon mcpaciincqreated



I~lated rat heart was preperfused for 15 min in the pre~nce or absence of mepa¢tin¢. Heart was then made ischemlc for 60 rain, followed by 60 lain reperfuslon FA was estimated from heart. Seven experinlents wereperformed at each time point. R~ults a~e¢xprcs~d as mean+ S.E. FA (nmol/g hcarO ~ ] - - 1 - ~ - . ' o ~8:0 18:1 IS:2 ControIlbaseline) ~.------~'~4~- 1~.5÷ ~--~'± ~ 3.S l.s 0.9 0.5 0.7

20:4 0.2++ 0.2

60 mla ischemia

-mepacdne +mepacrlne

6 8 . 9 ± 20.7± 20.8± 15.gfl= 4.1 2.3 1.2 * 2.2 * 46.3± 16.0± 11.9± 8.2++ 3.7" 3.2 2.1 1.4"

8.2:[. 4.9± 0.8 0.5 * 6.0++



0.3 •

15 ram reperfusion -mepacnne 9 2 . 7 ± 2S.0± 23.4± 19.2++ 18±± 5.6 * 3.2" 2.8" 3.6 * 23 ** +mepacrine 52.8± 19.8± 12.9j: 9.14- 8.3++ 4.6 " 1.S 2.3" 3.1 " 1.O "

7.8± t.2 *,t 1.6+ 0.4 "

60 rain reperfusion -mepa¢fin¢ 131.2± 38.5± 10.6 * 4.S *.t +mcpacline 58.6=b 19.55= 5.1" 2.3"

9.6± 0.8 *'t 1.8++ 0.3"

27.2++ 22.9++ 28.4++ 3.2 * 4.1 * 2.4 ** 15.1± 9.7++ 8.7++ 1.6" 2.2" L6"

• P < 0.05 ~mpared to baseline. t p < 0.05~mpared to rain ischemia. • P < 0.0'5 - mcp~fi~ v~ + mepacrine. FA by binding with them. Rccem studies demonstrated that a significant amount of H-FABP leaks out of the heart during ischemia and reperfusian [19,20], thus rendering the heart susceptible to the amphiphiles" attack. The present study confirms these previous reports and further demonstrates that H-FABP is released from heart durivg the initial 30 rain of reperfusion following an it,chemic insult. Release of H-FABP is minimal at the onset of reperfasion, enhances as reperfusion progresses, and finally lev01s off after 20 mill of ~perfusion. The amount of total release of H-FABP over the period of 60 rain of repetfttsion is about 107o of that of the tissue content. Using enzyme-finked immunosorbent assay (ELISA), it was previously shown that about 3% of total H-FABP was released during postischemie reperfusion when heart was perfused by the Langendorff technique using modified Tyrode buffer [19]. In another experiment, over 5O% of H-FABP was shown to be leaked out when postischemi¢ heart was perfused with blood [20]. In the present study, both tissue and perfusate H-FABP was measured as a function of duration of ischemia and reperfusion. Scanning of H-FABP band obtained from Western blot experiments showed excellent correlation between the loss of H-FABP from tissue and the appearance of the same protein in the petfusate.

400 A maximum amount of H-FABP leached out after about 20 rain of reperfusion. The FA level of heart was increased during reperfusion, as expected. A number of recent studies also revealed increased FA content during reperfusion of ischemic myoeardium [37,38]. There are two major sources for FA: (i) from breakdown of membrane phospholipids, and (ii) from the inhibited B-oxidation. Most of the studies demon s tr a te d accumulation of lysophosphoglycerides and arachidonic acid in the ischemic-reperfused heart [37,38]. Breakdown of membrane phospholipids in concert with defective reacyla]ion of lybophosphoglycerides has been attributed as the major cause for accumulation of this amphiphilic metabolites [6,10]. Loss of unsaturated fatty acids may subsequently result in modulation of membrane fluidity 139] and, thus, may enhance Ca 2+ permeability of the sarcoplasmic reticulum [40]. Ca 2÷ overloading may be the ultimate cause for cellular injury and tissue death observed in the ischemic-reperfused heart [41]. In view of the probable role of pbospholipid degradation in a Ca2+-permeability defect and subsequent cellular injury, Chien and his co-workers [4] demonstrated the efficacy of chlorpromazine, a phospholipase inhibitor, on ischemia-induced phospholipid degradation. Using mepacrine, also a phospholipase inhibitor, we were able to protect an ischemic heart from reperfusion injury [10,14,15]. Myocardial protection was attributed to the preservation of membrane phospholipids and the corresponding reduction in FA and lysophosphoglyceride accumulation. Besides its inhibitory action towards phospholipases, mepacrine can also bind to the membrane bilayers by its amphiphilic property to influence lipid packing, thus exerting a membrane stabilization effect. In this study, mepacrine significantly inhibited accumulation of FA and release of H-FABP from heart during ischemia and reperfusion. In previous experiments, mepaerine was found to reduce the release of creatine kinase (CK) from heart, a presumptive marker for cellular injury and tissue necrosis [10,15]. Taken together, these results suggest that mepacrine protected the ischemic-reperfused heart with a corresponding reduction in H-FABP release, further suggesting that loss of H-FABP (like loss of CK) may serve as a specific marker for cellular injury. However, whether the loss of H-FABP is the cause or consequence of the cell damage remains unclear. Recent studies from our laboratory indicated a specific physiological role of H-FABP in phospholipid biosynthesis in heart [41]. Thus, H-FABP enhanced acyl CoA monoacylgiycerophosphorylcholine acyltransferase and glycerophospbate acyltransferase in isehemic-reperfused heart to limit the loss of membrane phospholipids. Hence, it is likely that the loss of H-FABP from heart during ischemia and reperfusion may be iustru-

mental for th2 potentiation of phospholipid breakdown. In another study, H-FABP was found to scavenge oxygen-derived free radicals [42]. It is unequivocally established that reperfusion of iscbemic myocardium is associated with the generation of oxygen-derived free radicals [48-45], which may attack membrane phospholipids, causing their breakdown [46]. These free radicals, in physiologic concentrations, do not appear to affect the fatty acid binding capacities of H - F A B P [47]. Taken together, these results would tend to suggest that loss of H-FABP may have some contribution towards the reperfusion injury arising from the decrease in H-FABPlinked free radical scavenging activity, In summary, our results confirmed the previous reports on the release of H-FABP from heart during ischemia and reperfusion. It further demonstrated that the release of H-FABP may be considered as a nonspecific phenomenon resulting from ischemia/reperfnsion-induced cellular injury. Loss of H-FABP may be instrumental for the potentiation of membrane phospholipid breakdown, either via the free radical mediated process or by directly modulating the enzymes of the pbospholipid biosynthesis pathway. Acknowledgements These studies were supported by grants from N I H (HL22559, HL33899, and HL34360). The skillful secretarial assistance of Mrs. Joanna D'Aprile is gratefully acknowledged. References

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Release of fatty acid-binding protein from ischemic-reperfused rat heart and its prevention by mepacrine.

In an attempt to resolve the issue of whether there is a loss of fatty acid binding protein (H-FABP) from heart during ischemia and reperfusion, and t...
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