J

Mel Cell Cardiol 22, 1467-1475 (1990)

The

Effect

of Anoxia

Tar-Ame

on Lipid Cardiac

Hagve,

Department of Physiological

Howard

Metabolism Myocytes Sprecher

in Isolated

and

Chemistry, College of Medicine, Ohio 43210, 1JSA

Charlene

Adult

Rat

M. Hohl

The Ohio State Universi@, Columbus,

(Received 3 November 1989, accepted in revised form 29 June 1990) T.-A. HAGVE, H. SPRECHER AND C. M. HOHL. The Effect ofAnoxia on Lipid Metabolism in Isolated Adult Rat Cardiac Myocytes. Journal of Molecular and Cellular Cardiology (1990) 22, 1467-1475. In ischemic myocardium abnormal lipid metabolism results in accumulation of compounds that are deleterious to membrane structural integrity and membrane dependent functions. In this study isolated adult rat ventricular myocytes were used to investigate anoxia-induced alterations in cellular lipid composition and metabolism. Myocyte phospholipid content declined 197; on average during 60 min anoxia and intracellular arachidonic acid increased 3-fold, without affecting myocyte ATP content. Anaerobic incubation in the absence ofglucose depleted cellular ATP to 2 nmol/mg protein, elicited a 230/, decrease in phospholipids, and reduced triacylglycerol content by 519,. Intracellular levels of C1&& fatty acids were significantly elevated, especially palmitic and arachidonic acids. Myocytes presented with 0.08 mM [l-‘%I-palmitic or arachidonic acid acylated 857, (25-26 nmol/mgj of the fatty acid taken up into triacylglycerols. Anoxia decreased this esterification by 46-60%. Formation of [“ClCO2 was also depressed 70-90% by anaerobiosis. The results demonstrate that anoxia stimulates degradation of complrx lipids, with a concomitant increase in non-esterified fatty acids. especially arachidonic acid. KEY

WORDS:

Cardiac

myocyte;

Rat

heart

cell; Anoxia;

Lipid;

Fatty

acid;

Phospholipid;

Triacylglycerol:

j?-oxidation

Introduction

toyltransferase II activities, and elevated Derangements in cardiac lipid metabolism NADH inhibit /&oxidation (Pauly et al., 1987; manifest in ischemic or hypoxic myocardium Liedtke, 1981), resulting in high intracellular have been correlated to the inhibition of oxi- levels of long chain acyl-CoAs and acylcarnidative phosphorylation and resulting dimin- tines (Whitmer et al., 1978; Heathers et al.. ished supply of ATP in thesehearts (Katz and 1987). Ischemia is a complex entity consisting of Messineo, 1981; van B&en et al., 1989; Chien et al., 1985). Membrane phospholipids are hypoxia, acidosis, substrate deprivation and degraded, either as a result of increased phos- accumulation of metabolic wastes. It is diffithe contribution of each to the pholipase activity or in responseto reduced cult to assess phospholipid synthesisthat accompaniesnor- lossof contractile function that occurs during mal phospholipid turnover. Accordingly, lyso- an ischemic episode.The purpose of this study phospholipids and non-esterified fatty acids, was to examine the effects of anoxia, with and which have been implicated in cardiac mem- without ATP loss, on lipid metabolism in brane dysfunction and arrhythmogenesis, isolated adult rat ventricular cells. The results demonstrate that phospholipid content was accumulate [rev. Corr et al., 1984). Furthermore, decreased ATP-dependent fatty acid reduced approximately 20% by anaerobiosis, activation, lowered mitochondrial carnitine- irrespective of cellular ATP content, whereas acylcarnitine translocaseand carnitine palmi- triacylglycerols declined only in energ) Abbreviutzons;

nicotinamide

HEPES, 4. i2-hydroxyethyl)-1-piperazineethane adenine dinucleotide; ATP, adenosine triphosphate;

sulfonic ZAN.

acid; BSA, bovine serum albumin: total adenine nucleotides.

Plrase address all correspondence to: Charlene M. Hohl, Department of Physiological Univrrsity. 5170 Graves Hall, 333 W. 10th Avenue, Columbus, OH 43210, US.4. 0022-2828/90/

12 1467 + 09 $03.00/O

c

Chemistrv. 1990 Academic

NAD(

The Ohio

H:. Statr,

Press L.imited

1468

T.-A. Hagve et al.

depleted myocytes, with a concomitant the levels of free fatty acids.

Materials

rise in

and Methods Chemicals

[l-‘4C]-Palmitic acid was obtained from American Radiolabeled Chemicals Inc, St. Louis, MO. [ l-‘4C]-Arachidonic acid was made by total synthesis (Sprecher and Sankarappa, 1982). Eicosenoic acid and trieicosefrom Nu-Chek Preparations, noin were Elysian, MN. Essentially fatty acid free bovine serum albumin (BSA) was purchased from Sigma Chemical Co., St. Louis, MO. Collagenase was obtained from Worthington Biochemical Co., Freehold, NJ. BME and MEM amino acids were from Gibco Laboratories, Grand Island, NY. Cardiac myocyte isolation and incubation Cardiac myocytes were isolated from male Sprague-Dawley rats (30@350 g) as described previously (Li et al., 1988). Approximately 30 mg of protein was obtained from each heart and the viability was 80-90?,, as measured by trypan blue exclusion and by retention oflactate dehydrogenase (Murphy et al., 1982). Between 75 and 90% of the cells had the rod shape which is characteristic for cardiac myocytes in situ. Freshly isolated cells were incubated (37°C) in 4- (2-hydroxyethyl) - 1-piperazine ethanesulfonic acid (HEPES) buffer (pH 7.4) containing in mM: 25 HEPES, 118 NaCl, 4.8 KCl, 1.2 MgS04, 1.2 KHzP04, 1 CaCls, 5 glucose, 5 pyruvate, a complete mixture of amino acids (MEM and BME; Gibco Lab.) and 2% (w/v) essentially fatty acid free bovine serum albumin. Pyruvate (5 mM) was present in the incubation medium for all protocols but glucose was omitted where indicated in the text. In experiments designed to study the effect of anoxia on fatty acid uptake, 2.5 mg of cells in a total volume of 1 ml were incubated for 60 min with the albumin-bound [ 1-14C]-labeled fatty acid (specific activity: 7 mCi/mmol) in sealed vials flushed with either 950/b oxygen : 546 carbon dioxide (aerobic) or 1OOq/, argon (anoxic) .

Lipid analysis Measurement of [i4C]-labeled water soluble products and [i4C]-CO2 were performed as previously described (Hagve and Christopherson, 1984). Cells were separated from their suspending medium and washed rapidly with the incubation medium. The lipids in the suspending medium (plus supernatant from wash steps) and myocytes were extracted separately according to Folch et al. (1957). Lipid fractions were then separated by thin layer chromatography (Hagve and Sprecher, 1989), scraped into scintillation vials and counted. Non-labeled myocytes and their suspending medium were separately Folch extracted, and lipid fractions separated on Unisil columns as previously described (Voss and Sprecher, 1988). An aliquot of the phospholipid fraction was removed for total phosphorus analysis (Rouser et al., 1970). In some experiments, phospholipids were fractionated by thin layer chromatography using Whatman LK5 plates (Whatman, Clifton, NJ) with CHCls/MeOH/ 40% methylamine ( 120 : 40 : 10, v/v/v) as the solvent. Individual phospholipids were visualized by spraying with a 0.196 solution of 2’,7’dichloroflurescein in ethanol, and were scraped into test tubes for phosphorus analysis. Neutral lipids were separated into diacylglycerol, triacylglycerol and free fatty acid fractions by thin layer chromatography (Hagve and Sprecher, 1989). Each fraction was recovered by extracting the silica gel with CHCls/MeOH (2 : 1, v/v). All fractions were reacted with 3% (w/v) anhydrous HCl in methanol for 60 min at 80°C and the lipids subsequently extracted with hexane. Methyl esters were separated on a Varian Vista 6000 gas chromatograph equipped with a 10 ft by 2 mm id. glass column packed with lo:/, SP-2330 on lOO/ 120 mesh Supelcoport (Supelco, Bellefonte, PA). The carrier gas was helium (30 ml/min) and the temperature of the injector and detector were 240°C and 250°C respectively. The column temperature was initially 180°C and after 17 min increased S”C/min to 200°C which was maintained until the methyl ester of 22:6 (n-3) was eluted. Methyl esters were identified by comparing retention times with authentic standards. The concentrations of

Lipid

Changes

in Anoxic

the lipid fractions were determined by calculating the mass of each of the major fatty acids and correlating the peak areas to that of the internal standard. Analysis of adenine nucleotides and protein content Myocytes were separated from their incubation medium by sedimentation through a layer of bromododecane into 2N HC104. Nucleosides and nucleotides in the neutralized extract were separated on a Whatman Partisil 10 SAX HPLC column using a phosphate and pH gradient as described previously ( Altschuld et al., 1987). Protein was measured according to Lowry et al. (195 1) in an aliquot of the original cell suspension for lipid studies and in the protein pellet remaining after acid extraction for nucleotide analysis. Statistics All analytical procedures were performed in duplicate and the values averaged to obtain each individual data point for a given preparation of myocytes. The data given in the text represents the mean + standard deviation for 3-4 different cell preparations. Statistical analysis was performed using Student’s t-test when only two groups were being compared. In three group comparisons (Table 2 and Fig. 2), a one way analysis of variance (ANOVA) followed by Scheffe’ tests was performed (STATPAK 1987, Merrill Publishing Co, Columbus, OH). Values were considered significant when P < 0.05. Results

Endogenous lipid composition in myocytes Table 1 depicts the lipid content and fatty acid composition of isolated adult rat cardiomyocytes incubated in an oxygenated glucosecontaining medium. Over 90% of myocyte lipids were phospholipids (expressed as phosphorus equivalents per mg myocyte protein), with the major subclasses consisting of 57.1 + 4.1% choline-, 29.2 f 2.9% ethanolamine-, 3.4 + 0.4% serine-, and 5.0 + 2.9% inositol-containing glycerophospholipids. Cardiolipin plus sphingomyelin accounted for 3.3 + 1.2O/, of the phospholipid pool.

Cardiac

Myocytes

1m

Incubation of glucose-containing myocyte suspensions under argon for 60 min at 37°C resulted in a slight decrease in endogenous phospholipids compared to oxygenated cells (Table 2). This 19”/b decrease was statistically significant (P = 0.02) only when analyzed by a paired t-test comparing each anoxic preparation with its respective aerobic control. When data for separate myocyte preparations incubated anaerobically without glucose were included and the three groups compared by ANOVA, the decline was not statistically significant. The relative proportion of individual phospholipid subclasses was not altered under these conditions (results not shown). Except for a 3-fold increase in the level of arachidonic acid in the intracellular free fatty acid fraction (66.3 pmol/mg protein in aerobic myocytes; 224.4 pmol/mg protein in anoxic cells), the fatty acid composition of cellular lipids and of the free fatty acid fraction was not modified by anaerobiosis in the presence of glucose (results not shown). Adenine nucleotide content (Table 2) of myocytes was maintained through anaerobic glycolysis (up to 12 nmol lactate/min/mg protein produced under these Accordingly, NADH levels conditions). increased significantly (Table 2) without affecting total pyridine nucleotide content.

Effect of energy depletion on endogenous lipid content Cardiomyocytes incubated for 60 min under an argon atmosphere in a glucose-free medium lost 82% of their adenine nucleotides compared to respiring cells, but maintained a high pyridine nucleotide redox potential (Table Endogenous phospholipids 2). decreased 237, on average compared to normoxie cells (Table 2), without a change in phospholipid fatty acid composition. Triacylglycerols were reduced by 5 1 OjO, and the contents ofesterified palmitic, stearic, and linoleic acids were decreased in this lipid fraction [Fig. 1 (a)]. The levels of all intracellular free fatty acids rose significantly [Fig. 1 (b)]; especially notable were increases in palmitic and arachidonic acids. Approximately 8 nmol fatty acids per mg myocyte protein were released to the suspending medium of energy-depleted cardiac cells, apparently via back diffusion from

T.-A. Iiagve

1470 TABLE

1. Endogenous

lipid

composition

et al.

of aerobic

cardiac

myocytes.

Myocyte lipid content (nmollmg protein) Phospholipid

Fatty 16:0 16:1 18:0 18:1 18:2 20:4 22:5 22:6

acid

50

(“/$

of

composition

(n-6) (n-6) (n-3)

(n-6)

Triacylglycerol

299 f

11.6 0.4 21.4 7.7 19.6 17.4 1.9 11.6

Diacylglycerol

19.5 *

2.0

29.8 1.5 13.9 11.6 13.3 5.2

3.7 1.0 1.9 0.3 2.9 1.4

Free

3.4 * 0.9

fatty

acid

1.7 f 0.2

total)

+ 1.2 + 0.1 + 0.7 * 1.7 f 0.8 + 1.4 f 0.3 f 2.0

f f f + f +

18.7 + 3.3 + 20.7 + 20.2 & 9.7 &15.6 + N.D. 8.7 +

3.:&.1

2.1 0.8 2.4 5.1 1.7 3.2

27.5 6.7 20.6 21.4 6.4 3.9

+ f + + f &-

1.0 0.3 2.5 2.8 0.3 1.7

N.D.

3.2

4.5 + 1.9

Myocytes were incubated for 120 min under aerobic conditions (37°C) and then analyzed for lipid content as described in the methods. Phospholipid content is based on phosphorus equivalents per mg myocyte protein, whereas other lipid fractions were estimated based on their fatty acid content. Data are the mean f standard deviation of incubations from 4 myocyte preparations. ND denotes concentration was below detection limits. Fatty acids are indicated by their chemical notation: palmitic (16:0), palmitoleic (16: l), steak (18:0), oleic (18: I), linoleic (18:2 n-6), arachidonic (20:4 n-6), docosapentaenoic acid (22:5 n-3), docosatetraenoic acid (22:6 n-6).

myocytes. The distribution pattern of extracellular fatty acids closely paralleled that of the endogenous fatty acid fraction (data not shown). Viability

determined by their ability to retain lactate dehydrogenase. Viability decreased to 75% after 120 min aerobic incubation. Anaerobiosis resulted in a further loss in viability (10% reduction in the presence of glucose; 15% decreasein the absenceof glucose).

of myocytes

The observed differences in cellular lipid composition in anoxic cells may to someextent be attributed to changesin cell viability. Freshly isolated myocytes were 84 If: 3% viable as TABLE

2. Lipid

Glucose

(5 mM)

and

nucleotide

Deposition and oxidation of labeled fat@ acids by myocytes

In the previous section we have shown that

content

of aerobic

Aerobic

Anoxic

+

299.0 19.5 3.4 1.7 24.7 28.7 5.2

+ + * + f &+

49.8 2.0 0.9 0.2 5.0 5.4 0.9

242.7 19.5 2.4 2.2 22.3 29.6 0.71

anoxic

myocytes. Anoxic -

+ (nmol/mg

Phospholipid Triacylglycerol Diacylglycerol Free fatty acid ATP XAN NAD/NADH

and

cell protein) If: f f f + f +

45.8 2.4 0.8 0.6 1.0 2.0 0.13*

229.9 9.5 4.4 4.2 1.8 5.3 5.7

f f + + f + +

54.0 0.6* 0.8 1.2* l.l* 1.2* 0.4

Myocytes were incubated for 60 min at 37°C in an oxygenated glucose-containing medium (Aerobic), under argon in the presence of 5 rnM glucose (Anoxia + glucose), or under argon in the absence of glucose (Anoxia - glucose). Cellular lipids and nucleotide levels were determined as described in the methods. Phospholipids are nmol phosphorus equivalents/mg myocyte protein, other lipid fractions are nmol fatty acid equivalents/mg protein. Mean f standard deviation for S-4 preparations are given. * denotes significant difference compared to aerobic cells (P < 0.01).

Lipid

.f $ 2or & I L

73

cE

0

L

Changes

in Anoxic

-- -____-.

160 l&l

Triacylglycerol

(a) ~

18:O 18:l 18:2 20:4 22:6 (n-6) (n-6) (n-3)

Free Fatty Acids

(b)

F

\ 0.5 ‘0 E = 0.0

l6:O 16: I 18:O 18: I l8:2 20:4 22:6 (n-6)(/7-6) (n-3)

Cardiac

Myocytes

14il

Under anoxic conditions in the presence of glucose, cellular uptake of [l-‘4C]-palmitic acid was significantly decreased by 26Oh and [ 1 -‘4C]-arachidonic acid uptake was reduced by 43% (Table 3). Esterification of labeled fatty acid into triacylglycerols decreased substantially (46% reduction of palmitate and 61 y. reduction of arachidonate) in myocytes incubated under an argon atmosphere compared to oxygenated cells. The intracellular [“Cl-free fatty acid fraction increased 5 to IO-fold in anoxic cells compared to control, such that free fatty acids accounted for I l”,, (palmitic acid) or 23:/, (arachidonic acid, of the accumulated label. Fatty acid oxidation, measured as production of [14C]-C02, also declined during anaerobiosis (Table 3). Prelabeling

of

myocytes with radioactive.fatf

y acid

FIGURE 1. Fatty acid composition of endogenous triacylglycerol and free fatty acid fractions of cardiac myocytes. Myocytes were incubated in oxygenated HEPES-buffered medium for 60 min (37”C), followed by 60 min incubation in oxygenated medium in the presence of glucose 0 or under argon in the absence of extracellular glucose 8. (a) Fatty acid content of triacylglycerols; (b; Intracellular levels of non-esterified fatty acids. Results are the mean + standard deviation from 3-4 myocyte preparations and are expressed as nmol fatty acid/mg cell protein extracted in each fraction. * P < 0.05 compared to aerobic incubations. Fatty acids are indicated by their chemical notation: palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (la:]), linoleic acid (18:2 n-6), arachidonic acid (20:4 n6!, docosatetraenoic acid 122:6 n-3).

In order to study the effect of anoxia on intracellular fatty acid metabolism independent of the inhibitory action on fatty acid uptake, cardiac myocytes were prelabeled for one hour at 37°C with 0.08 mM [I-14C]palmitic acid or [I-‘4C]-arachidonic acid under aerobic conditions, resuspended in [14C]-fatty acid free medium, and subsequently subjected to normoxic or anoxic incubation. The intracellular distribution of the [14C]-labeled fatty acid immediately after the prelabeling period, but prior to further incubation, is therefore as indicated for Control cells in Table 3.

anoxic incubation of adult ventricular myocytes primarily affected the distribution of palmitic and arachidonic acids. Therefore, the metabolism of these two fatty acids by isolated adult cardiomyocytes was examined in more detail. Myocytes presented with 0.08 mM [ 1-‘4C]-palmitic or [ 1 -14C]-arachidonic acid incorporated or oxidized approximately 95% of the available labeled fatty acid within one hour under aerobic conditions (Table 3). Under these non-equilibrium labeling conditions, incorporated fatty acid was primarily esterified into triacylglycerols (85% of total uptake), with lesser amounts acylated into phospholipids (4-7%) or recovered as free fatty acids (l-2%).

Myocytes prelabeled with [l-‘4C]-palmitic acid Figure 2 shows the pattern of [14C]-fatty acid in myocyte lipids that were exposed to aerobic or anaerobic conditions after a one hour prelabeling period with either [ l-‘4C]-palmitic acid or [l- “C]-arachidoni c acid. There was a modest redistribution of [ 1 -‘4C]-palmitic acid in prelabeled myocytes incubated aerobically for an additional 60 min. Radioactive palmitic acid acylated into triacylglycerols decreased by 4.5 nmol/mg protein, whereas increased esterification into phospholipids and loss to the extracellular medium accounted for 1.2 nmol/mg protein each. Approximately 2.0 nmol [14C]- palmitic acid/mg protein was p-oxidized during the post-labeling aerobic incubation.

T.-A. Hagve et al.

1472

TABLE

3. Deposition

and oxidation

of labeled

fatty acid

in

aerobic

and

anoxic

cardiac

myocytes.

[I-‘“Cl-palmitate Anoxia

Control

Uptake B-Oxidation Phospholipid Triacylglycerol Diacylglycerol Free fatty acid

f 1.64 f 1.16 f 25.44 + 1.12 + 0.52 f 30.08

[l-‘4C]-arachidonate

0.72

0.24 0.20 0.60

0.12 0.04

22.20 0.64 2.56 13.64 0.64 2.52

f f f f f &

Myocytes (2.5 mg/ml) were incubated with 0.08 mM 60 min under an oxygen (Control) or argon (Anoxia) expressed as nmol/mg cell protein of labeled fatty acid to [14C]-C02. Mean + standard deviation of parallel significant difference compared to Control (P < 0.05).

Control

5.76* 0.24* 0.28* 0.76* 0.20* 0.76*

31.04 1.72 2.24 26.28 0.44 0.36

f f + f f +

Anoxia

0.44 0.56 0.12 0.56 0.04 0.08

17.76 0.16 2.16 10.72 0.56 4.04

+ f f + + +

3.48* 0.08* 0.32 2.08* 0.20 2.28*

[I-‘4C]-paImitic acid or [I-14C]-arachidonic acid for atmosphere in the presence ofglucose. The results are incorporated by myocyte lipid fractions or B-oxidized incubations from 3 preparations are given. * Denotes

In the presence of glucose, anaerobic incubation of myocytes prelabeled with [“Clpalmitic acid resulted in a significant decline in [14C]-palmitic acid esterified into triacylglycerols with corresponding increases in both intracellular (1.9 nmol/mg protein) and extracellular free fatty acids [2.9 nmol/mg; Fig. 2(a)]. These effects were more pronounced when glucose was absent from the suspending medium [2.4 nmol/mg intracellular free fatty acids and 6.8 nmol/mg free fatty acids in the suspending medium; Fig. 2 (a)]. B-Oxidation was decreased by 60% under anaerobic conditions.

Release of lipids by digitonin-treated myocytes In studies investigating myocardial lipid changes in response to anoxia or ischemia in the whole heart, it is difficult to assess the effects of cell death on the total measured response. We have therefore estimated the contribution that myocytes with broken sarcolemma1 membranes make to the suspending medium radioactivity. Myocytes prelabeled with [l- “C]-palmitic acid were lysed with digitonin (16 pg/mg protein), thus releasing cytosolic components and exposing mitochondria to detrimental levels of calcium (Murphy et al., 1982), and incubated further for 60 min. The suspending medium of lysed myocytes contained 4.2 f 0.2 nmol/mg cell protein of free [14C]-palmitic acid and 1.3 nmol/mg of [‘4C]-palmitic acid esterified into complex

0

PL

TG

FFA

SM

FIGURE 2. Content of labeled fatty acid in cellular lipids and suspending medium. Myocytes were prelabeled with 0.08 mM [I-‘4C]-palmitic acid (a) or [l-14C]arachidonic acid (b) for 60 min at 37°C. Cardiac cells were then resuspended in [“Cl-fatty acid free medium and incubated further for 60 min in oxygenated glucosecontaining medium 8, under argon in the presence of glucose q , or under argon in the absence of glucose @. Bars represent the content of labeled fatty acid recovered in each lipid fraction (phospholipid, PL; triacylglycerol, TG; non-esterified fatty acid, FFA) or extruded to the suspending medium (SM). Mean f S.D. for three different cell preparations or the average of two preparations (anoxic without glucose) are given. In the latter group, the range was 4-l 1 o/0 of the average in those fractions which were significantly different from respective aerobic controls. * P < 0.05 compared to aerobic incubations.

Lipid

Changes

in Anoxic

lipids. Thus, in anoxic incubations of intact cells less than 1 nmol/mg protein of the labeled fatty acid released to the suspending medium could be associated with cell death, and the remainder must be attributed to back diffusion from the cells. Myocytes prelabeled with [I-“Cl-arachidonic acid The intracellular distribution of [l-14C]arachidonic acid in complex lipids of prelabeled myocytes was not appreciably altered by an additional 60 min aerobic incubation [compare data in Table 3 with Fig. 2(b)]. A loss of isotope (2.0 nmol/mg) in the triacylglycerol fraction of myocytes was recovered as small increases in [‘4C]-arachidonic acid in the phospholipid and intracellular free fatty acid fractions (0.4-0.5 nmol/mg each), and the suspending medium [Fig. 2(b)]. [‘4C]-Arachidonic acid prelabeled myocytes incubated under argon in the presence of glucose showed a 16% decline in triacylglycerol-esterified arachidonic acid that was quantitatively recovered in the suspending medium as [14C]-labeled compounds [Fig. 2(b)]. The same pattern emerged when prelabeled cells were suspended in a glucosefree medium under anoxic conditions except that the loss of arachidonic acid from triacylglycerols was even greater (5.6 nmol/mg decrease) with a similar increase in suspending medium radioactivity. For all incubation conditions studied, 857; of the [14C]-labeled compounds recovered in the suspending medium were free fatty acids (results not shown!. [‘4C]-C02 production was not detected in anoxic myocytes prelabeled with [l-‘4C]-arachidonic acid. Discussion These results demonstrate that adult ventricular myocytes incubated under anoxic conditions, with or without cellular ATP loss, have depressed phospholipid levels. In this model activation of phospholipases having specific substrate preferences cannot be implicated since the composition of phospholipid subclasses, as well as, phospholipid fatty acid distribution remained unaltered. This generalized phospholipid reduction may, in part, reflect the small decline in viability observed

Cardiac

Myocytes

1473

under these conditions, but cannot be solely linked to de-energization. Recent investigations have suggested that there is a continuous remodeling of cardiac membrane phospholipids and that accumulation of free fatty acids, especially arachidonic acid, is due to a defective reacylation in ischemic heart (Chein et al., 1984). Elevation of non-esterified unsaturated fatty acids found primarily in phospholipids [Fig. 1 (b)], especially the 3-fold increase in intracellular arachidonic acid in anoxic myocytes would support this hypothesis. Endogenous triacylglycerol content was not affected by anaerobic incubation in the presence of glucose. By contrast, glucose-deprived anoxic myocytes showed a 51 y/b decline in triacylglycerol levels (Table 2). Under energy depleting conditions, the reduction of endogenous palmitic, stearic and linoleic acids esterified into triacylglycerol corresponded to the observed rise of these fatty acids intracellularly (Fig. 1) and in the suspending medium (data not shown). Experiments performed with myocytes where the triacylglycerol pool was prelabeled with radioactive fatty acids confirmed these findings (Fig. 2). In these studies, it is not possible to determine whether reduced triacylglycerol levels reflect increased triacylglycerol hydrolysis or decreased synthesis. However, triacylglycerol synthesis may be limited in these glucose-free incubations due to inadequate quantities of glycerol-3phosphate that woujd normally be generated by the glycolytic pathway. Additionally, insufficient ATP under these conditions (Table 2) would inhibit acyl-CoA synthetase activation of fatty acids. The accumulation of intracellular free fatty acids was not quantitative compared to their deacylation by complex lipids. This probably reflects a rise in fatty acyl-CoAs and acylcarnitines [Heathers et al., 1987; Whitmer et al., 1978) and a release of fatty acids to the suspending medium that has been reported under similar conditions, but were not measured in these studies. /?-Oxidation was inhibited 60-90?,, by anaerobiosis (Table 3). In the absence of electron transport, glycolyzing myocytes accumulate NADH (Table 2), which is a known inhibitor of j-hydroxy-acyl-CoA dehydrogenase (Witmer et al., 1978; Lledtke, 1981). In the absence of glucose, glycolytic

1474

T.-A. Hagve

activity is low and consequently NAD/NADH ratios remain high (Table 2). However, under these conditions the substantial fall in ATP content may limit fatty acid activation resulting in less substrate available for mitochondrial B-oxidation. Pauly et al. (1987) reported that carnitine-acylcarnitine translocase and carnitine palmitoyltransferase II activities were greatly reduced in the ischemic myocardium. Furthermore, evidence was provided that the decreased transport of fatty acids into the mitochondrial matrix was linked to the declining pool of reduced glutathione. However, glutathione levels and redox state were not altered by anaerobiosis in rat cardiac myocytes (Timerman et al., 1990); therefore sulfhydryl modification of enzymes was probably not a contributing factor in these incubations. Because of methodological limitations, it cannot be excluded that [‘4C]-COz production was, to some extent, decreased due to dilution of the labeled pool by endogenously released free fatty acids. Myocytes presented with exogenous [14C]palmitic or arachidonic acids esterified them efficiently into triacylglycerols (Table 3) but failed to appreciably label phospholipids.

et al.

Similar findings were reported by DeGrella and Light (1980) in adult rat heart cells, but in cultured neonatal cells equilibrium labeling of all lipid pools was achieved in a 24 h labeling period (Chien et al., 1985). Phospholipid synthesis (and consequently labeled fatty acid incorporation) and energy expenditure would be expected to be greater in beating, proliferating neonatal cells in comparison to quiescent terminally differentiated adult rat cardiac cells. In conclusion, the present study demonstrated that oxygen deprivation without concurrent ATP depletion can result in phospholipid degradation and subsequent accumulation of arachidonic acid. Triacylglycerol levels, however, are largely dependent on cellular ATP content. Acknowledgements This work was supported by NIH grants USPHS HL36240, DK18884 and DK20387, and a grant-in-aid from the Central Ohio Heart Association. We would like to thank Dr Ruth A. Altschuld for helpful advice during the preparation of this work.

References RA, GAMELIN LM, KELLEY RE, LAMBERT MR, APEL LE, BRIERLEY GP (1987) Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes. J Biol Chem 262: 13527-13533. CHIEN KR, HAN A, SEN A, BUJA LM, WILLERSON JT (1984) Accumulation of unesterified arachidonic acid in ischemic canine myocardium. Relationship to a phosphatidylcholine deacylation-reacylation cycle and the depletion of membrane phospholipids. Circ Res 54: 313-322. CHIEN KR, SEN A, REYNOLDS R, CHANC A, KIM Y, GUNN MD, BUJA LM, WILLERSON JT (1985) Release of arachidonate from membrane phospholipids in cultured neonatal rat myocardial cells during adenosine triphosphate depletion. J Clin Invest 75: 1770-I 780. CORR PB, GROSS RW, SOBEL BE (1984). Amphipathic metabolites and membrane dysfunction in ischemic myocardium. Circ Res 55: 135-154. DEGRELLA RF, LIGHT RJ (1980) Uptake and metabolism of fatty acids by dispersed adult rat heart myocytes. I. Kinetics of homologous fatty acids. J Biol Chem 255: 9731-9738. FOLCH J, LEES M, SLOANE-STANLEY GA (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226: 497-509. HAGVH TA, CHRISTOPHERSEN BO (1984) Effect of dietary fats on arachidonic acid and eicosapentaenoic acid biosynthesis and conversion to C as-fatty acids in isolated rat liver cells. Biochim Biophys Acta 796: 205217. HAGVE TA, SPRECHER H (1989) Metabolism of long-chain polyunsaturated fatty acids in isolated cardiac myocytes. Biochim Biophys Acta 1001: 338-344. HEATHERS GP, YAMADA KA, KANTER EM, CORR PB (1987) Long-chain acylcamitines mediate the hypoxia-induced increase in at-adrenergic receptors on adult canine myocytes. Circ Res 61: 735-746. KATZ AM, MESSINEO FC (1981) Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ Res 18: 1-16. LI Q ALTSCHULD RA, STOKES BT (1988) Myocyte deenergization and intracellular free calcium dynamics. Am J Physiol 255: Cl622168. L~EDTKE AJ (1981) Alterations ofcarbohydrate and lipid metabolism in the acutely ischemic heart. Prog Cardiovasc Dis 23: 321-333. LOWRY OH, ROSEBOROUGH NJ, FARR AL, RANDALL RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193: 265-275. ALTSCHULD

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MP, HOHL CM, BRIERLEY GP, ALTSCHULD RA (1982) Release ofenzymes from adult rat heart myocytes. (:irc Res 51: 560-568. PAULY DE‘, YOON SB, MCMILLIN JB (1987) Carnitine-acylcarnitine translocase in ischemia: evidence for sulfhydryl modification. Am J Physiol 253: H 1557-H 1565. ROUSER G, FLEISCER S, YAMAMOTO A (1970) Two dimensional thin-layer chromatographic separation of polar lipids and determination of phospholipids by phosphorous analysis of spots. Lipids 5: 494-496. SPRECHER H, SANKARAPPA SK (1982) Synthesis of radiolabeled fatty acids. Methods Enzymol86: 357-366. TIMERMAN AP, ALTSCHULD RA, HOHL CM. BRIERLEY GP, MEROLA AJ ( 1990) Cellular glutathionr and thv responsr of adult rat heart myocytes to oxidant stress. J Mol Cell Cardiol 22: 565-575. VAN BILSEN M, VAN DER VUSSE GJ, WILLEMSEN PHM, COUMANS WA, ROEMAN THM, RENEMAN KS I 1989’ Lipid alterations in isolated, working rats hearts during ischemia and reperfusion: its relation to myorardial damaRe (:irr Rcs 64: 304-3 14. WHITMER JT. IDELL-WECNER JA, ROVETTO MJ, NEELY JR (19783 Control of fatty acid metabolism iu ischrmic, and hypoxir hearts. J Biol Chem 253: 43054309. Voss AC, SPRECHER H (1988) Regulation of the metabolism of linoleic acid to arachidonic acid in rat hrpatocvtcs. Lipids 23: 660-665. MURPHY

The effect of anoxia on lipid metabolism in isolated adult rat cardiac myocytes.

In ischemic myocardium abnormal lipid metabolism results in accumulation of compounds that are deleterious to membrane structural integrity and membra...
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