Palmitic and erucic acid metabolism in isolated perfused hearts from weanling pigs

Biochimica et Biophysica Acta, 1004 (1989) 205-214 Elsevier

205

BBALIP 53168

Palmitic and erucic acid metabolism in isolated perfused hearts from weanling pigs F r a n k D. S a u e r ~, J o h n K . G . K r a m e r ~, G e o r g e V. F o r e s t e r 2 a n d K e i t h W. Butler 3 t Animal Research Centre, Research Branch, Agriculture Canada, Central Experimental Farm, 2 Division of Electrical Engineering, National Research Council and ~ Division of Biological Sciences, National Research Council, Ottawa (Canada)

(Received 28 April 1989)

Key words: Fatty acid metabolism; Paimitic acid; Erucic acid; (Pig heart)

Hearts from 4 week-old weanling pigs were capable of continuous work output when peffused with Krebs-Henseleit buffer containing 11 mM glucose. Peffused hearts metabolized either glucose or fatty acids, but optimum work output was achieved by a combination of g|ucose plus physiologica| concentrations (0.1 raM) of either palmita~.e or erucate. Higher concentrations of free fatty acids increased their rate of oxidation but also resulted in a large accumulation of neutral lipids in the myocardium, as well as a tendency to increased acetylation and acylation of coenzyme A and camitine. When hearts were peffused with 1 mM fatty acids, the work output declined below conUoI values. Emcic acid is known to be poorly oxidized by isolated rat heart mitochondria and, to a lesser degree, by peffused rat hearts, in addition, it has been reported that erucic acid acts as an uncoupler of oxidative phosphorylation. In isolated peffused pig hearts used in the present study, erucic acid oxidation rates were as high as pa|mitate oxidation rates. When energy coupling was measured by 31P-NMR, the steady-state levels of ATP and phosphocreatine during emcic acid peffusion did not change noticeably from those during glucose peffusion. It was concluded that the severe decrease in oxidation rates and ATP production resulting from the exposure of isolated pig and heart mitochondria to emcic acid are not replicated in the intact pig heart.

introduction

Erucic acid (22:1 ( n - 9)) is present in oilseeds and vegetables of the genus Brassica. Erucic acid, when fed to experimental animals in large quantities (4% by weight of the total diet), causes an accumulation of triacylglycerol as visible fat droplets in the myocardium [1]. A number of cardiotoxic effects have been ascribed to erucic acid. In the rat heart, these include decreased oxidation rates [2-4], a 70% depression in rates of ATP synthesis and a significant loss of respiratory control [2]. Experiments with perfused rat hearts have confirmed that erueic acid oxidation rates were somew'~at lower than those of palmitic acid but both fatty acids were readily incorporated into neutral and phospholipids during the time of perfusion [5,6]. It has also been shown that erucic acid, which by itself is poorly oxidized, may in addition inhibit palmitate oxidation [7].

Correspondence: F.D. Sauer, Animal Research Centre, Agriculture Canada, W.K. Neatby Building, Central Experimental Farm, Ottawa, Ontario KIA OR6, Canada.

In the rat, dietary erucic acid interferes with the work output of the heart [8]. Both left ventricular stroke work and maximal isometric contractile force in isolated papillary muscle were depressed in rats fed erucic acid as part of their diet [8]. While many studies have been conducted with isolated rat hearts and isolated rat heart mitochondria, relatively little information is available on the effects of erucic acid on heart preparations from other species. Mitochondria from human heart auricles showed slower /]-oxidation rates of erucic than oleic acid [9]. In pig heart mitochondria, erucic acid oxidation rates (measured against oleic acid) were depressed [10] but significantly less so than in the rat. In addition, erucic acid did not inhibit the oxidation rates of palmitic or oleic acids as it did in the rat [10]. Swine are good experimental model animals for biomedical research because their cardiovascular system and physiology closely resemble that of man [11]. Not only are swine omnivorous, but on a high fat, high cholesterol diet they show atherosclerotic lesiops commonly seen in western man [11]. Based on oxygen consumption rates and cardiac output profiles during

0005-2760/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Bio~edical Division)

206 exercise, it has been demonstrated that swine, and not dogs, resemble man more closely. In the present study, isolated perfused pig hearts were used to measure the effects of erucic and palmitic acids on oxidation rates, ATP synthesis and work output. Labeled fatty acids were used in the perfusion fluid in order to m~asure both their oxidation and incorporation into cardiac lipids. 3tP-NMR spectroscopy was used to measure ATP and phosphocreatine changes throughout the perfusion. Materials and Methods

Surgical preparation of pig hearts Weanling Yorkshire pigs (6-8 kg body weight) were presedated with Valium (0.4-3 ml) and anesthetized with ketamine (0.11-0.44 ml per kg body weight). They were then given Halothane by mask (3~) with 50/50~ O2/N2 at 2 I/min. A tracheotomy was performed and the animals were placed on a ventilator. The tidal volume was approx. 240 ml/min and the ventilation rate was 15-20 ml/min. Halothane was maintained at a level of 15. A midline sternotomy was performed and the pericardium was retracted; the great vessels were cleared of tissue, the aorta was isolated with a loose ligature and I ml of heparin (1000 IU) was injected into the aortic root. The heart was rapidly removed, plunged into ice-cold saline, and the aorta was cannulated and perfused retrograde in the Langendorff mode with a commercial cardioplegic solution (Plegisol, Abbott Labs., Chicago, IL) at 4 ° C The heart was then cleaned of tissue. A latex fluid-filled balloon was placed into the

left ventriclevia the left atriumin orderto follow left ventricular developed perfusion pressure. The heart was

then connected to the perfusion apparatus by attaching the tubing to the aortic inlet, as shown in Fig. 1. The hearts were equilibrated with Krebs-Henseleit solution containing (mM): NaCI, 119; KCI, 4.7; CaCI 2, 1.8; KH2PO 4, 1.2; MgSO4, 0.6; glucose, 11; NaHCO 3, 25; and 2~ albumin. The solution was oxygenated (95~ O2/5~ CO 2) in a Bentley 5 pediatrics blood oxygenator (Irving, CA). The test solution, which also contained fatty acids in addition to the above, was oxygenated in a second oxygenator. Both solutions were fed into the heart via two peristaltic pumps electronically metered so that the proportional flow of each could be regulated as required. Left ventricular pressure was measured with an Analog Medical System Corp. pressure transducer (Greenvale, NY). Heart rate and pressure were recorded with a Gould Brush 220 strip chart recorder (Cleveland, OH).

Perfusion medium Fatty acids were dissolved in water by warming with an equivalent amount of NaOH and added slowly with stirring to 4~ fatty acid-free albumin (Sigma Chemical Co., St. Louis, Me). The albumin-fatty acid solution was prepared at double the required concentration for mixing just before use with an equal volume of the Krebs-Henseleit solution, also made to twice the required concentration. The final Krebs-Henseleit solution contained fatty acid, 2~ albumin and 11 mM glucose.

NMR spectroscopy The thermostatted, water-jacketed chamber containing the pig heart was placed over the 4 cm coil of a Bruker 81/200 MHz surface coil probe which was then

intraventriculabal r loon waterbath

aorticperfusion--~ - ~ balloon i~l

37°C

I

I

~ I~

pressure transducer

peristalticpumps ] cOrOnarycOllectiOnfl~W -~

taperecorder

J

Fig. 1. Schematicof apparatus for retrograde per'fusion system for isolated pig hearts. Hearts are cannulated to the aorta and perfused from solutions in l)aediatric oxygenators at 37°C at constant flow via peristaltic pumps. The system allows for selection of the perfusion from oxygenator1 or 2, or calibratedmixturesfromboth. The coronary effluent from the coronary sinus was collected through an indwellingcatheter, ligated to the pulmonaryartery, from the right heart. Contractilityof the heart was monitoredby a fluid-filledballoon placed in the left ventricle; balloon pressurewas recorded both on a strip chart recorder and digital tape recorder. For gas analysesand t4CO2determinations,the coronary flowwascollecteddirectlyinto appropriatecontainerswithoutexposureto air.

207 inserted in a 4.7 T 30 cm horizontal bore magnet of a Bruker 4.7/30 Medspec spectrometer. The magnet was shimmed by observing the proton-free induction decay observed with the decoupling coil. Phosphorous spectra were acquired as a series of 60 accumulations each a~ a frequency of 81.03 MHz with 50 ~s CYCLOPS phase-cycles pulses and a recycle time of 2 s. The block size was 4096 data points and the sweepwidth was 10 kHz. Peak heights were used in all measurements. The pH was measured from the chemical shift of Pi [13]. The pH calibration curves were made using a standard solution containing 1 mM lactate, 1 mM Pi, 1 mM phosphocreatine, 0.5 t.~M ATP, 120 mM KCI, 2 mM MgCl 2, 598 D20 and 10 mM Pipes. The pH values were measured at room temperature and varied from 5.5 to 8.0. 3~p ~pectra were then recorded at the appropriate tempera*,ure (37 ° C).

Tissue analyses After perfusion, the hearts were immediately frozen in liquid N 2 and the top 1/3 was broken off and discarded. The muscle was crushed in liquid N 2 and one aliquot was removed for lipid analysis and another aliquot was put in a stainless steel percussion mortar cooled in liquid N 2 and pulverized. To the frozen powdered muscle was added three parts by weight of 698 perchloric acid, and the resulting mixture was sonicated for 1 rain to complete the extraction. After centrifugation, the supernatant fractions were combined and neutralized for enzymatic assays. Free CoASH was measured in the a-Ketoglutarate dehydrogenase assay by the reduction of NAD [14]. Acetyl-CoA was measured by the citrate synthase assay [15]. Free carnitine was measured by coupling the assay for free CoASH with the carnitine:acetyl-CoA transferase reaction [16] as described [17]. Acetyl-carnitine was measured by coupling the acetyl-CoA assay to the carnitine: acetyl-CoA transferase reaction [16,17]. The long-chain acyl-carnitine and acyl-CoA determinations were done after alkaline hydrolysis of the perchloric acid tissue pellet [17]. Assays were performed in a thermostatted cuvette holder in an Eppendorf photometer with fluorescence attachment (Netheler and Hinz GMBH, Hamburg) and recording adapter. Radioactive lipids synthesized by pig hearts during perfusion were extracted and separated as described previously [18,19]. After detection of lipids on the TLC plates, the radioactive spots were carefully scraped into scintillation vials for radioactivity determination in a Beckman model 380 liquid scintillation counter.

Gas analyses Oxygen, CO2 and pH measurements were made on a pH/blood gas analyzer (model 1304, Instrumentation Laboratory S.p.A., Milan). Oxygen consumption was

calculated as: ~tmol 0 2 (g dry h e a r t - l . m i n - l ) =

P 0 2 in - aDO'*out a. b 760 x 22.4

where POE in is the partial pressure of 02 in inflowing perfusate, PO 2 out is the partial pressure of O2 in the coronary outflow, a is the solubility of 02 in buffer (24.5 # l . ml-I at 35 ° C) and b is the rate of coronary flow in ml. rain-1, g dry wt.- 1. CO 2 production was calculated from the concentration of [CO2] + [HCO3] in the coronary outflow after subtracting the concentration of [CO2] + [HCO3] in the inflowing perfusate. Dissolved CO 2 was calculated as: ~mol C O 2 (g dry h e a r t - i. nfin- 1 ) = P C 0 2 in - P C 0 2 out × - -c' b 760 22.4

where P C O 2 iS the partial pressure pressure of C O 2 in the outflowing and inflowing perfusate, c is the solubility of CO 2 in buffer (592 #l. ml-I at 35°C) and b is the rate of coronary flow in ml. min-I .g dry wt.-l. The concentration of H C O ; was calculated from the Henderson-Hasselbach equation: p H = pK" + log

[HCO3 ]

[CO2]

where pH was measured directly and p K ' = 6.32.

~4C02 determination The coronary sinus effluent was collected by catheter from the right ventricle and 10 ml was collected at 5 min intervals directly inlo strong NaOH (final conc. 0.5 M) without exposure to air. To measured aliquots, excess BaCl 2 was added. The BaCO3 was precipitated by centrifugation, washed twice in water, once in ethanol, resuspended in scintillation fluid by brief sonication and monitored in a liquid scintillation counter.

Materials [14-14 ClErucic acid (47.2 Ci/mol) was obtained from Schwartz/Mann Radiochemical, Orangeburg, NY. [114C]Palmitic acid (5 Ci/mol) were obtained from Du Pont NEN Research Products, Markham, Ontario. Bovine albumin (98-9998, essentially fatty acid-free) was obtained from Sigma Chemical Company, St. Louis, MO. Results

Heart perfusion In separate experiments, right heart outflow (the coronary flow) was measured at different pump flow rates to the left side in pig hearts operating in the Langendorff mode. At pumping rates below 100 ml/min to the left heart, coronary flow increased. At pumping

208 rates above 100 ml/min, coronary flow again increased, thereby demonstrating that the hearts were operating in the range of coronary autoregulation. Within this range, the Langendorff perfused heart responds to an increased oxygen demand by increasing the coronary flow, and conversely, by decreasing coronary flow when oxygen demand is lessened [20]. Subsequently, the experimental hearts were perfused in the ascending part of the coronary flow curve, i.e., at pumping rates in excess of 100 ml/min. The mean pumping rate for all experiments was set at 147 + 4.2 ml/min.

Work output The heart rate in the pig hearts perfused with KrebsHenseleit solution containing 11 mM glucose was 119.9 ± 13.6 with peak systolic pressure of 98.9 ± 13.6 mm Hg. The pressure in the intraventricular balloon was set at 25 mm Hg and it was assumed that left ventricular end diastolic pressure would go to zero or near zero. The starting value of the rate pressure product, i.e., when the perfusate contained glucose but no fatty acids, was 11857 :t: 678.9 mm Hg. win-t. This value agrees well with values published for the perfused pig heart work output [21-23]. The rate pressure product obtained during the initial perfusion with glucose was set at 100~ (Fig. 2A and B) and the rate pressure product recorded during fatty acid perfusion was pIotted as percent change. Palmitate infusion at low concentration (0.1 mM) stimulated cardiac work output (Fig. 2A), while higher concentration (1 mM) depressed the work output below control values and significantly (P < 0.05)

TABLE Gas exchange and RQ values of hearts perfused either with 16: 0 or 22:1 (n -

9)

Expt. No.

Perfusate additions (mM)

1

glucose(ll) glucose + 16: 0 (0,l) glucose

2

glucose (! 1) glucose + 16:0 (! .0)

3

glucose (11) glucose + 22: I (n -9)(0.1)

4

glucose (1 !) glucos¢+ 22: ! (n -9)(1.0)

Gas exchange (pmol. min- i.g dry wt. - I)

Mean RQ

O 2 uptake

CO- ~rod.ced

values"

2.88±0.13b

2.99-e0.10

1.1

3.04±0.16 2.42 ± 0.10

1.82+0.23 2.59 ~ 0.27

0.7 1.0

5.29 ± 0.41

3.87 ± 0.40

0.8

6.22 ± 0.17

4.25 ± 0.39

0.7

4.14:~0.04

4.94±0.56

1.2

4.16±0.09

3.43±0.46

0.7

4,08 ±0,04

4.19±0.18

1.0

2,25±0.13

1.05±0.19

0.5

a RQ values were calculated from individual gas measurements taken at 5 min intervals. b Standard error of gas analyses taken at 5 win intervals of the same perfus,;on.

below values obtained with 0.1 mM 16:0. Similar results were obtained with perfusion of 22 : 1 (n - 9) (Fig. 2B). At concentrations of 22: 1 (n - 9) of 0.1 mM and 0,5 mM, rate pressure product values increased above control values; but a 22:1 ( n - 9) concentration of 1 mM resulted in a depression of rate pressure product.

o. ~,~0 A

~ 1,~o

B

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100

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z

-w < Z U

I,,,, ;Z w tj

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w

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io TIME (min)

2'5

3~

3'5

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~-

0

o

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~o TIME

~

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do

(min)

Fig. 2. Rate pressure product calculated as a percent of control (with only glucose as substrate) when ]6: 0 or 22:1 (n - 9) was added to perfusate. To the perfusate was added" palmitate, 0.1 mM (o) or 1.0 mM (O) (A), or 22:1 (n -9), 0.1 mM (/x), 0.5 mM (o) and 1.0 mM (e) (B). Data are expressed as the mean + S,E,, n = 3, All hearts were perfused for 15 min with Krebs-Henseleit buffer (11 mM glucose) before addition of fatty acids (time 0).

209 TABLE II

pH of heart perfusate taken by direct measurements and pH within the heart as estimated by 3tP.NMR spectroscopy

The imracellular myocardial pH was measured by 31P-NMR in individual hearts that were first perfused with 11 m M glucose for 15 rain, then switched to glucose plus the fatty acid indicated for the remainder of the experiment. * P < 0.05; * * P < 0.01. Substrate 11 mM glucose

0.1 mM 22:1 + 11 mM glucose

1.0 mM 22:1 + 11 mM glucose

0.1 m M 16 • 0 + 11 mM glucose

1.0 mM 16:0 + 11 mM glucose

7.38 + 0.026 7.27 5:0.050 -0.11

7.38 5:0.022 7.28 5:0.009 * 0.10

7.38 5:0.047 7.33 5:0.043 -0.05

7.33 5:0.020 7.28 + 0.041 -0.06

Perfusate (direct measurement) pH in

7.40 + 0.022 7.27 5:0.013 * * -0.13 •

pHout zlpH

Myocardial pH (estimated from 31P-NMR) 7.13 + 0.064 6.96 5:0.021 7.11 5:0.047 7.14 5:0.039

7.09 5:0.021 7.04 ± 0.029 -

-

-

7.11 5:0.035 -

7.11 5:0.027

in Expt. 1, Table I, when 16:0 was removed from the perfusate, the R.Q. returned to 1.0. Table lI shows the decrease in pH after the perfusate was collected from the coronary sinus. This decrease was most marked when glucose was the sole substrate and may be the result of metabolic CO 2, as well as organic acids produced from glucose. The intracellular pH of the heart when perfused with different substrates and as measured by 31P-NMR is shown in Table II. These values agree closely with the intracellular pH values reported for lamb, dog and cat heart [24]. There were no significant differences in oxidation rate of [1-14C]palmitate or [14-14C]erucate (Fig. 3A and B) in the isolated perfused young pig heart. The rates of

The greater work output with a low fatty acid concentration of 0.1 mM is not surpising in view of the fact that the physiological plasma free fatty acid concentrations of weanling pigs is quite low. Plasma free fatty acid concentrations of 0.048, 0.033 and 0.022 mM were recorded in 10-day-old piglets when fed either sows' milk, milk replacer prepared from soybean oil or milk replacer prepared from canola oil (unpublished data). The gas exchange data in Table I indicate that the working heart preparation was able to oxidize both long-chain- and intermediate-chain-length fatty acids. Mean R.Q. values decreased from approx. 1.0, where glucose was the sole substrate, to 0.7, when either 16 : 0 or 22:1 ( n - 9) was added to the perfusate. As shown

350-

300-

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115 20 PERFUSION

2'5 TIME

30 (min)

3J5 40

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0

5

10

15

PERFUSION

20 TIME

25

30

35

(rain)

Fig. 3. Oxidation rates with time of different concentrations of 16:0 (A) and 22:1 (n - 9) (B) added to perfusate. Perfusate contained 0.1 mM 16:0 (spec. act. 137 730 dpm//~mol) (o), 1 m M 16:0 (spec. act. 12 350 dpm//~mol) (~), 1 mM 16:0 (spec. act. 12250 d p m / # m o l ) (O), in the presence of decreasing glucose as indicated on the graph where 80% glucose = 9 m M glucose in perfusate (A). Perfusate contained 0.1 mM 22:1 (n - 9) (spec. act. 251080 d p m / # m o l ) (o), 0.5 m M 22:1 ( n - 9 ) (spec. act. 53260 dpm//~mol) (z~), 1.0 mM 22:1 ( n - 9 ) (spec. act. 21140 dpm//~ mol) (D) (B).

210 TABLE !II

Incorporation of 16:0 and 22:1 (n - 9)from per~mate into cardiac lipids during perfusion Values within rows with different superscripts are different ( P < 0.05). Three hearts were analyzed for each perfused fatty acid and at each concentration. Phosphofipids

Fatty acid concentration in perfusate (nmol. g dry w t - t ) 0.1 m M 16: 0

1.0 mM 16: 0

0.1 m M 22:1 ( n - 9 )

235±34 a 38 + 8 a 3 4 ± 13 47 ± 23 11 ± 4 a,b

4 9 3 ± 129 a 62 ± 7a 47 ± 21 139± 67 26 ± 11 ~,b

17± 3± 3:t: 3± 4±

Total

365

767

30

Neutral lipids Cholesterol + diacylglycerols Tria~lglycerols Cholesterol ester

54 ± 26 a 120 ± 15 a 5± ! a

! 608 ± 762 a,b 4 666 ± 1402 b 860 ± 322 b,~

31 ± 16 a 128 ± 65 a 21 :t: 9 a,,~

691 ± 156 b 1911 :t: 417 b 1524 ± 366 b

Total

179

7134

180

4126

Ratio: phospholipid/neutral lipid

2.04

oxidation with either fatty acid were concentration-dependent. However. the increasing specific activity of expired t4CO2 with [1-t4C]palmitate as substrate (Fig. 3A) probably was the result of an initial dilution of the labeled 16:0 by an endogenous pool of unlabeled 16: 0 present in the heart at zero time. This effect was not present with 22:1 ( n - 9) as substrate (Fig. 3B). It should be noted, however, that one can only equate t4CO2 production with fatty acid oxidation if the fatty acid is completely oxidized. The possibility that some short-chain acyl-CoA esters derived from 16: 0 or 22: 1 ( n - 9) escaped oxidation cannot be ruled out. The working heart preparation appears to have some flexibility in switching from glucose oxidation to fat oxidation. With the removal of glucose from the perfusate (Fig, 3A), 16:0 ~ m e s the principal substrate, its oxidation rate decreasing by approx. 30~ below control value. Data in Table III show the incorporation of 16:0 and 22:1 ( n - 9) into cardiac lipids after perfusion. When 0.I mM 16:0 was added to the perfusate, i.e., at approximately the concentration found in plasma, twice as much 16:0 was incorporated into the phospholipid as into the neutral lipid fraction. Compared to 16:0, significantly less 22:1 ( n - 9) was incorporated into phosphatidylcholine and phosphatidylethanolamine when perfused at 0.I mM concentration. At a con~ntration of 1.0 raM, there were, however, no sigaificant differences in incorporation of 16:0 and 22" 1 ( n - 9) into either the phospholipid or neutral lipid fraction. The ratios of incorporation of the perfused fatty acids into the neutral and phospholipid fractions are shown in Table III. For I mM 16: 0, and 0.1 and 1.0 mM 22:1 ( n - 9), 6- to 9-fold more fatty acids were incorporated into the neutral lipid than the phospho-

0,11

9b 1b 1 2 2b

1.0 m M 22:1 ( n - 9 )

Phosphatidylcholine+lyso-derivative Phosphatidylethanolamine Phosphatidylserine Phosphatdiylinositol Sphingomydin

245±121 a.b 37__, 13 a'b 6 7 ± 29 7 0 ± 31 20± 3 a 439

0,17

0.1 !

lipid fraction. This ratio was reversed for the 0.1 mM 16:0 perfusion, in which twice as much 16:0 was incorporated into the phospholipid fraction. The approximate ratios of fatty acids oxidized in 30 min to the quantity of fatty acid incorporated into lipids was also calculated. For both concentrations of 22 : 1 (n - 9) and 0.1 mM 16:0, the ratio of oxidation to lipid biosynTABLE IV

Concemration of metabolites in heart muscle after perfusion with 16: 0 and 22: I (n - 9) Metabolites

Fatty acid in perfusate '~ (nmol. g dry wt. - 1) 0.1 mM 16:0

CoenzymeA Acetyl-CoA

195 ± 11 48 ± 11

p h

1.0 mM 16:0 86 5q

± ±

6"* 5 *

< 0.001

Long-chain acyI-CoA

Carnitine Acetylcarnitine Long-chain acylcarnitine

113 ± 37 11099 ±189 * 782 ± 79 3193

± 933

0.1 mM 22:1 (n - 9 ) CJenzyme A Acetyl-CoA

218 ± 21 1265 ±119 * * * ] 166 ± 45 * 3 236

< 0.05 < 0.001 < 0.01

+ 590 *

1.0 mM 22:1 (n - 9 )

122.8 ± 27.36 37.7 ± 5.55

162.5 + 22.34 42.5 + 4 . 5 3

A~etylcarnitine

112.2+ 21.08 8 874.2 ± 38"/.25 912.4± 83.89

215.6+ 17.22 4 231.7 + 346.37 970.4± 68.49

Long-chain acylcarnitine

2651.9_+ 621.55

1836.2 + 555.93

Long-chain acyI-CoA

Carnitine

< 0.01 < 0.001

a Differences in concentration of the same metabolite within columns: * P < 0.05; ** P < 0.01, * * * P < 0.001. b Significant differences within a row. Three hearts were analyzed for each fatty acid at each concentration.

2il (b)

(c)

(d}

"

t

10

'

,

'

'

I

0

,

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o

-10

(e)

,

-

2 j0

pore

Fig. 4. Representative 31p-NMR spectrum of a pig heart perfused with Krebs-Henseleit buffer containing 11 mM glucose and 0.1 mM 22:1 (n - 9) bound to 2~ albumin. Phosphorous spectra were acquired as a series of 60 accumulations each at a frequency of 81.03 MHz with 50 #s CYCLOPS phase-cycled pulses and a recycle time of 2 s. Peak assignments from the downfield end of the spectrum are: inorganic phosphate (a); phosphocreatine (b); 7-phosphate of ATP (c); a-phosphate of ATP (d); B-phosphate of ATP (e).

thesis was 2:1. With 1 mM 16:0, this ratio decreased to 1 : 1, i.e., for every mol 16 : 0 oxidized, 1 mol of 16 : 0 was incorporated into lipoid esters. The results in Table IV give some insight into the reason why hearts perfused with low fatty acid concentrations (0.1 raM) have greater work output (as measured by rate pressure product) than the hearts perfused with higher, non-physiological fatty acid concentrations. Pig hearts perfused with 1 mM 16:0 contained significantly less free CoASH and free carnitine and significantly greater quantities of long-chain acylCoA and acetylcarnitine. The large difference in free carnitine concentration in hearts perfused with 0.1 mM 16 : 0 and 1.0 mM 16 : 0 cannot be accounted for by the sum of acetylcamitine and long-chain acylcarnitine. This raises the possibility that acylcarnitine(s) of intermediate-chain-length(s) were formed and that these were

not detected by the enzyme assay. Similar results were obtained with 22:1 ( n - 9) in the perfusate. The concentration of long-chain acyl-CoA increased and that of free carnitine decreased when 1 mM 22:1 ( n - 9) was added to the perfusate (Table IV). Free CoASH levels, however, did not decrease, which suggests that CoASH acetylation was slower with 22:1 ( n - 9) than with 16:0. This was also evident from a comparison of results with 1 mM 16:0 and 1 mM 22 : 1 (n - 9) (Table IV). With 1 mM 16:0, free CoASH and camitine levels were significantly decreased, while the acetylated form of these cofactors was significantly increased. Fig. 4 shows a typical 31p-NMR spectrum of a pig heart after 25 min perfusion. The resonances can be assigned to inorganic phosphate, phosphocreatine and to the 7, ot and fl phosphates of ATP. Phosphocreatine, ATP and inorganic phosphate a~p-NMR signal intensities in a pig heart perfused with Krebs-Henseleit buffer are shown in Fig. 5A. Over a 150 min perfusion period, phosphocreatine and ATP levels decrease. This suggests that for the perfused pig heart, 3 h may be the maximum time possible for useful observation. The phosphocreatine/ATP ratios, however, did not change significantly from the beginning to the end of the experiment. The ratio at the start was 2.36 ± 0.004 and at the end of the experiment, 2.10 + 0.144 (Fig. 5A). In pig hearts perfused with 1 mM 22:1 (n - 9) (Fig. 5B), the ratios at the beginning and end of the experiment were 2.31 + 0.079 and 2.01 + 0.050, respectively. In the perfusion experiment with 0.1 mM 22 : 1 (n - 9), the ratios were 1.89 + 0.062 and 2.11 + 0.082 (Fig, 5C) and in the experiment with 0.1 mM 16:0 the ratios were 1.59 ± 0.025 and 1.61 +0.071, respectively (Fig. 5D). These ratios are similar to those reported for the Langendorff perfused rat heart [25]. There was no evidence that the addition of either 16:0 or 22 : 1 (n - 9) to the perfusate in any way interfered with energy coupling in the perfused pig heart. Discussion It has long been accepted that free fatty acids are the primary energy source in the adult working heart. Some recent reports, however, suggest that this may be an oversimplification and that while glucose oxidation can be inhibited by free fatty acids, so can the oxidation of free fatty acids be inhibited by glucose, lactate or pyruvate. Werner et al. [21] showed that glucose oxidation in the newborn pig heart was suppressed by adding 16:0 or octanoate to the perfusate. Later they reported that lactate reduced 16 : 0 uptake and oxidation in pig hearts to the same extent that 16:0 reduced the uptake and oxidation of lactate and glucose [26]. Similar results have been observed with perfused rat hearts. Forsey et al. [27] showed that the oxidation of 18:1 ( n - 9) but not octanoate was inhibited by physiological levels of

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lactate, pyruvate or glucose plus insulin, thus clearly demonstrating that in the perfused rat heart, fats and carbohydrates can have reciprocal inhibitory effects on each other. The results of the present investigation show that the isolated perfused heart from a 4-week-old piglet can readily oxidize either glucose or fatty acids plus glucose with no decrease in rate pressure product if the fatty acid concentration in the perfusate is physiological. At a free fatty acid concentration of 1 raM, which is far greater than the normal plasma concentration, fatty acid oxidation rates increased, but the rate

pressure product declined below control values obtained with glucose alone. Similarly, Liedtke et al. [23] administered excess free fatty acids (1.5--2 raM) to globally perfused working pig hearts and recorded decreased aortic pressure and work output as compared to controis with < 0.5 mM free fatty acids. These effects were furter exaggerated if ischemia was superimposed on the treatment. From the work output data in the present study, it can be concluded that the weanling pig heart perfo_,'ms most efficiently with a combination of fatty acids and glucose. This was also indicated by the results

213 which showed that R.Q. values were approx. 1 with glucose, 0.7 with fatty acids and again increased to 1 when the perfused heart was switched back to glucose. For optimum work output by the perfused pig heart, it is important that the free fatty acids in the perfusate remain near physiological levels. If the perfusate contains 1 mM fatty acids, a concentration frequently used in perfusion experiments, the pig heart rapidly accumulates large quantities of neutral lipids. In the present experiments, heart perfusions with 1 mM fatty acid were accompanied by a significantly greater level of carnitine and CoASH acylation and, at least with 16 : 0, there was a decrease in both free CoASH and carnitine concentrations. Werner et al. [21] also noted depressed carnitine levels and increased levels of both acetyl- and acylcarnitine when neonatal pig hearts were perfused with 1.5 mM 16:0. Similarly, Liedtke et al. [23,28] observed increased levels of long-chain acyl-CoA and acylcarnitine esters in working hearts from adult (45 kg) swine when these were perfused with excess free fatty acids during periods of ischemia. The degree of acetylation/aeylation of these cofactors has regulatory implications. In heart, pyruvate oxidation is catalyzed by pyruvate dehydrogenase (EC 1.2.4.1), a large multie,nzyme complex. Its regulation by acetyl-CoA/CoASH ratios has been reviewed [291. Palmityl-CoA has been shown to inhibit carnitine palmitoyltransferase (EC 2.3.1.21) [30], long-chain acylCoA synthetase (EC 6.2.1.3) [31], Na+/K+-ATPase [32], and adenine nucleotide translocase [33]. It is therefore predictable that the cardiac work output or rate pressure product would decrease with 1 mM fatty acid in the perfusate since both 16:0 and 22:1 ( n - 9 ) increased the acyl-CoA concentration in the myocardium. Total carnitine levels (i.e., both free and esterified) were decreased when either 22:1 ( n - 9) or 16:0 were infused at 1 mM concentration. Some carnitine loss may occur in ischemia [34]; however, there was no evidence of isehemia in the perfused hearts used here. Carnitine, however, can be esterified to aliphatic shortchain acyl residues other than acetate [35]. These intermediate-chain-length carnitine esters would not have been detected in the acetylcarnitine assay and thus could account for some of the apparent carnitine loss. Both 22 : 1 (n - 9) and its isomer 22 : 1 (n - 11) are naturally occurring compounds which form part of the food chain; therefore, their effects on heart function and metabolism have been investigated extensively [1]. From numerous published reports it is clear that 22:1 ( n - 9) metabolism is species-dependent, and that resuits may vary depending on whether subcellular partitles or intact, perfused hearts are used in experiments. In mitochondria isolated from rat heart [4,10,36], the oxidation rate of 22:1 ( n - 9) is only a fraction of 16:0. Similar results were obtained with mitochondria from swine [10] and human myocardium [9]. Cultured

rat heart cells also showed low rates of 22:1 ( n - 9) oxidation, although there was evidence of active chain-shortening [37]. With intact, perfused rat hearts, the oxidation rate of 22:1 ( n - 9 ) is -,I~o depressed [5,38] but less so than with isolated mitochondria. Furthermore, this inhibition was largely overcome by prefeeding clofibrate, which apparently induced the peroxisomal system in rat heart necessary for the chainshortening process [38]. It was, therefore, of interest that the intact perfused pig heart, 22:1 ( n - 9 ) was oxidized to CO2 as readily as 16 : 0 even at the higher 1 mM perfusion level. With either fatty acid, the R.Q. decreased from 1 to 0.7, and there was no indication that more neutral lipid accumulated in the heart during perfusion with 22:1 ( n - 9) than with 16:0. The work output of the perfused pig heart was stimulated by both 22:1 ( n - 9) and 16:0 at low concentrations and inIfibited by both fatty acids at high concentrations. From this it must be concluded that the results which show that isolated pig heart mitochondria are severely inhibited by 22:1 ( n - 9) [10] are probably misleading. Isolated mitochondria may be directly uncoupled by long-chain free fatty acids. In addition, the oxidation of 22:1 ( n - 9) would be slowed, since chain shortening, normally carried out by peroxisomes, is missing from the isolated mitochondrial incubation system. Published reports have suggested that heart rnitochondria from rats fed a diet which contains 22:1 (n - 9 ) manifest depressed respiratory control, A D P / O ratios and rates of ATP synthesis [2,39,40]. These results indicate that 22:1 ( n - 9 ) may act as an uncoupling agent in vivo and have cardiotoxic properties. These reports have been challenged [36,41], but to date no definitive results are available to decide this question. The results of the present investigation show that 22 : 1 ( n - 9) not only serves as a substrate in the perfused, working pig heart but in no way interferes with the steady-state levels of ATP and phosphocreatine. 31p. NMR rapidly detects any change in the level of phosphocreatine and adenine nucleotides. In the perfused heart, even in a momentary ischemia, as reported [42], is followed by an immediate decrease in the recorded ATP and phosphocreatine levels (unpublished data). If the 22 : 1 (n - 9) had induced uncoupling of oxidative phosphorylation as reported for rat heart mitochondria [2,39,40], it certainly would have registered rapidly and clearly in the 31p-NMR spectrum. However, further experiments are needed to determine whether prolonged dietary exposure to 22 : 1 could alter the composition of mitochondrial membranes and account for these uncoupling defects, which might not occur in short-term perfusion experiments.

Acknowledgements The authors acknowledge partial financial support from the Canola Council of Canada and the technical

214

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