J Mol Cell Cardiol 16, 765-770 (1984)

Incorporation and Distribution of 3H Oleic Acid in the Isolated, Perfused Guinea-Pig Heart Made Hypoxic

Barbara Ward and Peter Harr i s*

Cardiothoracic Institute, 2 Beaumont Street, London, W1N 2DX, UK

(Received 20 June 1983, accepted in revised form 19 December 1983)

B. WARD AND P. HARRIS. Incorporation and Distribution of3H Oleic Acid in the Isolated, Perfused Guinea-Pig Heart Made Hypoxic. Journal of Molecular and Cellular Cardiology (1984) lg, 765 770. Improved methods of autoradiography and lipid extraction have been used to study the influence ofhypoxia on the fate of radioactive fatty acids in the isolated guinea pig heart. Evidence is provided that hypoxia causes a shift of the rate-limiting step from transport into the cell to oxidation in the mitochondria. This leads to an increased radioactivity in myocardial free fatty acid and in the cytosol. Radioactivity in the mitochondria is decreased and disappears more slowly. At the same time, there is an increased radioactivity in lipid droplets and in triacylglycerol. There is no evidence of a specific location of radioactivity in the sacroplasmic reticulum.

KEY WORDS: FFA; Myocardium; Hypoxia; Autoradiography.

Introduct ion

The fate of free fatty acids enter ing the card iac myocyte may be descr ibed in terms of b iochemical pa thways and mic roana tomica l pa thways . Previous studies on the incorpo- ra t ion of label led fat ty acids into l ipid [3, 11] have been carr ied out on tissue which has not been freeze-clamped, so tha t the cessation of l ipid enzyme act ivi ty is unlikely to have been ins tantaneous. Previous au to rad iog raph ic studies on t h e dis t r ibut ion of t r i t ia ted fat ty acid in the myoca rd ium [13] have used fix- a t ion procedures which we have shown to be accompan ied by a large loss of l ipid [15] and their analysis has not taken into account the effects of 'cross-fire' between organelles [1, 2, 10]. Conclusions concerning the subcel lular d is t r ibut ion of label led fat ty acid have also been d r a w n from the analysis of u l t racentr i fu- gal fractions, but we have found that there is such a transfer of rad ioac t ive mater ia l from one fract ion to another dur ing ul t ra- centr i fugat ion that the results must be inter- pre ted with grea t caut ion [4].

For all these reasons, we under took in a previous s tudy [16] a re -examina t ion of the

* To whom correspondence should be addressed.

0022-2828/84/080765 + 06 $03.00/0

dynamic pa thways of the label led fatty acid in the card iac myocyte under well oxygenated conditions. For this purpose we studied the b iochemical fate of the rad ioac t ive fat ty acid by rap id freezing of the hear ts before l ipid extract ion and measurement of rad ioac t iv i ty in l ipid classes. In para l le l experiments we used E M a u t o r a d i o g r a p h y to de termine the mic roana tomica l p a t h w a y of the radioact ive fat ty acid. In these exper iments we used a fix- a t ion p rocedure which limits the loss of l ipid [15] and the stereological analysis of the auto- rad iographs took 'cross-fire' into account [1, 2, 10]. Using these methods [16] we were able to trace more precisely the in t racel lu lar passage of label led fat ty acid in the hear t and showed that, con t ra ry to wha t had previously been repor ted [13], there is no preferential passage th rough the sarcoplasmic ret iculum.

The present s tudy ektends these observa- tions to the hypoxic heart . Biochemical studies suggest that , dur ing hypoxia, the ra te- l imi t ing step control l ing the passage of free fat ty acids shifts from t ranspor t into the cell to oxidat ion in the mi tochondr i a [9]. In add i t ion there is an increase in the incorpora t ion of fatty acid

�9 1984 Academic Press Inc. (London) Limited

766 B. Ward and P. Harris

into triacyglycerol [11]. Such effects should be readily apparent by changes in the anatom- ical pathways of tritiated fatty acid shown on autoradiography.

M a t e r i a l a n d M e t h o d s

Guinea pig hearts were perfused at 37~ using the Langendorff method with a perfusion pressure of 60 mmHg. The perfusion media, both unlabelled and radioactive, consisted of Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5 mM glucose and 0.2 m• oleic acid complexed to 0.2 mM bovine serum albumin [6]. The radioactive perfusate contained 100 #Ci/ml [9, 10-3H] oleic acid (Amersham International). The radiochemical purity was found to be 96% by thin layer chromatog- raphy.

Two reservoirs of unlabelled perfusate were used. The first was equilibrated with 95% 02: 5% CO 2 and was used for the initial 5 min perfusion. After this time perfusion was con- tinued from the other reservoir which had been equilibrated with 95% N2: 5% CO 2. Perfusion with the hypoxic solution was main- tained for 30 min.

This period was chosen after preliminary investigation had shown that it was long enough to cause biochemical changes but not long enough to cause gross ultrastructural changes which would complicate the analysis of the autoradiographs. Thus a period of 30 min hypoxia gave rise to more obvious bio- chemical changes than a period of 15 min, while ultrastructural damage was not appar- ent at 30 min but had become so after one hour.

At the end of the 30 min hypoxia the hearts were given a 15-s pulse with 2:5 ml (250 #Ci) of the radioactive perfusate which had pre- viously been bubbled with 95% N2: 5% CO 2. This radioactive solution was contained in a syringe attached to the aortic cannula by a 3-way tap and was given manually. The pulse perfusion was followed by an immediate return to perfusion with a 'chaser ~ of the unlabelled, hypoxic solution for a period of 15, 30, 60 or 120 s.

A recirculating perfusion was used for the 30-rain hypoxic period but a 'once-through' method of perfusion was used for the radioac- tive pulse and for the subsequent unlabelled 'chaser' perfusion.

Lipid analysis Four guinea-pig hearts were used for each of the periods of chaser perfusion. At the end of each period the hearts were frozen between two aluminium blocks which had been pre- cooled in liquid nitrogen [19]. The frozen hearts were wrapped in aluminium foil and rapidly pulverised with a hammer. The lipids were extracted from the frozen heart tissue by the method of Folch et al. (1957). Each powered frozen heart was transferred to 90 ml of a chloroform:methanol mixture (2:1 by volume). The tissue was left overnight in this mixture at room temperature. After filtering the extract, 20 ml 0.1 M NaC1 was added to each filtrate and the mixture was left over- night at 4~ to allow the phases to separate. The lipid-containing chloroform phase was taken to dryness using a rotary evaporator and redissolved in 2 ml chloroform. The lipids were separated into the main lipid classes by thin layer chromatography using the solvent system light petroleum ether (40 ~ to 60~ diethyl ether: glacial acetic acid in the ratio 85 : 15 : 2 by volume.

Separate bands corresponding to free fatty acid, triacylglycerol, cholesterol ester and phospholipid were transferred to Bray's solu- tion for scintillation counting. Measurements of the radioactivity of mono- and di- acylglycerol have not been included as we were unable to assess the contribution of radioactive acyl CoA to this fraction because it migrates to the same position on the thin- layer plate. Acyl carnitine remains at the origin with the phospholipids but, as the con- tribution of this substance was shown to be small [16] the phospholipid radioactivity has been included.

Measurement of radioactivity in CO 2

In order to assess the carbon dioxide produced by the oxidation of the radioactive free fatty acid, perfusions were carried out using [10, ~4C] oleic acid.

The hearts were perfused under liquid paraffin to prevent the diffusion of X4CO 2 into the surrounding atmosphere. Four guinea-pig hearts were used for the well oxygenated per- fusion and four for hypoxic perfusion. A 120-s chaser period was used to measure 14CO2 pro- duction and was chosen as the one most likely

Incorporation and Distribution of 3H Oleic Acid 767

to show a difference between the 14CO 2 pro- duced by the well oxygenated and hypoxic hearts.

The effluent from the pulse and chaser in each heart was collected under liquid paraffin in a flask containing 10 ml of 0.1 M K O H . The hearts were freeze-clamped at the end of the 120-s chaser period. The frozen tissue was shattered and extracted in chloro- form:methanol . The addition of an alkaline aqueous solution (pH 13) extracted the 14CO2 present. The method of determining the radioactivity of 14CO1 present in the effluent and myocardial extract was that of Vasdev and Kako [14] radioactivity being measured in aliquots before and after acidification with 1 N HC1 to release 14CO 2.

EM autoradiography A further series ofperfusions was performed in which the animals,.perfusates and perfusion procedure were as described in the freeze- clamping experiments. After the 'chaser' period the hearts were fixed by perfusion with 4% glutaraldehyde in 0.1 ~a sodium caco- dylate (pH 7.3 at 5~ for 10 rain. Four guinea-pig hearts were used for each chaser perfusion time. Pieces of the full thickness left ventricular free wall were processed for elec- tron microscopy using water-soluble Durcu- pan [12] which has been shown to possess good lipid-retaining properties [15]. In a pre- vious study [17] samples from the left ven- tricular wall were compared with samples from the interventricular septum and there was no significant difference between the dis- tribution of radioactive fatty acid in the two sites. At least 15 tissue pieces from each heart were processed and sections to cover at least one grid were taken from each block. At least 500 silver grains were photographed in a non- selective manner from each set of four hearts, with approximately equal numbers from each heart.

The method of autoradiography, including the analysis of micrographs by a modification of the method of Blackett and Parry [1, 2] and Parry and Blackett [10] was described pre- viously [16]. A hypothetical 'cross-fire' table was drawn up using an overlay screen pre- pared by the method of Blackett and Parry [1, 2, 10] except that the circles were reduced to

points so that a 'point-to-point ' analysis was carried out. This means that there were only single items and not junctional items. The hypothetical sources were arranged randomly on the screen by plotting coordinates from a list of random numbers. A chi-squared test was first carried out to determine whether the distribution of real grains was random or not. The 'relative intensity of sources' in each organelle was then calculated by solving a set of simultaneous equations which gave identi- cal results [16, 17] to the minimising prog- ramme of Blackett and Parry [1, 2, 10]. In this way calculations of the 'intensity of sources' were made in the following regions: myofib- rils, mitochondria, transverse tubules, sarco- plasmic reticulum, lipid droplets, nucleus and 'cytoplasm', i.e. areas of cytoplasm not occupied by myofibrils.

Tlae use of point-counting also allowed the calculation of the relative areas occupied by different regions of the cell.

R e s u l t s

Chemical distribution of radioactivity The radioactivity occurring in free fatty acid 15 s after the pulse was more than three times as high during hypoxia (Fig. 1). Radioactivity in free fatty acid decreased rapidly during the 2-min chaser period under both conditions.

10,000. - 2500

8000- 1 -2000 ~=

6000- -1500 ~m o~

4000- - 1000 -~

,,<

�9 2000 -4 \ -500 o

0 15 3~3 OlO 120' 0 Seconds of chaser

FIGURE l. Incorporation of radioactivity in free fatty acid and triglyceride during the first 2 rain after a pulse-perfusion with tritiated oleic acid. Interrupted lines represent well oxygenated conditions. Continuous lines represent hypoxic conditions. Bars represent standard deviation.

768 B. Ward and P. H a r r i s

Triacyclglycerol was only slightly labelled under both conditions at 15 s (Fig. 1). During the 2-min chaser period the radioactivity in triacylglycerol increased only slightly under well oxygenated conditions, but rose dramat- ically during hypoxia (Fig. 1). Incorporation of radioactivity into phospholipid was low throughout and relatively little affected by hypoxia.

The radioactivity in the perfusate attribut- able to CO 2 was approximately 11 times as great under well oxygenated conditions as under hypoxic conditions. Little radioactivity attributable to 14COz was found in the extract of the heart itself.

Ultrastructure

The 30 rain hypoxic perfusion did not produce any noticeable ultrastructural changes or oedema. The use of point-counting allowed a measurement of the relative areas (and therefore relative volumes) of the sub- cellular territories and organelles. An analysis of variance showed no significant difference in the distribution of areas between the well- oxygenated and hypoxic conditions. Neither did any significant change take place during the period of two minutes' chaser. These nega- tive data are not given.

F I G U R E 2. Autoradiograph of hypoxic myocardium showing preservation of structure. Four grains are included. Bar = 1 #m.

Anatomical distribution of radioactivity An autoradiograph of a hypoxic heart is shown in Figure 2.

The 'relative intensities of sources' in some of the subcellular organelles are shown in Figs 3 and 4. Radioactivity appeared earlier in lipid droplets under hypoxic conditions (Fig. 3) and the relative intensity of sources in lipid droplets was always considerably higher than under well oxygenated conditions. Under well oxygenated conditions the relative intensity of sources in mitochondria reached a maximum at 60 s and fell rapidly thereafter (Fig. 4). In the hypoxic hearts the relative intensity of sources in mitochondria was lower and did not show the sharp decrease at the end of the 2-rain chaser period. Under both conditions the intensity of sources in T-tubules was rela- tively low and largely confined to the first 30 s. Under well oxygenated conditions the intensity of sources in the myofibrils and 'cyto- plasm' was relatively low and of a similar

"6 ~ 5 "

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F I G U R E 3. Relative intensity of sources in lipid droplets during the first 2 min after a pulse-perfusion with tritiated oleic acid. The interrupted line represents well oxygenated conditions. The continuous line represents hypoxic conditions.

Incorporation and Distribution of 3H Oleic Acid 769

1.2-

1.0-

-

0 " 8 - a)

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0.6- -

e) > 0 " 4 -

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F I G U R E 4. R e l a t i v e i n t e n s i t y o f s o u r c e s i n m i t o c h o n -

dria (O) and cytoplasm (0) during the first 2 min after a pulse-perfusion with tritiated oleic acid. Interrupted lines represent well oxygenated conditions. Continuous lines represent hypoxic conditions.

magnitude (myofibri!s 0.085 -F 0.041 s.D.; cytoplasm 0.073 4- 0.033 S.D.). With hypoxia the relative intensity of sources in the 'cyto- plasm' was consistently higher throughout the chaser period (myofibrils 0.130 4- 0.041 S.D.; cytoplasm 0.283 4- 0.057 S.D.). The intensity of sources in the sarcoplasmic reticulum was inconstant and never as high as that in the cytoplasm.

D i s c u s s i o n

Both the biochemical and autoradiographic results are consistent with the view that, during hypoxia, the rate-limiting step in the utilisation of fatty acids changes from trans- port into the cell to oxidation in the mito- chondria [-9].

Reduction in oxidation was confirmed by the greatly reduced production of 14CO2. The large increase in tissue lipid radioactivity in hypoxia was initially overwhelmingly due to free fatty acid, the concentration of which in the cytoplasm would be expected to increase when oxidation becomes rate-limiting. The lower relative intensity of sources in mito- chondria together with the higher relative intensity of sources in the cytoplasm during hypoxia suggests that there is an inhibition of fatty acyl transfer. Similarly, the slower disap-

pearance of radioactivity from mitochondria during hypoxia is consistent with a slower rate of beta-oxidation.

During the 2-min chaser period there was a marked increase in radioactivity in tri- acylglycerol and a parallel increase in the relative intensity of radioactive sources in lipid droplets. These results could be explained by an increased availability of fatty acyl CoA together with an increased forma- tion ofglycerol-3-phosphate by glycolysis.

Our results lend no support to the sugges- tion by Stein and Stein [13] that fatty acids travel through the cell via the sarcoplasmic reticulum. The relative intensity of sources in the sarcoplasmic reticulum was inconstant, presumably because of the small number of grains lying over the organelle, but lower than that in the cytoplasm.

In the experiments of Stein and Stein [-13] there were no grains lying over the myofibrils. We believe that this is an indication of the degree of lipid loss during their preparative procedure. Under well oxygenated condi- tions, we found no difference between the relative intensity of sources in the myofibrils and that in the cytoplasm not occupied by contractile protein. During hypoxia, however, the intensity of sources seemed higher in the cytosol.

Since the areas of free cytoplasm occur most frequently in relation to mitochondria, we considered the possibility that our stereologi- cal technique had been inadequate to elimi- nate cross-fire in this region. The theoretical basis of the stereological analysis which we developed for this purpose [16] assumes a uniform distribution of radioactivity within any composite region. Thus, if there were a high intensity of sources associated with the mitochondrial outer membrane, this would have little effect on the over-all relative inten- sity of sources in the mitochondria, but it might give rise to a spuriously high relative intensity of sources in the small region of cyto- plasm surrounding the mitochondria. In order to investigate this possibility we carried out a circle analysis, using the method of Wil- liams [18]. For this purpose we considered only mitochondrial and extra-mitochondrial regions together with a region including the mitochondrial outer membrane. This analysis gave no evidence of a specifically high relative

770 B. Ward and P. Harr i s

in tens i ty of sources at the m i t o c h o n d r i a l ou t e r m e m b r a n e .

T h e r e seems, therefore , to be a p a r t i c u l a r loca l i sa t ion of r ad ioac t i v i t y to the c y t o p l a s m outs ide the m y o f i l a m e n t s d u r i n g hypox ia . Th i s m a y be because the con t rac t i l e p ro te ins o c c u p y space wh ich w o u l d o the rwise be

o c c u p i e d by a solut ion o f the h ighe r aff ini ty b i n d i n g prote ins [7, 8].

Acknowledgements This w o r k was s u p p o r t e d by a g r an t f rom the M e d i c a l Resea rch Counc i l .

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