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Metabolism of lipids in experimental hypertrophic hearts of rabbits
Metabolism Clinical and Experimental VOL. XXVIII, NO. 6
Metabolism
JUNE 1979
of Lipids in Experimental Hearts of Rabbits
Hypertrophic
N. W. Revis and A. J. V. Cameron Cardiac hypertrophy was induced in rabbits by subcutaneous injection of thyroxine or isoprenaline or by surgically constricting the abdominal aorta. Alterations in lipid metabolism were observed in these hypertrophic hearts. Thyroxine or isoprenaline treatment increased the fatty acids in the serum and stimulated a marked increase in total lipids, triglycerides, and fatty acids in the hypertrophied myocardium. Coarctation of the aorta, in contrast, induced a significant increase in these lipids without significantly affecting serum free fatty acids. Histochemical and morphological studies confirmed an increase in neutral lipids. It is suggested that the observed increase in fatty acids in the heart following thyroxine or isoprenaline treatment is related to the increase in serum free fatty acids, which is followed by an increase in the removal of serum fatty acids by the heart. However. the amount of serum fatty acids that is removed exceeds the amount that is oxidized, which leads to an increase in lipid stores. The increase in lipid stores in the heart following coarctation of the aorta probably corresponds to the decrease in myocardial concentrations of carnitine. Serum lipid levels following coarctation were not significantly different from those of controls.
U NDER lipids
NORMAL physiologic conditions, are considered to be the main substrate of cardiac metabolism.‘4 The arteriovenous difference in serum lipids in both humans and animals shows an important uptake of free and esterified fatty acids under normal conditions.“6 However, it has been suggested that this uptake may be appreciably larger than the amount that is oxidized by the heart, which suggests that the heart has a means of eliminating or putting into storage a part of the uptake.5*6 The accumulation of lipid droplets in the human hypertrophic heart at autopsy has frequently Metabolism, Vol.28, No. 6 (June). 1979
been observed,’ but the factor(s) responsible for this accumulation are presently unknown. Several factors, such as the rate of lipid oxidation, or the rate at which lipids are extracted versus the rate at which they are oxidized, will affect the accumulation of lipids in the heart. To determine what factor(s) contribute to the lipid stores in the hypertrophic heart, experimental cardiac hypertrophy was induced in rabbits. MATERIALS
AND
METHODS
Male Californian rabbits, 3 months described below.
old, were used as
Chemicals All chemicals were “analar grade” or its equivalent. L-Thyroxine and isoprenaline-HCI were purchased from Burroughs-Wellcome. Inc., England. Materials used for measuring triglycerides and carnitine were obtained from Boehringer Mannheim Corp., Mannheim, Germany.
Isoprenaline Treatment Isoprenaline-HCI dissolved in 0.9% saline and injected subcutaneously (SC) at an initial dose of 1 mg/kg body
From The University of Tennessee-Oak Ridge Graduate_ School of Biomedical Sciences, The Biology Division, Oak Ridge National Laboratory, Oak Ridge, Term. and Department of Cardiology, University of Glasgow, Glasgow, Scotland. Received for publicaiion June 12, 1978. Supported by the Division of Biomedical and Environmental Research, U.S. Department of Energy under contract W-74005-eng-26 with the Union Carbide Corporation. Address reprint requests to Dr. N. W. Revis. Biology Division, Oak Ridge National Laboratory, Post Office Box Y, Oak Ridge, Term. 37830. 0 1979 by Grune & Stratton, Inc. 0026-0495/79/2806~I$01.00/0
601
REVIS AND CAMERON
602
weight, on the first day. On each subsequent day the dose was increased by 1 mg/kg. so that on the fifteenth day the dose was 15 mg/kg body weight. Control rabbits were injected with 0.9% saline for 1S days.
tant were used to determine Bergmeyer, et al.“’
Thyroxine Treatment
Fatty acids were determined on an aliquot form extract by the method of Duncomb.”
Sodium L-Thyroxine was dissolved in 0.01 N NaOH and brought to pH 9.5 with 0.1 N HCI; daily SC/injections of 88 pgg/kg body weight were administered for 15 days. Control rabbits were injected daily with 0.9% saline adjusted to pH 10 with 0.01 N NaOH.
Coarctation of the Abdominal Aorta Animals were anesthetized with Nembutalo, and a l-mm silver clamp was placed around the descending aorta just beneath the diaphragm, according to the original method of Beznak as modified by Meerson.’ This procedure created a diminution of the transverse section of the aorta to about three-fourths of its original area. Sham-operated rabbits were treated identically except that aortic constriction was omitted.
Total Lipids Determination Lipids were extracted in a manner similar to that described by Folch, et al.9 Approximately 1 g of left ventricle was cut into small slices and placed in a volumetric flask containing 20 ml of chloroform-methanol (2:l v/v). After homogenization for 8 min in an “Ultra Turrax” homogenizer (setting of 120). the homogenate was filtered through Whatman No. 1 paper into a glass-stoppered test tube. The residue was rehomogenized in IO ml of chloroform-methanol mixture and again filtered. The combined extracts were shaken with 6 ml of 0.9% potassium chloride, and the two phases were separated by centrifugation at 400 g for 30 min. The upper phase was aspirated, and the lower phase was rinsed three times with 4.5 ml of a chloroform-methanolwater mixture (3:48:47 v/v/v). After each rinse the two phases were allowed to separate, and the upper phase was removed by aspiration. The lower phase was finally evaporated to dryness at 37“C under a stream of nitrogen. After drying, the sample was taken up in 7 ml of pure chloroform, filtered, and brought to a final volume of IO ml. The total amount of lipids present was measured by pipetting 2-ml aliquots of the chloroform extract into weighed tubes, evaporating the chloroform by heating at 37OC for 4 hr. and determining the weight of the residue.
Triglycerides Determination For triglyceride determination the chloroform-methanol extracts were taken to dryness with a stream of nitrogen and the residue was redissolved in chloroform and this mixture was passed through a column (0.8X I6 cm) that contained activated silicic acid mixture (0.5 g silicic acid/column). The column was then washed with chloroform. The elute was then evaporated to dryness at 37OC with a stream of nitrogen. The residue was saponified in preweighed tubes for 30 min in a 70°C water bath with 0.5 ml of 0.S N alcoholic KOH/mg of lipid. After saponification, 1 ml of 0.15 M magnesium sulfate was added, and the contents of the tubes were mixed and centrifuged. Then, 0.5-ml aliquots of the clear superna-
glycerol
by the method
of
Fatty Acid Determination of the chloro-
Cholesterol Determination An aliquot of the elute from the silicic acid column was evaporated to dryness with a stream of nitrogen, the residue was saponified, and total cholesterol was measured by the method of Zlatkis et al.”
Phospholipid Determination After the neutral lipids were eluted from the silicic column, the phospholipids were eluted with methanol.” A suitable volume of this methanol solution was evaporated to dryness at 65”-80°C. The lipid residue was then digested with nitrogen-free concentrated H,SO, and 70% perchloric acid, and assayed for phosphate by the method of Lindberg and Ernster.14
Serum Free Fatty Acid Determination Samples of blood were collected at 1 I a.m. from the marginal ear vein, the blood was allowed to coagulate, and serum was then removed. Fatty acids were extracted into chloroform-methanol (2: I v/v) and measured as described above.
Histochemical Procedures Sections of left ventricles were removed and fixed for 24 hr at 4°C in formal-calcium fixative containing 4% w/v formaldehyde and 1% anhydrous calcium chloride. Tissues were frozen and S-pm sections were cut by use of a cold microtome. These sections were either stained with Sudan black “B” as described by ChifTelle and PuttI and counterstained with hemalum, or stained with oil red “0” according to Lillie16 and counterstained with nuclear fast green.
Carnitine Determination A section of left ventricle was removed following the above treatments and frozen in dry ice. Total, bound, and free carnitine fractions were then determined by extraction as previously described.” The enzymatic assay was based on that of Marquis and Fritz” as modified by Boehringer Mannheim Corp. to measure the absorbance of free coenzyme A (CoA) at 233 nm.
Electron Microscope Studies Sections of the left ventricle were fixed for 4 hr in ice-cold 2.5% glutaraldehyde in 3% sucrose buffered at pH 7.2 with 0.1 M caccdylate; washed for 6 hr with cold 6% sucrose in 0. I M cacodylate (pH 7.2); postfixed with I% osmium tetroxide in 5% sucrose buffered with 0.1 M cacodylate (pH 7.2); dehydrated through an ethanol series and propylene oxide; and embedded in araldite. Silver-gray sections were cut and stained with uranyl acetate and lead citrate with an AEI electron
microscope.
and examined
LIPIDS IN HYPERTAOPHIC RABBIT HEARTS
Table 1. Effect of Coarctation,
603
Thyroxine.
or lsoprenaline
Treatment
on Body, Heart, and Left Ventricle
Wet Weights
at
Various Time Intervals Time of Treatment (days)
No.
of Animals
Treatment N0na
Thyroxine
lsoprenaline
Initial
Final
BodyWeight/ Heart Weight x loo
Left Ventricle Weight Ig)
Heart Weight (gl
-
2468
f
59
2791
IL 88
5.7 + 0.63
1.90 f
0.20
f
3
10
2541
f
63
2813
f
78
8.1
f
0.91.
3.44
+ o.ost
0.29
+ 0.031*
3
21
2400
+ 39
2700
f
51
10.4
f
1.3$
4.69
+ 0.1 J$
0.39
+ O.OSSS
3
5
2523
f
2510
f
44
0.77
1.93 + 0.08
0.22
+ 0.048
4
10
2488
+ 63
2489
zk 69
6.4
2.29
f
0.06
0.25
+ 0.053
3
15
2601
+ 48
2553
+ 60
8.3 f
2.81
f
0.10
0.32
+- 0.044$
4
5
2444
-c 37
2684
+ JO
9.4 + 0.59$
* 0.14t
0.35
f
0.066$
3
10
2541
f
44
2763
+ 30
9.3 f
0.83$
3.1 +_ 0.08t
0.34
f
0.040$
3
15
2600
f
63
2801
k 43
9.6
0.61$
3.6 + O.llt
0.34
f
0.051$
14
Coarctation
BodvWeight (a)
55
5.6 f
+ 0.68
f
0.10.
3.3
0.05
0.013
Values are means + SE.
lp <
0.05.
tp < 0.01. sp < 0.001. Statistical
and a progressive increase in heart weight of 40%. The peak increase in heart weight was significant, as were the ratios of heart weight to body weight following all three treatments. All three methods increased left ventricle weight. Following 5 to 15 days of thyroxine and isoprenaline treatment, the left ventricle still accounted for approximately one third of the heart weight, indicating that the rest of the heart had increased in the same proportion. In contrast to these results, left ventricle weights following coarctation accounted for almost half the weight of the heart, indicating that most of the growth occurred in the left ventricle. It is thus clear that all these methods produce an increase in both heart and left ventricle weights. The total amount of lipids in the left ventricle following these treatments is shown in Table 2.
Method
In the experiments for which statistical analysis was performed, results were analyzed by Student’s t test. The expressions p < 0.05, p < 0.01, and p < 0.001 are used to indicate significance at the 5%, I %, and 0.1% levels, respectively. RESULTS
Table 1 shows the changes in body, heart, and left ventricle wet weights following each of the three treatments. The saline-injected and shamoperated rabbits were grouped together because these treatments did not significantly affect either body, heart, or left ventricle weights. Both heart and body weights increased following isoprenaline treatment (maximum heart weight increase 60%) or coarctation (maximum heart weight increase 80%). Thyroxine treatment produced a progressive decrease in body weight Table 2.
Effect of Coarctation,
Thyroxine,
and lsoprenaline
the Left Ventricle
Treatment None Coarctation Thyroxine
lsoprenaline
No. of Animals 20
Time of Treatment (days)
Treatment
Total Lipids (mg)
0.05. 0.01. 0.001.
and Free Fatty Acids in
Triglycerides (pm&s glyceride glycerol)
Free Fatty Acids (rmoles)
-
20 + 2.1
1.622
f
0.133
5.9 + 0.33
3
10
32 + 2.6’
2.98
f
0.131*
8.9 + 0.64”
3
21
38 -I- 3.2t
2.89
rf: 0.097*
9.6
3
5
28 f
2.34
+ 0.063
7.3 + 0.40
4
10
41 + 5.4$
4.53
f
0.1 J3$
13.3
+ 0.73$
3
15
45 f
4.6$
4.73
f
0.166$
15.0
+ 0.78$
4
5
50 f
3.8$
4.80
f
0.213s
11.9 + 0.44$
3
10
38 + 2.6t
3.86
f
O.lOl$
12.2 + 0.60$
3
15
30 f
3.87
-c 0.096s
1.9
2.3t
Values are means + SE. All units are per g wet weight of the left ventricle.
*p < tp < sp <
on Total Lipids, Triglycerides,
at Various Time Intervals
+ 0.51t
9.1 f
0.53t
604
The saline-injected and sham-operated controls were grouped together because no significant difference was observed at the various time intervals. Significant increases in total lipids were observed following coarctation of the aorta for 10 or 21 days. Isoprenaline treatment produced an immediate increase at 5 days; total lipid levels gradually diminished as treatment was continued. In contrast, thyroxine treatment induced a progressive increase in total lipids; 15 days of thyroxine treatment more than doubled total lipids in these hearts. Table 2 shows the corresponding figures for glyceride glycerol (which may be taken to be proportionate to triglycerides) and for fatty acids. It is clear that these results are parallel to those for total lipids. The concentrations of cholesterol and phospholipid in the control hearts were 3.8 + 0.6 and 15.2 + 0.9 pmoles/g wet weight, respectively. The levels of choles-
REVIS AND
CAMERON
terol and phospholipid in the heart in the experimental groups were not significantly different from the controls, which suggests that the increase in total lipids reflects the increase in fatty acids and triglycerides. To confirm the chemical evidence of an increase in lipids in the hypertrophic hearts, histochemical and morphological studies were performed (Fig. 1). Sections of the left ventricle were stained with oil red “0,” which shows lipids as discrete red droplets, or with Sudan black black “B,” which shows lipids as discrete droplets. A normal heart is shown in Figs. 1A and 1B; lipid droplets were occasionally observed. Hypertrophic hearts are shown in Figs. lC-IG. A moderate increase in the number of lipid droplets was observed with coarctation (Fig. 1F). A progressive increase that became more marked was seen with thyroid treatment (Figs. 1C and 1D). The large number of lipid
Fig. 1. Sections of left ventricles from the following animals: (A) Rabbit injected with saline for 15 days. Stained with oil red “0” and counterstained with nuclear fast green. Note occasional lipid droplets that appear as discrete red droplets. x 150. (6) Sham-operated rabbit 21 days postoperative. Stained with Sudan black “B” and counterstained with hemalum. Occasional black discrete lipid droplets were observed. x 150. (Cl Rabbit injected with thyroxine for 10 days. Note the increase in red droplets inside the myocardial cells. x 400. (D) Rabbit injected with thyroxine for 15 days. Note the increase in black droplets over the number in (Cl. x 250. jE1 Rabbit injected with isoprenaline for 5 days. Discrete red droplets ere markedly increased. x 250. (Fl Rabbit in which aortic constriction was continued for 21 days. The number of discrete black droplets is increased over that in (B). x 200. (G) Rabbit injected with isoprenaline for 15 days. The number of discrete black droplets is decreased from that observed in (Cl. x 1CID.
JPIDS IN HYPERTROPHIC RABBIT HEARTS
605
Fig. 1 B
Fig. 1 C
REVIS AND CAMERON
606
Fig. 1 D
Fig. 1E
LIPIDS IN HYPERTROPHIC RABBIT HEARTS
607
Fig. 1F
Fig. 1G
606
droplets initially observed with isoprenaline treatment (Fig. 1E) decreased after longer treatment (Fig. 1G). Electron-microscope studies are shown in Fig. 2. In the normal heart (Fig. 2A), lipid droplets were occasionally observed. In hearts from isoprenaline-treated rabbits (Figs. 2B and 2C), the number of lipid droplets was increased; they were frequently found inside mitochondria that appeared to be degenerating. In hearts from rabbits treated surgically by constricting the aorta (Fig. 2D), or from thyroxine-treated rabbits (Fig. 2E), the number of lipid droplets was increased in the sarcoplasm in close association with the mitochondria. Occasionally, lipid inclusion in the mitochondria was observed in the heart following coarctation of the aorta. Thus, chemical, histochemical, and morphological studies confirmed the increase in lipids in the hypertrophic myocardium. In an attempt to
REVIS AND
CAMERON
explain the observed change in lipids in the hypertrophic hearts, serum free fatty acids (FFA) and tissue carnitine were measured. Results of the serum FFA determination are shown in Figure 3. Coarctation of the aorta for 10 days stimulated a small increase in serum FFA; however, the increase was not significant. Thyroxine had no effect for the first 6 days, but thereafter a modest but consistent increase @ < 0.05) in serum FFA was observed. Isoprenaline induced a significant (p < 0.05) increase in a matter of hours, which was sustained up to the sixth day, after which there was a return to the control level. The fact that thyroxine and isoprenaline both stimulated a significant increase in serum FFA, whereas coarctation did not, may be related to the previous observation (Table 2) that although all three treatments caused lipid accumulation in the heart, thyroxine and isoprenaline did so to a
Fig. 2. W Section of control heart from saline-injected rabbit. Note the normal appearance of the myofibrils (MY). mitochondria and transveraa tubular system Iarrows). and intercalated disc (ID). No lipid droplets appear in thii section. x lB,ooO. (B) Section of the left ventricle from a rabbit that was injected wlth iaopronalina for 6 days. Note the number of degenereting mitochondria end the number of mitochondria with lipid inclusion bodies. IF) fat droplet; (MI mitochondria. x 20,000. (C) Another section of left ventricle from aame rabbit as in (B). x 18,ooO (D) Sectii of the left ventricle following coarctation of the aorta for 21 days. Note the increase in lipid droplets in the aerooplasm and the fusing of the two 2 bands farrows). (GV) Golgi vesicles: fN) nucleus. x 21,000. (E) Section of heart from a rabbit injected with thyroxine for 10 days. Note the large lipid droplets that are surrounded by mitoohondria. x 26,000.
609
LIPIDS IN HYPERTROf’HlC RABBIT HEARTS
Fig. 28
Fig. 2C
REVIS AND
610
Fig. 2D
--
Fig. 2E
__ll_l----.
.._
CAMERON
LIPIDS IN HYPERTROPHlC RABBIT HEARTS
‘i”
4
6
HOURS
8
611
10
12
14
lipids, triglycerides, and FFA were all increased. That triglyceride levels were increased in these hypertrophic hearts suggests that serum triglycerides are removed at a rate which is greater than the amount oxidized, or that esterification of fatty acids is increased, or that oxidation of triglycerides is depressed. Previous studies have shown that catecholamines and thyroxine each stimulate an increase in serum triglycerides and the oxidation of triglycerides by the myocardium.‘6*‘s Thus the increase in myocardial triglycerides reported here may be the result of an increase in the uptake of serum triglycerides. With regard to triglyceride uptake, several conflicting results have been reported,‘7,‘9 and to date the question regarding triglyceride uptake remains unsolved. Nevertheless, these studies suggest that triglyceride uptake by the hypertrophied myocardium may explain the observed increase in triglycerides. An increase in the esterification cannot be ruled out. The present results suggest that several different mechanisms affect the metabolism of myocardial lipids following these treatments. It was shown that thyroxine treatment led to a gradual increase in myocardial lipids that was paralleled by a gradual increase in serum FFA. Several investigators have reported that thyroxine treatment leads to an increase in serum FFA20*2’and an increase in the uptake of serum FFA.22 Furthermore, it has been observed that in dogs receiving thyroxine, the amount of plasma fatty acids removed by the heart is higher than the amount oxidized by the heart.22 The increase in myocardial lipids observed in these studies following thyroxine treatment is most likely related to the fact that
16
DAYS
Concentrations of serum FFA in the controls Fig. 3. (0). or following coarctation of the aorta (A), thyroxine (0) or isopranslina treatments W. The increase following isoprenslina treatment for 5 days was significant (p < 0.05). as was the increase following thyroxine treatment from B to 16 days (p < 0.05).
greater extent than did coarctation. However, the fact that lipid accumulation does occur after coarctation may show that the accumulation is not entirely a result of raised serum FFA levels but is to some extent a feature of the process of hypertrophy itself. This possibility was further explored by measuring the concentration of carnitine. As shown in Table 3, carnitine levels were significantly depressed in the heart after 2 1 In contrast, carnitine days of coarctation. concentration was significantly increased after 15 days of thyroxine treatment. Carnitine levels following isoprenaline treatment were similar to those of controls. DISCUSSION
The present studies were performed to determine what factor(s) would affect the accumulation of lipids in the hypertrophied heart. When cardiac hypertrophy was induced by thyroxine or isoprenaline injections or by surgically constricting the abdominal aorta, concentrations of total Table 3.
Effect of Coarctation,
Thyroxine,
or lsopranalina
Treatment
on the Carnitine
Concentrations
in the Left Ventricle
at Various Time Intervals Time of No. of Treatment
Animals
None
14
Coarctation Thyroxine
lsoprenaline
Values are means f
lp
< 0.06.
tp <
0.01.
SE.
Carnitine Concentration
(pmoleslg
wet wtl
Treatment Bound
Free
(days)
Total
-
2.34
f
0.08
0.49
f
3
10
1.50
f
0.09.
0.36
+ 0.01
3
21
1.06
f
0.07T
0.30
* 0.02’
1.36 zk 0.09T
3
5
2.77
f
0.05
0.61
+ 0.06’
3.38
4
10
3.49
f
O.lOT
0.74
f
0.06.
4.23
f
3
15
4.01
+ 0.16T
0.86
+ o.ost
4.87
-c 0.1 lt
4
5
2.50
+ 0.04
0.33
* 0.04
2.83
+ 0.08
3
10
2.30
+ 0.06
0.39
+ 0.06
2.69
f
0.11
3
15
2.44
f
0.39
f
2.83
f
0.10
0.03
0.03
0.02
2.83
+ 0.14
1.86 rf: 0.19. +_ 0.10 O.lBT
REVIS AND CAMERON
612
following the increase in serum FFA, the heart removes more fatty acids than are oxidized, which results in the accumulation of lipid droplets in the myocardium. A similar explanation is provided for the increase in lipids in the hearts of isoprenalinetreated rabbits. Treatment with isoprenaline resulted in an immediate increase in both serum FFA and myocardial lipids during the first 5 days of treatment. Epinephrine and norepinephrine are known to stimulate both serum fatty acids and myocardial uptake and oxidation of fatty acids.23,24The observed increase in apparently degenerating mitochondria may be an additional reason for the increase in lipid droplets seen in the hearts of isoprenalinetreated rabbits. It is interesting to note that although isoprenaline treatment was continued for 15 days, serum FFA returned to normal levels following 7 days of treatment, and myocardial lipids decreased from the observed levels at day 5. These results suggest that the increase in myocardial fatty acids is related to the rise in serum FFA and that the mechanism responsible for the mobilization of serum FFA is altered as isoprenaline treatment is continued. Coarctation of the aorta induced a moderate but significant increase in myocardial lipids. Unlike thyroxine and isoprenaline treatments, coarctation had no significant effect on serum FFA. Thus, the increase in myocardial lipids following aortic constriction does not appear to be related to changes in serum FFA, as it was
following the other two treatments. If the removal of serum fatty acids by the heart exceeds the amount oxidized, then the increase in lipids in the heart after aortic constriction may have an explanation.5*6 It was observed that the concentrations of carnitine were significantly depressed following coarctation. Several investigators have shown that carnitine plays an important role in mitochondrial oxidation of fatty acids.25326 Thus, the decrease in carnitine and the possible disproportionate uptake-to-oxidation ratio of serum fatty acids in the hypertrophic heart following coarctation may be the mechanisms responsible for the observed increase in lipid stores in these hearts. It has been previously reported that carnitine concentrations are significantly reduced in the hypertrophic heart following coarctation.*’ The conclusion to be drawn from these studies is that lipid metabolism is altered in the hypertrophic heart regardless of the method used to induce hypertrophy. Furthermore, it is suggested that the factors contributing to the increase in lipid stores observed in these studies are (1) increased levels of serum FFA combined with an increase in the extraction by the heart, and (2) decreased ability of the heart to metabolize fatty acids due to the decrease in carnitine. These factors may be important in explaining the increase in lipid stores observed in the human hypertrophic heart at autopsy.
REFERENCES 1. Bing RJ: Cardiac metabolism. Physiol Rev 45:171198, 1965. 2. Opie LH: Metabolism of the heart in health and disease. Am Heart J 77:100-201. 1969 3. Evans JR: Cellular transport of fatty acids. Can J Biochem 42:955-961, 1964 4. Calsten LA, Hallgren R, Jugenburg A, et al: Myocardial metabolism of glucose, lactic acid, amino acids and fatty acids in healthy human individuals at rest and at different work loads. Stand J Clin Lab Invest 13:418--125, 1971 5. Gordon RW, Cherkes A: Unesterified fatty acids in human blood plasma. J Clin Invest 35:206-216, 1956 6. Dagenais CR, Marquis Y, Gailis L: Assessment of myocardial FFA metabolism in humans during heparin infusion. Seventh Annual Meeting of the International Study Group for Research in Cardiac Metabolism, Quebec, June 18-21, pp 35-38, 1972 (Abstract) 7. Pearse AGE: Cardiomyopathies. Ciba Foundation Symposium, 132.1964
8. Meerson FZ: The myocardium in hyperfunction, hypertrophy and heart failure. Circ Res 25 (Suppl 2):1-I 63, 1969 9. Folch J, Lees M, Sloane-Stanley GH: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497-509, 1957 10. Bergmeyer HU, Holz G, Kauder EM, et al: Cmxhfu myoccderma. Biochem 2 333:471-480, 1961 1I. Duncomb WG: The calorimetric micro-determination of long-chain fatty acids. Biochem J 88:7-10, 1963 12. ZIatkis A, Zak B, Boyle AJ: A new method for the direct determination of serum cholesteroL J Lab Ciin Med 41:486-492,1953 13. Patton S, Thomas AJ: Composition of lipid foams from win bladders of deep ocean fish species. J Lipid Res 12:331-335, 1971 14. Lindberg 0, Ernster L: Quantitative analysis of phospholipids. Methods Biochem Anal 3:1-7, 1956 15. ChifTelle TF, Putt FA: Propylene and ethylene glycol
LIPIDS IN HYPERTROPHIC RABBIT HEARTS
as solvent for Sudan IV and Sudan black B. Stain Technol 26:51-56.1951 16. Lillie RD: Study of certain oil soluble dyes for use as fat stains. J Technol Methods 24:37-42, 1944 17. Marquis NR, Fritz IB: Enzymological determination of free camitine concentrations in rat tissues. J Lipid Res 5:184-190.1964 18. Pearson DL, Tubbs PK: Tissue levels of acid insoluble carnitine in rat heart Biochim Biophys Acta 84:772-779, 1964 19. Crass MF III, Shipp JC, Pieper GM: Effects of catecholamines on myocardial endogenous substrates and contractility. Am J Physiol 228:618-627, 1975 20. Delcher HK, Fried M, Shipp SC: Metabolism of lipoprotein lipid in the isolated perfused rat heart. Biochim BiophysActa 106:1t&18, 1965 21. Opie LH: Metabolism of the heart in health and disease. Am Heart J 100:77-123, 1969 22. Gousios A, Felts JM, Have1 RJ: Metabolism of serum triglycerides and free fatty acids by the myocardium. Metabolism 12:75-88, 1963 23. Rich CE. Bierman L, Schwartz IL: Plasma nonesterified fatty acids in hyperthyroid states. J Clin Invest 38:275-278. 1959
613
24. Rosenfeld PS, Rosenberg IW: Effect of altered thyroid status upon epinephrine induced lipolysis in vitro. Proc Sot Exp Biol Med 118:221-225,1965 25. Gold M, Scott JC, Spitzer JJ: Myocardial metabolism of free fatty acids in control, hyperthyroid and hypothyroid dogs. Am J Physio123 1:239-244, 1967 26. Gold MH, Attar JH, Scott JC, Spitzer JJ: Effect of norepinephrine on myocardial free fatty acid uptake and oxidation. Proc Sot Exp Biol Med 118:876-879, 1965 27. Mayers SE: E&t of catecholamines on cardiac metabolism. Circ Res 34.35 (Suppl III):1 29-l 35, 1974 28. Fritz IB, Yue KTK: Long chain carnitine acyltransferase and the role of acyl carnitine derivatives in the catalytic increase of fatty acid oxidation induced by carnitine. J Lipid Res 4:279-288, 1963 29. Fritz IB: Hypothesis concerning the role of carnitine in the control of interrelations between fatty acids and carbohydrate metabolism. Perspect Biol Med 10:643-677, 1967 30. Wittels B, Spann JF: Defective lipid metabolism in the failing heart. J Clin Invest 47:1787-1793, 1968
Metabolism
JUNE 1979
of Lipids in Experimental Hearts of Rabbits
Hypertrophic
N. W. Revis and A. J. V. Cameron Cardiac hypertrophy was induced in rabbits by subcutaneous injection of thyroxine or isoprenaline or by surgically constricting the abdominal aorta. Alterations in lipid metabolism were observed in these hypertrophic hearts. Thyroxine or isoprenaline treatment increased the fatty acids in the serum and stimulated a marked increase in total lipids, triglycerides, and fatty acids in the hypertrophied myocardium. Coarctation of the aorta, in contrast, induced a significant increase in these lipids without significantly affecting serum free fatty acids. Histochemical and morphological studies confirmed an increase in neutral lipids. It is suggested that the observed increase in fatty acids in the heart following thyroxine or isoprenaline treatment is related to the increase in serum free fatty acids, which is followed by an increase in the removal of serum fatty acids by the heart. However. the amount of serum fatty acids that is removed exceeds the amount that is oxidized, which leads to an increase in lipid stores. The increase in lipid stores in the heart following coarctation of the aorta probably corresponds to the decrease in myocardial concentrations of carnitine. Serum lipid levels following coarctation were not significantly different from those of controls.
U NDER lipids
NORMAL physiologic conditions, are considered to be the main substrate of cardiac metabolism.‘4 The arteriovenous difference in serum lipids in both humans and animals shows an important uptake of free and esterified fatty acids under normal conditions.“6 However, it has been suggested that this uptake may be appreciably larger than the amount that is oxidized by the heart, which suggests that the heart has a means of eliminating or putting into storage a part of the uptake.5*6 The accumulation of lipid droplets in the human hypertrophic heart at autopsy has frequently Metabolism, Vol.28, No. 6 (June). 1979
been observed,’ but the factor(s) responsible for this accumulation are presently unknown. Several factors, such as the rate of lipid oxidation, or the rate at which lipids are extracted versus the rate at which they are oxidized, will affect the accumulation of lipids in the heart. To determine what factor(s) contribute to the lipid stores in the hypertrophic heart, experimental cardiac hypertrophy was induced in rabbits. MATERIALS
AND
METHODS
Male Californian rabbits, 3 months described below.
old, were used as
Chemicals All chemicals were “analar grade” or its equivalent. L-Thyroxine and isoprenaline-HCI were purchased from Burroughs-Wellcome. Inc., England. Materials used for measuring triglycerides and carnitine were obtained from Boehringer Mannheim Corp., Mannheim, Germany.
Isoprenaline Treatment Isoprenaline-HCI dissolved in 0.9% saline and injected subcutaneously (SC) at an initial dose of 1 mg/kg body
From The University of Tennessee-Oak Ridge Graduate_ School of Biomedical Sciences, The Biology Division, Oak Ridge National Laboratory, Oak Ridge, Term. and Department of Cardiology, University of Glasgow, Glasgow, Scotland. Received for publicaiion June 12, 1978. Supported by the Division of Biomedical and Environmental Research, U.S. Department of Energy under contract W-74005-eng-26 with the Union Carbide Corporation. Address reprint requests to Dr. N. W. Revis. Biology Division, Oak Ridge National Laboratory, Post Office Box Y, Oak Ridge, Term. 37830. 0 1979 by Grune & Stratton, Inc. 0026-0495/79/2806~I$01.00/0
601
REVIS AND CAMERON
602
weight, on the first day. On each subsequent day the dose was increased by 1 mg/kg. so that on the fifteenth day the dose was 15 mg/kg body weight. Control rabbits were injected with 0.9% saline for 1S days.
tant were used to determine Bergmeyer, et al.“’
Thyroxine Treatment
Fatty acids were determined on an aliquot form extract by the method of Duncomb.”
Sodium L-Thyroxine was dissolved in 0.01 N NaOH and brought to pH 9.5 with 0.1 N HCI; daily SC/injections of 88 pgg/kg body weight were administered for 15 days. Control rabbits were injected daily with 0.9% saline adjusted to pH 10 with 0.01 N NaOH.
Coarctation of the Abdominal Aorta Animals were anesthetized with Nembutalo, and a l-mm silver clamp was placed around the descending aorta just beneath the diaphragm, according to the original method of Beznak as modified by Meerson.’ This procedure created a diminution of the transverse section of the aorta to about three-fourths of its original area. Sham-operated rabbits were treated identically except that aortic constriction was omitted.
Total Lipids Determination Lipids were extracted in a manner similar to that described by Folch, et al.9 Approximately 1 g of left ventricle was cut into small slices and placed in a volumetric flask containing 20 ml of chloroform-methanol (2:l v/v). After homogenization for 8 min in an “Ultra Turrax” homogenizer (setting of 120). the homogenate was filtered through Whatman No. 1 paper into a glass-stoppered test tube. The residue was rehomogenized in IO ml of chloroform-methanol mixture and again filtered. The combined extracts were shaken with 6 ml of 0.9% potassium chloride, and the two phases were separated by centrifugation at 400 g for 30 min. The upper phase was aspirated, and the lower phase was rinsed three times with 4.5 ml of a chloroform-methanolwater mixture (3:48:47 v/v/v). After each rinse the two phases were allowed to separate, and the upper phase was removed by aspiration. The lower phase was finally evaporated to dryness at 37“C under a stream of nitrogen. After drying, the sample was taken up in 7 ml of pure chloroform, filtered, and brought to a final volume of IO ml. The total amount of lipids present was measured by pipetting 2-ml aliquots of the chloroform extract into weighed tubes, evaporating the chloroform by heating at 37OC for 4 hr. and determining the weight of the residue.
Triglycerides Determination For triglyceride determination the chloroform-methanol extracts were taken to dryness with a stream of nitrogen and the residue was redissolved in chloroform and this mixture was passed through a column (0.8X I6 cm) that contained activated silicic acid mixture (0.5 g silicic acid/column). The column was then washed with chloroform. The elute was then evaporated to dryness at 37OC with a stream of nitrogen. The residue was saponified in preweighed tubes for 30 min in a 70°C water bath with 0.5 ml of 0.S N alcoholic KOH/mg of lipid. After saponification, 1 ml of 0.15 M magnesium sulfate was added, and the contents of the tubes were mixed and centrifuged. Then, 0.5-ml aliquots of the clear superna-
glycerol
by the method
of
Fatty Acid Determination of the chloro-
Cholesterol Determination An aliquot of the elute from the silicic acid column was evaporated to dryness with a stream of nitrogen, the residue was saponified, and total cholesterol was measured by the method of Zlatkis et al.”
Phospholipid Determination After the neutral lipids were eluted from the silicic column, the phospholipids were eluted with methanol.” A suitable volume of this methanol solution was evaporated to dryness at 65”-80°C. The lipid residue was then digested with nitrogen-free concentrated H,SO, and 70% perchloric acid, and assayed for phosphate by the method of Lindberg and Ernster.14
Serum Free Fatty Acid Determination Samples of blood were collected at 1 I a.m. from the marginal ear vein, the blood was allowed to coagulate, and serum was then removed. Fatty acids were extracted into chloroform-methanol (2: I v/v) and measured as described above.
Histochemical Procedures Sections of left ventricles were removed and fixed for 24 hr at 4°C in formal-calcium fixative containing 4% w/v formaldehyde and 1% anhydrous calcium chloride. Tissues were frozen and S-pm sections were cut by use of a cold microtome. These sections were either stained with Sudan black “B” as described by ChifTelle and PuttI and counterstained with hemalum, or stained with oil red “0” according to Lillie16 and counterstained with nuclear fast green.
Carnitine Determination A section of left ventricle was removed following the above treatments and frozen in dry ice. Total, bound, and free carnitine fractions were then determined by extraction as previously described.” The enzymatic assay was based on that of Marquis and Fritz” as modified by Boehringer Mannheim Corp. to measure the absorbance of free coenzyme A (CoA) at 233 nm.
Electron Microscope Studies Sections of the left ventricle were fixed for 4 hr in ice-cold 2.5% glutaraldehyde in 3% sucrose buffered at pH 7.2 with 0.1 M caccdylate; washed for 6 hr with cold 6% sucrose in 0. I M cacodylate (pH 7.2); postfixed with I% osmium tetroxide in 5% sucrose buffered with 0.1 M cacodylate (pH 7.2); dehydrated through an ethanol series and propylene oxide; and embedded in araldite. Silver-gray sections were cut and stained with uranyl acetate and lead citrate with an AEI electron
microscope.
and examined
LIPIDS IN HYPERTAOPHIC RABBIT HEARTS
Table 1. Effect of Coarctation,
603
Thyroxine.
or lsoprenaline
Treatment
on Body, Heart, and Left Ventricle
Wet Weights
at
Various Time Intervals Time of Treatment (days)
No.
of Animals
Treatment N0na
Thyroxine
lsoprenaline
Initial
Final
BodyWeight/ Heart Weight x loo
Left Ventricle Weight Ig)
Heart Weight (gl
-
2468
f
59
2791
IL 88
5.7 + 0.63
1.90 f
0.20
f
3
10
2541
f
63
2813
f
78
8.1
f
0.91.
3.44
+ o.ost
0.29
+ 0.031*
3
21
2400
+ 39
2700
f
51
10.4
f
1.3$
4.69
+ 0.1 J$
0.39
+ O.OSSS
3
5
2523
f
2510
f
44
0.77
1.93 + 0.08
0.22
+ 0.048
4
10
2488
+ 63
2489
zk 69
6.4
2.29
f
0.06
0.25
+ 0.053
3
15
2601
+ 48
2553
+ 60
8.3 f
2.81
f
0.10
0.32
+- 0.044$
4
5
2444
-c 37
2684
+ JO
9.4 + 0.59$
* 0.14t
0.35
f
0.066$
3
10
2541
f
44
2763
+ 30
9.3 f
0.83$
3.1 +_ 0.08t
0.34
f
0.040$
3
15
2600
f
63
2801
k 43
9.6
0.61$
3.6 + O.llt
0.34
f
0.051$
14
Coarctation
BodvWeight (a)
55
5.6 f
+ 0.68
f
0.10.
3.3
0.05
0.013
Values are means + SE.
lp <
0.05.
tp < 0.01. sp < 0.001. Statistical
and a progressive increase in heart weight of 40%. The peak increase in heart weight was significant, as were the ratios of heart weight to body weight following all three treatments. All three methods increased left ventricle weight. Following 5 to 15 days of thyroxine and isoprenaline treatment, the left ventricle still accounted for approximately one third of the heart weight, indicating that the rest of the heart had increased in the same proportion. In contrast to these results, left ventricle weights following coarctation accounted for almost half the weight of the heart, indicating that most of the growth occurred in the left ventricle. It is thus clear that all these methods produce an increase in both heart and left ventricle weights. The total amount of lipids in the left ventricle following these treatments is shown in Table 2.
Method
In the experiments for which statistical analysis was performed, results were analyzed by Student’s t test. The expressions p < 0.05, p < 0.01, and p < 0.001 are used to indicate significance at the 5%, I %, and 0.1% levels, respectively. RESULTS
Table 1 shows the changes in body, heart, and left ventricle wet weights following each of the three treatments. The saline-injected and shamoperated rabbits were grouped together because these treatments did not significantly affect either body, heart, or left ventricle weights. Both heart and body weights increased following isoprenaline treatment (maximum heart weight increase 60%) or coarctation (maximum heart weight increase 80%). Thyroxine treatment produced a progressive decrease in body weight Table 2.
Effect of Coarctation,
Thyroxine,
and lsoprenaline
the Left Ventricle
Treatment None Coarctation Thyroxine
lsoprenaline
No. of Animals 20
Time of Treatment (days)
Treatment
Total Lipids (mg)
0.05. 0.01. 0.001.
and Free Fatty Acids in
Triglycerides (pm&s glyceride glycerol)
Free Fatty Acids (rmoles)
-
20 + 2.1
1.622
f
0.133
5.9 + 0.33
3
10
32 + 2.6’
2.98
f
0.131*
8.9 + 0.64”
3
21
38 -I- 3.2t
2.89
rf: 0.097*
9.6
3
5
28 f
2.34
+ 0.063
7.3 + 0.40
4
10
41 + 5.4$
4.53
f
0.1 J3$
13.3
+ 0.73$
3
15
45 f
4.6$
4.73
f
0.166$
15.0
+ 0.78$
4
5
50 f
3.8$
4.80
f
0.213s
11.9 + 0.44$
3
10
38 + 2.6t
3.86
f
O.lOl$
12.2 + 0.60$
3
15
30 f
3.87
-c 0.096s
1.9
2.3t
Values are means + SE. All units are per g wet weight of the left ventricle.
*p < tp < sp <
on Total Lipids, Triglycerides,
at Various Time Intervals
+ 0.51t
9.1 f
0.53t
604
The saline-injected and sham-operated controls were grouped together because no significant difference was observed at the various time intervals. Significant increases in total lipids were observed following coarctation of the aorta for 10 or 21 days. Isoprenaline treatment produced an immediate increase at 5 days; total lipid levels gradually diminished as treatment was continued. In contrast, thyroxine treatment induced a progressive increase in total lipids; 15 days of thyroxine treatment more than doubled total lipids in these hearts. Table 2 shows the corresponding figures for glyceride glycerol (which may be taken to be proportionate to triglycerides) and for fatty acids. It is clear that these results are parallel to those for total lipids. The concentrations of cholesterol and phospholipid in the control hearts were 3.8 + 0.6 and 15.2 + 0.9 pmoles/g wet weight, respectively. The levels of choles-
REVIS AND
CAMERON
terol and phospholipid in the heart in the experimental groups were not significantly different from the controls, which suggests that the increase in total lipids reflects the increase in fatty acids and triglycerides. To confirm the chemical evidence of an increase in lipids in the hypertrophic hearts, histochemical and morphological studies were performed (Fig. 1). Sections of the left ventricle were stained with oil red “0,” which shows lipids as discrete red droplets, or with Sudan black black “B,” which shows lipids as discrete droplets. A normal heart is shown in Figs. 1A and 1B; lipid droplets were occasionally observed. Hypertrophic hearts are shown in Figs. lC-IG. A moderate increase in the number of lipid droplets was observed with coarctation (Fig. 1F). A progressive increase that became more marked was seen with thyroid treatment (Figs. 1C and 1D). The large number of lipid
Fig. 1. Sections of left ventricles from the following animals: (A) Rabbit injected with saline for 15 days. Stained with oil red “0” and counterstained with nuclear fast green. Note occasional lipid droplets that appear as discrete red droplets. x 150. (6) Sham-operated rabbit 21 days postoperative. Stained with Sudan black “B” and counterstained with hemalum. Occasional black discrete lipid droplets were observed. x 150. (Cl Rabbit injected with thyroxine for 10 days. Note the increase in red droplets inside the myocardial cells. x 400. (D) Rabbit injected with thyroxine for 15 days. Note the increase in black droplets over the number in (Cl. x 250. jE1 Rabbit injected with isoprenaline for 5 days. Discrete red droplets ere markedly increased. x 250. (Fl Rabbit in which aortic constriction was continued for 21 days. The number of discrete black droplets is increased over that in (B). x 200. (G) Rabbit injected with isoprenaline for 15 days. The number of discrete black droplets is decreased from that observed in (Cl. x 1CID.
JPIDS IN HYPERTROPHIC RABBIT HEARTS
605
Fig. 1 B
Fig. 1 C
REVIS AND CAMERON
606
Fig. 1 D
Fig. 1E
LIPIDS IN HYPERTROPHIC RABBIT HEARTS
607
Fig. 1F
Fig. 1G
606
droplets initially observed with isoprenaline treatment (Fig. 1E) decreased after longer treatment (Fig. 1G). Electron-microscope studies are shown in Fig. 2. In the normal heart (Fig. 2A), lipid droplets were occasionally observed. In hearts from isoprenaline-treated rabbits (Figs. 2B and 2C), the number of lipid droplets was increased; they were frequently found inside mitochondria that appeared to be degenerating. In hearts from rabbits treated surgically by constricting the aorta (Fig. 2D), or from thyroxine-treated rabbits (Fig. 2E), the number of lipid droplets was increased in the sarcoplasm in close association with the mitochondria. Occasionally, lipid inclusion in the mitochondria was observed in the heart following coarctation of the aorta. Thus, chemical, histochemical, and morphological studies confirmed the increase in lipids in the hypertrophic myocardium. In an attempt to
REVIS AND
CAMERON
explain the observed change in lipids in the hypertrophic hearts, serum free fatty acids (FFA) and tissue carnitine were measured. Results of the serum FFA determination are shown in Figure 3. Coarctation of the aorta for 10 days stimulated a small increase in serum FFA; however, the increase was not significant. Thyroxine had no effect for the first 6 days, but thereafter a modest but consistent increase @ < 0.05) in serum FFA was observed. Isoprenaline induced a significant (p < 0.05) increase in a matter of hours, which was sustained up to the sixth day, after which there was a return to the control level. The fact that thyroxine and isoprenaline both stimulated a significant increase in serum FFA, whereas coarctation did not, may be related to the previous observation (Table 2) that although all three treatments caused lipid accumulation in the heart, thyroxine and isoprenaline did so to a
Fig. 2. W Section of control heart from saline-injected rabbit. Note the normal appearance of the myofibrils (MY). mitochondria and transveraa tubular system Iarrows). and intercalated disc (ID). No lipid droplets appear in thii section. x lB,ooO. (B) Section of the left ventricle from a rabbit that was injected wlth iaopronalina for 6 days. Note the number of degenereting mitochondria end the number of mitochondria with lipid inclusion bodies. IF) fat droplet; (MI mitochondria. x 20,000. (C) Another section of left ventricle from aame rabbit as in (B). x 18,ooO (D) Sectii of the left ventricle following coarctation of the aorta for 21 days. Note the increase in lipid droplets in the aerooplasm and the fusing of the two 2 bands farrows). (GV) Golgi vesicles: fN) nucleus. x 21,000. (E) Section of heart from a rabbit injected with thyroxine for 10 days. Note the large lipid droplets that are surrounded by mitoohondria. x 26,000.
609
LIPIDS IN HYPERTROf’HlC RABBIT HEARTS
Fig. 28
Fig. 2C
REVIS AND
610
Fig. 2D
--
Fig. 2E
__ll_l----.
.._
CAMERON
LIPIDS IN HYPERTROPHlC RABBIT HEARTS
‘i”
4
6
HOURS
8
611
10
12
14
lipids, triglycerides, and FFA were all increased. That triglyceride levels were increased in these hypertrophic hearts suggests that serum triglycerides are removed at a rate which is greater than the amount oxidized, or that esterification of fatty acids is increased, or that oxidation of triglycerides is depressed. Previous studies have shown that catecholamines and thyroxine each stimulate an increase in serum triglycerides and the oxidation of triglycerides by the myocardium.‘6*‘s Thus the increase in myocardial triglycerides reported here may be the result of an increase in the uptake of serum triglycerides. With regard to triglyceride uptake, several conflicting results have been reported,‘7,‘9 and to date the question regarding triglyceride uptake remains unsolved. Nevertheless, these studies suggest that triglyceride uptake by the hypertrophied myocardium may explain the observed increase in triglycerides. An increase in the esterification cannot be ruled out. The present results suggest that several different mechanisms affect the metabolism of myocardial lipids following these treatments. It was shown that thyroxine treatment led to a gradual increase in myocardial lipids that was paralleled by a gradual increase in serum FFA. Several investigators have reported that thyroxine treatment leads to an increase in serum FFA20*2’and an increase in the uptake of serum FFA.22 Furthermore, it has been observed that in dogs receiving thyroxine, the amount of plasma fatty acids removed by the heart is higher than the amount oxidized by the heart.22 The increase in myocardial lipids observed in these studies following thyroxine treatment is most likely related to the fact that
16
DAYS
Concentrations of serum FFA in the controls Fig. 3. (0). or following coarctation of the aorta (A), thyroxine (0) or isopranslina treatments W. The increase following isoprenslina treatment for 5 days was significant (p < 0.05). as was the increase following thyroxine treatment from B to 16 days (p < 0.05).
greater extent than did coarctation. However, the fact that lipid accumulation does occur after coarctation may show that the accumulation is not entirely a result of raised serum FFA levels but is to some extent a feature of the process of hypertrophy itself. This possibility was further explored by measuring the concentration of carnitine. As shown in Table 3, carnitine levels were significantly depressed in the heart after 2 1 In contrast, carnitine days of coarctation. concentration was significantly increased after 15 days of thyroxine treatment. Carnitine levels following isoprenaline treatment were similar to those of controls. DISCUSSION
The present studies were performed to determine what factor(s) would affect the accumulation of lipids in the hypertrophied heart. When cardiac hypertrophy was induced by thyroxine or isoprenaline injections or by surgically constricting the abdominal aorta, concentrations of total Table 3.
Effect of Coarctation,
Thyroxine,
or lsopranalina
Treatment
on the Carnitine
Concentrations
in the Left Ventricle
at Various Time Intervals Time of No. of Treatment
Animals
None
14
Coarctation Thyroxine
lsoprenaline
Values are means f
lp
< 0.06.
tp <
0.01.
SE.
Carnitine Concentration
(pmoleslg
wet wtl
Treatment Bound
Free
(days)
Total
-
2.34
f
0.08
0.49
f
3
10
1.50
f
0.09.
0.36
+ 0.01
3
21
1.06
f
0.07T
0.30
* 0.02’
1.36 zk 0.09T
3
5
2.77
f
0.05
0.61
+ 0.06’
3.38
4
10
3.49
f
O.lOT
0.74
f
0.06.
4.23
f
3
15
4.01
+ 0.16T
0.86
+ o.ost
4.87
-c 0.1 lt
4
5
2.50
+ 0.04
0.33
* 0.04
2.83
+ 0.08
3
10
2.30
+ 0.06
0.39
+ 0.06
2.69
f
0.11
3
15
2.44
f
0.39
f
2.83
f
0.10
0.03
0.03
0.02
2.83
+ 0.14
1.86 rf: 0.19. +_ 0.10 O.lBT
REVIS AND CAMERON
612
following the increase in serum FFA, the heart removes more fatty acids than are oxidized, which results in the accumulation of lipid droplets in the myocardium. A similar explanation is provided for the increase in lipids in the hearts of isoprenalinetreated rabbits. Treatment with isoprenaline resulted in an immediate increase in both serum FFA and myocardial lipids during the first 5 days of treatment. Epinephrine and norepinephrine are known to stimulate both serum fatty acids and myocardial uptake and oxidation of fatty acids.23,24The observed increase in apparently degenerating mitochondria may be an additional reason for the increase in lipid droplets seen in the hearts of isoprenalinetreated rabbits. It is interesting to note that although isoprenaline treatment was continued for 15 days, serum FFA returned to normal levels following 7 days of treatment, and myocardial lipids decreased from the observed levels at day 5. These results suggest that the increase in myocardial fatty acids is related to the rise in serum FFA and that the mechanism responsible for the mobilization of serum FFA is altered as isoprenaline treatment is continued. Coarctation of the aorta induced a moderate but significant increase in myocardial lipids. Unlike thyroxine and isoprenaline treatments, coarctation had no significant effect on serum FFA. Thus, the increase in myocardial lipids following aortic constriction does not appear to be related to changes in serum FFA, as it was
following the other two treatments. If the removal of serum fatty acids by the heart exceeds the amount oxidized, then the increase in lipids in the heart after aortic constriction may have an explanation.5*6 It was observed that the concentrations of carnitine were significantly depressed following coarctation. Several investigators have shown that carnitine plays an important role in mitochondrial oxidation of fatty acids.25326 Thus, the decrease in carnitine and the possible disproportionate uptake-to-oxidation ratio of serum fatty acids in the hypertrophic heart following coarctation may be the mechanisms responsible for the observed increase in lipid stores in these hearts. It has been previously reported that carnitine concentrations are significantly reduced in the hypertrophic heart following coarctation.*’ The conclusion to be drawn from these studies is that lipid metabolism is altered in the hypertrophic heart regardless of the method used to induce hypertrophy. Furthermore, it is suggested that the factors contributing to the increase in lipid stores observed in these studies are (1) increased levels of serum FFA combined with an increase in the extraction by the heart, and (2) decreased ability of the heart to metabolize fatty acids due to the decrease in carnitine. These factors may be important in explaining the increase in lipid stores observed in the human hypertrophic heart at autopsy.
REFERENCES 1. Bing RJ: Cardiac metabolism. Physiol Rev 45:171198, 1965. 2. Opie LH: Metabolism of the heart in health and disease. Am Heart J 77:100-201. 1969 3. Evans JR: Cellular transport of fatty acids. Can J Biochem 42:955-961, 1964 4. Calsten LA, Hallgren R, Jugenburg A, et al: Myocardial metabolism of glucose, lactic acid, amino acids and fatty acids in healthy human individuals at rest and at different work loads. Stand J Clin Lab Invest 13:418--125, 1971 5. Gordon RW, Cherkes A: Unesterified fatty acids in human blood plasma. J Clin Invest 35:206-216, 1956 6. Dagenais CR, Marquis Y, Gailis L: Assessment of myocardial FFA metabolism in humans during heparin infusion. Seventh Annual Meeting of the International Study Group for Research in Cardiac Metabolism, Quebec, June 18-21, pp 35-38, 1972 (Abstract) 7. Pearse AGE: Cardiomyopathies. Ciba Foundation Symposium, 132.1964
8. Meerson FZ: The myocardium in hyperfunction, hypertrophy and heart failure. Circ Res 25 (Suppl 2):1-I 63, 1969 9. Folch J, Lees M, Sloane-Stanley GH: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497-509, 1957 10. Bergmeyer HU, Holz G, Kauder EM, et al: Cmxhfu myoccderma. Biochem 2 333:471-480, 1961 1I. Duncomb WG: The calorimetric micro-determination of long-chain fatty acids. Biochem J 88:7-10, 1963 12. ZIatkis A, Zak B, Boyle AJ: A new method for the direct determination of serum cholesteroL J Lab Ciin Med 41:486-492,1953 13. Patton S, Thomas AJ: Composition of lipid foams from win bladders of deep ocean fish species. J Lipid Res 12:331-335, 1971 14. Lindberg 0, Ernster L: Quantitative analysis of phospholipids. Methods Biochem Anal 3:1-7, 1956 15. ChifTelle TF, Putt FA: Propylene and ethylene glycol
LIPIDS IN HYPERTROPHIC RABBIT HEARTS
as solvent for Sudan IV and Sudan black B. Stain Technol 26:51-56.1951 16. Lillie RD: Study of certain oil soluble dyes for use as fat stains. J Technol Methods 24:37-42, 1944 17. Marquis NR, Fritz IB: Enzymological determination of free camitine concentrations in rat tissues. J Lipid Res 5:184-190.1964 18. Pearson DL, Tubbs PK: Tissue levels of acid insoluble carnitine in rat heart Biochim Biophys Acta 84:772-779, 1964 19. Crass MF III, Shipp JC, Pieper GM: Effects of catecholamines on myocardial endogenous substrates and contractility. Am J Physiol 228:618-627, 1975 20. Delcher HK, Fried M, Shipp SC: Metabolism of lipoprotein lipid in the isolated perfused rat heart. Biochim BiophysActa 106:1t&18, 1965 21. Opie LH: Metabolism of the heart in health and disease. Am Heart J 100:77-123, 1969 22. Gousios A, Felts JM, Have1 RJ: Metabolism of serum triglycerides and free fatty acids by the myocardium. Metabolism 12:75-88, 1963 23. Rich CE. Bierman L, Schwartz IL: Plasma nonesterified fatty acids in hyperthyroid states. J Clin Invest 38:275-278. 1959
613
24. Rosenfeld PS, Rosenberg IW: Effect of altered thyroid status upon epinephrine induced lipolysis in vitro. Proc Sot Exp Biol Med 118:221-225,1965 25. Gold M, Scott JC, Spitzer JJ: Myocardial metabolism of free fatty acids in control, hyperthyroid and hypothyroid dogs. Am J Physio123 1:239-244, 1967 26. Gold MH, Attar JH, Scott JC, Spitzer JJ: Effect of norepinephrine on myocardial free fatty acid uptake and oxidation. Proc Sot Exp Biol Med 118:876-879, 1965 27. Mayers SE: E&t of catecholamines on cardiac metabolism. Circ Res 34.35 (Suppl III):1 29-l 35, 1974 28. Fritz IB, Yue KTK: Long chain carnitine acyltransferase and the role of acyl carnitine derivatives in the catalytic increase of fatty acid oxidation induced by carnitine. J Lipid Res 4:279-288, 1963 29. Fritz IB: Hypothesis concerning the role of carnitine in the control of interrelations between fatty acids and carbohydrate metabolism. Perspect Biol Med 10:643-677, 1967 30. Wittels B, Spann JF: Defective lipid metabolism in the failing heart. J Clin Invest 47:1787-1793, 1968