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Studies in vitro on single beating rat heart cells
BIOCtIIMICA ET BIOPHYSICA ACTA
15
BBA 25459 S T U D I E S I N V I T R O ON S I N G L E B E A T I N G RAT H E A R T CELLS IX. T H E R E S T O R A T I O N OF B E A T I N G BY SERUM L I P I D S AND FATTY ACIDS ISAAC HARARY, RICHARD McCARL* AND BARBARA F A R L E Y
Laboratory of Nuclear Medicine and Radiation Biology, Department of Biophysics and Nuclear Medicine, and Department of Biological Chemistry, School of Medicine, Centerfor the Health Sciences, University of California, Los Angeles, Calif. (U.S.A .) (Received April 9th, 1965)
SUMMARY
I. Heart cells incubated in lipid-deficient media lose their ability to beat. 2. The presence of lipid-free serum proteins partially maintains growth but not beating. 3. The presence of serum lipids maintains beating but not growth. 4. The addition of lipid-free serum and serum lipids m a y reconstitute whole serum in its ability to maintain growth and beating. 5. Lipid-free albumin maintains cells without growth or beating. 6. The addition of serum lipids or f a t t y acids to cells, maintained either in lipidfree serum proteins or lipid-free albumin, and which have lost their beating, restores the ability to beat.
INTRODUCTION
The primary purpose in the isolation and study of functioning heart cells in our laboratory 1 has been for the development of a system in which the process of differentiation m a y be studied ~. It is our plan to v a r y the conditions of the cultured beating cells and to follow the resultant changes in metabolism and function. As measures of function we have used the intrinsic beating of the cellss, the activity of myosin adenosine triphosphatase 4, and the contractility of actomyosin models 5. We have also followed changes in enzymes and metabolic systems, which are related to the changes in function, such as respiration, the respiratory quotient, the levels of tricarboxylic and glycolytic enzymes and f a t t y acid oxidation s. The indications are that when cells lose their ability to beat, the myosin activity falls and the ability to oxidize lipids decreases markedly. T h a t the heart cell function is somewhat related to lipids in the medium, is indicated b y experiments with a normal medium supplemented with f a t t y acids. Addition of palmitate and linoleate, and * Present address: Pennsylvania State University, Frear Laboratories, University Park, Pa. (U.S.A.).
Biochim. Biophys. Aeta, 115 (1966) 15-22
16
I. HARARY, R. MCCARL, B. FARLEY
lowering the glucose concentration, led to maintenance of function in heart cells, as measured by the above parameters 7. Most studies of nutritional requirements of cultured cells have been concerned with the maintenance of growth of established cell lines. Growth has also been the main criterion in the consideration of primary cell explants. Several laboratories have attempted to achieve a chemically defined medium. LEIBERMAN8 has described a defined medium for the growth of kidney cells. RAPPAPORT9 has developed a chemically defined medium for cells grown in specially treated bottles. EAGLE1° uses a synthetic medium which is designed mainly for spinner cultures and this requires a dialyzable growth factor. A most promising chemically defined medium is that of NAGLE el al. n who also uses this for spinner cultures. Although this medium is free of serum protein, it still contains protein in the form of insulin. All of these attempts have been based upon substitution for serum of like compounds. HAM~ has succeeded in developing a serum-free medium and has found that the medium must be very specifically designed for the particular cells under study. In all of these cases, the major parameter studied has been growth. However, a medium designed specifically for maintaining growth may do so at the expense of function. Our primary interest is in the nutritional requirements for maintenance of function. Since the only source of lipids, which seems necessary for beating, is the serum, and since growth is dependent also on the presence of serum, we have initiated a study of the serum fractions required for both activities. This report deals mainly with the fractionation of the serum and the relation of these fractions to the activity of the cultured cells. Extraction of the serum protein with lipid solvents has resulted in the demonstration that the beating of heart cells is completely dependent on the presence of the lipid extract and that the growth depends upon the presence of serum protein. METHODS
The culture techniques used were those reported previously 1~with the exception that cells grown in experimental media were washed in complete growth medium minus serum and kept in this medium until the cells were plated. Complete growth medium minus serum was the common base to which the various additions were made. In later experiments, the cells were maintained in the interim, in o.o5 % anti-trypsin in Hank's saline. About 4"1o6 cells per 6o-mm polystyrene dish were used. Test medium was subsequently added to each dish and the cells attached to the dish in the test medium supplied. The medium was changed every 2-3 days. Protein was determined according to OYAMAAND EAGLE14. Lipids were removed from the serum by freeze-drying the serum and extracting the powder with a chloroform-methanol solution (2 : I, v/v) at 25 °, three times, then washing the protein fraction several times with the chloroform-methanol solution. The protein fraction was further extracted with ether, two times, and dried in a vacuum desiccator for 24 h. This removed the last traces of chloroform. This is designated the lipid-free serum. The ether and chloroform-methanol lipid fractions were combined, evaporated to dryness in a rotary evaporator, reextracted with chloroform-methanol, washed with H20, evaporated to dryness again and then taken up in a small quantity of ether and stored in a deep freeze until ready for use. These are the serum lipids. Biochim. Biophys. Acla, 115 (1966) 15-22
TIlE RESTORATION OF BEATING BY LIPIDS AND FATTY ACIDS
17
Albumin was obtained from Mann Research Co. and purified free of lipids according to the method of GOODMAN1~. Commercial fetuin was obtained from Colorado Serum Co. A crude fetuin fraction was prepared by ammonium sulfate fractionation according to the method of FISHER 16. RESULTS
Maintenance of beating Removal of the serum protein from the complete growth medium resulted in a medium that did not allow attachment of the cells. However, once the cells had become attached to the dishes in complete growth medium, they remained attached for several days in the presence of serum-less medium but they did not grow and stopped beating. RAPPAPORT'S medium and treatment of the culture bottles 9 proved unsuccessful in culturing beating rat-heart cells as did NAGLE'S medium n or the substitution for serum of glycoprotein, albumin or fetuin. Under our culture conditions, addition of extracted lipids to complete growth medium minus serum did not allow cell attachment. Only serum protein or fetuin promoted attachment of cells. Our first experiments were designed to investigate the effect of the two serum fractions, lipid-free serum and the serum lipids on growth and function when they substituted for whole serum. The experiment shown in Fig. i compares the growth and beating of cells incubated in medium containing serum lipids in place of whole serum. The cells were attached in complete growth medium which was replaced with the experimental medium after one day. The upper curve represents the growth on complete growth medium. The filled symbols indicate beating and the open symbols indicate no beating. Cells kept in complete growth medium for 24 h and then subsequently in complete growth medium minus serum plus lipid did not grow, but the beating was as good as the control for 9 days at which time the experiment was 20002000-
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IN C U L T U R E
Fig. i. The g r o w t h and beating of r a t h e a r t cells in various media, n - - m , complete g r o w t h m e d i u m ; ~k--~k, m e d i u m m i n u s s e r u m plus lipid; O - - O , m e d i u m m i n u s serum, I n this and the following figures showing g r o w t h curves (Figs. I-5), t h e solid s y m b o l s indicate beating, the open s y m b o l s indicate no beating. Lipids were added to the basic m e d i u m in a concentration equal to t h a t originally p r e s e n t in the intact serum. Fig. 2. The effect of lipid-free s e r u m on g r o w t h and beating. O - - O , complete g r o w t h m e d i u m ; /x - - & , m e d i u m m i n u s s e r u m plus lipid-free serum. The lipid-free s e r u m was added to the m e d i u m in a concentration equivalent to t h a t originally found in the intact serum.
l?iochim. Biophys. Acta, 115 (1966) 15-22
I8
I. HARARY, R. MCCARL, B. FARLEY
terminated. Lipids were added in a concentration equivalent to their concentration in complete growth medium. In contrast, the cells incubated in complete growth medium minus serum alone did not grow and eventually stopped beating. The time that it took cells to stop beating when the complete growth medium was replaced b y a serum-less medium was usually about 2-3 days but in rare instances it has taken up to 8 days. This was probably due to variation in the nutritional state of the cells, and variation in the number of attached cells. If the cells were attached and reincubated in complete growth medium minus serum plus the lipid-free serum, then quite another situation resulted (Fig. 2). The cells showed a lower growth rate than that of the complete growth medium and they 3000-
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Fig. 3. Growth and beating of rat-heart cells on reconstituted medium. The reconstituted medium was made up of the complete growth medium minus serum, plus lipid extract and lipid-free serum protein. 0 - - 0 , regular medium; A--Ak, reconstituted medium. The solid symbols indicate beating. Fig. 4. The effect of dialysis on complete growth medium. Jk--Ak, complete growth medium; O - - Q , complete growth medium made up with dialyzed serum.
did not beat. This again indicates that the lipids are essential for the beating phenomenon, but that some growth m a y occur in the absence of lipids. The amount of growth in lipid-free serum was variable so that a definite quantitative statement, about how much growth can be maintained, cannot be made. This m a y be due to the variation in the serum which could lead to varying amounts of lipid left complexed with the extracted proteins. That the beating is related to the presence of lipids is also demonstrated by the data in Fig. 3. The growth rate and the beating activity of the heart cells in complete growth medium m a y at times be approximated by the addition of lipids plus lipid-free serum to complete growth medium minus serum. Thus it would seem that the lipids and lipid-flee serum can partially reconstitute whole serum and that lipid-free serum m a y maintain partial growth without beating. The lipids m a y or m a y not be needed for growth, but are always necessary to maintain beating. These observations have been extended to studies on dialyzed preparations, and include experiments on dialyzed serum and dialyzed lipid-free serum. Dialyzed serum m a y be substituted for serum with no change in the growth rate or the beating (Fig. 4). I t would seem from this observation that small molecular weight dialyzable Biochim. Biophys. Acta, 115 (1966) 15-22
THE RESTORATION OF BEATING BY LIPIDS AND FATTY ACIDS
19
components of the serum are not significantly necessary for growth or beating. Dialyzed serum maintained b o t h growth and beating but lipid-extracted serum maintained only partial growth b u t no beating. I t seemed therefore t h a t dialysis of lipid-free serum should have no effect on the growth maintenance b y this fraction. Dialyzed lipid-free serum, however, can neither maintain growth nor beating. W h e n dialyzed lipid-free serum is supplemented with lipid, it does not reconstitute dialyzed serum in its ability to support both growth and beating although it can support beating (Fig. 5). This indicates t h a t the extraction of serum with chloroform and methanol changes the protein so t h a t subsequent dialysis removes components not able to be removed from the whole serum b y dialysis. 140 -
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Fig. 5. Growth and beating on extracted dialyzed serum, i - - H , complete growth medium; O - - O , lipid-free dialyzed serum; ~k--~k, lipid-free dialyzed serum plus serum lipids. Fig. 6. Restoration of beating with lipids. ©--©, medium with lipid-free dialyzed serum; &--&, medium with lipid-free serum. Cells on complete growth medium beat throughout the experimental period at IOO beats per rain. At the arrow tile lipid-deficient media were replaced with lipidsupplemented media. Beating rate in this and the following figures was determined at 35°.
Restoration of beating The previous experiments represent a t t e m p t s to maintain either growth or beating with the addition of various fractions at the beginning of the incubation period. The following experiments are an a t t e m p t to alter the state of the cells b y changing nutritional conditions during the incubation periods. T h e y are designed to investigate whether cells, which have lost the ability to beat on a medium deficient in lipids, could be altered b y the subsequent addition of a lipid or lipid extract. T h a t this is the case is demonstrated b y the experiment shown in Fig. 6. Three groups of cells were used, one grown on complete growth medium, the second on lipid-free serum, and the third on lipid-free dialyzed serum. The cells incubated on the lipiddeficient media stopped beating in 8 days. T h e y were allowed to stay in this state for 4 days with three medium changes. During this time, the controls on complete growth medium continued to beat. On the fourth day, the medium was changed and this time the medium used contained serum lipid extract. Thus Plate 2 was changed from lipid-free serum to lipid-free serum plus lipid, and Plate 3 was changed from lipid-free dialyzed serum to lipid-free dialyzed serum plus lipid extract. 2-4 h after
Biochim. Biophys. Acta, 115 (1966) 15-22
20
I. H A R A R Y ,
R. MCCARL, B. F A R L E Y
this medium change, the cells in these plates began to beat at a rate comparable to the rate of the control. Cells in dishes which contained the lipid-free medium did not beat. Thus, lipid extracts of serum were directly responsible for the restoration of beating in cells which had stopped beating because of lipid deficiency. These experiments have been repeated 8 times and in various ways. Addition of lipids to cells incubated in lipid-deficient medium, without a medium change, also restored beating. TABLE
I
RESTORATION
OF BEATING
"~VITH L I P I D S
AND
FATTY
ACIDS
Cells w e r e i n c u b a t e d i n a c o m p l e t e g r o w t h m e d i u m w i t h l i p i d f r e e d i a l y z e d s e r u m o r l i p i d - f r e e s e r u m till b e a t i n g s t o p p e d i n t h r e e d a y s . O n t h e f o u r t h d a y , t h e a d d i t i o n s l i s t e d w e r e m a d e . T h e c r o s s e s i n d i c a t e r e s t o r a t i o n of b e a t i n g b u t d o n o t i n d i c a t e d u r a t i o n o r s t r e n g t h o f b e a t i n g . T h e d a s h e s i n d i c a t e n o r e s t o r a t i o n of b e a t i n g .
Additions
Final concentration
Basal m e d i u m -lipid-free dialyzed serum
Basal m e d i u m + lipid-free serum
Complete growth medium Lipid extract Sodium pahnitate Sodium acetate Sodium linoleate NaC1 Lipid wash Fetuin
-* I o -4 M 2. 5 . 1o - a M 4' io 5 M 1. 5 . i o 3 M
+ + + + -----
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* These additions were made in a concentration equal to that present in the whole serum. " L i p i d w a s h " is t h e w a t e r - s o l u b l e e x t r a c t of t h e s e r u m l i p i d s i n c h l o r o f o r m . T h e m e d i u m w a s reconstituted omitting the serum but with the additions brought up to the volume ordinarily taken up by the serum.
Evidence that fatty acids present in the lipid extract are at least in part responsible for the restoration of beating is shown by the fact that palmitate can substitute for the lipid extract (Table I). After the cells have stopped beating, the addition of o.I mM palmitate, o.o4 mM linoleate or 2.5 mM acetate restored the beating. Complete growth medium with whole serum was most effective in restoring beating. Of the others, palmitate seemed most effective. The effect of acetate was more transient. In an attempt to clarify the role of the serum fractions, similar experiments were performed in a medium containing albumin instead of the lipid-free serum fraction. The lipids were complexed with albumin and thus solubilized. The cells were attached in a medium containing a fetuin fraction of serum and were changed, after attachment, to a medium containing in one case, lipid-free albumin, and in another, lipid-free albumin and serum lipid extract. The beating rate was followed for 8 days. The beating in the lipid-supplemented medium was maintained although there was no growth, as measured by protein. On albumin alone, the beating ceased in 4-6 days and here too, there was no growth (Fig. 7)- The cells were allowed to incubate in the medium for 2 days during which time they did not beat. After this period, the additions listed in Fig. 8 were made. Palmitate and linoleate restored beating after a lag period of 3 h, acetate after 4 h. The effect of acetate was slight and more Biochim. Biophys. Acta, 115 (1966) 1 5 - 2 2
THE RESTORATION OF BEATING BY LIPIDS AND FATTY ACIDS
21
transient. NaC1, fetuin or additional albumin had no effect. Changing the cells to complete growth medium was the most effective of all in restoring a high rate of beating well within I h after the change. In one case, complete growth medium restored beating after 5 min of the medium change. The effect of the lipid-supplemented media m a y last for up to 2 or 3 days. 250-
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Fig. 7. The loss of beating on media with albumin. 0 - - 0 , a l b u m i n alone.
a l b u m i n plus serum lipids; A - - A ,
Fig. 8. Restoration of beating of cells grown in albumin media; the cells were incubated in complete growth m e d i u m minus serum plus lipid-free albumin. T h e y stopped beating after 4 days. After 2 d a y s of no beating, media was changed a t the time indicated by the arrow. @ - - @ , complete growth m e d i u m ; A - - & , p a l m i t a t e - s u p p l e m e n t e d m e d i u m ; [ ] - - [ 2 , linoleate-supplemented m e d i u m ; O - - O , acetate-supplemented medium. The beating of cells kept t h r o u g h o u t the whole period on complete growth m e d i u m was approx. 200 beats per m i n and did not cease during the experimental period. DISCUSSION
That the intact heart utilizes lipids from the serum is now well documented 17. There is also evidence that the endogenous metabolism of the heart is mainly lipid and that, in the presence of lipid, it is the main energy source for the heart 18. Cultured rat heart cells have an endogenous R.Q. of 0.7 8, which also indicates that lipids are the main metabolites. I t is significant that when these cells lose their function and stop beating there is a shift from lipid to carbohydrate metabolism 6. We have evidence that if the glucose in the medium is considerably lowered and palmitic and linoleic acids are added, the heart cells maintain their ability to metabolize lipids and beat for a longer period 7. The conclusion we can draw from the evidence presented in this report is that lipids are primarily concerned with the beating of the heart cells. The time lag observed in the induction of beating strongly suggests that the lipids do not act directly. Time is perhaps necessary for the induction of enzymes, for the conversion of the f a t t y acids into a more active compound, or for their incorporation into an important structure. At this point the variation of time lag with palmitate and acetate cannot be considered very significant. The fact however that complete growth medium restores beating very quickly indicates that, if palmitate and acetate are converted to a more active intermediate, complete growth medium already contains the active intermediate. We cannot yet say whether acetate or palmitate is the more directly involved. Recent experiments on the maintenance of function with various substrates Biochim. Biophys. Acta, 115 (1966) 15-22
I. HARARY, R. MCCARL, 13. FARLEY
22
do not clarify the problem but indicate that acetate is extremely effective in the maintenance of beating. It is possible that the primary role of the f a t t y acids is to serve as a specific energy source but the evidence from studies of the relation of the source of ATP to the beating of cultured heart cells indicates that the beating is dependent upon ATP but not upon its source. The heart cells will continue to beat with ATP supplied from glycolysis alone, or from oxidative phosphorylation alone 19. It would seem likely, therefore, that the role of lipids in supporting beating is not as a specific energy source. However, from a quantitative viewpoint, the loss of ability to oxidize fatty acids could have an effect on the beating if as a consequence, the energy reserves of the cell were seriously depleted. This might be the case if f a t t y acid oxidation was the main source of acetyl-CoA. Further study on the relative efficiency of various f a t t y acids for forming acetyl-CoA in young and older cells is under way. Alternatively, the primary role of lipids in the heart cells m a y be in membrane function and a deficiency of lipids m a y have its first effect on the beating because of an alteration in membrane structure. It is also possible that the overall lipid economy in the heart m a y be instrumental in maintaining the correct conditions for the synthesis of heart proteins. Support for this possibility derives from work at present underway which indicates that f a t t y acids are necessary for maintaining conditions for the synthesis of myosin, enzymes of the tricarboxylic acid cycle, and enzymes of f a t t y acid oxidation. ACKNOWLEDGEMENTS
This work was supported in part by contract No. AT-o4-I-GEN-I2 between the U.S. Atomic Energy Commission and the University of California, Los Angeles, and b y research grant No. A-2135 from the National Institutes of Health, U.S. Public Health Service. REFERENCES I 2 3 4 5 6 7 8 9 Io II 12 13 14 15 16 17 18 19
I. HARARY AND B. FARLEY, Science, 131 (196o) 1674. I. HARARY, A. FUJIMOTO AND H . KURAMITSU, Natl. Cancer Inst. Monograph, 13 (1964) 257. I. HARARY AND B. FARLEY, Science, 132 (196o) 1839. H. KORAMITSU AND I. HARARY, Biochim. Biophys. Acta, 86 (1964) 65. I. HARARY AND E , SATO, Biochim. Biophys. Acta, 82 (1964) 614. A. FUJIMOTO AND I. HARARY, Biochim. Biophys. Acta, 86 (1964) 74. I. HARARY, Circulation Res. Suppl. 1I, 15 (1964) 12o. I. LEIBERMAN AND J. OVE, J. Biol. Chem., 234 (1959) 2754. C. RAPPAPORT, J. P. POOLE AND H . P. RAPPAPORT, Exptl. Cell Res., 20 (196o) 465 . H . EAGLE, Proc. Natl. Acad. Sci. U.S., 46 (196o) 427 . S. C. ~N~AGLE,H . R . TRIBLE, JR., R. E . ANDERSON AND N. D. GARY, Proc. Soc. Exptl. Biol. Med., 112 (1963) 34 ° . R. G. HAM, Biochem. Biophys. Res. Commun., 14 (1964) 34. I. HARARY AND B. FARLEY, Exptl. Cell Res., 29 (1963) 451. V. I. OYAMA AND H . EAGLE, Proc. Soc. Exptl. Biol. Med., 91 (1956) 305 • D. S. GOODMAN, Science, 125 (1957) 1296. H . W . FISHER, T. T. PUCK AND G. SATO, Proe. Natl. Acad. Sci, U.S., 44 (1958) 4. R . J. BING, A. SIEGEL, I. UNGAR AND M. GILBERT, Am. J. Med., 16 (1954) 504 . J. R . EVANS, L. H . OPIE AND J . C. SHIPP, Am. J. Physiol., 205 (1963) 766. I. HARARY AND E . C. SLATER, Biochim. Biophys. Acta, 9 9 (1965) 227.
Biochim. Biophys. Acta, 115 (1966) 15-22
15
BBA 25459 S T U D I E S I N V I T R O ON S I N G L E B E A T I N G RAT H E A R T CELLS IX. T H E R E S T O R A T I O N OF B E A T I N G BY SERUM L I P I D S AND FATTY ACIDS ISAAC HARARY, RICHARD McCARL* AND BARBARA F A R L E Y
Laboratory of Nuclear Medicine and Radiation Biology, Department of Biophysics and Nuclear Medicine, and Department of Biological Chemistry, School of Medicine, Centerfor the Health Sciences, University of California, Los Angeles, Calif. (U.S.A .) (Received April 9th, 1965)
SUMMARY
I. Heart cells incubated in lipid-deficient media lose their ability to beat. 2. The presence of lipid-free serum proteins partially maintains growth but not beating. 3. The presence of serum lipids maintains beating but not growth. 4. The addition of lipid-free serum and serum lipids m a y reconstitute whole serum in its ability to maintain growth and beating. 5. Lipid-free albumin maintains cells without growth or beating. 6. The addition of serum lipids or f a t t y acids to cells, maintained either in lipidfree serum proteins or lipid-free albumin, and which have lost their beating, restores the ability to beat.
INTRODUCTION
The primary purpose in the isolation and study of functioning heart cells in our laboratory 1 has been for the development of a system in which the process of differentiation m a y be studied ~. It is our plan to v a r y the conditions of the cultured beating cells and to follow the resultant changes in metabolism and function. As measures of function we have used the intrinsic beating of the cellss, the activity of myosin adenosine triphosphatase 4, and the contractility of actomyosin models 5. We have also followed changes in enzymes and metabolic systems, which are related to the changes in function, such as respiration, the respiratory quotient, the levels of tricarboxylic and glycolytic enzymes and f a t t y acid oxidation s. The indications are that when cells lose their ability to beat, the myosin activity falls and the ability to oxidize lipids decreases markedly. T h a t the heart cell function is somewhat related to lipids in the medium, is indicated b y experiments with a normal medium supplemented with f a t t y acids. Addition of palmitate and linoleate, and * Present address: Pennsylvania State University, Frear Laboratories, University Park, Pa. (U.S.A.).
Biochim. Biophys. Aeta, 115 (1966) 15-22
16
I. HARARY, R. MCCARL, B. FARLEY
lowering the glucose concentration, led to maintenance of function in heart cells, as measured by the above parameters 7. Most studies of nutritional requirements of cultured cells have been concerned with the maintenance of growth of established cell lines. Growth has also been the main criterion in the consideration of primary cell explants. Several laboratories have attempted to achieve a chemically defined medium. LEIBERMAN8 has described a defined medium for the growth of kidney cells. RAPPAPORT9 has developed a chemically defined medium for cells grown in specially treated bottles. EAGLE1° uses a synthetic medium which is designed mainly for spinner cultures and this requires a dialyzable growth factor. A most promising chemically defined medium is that of NAGLE el al. n who also uses this for spinner cultures. Although this medium is free of serum protein, it still contains protein in the form of insulin. All of these attempts have been based upon substitution for serum of like compounds. HAM~ has succeeded in developing a serum-free medium and has found that the medium must be very specifically designed for the particular cells under study. In all of these cases, the major parameter studied has been growth. However, a medium designed specifically for maintaining growth may do so at the expense of function. Our primary interest is in the nutritional requirements for maintenance of function. Since the only source of lipids, which seems necessary for beating, is the serum, and since growth is dependent also on the presence of serum, we have initiated a study of the serum fractions required for both activities. This report deals mainly with the fractionation of the serum and the relation of these fractions to the activity of the cultured cells. Extraction of the serum protein with lipid solvents has resulted in the demonstration that the beating of heart cells is completely dependent on the presence of the lipid extract and that the growth depends upon the presence of serum protein. METHODS
The culture techniques used were those reported previously 1~with the exception that cells grown in experimental media were washed in complete growth medium minus serum and kept in this medium until the cells were plated. Complete growth medium minus serum was the common base to which the various additions were made. In later experiments, the cells were maintained in the interim, in o.o5 % anti-trypsin in Hank's saline. About 4"1o6 cells per 6o-mm polystyrene dish were used. Test medium was subsequently added to each dish and the cells attached to the dish in the test medium supplied. The medium was changed every 2-3 days. Protein was determined according to OYAMAAND EAGLE14. Lipids were removed from the serum by freeze-drying the serum and extracting the powder with a chloroform-methanol solution (2 : I, v/v) at 25 °, three times, then washing the protein fraction several times with the chloroform-methanol solution. The protein fraction was further extracted with ether, two times, and dried in a vacuum desiccator for 24 h. This removed the last traces of chloroform. This is designated the lipid-free serum. The ether and chloroform-methanol lipid fractions were combined, evaporated to dryness in a rotary evaporator, reextracted with chloroform-methanol, washed with H20, evaporated to dryness again and then taken up in a small quantity of ether and stored in a deep freeze until ready for use. These are the serum lipids. Biochim. Biophys. Acla, 115 (1966) 15-22
TIlE RESTORATION OF BEATING BY LIPIDS AND FATTY ACIDS
17
Albumin was obtained from Mann Research Co. and purified free of lipids according to the method of GOODMAN1~. Commercial fetuin was obtained from Colorado Serum Co. A crude fetuin fraction was prepared by ammonium sulfate fractionation according to the method of FISHER 16. RESULTS
Maintenance of beating Removal of the serum protein from the complete growth medium resulted in a medium that did not allow attachment of the cells. However, once the cells had become attached to the dishes in complete growth medium, they remained attached for several days in the presence of serum-less medium but they did not grow and stopped beating. RAPPAPORT'S medium and treatment of the culture bottles 9 proved unsuccessful in culturing beating rat-heart cells as did NAGLE'S medium n or the substitution for serum of glycoprotein, albumin or fetuin. Under our culture conditions, addition of extracted lipids to complete growth medium minus serum did not allow cell attachment. Only serum protein or fetuin promoted attachment of cells. Our first experiments were designed to investigate the effect of the two serum fractions, lipid-free serum and the serum lipids on growth and function when they substituted for whole serum. The experiment shown in Fig. i compares the growth and beating of cells incubated in medium containing serum lipids in place of whole serum. The cells were attached in complete growth medium which was replaced with the experimental medium after one day. The upper curve represents the growth on complete growth medium. The filled symbols indicate beating and the open symbols indicate no beating. Cells kept in complete growth medium for 24 h and then subsequently in complete growth medium minus serum plus lipid did not grow, but the beating was as good as the control for 9 days at which time the experiment was 20002000-
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IN C U L T U R E
Fig. i. The g r o w t h and beating of r a t h e a r t cells in various media, n - - m , complete g r o w t h m e d i u m ; ~k--~k, m e d i u m m i n u s s e r u m plus lipid; O - - O , m e d i u m m i n u s serum, I n this and the following figures showing g r o w t h curves (Figs. I-5), t h e solid s y m b o l s indicate beating, the open s y m b o l s indicate no beating. Lipids were added to the basic m e d i u m in a concentration equal to t h a t originally p r e s e n t in the intact serum. Fig. 2. The effect of lipid-free s e r u m on g r o w t h and beating. O - - O , complete g r o w t h m e d i u m ; /x - - & , m e d i u m m i n u s s e r u m plus lipid-free serum. The lipid-free s e r u m was added to the m e d i u m in a concentration equivalent to t h a t originally found in the intact serum.
l?iochim. Biophys. Acta, 115 (1966) 15-22
I8
I. HARARY, R. MCCARL, B. FARLEY
terminated. Lipids were added in a concentration equivalent to their concentration in complete growth medium. In contrast, the cells incubated in complete growth medium minus serum alone did not grow and eventually stopped beating. The time that it took cells to stop beating when the complete growth medium was replaced b y a serum-less medium was usually about 2-3 days but in rare instances it has taken up to 8 days. This was probably due to variation in the nutritional state of the cells, and variation in the number of attached cells. If the cells were attached and reincubated in complete growth medium minus serum plus the lipid-free serum, then quite another situation resulted (Fig. 2). The cells showed a lower growth rate than that of the complete growth medium and they 3000-
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Fig. 3. Growth and beating of rat-heart cells on reconstituted medium. The reconstituted medium was made up of the complete growth medium minus serum, plus lipid extract and lipid-free serum protein. 0 - - 0 , regular medium; A--Ak, reconstituted medium. The solid symbols indicate beating. Fig. 4. The effect of dialysis on complete growth medium. Jk--Ak, complete growth medium; O - - Q , complete growth medium made up with dialyzed serum.
did not beat. This again indicates that the lipids are essential for the beating phenomenon, but that some growth m a y occur in the absence of lipids. The amount of growth in lipid-free serum was variable so that a definite quantitative statement, about how much growth can be maintained, cannot be made. This m a y be due to the variation in the serum which could lead to varying amounts of lipid left complexed with the extracted proteins. That the beating is related to the presence of lipids is also demonstrated by the data in Fig. 3. The growth rate and the beating activity of the heart cells in complete growth medium m a y at times be approximated by the addition of lipids plus lipid-free serum to complete growth medium minus serum. Thus it would seem that the lipids and lipid-flee serum can partially reconstitute whole serum and that lipid-free serum m a y maintain partial growth without beating. The lipids m a y or m a y not be needed for growth, but are always necessary to maintain beating. These observations have been extended to studies on dialyzed preparations, and include experiments on dialyzed serum and dialyzed lipid-free serum. Dialyzed serum m a y be substituted for serum with no change in the growth rate or the beating (Fig. 4). I t would seem from this observation that small molecular weight dialyzable Biochim. Biophys. Acta, 115 (1966) 15-22
THE RESTORATION OF BEATING BY LIPIDS AND FATTY ACIDS
19
components of the serum are not significantly necessary for growth or beating. Dialyzed serum maintained b o t h growth and beating but lipid-extracted serum maintained only partial growth b u t no beating. I t seemed therefore t h a t dialysis of lipid-free serum should have no effect on the growth maintenance b y this fraction. Dialyzed lipid-free serum, however, can neither maintain growth nor beating. W h e n dialyzed lipid-free serum is supplemented with lipid, it does not reconstitute dialyzed serum in its ability to support both growth and beating although it can support beating (Fig. 5). This indicates t h a t the extraction of serum with chloroform and methanol changes the protein so t h a t subsequent dialysis removes components not able to be removed from the whole serum b y dialysis. 140 -
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Fig. 5. Growth and beating on extracted dialyzed serum, i - - H , complete growth medium; O - - O , lipid-free dialyzed serum; ~k--~k, lipid-free dialyzed serum plus serum lipids. Fig. 6. Restoration of beating with lipids. ©--©, medium with lipid-free dialyzed serum; &--&, medium with lipid-free serum. Cells on complete growth medium beat throughout the experimental period at IOO beats per rain. At the arrow tile lipid-deficient media were replaced with lipidsupplemented media. Beating rate in this and the following figures was determined at 35°.
Restoration of beating The previous experiments represent a t t e m p t s to maintain either growth or beating with the addition of various fractions at the beginning of the incubation period. The following experiments are an a t t e m p t to alter the state of the cells b y changing nutritional conditions during the incubation periods. T h e y are designed to investigate whether cells, which have lost the ability to beat on a medium deficient in lipids, could be altered b y the subsequent addition of a lipid or lipid extract. T h a t this is the case is demonstrated b y the experiment shown in Fig. 6. Three groups of cells were used, one grown on complete growth medium, the second on lipid-free serum, and the third on lipid-free dialyzed serum. The cells incubated on the lipiddeficient media stopped beating in 8 days. T h e y were allowed to stay in this state for 4 days with three medium changes. During this time, the controls on complete growth medium continued to beat. On the fourth day, the medium was changed and this time the medium used contained serum lipid extract. Thus Plate 2 was changed from lipid-free serum to lipid-free serum plus lipid, and Plate 3 was changed from lipid-free dialyzed serum to lipid-free dialyzed serum plus lipid extract. 2-4 h after
Biochim. Biophys. Acta, 115 (1966) 15-22
20
I. H A R A R Y ,
R. MCCARL, B. F A R L E Y
this medium change, the cells in these plates began to beat at a rate comparable to the rate of the control. Cells in dishes which contained the lipid-free medium did not beat. Thus, lipid extracts of serum were directly responsible for the restoration of beating in cells which had stopped beating because of lipid deficiency. These experiments have been repeated 8 times and in various ways. Addition of lipids to cells incubated in lipid-deficient medium, without a medium change, also restored beating. TABLE
I
RESTORATION
OF BEATING
"~VITH L I P I D S
AND
FATTY
ACIDS
Cells w e r e i n c u b a t e d i n a c o m p l e t e g r o w t h m e d i u m w i t h l i p i d f r e e d i a l y z e d s e r u m o r l i p i d - f r e e s e r u m till b e a t i n g s t o p p e d i n t h r e e d a y s . O n t h e f o u r t h d a y , t h e a d d i t i o n s l i s t e d w e r e m a d e . T h e c r o s s e s i n d i c a t e r e s t o r a t i o n of b e a t i n g b u t d o n o t i n d i c a t e d u r a t i o n o r s t r e n g t h o f b e a t i n g . T h e d a s h e s i n d i c a t e n o r e s t o r a t i o n of b e a t i n g .
Additions
Final concentration
Basal m e d i u m -lipid-free dialyzed serum
Basal m e d i u m + lipid-free serum
Complete growth medium Lipid extract Sodium pahnitate Sodium acetate Sodium linoleate NaC1 Lipid wash Fetuin
-* I o -4 M 2. 5 . 1o - a M 4' io 5 M 1. 5 . i o 3 M
+ + + + -----
+ + + + + ----
i nlg/ml
* These additions were made in a concentration equal to that present in the whole serum. " L i p i d w a s h " is t h e w a t e r - s o l u b l e e x t r a c t of t h e s e r u m l i p i d s i n c h l o r o f o r m . T h e m e d i u m w a s reconstituted omitting the serum but with the additions brought up to the volume ordinarily taken up by the serum.
Evidence that fatty acids present in the lipid extract are at least in part responsible for the restoration of beating is shown by the fact that palmitate can substitute for the lipid extract (Table I). After the cells have stopped beating, the addition of o.I mM palmitate, o.o4 mM linoleate or 2.5 mM acetate restored the beating. Complete growth medium with whole serum was most effective in restoring beating. Of the others, palmitate seemed most effective. The effect of acetate was more transient. In an attempt to clarify the role of the serum fractions, similar experiments were performed in a medium containing albumin instead of the lipid-free serum fraction. The lipids were complexed with albumin and thus solubilized. The cells were attached in a medium containing a fetuin fraction of serum and were changed, after attachment, to a medium containing in one case, lipid-free albumin, and in another, lipid-free albumin and serum lipid extract. The beating rate was followed for 8 days. The beating in the lipid-supplemented medium was maintained although there was no growth, as measured by protein. On albumin alone, the beating ceased in 4-6 days and here too, there was no growth (Fig. 7)- The cells were allowed to incubate in the medium for 2 days during which time they did not beat. After this period, the additions listed in Fig. 8 were made. Palmitate and linoleate restored beating after a lag period of 3 h, acetate after 4 h. The effect of acetate was slight and more Biochim. Biophys. Acta, 115 (1966) 1 5 - 2 2
THE RESTORATION OF BEATING BY LIPIDS AND FATTY ACIDS
21
transient. NaC1, fetuin or additional albumin had no effect. Changing the cells to complete growth medium was the most effective of all in restoring a high rate of beating well within I h after the change. In one case, complete growth medium restored beating after 5 min of the medium change. The effect of the lipid-supplemented media m a y last for up to 2 or 3 days. 250-
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Fig. 7. The loss of beating on media with albumin. 0 - - 0 , a l b u m i n alone.
a l b u m i n plus serum lipids; A - - A ,
Fig. 8. Restoration of beating of cells grown in albumin media; the cells were incubated in complete growth m e d i u m minus serum plus lipid-free albumin. T h e y stopped beating after 4 days. After 2 d a y s of no beating, media was changed a t the time indicated by the arrow. @ - - @ , complete growth m e d i u m ; A - - & , p a l m i t a t e - s u p p l e m e n t e d m e d i u m ; [ ] - - [ 2 , linoleate-supplemented m e d i u m ; O - - O , acetate-supplemented medium. The beating of cells kept t h r o u g h o u t the whole period on complete growth m e d i u m was approx. 200 beats per m i n and did not cease during the experimental period. DISCUSSION
That the intact heart utilizes lipids from the serum is now well documented 17. There is also evidence that the endogenous metabolism of the heart is mainly lipid and that, in the presence of lipid, it is the main energy source for the heart 18. Cultured rat heart cells have an endogenous R.Q. of 0.7 8, which also indicates that lipids are the main metabolites. I t is significant that when these cells lose their function and stop beating there is a shift from lipid to carbohydrate metabolism 6. We have evidence that if the glucose in the medium is considerably lowered and palmitic and linoleic acids are added, the heart cells maintain their ability to metabolize lipids and beat for a longer period 7. The conclusion we can draw from the evidence presented in this report is that lipids are primarily concerned with the beating of the heart cells. The time lag observed in the induction of beating strongly suggests that the lipids do not act directly. Time is perhaps necessary for the induction of enzymes, for the conversion of the f a t t y acids into a more active compound, or for their incorporation into an important structure. At this point the variation of time lag with palmitate and acetate cannot be considered very significant. The fact however that complete growth medium restores beating very quickly indicates that, if palmitate and acetate are converted to a more active intermediate, complete growth medium already contains the active intermediate. We cannot yet say whether acetate or palmitate is the more directly involved. Recent experiments on the maintenance of function with various substrates Biochim. Biophys. Acta, 115 (1966) 15-22
I. HARARY, R. MCCARL, 13. FARLEY
22
do not clarify the problem but indicate that acetate is extremely effective in the maintenance of beating. It is possible that the primary role of the f a t t y acids is to serve as a specific energy source but the evidence from studies of the relation of the source of ATP to the beating of cultured heart cells indicates that the beating is dependent upon ATP but not upon its source. The heart cells will continue to beat with ATP supplied from glycolysis alone, or from oxidative phosphorylation alone 19. It would seem likely, therefore, that the role of lipids in supporting beating is not as a specific energy source. However, from a quantitative viewpoint, the loss of ability to oxidize fatty acids could have an effect on the beating if as a consequence, the energy reserves of the cell were seriously depleted. This might be the case if f a t t y acid oxidation was the main source of acetyl-CoA. Further study on the relative efficiency of various f a t t y acids for forming acetyl-CoA in young and older cells is under way. Alternatively, the primary role of lipids in the heart cells m a y be in membrane function and a deficiency of lipids m a y have its first effect on the beating because of an alteration in membrane structure. It is also possible that the overall lipid economy in the heart m a y be instrumental in maintaining the correct conditions for the synthesis of heart proteins. Support for this possibility derives from work at present underway which indicates that f a t t y acids are necessary for maintaining conditions for the synthesis of myosin, enzymes of the tricarboxylic acid cycle, and enzymes of f a t t y acid oxidation. ACKNOWLEDGEMENTS
This work was supported in part by contract No. AT-o4-I-GEN-I2 between the U.S. Atomic Energy Commission and the University of California, Los Angeles, and b y research grant No. A-2135 from the National Institutes of Health, U.S. Public Health Service. REFERENCES I 2 3 4 5 6 7 8 9 Io II 12 13 14 15 16 17 18 19
I. HARARY AND B. FARLEY, Science, 131 (196o) 1674. I. HARARY, A. FUJIMOTO AND H . KURAMITSU, Natl. Cancer Inst. Monograph, 13 (1964) 257. I. HARARY AND B. FARLEY, Science, 132 (196o) 1839. H. KORAMITSU AND I. HARARY, Biochim. Biophys. Acta, 86 (1964) 65. I. HARARY AND E , SATO, Biochim. Biophys. Acta, 82 (1964) 614. A. FUJIMOTO AND I. HARARY, Biochim. Biophys. Acta, 86 (1964) 74. I. HARARY, Circulation Res. Suppl. 1I, 15 (1964) 12o. I. LEIBERMAN AND J. OVE, J. Biol. Chem., 234 (1959) 2754. C. RAPPAPORT, J. P. POOLE AND H . P. RAPPAPORT, Exptl. Cell Res., 20 (196o) 465 . H . EAGLE, Proc. Natl. Acad. Sci. U.S., 46 (196o) 427 . S. C. ~N~AGLE,H . R . TRIBLE, JR., R. E . ANDERSON AND N. D. GARY, Proc. Soc. Exptl. Biol. Med., 112 (1963) 34 ° . R. G. HAM, Biochem. Biophys. Res. Commun., 14 (1964) 34. I. HARARY AND B. FARLEY, Exptl. Cell Res., 29 (1963) 451. V. I. OYAMA AND H . EAGLE, Proc. Soc. Exptl. Biol. Med., 91 (1956) 305 • D. S. GOODMAN, Science, 125 (1957) 1296. H . W . FISHER, T. T. PUCK AND G. SATO, Proe. Natl. Acad. Sci, U.S., 44 (1958) 4. R . J. BING, A. SIEGEL, I. UNGAR AND M. GILBERT, Am. J. Med., 16 (1954) 504 . J. R . EVANS, L. H . OPIE AND J . C. SHIPP, Am. J. Physiol., 205 (1963) 766. I. HARARY AND E . C. SLATER, Biochim. Biophys. Acta, 9 9 (1965) 227.
Biochim. Biophys. Acta, 115 (1966) 15-22