Journal
of
Molecular andCellular
Effects
Cardiology
(1976)
8,925-940
of Isoproterenol on Myocardial Lipid Metabolism in Rat Hearts Perfused with and without Exogenous Substrates
Department (Received
FUMIO TAKENAKA AND SATOSHI TAKE0 of Pharmacology, Kumamoto University Medical School, Japan 4 .November
1975,
accepted in revisedform
22 March
1976)
F. TAKENAKA AND S. TAKEO. Effects of Isoproterenol on Myocardial Lipid Metabolism in Rat Hearts Perfused with and without Exogenous Substrates. Journal of Molealar and Cellular Cardiology (1976) 8, 925-940. Effects of isoproterenol on myocardial lipid metabolism were studied in perfused rat hearts by the Langendorff apparatus without recirculation. Fatty acids in phospholipid, free fatty acid and partial glyceride fractions did not alter during the initial 30-n& perfusion. However, fatty acids in triglyceride fraction decreased along with the perfusion. Isoproterenol enhanced the decrease in triglyceride fatty acid in the presence ofglucose, but not in the absence of glucose. When hearts were perfused for 30 min with exogenous myristirate in the presence of glucose, triglyceride fatty acid did not decrease in the control perfusion, and incorporation of myristiric acid into triglyceride fraction increased in the presence of isoproterenol. When hearts were perfused with myristirate in the absence of glucose, fatty acid in triglyceride fraction slightly decreased during the control 30-min perfusion, and marked bradycardia or ventricular arrest occurred within 10 min after the start of perfusion with solution containing isopro terenol. Triglyceride content 10 min after perfusion was not different from the control value. Mechanical performance of hearts with myristirate and isoproterenol improved when the amount of glucose in the perfusing solution was increased. The findings indicate that glucose may play an important role in the mechanical performance of the heart perfused with a solution containing isoproterenol. WORDS: Myocardial lipid metabolism; Cardiac performance; Isolated perfused heart;
KEY
Fatty acid; Glucose.
Myristiric
acid;
Isoproterenol;
1. Introduction It is well established that glucose and fatty acid are utilized as the main substrates for myocardial energy source [II]. Shipp et al. [13, la] have demonstrated that the latter is the preferential substrate in the isolated perfused heart. Effects of catecholamines on the utilization of these substrates in myocardium have been studied extensively [I, 2, 5, 9, 16-j. According to Williamson [16], epinephrine, norepinephrine and isoproterenol increased glucose oxidation in the perfused heart by activating phosphorylase activity. Gousios and Felts [8] and Gold et al. [j7 have shown that epinephrine and norepinephrine increased myocardial extraction and oxidation of palmitate-1-[‘*Cl. Furthermore, Kreisberg [9] has reported that epinephrine increased the utilization of myocardial lipid in the perfused heart.
926
F. TAKENAKA
AND
S. TAKE0
The studies so far reported, however, have dealt mostly with the utilization of triglyceride and phospholipid fraction, but not with partial glyceride and free fatty acid fraction. In the present experiments, the concentration of each fatty acid component in myocardial lipid fractions was determined, and effects of isoproterenol as well as the influence of glucose on myocardial lipid metabolism were investigated in isolated perfused rat hearts.
2. Materials
and Methods
Male albino Wistar rats weighing 240 to 270 g were anesthetized and killed by decapitation. The heart was rapidly removed and chilled in modified LockeRinger solution of the following compositions (mM) : KC 1, 5.63; NaCI, 154; CaC12, 2.18; NaHCOs, 20.8; glucose, 5.5. All adipose and connective tissues were removed from the heart and aorta in cold solution. Then, the heart was transferred to a Langendorff apparatus and perfused with modified Locke-Ringer solution previously equilibrated with a gas mixture of 95% 02 + 5% COa. The perfusate was not recirculated. The heart was pre-perfused for 5 min to stabilize cardiac performance. During the experimental perfusion, three kinds of perfusates were used; (1) modified Locke-Ringer solution, (2) the solution without glucose and (3) the solution containing sodium myristirate (0.4 mM) with bovine albumin (0.5%). The solution was gassed for 30 min with 95% 02 + 5% COa. The pH was adjusted to 7.40 f 0.03 and temperature at 35°C. Isoproterenol was added to the solution just before the start of perfusion at a concentration of 4 x IO-6 M in the absence of free fatty acid-albumin and 4 x 10-7 M in its presence. In the latter experiments, 4 x 10-7 M of NaHSOa was used as an antioxidant ofisoproterenol. At 3, 15 and 30 min of the experimental perfusion, the heart was removed from the apparatus, chilled, blotted dry and subjected to lipid analysis. Lipids were extracted from the homogenated heart with 20 ml of chloroform-methanol (2 :l v/v) according to the method of Folch et al. [4]. Dry weight was calculated by measuring the residue on the filter paper which was used for separation of lipid extracts from the homogenate, after drying at 100°C for 2 h. The chloroform phase was dried in vacua with a rotary evaporator under reduced pressure. The residue of total lipids was dissolved in 0.7 ml of chloroform-methanol mixture and separated by t.1.c. with silica gel G as the absorbant and a solvent system of petroleum etherdiethylether-acetic acid (80 :20: 1). The separation procedure was carried out according to the method of Olson and Hoeshen [12]. The lipid zones on the thin layer plate were scraped and extracted with 25 ml of chloroform-methanol mixture. Each lipid fraction of phospholipid(PL), triglyceride(TG), free fatty acid(FFA) and partial glyceride(PG) was dried under reduced pressure and ester moieties of each lipid fraction were changed to their methylester by refluxing for 2 h with 3 ml of absolute methanol and 5 drops of cont. sulfuric acid. The yield through thin
MYOCARDIAL
LIPID
METABOLISM
927
layer separation and esterification was 83% according to the examination of reference samples by the same procedure. These methylesters were subjected to g.1.c. analysis to determine the fatty acid components by using arachidic acid as an internal standard. The main components of fatty acids in the lipid fractions were myristiric, pahnitic, pahnitoleic, stearic, oleic, linoleic and arachidonic acid. The total amount of fatty acids in each fraction was determined by adding each of these components. To determine lipid content in the perfusate, lipids were extracted from the overAown perfusate cohected over the experimental period with diethylether at pH 1 adjusted by HCl after salting out with excess sodium chloride. After evaporation of the solvent, the total residual lipids were determined by the same esterification and gas liquid chromatographic analysis as mentioned above. In some cases, to determine fractional distribution of the total lipids, the lipids remaining in the diethyle~er extract were separated by t.l.c., and fatty acids in each fraction were determined by the same procedure as described above. Coronary flow was measured by a drop counter and heart rate was monitored by pulse rate tachometer (Nihon Koden RT-2). Cardiac contraction was estimated by monitoring isometric tension development of the heart preloaded by 1.25 g through a hook attached to the apex of the heart by means of a farce-displacement transducer (Nihon Koden SB-IT), and displayed on an oscillographic pen-recorder (Nihon Koden RM-I 50). The gain of the recording system was adjusted so that 1.25 g-preload caused a 2%mm pen deflection. Chemical substances employed in the present experiments were isoproterenol b~rdrochlo~de (Nikk~n Kagaku) , bovine albumin (Armor Pharillac~utical Co.) and sodium my&irate (Nakarai Yakuhin). Bovine albumin contained 2.0 pmoljg of free fatty acid and the purity of sodium my&irate was over 99% according to the gas liquid chroma~o~aphic analysis.
3. Results
Cardiac performance of the heart perfused with modified Locke-Ringer solution is shown in Figure I and changes in fatty acid contents in the myocardium are shown in Table I. Within 30 min of the start of the control perfusion with modified Locke-Ringer solution containing glucose, coronary flow and heart rate gradually decreasedto 69 & 3% and 83 & 3% of the initial value, respectively, while contractile force gradually increased and reached 133 f 7%.
I. Effects
(12) (10) (9) (10)
9.184 1.901 1.716 0.088
PL TG FFA PG PF
25.81 0.837 0.243 0.156
Isoproterenol
& f f f 10.762 1.791 1.473 0.118
235.64 12.804 1.326 1.500
PL TG FFA PG PF
Control
8.333 1.328 1.399 0.142
0.804 0.053 0.080 0.023
f f & f
1.159 0.075 0.075 0.005
j, 1.217 f 0.092 f 0.143 f 0.007
f 1.006 f 0.058 f 0.103 f 0.014
& f & 3
3 min
glyceride;
(12) (8) (12) (11)
(7) (7)
ii;
*
f f & f
& f f &
& f f f
0.591 0.134 0.141 0.013
0.500 0.266 0.085 0.012
1.147 0.063 0.055 0.009
1.228 0.052 0.094 0.008
(14) (12) (14) (12)
(14) (15) (16) (13)
(7)
(8) 63)
(8)
(8) (7)
(8)
(8)
glucose
210.91 4.590 1.529 1.231 0.189
222.53 3.745 1.668 1.309 0.167
229.44 3.268 1.076 1.508 0.215
231.27 6.431 1.398 1.503 0.163
+ f f f &
& f -ly f f
f f f f f
+ & & 5 f
PF, fatty
**
acids in the perfusate.
(9) (9) (9) (9) (8)
(10) (10) (9) (10) (10)
3.41 0.485 0.174 0.079 0.008*
glucose.
(7) (11) (11) (11) (4)
5.62 (8) 0.442##(11) 0.220 (12) 0.106 (12) 0.005 (5)
7.17 0.435** 0.130 0.132 0.019*
3.40 0.627 0.131 0.107 0.014
30 min
P< 0.05 control vs isoproterenol. P< 0.01 control vs isoproterenol. ## P< 0.01 with glucose vs without
6.312 2.562 1.802 0.143
7.174 2.257 1.852 0.154
7.941 1.277 1.373 0.149
f & & f
15 min
or without
9.976 1.323 1.366 0.137
for 3, 15 and 30 min with
9.395 1.261 1.423 0.121
(8) (7) (7)
(8)
perfused
PL TG FFA PG PF
f 6.91 f 1.484 f 0.144 f 0.084
ofhearts
Isoproterenol
232.91 11.964 1.415 1.528
lipid
PL TG FFA PG PF
on myocardial
Control
of isoproterenol
Values represent mean fatty acid contents (umol/g dry weight) & S.E. except for PF values which represent total fatty acid amount (pmol) in the perfusate. The figures in parentheses indicate the number ofdeterminations. Abbreviations: PL, phospholipid; TG, triglyceride; FFA, free fatty acid; PG, partial
Without glucose
With glucose
TABLE
MYOCARDIAL
LIPID
929
METABOLISM
EO-
60 40I
1 0
I 5
I 15 Time
I I 30 (min)
FIGURE 1. Cardiac performances of the perfused heart without exogenous fatty acid. Upper column represents changes in coronary flow, middle one heart rate and lower one contractile force. The vertical lines indicate mean values f S.E. (a = 8-12). The initial values of coronary flow (ml/ min) and heart rate (beatslmin) were 6.3 f 0.6, 304 f 10 (control in the presence of glucose, -O--), 5.2 f 0.4,304 & 13 (isoproterenol in the presence ofglucose, -@--), 6.0 & 0.6,292 & 10 (control in the absence of glucose, -a--) and 5.6 f 0.5,281 f 10 (isoproterenol in the absence of glucose, *--), respectively. *, P < 0.05; **, P < 0.01, . ***, P < 0.001 control vs isoproterenol.
Under these conditions, the time course of changes in myocardial lipids was examined. Fatty acid contents of phospholipid (PL), free fatty acid (FFA) and different from the initial contents partial glyceride (PG) were no significantly within 30 min of the perfusion, while the content of triglyceride (TG) fatty acid decreased gradually and reached 54% of the initial amount at 30 min of the perfusion.
930
F. TAKENAKA
AND
S. TAKE0
After the administration of 4 x IO-6 M of isoproterenol, coronary flow showed an initial decrease, followed by a significant increase at 15 min (P < 0.001) and 30 min (P < 0.001) of the perfusion. Contractile force increased strikingly within 30 s, followed by a gradual decline, but finally reached 90 + 7% of the initial value after 30 min. The content of TG fatty acid decreased to 50.8% of the control value (P < 0.01). The decrease of TG fatty acid was observed in all components; especially, the decrease of oleic acid (39.3%) and linoleic acid (40.6%) was most prominent (Table 2). Isoproterenol did not produce any significant changes in fatty acid content in PL, FFA and PG fractions.
In theabsence of glucose The heart was perfused with glucose-free solution. Within 30 min of the control perfusion, coronary flow and heart rate gradually decreased and reached 79 -+ 5% and 85 f 4% of the initial value, respectively. The results obtained were similar to those observed in the presence of glucose. Contractile force slightly increased during initial perfusion for 15 min, followed by the decline to 88 & 7% after 30 min. The findings were different from those in the presence of glucose (P < 0.05 at 15 min and P < 0 .Ol at 30 min of the perfusion). The time course of changes in myocardial lipids showed that fatty acid content in PL, FFA and PG fractions was not different from each initial amount during 30 min of the perfusion. The results were identical to those in the presence of glucose. The fatty acid content in TG fraction decreased to 29% of the initial amount 30 min after the perfusion. There was a significant difference (P < 0.01) in the amount of the decrease in myocardial TG fatty acid contents between the hearts perfusing with glucose and without glucose. When isoproterenol (4 x IO-6 M) was administered to the heart, coronary flow and heart rate showed results similar to those in the presence of glucose. Changes in contractile force were quite different from those of the heart perfused with solution containing glucose. The contractile force transiently decreased, followed by a rapid decline, reaching 72 & 7% of the initial value at 15 min and 46 f 8% at 30 min of the perfusion. The significant decrease, compared with the control, in TG content induced by isoproterenol as seen in the presence of glucose was not observed in the absence of glucose. In other lipid fractions, however, the contents of fatty acid were not different from the initial amounts.
Lipids in theperfusate Lipids in the perfusate were studied in the present experiments, since there is a possibility that myocardial lipids may diffuse into the perfusate. Fatty acid moieties
0.076 f 0.009*
64.4
vs isoproterenol,
Isoproterenol
(% of control)
*P
< 0.05 control
0.118 & 0.015
c14:o
2. Effect ofisoproterenol (pmol/g dry weight)
Control
TABLE
53.9 * *
87.5
0.161 50.070 69.3
0.529 f 0.032
0.763 & 0.053
cm:0
of triglyceride
vs isoproterenol.
composition
0.184 f 0.034
cm:1
acid
PC 0.0 1 control
1.316 + 0.197**
2.444 f 0.218
C1e:o
on the fatty
39.3
0.584 f 0.121””
1.485 + 0.224
c1s:1
fraction
40.6
0.537 f 0.116””
1.324 + 0.197
cm:2
in perfused
56.6
0.064 & 0.025
50.8
*
of glucose
3.268 & 0.435*
6.43 1 &- 0.627
Total
in the presence
0.113 & 0.023
c20:4
rat hearts
932
F. TAKENAKA
AND
S. TAKE0
were detected in the perfusate (Table 1). The amount of fatty acids in the perfusate during perfusion for 30 min attained to 0.163 f 0.014 pmol in the presence of glucose and 0.167 f 0.005 pm01 in the absence of glucose. Lipids in the perfusate were mainIy composed of cholesterolester ( 15 %) , triglyceride (2 1%) , free fatty acid (42%) and phospholipid (21%), as examined by g.1.c. analysis. In the heart perfused with isoproterenol for 30 min, the amount of fatty acid in the perfusate increased significantly, compared with the control both in the absence and presence of glucose (P < 0.05). The total volume (ml) of the per&sate during 30min perfusion was 175.6 & 7.5 (control), 204.4 f 7.8 (isoproterenol) in the presence of glucose and 171.8 & 10.6 (control), 229.8 f 17.4 (isoproterenol) in the absence of glucose, respectively.
Sodium myristirate was used as a tracer of exogenous free fatty acid, since the physiological level of myristiric acid(Cr4:s) is very low in ali lipid fractions. The contents of myristiric acid in PL, TG, FFA and PG fractions were found to be O.S%, 1.5%, 8.9% and 2.5%, respectively. The heart was perfused for 30 min with solution containing 0.4 mM of sodium myristirate with O.5o/o bovine albumin. Cardiac performances of the perfused heart are shown in Figure 2, myocardial fatty acid contents in Table 3 and fatty acid components of TG fraction in Table 4.
In the presence ofglucose
Coronary flow and heart rate gradually decreasedto 52 rf: 3% and 85 & 5% of the initial value, respectiveIy. Contractile force rapidly decreased to 65 & 8% after 5 min, followed by a gradual increase to 113 + 16% 30 min after the perfusion. The myristiric acid content in TG, FFA and PG fraction after 30-min perfusion increased to 15.5-, 3.4- and 5.5-fold of the initial vahtes, respectively .The myristiric acid content in PL fraction remained unchanged. The total content of TG fatty acid in the heart perfused with solution containing my&&ate was almost the sameas the initial amount. The finding was quite different from that observed in the perfused heart without exogenousfatty acid. When isoproterenol was applied to the heart, there was a significant change in coronary flow throughout the perfusion, but no appreciable change in heart rate except for an initial transient rise. ContractiIe force was markedly enhanced ~roughout the perfusion. The total content of myocardial TG fatty acid significantly decreased (P < 0.05), while incorporation of myristiric acid into TG fraction markedly increased (P < 0.01). Th ere was no significant change in other lipid fractions.
MYOCARDIAL
LIPID
METABOLISM
933
In the absenceof glucose Coronary flow and heart rate gradually declined as observed in the presence of glucose. Contractile force was also depressed during the initial perfusion for 10 min, followed by a gradual augmentation. The myocardial TG content after 30 min of the perfusion declined to 63.774, which was significantly lower than the TG content in the presence of glucose (P < 0.01). The amount of myristiric acid
60
Time
(min)
FIGURE 2. Cardiac performances of the perfused heart with exogenous fatty acid. Upper column represents changes in coronary flow, middle one heart rate and lower one contractile force. The vertical lines indicate mean values & S.E. (a = 6-8). The initial values of coronary flow (ml/mm) and heart rate (beatslmin) were 5.7 & 0.2, 282 f 10 (control in the presence ofglucose, -o--), 5.2 f 0.3, 281 + 8 (isoproterenol in the presence of glucose, --+-) and 6.0 -& 0.5, 292 & 12 (control in the absence of glucose, -•--), respectively. *, P < O.O5;**,P < 0.01 controlvsisoproterenol.
PL TG FFA PG
PL TG FFA PG
PL TG FFA PG
PL TG FFA PG
Myristiric acid
Total
Myristiric acid
lipid
Total
3. Myocardial
1.791 0.181 0.068 0.034
235.64 12.804 1.326 1.500
1.421 0.187 0.126 0.039
232.91 11.964 1.415 1.528
contents
f rfr & f
f &f i
+ & f +
0.117 0.025 0.028 0.007
25.81 0.837 0.243 0.156
0.365 0.026 0.053 0.013
f 6.91 * 1.484 f 0.144 f 0.084
Initial
(10) (10) (9) (10)
(12) (10) (9) (10)
(8) (8) (7) (7)
(8) (8) (7) (7)
of the heart
f & & f
f i + +
2.176 1.020 0.385 0.191
0.105 0.112 0.041 0.049
8.64 1.686 0.158 0.176
(6) (6) (6) (6)
(6) (6) (6) (6)
PG, partial
2.491 1.398 0.403 0.184
215.35 13.654 1.618 1.699
f f f -&
& + + f
(6) (6) (6) (6)
3.132 1.661 0.650 0.106
* f f f
f $f &
& f * &
1.338 2.895 0.433 0.214 223.80 8.152 2.027 1.196
f f f &
195.25 12.905 1.618 1.374
0.205 0.204t.t 0.050 0.011
41.40 1.052t 0.114 0.063
0.445 0.244 0.074 0.045
5.94 0.742 0.189 0.127
Control
(6) (6) (6) (6)
(6) (6) (6) (6)
(8) (7) (8) (7)
(8) (7) (8) (7)
1.871 3.653 0.416 0.308
& & f +=
(8) (8) (7) (7)
(8) (8) (7) (6)
glucose.
0.496 0.335** 0.087 0.084
f 11.07 f 0.972* f 0.140 f 0.120
Isoproterenol 204.96 10.106 1.555 1.484
30 min
* P< 0.05, ** P< 0.01 control vs isoproterenol. t I’< 0.01 and tt P< 0.001 with glucose vs without glyceride.
0.143 0.168 0.094 0.049
3.55 1.311 0.326 0.218
(6) (6) (6) (6)
myristirate
Isoproterenol
containing
10 min
a solution
Control
with
205.44 10.496 1.750 1.643
perfused
Values represent mean fatty acid contents (pmol/g dry weight) f S.E. The figures in parentheses indicate the number of determinations. Abbreviations: PL, phospholipid; TG, triglyceride; FFA, free fatty acid;
Without glucose
With glucose
TABLE
PLATE I. Low power autoradiograph from a semi-thin demonstrating the atrioventricular node (AVN) adjacent and intervenwicularseptum (VS). x 2W.
(1 w thidr) section of m adult rat heart to the tricuspid valve (TV), atrium (A)
PLATE 2. High power light microscopic autoradiograph of a I p thick section of the atrioventricular node 2 h after intravenous injection of 2 mCi [aH]-leucine. Control and experimental tissues are qualitatively similar in appearance and in both the grains me evenly distributed over the entire cross section ofthe node. x 950.
2.356 & 0.257i-f
2.285 f 0.377”
3.614 f 0.250
c&:0
Cl%:1
fraction
0.161 & 0.015
0.123 & 0.032
0.302 * 0.075
in triglyceride
* P < 0.05, * * P < 0.01 with glucose vs without glucose. t P < 0.05, tt P < 0.01, ttt P < 0.001 control vs isoproterenol.
3.653 & 0.335Tf
1.661 & 0.204**
Without glucose
With glucose
2.895 f 0.244
With glucose
C14:0
4. Myocardial fatty acid compositions (qol/g dry weight)
Xsoproterenol
Control
TABLE C1e:1
1.093 1.351 & 0.119 & 0.22 ltft
1.098 1.452 & 0.080 f 0.342”
1.202 2.493 f 0.240 & 0.240
c1a:o
1.230 f O.O65$tt
1.237 rt: 0.227”
2.137 & 0.219
C1s:a
c&o:4
0.263 & 0.065
0.294 & 0.038
Total
myristirate
10.106 & 0.9727
8.152 jI 1.052””
12.905 j, 0.742
containing
0.262 & 0.038
of the heart perfused with a solution
936
F. TAKENAKA
AND
S. TAKE0
incorporated into TG fraction was significantly less than in the presence of glucose (P < 0.001). When isoproterenol (4 x IO-7 M) was added to the perfusate, marked bradycardia in four cases and ventricular arrest in three cases out of seven occurred within 10 min. The myocardial content of TG fatty acid at 10 min of the perfusion remained unchanged or showed a Iittle higher value than that of the control. Incorporation of myristiric acid was only slightly enhanced. There was no change in fatty acid content in other lipid fractions within 30 min of perfusion.
180 I
60 t II 0
1 5
I IO
I 15 Time
I
30
Cmin)
FIGURE 3. Cardiac performances of the perfused heart with isoproterenol (4 x 10-r MA) and various doses of glucose in the presence of exogenous my&irate (0.4 mnr>. Upper column represents changes in coronary flow, middle one heart rate and lower one contractile force. The initial values of coronary flow (ml/mm) and heart rate (beats/mm) were 5.7 rt: 0.2,282 4 10 (control with 5.5 rnM of glucose, -O-), 5.2 -+ 0.3, 281 ;t 8 (isoproterenol with 5.5 rnn of glucose, ---)> 5.6 Jt 0.4, 296 i 15 (isoproterenol with 1.7 rn~ of glucose, --I-) and 5.6 + 0.2, 294 + 11 (isoproterenol with 0.17 mM of glucose, --A--), respectively (a = 6-8). * P c 0.05, ** P < 0.01 compared with isoproterenol-0.17 rnn of glucose.
MYOCARDIAL
LIPID
METABOLISM
937
E$ects of glucose on cardiac performances in the heart perfused with solution containing myristirate
Cardiac performances of the heart were studied during perfusion with soIution containing myristirate (0.4 mu), isoproterenol(4 x IO-7 M) and glucosein dosesof 0.17, 1.7 and 5.5 mM. Results are shown in Figure 3. Coronary flow and heart rate decreasedduring 30 min of the perfusion. The rate of decrement in coronary flow and heart rate was proportional to the amount of glucosereduced. Thus, there was a close relation between glucose content in the perfusate and cardiac contractile force. Contractile force of the heart perfused with solution containing 5.5 rnM of glucose was augmented throughout the perfusion. Contractile force of the heart perfused with solution containing 1.7 mM of glucose reached 127 & 24%, while that of the heart with 0.17 mM of glucose was sig~~cantly reduced {P < 0.0 l} to reach 72 f 16% at 30 min of the perfusion.
4. Discussion
The present experiments using isolated perfused hearts have demonstrated that the myocardial content of TG fatty acid gradually declined along with the time of petiusion with fatty acid-free medium. Olson and Hoeshen [IZ] have reported that TG fatty acid content did not change during 30-tin perfusion with nutrient-free medium in the isolated perfused rat heart. Dhalla et al. [3] have shown a marked decrease(74%) in tissue triglyceride content of the heart perfused for 2 h without glucose and a slight decrease(22%) with glucose.The discrepancy between our results and theirs may be explained by the difference in perfusing systemsemployed. Lipids in the myocardium are continuously washed out into the per&sate in the present perfusion method, while the diffused lipids may be recirculated and retaken up into myocardium in the closed system employed by Olson et al. and Dhalla et al. Lipids present in the perfusate increased significantly (P < 0.05) in the heart perfused for 30 min with isoproterenol both in the absenceand presenceof glucose. There is the possibility that the fatty acid releasefrom the myocardial TG fraction may be enhanced by isoproterenol. However, the total volume of the perfusate during the 30-min perfusion with isoproterenol was significantIy larger than that of the control. Therefore, it would be premature to conclude that the increase in amounts of lipids in the perfusate when isoproterenol was applied to the heart, is due to an augmentation of the fatty acid releasefrom tissue triglyceride, since we can not rule out another possibility that an increased lipid content in the perfusate might be attributable merely to leakage of lipids from the perfused heart. In the control perfusion of 30 min, the myocardial content of TG in the absence of glucosewas sign~~tly lessthan that in the presenceof glucose, suggestingthat
938
F. TAKENAKA
AND S. TAKEi
TG fatty acid may be utilized much more in the absence of glucose than in its presence. A significant decrease in the TG fatty acid content was seen during perfusion with solution containing isoproterenol. The results are consistent with those obtained by previous authors in that epinephrine signi~~an~y decreased endogeno~ TG content in working rat heart during 30-min perfusion in the presence of glucose [2] and also in the non-working rat heart during 45-min perfusion without glucose [5, 61. In the absence of glucose, however, the TG content at 30-min perfusion with isoproterenol was not significantly different from the control. Contractile force at 30-min perfusion was markedly depressed, whilst it was enhanced in the presence of glucose. These findings suggest that the decrease in endogenous TG content may correlate with changes in contractility. However> Grass et al. [2] have reported that the rate of tissue TG mobilization is accelerated with increased ventricular pressure development [.!I, but not with an increase in contractile frequency [2]. Thus, the decrease in endogenous TG content by isoproterenol observed in the present experiment can not be explained solely by changes in cardiac performances. iteratively there is evidence to indicate the existence of a number of myocardial TG pools [I] and independent TG pools in supernatant mitochondrial and microsomal fractions [15]. Olson and Hoeshen have shown that non-working hearts perfused for 90 min without substrates and hormones until exhaustion, retained about 32% TG content [I,?]. In the control perfusion in our experiments, TG content at 30-min perfusion was S&y0 of the initial value in the presence of glucose and 29% in the absence of glucose, Therefore, isoproterenol did not decrease tissue TG content further in the absence of glucose, sinceapproximately 30% of the tissue TG at 30 min of the control perfusion may be “‘mobilization resistant TG”. When the heart was perfused with solution containing myristirate, there was an i~ibition of the decrease in TG fatty acid content observed during the perfusion with fatty acid-free medium, A significant difference was seen between TG content in the myocardium perfused with solution containing glucose and without glucose after the control 30-min perfusion. In addition, myristiric acid uptake into the TG fraction was significantly lower in the absence of glucose than that in its presence. These facts indicate that exogenous myristirate can be taken up by the perfused heart and the incorporation of the fatty acid into myocardial TG fraction was lower in the absence of glucose; the latter may be because exogenous fatty acid is oxidized rather than incorporated into the TG fraction. When isoproterenol was applied to the heart perfused with exogenous myristirate in the presence ofglucose, TG fatty acid content significantly decreased, compared with the control. The incorporation of myristiric acid into the TG fraction increased significantly. The results suggest that isoproterenol may enhance tissue TG turnover in the myocardium. The facts are not inconsistent with those described by previous authors that incorporation of exogenous p~tate-~-[Go*] into myom~ocaxxiial
MYOCARDIALLIPIDMETABOLISM
939
lipid turnover cardial lipids was increased by epinephrine [IO] and endogenous enhanced by epinephrine [9]. In the glucose-free medium, however, a marked bradycardia or ventricular arrest occurred within 10 min of perfusion and endogenous TG content was not different from the control or the initial value. Moreover, in the heart perfused without exogenous fatty acid, isoproterenol enhanced continuous cardiac contraction in the presence of glucose, but had less effect in the absence of glucose. These findings indicate that glucose may play an important role in the mechanism responsible for energy utilization in the cardiac performance of the heart perfused with solution containing isoproterenol. The aggravation of isoproterenol on cardiac performance in the absence of glucose might be due to lack of energy supply satisfying the commensurate increase in energy demand of the heart by isoproterenol. In fact, as shown in the present experiments, the increased cardiac performances due to isoproterenol were closely related to the amount of glucose added to the perfusing solution containing fatty acid.
The authors wish to express to gratitude cology, Kumamoto University Medical commenting on it.
1.
2.
3.
4.
5. 6.
7.
to Dr K. Nishi, Department of PharmaSchool, for reading the manuscript and
C&W, M. F. III, MCCASKILL, E. S., SI~P, J. C. & MURPHY, V. K. Metabolism endogenous lipids in cardiac muscle: effect of pressure development. American Journal Physiology220,428-435 CRASS, M. F. III,
of of
(1971). SHIPP, J. C. & PIEPER,
G. M. Effects of catecholamines on myocardial endogenous substrates and contractility. American Journal of Physiolog 228,618627(1975). DHALLA,
N. S., MATOUSHEK, R. F., SUN, C. N. & OLSON, R. E. Metabolic, ultrastructural and mechanical changes in the isolated rat heart perfused with aerobic medium in the absence or presence of glucose. Canadian Journal of Physiology and Pharmacolosy51,590-603 (1973). FOLCH, J., LEES, M. & STANLEY, G. H. S. A simple method for the isolation and purification of total lipids from animal tissues. Journal of 3iologi~a~ Country 226, 497-509 (1957). GARTNER, S. L. & VAHOUNY, G. V. Elects of epinephrine and cyclic 3’, Y-AMP in the peritsed rat hearts. American Journal ofPhysiology222, 1121-l 124 (1972). GARTNER, S. L. & VAWOUNY, G. V. Endogenous triglyceride and glycogen in the perfused rat hearts. Proceedings of the Society for E.+erimental Biology and Medicine 143,556-560 (1973). C-OLD,
M., ATLAS, 3% J., SCOTT, J. C. & SPITZEN, J. J. Effect of norepinephrine on myocardial free fatty acid uptake and oxidation. Proceedings of the Societyfor Ex~~~a~ Biology
and Medicine
118,876-879
(1965).
940 8. 9. IO. II. 12. 13. 14. 15. 16.
F.TAKENAKAANDS.TAKEO Gous~os, A. & FELTS, J. M. Effects ofepinephr~e, norepinephrine, glucose and insulin on extraction and oxidation offree fatty acid by myocardium. Ci~~~ti~n 28, 729 (1963). KREXSBERG, R. A. Effect of epinephrine on myocardial triglyceride and free fatty acid utilization. AmericanJournal of PhysioloQ 210,385-389 (1966). MATHUR, P. P. & MORLER, C. M. Subcellular distribution and incorporation of palmitate-U-[Clan into myocardial lipids: Role of endogenous catecholamines. Journal of Molecular a~~e~~alaT ~ard~~o~ 7, 17-26 (1975). OLSON, R. E. Utilization of exogenous and endogenous lipid by the isolated perfused rat heart. In Coronary Circulation and Energetics of the Myocardium. G. Marchetti & B. Taccardi, Eds. pp. 162-185. Basel: S. Kargel AG. (1967). OLSON, R. E. & HOESHEN, R. J. Utilization of endogenous lipid by the isolated perfused heart. B~~c~i~~3ou~~ 103,796-801 (1967). SHIPP, J. C. Interrelation between carbohydrate and fatty acid metabolism of isolated perfused rat hearts. Metabolic 13,852-866 (1964). SHIPP, J. C., OPIE, I,+. H. & CHALLONER, D. Fatty acid and glucose metabolism in the perfused heart.Nature 189, 1018-1019 (1961). STEIN, 0. & STEIN, Y. Lipid synthesis, intracellular transport and storage III. Electron microscopic radioautographic study of the rat heart perfused with tritiated oleic acid. JmznuE of Cell Bi&gy 36,63-77 ( 1968). W~LL~~SON, J. R. Metabolic effects of epinephrine in the isolated, perfused rat heart. Journal of Biological Chemistry 239,272 l-2729 (1964).
of
Molecular andCellular
Effects
Cardiology
(1976)
8,925-940
of Isoproterenol on Myocardial Lipid Metabolism in Rat Hearts Perfused with and without Exogenous Substrates
Department (Received
FUMIO TAKENAKA AND SATOSHI TAKE0 of Pharmacology, Kumamoto University Medical School, Japan 4 .November
1975,
accepted in revisedform
22 March
1976)
F. TAKENAKA AND S. TAKEO. Effects of Isoproterenol on Myocardial Lipid Metabolism in Rat Hearts Perfused with and without Exogenous Substrates. Journal of Molealar and Cellular Cardiology (1976) 8, 925-940. Effects of isoproterenol on myocardial lipid metabolism were studied in perfused rat hearts by the Langendorff apparatus without recirculation. Fatty acids in phospholipid, free fatty acid and partial glyceride fractions did not alter during the initial 30-n& perfusion. However, fatty acids in triglyceride fraction decreased along with the perfusion. Isoproterenol enhanced the decrease in triglyceride fatty acid in the presence ofglucose, but not in the absence of glucose. When hearts were perfused for 30 min with exogenous myristirate in the presence of glucose, triglyceride fatty acid did not decrease in the control perfusion, and incorporation of myristiric acid into triglyceride fraction increased in the presence of isoproterenol. When hearts were perfused with myristirate in the absence of glucose, fatty acid in triglyceride fraction slightly decreased during the control 30-min perfusion, and marked bradycardia or ventricular arrest occurred within 10 min after the start of perfusion with solution containing isopro terenol. Triglyceride content 10 min after perfusion was not different from the control value. Mechanical performance of hearts with myristirate and isoproterenol improved when the amount of glucose in the perfusing solution was increased. The findings indicate that glucose may play an important role in the mechanical performance of the heart perfused with a solution containing isoproterenol. WORDS: Myocardial lipid metabolism; Cardiac performance; Isolated perfused heart;
KEY
Fatty acid; Glucose.
Myristiric
acid;
Isoproterenol;
1. Introduction It is well established that glucose and fatty acid are utilized as the main substrates for myocardial energy source [II]. Shipp et al. [13, la] have demonstrated that the latter is the preferential substrate in the isolated perfused heart. Effects of catecholamines on the utilization of these substrates in myocardium have been studied extensively [I, 2, 5, 9, 16-j. According to Williamson [16], epinephrine, norepinephrine and isoproterenol increased glucose oxidation in the perfused heart by activating phosphorylase activity. Gousios and Felts [8] and Gold et al. [j7 have shown that epinephrine and norepinephrine increased myocardial extraction and oxidation of palmitate-1-[‘*Cl. Furthermore, Kreisberg [9] has reported that epinephrine increased the utilization of myocardial lipid in the perfused heart.
926
F. TAKENAKA
AND
S. TAKE0
The studies so far reported, however, have dealt mostly with the utilization of triglyceride and phospholipid fraction, but not with partial glyceride and free fatty acid fraction. In the present experiments, the concentration of each fatty acid component in myocardial lipid fractions was determined, and effects of isoproterenol as well as the influence of glucose on myocardial lipid metabolism were investigated in isolated perfused rat hearts.
2. Materials
and Methods
Male albino Wistar rats weighing 240 to 270 g were anesthetized and killed by decapitation. The heart was rapidly removed and chilled in modified LockeRinger solution of the following compositions (mM) : KC 1, 5.63; NaCI, 154; CaC12, 2.18; NaHCOs, 20.8; glucose, 5.5. All adipose and connective tissues were removed from the heart and aorta in cold solution. Then, the heart was transferred to a Langendorff apparatus and perfused with modified Locke-Ringer solution previously equilibrated with a gas mixture of 95% 02 + 5% COa. The perfusate was not recirculated. The heart was pre-perfused for 5 min to stabilize cardiac performance. During the experimental perfusion, three kinds of perfusates were used; (1) modified Locke-Ringer solution, (2) the solution without glucose and (3) the solution containing sodium myristirate (0.4 mM) with bovine albumin (0.5%). The solution was gassed for 30 min with 95% 02 + 5% COa. The pH was adjusted to 7.40 f 0.03 and temperature at 35°C. Isoproterenol was added to the solution just before the start of perfusion at a concentration of 4 x IO-6 M in the absence of free fatty acid-albumin and 4 x 10-7 M in its presence. In the latter experiments, 4 x 10-7 M of NaHSOa was used as an antioxidant ofisoproterenol. At 3, 15 and 30 min of the experimental perfusion, the heart was removed from the apparatus, chilled, blotted dry and subjected to lipid analysis. Lipids were extracted from the homogenated heart with 20 ml of chloroform-methanol (2 :l v/v) according to the method of Folch et al. [4]. Dry weight was calculated by measuring the residue on the filter paper which was used for separation of lipid extracts from the homogenate, after drying at 100°C for 2 h. The chloroform phase was dried in vacua with a rotary evaporator under reduced pressure. The residue of total lipids was dissolved in 0.7 ml of chloroform-methanol mixture and separated by t.1.c. with silica gel G as the absorbant and a solvent system of petroleum etherdiethylether-acetic acid (80 :20: 1). The separation procedure was carried out according to the method of Olson and Hoeshen [12]. The lipid zones on the thin layer plate were scraped and extracted with 25 ml of chloroform-methanol mixture. Each lipid fraction of phospholipid(PL), triglyceride(TG), free fatty acid(FFA) and partial glyceride(PG) was dried under reduced pressure and ester moieties of each lipid fraction were changed to their methylester by refluxing for 2 h with 3 ml of absolute methanol and 5 drops of cont. sulfuric acid. The yield through thin
MYOCARDIAL
LIPID
METABOLISM
927
layer separation and esterification was 83% according to the examination of reference samples by the same procedure. These methylesters were subjected to g.1.c. analysis to determine the fatty acid components by using arachidic acid as an internal standard. The main components of fatty acids in the lipid fractions were myristiric, pahnitic, pahnitoleic, stearic, oleic, linoleic and arachidonic acid. The total amount of fatty acids in each fraction was determined by adding each of these components. To determine lipid content in the perfusate, lipids were extracted from the overAown perfusate cohected over the experimental period with diethylether at pH 1 adjusted by HCl after salting out with excess sodium chloride. After evaporation of the solvent, the total residual lipids were determined by the same esterification and gas liquid chromatographic analysis as mentioned above. In some cases, to determine fractional distribution of the total lipids, the lipids remaining in the diethyle~er extract were separated by t.l.c., and fatty acids in each fraction were determined by the same procedure as described above. Coronary flow was measured by a drop counter and heart rate was monitored by pulse rate tachometer (Nihon Koden RT-2). Cardiac contraction was estimated by monitoring isometric tension development of the heart preloaded by 1.25 g through a hook attached to the apex of the heart by means of a farce-displacement transducer (Nihon Koden SB-IT), and displayed on an oscillographic pen-recorder (Nihon Koden RM-I 50). The gain of the recording system was adjusted so that 1.25 g-preload caused a 2%mm pen deflection. Chemical substances employed in the present experiments were isoproterenol b~rdrochlo~de (Nikk~n Kagaku) , bovine albumin (Armor Pharillac~utical Co.) and sodium my&irate (Nakarai Yakuhin). Bovine albumin contained 2.0 pmoljg of free fatty acid and the purity of sodium my&irate was over 99% according to the gas liquid chroma~o~aphic analysis.
3. Results
Cardiac performance of the heart perfused with modified Locke-Ringer solution is shown in Figure I and changes in fatty acid contents in the myocardium are shown in Table I. Within 30 min of the start of the control perfusion with modified Locke-Ringer solution containing glucose, coronary flow and heart rate gradually decreasedto 69 & 3% and 83 & 3% of the initial value, respectively, while contractile force gradually increased and reached 133 f 7%.
I. Effects
(12) (10) (9) (10)
9.184 1.901 1.716 0.088
PL TG FFA PG PF
25.81 0.837 0.243 0.156
Isoproterenol
& f f f 10.762 1.791 1.473 0.118
235.64 12.804 1.326 1.500
PL TG FFA PG PF
Control
8.333 1.328 1.399 0.142
0.804 0.053 0.080 0.023
f f & f
1.159 0.075 0.075 0.005
j, 1.217 f 0.092 f 0.143 f 0.007
f 1.006 f 0.058 f 0.103 f 0.014
& f & 3
3 min
glyceride;
(12) (8) (12) (11)
(7) (7)
ii;
*
f f & f
& f f &
& f f f
0.591 0.134 0.141 0.013
0.500 0.266 0.085 0.012
1.147 0.063 0.055 0.009
1.228 0.052 0.094 0.008
(14) (12) (14) (12)
(14) (15) (16) (13)
(7)
(8) 63)
(8)
(8) (7)
(8)
(8)
glucose
210.91 4.590 1.529 1.231 0.189
222.53 3.745 1.668 1.309 0.167
229.44 3.268 1.076 1.508 0.215
231.27 6.431 1.398 1.503 0.163
+ f f f &
& f -ly f f
f f f f f
+ & & 5 f
PF, fatty
**
acids in the perfusate.
(9) (9) (9) (9) (8)
(10) (10) (9) (10) (10)
3.41 0.485 0.174 0.079 0.008*
glucose.
(7) (11) (11) (11) (4)
5.62 (8) 0.442##(11) 0.220 (12) 0.106 (12) 0.005 (5)
7.17 0.435** 0.130 0.132 0.019*
3.40 0.627 0.131 0.107 0.014
30 min
P< 0.05 control vs isoproterenol. P< 0.01 control vs isoproterenol. ## P< 0.01 with glucose vs without
6.312 2.562 1.802 0.143
7.174 2.257 1.852 0.154
7.941 1.277 1.373 0.149
f & & f
15 min
or without
9.976 1.323 1.366 0.137
for 3, 15 and 30 min with
9.395 1.261 1.423 0.121
(8) (7) (7)
(8)
perfused
PL TG FFA PG PF
f 6.91 f 1.484 f 0.144 f 0.084
ofhearts
Isoproterenol
232.91 11.964 1.415 1.528
lipid
PL TG FFA PG PF
on myocardial
Control
of isoproterenol
Values represent mean fatty acid contents (umol/g dry weight) & S.E. except for PF values which represent total fatty acid amount (pmol) in the perfusate. The figures in parentheses indicate the number ofdeterminations. Abbreviations: PL, phospholipid; TG, triglyceride; FFA, free fatty acid; PG, partial
Without glucose
With glucose
TABLE
MYOCARDIAL
LIPID
929
METABOLISM
EO-
60 40I
1 0
I 5
I 15 Time
I I 30 (min)
FIGURE 1. Cardiac performances of the perfused heart without exogenous fatty acid. Upper column represents changes in coronary flow, middle one heart rate and lower one contractile force. The vertical lines indicate mean values f S.E. (a = 8-12). The initial values of coronary flow (ml/ min) and heart rate (beatslmin) were 6.3 f 0.6, 304 f 10 (control in the presence of glucose, -O--), 5.2 f 0.4,304 & 13 (isoproterenol in the presence ofglucose, -@--), 6.0 & 0.6,292 & 10 (control in the absence of glucose, -a--) and 5.6 f 0.5,281 f 10 (isoproterenol in the absence of glucose, *--), respectively. *, P < 0.05; **, P < 0.01, . ***, P < 0.001 control vs isoproterenol.
Under these conditions, the time course of changes in myocardial lipids was examined. Fatty acid contents of phospholipid (PL), free fatty acid (FFA) and different from the initial contents partial glyceride (PG) were no significantly within 30 min of the perfusion, while the content of triglyceride (TG) fatty acid decreased gradually and reached 54% of the initial amount at 30 min of the perfusion.
930
F. TAKENAKA
AND
S. TAKE0
After the administration of 4 x IO-6 M of isoproterenol, coronary flow showed an initial decrease, followed by a significant increase at 15 min (P < 0.001) and 30 min (P < 0.001) of the perfusion. Contractile force increased strikingly within 30 s, followed by a gradual decline, but finally reached 90 + 7% of the initial value after 30 min. The content of TG fatty acid decreased to 50.8% of the control value (P < 0.01). The decrease of TG fatty acid was observed in all components; especially, the decrease of oleic acid (39.3%) and linoleic acid (40.6%) was most prominent (Table 2). Isoproterenol did not produce any significant changes in fatty acid content in PL, FFA and PG fractions.
In theabsence of glucose The heart was perfused with glucose-free solution. Within 30 min of the control perfusion, coronary flow and heart rate gradually decreased and reached 79 -+ 5% and 85 f 4% of the initial value, respectively. The results obtained were similar to those observed in the presence of glucose. Contractile force slightly increased during initial perfusion for 15 min, followed by the decline to 88 & 7% after 30 min. The findings were different from those in the presence of glucose (P < 0.05 at 15 min and P < 0 .Ol at 30 min of the perfusion). The time course of changes in myocardial lipids showed that fatty acid content in PL, FFA and PG fractions was not different from each initial amount during 30 min of the perfusion. The results were identical to those in the presence of glucose. The fatty acid content in TG fraction decreased to 29% of the initial amount 30 min after the perfusion. There was a significant difference (P < 0.01) in the amount of the decrease in myocardial TG fatty acid contents between the hearts perfusing with glucose and without glucose. When isoproterenol (4 x IO-6 M) was administered to the heart, coronary flow and heart rate showed results similar to those in the presence of glucose. Changes in contractile force were quite different from those of the heart perfused with solution containing glucose. The contractile force transiently decreased, followed by a rapid decline, reaching 72 & 7% of the initial value at 15 min and 46 f 8% at 30 min of the perfusion. The significant decrease, compared with the control, in TG content induced by isoproterenol as seen in the presence of glucose was not observed in the absence of glucose. In other lipid fractions, however, the contents of fatty acid were not different from the initial amounts.
Lipids in theperfusate Lipids in the perfusate were studied in the present experiments, since there is a possibility that myocardial lipids may diffuse into the perfusate. Fatty acid moieties
0.076 f 0.009*
64.4
vs isoproterenol,
Isoproterenol
(% of control)
*P
< 0.05 control
0.118 & 0.015
c14:o
2. Effect ofisoproterenol (pmol/g dry weight)
Control
TABLE
53.9 * *
87.5
0.161 50.070 69.3
0.529 f 0.032
0.763 & 0.053
cm:0
of triglyceride
vs isoproterenol.
composition
0.184 f 0.034
cm:1
acid
PC 0.0 1 control
1.316 + 0.197**
2.444 f 0.218
C1e:o
on the fatty
39.3
0.584 f 0.121””
1.485 + 0.224
c1s:1
fraction
40.6
0.537 f 0.116””
1.324 + 0.197
cm:2
in perfused
56.6
0.064 & 0.025
50.8
*
of glucose
3.268 & 0.435*
6.43 1 &- 0.627
Total
in the presence
0.113 & 0.023
c20:4
rat hearts
932
F. TAKENAKA
AND
S. TAKE0
were detected in the perfusate (Table 1). The amount of fatty acids in the perfusate during perfusion for 30 min attained to 0.163 f 0.014 pmol in the presence of glucose and 0.167 f 0.005 pm01 in the absence of glucose. Lipids in the perfusate were mainIy composed of cholesterolester ( 15 %) , triglyceride (2 1%) , free fatty acid (42%) and phospholipid (21%), as examined by g.1.c. analysis. In the heart perfused with isoproterenol for 30 min, the amount of fatty acid in the perfusate increased significantly, compared with the control both in the absence and presence of glucose (P < 0.05). The total volume (ml) of the per&sate during 30min perfusion was 175.6 & 7.5 (control), 204.4 f 7.8 (isoproterenol) in the presence of glucose and 171.8 & 10.6 (control), 229.8 f 17.4 (isoproterenol) in the absence of glucose, respectively.
Sodium myristirate was used as a tracer of exogenous free fatty acid, since the physiological level of myristiric acid(Cr4:s) is very low in ali lipid fractions. The contents of myristiric acid in PL, TG, FFA and PG fractions were found to be O.S%, 1.5%, 8.9% and 2.5%, respectively. The heart was perfused for 30 min with solution containing 0.4 mM of sodium myristirate with O.5o/o bovine albumin. Cardiac performances of the perfused heart are shown in Figure 2, myocardial fatty acid contents in Table 3 and fatty acid components of TG fraction in Table 4.
In the presence ofglucose
Coronary flow and heart rate gradually decreasedto 52 rf: 3% and 85 & 5% of the initial value, respectiveIy. Contractile force rapidly decreased to 65 & 8% after 5 min, followed by a gradual increase to 113 + 16% 30 min after the perfusion. The myristiric acid content in TG, FFA and PG fraction after 30-min perfusion increased to 15.5-, 3.4- and 5.5-fold of the initial vahtes, respectively .The myristiric acid content in PL fraction remained unchanged. The total content of TG fatty acid in the heart perfused with solution containing my&&ate was almost the sameas the initial amount. The finding was quite different from that observed in the perfused heart without exogenousfatty acid. When isoproterenol was applied to the heart, there was a significant change in coronary flow throughout the perfusion, but no appreciable change in heart rate except for an initial transient rise. ContractiIe force was markedly enhanced ~roughout the perfusion. The total content of myocardial TG fatty acid significantly decreased (P < 0.05), while incorporation of myristiric acid into TG fraction markedly increased (P < 0.01). Th ere was no significant change in other lipid fractions.
MYOCARDIAL
LIPID
METABOLISM
933
In the absenceof glucose Coronary flow and heart rate gradually declined as observed in the presence of glucose. Contractile force was also depressed during the initial perfusion for 10 min, followed by a gradual augmentation. The myocardial TG content after 30 min of the perfusion declined to 63.774, which was significantly lower than the TG content in the presence of glucose (P < 0.01). The amount of myristiric acid
60
Time
(min)
FIGURE 2. Cardiac performances of the perfused heart with exogenous fatty acid. Upper column represents changes in coronary flow, middle one heart rate and lower one contractile force. The vertical lines indicate mean values & S.E. (a = 6-8). The initial values of coronary flow (ml/mm) and heart rate (beatslmin) were 5.7 & 0.2, 282 f 10 (control in the presence ofglucose, -o--), 5.2 f 0.3, 281 + 8 (isoproterenol in the presence of glucose, --+-) and 6.0 -& 0.5, 292 & 12 (control in the absence of glucose, -•--), respectively. *, P < O.O5;**,P < 0.01 controlvsisoproterenol.
PL TG FFA PG
PL TG FFA PG
PL TG FFA PG
PL TG FFA PG
Myristiric acid
Total
Myristiric acid
lipid
Total
3. Myocardial
1.791 0.181 0.068 0.034
235.64 12.804 1.326 1.500
1.421 0.187 0.126 0.039
232.91 11.964 1.415 1.528
contents
f rfr & f
f &f i
+ & f +
0.117 0.025 0.028 0.007
25.81 0.837 0.243 0.156
0.365 0.026 0.053 0.013
f 6.91 * 1.484 f 0.144 f 0.084
Initial
(10) (10) (9) (10)
(12) (10) (9) (10)
(8) (8) (7) (7)
(8) (8) (7) (7)
of the heart
f & & f
f i + +
2.176 1.020 0.385 0.191
0.105 0.112 0.041 0.049
8.64 1.686 0.158 0.176
(6) (6) (6) (6)
(6) (6) (6) (6)
PG, partial
2.491 1.398 0.403 0.184
215.35 13.654 1.618 1.699
f f f -&
& + + f
(6) (6) (6) (6)
3.132 1.661 0.650 0.106
* f f f
f $f &
& f * &
1.338 2.895 0.433 0.214 223.80 8.152 2.027 1.196
f f f &
195.25 12.905 1.618 1.374
0.205 0.204t.t 0.050 0.011
41.40 1.052t 0.114 0.063
0.445 0.244 0.074 0.045
5.94 0.742 0.189 0.127
Control
(6) (6) (6) (6)
(6) (6) (6) (6)
(8) (7) (8) (7)
(8) (7) (8) (7)
1.871 3.653 0.416 0.308
& & f +=
(8) (8) (7) (7)
(8) (8) (7) (6)
glucose.
0.496 0.335** 0.087 0.084
f 11.07 f 0.972* f 0.140 f 0.120
Isoproterenol 204.96 10.106 1.555 1.484
30 min
* P< 0.05, ** P< 0.01 control vs isoproterenol. t I’< 0.01 and tt P< 0.001 with glucose vs without glyceride.
0.143 0.168 0.094 0.049
3.55 1.311 0.326 0.218
(6) (6) (6) (6)
myristirate
Isoproterenol
containing
10 min
a solution
Control
with
205.44 10.496 1.750 1.643
perfused
Values represent mean fatty acid contents (pmol/g dry weight) f S.E. The figures in parentheses indicate the number of determinations. Abbreviations: PL, phospholipid; TG, triglyceride; FFA, free fatty acid;
Without glucose
With glucose
TABLE
PLATE I. Low power autoradiograph from a semi-thin demonstrating the atrioventricular node (AVN) adjacent and intervenwicularseptum (VS). x 2W.
(1 w thidr) section of m adult rat heart to the tricuspid valve (TV), atrium (A)
PLATE 2. High power light microscopic autoradiograph of a I p thick section of the atrioventricular node 2 h after intravenous injection of 2 mCi [aH]-leucine. Control and experimental tissues are qualitatively similar in appearance and in both the grains me evenly distributed over the entire cross section ofthe node. x 950.
2.356 & 0.257i-f
2.285 f 0.377”
3.614 f 0.250
c&:0
Cl%:1
fraction
0.161 & 0.015
0.123 & 0.032
0.302 * 0.075
in triglyceride
* P < 0.05, * * P < 0.01 with glucose vs without glucose. t P < 0.05, tt P < 0.01, ttt P < 0.001 control vs isoproterenol.
3.653 & 0.335Tf
1.661 & 0.204**
Without glucose
With glucose
2.895 f 0.244
With glucose
C14:0
4. Myocardial fatty acid compositions (qol/g dry weight)
Xsoproterenol
Control
TABLE C1e:1
1.093 1.351 & 0.119 & 0.22 ltft
1.098 1.452 & 0.080 f 0.342”
1.202 2.493 f 0.240 & 0.240
c1a:o
1.230 f O.O65$tt
1.237 rt: 0.227”
2.137 & 0.219
C1s:a
c&o:4
0.263 & 0.065
0.294 & 0.038
Total
myristirate
10.106 & 0.9727
8.152 jI 1.052””
12.905 j, 0.742
containing
0.262 & 0.038
of the heart perfused with a solution
936
F. TAKENAKA
AND
S. TAKE0
incorporated into TG fraction was significantly less than in the presence of glucose (P < 0.001). When isoproterenol (4 x IO-7 M) was added to the perfusate, marked bradycardia in four cases and ventricular arrest in three cases out of seven occurred within 10 min. The myocardial content of TG fatty acid at 10 min of the perfusion remained unchanged or showed a Iittle higher value than that of the control. Incorporation of myristiric acid was only slightly enhanced. There was no change in fatty acid content in other lipid fractions within 30 min of perfusion.
180 I
60 t II 0
1 5
I IO
I 15 Time
I
30
Cmin)
FIGURE 3. Cardiac performances of the perfused heart with isoproterenol (4 x 10-r MA) and various doses of glucose in the presence of exogenous my&irate (0.4 mnr>. Upper column represents changes in coronary flow, middle one heart rate and lower one contractile force. The initial values of coronary flow (ml/mm) and heart rate (beats/mm) were 5.7 rt: 0.2,282 4 10 (control with 5.5 rnM of glucose, -O-), 5.2 -+ 0.3, 281 ;t 8 (isoproterenol with 5.5 rnn of glucose, ---)> 5.6 Jt 0.4, 296 i 15 (isoproterenol with 1.7 rn~ of glucose, --I-) and 5.6 + 0.2, 294 + 11 (isoproterenol with 0.17 mM of glucose, --A--), respectively (a = 6-8). * P c 0.05, ** P < 0.01 compared with isoproterenol-0.17 rnn of glucose.
MYOCARDIAL
LIPID
METABOLISM
937
E$ects of glucose on cardiac performances in the heart perfused with solution containing myristirate
Cardiac performances of the heart were studied during perfusion with soIution containing myristirate (0.4 mu), isoproterenol(4 x IO-7 M) and glucosein dosesof 0.17, 1.7 and 5.5 mM. Results are shown in Figure 3. Coronary flow and heart rate decreasedduring 30 min of the perfusion. The rate of decrement in coronary flow and heart rate was proportional to the amount of glucosereduced. Thus, there was a close relation between glucose content in the perfusate and cardiac contractile force. Contractile force of the heart perfused with solution containing 5.5 rnM of glucose was augmented throughout the perfusion. Contractile force of the heart perfused with solution containing 1.7 mM of glucose reached 127 & 24%, while that of the heart with 0.17 mM of glucose was sig~~cantly reduced {P < 0.0 l} to reach 72 f 16% at 30 min of the perfusion.
4. Discussion
The present experiments using isolated perfused hearts have demonstrated that the myocardial content of TG fatty acid gradually declined along with the time of petiusion with fatty acid-free medium. Olson and Hoeshen [IZ] have reported that TG fatty acid content did not change during 30-tin perfusion with nutrient-free medium in the isolated perfused rat heart. Dhalla et al. [3] have shown a marked decrease(74%) in tissue triglyceride content of the heart perfused for 2 h without glucose and a slight decrease(22%) with glucose.The discrepancy between our results and theirs may be explained by the difference in perfusing systemsemployed. Lipids in the myocardium are continuously washed out into the per&sate in the present perfusion method, while the diffused lipids may be recirculated and retaken up into myocardium in the closed system employed by Olson et al. and Dhalla et al. Lipids present in the perfusate increased significantly (P < 0.05) in the heart perfused for 30 min with isoproterenol both in the absenceand presenceof glucose. There is the possibility that the fatty acid releasefrom the myocardial TG fraction may be enhanced by isoproterenol. However, the total volume of the perfusate during the 30-min perfusion with isoproterenol was significantIy larger than that of the control. Therefore, it would be premature to conclude that the increase in amounts of lipids in the perfusate when isoproterenol was applied to the heart, is due to an augmentation of the fatty acid releasefrom tissue triglyceride, since we can not rule out another possibility that an increased lipid content in the perfusate might be attributable merely to leakage of lipids from the perfused heart. In the control perfusion of 30 min, the myocardial content of TG in the absence of glucosewas sign~~tly lessthan that in the presenceof glucose, suggestingthat
938
F. TAKENAKA
AND S. TAKEi
TG fatty acid may be utilized much more in the absence of glucose than in its presence. A significant decrease in the TG fatty acid content was seen during perfusion with solution containing isoproterenol. The results are consistent with those obtained by previous authors in that epinephrine signi~~an~y decreased endogeno~ TG content in working rat heart during 30-min perfusion in the presence of glucose [2] and also in the non-working rat heart during 45-min perfusion without glucose [5, 61. In the absence of glucose, however, the TG content at 30-min perfusion with isoproterenol was not significantly different from the control. Contractile force at 30-min perfusion was markedly depressed, whilst it was enhanced in the presence of glucose. These findings suggest that the decrease in endogenous TG content may correlate with changes in contractility. However> Grass et al. [2] have reported that the rate of tissue TG mobilization is accelerated with increased ventricular pressure development [.!I, but not with an increase in contractile frequency [2]. Thus, the decrease in endogenous TG content by isoproterenol observed in the present experiment can not be explained solely by changes in cardiac performances. iteratively there is evidence to indicate the existence of a number of myocardial TG pools [I] and independent TG pools in supernatant mitochondrial and microsomal fractions [15]. Olson and Hoeshen have shown that non-working hearts perfused for 90 min without substrates and hormones until exhaustion, retained about 32% TG content [I,?]. In the control perfusion in our experiments, TG content at 30-min perfusion was S&y0 of the initial value in the presence of glucose and 29% in the absence of glucose, Therefore, isoproterenol did not decrease tissue TG content further in the absence of glucose, sinceapproximately 30% of the tissue TG at 30 min of the control perfusion may be “‘mobilization resistant TG”. When the heart was perfused with solution containing myristirate, there was an i~ibition of the decrease in TG fatty acid content observed during the perfusion with fatty acid-free medium, A significant difference was seen between TG content in the myocardium perfused with solution containing glucose and without glucose after the control 30-min perfusion. In addition, myristiric acid uptake into the TG fraction was significantly lower in the absence of glucose than that in its presence. These facts indicate that exogenous myristirate can be taken up by the perfused heart and the incorporation of the fatty acid into myocardial TG fraction was lower in the absence of glucose; the latter may be because exogenous fatty acid is oxidized rather than incorporated into the TG fraction. When isoproterenol was applied to the heart perfused with exogenous myristirate in the presence ofglucose, TG fatty acid content significantly decreased, compared with the control. The incorporation of myristiric acid into the TG fraction increased significantly. The results suggest that isoproterenol may enhance tissue TG turnover in the myocardium. The facts are not inconsistent with those described by previous authors that incorporation of exogenous p~tate-~-[Go*] into myom~ocaxxiial
MYOCARDIALLIPIDMETABOLISM
939
lipid turnover cardial lipids was increased by epinephrine [IO] and endogenous enhanced by epinephrine [9]. In the glucose-free medium, however, a marked bradycardia or ventricular arrest occurred within 10 min of perfusion and endogenous TG content was not different from the control or the initial value. Moreover, in the heart perfused without exogenous fatty acid, isoproterenol enhanced continuous cardiac contraction in the presence of glucose, but had less effect in the absence of glucose. These findings indicate that glucose may play an important role in the mechanism responsible for energy utilization in the cardiac performance of the heart perfused with solution containing isoproterenol. The aggravation of isoproterenol on cardiac performance in the absence of glucose might be due to lack of energy supply satisfying the commensurate increase in energy demand of the heart by isoproterenol. In fact, as shown in the present experiments, the increased cardiac performances due to isoproterenol were closely related to the amount of glucose added to the perfusing solution containing fatty acid.
The authors wish to express to gratitude cology, Kumamoto University Medical commenting on it.
1.
2.
3.
4.
5. 6.
7.
to Dr K. Nishi, Department of PharmaSchool, for reading the manuscript and
C&W, M. F. III, MCCASKILL, E. S., SI~P, J. C. & MURPHY, V. K. Metabolism endogenous lipids in cardiac muscle: effect of pressure development. American Journal Physiology220,428-435 CRASS, M. F. III,
of of
(1971). SHIPP, J. C. & PIEPER,
G. M. Effects of catecholamines on myocardial endogenous substrates and contractility. American Journal of Physiolog 228,618627(1975). DHALLA,
N. S., MATOUSHEK, R. F., SUN, C. N. & OLSON, R. E. Metabolic, ultrastructural and mechanical changes in the isolated rat heart perfused with aerobic medium in the absence or presence of glucose. Canadian Journal of Physiology and Pharmacolosy51,590-603 (1973). FOLCH, J., LEES, M. & STANLEY, G. H. S. A simple method for the isolation and purification of total lipids from animal tissues. Journal of 3iologi~a~ Country 226, 497-509 (1957). GARTNER, S. L. & VAHOUNY, G. V. Elects of epinephrine and cyclic 3’, Y-AMP in the peritsed rat hearts. American Journal ofPhysiology222, 1121-l 124 (1972). GARTNER, S. L. & VAWOUNY, G. V. Endogenous triglyceride and glycogen in the perfused rat hearts. Proceedings of the Society for E.+erimental Biology and Medicine 143,556-560 (1973). C-OLD,
M., ATLAS, 3% J., SCOTT, J. C. & SPITZEN, J. J. Effect of norepinephrine on myocardial free fatty acid uptake and oxidation. Proceedings of the Societyfor Ex~~~a~ Biology
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(1965).
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F.TAKENAKAANDS.TAKEO Gous~os, A. & FELTS, J. M. Effects ofepinephr~e, norepinephrine, glucose and insulin on extraction and oxidation offree fatty acid by myocardium. Ci~~~ti~n 28, 729 (1963). KREXSBERG, R. A. Effect of epinephrine on myocardial triglyceride and free fatty acid utilization. AmericanJournal of PhysioloQ 210,385-389 (1966). MATHUR, P. P. & MORLER, C. M. Subcellular distribution and incorporation of palmitate-U-[Clan into myocardial lipids: Role of endogenous catecholamines. Journal of Molecular a~~e~~alaT ~ard~~o~ 7, 17-26 (1975). OLSON, R. E. Utilization of exogenous and endogenous lipid by the isolated perfused rat heart. In Coronary Circulation and Energetics of the Myocardium. G. Marchetti & B. Taccardi, Eds. pp. 162-185. Basel: S. Kargel AG. (1967). OLSON, R. E. & HOESHEN, R. J. Utilization of endogenous lipid by the isolated perfused heart. B~~c~i~~3ou~~ 103,796-801 (1967). SHIPP, J. C. Interrelation between carbohydrate and fatty acid metabolism of isolated perfused rat hearts. Metabolic 13,852-866 (1964). SHIPP, J. C., OPIE, I,+. H. & CHALLONER, D. Fatty acid and glucose metabolism in the perfused heart.Nature 189, 1018-1019 (1961). STEIN, 0. & STEIN, Y. Lipid synthesis, intracellular transport and storage III. Electron microscopic radioautographic study of the rat heart perfused with tritiated oleic acid. JmznuE of Cell Bi&gy 36,63-77 ( 1968). W~LL~~SON, J. R. Metabolic effects of epinephrine in the isolated, perfused rat heart. Journal of Biological Chemistry 239,272 l-2729 (1964).
