Effect of propranolol on the metabolism and function of the rat heart

Jownul

of Moleouksr and CelLalar

Effect of Propranolol

Cardidopy

(1971)

2, 71-89

on the Metabolism Rat Heart

and Function

of the

ANDRIES J. BRINK, ANDRli J. BESTER AND AMANDA LOCHNER Molecular and Cellular Cardiology Unit of the M.R.C. and University of Stellenbosch, Departnwnt of Medicine, Univemity of SteUenbosch Medical School, Karl Rremer Hoepital, BeUrn&, Republic of South Africa (Received 1 April

1970, and accepted in retied

form

15

February

1971)

A. J. BRINK, A. J. BESTER AND A. LOCENER. Effect of Proprenolol on the Metabolism and Function of the Rat Heart. Journal of Mokxular and Cellluar Cardiology (1971) 2, 71-89. The effect of propmnolol, 8 betaadrenergic blocking agent, was studied on the metabolism 8nd function of the isolated, perfused rat heart. Proprmnolol (20 q) depressed the oxygen consumption, coronary flow rate, p8hnitSte uptake and oxidation 8s well 8s the high energy phosphate oontents of the perfused heart. Propm110101 signi&antty reduced heart rete while the time to pesk height of developed tension period wes increased. The effect of propranolol was also studied on the metabolism of he8rt muscle slices end the eemosomal oxidetive phosphory&ion process. With pyruv8te or glucose as substrete, propranolol stimulmted the IWipir8tiOn and metabolism of heart slices, while it depressed oxidative phosphoryl&ion. In order to eliminste the effect of the reduction in heart reta on substmte metabolism in the perfused heert, additional studies were performed in which the effect of proprmnolol ~8s studied in hearts driven at a controlled hesrt r&e, by 8 pacemaker. The results showed th8t the chronotropic effect of proprmnolol h8d a significant effect on the reduction in substrate metabolism.

1. Introduction The depressant effects of propranolol, a j?-adrenergic blocking agent on heart rate [S, 11, 16, 21, 26, 39, 42, 44, 461, cardiac output [9, 11, 16, 17, 26, 281, myooardial oxygen consumption [9,11,16,17,21,27,28,37,38,43,46] and coronary flow rate [21,27,44,46] have been well established.It hasalso been shown that it reducesthe rate of left ventricular isometric tension development in humans [21,27,4s] as well as tension development in papillary musclesin vitro [30, 31, 331. Other studies have shown that besidesits t%blocking potency, propranolol impaired lipid-facilitated transport of C&s+,while it had no effect on myofibrillar ATPase activity [29, 311. In this study on the isolated perfused rat heart, the effect of propranolol was studied on myocardial cellular metabolism and brought into relation with the mechanical events. The findings suggest that the negative chronotropic effect of propranolol plsys an important role in determining its effect on myocardial metabolism.

2. Material

and Methods

Female albino rats of the Wistar strain weighing about 260 g were used. All animals were fed ad lib&urn until decapitation. 6 71

72

A. J. BRINK,

A. J. BESTER Metdolic

AND

A. LOCIINRR

studies

The isolated rat heart was perfused with 15 ml of a modified Krebs-Henseleit bicarbonate buffer (pH 7.4) for 30 min in a modifled Langendorff perfusion system as described previously [32]. Substrates used were [U-rK!]glucose (10 mM), [l-r%]palmitate (0.7 mM) and [lJ4C]- and [3J’%]pyruvate (6 mM) (obtained from the Radiochemical Centre, Amersham, England). Measurements and calculations of glucose uptake, lactate production, pyruvate uptake or production, fitratable and 1%fatty acid uptake, incorporation of 1% into tissue lipids and residual cardiac glycogen were carried out as before [32]. For determination of myocardial ATP, ADP, AMP, CrP and Pi contents, the hearts were immediately after perfusion clamped with Wollenberger tongs, precooled in liquid Nz, immersed in liquid nitrogen and ground into a fine powder. The ATP contents were determined enzymically by a standard method as modified by Opie eE al. [32]. The ADP and AMP contents were determined enzymically as described by Bergmeyer [I]. The phosphocreatine (CrP) and inorganic phosphate (Pi) contents were determined according to the method of Furchgott and de Gubareff [13]. Measurements of myocardial oxygen uptake and coronary flow rate were made over a period of 30 min as described before [a]. Since propranolol caused a significant fall in heart rate, substrate metabolism and oxygen consumption were also studied in a series of rat hearts treated with propranolol, driven at a rate of 190 to 224 beats/mm by a Medtronic Pulse Generator (Model 5837) to approach the average rate of the control isolated beating heart. A square wave impulse of 2 msec duration and output of 3 mA was used. In both series of experiments nn-propranolol (1 -isopropylamino-3-( 1naphthyloxy) propan-2-01 hydrochloride) was added to the perfusate in the concentration of 20 or 40 pM. Mechanical studies Measurements of tension development were made by means of the system with the isolated beating rat heart using a differential myographic force transducer and Beckman 6 channel polygraph (Type R Dynograph, Type 504A), as described by Brink and Lochner [5, 61. The hearts were perfused with 30 ml of per&sate at a constant pressure of 60 mmHg over a period of 45 min. Glucose (10 mM) was used as substrate. All measurements of tension development were made with a constant preload of a 10 g weight. Tension development was measured as peak height of developed tension (PH) in millimetres and tension time index (TTI) in milligram-seconds, obtained by planimetric integration of the area under the systolic curve. The maximum rate of rise of developed tension (D.T./d&,& in milligram-seconds and time to peak height of developed tension (t-PH) in psec, were also measured. The degree of stress relaxation (SR) was measured on the recording as an increase in

EFFECT

OF PROPRANOLOL

ON

PERFUSED

RAT

HEART

73

resting length [5, 61. Observations were made at 6-min intervals and mean values calculated for each 15-min period. To determine the stability of the mechanical performance of the isolated heart, a series of control rat hearts without addition of propranolol was studied in this fashion for a 45-min period. A second series was perfused without propranolol for the first 15 min, serving as a double control, after which propranolol(20 PM) was added and the effect on muscle mechanics was observed over the next 30 min. In order to eliminate the influence of reduced heart rate on the parameters of tension development, the hearts were paced at a rate of 190 to 224 beats/min, using a Medtronic Pulse Generator. In these studies propranolol was added (20 PM) to the perfusate of each heart at zero time and the mechanical performance recorded for a period of 15 min, whereafter it was paced for a further period of 30 min. Evaluation of the technical recording system The modified Langendorff perfusion system with myographic force transducer is sufficiently sensitive to register small changes in inotropism of the isolated, perfused rat heart. This was shown in studies on the effect of emetme [a], alcohol [Z4] and oligomycin and 2,4dinitrophenol[22] on the mechanical activity of the perfused rat heart. This system measures tension development in the myocardium between the aorta and apex. The position of both aorta and apex is fixed during perfusion; the aorta is tied onto the aortic cannula and the apex is fixed to a small hook [5]. Although a preload of 10 g is attached to the apex, the system measures tension development only between these fixed points. The calibration curve of the tension measuring system is linear. &4dies of oxdative phosphorylution Preparation of sarcosomes and measurement of phosphorylation/oxidation ratios (P : 0) were done as described previously [23, 241. Substrates used were malate, pyruvate and succinate (30 ,umole/flask). Phosphorylation associated with transfer of electrons in the region NADHcytochrome G was studied using pyruvate as electron donor and KsFe(CN)e as electron acceptor [25, 361. The region cytochrome c-oxygen was studied using a catalytic amount of tetramethyl-p-phenylenediamine (TMPD) as mobile electron carrier between external aecorbate and members of the respiratory chain 1191. SWies of glycolysis The metabolism of [U-Wlglucose (10 mM) and [l-14C]pyruvate (5 mM) by rat heart slices was studied according to the method of Lochner et al. [25]. Experiments of oxidative phosphorylation and of glycolysis were carried out in a Gibson Differential Respirometer. Two concentrations of propranolol were studied, viz. 20 and 40 /&M.

74

A. J. BRINK,

A. J. BESTER

All results are expressed as means f are derived from Student’s t-text [40].

S.E.M.

AND

A. LOCENEB

(number of observations).

P values

3. Results Effect of propranolol

012the mechanical performance of the perfused rat heart

without pa&g Investigation of the mechanical activity of the control hearts over a period of 46 mm showed that coronary flow and heart rate decreased, whereas tension development and contractility tended to increase during perfusion, achieving a fairly steady state after the first 15 mm. It was therefore decided to compare the mechanical activity of the propranolol perfused hearts with the last 30 min of perfusion of the control hearts (Table 1). Propranolol resulted in a significant depression in heart rate throughout the perfusion period (P < 0.001). This depression in heart rate was significant within 5 mm after addition of propranolol (control 225 f 8; propranolol 199 f 8). Although propranolol resulted in a transient reduction in stress relaxation within 5 mm the mean values obtained for the two 15min periods did not differ significantly from those of the control hearts. Propranolol had no effect on peak height of developed tension and the maximum rate of rise of developed tension, while tension time index was significantly elevated during the last 15 min of perfusion. The period taken to reach peak height of developed tension was significantly prolonged throughout the perfusion period.

With pacing To eliminate the effect of the reduction in heart rate produced by propranolol, the hearts were perfused with propranolol over a period of 45 min at an increased rate of 190 to 224 beats/min, 15 min after commencement of perfusion. Parameters of tension development during perfusion with addition of propranolol and with pacing were compared with the last 30 mm of perfusion with propranolol only (Table 1). Stress relaxation was signif?cantly enhanced throughout perfusion when the heart was paced. Peak height of developed tension was significantly improved by the increased heart rate during the first 16 mm, but depressed during the last 16 min. No significant change was obtained in tension time index and maximum rate of rise of developed tension during the first 16 min, but both became significantly less during the last 16 min. Time to peak height of developed tension, on the other hand, was significantly (P < 0.001) reduced by increasing heart rate throughout the perfusion period.

Proprltnolol f Pacing (7) pa

Pl

Tim 30-45 min Control (IO) Propranolol (10)

Propranolol + Pkine; (7) Pa

Pl

-Ti?ne 15-30 mh Control (10) PropraSnolol (IO)

TABLE


104.37f8.21 123.82f8.29 NS 75.16&8.41

-CO.06

104.69f7.17 102.0lf7.03 NS 127.45k8.64

Peak height (=)

activity


1474&70 1749fll8
NS

1546f81 14335103 NS 1574&119

Tension time index (mg-=)

(20 PM) on the mechanical

<0.025

309,285fl3,286 279,769&14,772 NS 213,601&21,223

<0.02

333,006&12,696 266,640&14,904
Tension time permin (mg-aec/min)

of the perfused

Meaeurements

were

made

every

6 min

and mean

values

oelculetad

for each

15-min

Numbers in perentheees indicete number of hearts. P, indic&as signScanoe of difference between control andpropmnolol hearts. P, indicates signikance of diEerence between propranolol and propranolol + paoing.

<0.02




66.78f8.73 57.05&7.16 NS 85.30&7.60

67.66f7.94 44.48&5.19 NS 71.97h2.6

222f6 172fS
212&-4 169f7
relaxetion @m

Hsart rate/min

stress

1. Effect of propranolol

period.


530&46 572f39 NS 301&35


89&l 97f2 <0.006 8lfl


91&-l QQf2 <0.006 84fl

603&38 461&33 NS 512f36 NS

t-PH (P-4

D.T./&.x (m&W

rat heart

76

A. J. BRINK,

A.

J. BESTER

AND

A. LOCHNER

Effed of propranolol on metabolicpatterns of the perfused rat heart Without pacing Propranolol had no effect on the metabolism of glucoseby the perfused rat heart, with exception of a reduction in 14COsproduction and an increase in pyruvate production (Table 2). Titratable as well as [14C]palmitate uptake, 14COsproduction and percentage conversion of palmitate uptake to 1WOs were decreasedby propranolol (Table 3) while the percentage conversion of palmitate uptake to lipids was increased by propranolol (P < 0.001). TABLE

2. Effect of propranolol (20 PM) on the metabolism by the perfused rat heart

of [U-lW]glucose

(10 mm)

[U-14C]-

Glucose uptake

production

production

produotion

glucose incorporation into glycogen

21.72 &I.69 18.63 jc1.74 NS 27.83 kO.94
3.23 kO.45 1.78 IfIO.26 <0.02 4.06 f0.63 <0.005

9.98 h1.79 6.79 50.71 NS 8.95 hO.86 NS

0.60 f0.05 0.76 f0.04 to.02 0.67 50.08 NS

3.65 50.41 3.51 f0.41 NS 5.38 50.20
14co2

---Control

( 18)

Propranolol

(23)

Pl

Propranolol + pacemaker (6) pa

Numbers in perentheses Ah results expressed as P, indicates signi6cance P, indicates significance

Lactate

Pyruvate

indioete number of hearts. (Imoles gluoose equivalents/g wet weight/30 min. of difference between control and propranolol. of difference between propranolol and propranolol

Residual glycogen content 12.65 f0.94 11.95 f0.82 NS -

f pacemaker.

Propranolol had no effect on the metabolism of [l-W]- and [3-W]pyruvate. Addition of propranolol when a combination of [U-l%]glucose and palmitate were used as substrates (Table 4), resulted in a significant depressionof glucose uptake, lactate and 1WOs production. Similarly WOs production was depressed with [l-W]palmitate and glucoseas substrates. Addition of 20 PM propranolol reduced oxygen uptake and coronary flow rate of the perfused heart throughout the perfusion period, becoming sign&ant after 10 min (Figure 1). With pacing The effect of the reduced heart rate on the metabolic patterns was studied in an additional seriesof experiments in which the hearts were driven at a rate of 190 to 224 beats/min by a pacemaker.

EFFECT

OF PROPRANOLOL

ON

PERFUSED

RAT

77

HEART

Although lactate and pyruvate production were not significantly altered, glucose uptake, 14COs production and incorporation of [Wlglucose into glycogen were increased significantly by increasing the heart rate (Table 2). The pattern of palmitate metabolism as produced by propranolol was altered by increasing the heart rate (Table 3). The significant reduction in palmitate uptake and 14CO~ production produced by propranolol, was reversed and no significant changes were observed in these parameters. The percentage conversion of [14C]palmitate uptake to tissue lipids was increased in the presence of propranolol, while increasing the heart rate reduced lipid production from [W]palmitate (P < 0.001). TABLE

3. Effect of proprauolol (20 PM) on the metabolism (0.7 maa) by the perfused rat heart [14C]-

P&I&&3 uptake (titration) Control

(7)

Propranolol Pl

Propranolol pacemaker PZ

3.16 fO.16 (15) 2.66 10.21 O.l>P>O.O5 + 3.41 (10) kO.41 NS

Numbers in parentheses All results expressed as P, indicates signnicanoe P, indicates significance

WIpabnitate uptake 4.37

kO.14 3.38 ho.18
14co2 production 1.88 hO.20 0.83 kO.09
palm&ate incorporation into tissne lipids 1.79 fO.12 1.89 AO.11 NS 1.18 kO.04 (0.001

of [ I-Wlpalmitate o/o Cony. Con- version of version of palmitate palmitate uptake to uptake to wo2

tissue lipids

42.49 h3.74 23.73 &1.69
41.73 f3.97 57.90 *4.26 <0.02 31.36 k1.37
indicate number of hearts. pmoles/g weight/30 min. of diierence between control and propranolol. of difference between propranolol and propranolol

+ pacemaker.

Increasing heart rate results in a reversal of the pattern of metabolism of the combined glucose and palm&ate substrates (Table 4). Elevating the heart rate now produced an increase in the uptake of glucose and production of lactate. Similarly, palmitate uptake and 14COs production were significantly increased. Increasing the heart rate had no effect on the depression in myocardial oxygen uptake produced by propranolol and the oxygen uptake remained depressed during the perfusion period. Coronary flow rate was significantly depressed by the increase in heart rate (Figure 1). Effect of propranold High-energy substrate.

phosphates

on the high-energy p?tmphute content of the perfused heart were determined

after perfusion

with glucose (10 mM)

as

Palm&ate uptake (titration)

wet

palmitate uptake

w-3

Parameters P, indicates P, indicates

of pahnitate significance signiflcanoe

metabolism of difference of difference

expressed between between

NS

W

(12)

<0.005 0.71 f0.08

as Foles/g wet weight/30 control and propranolol. propranolol and propranolol

+ pacemaker.

min.

weight/30

14.06 *1.04 (11) 16.68 &l.lS (13) NS 17.57 h-l.47

Residual glycogen content

min.

(12)

<0.05 1.99 kO.28 (10) <0.06

1.13 ho.23

(1%

1.82 10.22

w-apalmitate conversion to ‘4cO2

by the perfused

Numbers in parentheses indicate number of hearts. Parameters of glucose metabolism expressed as pmoies glucose equivalents/g

(‘3) ~0.001

(‘3)


(25)


17.40 &I.63 (24)
6.62 f0.79

(22)

(22)

4.06 f0.60 (11) 4.37 kO.99 (13) NS 8.10 f2.19 v-v NS

glucose convemion to glycogen

and [ 1-14ClpeJmitate

3.99 kO.58 (11) 4.36 fO.51 (11) NS 6.00 *0.33 (10) <0.02

1.61 10.27 w 0.61 f0.08

Paglucose conversion to woa

l?W

of [U-W]glucose

4.76 f0.19 (23) 4.07 f0.30 (24) O.l>P>O.O5 5.79 ho.23 63) (10)
13.66 k1.80

Lactate production

(20 PM’) on the metabolism

26.27 k3.36

Glucose uptake

4. Effect of propranolol

Propranolol + pacemaker

Propranolol

-Control

TABLE

NS 2.79 &O. 15 (10) NS

(1%

2.45 &O. 16

(12)

2.28 ho.16

pahnitate incorporation into tissue lipids

Pa-

rat heart

EFFECT

TABLE

OF PROPRANOLOL

6. Effect of propranolol rat heart. Substrate

---Control

(12)

20 j.a propranolol

(6)

P 40 px propritnolol

(6)

P

ON

PERFUSED

RAT

on the high energy phosphate : glucose (10 mar)

79

HEART

contents

of the perfused

*ATP

ADP

AMP

Pi

CrP

6.82 kO.67 0.42 f0.25 NS 4.97 kO.48 <0.026

1.89 f0.20 1.87 fO.16 NS 1.05 &O.lS
0.32 10.04 0.24 kO.03 NS 0.19 50.02 (0.02

0.95 50.60 6.22 fO.67 NS 8.06 ho.78 NS

5.52 ho.27 5.19 f0.29 NS 4.04 f0.14 to.001

* Result8 expressed IM pmoles/g wet weight. Numbers in parentheses indicata number of hearts. P values indioate signifioanoe of difference from oontrol. TABLE

Substrate ---PyTUvate

Melate

fhlCCiIl8te

6. Effect of propranolol

on sarcosomal

No. of experiments

Phosphorous uptake ( pmoles/mg prot/40 min)

prop’zoloi 0

6

20

9

P 40

9

P 0

6

20

9

P 40

9

P 0

6

20

9

P 40

9

P P

velues indicate signifkance

26.62 AO.49 26.04 zfo.15 <0.02 23.27 f0.24 (0.001 15.40 kO.25 14.60 &to. 12 (0.02 13.06 *o. 17
oxidative

phosphoryletion

Oxygen uptake (~atoms/mg prot/40 min) 10.94 kO.11 10.74 f0.17 N.S. 10.72 f0.29 N.S. 7.00 &0.26 7.09 hO.20 N.S. 7.18 10.21 N.S. 7.91 ho.48 8.06 &0.39 N.S. 8.34 &0.32 N.S.

difference from

oontrol.

P : 0 ratio 2.41 AO.03 2.33 f0.04 N.S. 2.17 +0.03 P>O.O6 1.02 f0.03
80

A. J. BRINK,

A. J. BESTER

AND

A. LOCHNER

Propranolol at a 20 ,uM level did not significantly affect ATP, ADP, AMP, CrP and Pi, but decreased the ATP, ADP, AMP and CrP contents significantly at, a concentration of 40 pM (Table 5). The inorganic phosphate content on the ot(her hand was elevated by 40 pM propranolol. Effect of propranolol on oxidative phosphorylation in rat heart sarcosomes In order to determine the causeof the changesin high-energy phosphate contents of the perfused rat heart, the effect of propranolol (20 and 40 pM) was studied on the sarcosomal oxidative phosphorylation process (Table 6). Pyruvate, malate and succinate were used as substrates. At 20 pM propranolol causeda significant reduction in P : 0 ratio with malate only assubstrate, but at 40 pM it was decreasedwith all these substrates. No change was observed in oxygen uptake, while inorganic phosphorous uptake was markedly decreased with increasing concentration of propranolol . Studies on localization of defect of oxidative phosphorylation In order to demonstrate the site of reduction of uncoupling of oxidative phosphorylation due to propranolol in the respiratory chain, phosphorylation between TABLE

7. (a)

Effect of propranolol oxidation of pyruvate sarcosomes

on with

phosphoryletion ferricyanide

JGJWCN)a ( pmole/mg prot/40 min)

Phosphorous uptake ( eolelmg prot/40 min)

P : 26 ratio

17.56 10.31 6.84 jO.61
11.29 50.30 3.61 10.34 to.001

1.29 10.001 1.05 f0.03
reduced No. of experiments Control

10

40 PM propranolol

12

P values TABLE

7.

(b)

Effect oxidation

of

indicate

significance

propranolol of ascorbate

by

No. of experiments Control

9

40 PM propranolol

9

associated with anaerobic acceptor in rat heart

as electron

on rat

of difference

from

control.

phosphorylation associated heart sarcosomes

with

aerobic

Phosphorous uptake (wol+g prot/20 min)

Oxygen uptake (+-f&g prot/20 min)

P : 0 ratio

2.90 &to.01 2.86 fO.04

3.59 jo.01 3.61 kO.03

0.81 10.01 0.80 *0.01

EFFECT

OF PROPRANOLOL

ON PERFUSED

81

RAT HEART

NADH ana cytochrome c wa5 StUdid, using pyruvate as electron donor and potassium ferricyanide as acceptor. The results are demonstrated in Table 7(a). Propranolol reduces potassium ferricyanide reduction and phosphorous uptake, with a significant reduction in the ratio between the uptake of phosphorous and electrons (P/2t! ratio). These studies suggest a reduction or uncoupling of oxidative phosphorylation in this region of the respiratory chain, due to the effect of 40 PM propranolol. The effect of propranolol on the phosphorylation associatedwith the transfer of electrons between cytochrome c and oxygen was further studied. The results are summarized in Table 7(a). Ascorbate was used as electron donor and oxygen as acceptor. From the data [Table 7(b)] 1‘t is clear that 40 PM propranolol had no effect on the P : 0 ratio. Thesefindings therefore suggestthat propranolol had no effect on the oxidative phosphorylation in the cytochrome oxidase region of the of the electron transport chain. Effect of propranolol on glywlysis in rat heart slices Met&ohm

of [ U-‘*C]glucose

The effect of propranolol, at two different levels (20 and 40 PM), on glycolysis was studied by deWmining the metabolic fates of [U-W]glucose (10 mu) in rat heart slices (Table 8). The oxygen uptake, glucose uptake and 14C02production were TABLE

8. Effect of propranolol heart slices

Control 20

/1M

propranolol

WOs production

Pyruvate production

La&ate production

Lactate pyruvate ratio

854. IO &78.02 (9) 1291.62 *48.74

8.74 50.91 (9) 13.76 10.74

1.65 50.14 (9) 2.87 f0.22

0.58 hO.005 (9) 0.68 AO.05

5.21 ho.476 (9) 5.58 10.40

9.52 51.35 (9) 8.85 hO.99

(12)

(12)

(12)

(12)

(12) (0.001 Oxygen Clucoee

uptake uptake,

equivalents/g Numbers P values

(10 mM) by rat

Glucose uptake

(0.001 1336.64 k-53.48

P

of [U-l%]glucose

Oxygen uptake

(12)

P 40 FM propranolol

on the metabolism

(12)

(0.001 17.93 61.14 (12) (0.001

expressed as g/g wet production of WO,,

<0.0010.1>P>0.05 3.33 0.77 &O.lS *0.005

(12)

(1%


weight/hr. lactate

and


wet weight&r. in parentheses indicate number indicate significance of difference

of experiments. from control.

expressed

NS 7.62 hO.43

NS 10.63 51.12

(12)

(1%

<0.005

as pmoles

NS glucose

82

A. J. BRINK,

A. J. BESTER

AND

A. LOCHNER

significantly enhanced at both levels of propranolol used. Pyruvate and lactate production did not increase significantly with 20 pM propranolol, but the production of these two metsbolites increased significantly with 40 PM propranolol. &letubolLwn of [1-‘4C]pyruvate The effects of propranolol (20 and 40 PM) on the metabolism of [1-W]pyruvate are summarized in Table 9. Oxygen uptake and 14COsproduction were significantly TABLE

9. Effect heart

of propranolol slices

on the

Oxygen uptake

metabolism

of [I-14Clpyruvate

(6 m&f)

FyrLlvate uptake

WO2 production

Lactate production

33.05 f3.58 (9) 40.66 51.38

24.81 f1.78 (9) 32.28 f3.00

10.50 10.78 (9) 12.64 hO.86

by

rat

---Control

20 PM propranolol

1320.67 klO0.94 (9) 1665.66 flO6.15

w-4) P 40 PM propranolol

(W


NS 43.39 f1.53

(1% P

(12)


<0.026

Oxygen uptake expressed aa d/g wet weightlhr. Pyruvate uptake, WO, production and lactate produotion weight&. P values indicate signScanoe of difference from control.

(1% <0.05 34.39 f3.03

(1% (0.02

expressed

(1.3 NS 14.07 f0.90

(12)
MI pmoles/g

wet

increased at both levels of propranolol used. Propranolol at a concentration of 20 ,UM had no effect on pyruvate uptake and lactate production, but at s,concentration of 40 PM it significantly increased pyruvate uptake and lactate production. The results in Tables 8 and 9 clearly demonstrated that glycolysis was increased by propranolol. 4.

Discussion

The concentration of propranolol usedin thesestudies (20 and 40 pM) is higher than those used by Nayler et al. [31] (6, 5 and 13 @K),while the concentrations used by Parmley and Braunwald [33] on papillary musclesvaried between 0.01 and 100 ,UM. The studies on the isolated, perfused rat heart emphasizethe immediate negative chronotropic action of propranolol, which had a profound effect on the metabolic patterns. In order to eliminate this effect, the hearts were paced at a control rate (190to 224 beats/mm) and the subsequentmetabolism and mechanical activity compared to those of spontaneously beating control hearts. Although it has been shown

EFFECT

OF PROPRANOLOL

ON

PERFUSED

RAT

HEART

83

that field stimulation caused liberation of norepinephrine in isolated heart muscle [2], additional experiments in this laboratory showed that electrical pacing had no effect on the metabolism of the isolated perfused rat heart in the presence of propranolol. It has also been shown by Blinks [2] that propranolol markedly reduces the adrenergic response to pacing and it is possible that propranolol blocked the action of any liberated norepinephrine in the present studies. Furthermore, it has been shown that the cardiac catecholamine stores are of little, if any, importance in regulating the intrinsic contractile state and rhythmicity of heart muscle [43]. It was therefore decided to use spontaneously beating hearts as controls. Effect of propran& 012the mechanical activity of the perfused rat heart The exact mechanism of the negative chronotropic effect of propranolol is uncertain; it may be partially or wholly through its blocking effect on the adenyl cyclase mechanism. However, it may be due to some other effect as suggested by the tinding that the depression of force and velocity of contraction of papillary muscle as well as the spontaneous atria1 rate in vitro were similar for D- and nn-propranolol[33]. Since the /l-blocking effect of n-propranolol is very slight compared to that of DLpropranolol [18, 201 it would appear that mechanisms other than p-blockade are involved. It has been postulated that the ability of propranolol to impede lipidfacilitated transport of ionized calcium across an aqueous lipid-solvent interface is primarily responsible for its effect on myooardial function [29, 311. The negative inotropic action of propranolol, as was shown in clinical [lo, 11,21, 421 and in vit-ro studies on papillary muscle [30, 31, 331 has also been demonstrated in this study (Table 1). The most sensitive indication of negative inotropism in this system was impairment of tension development as shown by prolongation of the time period to develop peak systolic tension (t-PH). Wolfson et al. [46] suggested that the chronotropic action of propranolol is responsible for its effect on the mechanical activity of the heart, since by pacing the heart at constant rate, no change was found in ventricular volume, mean systolic tension, A-V oxygen extraction and mean fibre shortening rate. However, other studies from this laboratory showed that a change in heart rate from 150 to 220 beatsJmin had no significant effect on the various parameters of tension development. By pacing the isolated rat heart at constant rate in the presence of propranolol, the parameters of tension development improved during the flrst 16 min, but a sign&ant depression occurred during the last 15 min of perfusion (Table 1). The significant change in D.T./dt,,, after pacing is also evidence of a negative inotropic action of propranolol, when heart rate is kept constant. These results suggest that propranolol has a negative inotropic effect unassociated with changes in heart rate. The significant reduction in coronary flow rate or increase in stress relaxation may be relevant in the reduction in mechanical activity. The greater degree of stress relaxation, in particular, would explain the initial increase of the functional measurements and subsequent decline if overstretching occurred.

84

A. .I. BRINK,

A. J. BESTER

AND

A. LOCHNER

As in the intact human heart [Zl, 27, 44, 461 propranolol caused a reduction in coronary flow rate of the perfused rat heart. This phenomenon was attributed to increased vascular resistance by several workers [IZ, 14, 451. Wolfson eEal. [46] proposed that propranolol affects coronary flow rate solely through the mechanical events, since keeping the heart rate constant between control and propranolol states, resulted in no change in caronary flow rate. However, Whit&t and Lucchesi [45] found that the decreasein heart rate was responsible for two-thirds of the

FIGURE perfused rat

1.EfGat

of

propranoloi(20 paf)on the oxygenupt&e (0-O) (e---e) ( n *-.*m)

increase

in

and coronary

flow r&e

of the

heart.

coronary

vascular

Control: 11 hearts Propranolol: 13 hearts Propr&nolol + pacing: resistance,

while

6 hearts the

remaining

one-third,

which

occurs during a constant heart rate, might be due to a non-specific action of propranolol, unrelated to beta-blockade. The results obtained in this study also indicate that propranolol might indeed have a vasoconstrictor action, since keeping the heart rate con&ant during perfusion with propranolol, resulted in a further decline in coronary flow rate (Figure 1). This is substantiated by the finding that increased heart rate is usually associatedwith increased coronary flow rate [3]. The reduction in coronary flow rate with pacing after propranolol administration, is in contrast to the results obtained in the unanaesthetized, consciousdog [7, 341. It was found that with increasing heart rate after propranolol administration, mean coronary blood flow was comparable to that before beta-receptor blockade. Effect of propranolol

on myocmdial

metabolism

Administration of propranolol results in a significant depression of the oxygen uptake of the perfused rat heart (Figure 1). This might be attributed to (i) a direct

EFFECT OFPROPRANOLOLON

PERFUSED RATHEART

85

depressanteffect on Krebs cycle oxidation, (ii) the effect on heart rate, or (iii) the reduction in coronary flow rate. The first possibility can be ruled out, since propranolol actually increasesthe oxygen uptake of rat heart slices, while it has no effect on sarcosomaloxygen uptake [Tables 6, 7(b)]. It is unlikely that propranolol reduced oxygen uptake by virtue of its negative chronotropic effect, since keeping the heart rate at control levels during propranolol administration had no effect on the depressionin oxygen uptake produced by propranolol. This is substantiated by the finding that increasing the frequency of contraction usually results in an increased oxygen consumption [3]. Therefore it would seemthat the reduction in coronary flow rate is the most significant factor in the depressanteffect of propranolol on the oxygen uptake of the perfused rat heart. The chronotropic effect of propranolol appears to play an important role in determining its effect on myocardial substrate metabolism. Using a non-beating preparation, viz. heart slices, propranolol stimulates both glucose and pyruvate metabolism while with exception of a reduction in i4COz production from [U-iW]glucose, it has no effect on the metabolism of these substrates by the perfused rat heart. Increasing the heart rate to control levels, 1WOz production from [U-14C]glucose was enhanced. The reduction in palmitate uptake and i*COs production produced by propranolol, was also eliminated by increasing the heart rate. Furthermore, the depressanteffect of propranolol on both glucoseand palmitate metabolism when a combination of glucoseand palmitate was used as substrates, was reversed with increased heart rate. The results obtained from the isolated, perfused rat heart indicate that the increase in myocardial glucose consumption, was due to the increase in arterial blood glucose levels [21]. Furthermore, the shift to carbohydrate metabolism and reduction in free fatty acid oxidation and turnover also appear to be due to this increasein blood glucose concentration, since propranolol depressedthe metabolism of both substrates when glucoseplus palmitate were used as substrates for the perfused heart. The reduced free fatty acid turnover and oxidation in the human heart may alsobe partially due to the effect of the reduced heart rate, rather than to direct blockade of myocardial free fatty acid extraction [15]. The depressionin high energy phosphate contents of the perfused rat heart by propranolol probably results from:the reduction in sarcosomaloxidative phosphorylation. It is unlikely that the reduction in oxygen uptake is responsible for this phenomenonsinceit has been shown that hypoxia causeslittle changein high energy phosphate content [35]. The reduction in P : 0 ratio is due to a significant decrease in inorganic phosphate uptake while sarcosomal oxygen uptake remained unchanged (Table 6). This might be an explanation for the increasedarterial inorganic phosphate content observed in patients after propranolol administration [21], indicating that body tissue respiration might be uncoupled to a certain extent after propranolol administration.

86

A. J. BRINK,

A.

J. BESTER

AND

A. LOCHNER

Studies on localization of the depressant effect of propranolol, showed that it had no effect on phoaphorylation in the cytochrome c-oxygen region of the electron transport chain. Phosphorylation between NADH and cytoohrome c was significantly reduced by 40 PM propranolol. Further studies using succinate, a substrate which does not depend on NADH for electron transfer, also showed a depressant effect of propranolol on phosphorylation. These studies indicate that propranolol probably exerts its inhibitory effect on the second phosphorylation site of the electron transfer chain. However, further studies are needed to elucidate the exact mechanism of propranolol action on oxidative phosphorylation. In contrast to the present findings, Sobel et al. [U] reported that propranolol (1000 FM) had no effect on sarcosomaloxidative phosphorylation. In contrast to the perfused heart, Nayler et al. [31] found no change in myocardial ATP and CrP contents after propranolol administration to dogs; this might be due to a low dosage. In summary, propranolol affects the perfused rat heart by both its negative chronotropic and inotropic action. These factors are partially responsible for the reduction in coronary flow rate and the depression in myocardial substrate metabolism.

We wish to thank the C.S.I.R., the University of Stellenbosch and the South African Atomic Energy Board for financial support. We gratefully acknowledge the expert technical assistanceof the following persons: J. C. N. Hot&, B. Rehder, S. Schoeman and M. Piek.

1. BERWEYER,

H. U. Meti

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26.

27.

28.

29.

30.

31.

32. 33.

34. 35.

36. 37.

38.

39. 40. 41.

42.

in

hereditary

EFFECT

OF PROPRANOLOL

ON PERFUSED

SPAN-N, J. F., JR., SONNENBLICK, E. H., COOPER, T., CIXIDSEY, V. L. & BRAUNW~, E. Cardiac norepinephrine stores and the heart muscle. Circuldion R~rch 19, 317-325 (1966). 44. STEIN, P. D., BROOKS, H. L., MATSON, J. L. & HYLAND, J. adrenergic blockade on coronary blood flow. Cardiovamuh(1968). 45. WHITSITT, L. S. & LUCCHESI, B. R. Effects of propranolol and its coronary vascular resistance. CirceLlcction Research 21, 305-317 46. WOLFSON, S., HEINLE, R. A., HER-, M. V., KEY, H. G., GORLIN, R. Propranolol and angina pectoris. Awwican Journal 345-353 (1966). 43.

89

RAT HEART

C. A., WILLMAN, contractile state of

W. Effect of betaResearch 2, 63-76 stereoisomers (1967). SULLIVAN,

upon

J. M.

of Cardiology

&

18.