A protective effect of verapamil on the calcium paradox in the isolated perfused rat heart

of Molecular

Joumnl

and Cellular

A Protective

i 1982 ) 14, 13320

Cardiology

Effect of Verapamil on the Calcium in the Isolated Perfused Rat Heart*

Hideto

Ohhara,

Hideo

Kanaide

and

Motoomi

Paradox

Nakamura

Research Institute of Angiocardiolopy and Cardiovascular Clinic, Kyushu University 3- 1- 1 Maidashi, Higashi-ku, Fukuoka, Japan 812 (Received 2 1 April

198 1, accepted in revised form 8 October

Medical

School.

198 1)

H. OHHARA, H. KANAIDE AND M. NAKAMURA. A Protective Effect of Verapamil on the Calcium Paradox in the Isolated Perfused Rat Heart. Journal of Molecular and Cellular Cardiology (1982) 14, 13-20. Experiments were undertaken to examine whether and how verapamil, one of the well known slow-channel calcium-antagonists, protected cardiac muscles against the calcium paradox in isolated perfused rat hearts. Following a 5-min perfusion with calcium-free buffer at pressure of 80 cm H,O (high flow) and 40 cm HsO (low flow), severe and mild calcium paradoxical injuries were produced, respectively, by reperfusion with buffer containing 2.5 mM of calcium for 10 min at 80 cm H,O. When verapamil (I mg/l) was added to the perfusion medium during both the calcium depletion and repletion periods, a marked protective effect on the mild calcium paradox was observed, evidenced by a higher recovery in the contractility, an about 50% reduction in development of the contracture, an almost complete prevention in release of creatine kinase, an inhibition of tissue calcium accumulation, and much larger tissue stores of high energy phosphates; whereas no protective effect was observed on the severe calcium paradox. In the mild calcium paradox, administration of verapamil up to 2 min after the readmission of calcium was sufficient for protection against the calcium paradox. It was suggested that verapamil could substantially protect heart muscles from injuries associated with the calcium paradox probably due to an inhibition of sudden calcium influx into the cardiac cells in early calcium repletion period. KEY

WORDS:

Calcium

paradox;

V’erapamil;

Creature

kinasc;

High

energy

phosphate;

C:alcium

ac‘cumu-

lation.

Introduction Myocardial injuries associated with the calcium paradox was first reported by Zimmerman and Hiilsmann [21]. The damaging effect of the calcium paradox has been postulated to be the consequence of a sudden and massive influx of calcium into the cells upon calcium repletion [I, 5, 17, 21, 221. If the route of a sudden and massive influx of calcium is, at least in part, the slow calcium channels, it can be expected that the slow-channel calcium-antagonist drugs protect heart muscles from injuries associated with the calcium paradox. Recently, it was demonstrated that verapamil, one of the slow-channel calciumantagonists, protected heart muscles from protein release during the calcium paradox [4]. A protec-

* This 0022-2828/82/O

study

was

supported,

100 13 + 08

in part, 000.00/O

by

the

grant

tive effect of these drugs against the calcium paradox may also be expected if the high energy phosphates of myocardium is preserved by these drugs to such an extent that more energy is available to maintain ionic homeostasis [II]. However, there were also reports that verapamil could not reveal protective effect on the calcium paradox [I, 12, ICY]. The present study was performed to investigate whether and how verapamil could protect cardiac muscles from the calcium paradox; severe and mild ones. Severe and mild injuries of the calcium paradox were produced by regulating coronary flow rate during calcium depletion period [7].

from

the

Naito

Foundation.

Q 1982 Academic

Press Inc.

(l,ondon

I Limited

1-l

Hideto

Ohhara

Materials

and

Perjiision Male Wistar rats (280 to 320 g) were heparinized (500 iu/head) and killed by a blow on the head. The hearts were quickly excised and subsequently perfused by the Langendorff technique. The normal perfusion medium was Krebs-Henseleit solution (pH 7.4) containing in mM NaCI, 120: NaHCO,, 25; KCl, 4.8; KH,PO,, 1.2; MgSO,, 1.2; CaCI,, 2.5; glucose, 5.5. The medium was maintained at 37°C and continuously gassed with a mixture of 957; 0, and 596 CO,, providing a p0, of over 500 mmHg. No correction was made

Experimental

Coronary effluent was collected at timed intervals of 2 to 5 min. Creatine kinase (CK) activity of the effluent was measured by the method of Rosalki

for the osmolarity when calcium was omitted liionl the normal medium. The hearts were electrically paced at 280 beats/min. The contractile force was recorded via a strain gauge (SB-IT, Nihon Kohden) connected to the apex of the heart. Iit the start of each experiment, the resting tension was set at about 1 g. Oxygen saturation of myoglobin of perfused hearts was continuously monitored by the dual wave-length spectrophotomctries (581 to 592 nm) of the reflected li,ght of epicardium [S].

perfusion medium during both the calcium depletion period and repletion period. In the low flow experimental system, the drug was added to the perfusion medium during both the calcium depletion and repletion periods (verapamil-I group), or during the calcium depletion period and only an initial 2 min of the calcium repletion period (verapamil-II group) (Figure 1).

procedures [16]. CK iu/min. At the

release end

of

from 10 min

tissue

was

of

calcium

-

+

-

+

+

I

-

+

II

-

+

High flow

LOW flow

FIGURE

technique

+

Calcium

Verapamil

Methods

time course

For the equilibration, the hearts were perfused for an initial 20 min with the normal medium at a pressure of 80 cm H,O. The hearts were then subjected to 5 min of calcium-free perfusion at the same pressure (high flow) or 40 cm H,O (low flow) followed by a 10 min of calcium repletion at a pressure of 80 cm H,O. In the high flow experimental system, verapamil was present in the

.gnalytical

et al.

1. Experimental

time

course.

0 timr:

starting

+ t

of the calcium-fiiee

-

perfusion.

expressed repletion,

as

Effect hearts analysis liquid assayed nosine phosphate

of Verapamil

on the

were quickly frozen for the biochemical by a Wollenberger clamp precooled in nitrogen [19]. The frozen tissues were for adenosine triphosphate (ATP), adediphosphate (ADP), adenosine mono(AMP), creatine phosphate (CP),

(+s.E.M.\

were expressed as standard of n experiments. Test

1i

Paradox

nicotinamide adenine dinucleotide (NAD) procedures described previously [ 14, 151. calcium contents were measured by absorption spectrophotometer in a Nippon Ash apparatus (model AA-782) [9. 20, 121.

Statistical Results

Calcium

error means of significance

by the Tissue atomic Jarrel-

analysis was calculated by Student’s P 7 0.05 as the limit of significance.

1

test.

taking:

Results Effect of verapamil

on recovery

in the contractile

force

and chan,ge in the resting

Time courses of recoveries in the contractile tension development, changes in the resting tension and the coronary flow rate of calcium reperfused hearts under two experimental systems, high flow and low flow, were shown in Figures 2 and 3.

fyL.rlfi. ji;;;;l’ 0 2 Time after

4 Co’+

6 6 IO readmlssion (min)

FIGURE 2. Effect at’ verapamil on recovery in the contractile tension development (a), changes in the resting tension (b) and the coronary flow rate (c) upon reperfusion with the calcium-containing medium after 5 min of calcium-free perfusion in the high flow experimental system. Perfusion pressure during the calcium-free period was 80 cm H,O as shown in Figure I. No verapamil group [(e) n = 4, the coronary flow rate during the calcium-free perfusion: 55.9 & 1.6 ml/5 min]; verapamil group [(Q), n = 4, 61.8 + 2.5 ml/5 min]. Each value is a mean + S.E. of n cxprrimrnts.

tenTion upon the calcium

repletion

respectively. In the high flow experimental system, the coronary flow rate during the calcium depletion was 55.9 3 1.6 ml/5 min ia the absence and 61.8 & 2.5 ml/5 min in the presence of verapamil. There was-no significant difference in the coronary flow rate during the calcium depletion between these two groups. Addition of verapamil to the medium had no effect on recovery in the contractility, elevation of the resting tension and decrease in the coronary flow rate upon the calcium repletion in the high flow experimental system as shown in Figure 2. In the low Row rxperimental system, the coronary flow rate during the calcium depletion was 30.7 f 0.6 ml/5 min jno verapamil 32.8 h 1.8 ml/5 min group), (verapamil-I group) and 31.8 + 1.6 ml/5 min (verapamil-II group). There was no significant difference in the coronary flow rate during the calcium depletion among these three low flow groups. There was no recovery in the contractile tension development upon the calcium repletion in no verapamil group: however, there was a slight recovery in the presence of verapamil [Figure 3(a)]. Furthermore, a significantly higher recovery was found 10 min after the calcium repletion in verapamil-II group than in verapamilI group (P < 0.05) as shown in Figure 3(a). In both verapamil-I and verapamil-II groups the marked rise in the resting tension upon the calcium repletion was significantly reduced throughout 10 min of the calcium repletion period (P < 0.01, as shown in Figure 3(b). The coronary flow rat<> was significantly higher in both verapamil-I and verapamil-II groups than in no verapamil group (P < 0.001) as shown in Figure 3(c). Since there was no change in oxygen saturation of myoglobin during the calcium depletion period. it was suggested that myocardial oxygen tension was kept at aerobic level during that period. even in the low flow experimental system.

16

Hideto

Ohhara

al.

et

60-

I

I

I

I

4

6

,

I

14 (b) ^,

5-,(b)

s ‘S i

4-

f

2-

IO-

3-

a-

1'X 'D 5

T

12-

o-

6I

0 Time after

0

2 Time

4 after

6 Co’*readmission

8

10

(min)

FIGURE 3. Effect of verapamil on recovery in the contractile tension development (a), changes in the resting tension (b) and the coronary flow rate (c) upon reperfusion with calcium after 5 min of calcium-free perfusion in the low flow experimental system. Perfusion pressure during the calcium-free period was 40 cm HsO as shown in Figure 1. No verapamil group [(O), n = 8, the coronary flow rate during the calcium-free perfusion: 30.7 += 0.6 ml/5 min] ; verapamil-I group [(O), n = 4, 32.8 + 1.8 ml/5 min]; verapamil-II group [(o), n = 4, 31.8 & 1.6 ml/

2

Co2+raodmission

8

10

(min)

FIGURE 4. Effect of verapamil on release of creatinc kinase from tissues into coronary effluent upon reperfusion with calcium after 5-min of calcium-free perfusion. Upper panel (a): data in the high llow experimental system. No verapamil group [(a), n = 41; verapamil group [(O), n = 41. Lower panel (b) : data in the low flow experimental system. No verapamil group [(a), n = 51; verapamil-I group [(O), n = 41; verapamil-II group [(a), R = 41. Each value is a mean i S.E. of n experiments. *P < 0.01; **P < 0.001 compared with no verapamil group.

5 min]. sented Each

Recoveries of the developed tension arc repreas percentage of the values of control periods. value is a mean & S.E. of n experiments. +**P < 0.001 compared *P < 0.05; **p < 0.01; with no verapamil group. AP < 0.05 compared with verapamil-I group.

Effect

of Verapamil

of verapamil

Effect

on the

on myocardial

NO

contents of high energy phosphates, repletion for IO min

Vempomil

NAD

and calcium

after

the calcium

control. Verapamil had no effect on the reduction in the above myocardial constituents. In the low flow experimental system, smaller degrees of the reduction in myocardial high energy phosphates and NAD stores than those in the high flow experimental system were observed. The ;\TP. total adenine nucleotide, CP and NAD stores of

Veropomd High

repletion

tal system, on the other hand, the presence of verapamil in the medium revealed a clear protection of myocardium against CK release [P < 0.001, Figure 4(b)]. The total amounts of CK release during 10 min of the calcium repletion in both verapamil-I and II groups were less than 1 o/0 of those in no verapamil group.

Myocardial stores of adenine nucleotides, CP and NAD at the end of 10 min of the calcium repletion period following the calcium depletion were shown in Figure 5. In the high flow experimental system, myocardiaP stores of ATP, ADP, total adenine nucleotide, CP and NAD markedly decreased compared to those of the normal

Normal

17

Paradox

on release of CK upon the calcium

During the stabilization and the calcium-free perfusion periods the hearts released negligible amounts of CK into the coronary effluent. Figure 4 shows the time courses of CK release during 10 min of the calcium repletion. In the high flow experimental system, verapamil had no protective effect on the massive release of CK as shown in Figure 4(a). In the low flow experimen-

Effect of uerapamil

Calcium

NO

Vnopamil

flow

Ver~mil-I

Vempomil-II

LOW flow Ca’+-free

(5

min)+

Cd+-reperfuston

(IOmml

FIGURE 5. Effect of verapamil on myocardial high energy phosphates and NAD stores at the end of 10 min of reperfusion with calcium after 5 min of calcium-free perfusion on the high and low flow experimental systems. (m) ATP; (a) ADP; (m) AMP; (0) total adenine nucleotide; (l?Jj) creatine phosphate; (a) NAD. Numbers in parentheses indicate number of hearts. Data are expressed as mean f S.E. *P 4 0.05: **P C’ 0.01 : .**p ‘H 0.001 compared with no verapamil group in the low flow experimental system.

18

Hideto

Ohhara

both verapamil-I and II groups were significantl) higher than those of no verapamil group (P < 0.01). Myocardial calcium contents at the end of 10 min of the calcium repletion period were shown in Figure 6. Verapamil revealed a marked reduction in increased calcium contents upon the calcium repletion in the low flow experimental system (P < 0.001) although it had no effect in the high flow experimental system.

et al.

3 $’ 1” g ,i g g

50 40 30 20

FIGURE 6. Effect of verapamil on myocardial calcium contents at the end of 10 min of reperfusion with calcium after 5 min of calcium-free perfusion in the high and low flow experimental systems. Numbers in parentheses indicate number of hearts. Data are expressed as mean $ S.E. *P < 0.01; **P < 0.001 compared with no verapamil group in the low flow experimental system.

IO 0

Ca2+-free

(5 min)+Ca2+I~IC.LKK

reperfusian

(lOmid

b.

Discussion Recently, it was reported that the severity in myocardial damage in the calcium paradox depends upon coronary flow rate during the calcium depletion period [7]. In the present study, severe and mild calcium paradoxical injuries were produced by two coronary flow rates (high flow and low flow) during the calcium depletion period, and protective effects of verapamil, a slow-channel antagonist, were examined under these two experimental systems. The dose of verapamil used in the present study was decided at 1 mg/l of perfusion medium because it was shown to protect heart muscles against hypoxic damages [9] and used in the previous studies of the calcium paradox by other authors [I, 12, 181. The present study revealed that verapamil had a protective effect on mild myocardial injuries occurring during the calcium paradox. The presence of verapamil in perfusion medium during the calcium depletion and repletion periods reduced about 50:/, in increased resting tension, and markedly prevented CK release, tissue accumulation of calcium and depletion in tissue stores of high energy phosphates. Furthermore, recoveries in the contractility upon the calcium repletion were observed in the hearts perfused with verapamil. On the contrary, verapamil had no effect on severe myocardial injuries occurring during the calcium paradox. It is known that verapamil binds to cardiac muscles and reveals a strong negative inotropic effect [13]. The amounts of verapamil bound to myocardial tissues under both the high flow and low flow conditions were unknown in the present study. The severe calcium paradox induced by

the high flow condition during the calcium depletion could not be protected by verapamil even with the exposure to a higher concentration of verapamil which probably resulted in more verapamil bound to tissues. It might be possible to conclude that the difference between the successful protection by verapamil under the low flow condition and the unsuccessful one under the high flow condition in the present study might not relate to the amounts of verapamil bound. However, detailed correlations among the severity of the calcium paradox, the amounts of verapamil bound to tissues and its protective effects were unknown in the present study. Present findings essentially coincide with those of Hearse et al. [4] who reported greater protective effects of verapamil on mild protein release induced by reperfusion with lower concentration of calcium than on severe one induced by reperfusion with higher concentration of calcium. The studies reporting the negative effect of verapamil on the calcium paradox [I, 1.2, 181 seemed to be carried out under the condition of hearts which corresponded to severe damage produced by high coronary flow rate during the calcium depletion period in the present study. In the present study, it was found that the presence of verapamil in the medium during only an initial 2 min of calcium repletion in addition to the calcium depletion period was sufficient fat protection against the calcium paradox. The greater recovery in the contractile tension development of verapamil-II group than that of verapamil-I group was supposed to be simply due to removal of the negative inotropic effect of the

Effect

of Verapamil

on the

wish

to thank

IMiss

M.

Okada,

Miss

Paradox

least in part, the slow calcium channels. Besides, since it is agreed that there are changes in cell membrane including separation of intercalated discs during the calcium depletion period, which is prior to a sudden and massive influx of calcium [Z, 3, 5, 201, verapamil may also exert an action, other than slow channel blockade, on the cardiac cell membrane. Preservation of high energy phosphates by verapamil may contribute, in part. to maintain energy dependent ionic homeostasis which is one of the important functions of thr cell membrane [ 8. I I 1.

drug. Since the damaging effect of the calcium paradox has been postulated to be due to a sudden and massive influx of calcium into the cells [I, 51 and since the tissue accumulation of calcium upon the calcium repletion was found to be preventrd by verapamil in this study, it was suggested that heart muscles were protected from the calcium paradox by verapamil via inhibition of a sudden and massive influx of calcium into the cells, Furthermore, the findings that verapamil inhibited tissue accumulation of calcium suggested that the route of a sudden influx of calcium associated with the calcium paradox might be, at

‘rhe authors assistance.

Calcium

R. Maeda

and

Miss

K.

Fujiyoshi

for

thrir

technical

paradox.

.41ne&nn

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21.

Gewebstiicke.

after