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Myocardial action potential prolongation by calcium channel activation under calcium free-EGTA condition in rats: Developmental and regional variations
Gen.
Pergamon
Pharmar. Vol. 26, No. I, pp. 3943, 1995 Copyright 0 1995 Elsevier Science Ltd
Prinled in Great Britain. All rights reserved 0306-3623/95 $9.50 + 0.00
Myocardial Action Potential Prolongation by Calcium Channel Activation Under Calcium Free-EGTA Condition in Rats: Developmental HIKARU
TANAKA,
Department Miyama 2-2-l
and Regional KAZUO
NOGUCHI
and
Variations KOKI
SHIGENOBU
of Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashr, Chiba 274, Japan [Tel.: (0474) 72-2092 Fax: (0474) 72-21131 (Received
I9 July 1994)
Prolongation of action potentials upon the addition of isoproterenol, forskolin, isobutylmethyl-xanthine (IBMX) and dibutyril CAMP (dbcAMP) under Ca-free EGTA condition was examined in isolated myocardial preparations from neonatal and adult rats, whose action potential configuration greatly differ. 2. The prolongation of the action potential was previously suggested to be produced by persistent sodium influx through calcium channel due to the lack of calcium-mediated inactivation of calcium channels under such experimental condition. 3. Preparations used were papillary muscles and free walls of the right and left ventricles from neonatal and adult rats. 4. In adult preparations, the prolongation produced by isoproterenol, forskolin and IBMX in the right free wall was smaller than those in the other three regions, while no regional difference was observed with dbcAMP. 5. The degree of prolongation by all of the four drugs were smaller in the neonate than in the adult. No regional difference was observed with any of the drugs in the neonate. 6. Our present results suggest that contribution of calcium-mediated inactivation of calcium channels to the repolarization of rat myocardium may increase postnatally to produce the developmental shortening of its action potential. Also, regional difference in the CAMP related mechanisms may appear postnatally. Abstract-l.
Key Words: Myocardium,
action
potential
prolongation,
INTRODUCTION
of the Ca channel
seems to be different
inactivation,
Sr and Na pass through
development
the channel as charge carriers
(Hess and Tsien, 1984; Kass and Sanguinetti, 1984). In intact myocardial tissue, Ca reduction by the use
The myocardial action potential duration is determined by the time-course of inward Ca current decay and outward K current activation. The inactivation process
Ca’+ channels,
of chelating agents such as EDTA or EGTA will evoke prolonged action potentials (Tritthart et al., 1973). Although this can be ascribed to decreased K
from
conductance (Miller and Morchen, 1978), persistent activation of Ca channels can also explain the
that of the Na channel as was shown by voltage clamp analyses of isolated myocardial cells (Tseng, 1988). The process is not only voltage dependent but Ca
prolongation (Linden and Brooker, 1982). In our previous study (Tanaka et al., 1994), we applied
ions. Intracellular application of Ca chelating agents (Josephson et al., 1984; Bechem and Pott, 1985) or clamping membrane potentials at high voltages beyond the reversal potential for Ca currents (Marban and Tsien, 1981; Mentrard et al., 1984) were shown to reduce inactivation. Inactivation of the Ca channel is also reduced when ions other than Ca such as Ba,
isoproterenol (ISO) or dibutyril cyclic adenosine monophosphate (dbcAMP) under Ca free EGTA condition and observed prolongation of the myocardial action potential. This prolongation was pharmacologically demonstrated to be due to persistent influx of Na ions through Ca channels. We also demonstrated that the action potential prolongation
also seems to require
GP26,I--D
a build up of intracellular
39
40
Hikaru
Tanaka
et al.
in the rat, which has an action potential with an extremely short duration and a highly developed sarcoplasmic reticulum (SR) (Fabiato and Fabiato, 1978; Penefski, 1974; Sutko and Willerson, 1980; Tanaka and Shigenobu, 1989) is much larger than
whether increased Ca-mediated inactivation of Ca channels contributes to the developmental shortening of its action potential. Further, as regional differences
that in the guinea
be important for understanding abnormal electrophysiology of the heart (reviewed by Antzelevitch
pig, which
has an action
poten-
in myocardial action potential characteristics and their ionic mechanisms are recently considered to
tial with a normal plateau phase and a moderately developed SR (Agata et al., 1992; Bers et al., 1981). We concluded that contribution of calcium-
et al., 1991) we performed our experiments with preparations from four different regions of the
mediated
ventricle.
inactivation
of calcium
channels
repolarization of normal myocardium in the rat than in the guinea pig.
to the
may be larger MATERIALS
In the present study, we compared the action potential prolongation under the same experimental
Right
condition between neonatal and adult rat myocardia, whose action potential configuration and excitation contraction mechanisms greatly differ. The adult rat ventricle, as mentioned above, has an action potential of short duration with little evidence of a plateau and a highly rat
developed
myocardium
SR. has
In contrast,
an
action
the neonatal
potential
with
a
normal plateau phase and a less developed SR when compared with its adult (Cohen and Lederer, 1988; Fabiato and Fabiato, 1978; Tanaka and Shigenobu, 1989). Therefore, we compared the effect of Ca channel between
activation under Ca free EGTA condition neonatal and adult rat myocardia to find out
and
/:
b-
i\
7%ms
-j
I i\
CaCl, 2, MgCl,
1, NaHCO,
7.4). The nutrient
solution
5% CO* and maintained by a pair of platinum
15 and glucose was aerated
at 36.5”C. They were driven electrodes with rectangular
System,
DPS-165B).
Conventional
-
microelectrode
100 ms
c-1 _ \
-l
i -
-
1 set
5.5 (pH
with 95% O,-
current pulses (0.5 Hz, 5 msec 1.2 x threshold voltage) generated from an electronic stimulator (Dia Medical
100 ms
1 set
-
and
containing physiological salt solution of the following composition (mM concentration): NaCl 135, KC1 5,
-I
-v -7
strips
Wistar strain rats. Preparations were pinned down horizontally on a silicon block in a 30 ml organ bath
100 ms
d
wall
50 mV
! ‘L
c_[-------_-_ -I
free
LP
il
_JL-_
ventricular
papillary muscles were rapidly isolated from neonatal (l-3 days after birth) and adult (200-250 g)
RF
a-\,
left
AND METHODS
d-Y -I
L 1 set
Fig. I. Action potential prolongation in adult rat myocardium. Action potentials of right ventricular free wall (RF) and left ventricular papillary muscle (LP) were recorded under normal condition (a); Ca free-EGTA condition (b) and after further addition of either 10eh M isoproterenol (c) or 10-j M dbcAMP (d). Short horizontal bars indicate 0 mV and the longer horizontal bars indicate time. Note that the time scale in (c) is different between RF and LP.
Calcium free-EGTA condition in rats Table
I. Action
potential
RF Adult
The
time
*P
< 0.05
100.0 i required or
(n = 7)
ventricular duration
for
8.0.
free
104.4 f
repolarization
difference
wall;
RP = right
3.8*
of the normal
from
potential
adult
papillary
116.3 f
values.
muscle;
were
expressed
Regional
LF = left
7.8’
30.4 f
4.9
(n = 14)
(n = 8)
115.0 f
6.5*
(n = IO)
as mean f
differences
ventricular
LP (n = 9)
were
free
SE.
not
wall;
statistically
2. Action
Dotential
377. I + 9.9
(n = 7)
Neonate
103.8 f
(n = 8)
required
l2.2t
for 50%
were expressed
duration
oroloneed
4392.6
1720.2*
LF (n = IO)
4583.9
(n = 8)
145.0 & 17.9t
repolarization
as mean f
I
of the prolonged
action
*P
< 0.05
for significant
difference
from
corresponding
RF
< 0.05
for significant
difference
from
corresponding
adult
Table
i
131.9t
potential
after
LP
1729.0’
(n = 9)
13.3t
(n = 8)
addition
5751.0
565.3 f
IBMX
394.8
dbcAMP
required
for
condition
149.5
3. Action
potential
of isoproterenol
under
50%
duration
prolonged
2745.5*
1,‘ $ :::;; ,g:; _
(n = 6)
repolarization expressed
f
of the prolonged
as mean i
by forskolin,
IBMX
and dbcAMP
< 0.05
for significant
difference
from
corresponding
RF
< 0.05
for significant
difference
from
corresponding
adult
were made into the endocardial
with
and capacity neutralization cathode ray oscilloscope
high
(n = 7)
potential
after
LP
164.4 k 25.2t
(n = 8)
185.6 F 26.5t
131.9 + 24.0t
(n = 8)
152.5 i
1016.9 + 247.7t
(n = 8)
1763.1 f
addition
of forskolin,
IBMX
surface
input
impetance
was fed into a dual beam (Nihon Kohden, VC-11)
the bathing medium solution containing
(n = 8)
548.3t
(n = 8)
or dbcAMP
under
Ca free
values
LP
a -
or a thermal array recorder (Nihon Kohden, RTA-1100). After confirming normal action potential generation, calcium-free
(n = 8)
24.11
values.
using glass microelectrodes filled with 3 M KCI. The output of a microelectrode preamplifier (Nihon MEZ-8201)
______._
RF
En” I f:;
action
*P
Kohden,
condition
SE.
tP
penetrations
(n = IO)
Ca free EGTA
Neonate
6949.1
(n = IO)
3827.9
were
(n = 14)
values.
LP (n = I I)
k 98.2
12,020.0?
885.6;
values.
RF Forskolin
f
118.0 k l2.7t
Adult
time
muscle;
SE.
tP
EGTA
RF = right
papillary
bv isooroterenol
RP
Adult
The
significant.
LP = left ventricular
in msec.
Table
time
conditions
19.8 f 0.9
(n = 8)
action
corresponding
ventricular
(n = IO)
RF
The
normal
LF
19.8 + 2.5
(n = 8)
50%
significant
under
RP
13.4k2.1
Neonate
duration
41
was changed into 1 mM EGTA. In
this solution, prolongation of the action potential was observed upon the addition of isoproterenol (ISO),
b
forskolin, isobutyl-methylaxanthine (IBMX) or dibutyril cyclic adenosine monophosphatc (dbcAMP). The time required for 50% repolarization was measured and presented in mean f SEM. Significant difference between means were evaluated by Williams’ multiple-range test. A P value less than 0.05 was considered significant.
b \
II P----_!
I
_I‘\._ C
-
A- ,1 \:\
RESULTS The normal ventricular action potential of the adult rat had a short action potential with little evidence of a plateau phase (Fig. 1). The action potential duration expressed in terms of time required for 50% repolarization tended to be shorter in the
50 mV
100
ms
Fig. 2. Action potential prolongation in neonatal rat myocardium. Action potentials of right ventricular free wall (RF) and left ventricular papillary muscle (LP) were recorded under normal conditions (a); Ca free-EGTA conditions (b) and after further addition of IO-” M isoproterenol (c). Short horizontal bars indicate 0 mV and the longer horizontal bars indicate time.
Hikaru Tanaka et al.
42
right ventricular free wall (RF) than in the right ventricular papillary muscle (RP), left ventricular free wall (LF) and left ventricular papillary muscle (LP), but the difference was not significant (Table 1). Changing the bathing solution into Ca free-EGTA
the neonate results in a slower rate of inactivation of Ca channels and an action potential with a normal plateau phase. The adult rat myocardium is known to
solution duration
resulted in a decrease in action potential in RF while it resulted in an increase in RP,
Willerson, 1980; Tanaka and Shigenobu, also its electrophysiological properties
LP. Upon
et al., 1984a, b) are known to be affected by ryanodine. Whether intracellularly released Ca from the
LF and IBMX plateau
the addition
of ISO, forskolin,
or dbcAMP, extreme prolongation of the phase was observed. In the case of ISO, the
have a highly developed sarcoplasmic reticulum. Not only its contractile force (Penefski, 1974; Sutko and
highly
developed
SR of the adult
prolongation in RF was significantly smaller than in other three regions (Fig. 1, Table 2). Also in the cases
accelerates Ca channel unique action potential
of forskolin and IBMX, the prolongation plateau phase by the drugs were significantly
interest.
of the larger in
LP when compared with RF. In the case of dbcAMP, however, no regional difference was observed (Fig. 1, Table 3). The action
potential
of
neonatal
ventricular
preparations had a plateau phase of about 200 msec. Changing the bathing solution into Ca free-EGTA solution
resulted
in a decrease
in action
potential
duration. Upon the addition of ISO, forskolin, IBMX or dbcAMP, extreme prolongation of the plateau phase was observed, which was qualitatively similar to that observed in adult preparations. The extent of prolongation, however, was significantly smaller in the neonate examined difference
than in the adult for all of the drugs
(Fig. 2, Tables 2 and 3). No regional between RF and LP was observed with any
of the drugs in the neonate
(Tables
2 and 3).
has a short
action
potential
with little evidence of a plateau, which has been attributed not only to a large contribution of repolarizing K currents such as I,, (Kilborn and Fedida, 1990) and I,,] (Wahler, 1992) but also to the kinetics of the Ca current in this species (Cohen and Lederer, 1988). In dbcAMP observed potential
our previous study, we applied IS0 or under Ca free EGTA condition and prolongation of the myocardial action of rats and guinea pigs. We pharmaco-
logically
demonstrated
that
rat myocardium
inactivation configuration
to produce its is a matter of
In adult myocardia, action potential prolongation under Ca free EGTA condition by ISO, forskolin or IBMX was smaller in RF than in LP. The concentrations of these three drugs were those previously reported to produce in isolated ventricular
maximum inotropic responses myocardia from neonatal and
adult rats (Shigenobu
et al., 1988). A common
of these three drugs
is their action
CAMP related mechanisms.
feature
on endogenous
In contrast
to these three
agents, no regional difference was observed with dbcAMP, which directly liberates CAMP intracellularly. These results suggest that regional differences may be present in the mechanisms prior to the CAMP production, e.g. lower basal CAMP turnover in RF, but direct evidence awaits further investigation. Contrary observed
to the adult, regional in neonatal myocardia
difference indicating
was not that the
difference may occur during postnatal development. The developmental increase in the degree of prolongation and the appearance of regional difference
DISCUSSION
The rat myocardium
1989) but (Mitchell
this prolongation
was
due to persistent influx of Na ions through Ca channels due to lack of its inactivation by intracellular Ca under such condition (Tanaka et al., 1994). The large degree of action potential prolongation in the adult rat observed in the present study suggests that a large degree of Ca inactivation of Ca channels contributes to the rapid repolarization of its action potential. In contrast, the degree of action potential prolongation under Ca free EGTA condition in the neonate was much smaller than in the adult. This suggests that a smaller degree of Ca inactivation in
may reflect developmental properties
variations
in the functional
of these muscles.
REFERENCES
Agata N., Tanaka H. and Shigenobu K. (1992) Developmental changes in the pharmacological properties of guinea pig myocardial contraction. Folia Phnrmac. Jap. 100, 14. Antzelevitch C., Sicouri S., Litovsky S. H., Lukas A., Krishnan S. C., Di Diego J. M., Gintant G. A. and Liu D.-W. (1991) Heterogeneity within the ventricular wall: electrophysiology and pharmacology of epicardial, endocardial and M cells. Circ. Res. 69, 1427-1449. Bechem M. and Pott L. (1985) Removal of Ca current inactivation in dialysed guinea-pig atria1 cardioballs by Ca chelators. Pflugks A&hs 404,10-20. Bers D. M.. Philiason K. D. and Lancer G. A. (1981) Cardiac contra&ty and sarcolemmal calcium binding in several cardiac muscle preparations. Am. J. Physiol. 240, H5766H583. Cohen N. M. and Lederer W. J. (1988) Changes in the calcium current of rat heart ventricular myocytes during development. J. Physiol. 406, 115-146. _ Fabiato A. and Fabiato F. (1978) Calcium-induced release of calcium from the sarcbplasmic reticulum of skinned cells from adult human, dog, cat, rat and frog hearts and
Calcium
free-EGTA
from fetal and newborn rat ventricles. Ann. N.Y. Acad. Sci. 307, 491-522. Hess P. and Tsien R. W. (1984) Mechanism of ion permeation through calcium channels. Nafure 309, 453-456. Josephson I. R., Sanchez-Chapula J. and Brown A. M. (1984) A comparison of Ca currents in rat and guinea-pig single ventricular cells. Circ. Res. 54, 144&156. Kass R. S. and Sanguinetti M. C. (1984) Inactivation of Ca channel current in the calf cardiac Purkinje fiber: evidence for voltage and Ca mediated mechanisms. J. gen. Physiol. 84, 705-726. Kilborn M. J. and Fedida D. (1990) A study of the developmental changes in outward currents of rat ventricular myocytes. J. Physiol. 430, 37-60. Linden J. and Brooker G. (1982) Evidence for persistent activation of cardiac slow channels in low-calcium solutions. Am. J. Physiol. 242, H827-H833. Marban and Tsien (1981) Is the slow inward calcium current of heart muscle inactivated by calcium? Biophys. J. 33, 143a. Mentrard D., Vassort G. and Fischmeister R. (1984) Calcium-mediated inactivation of calcium conductance in cesium-loaded frog heart cells. J. gen. Physiol. 85, 105-131. Miller D. J. and Morchen A. (1978) On the effects of divalent cations and ethylene glycol-bis-(p-aminoethyl ether)N,N,N’,N’-tetraacetate on action potential duration in frog heart. J. gen. Physiol. 71, 47-67. Mitchell M. R., Powell T., Terrar D. A. and Twist V. W. (1984a)The effects of ryanodine, EGTA, and low-sodium on action potentials in rat and guinea-pig ventricular myocytes: evidence for two inward currents during the plateau Br. J. Pharmac. 81, 543-550. Mitchell M. R., Powell T., Terrar D. A. and Twist V. W. (1984b) Possible association between an inward
condition
in rats
43
current and the late plateau of action potentials in ventricular cells isolated from rat heart. J. Physiol. 351, 4OP. Penefski Z. J. (1974) Studies on mechanism of inhibition of cardiac muscle contractile tension by ryanodine. P~¶ugers Archs 347, 173-I 84. Shigenobu K., Tanaka H. and Kasuya Y. (1988) Changes in sensitivity of rat heart to norepinephrine and isoproterenol during pre- and postnatal development and its relation to sympathetic innervation. Dev. Pharmac. Thu. 11, 226-236. Sutko J. L. and Willerson J. T. (1980) Ryanodine alteration of the contractile state of rat ventricular myocardium. Comparison with dog, cat, and rabbit ventricular tissues. Circ. Res. 46, 332-343. Tanaka H. and Shigenobu K. (1989) Effect of ryanodine on neonatal and adult rat heart: developmental increase in sarcoplasmic reticulum function. J. Molec. Cell. Cardiol. 21, 1305-1313. Tanaka H., Noguchi K. and Shigenobu K. (1994) Myocardial action potential prolongation by calcium channel activation under calcium free-EGTA condition in guinea pigs and rats. Gen. Pharmac. 25, 475-480. Tseng G.-N. (1988) Calcium current restitution in mammalian ventricular myocytes is modulated by intracellular calcium. Circ. Res. 63, 4688482. Tritthart H., MacLeod D. P., Stierle H. E. and Krause H. (1973) Effects of Ca-free and EDTA-containing Tyrode solution on transmembrane electrical activity and contraction in guinea pig papillary muscle. Pflugers Archs 338, 361-376. Wahler G. M. (1992) Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes. Am. J. Physiol. 262, Cl266-C1272.
Pergamon
Pharmar. Vol. 26, No. I, pp. 3943, 1995 Copyright 0 1995 Elsevier Science Ltd
Prinled in Great Britain. All rights reserved 0306-3623/95 $9.50 + 0.00
Myocardial Action Potential Prolongation by Calcium Channel Activation Under Calcium Free-EGTA Condition in Rats: Developmental HIKARU
TANAKA,
Department Miyama 2-2-l
and Regional KAZUO
NOGUCHI
and
Variations KOKI
SHIGENOBU
of Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashr, Chiba 274, Japan [Tel.: (0474) 72-2092 Fax: (0474) 72-21131 (Received
I9 July 1994)
Prolongation of action potentials upon the addition of isoproterenol, forskolin, isobutylmethyl-xanthine (IBMX) and dibutyril CAMP (dbcAMP) under Ca-free EGTA condition was examined in isolated myocardial preparations from neonatal and adult rats, whose action potential configuration greatly differ. 2. The prolongation of the action potential was previously suggested to be produced by persistent sodium influx through calcium channel due to the lack of calcium-mediated inactivation of calcium channels under such experimental condition. 3. Preparations used were papillary muscles and free walls of the right and left ventricles from neonatal and adult rats. 4. In adult preparations, the prolongation produced by isoproterenol, forskolin and IBMX in the right free wall was smaller than those in the other three regions, while no regional difference was observed with dbcAMP. 5. The degree of prolongation by all of the four drugs were smaller in the neonate than in the adult. No regional difference was observed with any of the drugs in the neonate. 6. Our present results suggest that contribution of calcium-mediated inactivation of calcium channels to the repolarization of rat myocardium may increase postnatally to produce the developmental shortening of its action potential. Also, regional difference in the CAMP related mechanisms may appear postnatally. Abstract-l.
Key Words: Myocardium,
action
potential
prolongation,
INTRODUCTION
of the Ca channel
seems to be different
inactivation,
Sr and Na pass through
development
the channel as charge carriers
(Hess and Tsien, 1984; Kass and Sanguinetti, 1984). In intact myocardial tissue, Ca reduction by the use
The myocardial action potential duration is determined by the time-course of inward Ca current decay and outward K current activation. The inactivation process
Ca’+ channels,
of chelating agents such as EDTA or EGTA will evoke prolonged action potentials (Tritthart et al., 1973). Although this can be ascribed to decreased K
from
conductance (Miller and Morchen, 1978), persistent activation of Ca channels can also explain the
that of the Na channel as was shown by voltage clamp analyses of isolated myocardial cells (Tseng, 1988). The process is not only voltage dependent but Ca
prolongation (Linden and Brooker, 1982). In our previous study (Tanaka et al., 1994), we applied
ions. Intracellular application of Ca chelating agents (Josephson et al., 1984; Bechem and Pott, 1985) or clamping membrane potentials at high voltages beyond the reversal potential for Ca currents (Marban and Tsien, 1981; Mentrard et al., 1984) were shown to reduce inactivation. Inactivation of the Ca channel is also reduced when ions other than Ca such as Ba,
isoproterenol (ISO) or dibutyril cyclic adenosine monophosphate (dbcAMP) under Ca free EGTA condition and observed prolongation of the myocardial action potential. This prolongation was pharmacologically demonstrated to be due to persistent influx of Na ions through Ca channels. We also demonstrated that the action potential prolongation
also seems to require
GP26,I--D
a build up of intracellular
39
40
Hikaru
Tanaka
et al.
in the rat, which has an action potential with an extremely short duration and a highly developed sarcoplasmic reticulum (SR) (Fabiato and Fabiato, 1978; Penefski, 1974; Sutko and Willerson, 1980; Tanaka and Shigenobu, 1989) is much larger than
whether increased Ca-mediated inactivation of Ca channels contributes to the developmental shortening of its action potential. Further, as regional differences
that in the guinea
be important for understanding abnormal electrophysiology of the heart (reviewed by Antzelevitch
pig, which
has an action
poten-
in myocardial action potential characteristics and their ionic mechanisms are recently considered to
tial with a normal plateau phase and a moderately developed SR (Agata et al., 1992; Bers et al., 1981). We concluded that contribution of calcium-
et al., 1991) we performed our experiments with preparations from four different regions of the
mediated
ventricle.
inactivation
of calcium
channels
repolarization of normal myocardium in the rat than in the guinea pig.
to the
may be larger MATERIALS
In the present study, we compared the action potential prolongation under the same experimental
Right
condition between neonatal and adult rat myocardia, whose action potential configuration and excitation contraction mechanisms greatly differ. The adult rat ventricle, as mentioned above, has an action potential of short duration with little evidence of a plateau and a highly rat
developed
myocardium
SR. has
In contrast,
an
action
the neonatal
potential
with
a
normal plateau phase and a less developed SR when compared with its adult (Cohen and Lederer, 1988; Fabiato and Fabiato, 1978; Tanaka and Shigenobu, 1989). Therefore, we compared the effect of Ca channel between
activation under Ca free EGTA condition neonatal and adult rat myocardia to find out
and
/:
b-
i\
7%ms
-j
I i\
CaCl, 2, MgCl,
1, NaHCO,
7.4). The nutrient
solution
5% CO* and maintained by a pair of platinum
15 and glucose was aerated
at 36.5”C. They were driven electrodes with rectangular
System,
DPS-165B).
Conventional
-
microelectrode
100 ms
c-1 _ \
-l
i -
-
1 set
5.5 (pH
with 95% O,-
current pulses (0.5 Hz, 5 msec 1.2 x threshold voltage) generated from an electronic stimulator (Dia Medical
100 ms
1 set
-
and
containing physiological salt solution of the following composition (mM concentration): NaCl 135, KC1 5,
-I
-v -7
strips
Wistar strain rats. Preparations were pinned down horizontally on a silicon block in a 30 ml organ bath
100 ms
d
wall
50 mV
! ‘L
c_[-------_-_ -I
free
LP
il
_JL-_
ventricular
papillary muscles were rapidly isolated from neonatal (l-3 days after birth) and adult (200-250 g)
RF
a-\,
left
AND METHODS
d-Y -I
L 1 set
Fig. I. Action potential prolongation in adult rat myocardium. Action potentials of right ventricular free wall (RF) and left ventricular papillary muscle (LP) were recorded under normal condition (a); Ca free-EGTA condition (b) and after further addition of either 10eh M isoproterenol (c) or 10-j M dbcAMP (d). Short horizontal bars indicate 0 mV and the longer horizontal bars indicate time. Note that the time scale in (c) is different between RF and LP.
Calcium free-EGTA condition in rats Table
I. Action
potential
RF Adult
The
time
*P
< 0.05
100.0 i required or
(n = 7)
ventricular duration
for
8.0.
free
104.4 f
repolarization
difference
wall;
RP = right
3.8*
of the normal
from
potential
adult
papillary
116.3 f
values.
muscle;
were
expressed
Regional
LF = left
7.8’
30.4 f
4.9
(n = 14)
(n = 8)
115.0 f
6.5*
(n = IO)
as mean f
differences
ventricular
LP (n = 9)
were
free
SE.
not
wall;
statistically
2. Action
Dotential
377. I + 9.9
(n = 7)
Neonate
103.8 f
(n = 8)
required
l2.2t
for 50%
were expressed
duration
oroloneed
4392.6
1720.2*
LF (n = IO)
4583.9
(n = 8)
145.0 & 17.9t
repolarization
as mean f
I
of the prolonged
action
*P
< 0.05
for significant
difference
from
corresponding
RF
< 0.05
for significant
difference
from
corresponding
adult
Table
i
131.9t
potential
after
LP
1729.0’
(n = 9)
13.3t
(n = 8)
addition
5751.0
565.3 f
IBMX
394.8
dbcAMP
required
for
condition
149.5
3. Action
potential
of isoproterenol
under
50%
duration
prolonged
2745.5*
1,‘ $ :::;; ,g:; _
(n = 6)
repolarization expressed
f
of the prolonged
as mean i
by forskolin,
IBMX
and dbcAMP
< 0.05
for significant
difference
from
corresponding
RF
< 0.05
for significant
difference
from
corresponding
adult
were made into the endocardial
with
and capacity neutralization cathode ray oscilloscope
high
(n = 7)
potential
after
LP
164.4 k 25.2t
(n = 8)
185.6 F 26.5t
131.9 + 24.0t
(n = 8)
152.5 i
1016.9 + 247.7t
(n = 8)
1763.1 f
addition
of forskolin,
IBMX
surface
input
impetance
was fed into a dual beam (Nihon Kohden, VC-11)
the bathing medium solution containing
(n = 8)
548.3t
(n = 8)
or dbcAMP
under
Ca free
values
LP
a -
or a thermal array recorder (Nihon Kohden, RTA-1100). After confirming normal action potential generation, calcium-free
(n = 8)
24.11
values.
using glass microelectrodes filled with 3 M KCI. The output of a microelectrode preamplifier (Nihon MEZ-8201)
______._
RF
En” I f:;
action
*P
Kohden,
condition
SE.
tP
penetrations
(n = IO)
Ca free EGTA
Neonate
6949.1
(n = IO)
3827.9
were
(n = 14)
values.
LP (n = I I)
k 98.2
12,020.0?
885.6;
values.
RF Forskolin
f
118.0 k l2.7t
Adult
time
muscle;
SE.
tP
EGTA
RF = right
papillary
bv isooroterenol
RP
Adult
The
significant.
LP = left ventricular
in msec.
Table
time
conditions
19.8 f 0.9
(n = 8)
action
corresponding
ventricular
(n = IO)
RF
The
normal
LF
19.8 + 2.5
(n = 8)
50%
significant
under
RP
13.4k2.1
Neonate
duration
41
was changed into 1 mM EGTA. In
this solution, prolongation of the action potential was observed upon the addition of isoproterenol (ISO),
b
forskolin, isobutyl-methylaxanthine (IBMX) or dibutyril cyclic adenosine monophosphatc (dbcAMP). The time required for 50% repolarization was measured and presented in mean f SEM. Significant difference between means were evaluated by Williams’ multiple-range test. A P value less than 0.05 was considered significant.
b \
II P----_!
I
_I‘\._ C
-
A- ,1 \:\
RESULTS The normal ventricular action potential of the adult rat had a short action potential with little evidence of a plateau phase (Fig. 1). The action potential duration expressed in terms of time required for 50% repolarization tended to be shorter in the
50 mV
100
ms
Fig. 2. Action potential prolongation in neonatal rat myocardium. Action potentials of right ventricular free wall (RF) and left ventricular papillary muscle (LP) were recorded under normal conditions (a); Ca free-EGTA conditions (b) and after further addition of IO-” M isoproterenol (c). Short horizontal bars indicate 0 mV and the longer horizontal bars indicate time.
Hikaru Tanaka et al.
42
right ventricular free wall (RF) than in the right ventricular papillary muscle (RP), left ventricular free wall (LF) and left ventricular papillary muscle (LP), but the difference was not significant (Table 1). Changing the bathing solution into Ca free-EGTA
the neonate results in a slower rate of inactivation of Ca channels and an action potential with a normal plateau phase. The adult rat myocardium is known to
solution duration
resulted in a decrease in action potential in RF while it resulted in an increase in RP,
Willerson, 1980; Tanaka and Shigenobu, also its electrophysiological properties
LP. Upon
et al., 1984a, b) are known to be affected by ryanodine. Whether intracellularly released Ca from the
LF and IBMX plateau
the addition
of ISO, forskolin,
or dbcAMP, extreme prolongation of the phase was observed. In the case of ISO, the
have a highly developed sarcoplasmic reticulum. Not only its contractile force (Penefski, 1974; Sutko and
highly
developed
SR of the adult
prolongation in RF was significantly smaller than in other three regions (Fig. 1, Table 2). Also in the cases
accelerates Ca channel unique action potential
of forskolin and IBMX, the prolongation plateau phase by the drugs were significantly
interest.
of the larger in
LP when compared with RF. In the case of dbcAMP, however, no regional difference was observed (Fig. 1, Table 3). The action
potential
of
neonatal
ventricular
preparations had a plateau phase of about 200 msec. Changing the bathing solution into Ca free-EGTA solution
resulted
in a decrease
in action
potential
duration. Upon the addition of ISO, forskolin, IBMX or dbcAMP, extreme prolongation of the plateau phase was observed, which was qualitatively similar to that observed in adult preparations. The extent of prolongation, however, was significantly smaller in the neonate examined difference
than in the adult for all of the drugs
(Fig. 2, Tables 2 and 3). No regional between RF and LP was observed with any
of the drugs in the neonate
(Tables
2 and 3).
has a short
action
potential
with little evidence of a plateau, which has been attributed not only to a large contribution of repolarizing K currents such as I,, (Kilborn and Fedida, 1990) and I,,] (Wahler, 1992) but also to the kinetics of the Ca current in this species (Cohen and Lederer, 1988). In dbcAMP observed potential
our previous study, we applied IS0 or under Ca free EGTA condition and prolongation of the myocardial action of rats and guinea pigs. We pharmaco-
logically
demonstrated
that
rat myocardium
inactivation configuration
to produce its is a matter of
In adult myocardia, action potential prolongation under Ca free EGTA condition by ISO, forskolin or IBMX was smaller in RF than in LP. The concentrations of these three drugs were those previously reported to produce in isolated ventricular
maximum inotropic responses myocardia from neonatal and
adult rats (Shigenobu
et al., 1988). A common
of these three drugs
is their action
CAMP related mechanisms.
feature
on endogenous
In contrast
to these three
agents, no regional difference was observed with dbcAMP, which directly liberates CAMP intracellularly. These results suggest that regional differences may be present in the mechanisms prior to the CAMP production, e.g. lower basal CAMP turnover in RF, but direct evidence awaits further investigation. Contrary observed
to the adult, regional in neonatal myocardia
difference indicating
was not that the
difference may occur during postnatal development. The developmental increase in the degree of prolongation and the appearance of regional difference
DISCUSSION
The rat myocardium
1989) but (Mitchell
this prolongation
was
due to persistent influx of Na ions through Ca channels due to lack of its inactivation by intracellular Ca under such condition (Tanaka et al., 1994). The large degree of action potential prolongation in the adult rat observed in the present study suggests that a large degree of Ca inactivation of Ca channels contributes to the rapid repolarization of its action potential. In contrast, the degree of action potential prolongation under Ca free EGTA condition in the neonate was much smaller than in the adult. This suggests that a smaller degree of Ca inactivation in
may reflect developmental properties
variations
in the functional
of these muscles.
REFERENCES
Agata N., Tanaka H. and Shigenobu K. (1992) Developmental changes in the pharmacological properties of guinea pig myocardial contraction. Folia Phnrmac. Jap. 100, 14. Antzelevitch C., Sicouri S., Litovsky S. H., Lukas A., Krishnan S. C., Di Diego J. M., Gintant G. A. and Liu D.-W. (1991) Heterogeneity within the ventricular wall: electrophysiology and pharmacology of epicardial, endocardial and M cells. Circ. Res. 69, 1427-1449. Bechem M. and Pott L. (1985) Removal of Ca current inactivation in dialysed guinea-pig atria1 cardioballs by Ca chelators. Pflugks A&hs 404,10-20. Bers D. M.. Philiason K. D. and Lancer G. A. (1981) Cardiac contra&ty and sarcolemmal calcium binding in several cardiac muscle preparations. Am. J. Physiol. 240, H5766H583. Cohen N. M. and Lederer W. J. (1988) Changes in the calcium current of rat heart ventricular myocytes during development. J. Physiol. 406, 115-146. _ Fabiato A. and Fabiato F. (1978) Calcium-induced release of calcium from the sarcbplasmic reticulum of skinned cells from adult human, dog, cat, rat and frog hearts and
Calcium
free-EGTA
from fetal and newborn rat ventricles. Ann. N.Y. Acad. Sci. 307, 491-522. Hess P. and Tsien R. W. (1984) Mechanism of ion permeation through calcium channels. Nafure 309, 453-456. Josephson I. R., Sanchez-Chapula J. and Brown A. M. (1984) A comparison of Ca currents in rat and guinea-pig single ventricular cells. Circ. Res. 54, 144&156. Kass R. S. and Sanguinetti M. C. (1984) Inactivation of Ca channel current in the calf cardiac Purkinje fiber: evidence for voltage and Ca mediated mechanisms. J. gen. Physiol. 84, 705-726. Kilborn M. J. and Fedida D. (1990) A study of the developmental changes in outward currents of rat ventricular myocytes. J. Physiol. 430, 37-60. Linden J. and Brooker G. (1982) Evidence for persistent activation of cardiac slow channels in low-calcium solutions. Am. J. Physiol. 242, H827-H833. Marban and Tsien (1981) Is the slow inward calcium current of heart muscle inactivated by calcium? Biophys. J. 33, 143a. Mentrard D., Vassort G. and Fischmeister R. (1984) Calcium-mediated inactivation of calcium conductance in cesium-loaded frog heart cells. J. gen. Physiol. 85, 105-131. Miller D. J. and Morchen A. (1978) On the effects of divalent cations and ethylene glycol-bis-(p-aminoethyl ether)N,N,N’,N’-tetraacetate on action potential duration in frog heart. J. gen. Physiol. 71, 47-67. Mitchell M. R., Powell T., Terrar D. A. and Twist V. W. (1984a)The effects of ryanodine, EGTA, and low-sodium on action potentials in rat and guinea-pig ventricular myocytes: evidence for two inward currents during the plateau Br. J. Pharmac. 81, 543-550. Mitchell M. R., Powell T., Terrar D. A. and Twist V. W. (1984b) Possible association between an inward
condition
in rats
43
current and the late plateau of action potentials in ventricular cells isolated from rat heart. J. Physiol. 351, 4OP. Penefski Z. J. (1974) Studies on mechanism of inhibition of cardiac muscle contractile tension by ryanodine. P~¶ugers Archs 347, 173-I 84. Shigenobu K., Tanaka H. and Kasuya Y. (1988) Changes in sensitivity of rat heart to norepinephrine and isoproterenol during pre- and postnatal development and its relation to sympathetic innervation. Dev. Pharmac. Thu. 11, 226-236. Sutko J. L. and Willerson J. T. (1980) Ryanodine alteration of the contractile state of rat ventricular myocardium. Comparison with dog, cat, and rabbit ventricular tissues. Circ. Res. 46, 332-343. Tanaka H. and Shigenobu K. (1989) Effect of ryanodine on neonatal and adult rat heart: developmental increase in sarcoplasmic reticulum function. J. Molec. Cell. Cardiol. 21, 1305-1313. Tanaka H., Noguchi K. and Shigenobu K. (1994) Myocardial action potential prolongation by calcium channel activation under calcium free-EGTA condition in guinea pigs and rats. Gen. Pharmac. 25, 475-480. Tseng G.-N. (1988) Calcium current restitution in mammalian ventricular myocytes is modulated by intracellular calcium. Circ. Res. 63, 4688482. Tritthart H., MacLeod D. P., Stierle H. E. and Krause H. (1973) Effects of Ca-free and EDTA-containing Tyrode solution on transmembrane electrical activity and contraction in guinea pig papillary muscle. Pflugers Archs 338, 361-376. Wahler G. M. (1992) Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes. Am. J. Physiol. 262, Cl266-C1272.