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Junctional permeability in heart muscle is independent upon the non-junctional membrane potential
Cell Biology
International
Reports,
JUNCTIONAL PERMEABILITY NON-JUNCTIONAL MErlBRANE !;Jalmor
Vol. 7 1, No. 1, January
IN HEAIIT ?IUSCLE POTENTIAL
C.
De Mello
Department of Pharmacoloqy, 5367, San Juan, Puerto Rico
Medical 00936
and
IS
P.
Il'!DEPENDENT
van
Sciences
1987
UPON THE
Loon* Campus,
G.P.O.
Box
ABSTRACT: The influence of high K solution on the longitudinal movement of Lucifer Yellow CH along doq atria1 trabeculae was investigated. It was found that in normal heart muscle the dye diffused from cell -to-cell and the average diffusion coefficient (D) was 4.3 f 1.3 x 10-7 cm2/s. In muscles exposed to 20, 40 or 60 m"! K solution the resting potential was reduced from -78 mV (S.E. f 0.71) (control) to -41 mV (S.E. f 0.95), -30 mV (S.E. f 0.64) and -22.5 mV (S.E. f 0.64), respectively. Despite the maintained depolarization the cell-to-cell diffusion of Lucifer Yellow CH did not chanqe. These findings indicate that the ;junctional permeability in heart muscle is not influenced by the non-jucntional membrane potential. Key
!,;ords:
,junctional
permeability,
depolarized
heart
Cardiac cells are connected through low resistance making possible the exchanqe of electrical and chemical between the apposinq myocytes (see De Flello, 1982).
junctions messages
Evidence is available that the permeability of the intercellular channels is greatly influenced by hiqh free {Ca2+)i (De 'lello. 1975; Rose and Loewenstein, 1975), low pHi (Turin and I,'arner, 1977, 1980; De plello, 1980; Spray et al., 1931) and by cl\.'lP (De :lello, 1984; Flagg-Newton et al., 1981). It is known that cardiac muscle is a heteroqeneous structure with areas showing a wide variation of resting potentials (see tioffman and Cranefield, 1960; De Mello, 1972). Indeed, the electrical impulse which is originated in cells of the sinoatrial node !dith a maximal diastolic potential of about -60 mV flows into atria1 cells with a much hiqher internal neqativity (-80 mV) and then crosses the atrioventricular node (restinq potential 60-65 mV) to reach the His bundle (resting potential -80 mV). *Department San Juan,
of Physiology, Yedical Puerto Rico 00936
0309-1651'87~010001-11~$03.00/0
Sciences
@ 1987
Campus,
Academic
GPO Box
Press
Inc.
5067,
(London)
Ltd.
Cell Biology
International
Reports,
Vol. 7 1, No. 1, January
1987
Although it is known that the conduction velocity depends on t.je intracellular longitudinal resistance (ri) and is closely related to the fiber diameter, no information is available if the resting potential, per se, is able to influence the junctional permeability in cardiac fibers. In the present work this problem was investigated. MATERIALS AND METHODS Muscle trabeculae were dissected from left atrium of dog's heart. The animals were anesthetized with sodium pentobarbital, the heart immediately removed and immersed in a cooled oxygenated Tyrode solution. Muscle trabeculae (5 to 6 mm in length; 1 mm in diameter) were excised from the left atrium and transferred to a Perspex bath through which Tyrode solution at 37°C saturated with a gas mixture (95% 02, 5% CO2). flowed at a rate of 0.5 ~1 solution mg-I of tissue, s-I. The composition of Tyrode solution was as follows (mll): NaCl - 137; KC1 - 5.4; NaHC03 - 12; MgCl - 0.5; NaH2PD4 - 3.6; CaC12 - 2.7; dextrose - 5.5 (pH - 7.1 7 . Cut-end
method.
The longitudinal movement of Lucifer Yellow along quiescent muscle trabeculae was followed with the cut-end method (Imanaga, 1974). The preparations were kept in normal Tyrode solution at 37°C to heal-over, and then pulled through a tight-fitting hole in a rubber membrane which separated a Perspex chamber into two Normal Tyrode solution flowed continously through compartments. both compartments. Forty min. later the right segment of the trabeculae was exposed to Ca-free solution plus EGTA (0.5 m'l) for 10 min. to avoid healing-over (De "lello, 1972) and then cut with a fine pair of scissors within 1 mm from the rubber partition. After lesion the compartment was filled with Ca-free EGTA solution containing 0.1% of Lucifer Yellow and the damaged tissue was Samples of Tyrode solution were exposed to the dye for 10 min. taken from the left compartment every 3 min. after the introduction of Lucifer Yellow into the right compartment to check the possible leak of the dye to the left side. The dye was then removed from the right compartment and the cut-end exposed to normal Tyrode solution for 45 min. Toluidine blue (5 mg/lOO ml) added made the site of the partition visible. to determine the length of the loading The exposure of the muscle to this dye cal or mechanical properties of cardiac The preparation was pulled out of the frozen stretch in small rubber plate, cut into slices of 0.5 mm. The slices
to the right compartment This procedure was used region (h in equation 1). does not alter the electrifibers (Ia!eidmann, 1966). rubber membrane, laid without in liquid nitrogen and then were weighed and transferred
Cell Biology
International
Reports,
Vol. 11, No. 1, January
1987
to vials containing 4 ml of distilled water and the cells were The vials were kept in the dark overdisrupted by boiling water. night. The samples were then passed through a millipore filter and the fluorescence WIS read in a spectrofluorometer (430 mm excitation; 540 nm emission). To investigate the possible influence of membrane potential on the cell-to-cell diffusion of Lucifer Yellow both segments of the trabecula were exposed to Tyrode solution containing 20, 40 or 60 mtl K, respectively. In these solutions the K concentration was increased, but the concentration of the other salts was kept constant. As the depolarization of heart cell membrane by hiqh K solutions enhances the free {Ca2+}i and the resting tensi n, I decided to follow the time-course of K contractures in order to determine the time required for the total reversion of the increase in tension. This allows us to study the longitudinal diffusion of Lucifer Yellow CH in depolarized fibers but only when the increase in free {Ca2+3i was totally reversed, Fiq. 1 shows the time-course of the K contracture exposing the trabecula to 60 ml1 K solution. As it increment in resting tension was totally reversed Considering that an increase in free (Ca2f)i might communication (De Mello, 1975) the loading of the Lucifer Yellow CH was initiated only after 45 min. high K solution, that is, when the resting tension to normal levels.
achieved by can be seen the n about 30 min. impair cell ibers with of exposure to already returned
Theory The diffusion of a substance in a cylinder of length 1 which was initially confined to the region - h < x i h is described by the following equation assuming no loss of tracer through the surface cell membrane
c (x,t)
5 l/2
co (t
= 0)
n=l --I J/A
e~f(~!J~~~Z!?l~~-x -)+ erf( h~Z!Jl-t-~ \ 2 nt / ’ ) 2 Dt
n =-1 see Crank (1956) where C (x,t) fusing substance at a distance (diffusion time).
is the concentrat x from the origin
ion of the difafter a t ime t
Cell Biology
the
D is
the
diffusion
n is ends
the has
integer, taken
Co is
initial
International
Reports,
coefficient
in
increasing place. concentration
the of
1987
cm*/s. number
the
Vol. 11, No. 1, January
dye
of in
times the
reflection
loading
at
compart-
ment. For reflection to take place the concentration at the end of the cylinder has to be significantly different from zero. Since our data show the concentration of Lucifer Yellow at the end of the trabeculae was less than 5% of the concentration at the origin we took one reflection into consideration (n = 1). The value of D was obtained by an interactive computer program for non-linear regression. According to this procedure the value of D corresponds to the curve for which the sum of squares of the deviation is minimal. Electrical
recording.
Measurements of resting potential from left and right segments of the trabeculae were made 40 min after mounting the muscle into the chamber and at the end of 80 min of equilibration in high K solution. Only muscles with normal membrane potential were used. The resting potential was measured with intracellular microelectrodes filled with 3 tl KC1 (Ling and Gerard, 1949) connected to a high impedance DC amplifier (!J.P. Instruments). The voltage changes were displayed on both on oscilloscope and pen recorder (Gould Instruments). Tension
recording.
Strips of dog right atrium muscle were mounted vertically in a transparent chamber through which oxygenated Tyrode solution at The muscles were stimulated through 37°C flowed continuously. platinum plate electrodes mounted vertically inside the bath. Rectangular pulses of 5 msec duration of thresfold strenqth, were delivered at 1 Hz by an electronic stimulator through an isolation unit. The muscle strips were connected via a stainless steel wire At the beginning of the experiment, a to a Grass transducer F03. resting tension of about 350 mq was established by gently stretchThe contractile responses ing the preparation with the transducer. were recorded isometrically and displayed on-a Grass recorder, at the same time they were observed with a Tektronix oscilloscope and photographed. Efflux tionDZEl
studies. SSQ~e!~~aZOw~!~.di?2
ected and kept e preparations
in noryal were t,en
Tvrod? t ans
sol!erre
Cell Biology
international
Reports,
Vol. 11, No. 1, January
1987
5
to an oxygenated Ca-free EGTA (C.5 mM) solution for 10 min cut and loaded with Lucifer Yellow CH for 10 min. The muscles were then immersed in normal Tyrode with the dye for 20 min., transferred to saline solution free of the dye for 5.s to wash Lucifer Yellow CH from the external surface of the cells and then to a series of vials containing 5 ml oxygenated Tyrode solution for periods of 1 to 20 min. To investigate curves damaged sclution were
the possible effect muscles equilibrated loaded with Lucifer
of for Yellow
high K solutions on efflux 80 min in 60 mM K CH as described above.
The amount of Lucifer Yellow CH in the rinsing solution was determined in aspectrofluorometer. The muscles were weighed blotted on filter paper and the cells destroyed in boiling distilled water. From the amount of Lucifer remaining in the fibers and that lost in each vial the total fluorescence in the Efflux studies fibers was determined at each collecting period. were also performed in non-injured muscles exposed to high K solution and loaded with Lucifer Yellow CH for the period of time (see above).
Fig. 1 - Contracture produced by high K (60 mM) solution. As it can be seen the increase in resting tension was completely reversed in about 25 min. Temperature - 37°C.
Cell Biology
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RESULTS
Reports,
Vol. 11, No. 1, January
1987
AND DISCUSSION
Fig. 2 shows the typical distribution of Lucifer Yellow CH along a single dog trabecula superfused in normal Tyrode solution. As it can be seen the redistribution of the dye took place over distances much larger than the length of a single heart cell (125,#m) (Marceau, 1904). Efflux studies performed on trabeculae exposed to 60 mM K solution for 80 min indicated: 1) in non-damaged muscles the washout curve wa characterized by a quick reduction of fluorescence 90 (half-life 1 min.) suggesting a fast removal of the dye from the extracellular space. In damaged muscles exposed to 602p K, loaded with Lucifer Yellow CH and then healed with normal fCa jo in presence of the dye the "56 lux . curve showed not only an initial min.) but also a second very slow -;-,;;P;p$ ct1/2 = 3 - 2 x 10 min.) which can be ascribed to an extremely small diffusion of the compound through the surface cell membrane, These results are identical to those described in normal fibers (De Mello et al., 1983). Considering that the permeability of the surface cell membrane to Lucifer Yellow CH is extremely low (De Mello et al., 1983) the present results support the view that cardiac cells are connected through permeable channels. The quantitative evaluation of the diffusion was made by fitting the experimental results as described in Methods. Th? regults showed the mean) of 4.3 _t 1.3 x locm /s (see Fig.
coefficient (D) to theoretical points a D (mean _t S.E. of 2 and Table I).
As sealing of the cut-end is essential for the applicability 1974) control measurements of of the cut-end method (Imanaga, Twenty resting potential performed immediately after lesion. minutes after damage the resting potential recorded very near n = 10) was similar (0.05 cm) the cut-end (31 mV; S.E. ;t 0.70; that recorded far away (0.17 cm) (33 mV; S.E. t 0.79; n = 12) the lesion (see Weidmann, 1952; De Mello, 1983).
to
from
Table 1 shows values of diffusion coefficient (d) determined from control preparations exposed to 40 and 60 mM K solution. AS it can be seen the diffusion coefficient was not different from the controls (P70.05). As stressed in Methods these experiments were performed when the increment in resting tension produced by high K This means that the longitudinal solutions was totally reversed. in depolarized heart movement of Lucifer Yellow Cg+ was followed . . )i similar to that present in normal fibers but with the free fCa fibers.
Cell Biology
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Reports,
Vol. 11, No. 1, January
1987
MO=60 mM D = 3.9 x 10~7Cmz/s t
= 45min
h = lmm
mm.
2 - Longitudinal redistribution of Lucifer Yellow CH Fig. along a dog trabecula immersed in high K. solution (60 mM). The fluorescence from 0.5 mm slices are normalized by total fluorescence and plotted against distance from cut-end. The points represent experimental values which ar_e7fitsed by a theoretical diffusion curve with D = 3.9 x 10 The cm /s. dotted line separates the loaded (left) from the unloaded segment. Diffusion period - 45 min. Length of the preparation - 4 mm. Temperature - 3i"C.
8
Cell Biology
International
Reports,
Vol. 11, No. 1, January
1987
TABLE 1 Lack of influence of high K solutions on the longitudinal of Lucifer Yellow CH along dog trabeculae Experiment
Resting potential
{KIo
Diffusion period (min.)
(mv) 5.4 5.4 5.4 5.4 5.4
-80 (n = 15) -76 (n = 20) -78 (n = 16) -79
-77 X
(n = 18) (n = 20)
45 45 45 45 45
1 1 1
-
-
45 45 45 45
1.1 1 1 1
1 1.1
= -78 + 0.71
-
20 20 20 20
Length of loaded segment (mm)
-40 -42 -44 -41
(n (n (n (n
= = = =
14) 20) 20) 20)
-31 -30 -32 -30
(n (n (n (n
= = = =
15) 20) 20) 20)
-
-
45 45 45 45
1 1.1 1.1 1
X = -30 + 0.64
+ S.E.
of mean
-24 (n = 21) -21 (n = 20) -23 (n = 18) -22 (n = 16) X = 22.5 + 0.64
1.8 9
6 1.8 3.2 x = 4.3 + 1.3 7.4 1.9
4.8 3.9
5 3.6 4.9 5.9
X = 4.85 f 0.47 ---
60 60 60 60
Diffusion coefficient j;17jcm2/s
x = 4.5 + 1.14
x = -41 f 0.95
40 40 40 40
movement
-
-
45
1
4.8
t: 45
1.1 1.1 1
5.9
7.3 3 X = 5.2 f 0.90
x
Cell Biology
International
Reports,
Vol. 11, No. 7, January
7987
9
The present results indicate that prolonged depolarization of the surface cell membrane, per se, has no influence on junctional These findings are in agreement permeability in cardiac fibers. with studies on the influence of membrane potential on junctional conductance performed in cell pairs isolated from the guinea-pigs or rat hearts (Kameyama, 1983; White et al., 1984; Metzger and Weingart, 1985; Weingart, 1986). In Chironomus salivary glands the junctional conductance is strongly influenced by the voltage between the bath and cytoplasm (Obaid et al., 1983). In this tissue equal depolarization of the two apposing cells reduces the junctional conductance while equal hyperpolarization of both cells enhances the conductance (Obaid et al .,1983). In squid blastomers the junctional conductance is affected by both transjunctional and the membrane potential (see Spray et al., 1981). The lack of influence of prolonged cell-to-cell diffusion of Lucifer indicates that variations in the ance found in normal heart (Spach differences in values of membrane in fiber diameter, number and size
depolarization (per se) on the Yellow CH along heart fibers intracellular longitudinal resistet al., 1982) are not due to potential per se, but no changes of gap junctions.
ACKNOWLEDGMENTS
This We are grateful to Maria Gonzglez for her technical work. work was supported by Grant Nos. HL-34353 and HL-34148 from the National Heart Institutes, Bethesda, Maryland.
REFERENCES
Crank, J. Claredon
(1956) Press.
The
De Mello, W. C. (1972) De Mello. New York:
Mathematics
of
Electrical Academic
Press,
Diffusion.
Phenomena in pp 323-351.
De Mello, W. C. (1975 and strontium on ce 245
Effect of intracellular 1 communication in heart.
De Me 110, W. C. (1980 the electrical coup Rep . 4, 51-57.
ing
Influence of in cardiac
1st
intracellular Purkinje
the
edn.
Heart,
injection of J. Physiol.
fibres.
injection Cell
Oxford:
ed.
W. C.
calcium 250, 231,
of H+ on Biol. Int
10
De Mello, tissues.
Cell Biology
international
W. C. (1982) Cell-to-cell Prog. Biophys. Molec.
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communication in heart Biol. 39, 147-182.
1987
and other
De Mello, W. C. (1983) The influence of pH on the healing-over mammalian cardiac muscle. J. Physiol. 339, 299-307.
of
De Mello, W. C. (1984) Effect of intracellular injection of CAMP on the electrical coupling of mammalian cardiac cells. Biochem. Biophys. Res. Comm. 119, 1001-1007. De Mello, cellular J. Mol.
W. C., Gonzalez-Castillo, M. and van Loon, P. (1983) diffusion of Lucifer Yellow CH in mammalian cardiac Cell Cardiol. 15, 637-643.
Interfibers.
Flagg-Newton, J. L., Dahl, G. and Loewenstein, W. R. (1981) Cell junction and cyclic AMP: I. Upregulation of junctional membrane permeability and junctional membrane particles by administration of cylic nucleotide or phosphodiesterase inhibitor. J. Membrane Biol. 63, 105121. Hoffman, B. F. and Cranefield, P. (1960) McGraw-Hill Book Co., New York.
Electrophysiology
Kameyama, M. (1983) Electrical coupling between isolated from guinea-pig hearts. J. Physiol., Ling, G. and Gerard, sartorius fibers.
Heart.
ventricular paired 336, 345-357.
cells
R. W. (1949) The normal membrane potential J. Cell Comp. Physiol. 34, 383-396.
Marceau, F. (1904) Recherches sur la structure compares des fibres cardiaques dans la se'rie Annls Sci. nat. (Zool.). 19,191-365. Metzger, pairs 195.
of the
of frog
et de development des vertebres.
P. and Weingart, R. (1985) Electric current isolated from adult rat hearts. J. Physiol.
flow 366,
in cell 177-
Obaid, A. L., Socolar, S. J. and Rose, B. (1983) Cell-to-cell channels with two independent regulated gates in series: analysis of junctional channel modulation by membrane potential, calcium and pH. J. Membr. Biol. 73 69-89. --) Rose, B. and Loewenstein, W. R. (1975) Calcium ion distribution in cytoplasm visualized by aequorin: diffusion in cytosol restricted by energized sequestering. Science, 190, 1204-1206. Spach, M. S., Miller, role of structural tion in the atrium to discontinuiti-s 175-191.
W. T., Dolber, D. C. (1982) The functional complexities in the propagation of depolarizaof the dog. Cardiac conduction disturbances of effective axial resistivity. Circ. Res.
due 50,
Cell Biology
International
Reports,
Vol. 11, No. 1, January
Spray, D. C., Harris, A. L. and Bennett, M. V. L. (1981). properties of a voltage dependent junctional conductance. Physiol. 77, 77-93. Turin, L, and Warner, A. E. (1977) ionic communication between cells Nature (Lond.), 270, 56-57.
11
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Equilibrium J.gen.
Carbon dioxide reversibly abolishes of early amphybian embryon.
Intracellullar pH in early Xenopus Turin, L., and Warner, A. E. (1980) embryos: its effect on current flow between blastomeres. J. Physiol. 300, 489-504. Weidmann, Physiol.
S. (1952) The electrical (Lond.) 118, 348-360.
constants
of
Purkinje
fibres.
J.
Weidmann, S. (1966) discs of mammalian
The diffusion of radiopotassium across intercalated cardiac muscle. J. Physiol. (Lond.) 187, 323-342.
Weingart, in rat
Electrical cell pairs.
R. (1986) ventricular
properties of the nexal membrane studied J. Physiol. (Lond.) 370, 267-284.
White, R. L., Spray, D. C., Schwartz, G. J. ,.bJittenberg, B. A. and Bennett, M. V. L. (1984) Some physiological and pharmacological properties of cardiac gap junctions. Biophys. J. -’45 279a.
Received
6.10.86
Accepted
11.11.86
International
Reports,
JUNCTIONAL PERMEABILITY NON-JUNCTIONAL MErlBRANE !;Jalmor
Vol. 7 1, No. 1, January
IN HEAIIT ?IUSCLE POTENTIAL
C.
De Mello
Department of Pharmacoloqy, 5367, San Juan, Puerto Rico
Medical 00936
and
IS
P.
Il'!DEPENDENT
van
Sciences
1987
UPON THE
Loon* Campus,
G.P.O.
Box
ABSTRACT: The influence of high K solution on the longitudinal movement of Lucifer Yellow CH along doq atria1 trabeculae was investigated. It was found that in normal heart muscle the dye diffused from cell -to-cell and the average diffusion coefficient (D) was 4.3 f 1.3 x 10-7 cm2/s. In muscles exposed to 20, 40 or 60 m"! K solution the resting potential was reduced from -78 mV (S.E. f 0.71) (control) to -41 mV (S.E. f 0.95), -30 mV (S.E. f 0.64) and -22.5 mV (S.E. f 0.64), respectively. Despite the maintained depolarization the cell-to-cell diffusion of Lucifer Yellow CH did not chanqe. These findings indicate that the ;junctional permeability in heart muscle is not influenced by the non-jucntional membrane potential. Key
!,;ords:
,junctional
permeability,
depolarized
heart
Cardiac cells are connected through low resistance making possible the exchanqe of electrical and chemical between the apposinq myocytes (see De Flello, 1982).
junctions messages
Evidence is available that the permeability of the intercellular channels is greatly influenced by hiqh free {Ca2+)i (De 'lello. 1975; Rose and Loewenstein, 1975), low pHi (Turin and I,'arner, 1977, 1980; De plello, 1980; Spray et al., 1931) and by cl\.'lP (De :lello, 1984; Flagg-Newton et al., 1981). It is known that cardiac muscle is a heteroqeneous structure with areas showing a wide variation of resting potentials (see tioffman and Cranefield, 1960; De Mello, 1972). Indeed, the electrical impulse which is originated in cells of the sinoatrial node !dith a maximal diastolic potential of about -60 mV flows into atria1 cells with a much hiqher internal neqativity (-80 mV) and then crosses the atrioventricular node (restinq potential 60-65 mV) to reach the His bundle (resting potential -80 mV). *Department San Juan,
of Physiology, Yedical Puerto Rico 00936
0309-1651'87~010001-11~$03.00/0
Sciences
@ 1987
Campus,
Academic
GPO Box
Press
Inc.
5067,
(London)
Ltd.
Cell Biology
International
Reports,
Vol. 7 1, No. 1, January
1987
Although it is known that the conduction velocity depends on t.je intracellular longitudinal resistance (ri) and is closely related to the fiber diameter, no information is available if the resting potential, per se, is able to influence the junctional permeability in cardiac fibers. In the present work this problem was investigated. MATERIALS AND METHODS Muscle trabeculae were dissected from left atrium of dog's heart. The animals were anesthetized with sodium pentobarbital, the heart immediately removed and immersed in a cooled oxygenated Tyrode solution. Muscle trabeculae (5 to 6 mm in length; 1 mm in diameter) were excised from the left atrium and transferred to a Perspex bath through which Tyrode solution at 37°C saturated with a gas mixture (95% 02, 5% CO2). flowed at a rate of 0.5 ~1 solution mg-I of tissue, s-I. The composition of Tyrode solution was as follows (mll): NaCl - 137; KC1 - 5.4; NaHC03 - 12; MgCl - 0.5; NaH2PD4 - 3.6; CaC12 - 2.7; dextrose - 5.5 (pH - 7.1 7 . Cut-end
method.
The longitudinal movement of Lucifer Yellow along quiescent muscle trabeculae was followed with the cut-end method (Imanaga, 1974). The preparations were kept in normal Tyrode solution at 37°C to heal-over, and then pulled through a tight-fitting hole in a rubber membrane which separated a Perspex chamber into two Normal Tyrode solution flowed continously through compartments. both compartments. Forty min. later the right segment of the trabeculae was exposed to Ca-free solution plus EGTA (0.5 m'l) for 10 min. to avoid healing-over (De "lello, 1972) and then cut with a fine pair of scissors within 1 mm from the rubber partition. After lesion the compartment was filled with Ca-free EGTA solution containing 0.1% of Lucifer Yellow and the damaged tissue was Samples of Tyrode solution were exposed to the dye for 10 min. taken from the left compartment every 3 min. after the introduction of Lucifer Yellow into the right compartment to check the possible leak of the dye to the left side. The dye was then removed from the right compartment and the cut-end exposed to normal Tyrode solution for 45 min. Toluidine blue (5 mg/lOO ml) added made the site of the partition visible. to determine the length of the loading The exposure of the muscle to this dye cal or mechanical properties of cardiac The preparation was pulled out of the frozen stretch in small rubber plate, cut into slices of 0.5 mm. The slices
to the right compartment This procedure was used region (h in equation 1). does not alter the electrifibers (Ia!eidmann, 1966). rubber membrane, laid without in liquid nitrogen and then were weighed and transferred
Cell Biology
International
Reports,
Vol. 11, No. 1, January
1987
to vials containing 4 ml of distilled water and the cells were The vials were kept in the dark overdisrupted by boiling water. night. The samples were then passed through a millipore filter and the fluorescence WIS read in a spectrofluorometer (430 mm excitation; 540 nm emission). To investigate the possible influence of membrane potential on the cell-to-cell diffusion of Lucifer Yellow both segments of the trabecula were exposed to Tyrode solution containing 20, 40 or 60 mtl K, respectively. In these solutions the K concentration was increased, but the concentration of the other salts was kept constant. As the depolarization of heart cell membrane by hiqh K solutions enhances the free {Ca2+}i and the resting tensi n, I decided to follow the time-course of K contractures in order to determine the time required for the total reversion of the increase in tension. This allows us to study the longitudinal diffusion of Lucifer Yellow CH in depolarized fibers but only when the increase in free {Ca2+3i was totally reversed, Fiq. 1 shows the time-course of the K contracture exposing the trabecula to 60 ml1 K solution. As it increment in resting tension was totally reversed Considering that an increase in free (Ca2f)i might communication (De Mello, 1975) the loading of the Lucifer Yellow CH was initiated only after 45 min. high K solution, that is, when the resting tension to normal levels.
achieved by can be seen the n about 30 min. impair cell ibers with of exposure to already returned
Theory The diffusion of a substance in a cylinder of length 1 which was initially confined to the region - h < x i h is described by the following equation assuming no loss of tracer through the surface cell membrane
c (x,t)
5 l/2
co (t
= 0)
n=l --I J/A
e~f(~!J~~~Z!?l~~-x -)+ erf( h~Z!Jl-t-~ \ 2 nt / ’ ) 2 Dt
n =-1 see Crank (1956) where C (x,t) fusing substance at a distance (diffusion time).
is the concentrat x from the origin
ion of the difafter a t ime t
Cell Biology
the
D is
the
diffusion
n is ends
the has
integer, taken
Co is
initial
International
Reports,
coefficient
in
increasing place. concentration
the of
1987
cm*/s. number
the
Vol. 11, No. 1, January
dye
of in
times the
reflection
loading
at
compart-
ment. For reflection to take place the concentration at the end of the cylinder has to be significantly different from zero. Since our data show the concentration of Lucifer Yellow at the end of the trabeculae was less than 5% of the concentration at the origin we took one reflection into consideration (n = 1). The value of D was obtained by an interactive computer program for non-linear regression. According to this procedure the value of D corresponds to the curve for which the sum of squares of the deviation is minimal. Electrical
recording.
Measurements of resting potential from left and right segments of the trabeculae were made 40 min after mounting the muscle into the chamber and at the end of 80 min of equilibration in high K solution. Only muscles with normal membrane potential were used. The resting potential was measured with intracellular microelectrodes filled with 3 tl KC1 (Ling and Gerard, 1949) connected to a high impedance DC amplifier (!J.P. Instruments). The voltage changes were displayed on both on oscilloscope and pen recorder (Gould Instruments). Tension
recording.
Strips of dog right atrium muscle were mounted vertically in a transparent chamber through which oxygenated Tyrode solution at The muscles were stimulated through 37°C flowed continuously. platinum plate electrodes mounted vertically inside the bath. Rectangular pulses of 5 msec duration of thresfold strenqth, were delivered at 1 Hz by an electronic stimulator through an isolation unit. The muscle strips were connected via a stainless steel wire At the beginning of the experiment, a to a Grass transducer F03. resting tension of about 350 mq was established by gently stretchThe contractile responses ing the preparation with the transducer. were recorded isometrically and displayed on-a Grass recorder, at the same time they were observed with a Tektronix oscilloscope and photographed. Efflux tionDZEl
studies. SSQ~e!~~aZOw~!~.di?2
ected and kept e preparations
in noryal were t,en
Tvrod? t ans
sol!erre
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1987
5
to an oxygenated Ca-free EGTA (C.5 mM) solution for 10 min cut and loaded with Lucifer Yellow CH for 10 min. The muscles were then immersed in normal Tyrode with the dye for 20 min., transferred to saline solution free of the dye for 5.s to wash Lucifer Yellow CH from the external surface of the cells and then to a series of vials containing 5 ml oxygenated Tyrode solution for periods of 1 to 20 min. To investigate curves damaged sclution were
the possible effect muscles equilibrated loaded with Lucifer
of for Yellow
high K solutions on efflux 80 min in 60 mM K CH as described above.
The amount of Lucifer Yellow CH in the rinsing solution was determined in aspectrofluorometer. The muscles were weighed blotted on filter paper and the cells destroyed in boiling distilled water. From the amount of Lucifer remaining in the fibers and that lost in each vial the total fluorescence in the Efflux studies fibers was determined at each collecting period. were also performed in non-injured muscles exposed to high K solution and loaded with Lucifer Yellow CH for the period of time (see above).
Fig. 1 - Contracture produced by high K (60 mM) solution. As it can be seen the increase in resting tension was completely reversed in about 25 min. Temperature - 37°C.
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RESULTS
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Vol. 11, No. 1, January
1987
AND DISCUSSION
Fig. 2 shows the typical distribution of Lucifer Yellow CH along a single dog trabecula superfused in normal Tyrode solution. As it can be seen the redistribution of the dye took place over distances much larger than the length of a single heart cell (125,#m) (Marceau, 1904). Efflux studies performed on trabeculae exposed to 60 mM K solution for 80 min indicated: 1) in non-damaged muscles the washout curve wa characterized by a quick reduction of fluorescence 90 (half-life 1 min.) suggesting a fast removal of the dye from the extracellular space. In damaged muscles exposed to 602p K, loaded with Lucifer Yellow CH and then healed with normal fCa jo in presence of the dye the "56 lux . curve showed not only an initial min.) but also a second very slow -;-,;;P;p$ ct1/2 = 3 - 2 x 10 min.) which can be ascribed to an extremely small diffusion of the compound through the surface cell membrane, These results are identical to those described in normal fibers (De Mello et al., 1983). Considering that the permeability of the surface cell membrane to Lucifer Yellow CH is extremely low (De Mello et al., 1983) the present results support the view that cardiac cells are connected through permeable channels. The quantitative evaluation of the diffusion was made by fitting the experimental results as described in Methods. Th? regults showed the mean) of 4.3 _t 1.3 x locm /s (see Fig.
coefficient (D) to theoretical points a D (mean _t S.E. of 2 and Table I).
As sealing of the cut-end is essential for the applicability 1974) control measurements of of the cut-end method (Imanaga, Twenty resting potential performed immediately after lesion. minutes after damage the resting potential recorded very near n = 10) was similar (0.05 cm) the cut-end (31 mV; S.E. ;t 0.70; that recorded far away (0.17 cm) (33 mV; S.E. t 0.79; n = 12) the lesion (see Weidmann, 1952; De Mello, 1983).
to
from
Table 1 shows values of diffusion coefficient (d) determined from control preparations exposed to 40 and 60 mM K solution. AS it can be seen the diffusion coefficient was not different from the controls (P70.05). As stressed in Methods these experiments were performed when the increment in resting tension produced by high K This means that the longitudinal solutions was totally reversed. in depolarized heart movement of Lucifer Yellow Cg+ was followed . . )i similar to that present in normal fibers but with the free fCa fibers.
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MO=60 mM D = 3.9 x 10~7Cmz/s t
= 45min
h = lmm
mm.
2 - Longitudinal redistribution of Lucifer Yellow CH Fig. along a dog trabecula immersed in high K. solution (60 mM). The fluorescence from 0.5 mm slices are normalized by total fluorescence and plotted against distance from cut-end. The points represent experimental values which ar_e7fitsed by a theoretical diffusion curve with D = 3.9 x 10 The cm /s. dotted line separates the loaded (left) from the unloaded segment. Diffusion period - 45 min. Length of the preparation - 4 mm. Temperature - 3i"C.
8
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1987
TABLE 1 Lack of influence of high K solutions on the longitudinal of Lucifer Yellow CH along dog trabeculae Experiment
Resting potential
{KIo
Diffusion period (min.)
(mv) 5.4 5.4 5.4 5.4 5.4
-80 (n = 15) -76 (n = 20) -78 (n = 16) -79
-77 X
(n = 18) (n = 20)
45 45 45 45 45
1 1 1
-
-
45 45 45 45
1.1 1 1 1
1 1.1
= -78 + 0.71
-
20 20 20 20
Length of loaded segment (mm)
-40 -42 -44 -41
(n (n (n (n
= = = =
14) 20) 20) 20)
-31 -30 -32 -30
(n (n (n (n
= = = =
15) 20) 20) 20)
-
-
45 45 45 45
1 1.1 1.1 1
X = -30 + 0.64
+ S.E.
of mean
-24 (n = 21) -21 (n = 20) -23 (n = 18) -22 (n = 16) X = 22.5 + 0.64
1.8 9
6 1.8 3.2 x = 4.3 + 1.3 7.4 1.9
4.8 3.9
5 3.6 4.9 5.9
X = 4.85 f 0.47 ---
60 60 60 60
Diffusion coefficient j;17jcm2/s
x = 4.5 + 1.14
x = -41 f 0.95
40 40 40 40
movement
-
-
45
1
4.8
t: 45
1.1 1.1 1
5.9
7.3 3 X = 5.2 f 0.90
x
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7987
9
The present results indicate that prolonged depolarization of the surface cell membrane, per se, has no influence on junctional These findings are in agreement permeability in cardiac fibers. with studies on the influence of membrane potential on junctional conductance performed in cell pairs isolated from the guinea-pigs or rat hearts (Kameyama, 1983; White et al., 1984; Metzger and Weingart, 1985; Weingart, 1986). In Chironomus salivary glands the junctional conductance is strongly influenced by the voltage between the bath and cytoplasm (Obaid et al., 1983). In this tissue equal depolarization of the two apposing cells reduces the junctional conductance while equal hyperpolarization of both cells enhances the conductance (Obaid et al .,1983). In squid blastomers the junctional conductance is affected by both transjunctional and the membrane potential (see Spray et al., 1981). The lack of influence of prolonged cell-to-cell diffusion of Lucifer indicates that variations in the ance found in normal heart (Spach differences in values of membrane in fiber diameter, number and size
depolarization (per se) on the Yellow CH along heart fibers intracellular longitudinal resistet al., 1982) are not due to potential per se, but no changes of gap junctions.
ACKNOWLEDGMENTS
This We are grateful to Maria Gonzglez for her technical work. work was supported by Grant Nos. HL-34353 and HL-34148 from the National Heart Institutes, Bethesda, Maryland.
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Received
6.10.86
Accepted
11.11.86