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Characterization of isolated ventricular myocytes: Two levels of resting potential
j M o l Cell C a r d i o l 19, 831-839 (1987)
Characterization o f Isolated Ventricular Myocytes: Two Levels o f Resting Potential* Masako Oya Masuda, t wGustavo de Magalhies Engel~ and Ana Paula Barbosa Moreira~ Instituto de Biof'zsica Carlos Chagas Filho Centro de Ci~nciasda Sailde-Bloco "G' UniversidadeFederal do Rio de Janeiro Ilha do F u n ~ o ~ 21941 Rio de Janeiro, Brasil (Received27January 1986, acceptedin revisedform 31 March 1987) M. O. MASUDA,G. DE MAGALHAESENGEL AND A. P. BARBOSAMOREIRA. Characterization of Isolated Ventricular Myocytes: Two Levels of Resting Potential. Journalof Molecularand CellularCardiology(1987) 19, 831 839. Standard microelectrode and whole cell patch clamp techniques were used to characterize the resting properties of ventricular myocytes obtained by enzymatic dispersion from rabbit hearts. In 37 cells studied under current clamp using intracellular microelectrodes, three distinct group of cells can be recognized. The first group (23 cells) is constituted by permanently depolarized cells (RP = - 2 4 _+ 5 mV; R m = 8.1 _ 3.4 Kfl cm2; Cm = 2.4 + 1.2 ,uF/cm2). A second group (13 cells) is constituted by cells able to be in two distinct states: D state (RP = - 2 8 ___ 11 mV; R m = 15.2 + 9.1 kfl cm2; Cm = 3.8 _+ 0.8 ,uF/cm 2) and H state (RP = - 7 4 + 8 mV; R m = 3.2 +_ 2.3 Kfl cm 2 and Cm = 2.9 + 2.3 #F/cm2). Some of these cells were able to switch between the two states. The third group is represented by one cell and had only one resting potential in the H state range. Resting potential of permanently depolarized cells and cells in D state did not depend on extracellular potassium concentration. In H state however, it varied with extracellular potassium for concentrations above 2.5 mm as expected for a potassium electrode; for lower concentrations a strong deviation was observed. Accordingly, the steady state current voltage plot obtained under whole cell voltage clamp conditions showed either one zero current potential with positive slope conductance at the D state resting range; two zero current voltages corresponding to D and H state resting levels or only one zero current potential at H state resting level. We could not determine the exact reason for this heterogeneity but suggestion is made that it could be due either to varying degrees of a very small leak and/or to some alteration in the anomalous rectifier. KEY WORDS: Isolated myocytes ; Resting properties ; Two levels of resting potential.
Introduction During the last'five years, isolated cardiac myocytes have become a subject of choice on s t u d i e s o f e l e c t r o p h y s i o l o g i c a l as w e l l as m e c h a n i c a l p r o p e r t i e s o f t h e h e a r t [1, 2, 3, 5, 6, 7, 9-15, 19]. H o w e v e r , s y s t e m a t i c i n v e s t i g a t i o n o f t h e r e s t i n g c h a r a c t e r i s t i c s o f t h e s e isol a t e d cells a r e r e s t r i c t e d to a f e w s t u d i e s [1, 3, 5, 10]. T h e p u r p o s e o f t h e p r e s e n t r e p o r t is t o c h a r acterize the resting electrical properties of single ventricular myocytes isolated by a m e t h o d w e h a v e a d a p t e d to r a b b i t h e a r t s o n
the basis of previously described work in other p r e p a r a t i o n s [6, 18].
Methods Y o u n g r a b b i t s o f a b o u t 1.5 K g w e r e h e p a r i n ized and killed by a blow on the neck. The heart was rapidly removed, cannulated through the aorta, and perfused with a T y r o d e ' s s o l u t i o n ( T a b l e 1). When no visible trace of blood was present i n t h e e f f l u e n t , a s e c o n d s o l u t i o n ( s o l u t i o n A, T a b l e 1) w a s p e r f u s e d t h r o u g h t h e s y s t e m u n t i l t h e v e n t r i c l e s s t o p p e d b e a t i n g (5 to 10 rains).
* Research supported by grants from C E P G - U F R J , MCT/CNPq and MCT/FINEP. "~ Career Investigators, MCT/CNPq. Recipients of a Fellowship from MCT/CNPq. w To whom all correspondence should be sent at the above address. 0022-2828/87/090831 + 09 $03.00/0
9 1987 Academic Press Limited
832
M a s u d a et al.
TABLE 1. Composition of solutions (raM) Tyrode's NaC1 KC1 CaC1 z MgC12 GLUCOSE HEPES NaHCO 3 EGTA pH
137 5 2.5 0.5 6 12 7.2
Sol. A
Sol. B
Sol. C
140 5
15 I00 3.5 20 10 6.9
5 140 0.2 2.0 I0 0.6 7.2
1 20 10 7.0
T h e n , solution B ( T a b l e 1) was perfused for a p p r o x i m a t e l y 5 mins in o r d e r to expose the e x t r a c e l l u l a r m e d i u m to high potassium. Finally, collagenase ( W o r t h i n g t o n C L C I I ) was a d d e d to 45 ml of solution B to a final c o n c e n t r a t i o n of 0.3 to 0.5 mg/ml, a n d the m i x t u r e recirculated for 25 mins. After this p r o c e d u r e the ventricles were cut into small pieces a n d kept for 5 mins in the same e n z y m e - c o n t a i n i n g solution, to which CaC12 was g r a d u a l l y a d d e d to a final c o n c e n t r a t i o n of 2.5 mM. T h e pieces of tissue were then transfered to solution A with CaC12 (2.5 mM) a d d e d . Free cells were o b t a i n e d by gentle shaking. T h e whole perfusion p r o c e d u r e was c a r r i e d out at 35~ and at a flow rate of 10 ml/min. T y r o d e ' s solution was equilibrated with a m i x t u r e of 95% 0 2 a n d 5% C O 2 (pH 7.2), a n d solutions A and B were e q u i l i b r a t e d with 0 2. A s a m p l e of the cell suspension was p l a c e d in a culture dish on the stage of an i n v e r t e d microscope ( P R I O R ) e q u i p p e d with phase constrast optics. H e a l t h y looking cells with a p p r o x i m a t e l y r e c t a n g u l a r shape, t r a n s p a r e n t cytoplasm, a n d clear cross striations were chosen. M o s t of the cells were quiescent in solution A containing 2.5 mM CaC12 which was our control m e d i u m . U n d e r c u r r e n t c l a m p conditions, the transm e m b r a n e voltage was m o n i t o r e d t h r o u g h conventional 2.5 M KC1 filled glass microelectrodes, connected to a high i n p u t i m p e d a n c e amplifier ( A M - 2 , Biodyne Instruments). T h e whole cell p a t c h c l a m p experiments were performed with borosilicate p a t c h pipettes filled with solution C connected to a $7050 A p a t c h c l a m p amplifier ( W P I ) . C u r r e n t was delivered by an isolated stimu-
lator (DS3 D i g i t i m e r Inst.). C u r r e n t a n d t r a n s m e m b r a n e voltage were both m o n i t o r e d on the screen o f a 565 T e k t r o n i x oscilloscope a n d on a c h a r t recorder (Gould Brush 2200). All experiments were p e r f o r m e d at room temp e r a t u r e (26 to 30~ a n d the solution in the culture dish was continuously changed at 0.5 m!/min. Results
W e will first describe the results o b t a i n e d u n d e r c u r r e n t c l a m p conditions. Figure 1 shows the distribution of the resting potentials in a total of 37 cells. O n e g r o u p of cells (open bars) h a d resting potentials r a n g i n g between - - 1 5 and - - 3 5 m V ( - - 2 4 _+ 5 m V , m e a n __+S.D. n = 23) stable for at least 30 mins d u r i n g which h y p e r p o l a r i z i n g a n d d e p o l a r i z i n g pulses were applied. A second g r o u p (stippled bars) h a d two distinct values for resting p o t e n t i a l : usually soon after i m p a l e m e n t , these cells exhibited a resting potential r a n g i n g from - - 1 0 to - - 5 0 m V ( - - 2 8 __+ 11 m V , n = 13); after a while the resting p o t e n t i a l suddenly increased to a m u c h more polarized value, r a n g i n g from - 6 5 to - 9 0 m V (Table 2). O n e cell h a d a single resting potential of - 80 mV. Figure 2 shows results of a typical experim e n t in a cell of the first group, with a resting
12 "6
8-
z
0
-50 Resting potential (mV)
-I00
FIGURE 1. Histogram showing the distribution of resting potential values in 37 isolated ventricular cells measured with conventional microelectrode. Clear bars represent the depolarized population (23 cells). Stippled bars represent the population with higher resting potential (14 cells) which except for one cell, was able to show a second more depolarized resting potential.
Isolated Myocytes:
833
Two Levels of Resting Potential
TABLE 2. Electrical constants of isolated rabbit ventricular myocytes (expressed as mean _+ S.D.) Cell population Permanently depolarized cells Cells with two stable resting potentials
D H
RP (mV)
Ri,put Mf~
9 (ms)
Apparent surface area (/~m2)
Rm ( K ~ cm z)
Cm (pF/cm z)
-24-t-5 (n = 23)
117_+48 (n - 23)
19_+ 11 (n = 23)
6.836_+910 (n = 22)
8.1 _+3.4 (n = 22)
2.4_+ 1,2 (n = 22)
--28 + I1 (n = 13) -74 _ 8 (n = 14)
193 _ (n = 41 _ (n =
48 (n 4.7 (n
7.047_+ 1.339 (n = 12)
15.2___9.1 (n = 9) 3.2 + 2.3 (n = 11)
3.8__+0.8 (n = 7) 2.9 _ 2.3 (n = 6)
144 10) 23 12)
+ = + =
11 7) 2.0 8)
1. The differences in Rinput, T, R m and Cm between the permanently depolarized cells and D state in second group are significant (P < 0.05) 2. Differences between D and H s t a t e are all significant (P < 0.01) except for Cm .
p o t e n t i a l of - - 2 5 mV. Small d e p o l a r i z i n g and h y p e r p o l a r i z i n g c u r r e n t pulses p r o d u c e d voltage changes with time courses t h a t could be fitted by simple exponential with a time constant of 35 ms (a, b). F o r stronger hyperpolarizing current pulses the onset of the voltage response d e p a r t e d from the exponential function, either not reaching a stable level (c) or doing so along a time course resembling phase 3 of the n o r m a l v e n t r i c u l a r action potential (d). T h e h y p e r p o l a r i z a t i o n was faster the stronger the current pulse a p p l i e d (not shown). A n o d a l b r e a k excitation was also observed with stronger h y p e r p o l a r i z a t i o n (d) a n d it caused a synchronous c o n t r a c t i o n in the whole cell. F i g u r e 2(e) is the c u r r e n t voltage plot for the same cell. Voltages were
(o)
(b)
m e a s u r e d at the end of 300 ms, w h e t h e r or not the m e m b r a n e voltage h a d reached a steady value (see legend). F o r voltages n e a r the resting potential, the relationship between a p p l i e d current a n d voltage was almost linear with a slope of 184 Mf~. Between - 4 0 and - 80 m V the curve becomes almost parallel to the voltage axis. F i n a l l y for voltages negative to - - 8 0 m V the c u r r e n t - v o l t a g e relationship a g a i n becomes almost linear, with a very small slope. T h e surface a r e a of this cell was 5.500 /~m 2. This value was o b t a i n e d from m e a s u r e m e n t s of the length a n d w i d t h of the cell, a n d taking the height to be one third of the w i d t h (considering the cells 'brick s h a p e d ' ) . F r o m these figures a specific m e m b r a n e resistance Rm = 10 K ~ cm 2 and a spe-
(e)
o
I
-40
I -20
-
(d)
/
/
L_, (c)
2o
-
/
-
f
20 IO-"A
-50
13
o__o_._o-O/ - -I00 F I G U R E 2. Typical I-clamp experiment in a cell of the permanently depolarized population. F r o m (a) to (d) ; upper trace shows applied current and lower trace, t r a n s m e m b r a n e voltage. At right, the current-voltage curve for the same experiment, voltage measured at the end of a 300 ms pulse. Squares denote the situation in which the plotted voltage had not stabilized (as in (c)). Calibrations : vertical bar 40 mV, 40 • 10- I x A and horizontal b a r 100 ms.
834
M a s u d a et aL
cific membrane capacitance, C m = 3.5 pF/cm 2 (calculated from a time constant = 35 ms) w e r e o b t a i n e d . T a b l e 2 gives the m e a n v a l u e s o f all p a r a m e t e r s for 23 cells in this first g r o u p . F i g u r e 3 illustrates the t y p i c a l b e h a v i o u r of a cell a b l e to s h o w two levels o f resting p o t e n tial w i t h transitions b e t w e e n t h e m . I n (a) a c h a r t r e c o r d e r t r a c i n g shows t h a t resting p o t e n t i a l soon after cell i m p a l e m e n t was a p p r o x i m a t e l y - - 2 5 m V , after a while a sudden hyperpolarization to --65 mV o c c u r r e d . I n s o m e cells, o n c e this t r a n s i t i o n f r o m the d e p o l a r i s e d (D state) to h y p e r p o l a r ized p o t e n t i a l (H state) h a d o c c u r r e d , the resting p o t e n t i a l stayed at the H level for hours, i n d e p e n d e n t o f the p o l a r i t y , a m p l i t u d e a n d d u r a t i o n of the c u r r e n t pulses a p p l i e d thereafter. I n o t h e r cells (five o u t o f thirteen), h o w e v e r , transitions b e t w e e n D a n d H states c o u l d be o b s e r v e d all a l o n g . F i g u r e 3(b) shows a t r a n s i t i o n f r o m H to D state. T h i s is usually b r o u g h t a b o u t by a n e v o k e d a c t i o n p o t e n t i a l . I n the p r e s e n t e x a m p l e , after 6 s, a h y p e r p o l a r i z i n g pulse i n d u c e d the t r a n s i t i o n (a)
b a c k to the H state. F i g u r e s 3(c) a n d (d) s h o w a t r a n s i t i o n f r o m D to H state in an e x p a n d e d t i m e scale. I n 3(c) the cell i n D state r e s p o n d e d to a s t r o n g h y p e r p o l a r i z i n g pulse in a w a y s i m i l a r to t h a t in F i g u r e 2(b), e x c e p t t h a t the a n o d a l b r e a k e x c i t a t i o n is m u c h d e l a y e d . I n 3(d) the h y p e r p o l a r i z a t i o n was n o t followed b y the a n o d a l b r e a k e x c i t a t i o n , thus m a k i n g t r a n s i t i o n f r o m D to H state. F i g u r e 4 shows a typical c u r r e n t - c l a m p e x p e r i m e n t in a cell e x h i b i t i n g two levels of resting p o t e n t i a l . (a) a n d (b) s h o w the result o f passing h y p e r p o l a r i z i n g pulses at the resting p o t e n t i a l o f - 2 5 m V (D state). F o r small depolarizations and hyperpolarizations the onset o f the v o l t a g e c h a n g e is a single e x p o n e n t i a l . N o t e in (a), 2nd trace, t h a t w i t h a l a r g e r h y p e r p o l a r i z a t i o n the v o l t a g e c h a n g e was n o t e x p o n e n t i a l a n d did n o t r e a c h a s t e a d y level. W i t h f u r t h e r increases in the h y p e r p o l a r i z i n g pulse a m p l i t u d e , a steady v o l t a g e was r e a c h e d faster for s t r o n g e r c u r r e n t pulses, f o l l o w i n g a c o m p l e x t i m e course [-(a), 3rd trace a n d (b)]. T h e s a m e cell, n o w w i t h a resting p o t e n t i a l o f - - 6 5 m V , shows a corn(b)
9
(c)
(d)
I
II
FIGURE 3. Transitions between the two resting potentials. (a) and (b) show records in a chart recorder, voltage in the lower trace and current in the upper trace. Current pulses were applied at 2 s intervals. Zero voltage level is indicated by an arrow in (a) and (b). (a) in the beginning, the electrode was out of the cell; upon impalement by electronic oscillation, resting potential of approximately -- 25 mV was recorded. In sequence, a slow and progressive increase in resting potential is seen. Close to the end of the panel, a sudden change of the resting potential to approximately - 6 5 mV is shown. (b) shows transition from the more polarized (H state) to the depolarized resting potential (D state) following a stimulated action potential. After 6 s a hyperpolarizing pulse brought it back to the H state. (c) and (d) are pictures taken from the screen of an oscilloscope showing transition from D to H state (upper trace, current and lower trace, voltage). In (c), a strong hyperpolarizing current was applied to a cell in the D state. At the end of this pulse a delayed anodal break excitation was seen, bringing the membrane potential back to the previous depolarized level (D state). In (d), after several cycles, the same hyperpolarizing pulse did not stimulate anodal break excitation, thus switching the cell to H state. Calibration in A, corresponds to 50 x 10 -11 A and 50 mV. For (c) and (d) the vertical bar corresponds to 40 mV and 40 x 10 T M A and the horizontal bar to 100 ms.
Isolated Myocytes: T w o Levels o f R e s t i n g Potential (a)
835
(b)
(c)
_r-
_J
Q
I
~ 2o
I
I 50
-50
lO-lIA
/
/ o/
-IOO
FIGURE 4. A typical I-clamp experiment in a cell with two levels of resting potential. (a) and (b) effects of current pulses applied to the cell in the D state (resting potential --25 mV) and (e) and (d), in the H state (resting potential - 6 5 mV). Observe the non exponential time course in response to the stronger current pulses in (a) and (b). The current voltage plot shows the curve for D (O) and H state (O). Calibration : 40 mV, 40 x 10-11 A and 100 ms. pletely passive b e h a v i o u r for h y p e r p o l a r izations of all a m p l i t u d e s tested (c) a n d for small depolarizations. A d e q u a t e l y strong d e p o l a r i z i n g pulses evoked n o r m a l action potentials, (d) followed by synchronous contraction of the whole cell. T h e current-voltage plot for the same cell is shown at the b o t t o m of Figure 4. O b s e r v e the similarity between the curve o b t a i n e d for the cell in D state, with a resting potential of - - 2 5 m V (filled circles) and the one shown for a perm a n e n t l y d e p o l a r i z e d cell (Fig. 2). T h e c u r r e n ~ v o l t a g e plot in the H state is almost linear for potentials negative to - 7 0 m V , with a small slope. So it is possible to associate the D state not only with a d e p o l a r i z e d resting potential, b u t also with a c u r r e n t - v o l t a g e plot with a steep region between - - 4 0 to - 8 0 mV.
O n the other h a n d the H state m a y be associated with an almost linear, shallow c u r r e n t voltage plot a n d resting potentials negative to - 6 0 m V (in the presence of 5 mM KC1). I n the cell shown in F i g u r e 4, the measured i n p u t resistance, R i n p u t , and the time constant z, for the D state were respectively, 243 Mf~ a n d 50 ms, resulting in calculated values o f R m = 23.3 Kf2 cm 2 a n d Cm = 2.0 ~ F / c m 2. F o r measurements m a d e with small h y p e r p o l a r i z i n g pulses, the same cell in H state h a d a Rinput -87 M f l a n d R m = 9.5 K f l cm 2. T h e cell disp l a y i n g only the h y p e r p o l a r i z e d resting p o t e n t i a l behaves in all aspects as the cells in H state. T a b l e 2 summarizes the values of all p a r a m e t e r s d e t e r m i n e d in 14 cells in H state, including the cell with only high resting
836
M a s u d a et aL
potential. It can be observed that D and H states have completely different membrane resistances (Rm). It is also important to point out that despite the qualitative similarity between the cells in D state and the permanently depolarized cells, there were significant differences in the measured parameters between them. Figure 5(a) shows current-voltage plots from a cell in H state exposed to 5, 10, 25, 50 and 100 mm potassium. Observe that: (1) the resting potential varies with extracellular potassium and (2) the shape of the c u r r e n ~ voltage plot does not necessarily change in different potassium concentrations, despite the changes in resting potential. Additional interesting observation was that transitions between the two states, D and H, occurred at different potassium concentrations from 1 to 50 mM. The transition from H to D state was characterized by a change in resting potential and in the shape of the curren~voltage plot as already mentioned for 5 mM potassium. This is shown in Figure 5(b) for 25 mM extracellular potassium. Figure 6 shows the effect of extracellular potassium concentration on the resting potential both in H (open symbols) and D (filled symbols) states. In the H state, a clear dependence of resting potential on extracellular potassium is seen. The isolated cells behave in a way very similar to that already described for
~2
(o)
0
50
ventricular tissue. For potassium concentrations above 2.5 mM a linear relationship with a slope close to that expected for a potassium electrode was observed. For lower concentrations a clear deviation is seen. In the D state no relation exists between resting potential and extracellular potassium (filled symbols). In permanently depolarized cells, the resting potential did not depend at all on extracellular potassium concentration just as the cells in D state. "Whole cell patch clamp" experiments were successfully performed in seven cells maintained in sol. A in order to analyse the steady state current-voltage relationship. The holding potential was 0 mV. The resting potential soon after the breakage of the patch membrane was - 77 4- 10 mV. Although all of them had current-voltage relations expressed by roughly N shaped curves as seen in ventricular tissue and also in isolated cells, some differences could be distinguished between them. Figure 7, left, shows the three types of current-voltage plots observed. A (open circles), is a plot seen in two of the cells, and is similar to the previously described by Sakmann and Trube, in that, the only one zero current potential observed corresponded to the resting potential seen under current clamp conditions. Four cells had curves similar to that shown in B (triangles). In this
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~A~ ~
~,Z
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/150 [K] o -- 25 mM
-100 F I G U R E 5. Effects ofextracellular potassium concentration on resting potential in H state. (a) current-voltage plot in a cell exposed to 5 ( 9 10 (O), 25 (A), 50 (A) and 100 (D) mM KC1. Observe the parallelism between the 5 curves, despite change in resting potential. (b) transitions between D (A) and H (A) states in presence of 25 mM extracellular potassium.
Isolated
Myocytes:
Two
Levels
of Resting
837
Potential
O S
o
o/ @
@
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7
9
fil
-50
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0.1
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I I Illl
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[K]e (mM)
I00
FIGURE 6. Effect of extracellular potassium concentration on resting potential in H (open symbols) and D (filled symbols) states. Observe clear dependence in the H state and complete independence in the D state, of the resting potential on Ke . Straight line is for a potassium electrode. Same symbols (open and full) were used to represent the same cell.
g r o u p , the c u r r e n t - v o l t a g e c u r v e for p o t e n tials b e t w e e n - - 3 0 a n d 0 m V b e c o m e s a l m o s t p a r a l l e l to the v o l t a g e axis, v e r y close to o r at the z e r o c u r r e n t level. So, at least t w o regions o f z e r o c u r r e n t v o l t a g e c o u l d be d i s t i n g u i s h e d , o n e at the resting p o t e n t i a l level seen u n d e r c u r r e n t c l a m p a n d a n o t h e r , at m o r e d e p o l a r ized level. O n e cell t h a t initially h a d a resting
(a)
i0-i2 A A
I - 200
p o t e n t i a l of - - 6 0 m V , w h e n c l a m p e d to 0 m V s h o w e d a slow drift in h o l d i n g c u r r e n t t o w a r d s z e r o a n d t h e n it stabilized (Fig. 7, left, C, squares). T h e o n l y zero c u r r e n t v o l t a g e for this cell n o w is close to zero. F i g u r e 7, right, shows the effect of 5 mM CsC1 on s t e a d y state c u r r e n t v o l t a g e plot. T h e blocki n g effect is c l e a r for p o t e n t i a l s n e g a t i v e to
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V (mV)
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50
V (mY) - -IOI3
- -IO0
/ zx
A
-200
Control o
Cs CI 5 ml
- -200
A
FIGURE 7. Steady-state current voltage curves obtained from whole cell patch-clamp experiments. Curves A, (O), B (A) and C (IN) were obtained from three different cells, maintained in control solution, at same holding potential (0 mV). Right: Steady state current voltage relation under control solution (A) and in presence of 5 mM CsCI (0) HP = 0 mg.
838
M a s u d a et al.
- 4 0 mV. The slope resistance around the resting potential increases from 47 to 180 Mf~ in presence of CsC1. Discussion
The results presented in present paper, show that three distinct groups of cell can be found among enzymatically dissociated ventricular myocytes. One group is characterized by low resting potential, as shown in Figures 1, 2 and Table 2 (permanently depolarized cells). Presence of depolarized cells among the isolated cardiac myocytes has previously been described by other authors [6, 16] but a distinction exists between the cells they described and present ones; these cells have much higher membrane resistance. For instance, Isenberg & Klockner found 10 __+4 Mf~ in input resistance for rat myocytes of approximately same size compared to 117 __ 47 Mf~ in present paper. They are also characterized by the fact that their resting potentials are not dependent on the extracellular potassium concentrations. These cells can be identified with the cell which current-voltage relationship obtained under whole cell voltage clamp is represented by curve C in Figure 7, left, which had only one region of zero current potential ranging between - 2 5 and 0 mV. No zero current-voltage was found in the hyperpolarized range usually corresponding to resting potential in ventricular cells in tissue. A second group of cells had two different states with distinct resting potentials: H and D states (Fig. 1 and Table 2) as previously described for Purkinje fibers by Wiggins and Cranefield, 1976 and Gadsby and Cranefield, 1977. Some of these cells were able to switch from one state to the other as a consequence of imposed de- or hyperpolarization (Figs 3 and 4). In the D state, this cell behaves in all aspects as the permanently depolarized cells described above despite the significant difference in membrane resistance (Fig. 4, Table 2). In the H state, however, they had completely different properties: the membrane resistance was much smaller being the input resistances found comparable to values described for other isolated myocytes; for instance Isenberg and Klockner [6] found 33 _+ 8 Mf~; 36 __ 9 Mf~ and 41 ___ 10 M ~ for rat, guinea-pig and bovine ventricular cells and Sheets et al. [13] found Rinput = 21 ___8 M ~ in canine Purkinje fibres with comparable resting potentials. The resting potential
depended on extracellular potassium concentration in a way very close to that predicted by Nernst Equation (Figs 5 and 6) as found by Isenberg and Klockner [9]. Curve B in Figure 7, left, represents this group of cells. As the current-voltage plot under voltage clamp is N shaped and has two ranges of zero current voltages, it can be expected that when the cell is in H state, depolarizing currents strong enough to bring the transmembrane potential to or beyond the negative conductance region, would make the switch to D state upon the turning off of the current pulse. On the other hand, a cell in D state could suddenly change to H state by application of a hyperpolarizing pulse strong enough to overcome the negative conductance range. The above described behaviour corresponds exactly to what we have shown in Figures 3 and 4. A third group of cells, had only one resting potential in the hyperpolarized range usually found in tissue. This group corresponds to a current-voltage plot shown in curve A, Figure 7 left, with only one zero curren~voltage in the hyperpolarized region. Similar results had previously been described in isolated cells by Sakmann and T r u b e , 1984. The resting properties in these cells are all similar to the second group of cells in H state. The dependence of the hyperpolarized resting potential on extracellular potassium concentration confirms previous observations both in tissue and in isolated cells, that at the usual resting potential, the ventricular cell membrane is highly selective to potassium (4, 12 and others). The independence of the resting potential in the D state and in permanently depolarized cells to external potassium and the much higher membrane resistance seen in D state as compared to H state, suggests the presence of an anomalous rectifier, which is open at more negative potentials but shut off in D state. Transition from D to H state could be promoted by the turning on of the anomalous rectifier channel leading to the observed decrease in membrane resistance. The effect of CsC1, on the current voltage plot in these cells, increasing the slope resistance around the hyperpolarized resting potential (Fig. 7, right) is additional support for the above hypothesis. We do not know yet, what is responsible for the differences in the current voltage relation-
Isolated Myocytes: Two Levels of Resting Potential ship a m o n g different isolated cells a n d therefore, w h a t d e t e r m i n e s the described h e t e r o g e n e i t y in resting p o t e n t i a l s a m o n g them. O n e possibility is t h a t a v e r y small yet i m p o r t a n t difference in a n i n w a r d b a c k g r o u n d c u r r e n t exists a m o n g them. T h i s c o u l d e x p l a i n the difference b e t w e e n curves A a n d B in F i g u r e 7 left, specially for p o t e n t i a l s positive to - - 5 0 m V . T h i s could also e x p l a i n the difference in m e m b r a n e resistance observed b e t w e e n cells in D state a n d p e r m a n e n t l y d e p o l a r i z e d cells. A n o t h e r possibility c o u l d be a difference in the properties of the a n o m a l o u s rectifier c h a n nels. T h i s idea was raised u p from the observ a t i o n t h a t in p e r m a n e n t l y d e p o l a r i z e d cells s t u d i e d u n d e r c u r r e n t c l a m p , while a s t r o n g h y p e r p o l a r i z i n g pulse is m a i n t a i n e d , the t r a n s m e m b r a n e v o l t a g e b e h a v e s in a w a y v e r y similar to those cells in D state. After the t u r n i n g o f f o f the pulse, in the first case, t r a n s m e m b r a n e p o t e n t i a l comes b a c k to the s a m e previous d e p o l a r i z e d level, while cells i n D
839
state c h a n g e its resting p o t e n t i a l to a n e w h y p e r p o l a r i z e d value, s w i t c h i n g to the H state. I t seems as if i n p e r m a n e n t l y d e p o l a r ized cells the c h a n n e l s responsible for the h y p e r p o l a r i z e d resting p o t e n t i a l ( a n o m a l o u s rectifier) t h o u g h present, were n o t able to stay open, o n c e the a p p l i e d pulse is t u r n e d off. I t seems also m e a n i n g f u l t h a t the slope resistance in c o m p a r a b l e voltages i n the h y p e r p o l a r i z e d r a n g e is l a r g e r i n cells w i t h o n l y the d e p o l a r ized zero c u r r e n t p o t e n t i a l , t h a n in those w i t h two zero c u r r e n t potentials ( c o m p a r e curves C a n d B, Fig. 7 left).
Acknowledgements T h e a u t h o r s wish to a c k n o w l e d g e E d m i l s o n A n t o n i o P e r e i r a for the excellent t e c h n i c a l assistance a n d Drs A. C. C a m p o s de C a r valho, S. C u k i e r m a n a n d A. Paes de C a r v a l h o for helpful discussion a n d c o m m e n t s o n the manuscript.
References 1 BROWN,A. M., LEE, K. S., POWELL,T. Voltage clamp and internal perfusion of single rat heart cells. J Physiol 318, 455-477 (1981). 2 BROWN,A. M., LEE, K. S., POWELL,T. Sodium current in single rat heart muscle cells. J Physiol 318, 479-500 (1981). 3 CALLEWAERT,G., CARMEEIET,E., VEREECKE,J. Single cardiac Purkinje cells: General electrophysiology and voltage-clamp analysis of the pacemaker current.J Physio1349, 643-661 (1984). 4 GADSBY,D. C., CRANEFIELD,P. F. Two levels of resting potential in cardiac Purkinje fibers. J Gen Physiol 701, 725-746 (1977). 5 HuME,J. R., GILES,W. Ionic currents in single isolated bull-frog cells.J Gen Physio181,153-194 (1983). 6 ISENBERO,G., KLOCKNER, U. Calcium tolerant ventricular myocytes prepared by preincubation in a 'KB Medium'. Pfliigers Arch, EurJ Physio1395, 6-18 (1982). 7 ISENBERG,G., KLOCKNER,U. Isolated bovine ventricular myocytes: characterization of the action potential. Pfluge'i-sArch, EurJ Physio1395, 19-29 (1982) 8 ISENBERO,G., KLOCKNER,U. Calcium currents of isolated bovine ventricular myocytes are fast and of large amplitude. Pflilgers Arch, EurJ Physio1395, 30~1 (1982). 9 LEE,K. S., TSXEN,R. W. Reversal of current through calcium channels in dialysed single heart cells. Nature 297, 498-501 (1982). 10 LENTO,H., TIRm, R. Characterization of contractions in mechanically disaggregated myocardial cells from the rat. Acta Physiol Scand 110, 385-389 (1980). 11 POWELL,T., TERRAR,D. A., TwisT, V. W. Electrical properties of individual cells isolated from adult rat ventricular miocardium. J Physio13112, 131-153 (1980). 12 SAKMANN,B., TRUaE, G. Conductance properties of single inwardly rectifying potassium channels in ventrlcular cells from guinea pig heart. J Physio1347:642-658 (1984). 13 SHEETS,M.F., JANUARY,C. Z., FOZZARD,H. A. Isolation and characterization of single canine cardiac Purkinje cells. Circ Res 53, 544-548 (1983). 14 TARR,M., TRANK,J. W. Preparation of isolated single cardiac cells from adult frog atrial tissue. Experimentia 32, 338-340 (1976). 15 TRAtJTWEIN,W., TANIOUCnLJ., NOMA,A. The effect ofintracellular cyclic nucleotides and calcium on the action potential and acetylcholine response of isolated cardiac cells. Pfliigers Arch EurJ Physio1392:307 314 (1982). 16 WATANAaE,T., DELBRIDOE,L. M., BUSTAMANTE,J. O., McDoNALD, T. Heterogeneity of the action potential in isolated rat ventricular myocytes and tissue. Circ Res 52, 280-290 (1983). 17 WmOXNS,J. R., CRANEFIELD,P. F. Two levels of resting potential in canine cardiac Purkinje fibers exposed to sodium-free solutions. Circ Res 39, 466~-74 (1976). 18 WIT~rENBERa,B. A., ROmNSON,T. F. Oxygen requirements, morphology, cell coat and membrane permeability of calcium-tolerant myocytes from hearts of adult rats. Cell Tissue Res 216, 231-251 (1981). 19 ZILBETER,Yu. I., TIMIM,E. N., BENDUKIDZE,Z. A., BURNASltEV,N. A. Patch-voltage clamp method for measuring fast inward current in single rat heart muscle cells. Pfliigers Arch EurJ Physio1311,1,150-155 (1982).
Characterization o f Isolated Ventricular Myocytes: Two Levels o f Resting Potential* Masako Oya Masuda, t wGustavo de Magalhies Engel~ and Ana Paula Barbosa Moreira~ Instituto de Biof'zsica Carlos Chagas Filho Centro de Ci~nciasda Sailde-Bloco "G' UniversidadeFederal do Rio de Janeiro Ilha do F u n ~ o ~ 21941 Rio de Janeiro, Brasil (Received27January 1986, acceptedin revisedform 31 March 1987) M. O. MASUDA,G. DE MAGALHAESENGEL AND A. P. BARBOSAMOREIRA. Characterization of Isolated Ventricular Myocytes: Two Levels of Resting Potential. Journalof Molecularand CellularCardiology(1987) 19, 831 839. Standard microelectrode and whole cell patch clamp techniques were used to characterize the resting properties of ventricular myocytes obtained by enzymatic dispersion from rabbit hearts. In 37 cells studied under current clamp using intracellular microelectrodes, three distinct group of cells can be recognized. The first group (23 cells) is constituted by permanently depolarized cells (RP = - 2 4 _+ 5 mV; R m = 8.1 _ 3.4 Kfl cm2; Cm = 2.4 + 1.2 ,uF/cm2). A second group (13 cells) is constituted by cells able to be in two distinct states: D state (RP = - 2 8 ___ 11 mV; R m = 15.2 + 9.1 kfl cm2; Cm = 3.8 _+ 0.8 ,uF/cm 2) and H state (RP = - 7 4 + 8 mV; R m = 3.2 +_ 2.3 Kfl cm 2 and Cm = 2.9 + 2.3 #F/cm2). Some of these cells were able to switch between the two states. The third group is represented by one cell and had only one resting potential in the H state range. Resting potential of permanently depolarized cells and cells in D state did not depend on extracellular potassium concentration. In H state however, it varied with extracellular potassium for concentrations above 2.5 mm as expected for a potassium electrode; for lower concentrations a strong deviation was observed. Accordingly, the steady state current voltage plot obtained under whole cell voltage clamp conditions showed either one zero current potential with positive slope conductance at the D state resting range; two zero current voltages corresponding to D and H state resting levels or only one zero current potential at H state resting level. We could not determine the exact reason for this heterogeneity but suggestion is made that it could be due either to varying degrees of a very small leak and/or to some alteration in the anomalous rectifier. KEY WORDS: Isolated myocytes ; Resting properties ; Two levels of resting potential.
Introduction During the last'five years, isolated cardiac myocytes have become a subject of choice on s t u d i e s o f e l e c t r o p h y s i o l o g i c a l as w e l l as m e c h a n i c a l p r o p e r t i e s o f t h e h e a r t [1, 2, 3, 5, 6, 7, 9-15, 19]. H o w e v e r , s y s t e m a t i c i n v e s t i g a t i o n o f t h e r e s t i n g c h a r a c t e r i s t i c s o f t h e s e isol a t e d cells a r e r e s t r i c t e d to a f e w s t u d i e s [1, 3, 5, 10]. T h e p u r p o s e o f t h e p r e s e n t r e p o r t is t o c h a r acterize the resting electrical properties of single ventricular myocytes isolated by a m e t h o d w e h a v e a d a p t e d to r a b b i t h e a r t s o n
the basis of previously described work in other p r e p a r a t i o n s [6, 18].
Methods Y o u n g r a b b i t s o f a b o u t 1.5 K g w e r e h e p a r i n ized and killed by a blow on the neck. The heart was rapidly removed, cannulated through the aorta, and perfused with a T y r o d e ' s s o l u t i o n ( T a b l e 1). When no visible trace of blood was present i n t h e e f f l u e n t , a s e c o n d s o l u t i o n ( s o l u t i o n A, T a b l e 1) w a s p e r f u s e d t h r o u g h t h e s y s t e m u n t i l t h e v e n t r i c l e s s t o p p e d b e a t i n g (5 to 10 rains).
* Research supported by grants from C E P G - U F R J , MCT/CNPq and MCT/FINEP. "~ Career Investigators, MCT/CNPq. Recipients of a Fellowship from MCT/CNPq. w To whom all correspondence should be sent at the above address. 0022-2828/87/090831 + 09 $03.00/0
9 1987 Academic Press Limited
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M a s u d a et al.
TABLE 1. Composition of solutions (raM) Tyrode's NaC1 KC1 CaC1 z MgC12 GLUCOSE HEPES NaHCO 3 EGTA pH
137 5 2.5 0.5 6 12 7.2
Sol. A
Sol. B
Sol. C
140 5
15 I00 3.5 20 10 6.9
5 140 0.2 2.0 I0 0.6 7.2
1 20 10 7.0
T h e n , solution B ( T a b l e 1) was perfused for a p p r o x i m a t e l y 5 mins in o r d e r to expose the e x t r a c e l l u l a r m e d i u m to high potassium. Finally, collagenase ( W o r t h i n g t o n C L C I I ) was a d d e d to 45 ml of solution B to a final c o n c e n t r a t i o n of 0.3 to 0.5 mg/ml, a n d the m i x t u r e recirculated for 25 mins. After this p r o c e d u r e the ventricles were cut into small pieces a n d kept for 5 mins in the same e n z y m e - c o n t a i n i n g solution, to which CaC12 was g r a d u a l l y a d d e d to a final c o n c e n t r a t i o n of 2.5 mM. T h e pieces of tissue were then transfered to solution A with CaC12 (2.5 mM) a d d e d . Free cells were o b t a i n e d by gentle shaking. T h e whole perfusion p r o c e d u r e was c a r r i e d out at 35~ and at a flow rate of 10 ml/min. T y r o d e ' s solution was equilibrated with a m i x t u r e of 95% 0 2 a n d 5% C O 2 (pH 7.2), a n d solutions A and B were e q u i l i b r a t e d with 0 2. A s a m p l e of the cell suspension was p l a c e d in a culture dish on the stage of an i n v e r t e d microscope ( P R I O R ) e q u i p p e d with phase constrast optics. H e a l t h y looking cells with a p p r o x i m a t e l y r e c t a n g u l a r shape, t r a n s p a r e n t cytoplasm, a n d clear cross striations were chosen. M o s t of the cells were quiescent in solution A containing 2.5 mM CaC12 which was our control m e d i u m . U n d e r c u r r e n t c l a m p conditions, the transm e m b r a n e voltage was m o n i t o r e d t h r o u g h conventional 2.5 M KC1 filled glass microelectrodes, connected to a high i n p u t i m p e d a n c e amplifier ( A M - 2 , Biodyne Instruments). T h e whole cell p a t c h c l a m p experiments were performed with borosilicate p a t c h pipettes filled with solution C connected to a $7050 A p a t c h c l a m p amplifier ( W P I ) . C u r r e n t was delivered by an isolated stimu-
lator (DS3 D i g i t i m e r Inst.). C u r r e n t a n d t r a n s m e m b r a n e voltage were both m o n i t o r e d on the screen o f a 565 T e k t r o n i x oscilloscope a n d on a c h a r t recorder (Gould Brush 2200). All experiments were p e r f o r m e d at room temp e r a t u r e (26 to 30~ a n d the solution in the culture dish was continuously changed at 0.5 m!/min. Results
W e will first describe the results o b t a i n e d u n d e r c u r r e n t c l a m p conditions. Figure 1 shows the distribution of the resting potentials in a total of 37 cells. O n e g r o u p of cells (open bars) h a d resting potentials r a n g i n g between - - 1 5 and - - 3 5 m V ( - - 2 4 _+ 5 m V , m e a n __+S.D. n = 23) stable for at least 30 mins d u r i n g which h y p e r p o l a r i z i n g a n d d e p o l a r i z i n g pulses were applied. A second g r o u p (stippled bars) h a d two distinct values for resting p o t e n t i a l : usually soon after i m p a l e m e n t , these cells exhibited a resting potential r a n g i n g from - - 1 0 to - - 5 0 m V ( - - 2 8 __+ 11 m V , n = 13); after a while the resting p o t e n t i a l suddenly increased to a m u c h more polarized value, r a n g i n g from - 6 5 to - 9 0 m V (Table 2). O n e cell h a d a single resting potential of - 80 mV. Figure 2 shows results of a typical experim e n t in a cell of the first group, with a resting
12 "6
8-
z
0
-50 Resting potential (mV)
-I00
FIGURE 1. Histogram showing the distribution of resting potential values in 37 isolated ventricular cells measured with conventional microelectrode. Clear bars represent the depolarized population (23 cells). Stippled bars represent the population with higher resting potential (14 cells) which except for one cell, was able to show a second more depolarized resting potential.
Isolated Myocytes:
833
Two Levels of Resting Potential
TABLE 2. Electrical constants of isolated rabbit ventricular myocytes (expressed as mean _+ S.D.) Cell population Permanently depolarized cells Cells with two stable resting potentials
D H
RP (mV)
Ri,put Mf~
9 (ms)
Apparent surface area (/~m2)
Rm ( K ~ cm z)
Cm (pF/cm z)
-24-t-5 (n = 23)
117_+48 (n - 23)
19_+ 11 (n = 23)
6.836_+910 (n = 22)
8.1 _+3.4 (n = 22)
2.4_+ 1,2 (n = 22)
--28 + I1 (n = 13) -74 _ 8 (n = 14)
193 _ (n = 41 _ (n =
48 (n 4.7 (n
7.047_+ 1.339 (n = 12)
15.2___9.1 (n = 9) 3.2 + 2.3 (n = 11)
3.8__+0.8 (n = 7) 2.9 _ 2.3 (n = 6)
144 10) 23 12)
+ = + =
11 7) 2.0 8)
1. The differences in Rinput, T, R m and Cm between the permanently depolarized cells and D state in second group are significant (P < 0.05) 2. Differences between D and H s t a t e are all significant (P < 0.01) except for Cm .
p o t e n t i a l of - - 2 5 mV. Small d e p o l a r i z i n g and h y p e r p o l a r i z i n g c u r r e n t pulses p r o d u c e d voltage changes with time courses t h a t could be fitted by simple exponential with a time constant of 35 ms (a, b). F o r stronger hyperpolarizing current pulses the onset of the voltage response d e p a r t e d from the exponential function, either not reaching a stable level (c) or doing so along a time course resembling phase 3 of the n o r m a l v e n t r i c u l a r action potential (d). T h e h y p e r p o l a r i z a t i o n was faster the stronger the current pulse a p p l i e d (not shown). A n o d a l b r e a k excitation was also observed with stronger h y p e r p o l a r i z a t i o n (d) a n d it caused a synchronous c o n t r a c t i o n in the whole cell. F i g u r e 2(e) is the c u r r e n t voltage plot for the same cell. Voltages were
(o)
(b)
m e a s u r e d at the end of 300 ms, w h e t h e r or not the m e m b r a n e voltage h a d reached a steady value (see legend). F o r voltages n e a r the resting potential, the relationship between a p p l i e d current a n d voltage was almost linear with a slope of 184 Mf~. Between - 4 0 and - 80 m V the curve becomes almost parallel to the voltage axis. F i n a l l y for voltages negative to - - 8 0 m V the c u r r e n t - v o l t a g e relationship a g a i n becomes almost linear, with a very small slope. T h e surface a r e a of this cell was 5.500 /~m 2. This value was o b t a i n e d from m e a s u r e m e n t s of the length a n d w i d t h of the cell, a n d taking the height to be one third of the w i d t h (considering the cells 'brick s h a p e d ' ) . F r o m these figures a specific m e m b r a n e resistance Rm = 10 K ~ cm 2 and a spe-
(e)
o
I
-40
I -20
-
(d)
/
/
L_, (c)
2o
-
/
-
f
20 IO-"A
-50
13
o__o_._o-O/ - -I00 F I G U R E 2. Typical I-clamp experiment in a cell of the permanently depolarized population. F r o m (a) to (d) ; upper trace shows applied current and lower trace, t r a n s m e m b r a n e voltage. At right, the current-voltage curve for the same experiment, voltage measured at the end of a 300 ms pulse. Squares denote the situation in which the plotted voltage had not stabilized (as in (c)). Calibrations : vertical bar 40 mV, 40 • 10- I x A and horizontal b a r 100 ms.
834
M a s u d a et aL
cific membrane capacitance, C m = 3.5 pF/cm 2 (calculated from a time constant = 35 ms) w e r e o b t a i n e d . T a b l e 2 gives the m e a n v a l u e s o f all p a r a m e t e r s for 23 cells in this first g r o u p . F i g u r e 3 illustrates the t y p i c a l b e h a v i o u r of a cell a b l e to s h o w two levels o f resting p o t e n tial w i t h transitions b e t w e e n t h e m . I n (a) a c h a r t r e c o r d e r t r a c i n g shows t h a t resting p o t e n t i a l soon after cell i m p a l e m e n t was a p p r o x i m a t e l y - - 2 5 m V , after a while a sudden hyperpolarization to --65 mV o c c u r r e d . I n s o m e cells, o n c e this t r a n s i t i o n f r o m the d e p o l a r i s e d (D state) to h y p e r p o l a r ized p o t e n t i a l (H state) h a d o c c u r r e d , the resting p o t e n t i a l stayed at the H level for hours, i n d e p e n d e n t o f the p o l a r i t y , a m p l i t u d e a n d d u r a t i o n of the c u r r e n t pulses a p p l i e d thereafter. I n o t h e r cells (five o u t o f thirteen), h o w e v e r , transitions b e t w e e n D a n d H states c o u l d be o b s e r v e d all a l o n g . F i g u r e 3(b) shows a t r a n s i t i o n f r o m H to D state. T h i s is usually b r o u g h t a b o u t by a n e v o k e d a c t i o n p o t e n t i a l . I n the p r e s e n t e x a m p l e , after 6 s, a h y p e r p o l a r i z i n g pulse i n d u c e d the t r a n s i t i o n (a)
b a c k to the H state. F i g u r e s 3(c) a n d (d) s h o w a t r a n s i t i o n f r o m D to H state in an e x p a n d e d t i m e scale. I n 3(c) the cell i n D state r e s p o n d e d to a s t r o n g h y p e r p o l a r i z i n g pulse in a w a y s i m i l a r to t h a t in F i g u r e 2(b), e x c e p t t h a t the a n o d a l b r e a k e x c i t a t i o n is m u c h d e l a y e d . I n 3(d) the h y p e r p o l a r i z a t i o n was n o t followed b y the a n o d a l b r e a k e x c i t a t i o n , thus m a k i n g t r a n s i t i o n f r o m D to H state. F i g u r e 4 shows a typical c u r r e n t - c l a m p e x p e r i m e n t in a cell e x h i b i t i n g two levels of resting p o t e n t i a l . (a) a n d (b) s h o w the result o f passing h y p e r p o l a r i z i n g pulses at the resting p o t e n t i a l o f - 2 5 m V (D state). F o r small depolarizations and hyperpolarizations the onset o f the v o l t a g e c h a n g e is a single e x p o n e n t i a l . N o t e in (a), 2nd trace, t h a t w i t h a l a r g e r h y p e r p o l a r i z a t i o n the v o l t a g e c h a n g e was n o t e x p o n e n t i a l a n d did n o t r e a c h a s t e a d y level. W i t h f u r t h e r increases in the h y p e r p o l a r i z i n g pulse a m p l i t u d e , a steady v o l t a g e was r e a c h e d faster for s t r o n g e r c u r r e n t pulses, f o l l o w i n g a c o m p l e x t i m e course [-(a), 3rd trace a n d (b)]. T h e s a m e cell, n o w w i t h a resting p o t e n t i a l o f - - 6 5 m V , shows a corn(b)
9
(c)
(d)
I
II
FIGURE 3. Transitions between the two resting potentials. (a) and (b) show records in a chart recorder, voltage in the lower trace and current in the upper trace. Current pulses were applied at 2 s intervals. Zero voltage level is indicated by an arrow in (a) and (b). (a) in the beginning, the electrode was out of the cell; upon impalement by electronic oscillation, resting potential of approximately -- 25 mV was recorded. In sequence, a slow and progressive increase in resting potential is seen. Close to the end of the panel, a sudden change of the resting potential to approximately - 6 5 mV is shown. (b) shows transition from the more polarized (H state) to the depolarized resting potential (D state) following a stimulated action potential. After 6 s a hyperpolarizing pulse brought it back to the H state. (c) and (d) are pictures taken from the screen of an oscilloscope showing transition from D to H state (upper trace, current and lower trace, voltage). In (c), a strong hyperpolarizing current was applied to a cell in the D state. At the end of this pulse a delayed anodal break excitation was seen, bringing the membrane potential back to the previous depolarized level (D state). In (d), after several cycles, the same hyperpolarizing pulse did not stimulate anodal break excitation, thus switching the cell to H state. Calibration in A, corresponds to 50 x 10 -11 A and 50 mV. For (c) and (d) the vertical bar corresponds to 40 mV and 40 x 10 T M A and the horizontal bar to 100 ms.
Isolated Myocytes: T w o Levels o f R e s t i n g Potential (a)
835
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FIGURE 4. A typical I-clamp experiment in a cell with two levels of resting potential. (a) and (b) effects of current pulses applied to the cell in the D state (resting potential --25 mV) and (e) and (d), in the H state (resting potential - 6 5 mV). Observe the non exponential time course in response to the stronger current pulses in (a) and (b). The current voltage plot shows the curve for D (O) and H state (O). Calibration : 40 mV, 40 x 10-11 A and 100 ms. pletely passive b e h a v i o u r for h y p e r p o l a r izations of all a m p l i t u d e s tested (c) a n d for small depolarizations. A d e q u a t e l y strong d e p o l a r i z i n g pulses evoked n o r m a l action potentials, (d) followed by synchronous contraction of the whole cell. T h e current-voltage plot for the same cell is shown at the b o t t o m of Figure 4. O b s e r v e the similarity between the curve o b t a i n e d for the cell in D state, with a resting potential of - - 2 5 m V (filled circles) and the one shown for a perm a n e n t l y d e p o l a r i z e d cell (Fig. 2). T h e c u r r e n ~ v o l t a g e plot in the H state is almost linear for potentials negative to - 7 0 m V , with a small slope. So it is possible to associate the D state not only with a d e p o l a r i z e d resting potential, b u t also with a c u r r e n t - v o l t a g e plot with a steep region between - - 4 0 to - 8 0 mV.
O n the other h a n d the H state m a y be associated with an almost linear, shallow c u r r e n t voltage plot a n d resting potentials negative to - 6 0 m V (in the presence of 5 mM KC1). I n the cell shown in F i g u r e 4, the measured i n p u t resistance, R i n p u t , and the time constant z, for the D state were respectively, 243 Mf~ a n d 50 ms, resulting in calculated values o f R m = 23.3 Kf2 cm 2 a n d Cm = 2.0 ~ F / c m 2. F o r measurements m a d e with small h y p e r p o l a r i z i n g pulses, the same cell in H state h a d a Rinput -87 M f l a n d R m = 9.5 K f l cm 2. T h e cell disp l a y i n g only the h y p e r p o l a r i z e d resting p o t e n t i a l behaves in all aspects as the cells in H state. T a b l e 2 summarizes the values of all p a r a m e t e r s d e t e r m i n e d in 14 cells in H state, including the cell with only high resting
836
M a s u d a et aL
potential. It can be observed that D and H states have completely different membrane resistances (Rm). It is also important to point out that despite the qualitative similarity between the cells in D state and the permanently depolarized cells, there were significant differences in the measured parameters between them. Figure 5(a) shows current-voltage plots from a cell in H state exposed to 5, 10, 25, 50 and 100 mm potassium. Observe that: (1) the resting potential varies with extracellular potassium and (2) the shape of the c u r r e n ~ voltage plot does not necessarily change in different potassium concentrations, despite the changes in resting potential. Additional interesting observation was that transitions between the two states, D and H, occurred at different potassium concentrations from 1 to 50 mM. The transition from H to D state was characterized by a change in resting potential and in the shape of the curren~voltage plot as already mentioned for 5 mM potassium. This is shown in Figure 5(b) for 25 mM extracellular potassium. Figure 6 shows the effect of extracellular potassium concentration on the resting potential both in H (open symbols) and D (filled symbols) states. In the H state, a clear dependence of resting potential on extracellular potassium is seen. The isolated cells behave in a way very similar to that already described for
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ventricular tissue. For potassium concentrations above 2.5 mM a linear relationship with a slope close to that expected for a potassium electrode was observed. For lower concentrations a clear deviation is seen. In the D state no relation exists between resting potential and extracellular potassium (filled symbols). In permanently depolarized cells, the resting potential did not depend at all on extracellular potassium concentration just as the cells in D state. "Whole cell patch clamp" experiments were successfully performed in seven cells maintained in sol. A in order to analyse the steady state current-voltage relationship. The holding potential was 0 mV. The resting potential soon after the breakage of the patch membrane was - 77 4- 10 mV. Although all of them had current-voltage relations expressed by roughly N shaped curves as seen in ventricular tissue and also in isolated cells, some differences could be distinguished between them. Figure 7, left, shows the three types of current-voltage plots observed. A (open circles), is a plot seen in two of the cells, and is similar to the previously described by Sakmann and Trube, in that, the only one zero current potential observed corresponded to the resting potential seen under current clamp conditions. Four cells had curves similar to that shown in B (triangles). In this
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-100 F I G U R E 5. Effects ofextracellular potassium concentration on resting potential in H state. (a) current-voltage plot in a cell exposed to 5 ( 9 10 (O), 25 (A), 50 (A) and 100 (D) mM KC1. Observe the parallelism between the 5 curves, despite change in resting potential. (b) transitions between D (A) and H (A) states in presence of 25 mM extracellular potassium.
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837
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FIGURE 6. Effect of extracellular potassium concentration on resting potential in H (open symbols) and D (filled symbols) states. Observe clear dependence in the H state and complete independence in the D state, of the resting potential on Ke . Straight line is for a potassium electrode. Same symbols (open and full) were used to represent the same cell.
g r o u p , the c u r r e n t - v o l t a g e c u r v e for p o t e n tials b e t w e e n - - 3 0 a n d 0 m V b e c o m e s a l m o s t p a r a l l e l to the v o l t a g e axis, v e r y close to o r at the z e r o c u r r e n t level. So, at least t w o regions o f z e r o c u r r e n t v o l t a g e c o u l d be d i s t i n g u i s h e d , o n e at the resting p o t e n t i a l level seen u n d e r c u r r e n t c l a m p a n d a n o t h e r , at m o r e d e p o l a r ized level. O n e cell t h a t initially h a d a resting
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p o t e n t i a l of - - 6 0 m V , w h e n c l a m p e d to 0 m V s h o w e d a slow drift in h o l d i n g c u r r e n t t o w a r d s z e r o a n d t h e n it stabilized (Fig. 7, left, C, squares). T h e o n l y zero c u r r e n t v o l t a g e for this cell n o w is close to zero. F i g u r e 7, right, shows the effect of 5 mM CsC1 on s t e a d y state c u r r e n t v o l t a g e plot. T h e blocki n g effect is c l e a r for p o t e n t i a l s n e g a t i v e to
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FIGURE 7. Steady-state current voltage curves obtained from whole cell patch-clamp experiments. Curves A, (O), B (A) and C (IN) were obtained from three different cells, maintained in control solution, at same holding potential (0 mV). Right: Steady state current voltage relation under control solution (A) and in presence of 5 mM CsCI (0) HP = 0 mg.
838
M a s u d a et al.
- 4 0 mV. The slope resistance around the resting potential increases from 47 to 180 Mf~ in presence of CsC1. Discussion
The results presented in present paper, show that three distinct groups of cell can be found among enzymatically dissociated ventricular myocytes. One group is characterized by low resting potential, as shown in Figures 1, 2 and Table 2 (permanently depolarized cells). Presence of depolarized cells among the isolated cardiac myocytes has previously been described by other authors [6, 16] but a distinction exists between the cells they described and present ones; these cells have much higher membrane resistance. For instance, Isenberg & Klockner found 10 __+4 Mf~ in input resistance for rat myocytes of approximately same size compared to 117 __ 47 Mf~ in present paper. They are also characterized by the fact that their resting potentials are not dependent on the extracellular potassium concentrations. These cells can be identified with the cell which current-voltage relationship obtained under whole cell voltage clamp is represented by curve C in Figure 7, left, which had only one region of zero current potential ranging between - 2 5 and 0 mV. No zero current-voltage was found in the hyperpolarized range usually corresponding to resting potential in ventricular cells in tissue. A second group of cells had two different states with distinct resting potentials: H and D states (Fig. 1 and Table 2) as previously described for Purkinje fibers by Wiggins and Cranefield, 1976 and Gadsby and Cranefield, 1977. Some of these cells were able to switch from one state to the other as a consequence of imposed de- or hyperpolarization (Figs 3 and 4). In the D state, this cell behaves in all aspects as the permanently depolarized cells described above despite the significant difference in membrane resistance (Fig. 4, Table 2). In the H state, however, they had completely different properties: the membrane resistance was much smaller being the input resistances found comparable to values described for other isolated myocytes; for instance Isenberg and Klockner [6] found 33 _+ 8 Mf~; 36 __ 9 Mf~ and 41 ___ 10 M ~ for rat, guinea-pig and bovine ventricular cells and Sheets et al. [13] found Rinput = 21 ___8 M ~ in canine Purkinje fibres with comparable resting potentials. The resting potential
depended on extracellular potassium concentration in a way very close to that predicted by Nernst Equation (Figs 5 and 6) as found by Isenberg and Klockner [9]. Curve B in Figure 7, left, represents this group of cells. As the current-voltage plot under voltage clamp is N shaped and has two ranges of zero current voltages, it can be expected that when the cell is in H state, depolarizing currents strong enough to bring the transmembrane potential to or beyond the negative conductance region, would make the switch to D state upon the turning off of the current pulse. On the other hand, a cell in D state could suddenly change to H state by application of a hyperpolarizing pulse strong enough to overcome the negative conductance range. The above described behaviour corresponds exactly to what we have shown in Figures 3 and 4. A third group of cells, had only one resting potential in the hyperpolarized range usually found in tissue. This group corresponds to a current-voltage plot shown in curve A, Figure 7 left, with only one zero curren~voltage in the hyperpolarized region. Similar results had previously been described in isolated cells by Sakmann and T r u b e , 1984. The resting properties in these cells are all similar to the second group of cells in H state. The dependence of the hyperpolarized resting potential on extracellular potassium concentration confirms previous observations both in tissue and in isolated cells, that at the usual resting potential, the ventricular cell membrane is highly selective to potassium (4, 12 and others). The independence of the resting potential in the D state and in permanently depolarized cells to external potassium and the much higher membrane resistance seen in D state as compared to H state, suggests the presence of an anomalous rectifier, which is open at more negative potentials but shut off in D state. Transition from D to H state could be promoted by the turning on of the anomalous rectifier channel leading to the observed decrease in membrane resistance. The effect of CsC1, on the current voltage plot in these cells, increasing the slope resistance around the hyperpolarized resting potential (Fig. 7, right) is additional support for the above hypothesis. We do not know yet, what is responsible for the differences in the current voltage relation-
Isolated Myocytes: Two Levels of Resting Potential ship a m o n g different isolated cells a n d therefore, w h a t d e t e r m i n e s the described h e t e r o g e n e i t y in resting p o t e n t i a l s a m o n g them. O n e possibility is t h a t a v e r y small yet i m p o r t a n t difference in a n i n w a r d b a c k g r o u n d c u r r e n t exists a m o n g them. T h i s c o u l d e x p l a i n the difference b e t w e e n curves A a n d B in F i g u r e 7 left, specially for p o t e n t i a l s positive to - - 5 0 m V . T h i s could also e x p l a i n the difference in m e m b r a n e resistance observed b e t w e e n cells in D state a n d p e r m a n e n t l y d e p o l a r i z e d cells. A n o t h e r possibility c o u l d be a difference in the properties of the a n o m a l o u s rectifier c h a n nels. T h i s idea was raised u p from the observ a t i o n t h a t in p e r m a n e n t l y d e p o l a r i z e d cells s t u d i e d u n d e r c u r r e n t c l a m p , while a s t r o n g h y p e r p o l a r i z i n g pulse is m a i n t a i n e d , the t r a n s m e m b r a n e v o l t a g e b e h a v e s in a w a y v e r y similar to those cells in D state. After the t u r n i n g o f f o f the pulse, in the first case, t r a n s m e m b r a n e p o t e n t i a l comes b a c k to the s a m e previous d e p o l a r i z e d level, while cells i n D
839
state c h a n g e its resting p o t e n t i a l to a n e w h y p e r p o l a r i z e d value, s w i t c h i n g to the H state. I t seems as if i n p e r m a n e n t l y d e p o l a r ized cells the c h a n n e l s responsible for the h y p e r p o l a r i z e d resting p o t e n t i a l ( a n o m a l o u s rectifier) t h o u g h present, were n o t able to stay open, o n c e the a p p l i e d pulse is t u r n e d off. I t seems also m e a n i n g f u l t h a t the slope resistance in c o m p a r a b l e voltages i n the h y p e r p o l a r i z e d r a n g e is l a r g e r i n cells w i t h o n l y the d e p o l a r ized zero c u r r e n t p o t e n t i a l , t h a n in those w i t h two zero c u r r e n t potentials ( c o m p a r e curves C a n d B, Fig. 7 left).
Acknowledgements T h e a u t h o r s wish to a c k n o w l e d g e E d m i l s o n A n t o n i o P e r e i r a for the excellent t e c h n i c a l assistance a n d Drs A. C. C a m p o s de C a r valho, S. C u k i e r m a n a n d A. Paes de C a r v a l h o for helpful discussion a n d c o m m e n t s o n the manuscript.
References 1 BROWN,A. M., LEE, K. S., POWELL,T. Voltage clamp and internal perfusion of single rat heart cells. J Physiol 318, 455-477 (1981). 2 BROWN,A. M., LEE, K. S., POWELL,T. Sodium current in single rat heart muscle cells. J Physiol 318, 479-500 (1981). 3 CALLEWAERT,G., CARMEEIET,E., VEREECKE,J. Single cardiac Purkinje cells: General electrophysiology and voltage-clamp analysis of the pacemaker current.J Physio1349, 643-661 (1984). 4 GADSBY,D. C., CRANEFIELD,P. F. Two levels of resting potential in cardiac Purkinje fibers. J Gen Physiol 701, 725-746 (1977). 5 HuME,J. R., GILES,W. Ionic currents in single isolated bull-frog cells.J Gen Physio181,153-194 (1983). 6 ISENBERO,G., KLOCKNER, U. Calcium tolerant ventricular myocytes prepared by preincubation in a 'KB Medium'. Pfliigers Arch, EurJ Physio1395, 6-18 (1982). 7 ISENBERG,G., KLOCKNER,U. Isolated bovine ventricular myocytes: characterization of the action potential. Pfluge'i-sArch, EurJ Physio1395, 19-29 (1982) 8 ISENBERO,G., KLOCKNER,U. Calcium currents of isolated bovine ventricular myocytes are fast and of large amplitude. Pflilgers Arch, EurJ Physio1395, 30~1 (1982). 9 LEE,K. S., TSXEN,R. W. Reversal of current through calcium channels in dialysed single heart cells. Nature 297, 498-501 (1982). 10 LENTO,H., TIRm, R. Characterization of contractions in mechanically disaggregated myocardial cells from the rat. Acta Physiol Scand 110, 385-389 (1980). 11 POWELL,T., TERRAR,D. A., TwisT, V. W. Electrical properties of individual cells isolated from adult rat ventricular miocardium. J Physio13112, 131-153 (1980). 12 SAKMANN,B., TRUaE, G. Conductance properties of single inwardly rectifying potassium channels in ventrlcular cells from guinea pig heart. J Physio1347:642-658 (1984). 13 SHEETS,M.F., JANUARY,C. Z., FOZZARD,H. A. Isolation and characterization of single canine cardiac Purkinje cells. Circ Res 53, 544-548 (1983). 14 TARR,M., TRANK,J. W. Preparation of isolated single cardiac cells from adult frog atrial tissue. Experimentia 32, 338-340 (1976). 15 TRAtJTWEIN,W., TANIOUCnLJ., NOMA,A. The effect ofintracellular cyclic nucleotides and calcium on the action potential and acetylcholine response of isolated cardiac cells. Pfliigers Arch EurJ Physio1392:307 314 (1982). 16 WATANAaE,T., DELBRIDOE,L. M., BUSTAMANTE,J. O., McDoNALD, T. Heterogeneity of the action potential in isolated rat ventricular myocytes and tissue. Circ Res 52, 280-290 (1983). 17 WmOXNS,J. R., CRANEFIELD,P. F. Two levels of resting potential in canine cardiac Purkinje fibers exposed to sodium-free solutions. Circ Res 39, 466~-74 (1976). 18 WIT~rENBERa,B. A., ROmNSON,T. F. Oxygen requirements, morphology, cell coat and membrane permeability of calcium-tolerant myocytes from hearts of adult rats. Cell Tissue Res 216, 231-251 (1981). 19 ZILBETER,Yu. I., TIMIM,E. N., BENDUKIDZE,Z. A., BURNASltEV,N. A. Patch-voltage clamp method for measuring fast inward current in single rat heart muscle cells. Pfliigers Arch EurJ Physio1311,1,150-155 (1982).