627

Journal of Physiology (1991), 434, pp. 627-645 With 10 figures Printed in Great Britain

THE CALCIUM PARADOX IN ISOLATED GUINEA-PIG VENTRICULAR MYOCYTES: EFFECTS OF MEMBRANE POTENTIAL AND INTRACELLULAR SODIUM

BY GLENN C. RODRIGO* AND R. A. CHAPMAN From the Department of Physiology, School of Veterinary Science, Park Row, Bristol BS1 5LS

(Received 3 May 1990) SUMMARY

1. Guinea-pig ventricular myocytes, isolated enzymatically without the aid of special media, show a similar sensitivity to the calcium paradox as Langendorffperfused hearts. 2. Measurement of the intracellular activities of Na+ and Ca2+ ions, with a suctiontype ion-sensitive microelectrode at rest, during calcium depletion and during inhibition of the Na+ pump (under both current and voltage clamp) yield values similar to those obtained from multicellular preparations and from isolated myocytes by other means. 3. In voltage-clamped myocytes bathed by media free of divalent cations, an inward sodium current that flows through the L-type Ca21 channels, the rate of rise of a'a and the strength of the contraction induced by return to normal Tyrode solution, show a similar bell-shaped dependence on the membrane potential during the period of Ca2+ deprivation. 4. The rise in a'a that occurs in Ca2+-free, Mg2+-free media, induces an outward current which is composed of currents due to activation of the Na+ pump and K+ channels. 5. On Ca2+ repletion the loading of the cells with Ca2+ does not generate an inward current and the contracture can be reduced, in a dose-dependent way, by the introduction of BAPTA into the sarcoplasm from the solution in the voltage electrode. When [Ca21]i is buffered by added BAPTA, the estimated amount of Ca21 which can enter on Ca2+ repletion is sufficient to bind up to 10 mm of the BAPTA. This change in concentration is similar to that expected from the rise and fall in aNa, seen on Ca2+ depletion and repletion, if a 3 Na+: 1 Ca2+ exchange is responsible for the Ca2+ influx. 6. These data offer support for the so-called intracellular sodium hypothesis for the origin of the calcium paradox in the heart. As the effects of Ca2+ repletion can be prevented by clamping the membrane potential so that a'a does not rise, the contribution of the other effects of Ca2+ depletion to the initiation of the calcium paradox would seem to be less important. Present address: Department of Physiological Sciences, University of Newcastle, Newcastle upon Tyne N2 4HH. *

MS 8470

628

G. C. RODRIGO AND R. A. CHAPMAN INTRODUCTION

In a recent review Chapman & Tunstall (1987) put forward an hypothesis for the mechanism by which the calcium paradox develops in the heart. It proposes that, upon reduction of the bathing Ca2" to below 1 IM, the cell membrane depolarizes to a potential that activates the L-type Ca2" channels and because the ionic selectivity of these channels and their ability to inactivate are lost, a sustained entry of Na+ occurs. These changes result in prolonged action potentials and a rise in the measured intracellular activity of Na+ ions (aNa). The distribution of Na+ across the cell membrane, in Ca2+-free Mg2+-free media, approaches electrochemical equilibrium only when the Na+ pump is also inhibited. This suggests that when the pump is active, the Na+ entry via the Ca2+ channels is eventually balanced by an increased Na+ efflux via the Na+ pump. A large influx of Ca2 , on Ca2+ repletion, would seem to be via the Na+-Ca2+ exchange driven by the elevated aNa, because the severity of various symptoms that occur on Ca2+ repletion show a clear correlation with the level to which a'a has risen. The marked rise in intracellular Ca2` then induces cellular damage, protein loss etc. Chapman & Tunstall (1987) list a wide range of observations that are consistent with this 'intracellular sodium hypothesis' such as the ameliorating effects of procedures that reduce the entry of Na+ via the Ca2+ channels and the exacerbating effects caused by inhibition of the active Na+ efflux. An earlier hypothesis suggests that a weakening of the cell membrane, or more likely the junction between cells, is the critical change to occur during Ca2+ deprivation. On Ca2+ repletion, these changes, together with the contraction of the cells, cause a mechanical disruption of the cell membrane which leads to the cellular damage, i.e. the calcium paradox arises from the multicellular nature of the heart (e.g. Grinwald & Nayler, 1982; Ganote, Sims & Van der Heide, 1983; Nayler, Elz, Perry & Daly, 1983). This hypothesis would seem to be supported by reports that ventricular myocytes, isolated by enzymatic treatment of the heart, do not readily develop symptoms typical of the calcium paradox (Piper, Spahr, Hutter & Spieckermann, 1985; Ruigrok, 1990). Although this contrasts with a number of other studies (Goshima, Wakabayashi & Matsuda, 1980; Slade, Severs, Powell & Twist, 1983; Lambert, Johnson, Lamka, Brierley & Altschuld, 1986), it is clear that a failure of isolated myocytes to develop a calcium paradox would put the 'intracellular sodium hypothesis' in jeopardy. In isolated myocytes, the membrane potential can be readily controlled even when large ionic currents flow and substances can be introduced into the sarcoplasm, through a penetrating microelectrode, while changes in the contraction and physical appearance of the myocytes can be observed (such as the granulation of the cytoplasm and the development of blebs on the cell membrane). This preparation therefore presents an opportunity to test some of the critical predictions of the intracellular sodium hypothesis. These are that the Na+ current through the Ca2+ channels and hence the rise in intracellular Na+ in Ca2+-free media and the effects on the repletion of Ca2+, should show the same dependence on the membrane potential during the Ca2+-free period and that the rise in intracellular free [Ca2+] is necessary for damage to develop on Ca2+ repletion. A number of preliminary reports of this work have appeared as abstracts

THE CALCIUM PARADOX IN ISOLATED MYOCYTES 629 (Chapman & Rodrigo, 1987, 1988, 1989 a, b, 1990 a; Rodrigo, 1988; Chapman, Jones & Rodrigo, 1989; Rodrigo & Chapman 1990a). METHODS

Cell isolation. Ventricular myocytes were isolated from the hearts of adult male guinea-pigs (400-600 g). The method of isolation is essentially the same as that described by Mitra & Morad (1985), with the inclusion of 1 % albumin (Sigma, fraction V) in the enzyme-containing solution. After exposure to the enzyme-containing solution, the isolated heart was perfused for 5 min with normal Tyrode solution containing 2 mM-CaCl2. The heart was then removed from the cannula, cut into small pieces and the cells separated by gently agitating the heart in a small volume of normal Tyrode solution in a shaking water bath at 35 'C. The yield of quiescent, striated rod-shaped cells varied between 50 and 80% and these were stored at room temperature in Tyrode solution and used within 24 h. Experimental procedure. Myocytes were placed in a bath which had a volume of 0 5 ml, heated to 32 + 2 'C by fine heating elements fixed to a glass cover-slip which forms its base and left to settle for 5-10 min. The flow of perfusing fluid was then established at 1 ml min-' and the experiment was started after a further 20-30 min. Cells were observed using an inverting microscope (Nikon, TMS) incorporating a CCD camera (Pulnix, TM-460). In the early experiments myocytes were impaled with a conventional microelectrode filled with 3 M-KCl (tip resistance 30 MQ) to record the membrane potential. These microelectrodes proved unsuitable for single-electrode voltage clamp when large inward currents flow during Ca2+ depletion. Lowering the resistance of the microelectrode improved the voltage control but viability of the cells was adversely affected. If microelectrodes are filled with 1 M-KCl solution (resistance 20 MCI), good voltage control was achieved without an effect on the viability of the myocytes. In other experiments, a good voltage clamp was achieved using a patch-type electrode (resistance of 6-8 MCI) filled with an electrode solution consisting of (in mM): 150 KCl, 5 Na2ATP, 1 0 MgCl2 and 10 HEPES/KOH, at a pH of 7-2. In some experiments chemicals were added to the solution filling the voltage electrode and allowed to diffuse into the sarcoplasm: the fast Ca2+-chelating agent BAPTA (Sigma, chemicals) was added as the potassium salt up to a concentration of 40 mm and in other experiments either tetraethylammonium chloride or CsCl (at 50 mM) was added to block the outward K+ currents, with tonicity maintained by the removal of KCl. Membrane and action potentials were recorded with an Axoclamp-2A amplifier (Axon Instruments) and the cells were voltage clamped using a single-microelectrode switch clamp, with a sampling rate of 3-5 kHz. Cell length. Cell contraction was indirectly monitored by measuring cell length with a video edgedetecting system either at the time of the experiment or later from a video recording (Steadman, Moore, Spitzer & Bridge, 1989). Measurement of intracellular ionic activities. The activities of intracellular Ca2+ and Na+ ions were measured with a new kind of ion-sensitive microelectrode as described by Rodrigo & Chapman (1990b). The quality of subtraction, between an ion-sensitive and membrane potential electrode, was checked by applying a weak depolarizing voltage clamp pulse, of short duration (Fig. 1A). Experiments were continued only if an imposed step in membrane potential resulted in the difference trace returning to within + 1 mV within 500 ms. Imposed changes in holding potential, greater than 10 s in duration, induced time-dependent changes in a'a (Fig. 1B). Experimental solutions. Normal Tyrode solution contained (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 10 MgCl2, 0 33 NaH2PO4, 5 sodium pyruvate, 10 glucose and 10 HEPES (pH adjusted to 7-3 with NaOH); however, in the initial experiments [Mg]. was 0-5 mm. The free Ca2+ in Ca2+-depleted solution was buffered to the required activity, by mixing Ca-EGTA and Tris-EGTA in the appropriate ratios at a total concentration of 4 mm, and the pCa was calculated, assuming a binding constant of EGTA for Ca2+ of 6-44 mol-' at a pH of 7-4. In some experiments, the concentration of NaCl in the Tyrode solution was reduced and replaced with an equimolar amount of either tetramethylammonium chloride or LiCl. Acetylstrophanthidin (Sigma chemicals) was added from a 10 mm stock solution made up in ethanol. Data recording and analysis. Records were made on a Phillips PM3305 digital storage scope and later printed out on a X-Y plotter. Alternatively chart or scope records are digitized and stored on disc using the MacLab system (WPI) together with a Macintosh Plus or SE/30 microcomputer.

G. C. RODRIGO AND R. A. CHAPMAN

630

Trypan Blue experiments. Isolated myocytes were introduced into a small experimental chamber and exposed to Tyrode solution in which the bathing free Ca2+ was buffered, with the aid of EGTA, to a pCa of between 8 and 5. After a period of 10 min, the solution was switched to normal Tyrode solution. After a further 5 min perfusion with Tyrode solution, the solution was changed to one of B A

E

>+i40

48h

-60 -0.81 ~ ~ ~ ~ ~~~~~~~.i8.3. IE -20E E -60 :> 3-8 -100

100ms

c

60s

C

z

Fig. 1. The responses of the new design intracellular ion-sensitive electrodes to changes in membrane potential imposed on a single ventricular myocyte using a single-electrode voltage clamp. A, the membrane is depolarized from a holding potential of -75 mV to 0 mV for 500 ms (upper trace). The global membrane current (middle trace) and the difference between the imposed change in membrane potential and that recorded by a Na+-sensitive microelectrode is shown (lower trace). The electrical response of the Na+sensitive electrode is slower than the voltage electrode but the difference between the potentials recorded by each of the microelectrodes returns to baseline after 100 ms. B, sustained changes in holding potential (upper trace) induce a slow change in intracellular Na+ activity, which stabilize after several minutes (lower trace).

Tyrode solution containing 0 1 % Trypan Blue and 5 min later, the proportion of myocytes unable to exclude the Trypan Blue was then determined. Cell dimension measurements. The mean dimensions of the isolated myocytes were measured under high magnification with an eyepiece micrometer, and the capacity of each myocyte was determined from the membrane time constant and the membrane resistance or by integration of the capacity transient induced by a weak hyperpolarzing voltage clamp pulse. RESULTS

The physical properties of the preparations used Care was taken to select preparations for study that were composed of a single myocyte. The most reliable method arose because experiments invariably end when the myocyte contracts irreversibly and in multicellular preparations one cell can be seen to contract before the other. The dimensions of those preparations used were 171 + 33 ,tm by 32 + 7 /sm by 12 + 2-5 ,um which yielded a volume of 50 + 25 pl and the mean capacitance of the myocytes was 131+40 pF (mean and S.D.).

The initiation of the calcium paradox in isolated ventricular myocytes Myocytes, isolated using a modified version of the method described by Mitra & Morad (1985), remain quiescent on exposure to nominally Ca2+-free Tyrode solution. When returned to normal Tyrode solution, after 5-10 min, the majority of myocytes show a bout of spontaneous mechanical activity. After a further 10 min, 20+ 11 % of the cells are found to be hypercontracted and unable to exclude Trypan Blue. On

THE CALCIUM PARADOX IN ISOLATED MYOCYTES

631

exposure to Mg2+-free Tyrode solution, in which the free Ca21 is buffered with EGTA, a sigmoidal relationship is found between the number of cells unable to exclude Trypan Blue on Ca2+ repletion and the pCa of the fluid during Ca2+ depletion. Less than 15 % of the myocytes are affected by pre-incubation at pCa 4 or 5; 50 + 11 % are irreversibly damaged at a pCa of 6 which rises to 95 + 5 % at pCa 8.

Ca-

E2

0 ~ -1.6

3

pCa 7, 0 Mg2+ 1 > 0 Z

i

0 K+

t

20 -20

mM-Mg2+

mM-Ca2+

_

_

_

_

_

_

_

_

_

_

_

_

EC-40 -60

X -80 10 13 19 16 Time (min) Fig. 2. The membrane current (upper trace) recorded from an isolated myocyte at a holding potential of -32 mV (lower trace). The changes in the composition of the bathing fluid and their duration are indicated by the labelled horizontal bars. Initially, the bathing fluid is changed to one free of Mg2+ and with the free Ca2` buffered to 0-1 UM, which induces a large inward current which decays slowly over the next few minutes. When the bathing [Mg] is raised to 1 mm for 5 min, the inward current is blocked and a decaying extra outward current is revealed. The subsequent return to Mg2+-free fluid reactivates the inward current and the further removal of the bathing K+ induces a brief increase in inward current followed by a large outward current. This outward current, which goes off-scale, declines slowly on return to normal Tyrode solution. No contraction or damage is induced in the myocyte because 20 mM-BAPTA was present in the solution filling the voltage electrode.

1.0

4*0

7.0

Changes in membrane potential or current observed during calcium depletion Isolated myocytes, exposed to Tyrode solution containing 05 mM-Mg2+ and nominally free Ca2+ or in which the free Ca2+ is buffered with EGTA to less than 1 ,UM, slowly depolarize until spontaneous action potentials develop; these progressively prolong until one fails to repolarize and the membrane potential stabilizes between -15 and -25 mV. If the bathing Mg2+ is also absent, the rate of the initial depolarization is increased and a single action potential develops which fails to repolarize as the membrane potential stabilizes at around -10 mV. A prolonged action potential fails to develop if Ca2+ is removed in the presence of 1-2 mM_Mg2+, although the membrane is depolarized to about -60 mV. Once a prolonged action potential has developed, the membrane will repolarize on elevation of the [Mg2+]o or reduction of the [Na+]o by more than 75 % (when Na+ is replaced by TMA+ but not by Li+). When the cell is voltage clamped at -80 mV, the removal of the bathing divalent cations results in the development of a small inward holding current. At holding potentials positive to -50 mV a larger inward current is induced, which can

G. C. RODRIGO AND R. A. CHAPMAN be carried by either Na+ or Li+ ions but not by TMA+ and which is blocked by elevation of the [Mg2+]o above 1 mm. The current-voltage relations for this inward current can be determined in a number of ways which require activation of the current (either by removal of divalent cations at various holding potentials (e.g. Fig. 5) or by 632

B

A

pCa 6 QK 2 4 1 mM-M2+ < 2.41mMM 40 mm-TEACI

~0.0

Membrane potential (mV) -40 -30 -20 -10

20 10

-2.4

50 mM-CsCI E 0.0 n.0 °e -24

~E

/-

-145

0 -40 4) -80

0

z

Ee0 __

1 2

_

_

_

_

3 4 5 Time (min)

_

_

6

-2.5J

Fig. 3. The effects of Ca2+ depletion and Na+ pump inhibition on membrane current of an isolated myocyte into which 50 mm-CsCl or 40 mM-TEACl has been allowed to diffuse from the solution in the voltage electrode. A, current traces induced at a holding potential of -10 mV by the lowering of the bathing [Ca2+] to 1 ,UM (in the absence of Mg2+) induces an inward current which unlike the trace in Fig. 2 is sustained. The subsequent removal of bathing K+ increases the sustained inward current. B, the collected data for twelve myocytes in when the K+ currents are blocked by the introduction of Cs+ into the sarcoplasm from the solution in the suction voltage electrode, where the steady inward current, in the presence (0) and absence of bathing K+ (I), is plotted against the membrane potential. The lines are linear regression lines (r > 0 98).

depolarization from a holding potential negative to -50 mV, in the absence of bathing divalent cations. A typical current-voltage relationship, uncorrected for outward currents, determined by the removal of divalent actions from the bathing fluid is shown in Fig. 6A. When the K+ currents are blocked by the diffusion of 50 mM-TEA+ or Cs+ from the voltage electrode the apparent reversal potential of the inward Na+ current is shifted towards less positive potentials as compared to that for current when Ca2+ is the principal charge carrier (Fig. 3B). A shift of this magnitude has been reported previously (Almers, McCleskey & Palade, 1984; Hess & Tsien, 1984; Imoto, Ehara & Goto, 1985). The recorded inward current, elicited by exposure to Ca2+-free, Mg2+-free fluid at holding potentials between -30 and 0 mV, decays spontaneously (Fig. 2). On addition of 1-2 mM-Mg2+ to the bathing fluid, the inward current is blocked, and the current through the membrane reverses to exceed the original holding current. The size of this extra outward current increases with the time spent in Ca2+-free medium.

THE CALCIUM PARADOX IN ISOLATED MYOCYTES

633

Both the spontaneous decay of the inward current and the extra outward current are absent if either 50 mM-TEA+ or Cs+ is present in the voltage electrode or 4 mM-4-aminopyridine is added to the bathing fluid (Fig. 3A). This suggests that an outward current, carried by K+, is gradually activated during expose to Ca2+-free, A

B

cE-2|

cE

20

l

.0 Ca

0 -40 o -80 Q.

E

3 4.7

, u

6.4

60. 7.9

pCa 7, 0 Mg2+

Fig. 7. The effects of the exposure of an isolated myocyte to Tyrode solution free of Mg2+ and with the pCa buffered to 7 (as indicated by the labelled bar), on the membrane potential (upper trace) and the intracellular activity of Ca ions measured with a second ion-sensitive microelectrode (lower trace). On Ca2+ repletion a'a rises in two phases. The first was accompanied by a severe shortening of the myocyte, the second by the disruption of the cell membrane.

Changes in aia during calcium depletion Lambert et al. (1986) have shown, using Quin-2, that [Ca21]i falls from 150 to 60 nM on exposure of rat myocytes to a bathing pCa greater than 6. The response of the intracellular Ca2+-sensitive electrodes rarely showed a change in potential indicative of a fall in a'5 on Ca2+ depletion (Fig. 7). This may be an artifact, due to the combined effects of the rise in ala and the poor selectivity of the resin for Na+ as against Ca2+. The effect of membrane potential during the period of Ca2+ depletion on the contracture seen on Ca2+ repletion The above results show a close correlation between the size of the inward current and the rise in a'a recorded in Ca2+-free, Mg2+-free Tyrode solution, in voltageclamped myocytes. If the level to which aia rises is the factor that determines the loading of the cells with Ca2 , it would be anticipated that the effects of the repletion of Ca2+ will depend on the membrane potential during the period of Ca2' depletion, in a similar way to the rise in aia and the size of the sustained inward current. Figure 8 shows a typical experiment where the membrane potential of a single myocyte is clamped at various values during Ca2+ depletion and returned to the holding potential of -80 mV just prior to Ca2+ repletion. The sequence of holding potentials during Ca2+ depletion was -80, 0, + 20 and -20 mV. At -80 and + 20 mV, no inward current is recorded on Ca2+ depletion and little or no contraction is induced by Ca2+ repletion. At 0 mV, a phasic contraction develops but the mechanical and electrical properties of the cell recover fully. At -20 mV, a large inward current is evoked during Ca2+ depletion and sustained hypercontracture is observed on Ca2+ repletion.

THE CALCIUM PARADOX IN ISOLATED MYOCYTES A

Em (mV)

/m (nA) Cell shortening M

0 -80 1*2. 0 J

Cell

shortening

_

60 s

0

/m (nA)

pa 6, 0 Mg2 pCa____6_______Mg

0 10

B

Em (mV)

-78 mV

0 mV

-80

0.8 0 -0.8 0 6

(%)

pCa 6, 0

Mg2+

60 s

C

+25 mV

Em (mV)

40

/m (nA)

1.2 0

Cell

shortening

-1.2 0 10

(%) D

0

Em,

pCa 6, 0 Mg2+ 60 s

-20 mV

(mV) -60

1.2 0 -1.2 Cell 0 25 shortening 50 (%)

Im (nA)

Mg2+ pCa 6, 0 Mg 60 s

Fig. 8. Experimental traces of the membrane potential (upper traces), membrane current (middle traces) and cell length (lower traces) of a myocyte under voltage clamp. The periods during which the myocyte is perfused by Tyrode solution containing no Mg2+ and with a pCa buffered to 6 are indicated by the horizontal bars. Note that the membrane potential was clamped to -80 mV just before the return to normal Tyrode solution. The experimental traces were obtained in the order, A, B, C, and D to increase the likelihood of a complete experiment. In the same myocyte, as seen in D, when the driving force on Na+ entry and hence the inward Na+ current is largest, the subsequent repletion of Ca2+ caused the cell to supercontract and lose its membrane integrity.

637

G. C. RODRIGO AND R. A. CHAPMAN

638

B

A

20 mM-BAPTA

40

5 mM-BAPTA

40

c

c

01-

.

c

-0

E

-40

E

-

.* -40 -

' °0. -80

C

-80 _

_

-0 a)

E

0

a) 4c

60

0)

s

_

2 201

60 s

20

c 20 mM-BAPTA 0 0

r

-0

E

.' _40

'0°80

= az

Ec 0

Fig. 9. The membrane potential (upper traces) and cell length (lower traces) or three myocytes exposed to Mg2+-free fluid with pCa buffered at 7. Each myocyte was penetrated with a conventional 1 M-KCl-filled high-resistance microelectrode with contained between 5 mM (A) and 20 mM (B and C) BAPTA in the electrode-filling solution (as indicated above each trace). In each case removal of the bathing divalent cations (the timing of which is indicated by the horizontal bar) caused a prompt and sustained depolarization of the membrane. A, the return to normal Tyrode solution causes repolarization and a sustained resting potential in spite of a hypercontracture at 5 mM-BAPTA; B, at 20 mM-BAPTA contraction is almost fully suppressed as the membrane potential repolarizes; C, a

THE CALCIUM PARADOX IN ISOLATED MYOCYTES

639

Once the contracture had developed fully, evidence of damage to the cell membrane became apparent; blebs rapidly appeared and the membrane potential and resistance fell to zero as cytoplasm was seen to spill from the cell. Figure 6A shows data collected from a number of experiments of this sort which are compared to other experiments where the rate of rise of a'a and the inward current were measured. It shows that the dependence of the degree of shortening on Ca2+ repletion closely parallels the potential dependence of both the size of the inward current and the rise of a'a observed during the period of Ca2' depletion.

Other changes seen on Ca2+ repletion Intracellular Na+ and Ca2+ A steep rise in aia has been reported on repletion of Ca2+ in cardiac cells (Lambert et al. 1986), which may or may not be accompanied by a fall in a'a (Chapman, Fozzard, Friedlerlander & January, 1986). In the present experiments the exact values of a' are uncertain because the rise in a' that accompanies Ca2+ depletion affects the response of the Ca2+ electrode. If it is assumed that a"a rises to between 30 and 40 mm then a'a would approach 10 /IM during the first minute of Ca2+ repletion. When a myocyte recovers and maintains both resting and action potentials, measurements in separate myocytes suggest that aia and aia fall back to return to control levels over the next 3-5 min. If the cell membrane shows signs of damage, the measured values of aia and aia rapidly approach those of the bathing fluid (Fig. 7). When Ca2+ repletion induces a contracture in voltage-clamped myocytes, apart from the currents associated with the preceding clamp back to -80 mV, no other inward current is observed (e.g. Fig. 8B).

The effects of increasing intracellular calcium buffering with BAPTA If BAPTA is included in the solution filling either a microelectrode or a suction electrode the action potential is prolonged while under voltage clamp the slow inward Ca2+ current is seen to increase in amplitude, and its inactivation is slowed. The presence of 2 mM-BAPTA in the voltage electrode is sufficient to abolish all shortening associated with the action potential within 5 min. Figure 9 shows the effect of Ca2+ repletion, upon membrane potential and cell length of myocytes exposed to Tyrode solution free of divalent cations for 5 min. The cells are penetrated with conventional microelectrodes containing different concentrations of BAPTA (20 min is allowed for equilibration between the solution in the electrode and the sarcoplasm). The degree of shortening on Ca2+ repletion decreases with increased [BAPTA] in the electrode. Little or no shortening occurs at a concentration of 20 mM (Fig. 9B) while at 10 mM-BAPTA the cell contracts strongly but then relaxes myocyte, containing 20 mM-BAPTA, is exposed to media free of divalent cations which also contained 50 ,uM-stropilanthidin. On return to Tyrode solution the membrane promptly repolarizes and action potentials can be evoked. After less than 1 min the cell suddenly supercontracted and shortly afterwards the integrity of the cell membrane is lost as evidenced by the loss of membrane potential and resistance. (Note that the action potentials at the beginning of the trace are much affected by the aliasing of the A-D convertors, but note that there is no accompanying twitch, although the cell relaxes slightly on exposure to the Ca2+-free solution.

G. C. RODRIGO AND R. A. CHAPMAN completely. At 5 mm and in the absence of BAPTA the myocytes shorten irreversibly, but with 5 mM-BAPTA the membrane potential repolarizes and action potentials can be evoked even though the cell remains hypercontracted (Fig. 9A). A similar set of results are obtained with suction electrodes. If a myocyte, with 20 mm-BAPTA in the sarcoplasm is subjected to Ca2+ deprivation and repletion in the presence of 50 ftMstrophanthidin, the cell repolarizes briefly on Ca2+ repletion and action potentials can be evoked. The duration of each successive action potential is progressively shortened until the myocyte contracts vigorously and some seconds later the recorded membrane potential falls to zero (in Fig. 9 C the action potentials were too brief to be captured adequately by the A/D converters). In some experiments an ion-sensitive microelectrode was used to measure the changes in a'a or aia in myocytes into which BAPTA had been allowed to diffuse before exposure to Ca2+ depletion and repletion. The removal of bathing divalent cations caused a'a to rise, and Ca2+ repletion lead to the recovery of a a (Fig. 4B). In a few experiments, aia was measured and if it is assumed that a'a reaches between 30 and 40 mm, the rise in aia would be equivalent to 1 /UM (with 5 or 10 mM-BAPTA in the voltage electrode). Irrespective of the exact value to which a'. rises, these results show that a Ca2+ influx, sufficient to overcome a substantial part of the buffering offered by the BAPTA, can occur on Ca2+ repletion. Furthermore, they show that the diffusion of unbound BAPTA from the microelectrode into the sarcoplasm is too slow to counteract this. 640

DISCUSSION

Myocytes isolated from guinea-pig ventricles using the method of Mitra & Morad (1985) show a sensitivity to the calcium paradox similar to Langendorff-perfused hearts, in that a significant proportion show an irreversible contracture and fail to exclude Trypan Blue, on return to Ca2+-containing Tyrode solution after only 5 min in nominally Ca2+-free Tyrode solution. A sensitivity to the calcium paradox similar to that of intact hearts has been reported previously for isolated cardiac muscle cells, when a variety of methods have been used to assess the consequences of Ca2+ repletion (Goshima et al. 1980; Slade et al. 1983; Lambert et al. 1986). However, Piper et al. (1985) obtain myocytes which show a marked resistance to both the calcium and oxygen paradoxes. This increased resistance would seem to derive from the method used to isolate the cells, which involves the exposure, during the final stages of isolation, to media rich in certain amino acids, which produces myocytes with a greater ability to regulate a'Na (Chapman & Rodrigo, 1990b). As a consequence, myocytes, isolated without the aid of agents to increase their calcium tolerance, have been used in the present study. When the measurement of intracellular ionic activities in isolated myocytes with ion-sensitive microelectrodes is carefully controlled (as discussed in detail elsewhere; Rodrigo & Chapman, 1990 b), the levels of intracellular Na+ and Ca2+ ions, measured in resting myocytes bathed by normal Tyrode solution, are similar to those found in multicellular preparations (Lee, 1981; Bers & Ellis, 1982; Sheu & Fozzard, 1982; Chapman, 1986). Furthermore, the magnitude of the rise in aia on removal of bathing divalent cations and rise in aia on the repletion of Ca2+ agree well with those reported for multicellular preparations when measured with ion-sensitive electrodes

THE CALCIUM PARADOX IN ISOLATED MYOCYTES

641

and for isolated myocytes when other methods are used (Goshima et al. 1980; Chapman, Rodrigo, Tunstall, Yates & Busselen, 1984; Lambert et al. 1986; Chapman et al. 1986; Tunstall, Busselen, Rodrigo & Chapman, 1986; Chapman & Rodrigo, 1987; Donoso, Eisner, & O'Neill, 1990). In addition the effects of holding potential upon aNa are similar to those reported for multicellular preparations (Eisner, Lederer & Vaughan-Jones, 1983; January & Fozzard, 1984). The main purpose of this study was to test some predictions of the intracellular sodium hypothesis for the origin and development of the calcium paradox. The inward current, evoked by exposure to media free of divalent cations, fails to develop when TMA+ but no Li+ replaces the Na+ in the bathing fluid and is blocked by a [Mg2+]. of 1 mm or above. It therefore closely resembles the monovalent cation current through the L-type Ca2+ channels described previously (Almers et al. 1984; Hess & Tsien, 1984; Imoto et al. 1985). The inward membrane current and the rate of rise of aia during Ca2+ depletion, show a similar bell-shaped dependence on the membrane potential except at potentials near to + 20 mV when the recorded current is outward and a slow rise in a'Na persists. As further depolarization is required to block completely the rise in aia the true reversal potential for the Na+ current through the Ca2+ channels is around +30 mV, indicating that the recorded current includes currents associated with activation of outward K+ currents and the electrogenic Na+ pump. Figure 6B shows a linear relationship between the peak inward Na+ current and the rate of rise of a'a, for all the data when the Ca2+ channels are unblocked by the removal of Mg2+ from the bathing media over a range of holding potentials. The mean slope of the regression line, at 5-2 mm nA-' min-' is about half of what would be expected from Faraday's constant and the mean volume of the myocytes at 50 + 25 pl. However, no allowance has been made for a contribution of the Na+ pump to membrane current and the rise in a'a or for the activation of other outward membrane currents. The activity of the Na+ pump will reduce both the rise in aiN. and the net inward current but by unequal amounts, while the activation of K+ currents will contribute to the outward current under these circumstances. For these reasons, it is not surprising that a discrepancy between the size of the inward current and the rate of rise of aia is found. When the outward K+ currents are blocked the recorded inward current is sustained during Ca2+ depletion and the contribution of the Na+ pump to the membrane current can be revealed by the removal of bathing K+ (Fig. 3A). The maintained nature of the inward current, with and without bathing K+, suggests that the influx of Na+ is sustained but opposed by an efflux via the Na+ pump. When the influx and efflux are equal, if it is assumed that no other currents are flowing an estimate of the stoichiometry of the Na+ pump can be made. The 3/2 ratio, seen in Fig. 3B, would therefore be consistent with the known stoichiometry of the Na+ pump. At larger inward currents, the ratio exceeds 3/2 suggesting that the Na+ pump does not balance the Na+ influx through the Ca2+ channels either because the driving force is high or because there is an effect of membrane potential on the Na+ pump. The current-voltage relations for the two conditions can be fitted by straight lines, which extrapolate to a common intercept with the voltage axis (Fig. 3B), i.e. when there is no extra Na+ influx there is no extra Na+ pump current. These data suggest that the rise in a4a (mainly due to the influx of Na+ through the L-type Ca2+ channels) is opposed and eventually balanced by an increased efflux via the Na+ pump (as diagrammatically represented in Fig. lOA). 21

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The data in Fig. 6A suggest strongly that it is the rise in aka that occurs during Ca2+ depletion, which determines the magnitude of Ca2+ loading on Ca2+ repletion, because the strength of the contracture that develops on Ca2+ repletion closely parallels the relationship for the rise in aNa. Particularly convincing are the data at B

A

Ca2+ depletion

Ca2+ repletion

L-type

Ca2+ channel Na+ 3 Na+-

Ca2+

P

Na+

ATP_4

2 K+

3

Na+.*?

3 Na+ _AT Na++ C2 C aa Ca22+

Nal ~~~~~~~~3 P

2 K+

Fig. 10. A schematic representation of the events that occur when a cardiac myocyte is exposed to media free of divalent cations (Ca2+ depletion) followed by return to normal Tyrode solution (Ca2+ repletion). A, on Ca2+ depletion the probable pathways by which Na+ enters the sarcoplasm are the Na+-Ca2+ exchange and the L-type Ca2+ channels. This entry of Na+ is not immediately balanced by the increased activity of the dNa+ pump so the a'a rises. B, on Ca2+ repletion the Ca2+ channels close and inactivate as the membrane repolarizes and [Ca2+]i rises. Ca2` entry is therefore principally via the Na+-Ca2+ exchange driven by the elevated aia. The rise in [Ca2+]i will be maintained until the Na+ pump has reduced a'a to levels where the Na+-Ca2+ exchange reverses.

potentials negative to -50 mV and positive to + 20 mV, when there is no inward current, little rise in a'a and no shortening of the myocytes on the repletion of Ca2+ (when the holding potential is -80 mV), i.e. at potentials that would have very different effects on the Na+-Ca2+ exchange. The isolated myocytes not only contract on Ca2+ repletion, but show symptoms similar to those seen in multicellular preparations and intact hearts such as the loss of membrane potential and resistance and the disruption of the cell membrane. These results not only confirm that a calcium paradox can be elicited in isolated myocytes but also show that the effects of Ca2+ depletion, other than those that led to a rise in aNa, are not critical in determining many of the effects seen on Ca2+ repletion. The introduction of Ca2+-chelating agents into the sarcoplasm of isolated myocytes not only affects the action potential and contraction (Terrar & White, 1988) but can alleviate the effects of the calcium depletion followed by Ca2+ repletion, even when aka has risen (> 50 mm) during the Ca2+-free perfusion (Fig. 4B). This would seem to indicate that the rise in Ca2+ in the sarcoplasm induces the cellular damage on the return of Ca2+ to the bathing fluid. Even if the concentration of BAPTA established in the sarcoplasm is lower than in the voltage electrode, it is clear that total free and bound calcium in the cell must increase by several millimolar, on the bathing repletion of the Ca2+ for contraction to occur and for the a'a to rise. This means that a substantial influx of Ca2+ through the cell membrane occurs on Ca2+ repletion as suggested by previous work (Goshima et al. 1980; Nayler, Perry, Elz & Daly, 1984). This Ca2+ influx would seem not to be through ionic channels because the membrane

THE CALCIUM PARADOX IN ISOLATED MYOCYTES

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potential repolarizes when the [BAPTA] in the suction electrode is greater than 5 mm, and under voltage clamp an inward current is never seen (Figs 4B, 8 and 9). A mechanism that could carry Ca2" into the cell without generating an inward current is the Na+-Ca2+ exchange. It is possible to obtain an estimate of the rise in total cellular Ca2+ that occurs on the repletion of Ca2+ when BAPTA is present inside the cell and it is assumed that its concentration is close to that in the voltage electrode, and that diffusion is not sufficiently rapid as to add significantly to the free BAPTA when [Ca2+]i rises. When 20 mM-BAPTA is allowed to diffuse into the sarcoplasm, myocytes show only little shortening on repletion; as the apparent binding constant of BAPTA for Ca2+ is close to the contractile threshold for the contractile proteins (Tsein, 1980), it would be expected that half the BAPTA should be bound to calcium. At a [BAPTA] of 10 mm, the myocytes contract strongly but then relax and [Ca2+]i rises to about 1 /tM. At this [Ca2+], 90% of the BAPTA should be bound to calcium. These estimates suggest that the gain in total intracellular calcium that occurs on Ca2+ repletion is sufficient to bind between 9 and 10 mm of BAPTA in the cytoplasm. However, a rise of 10 mm in total cell Ca2+ would have to exchange for 30 mm of Na+ via the Na+-Ca2+ exchange, assuming a 3Na+: 1Ca2+ stoichiometry. This is generally somewhat smaller than the rise and fall of a' that occurs on Ca2+ depletion and repletion in unclamped myocytes. A contribution of the Na+ pump to the fall in aiNa is suggested by the observation that with 20 mM-BAPTA in the voltage electrode and the Na+ pump blocked, on Ca2+ repletion the membrane potential repolarizes only briefly and after a few seconds the cell hypercontracts and the membrane potential and resistance are lost suggesting that the buffering offered by the BAPTA is exceeded (Fig. 9). In myocytes without additional intracellular Ca2+ buffering, the influx of Ca2+ will be much less because of the self-regulatory nature of the Na+-Ca2+ exchange, so that a new steady state at a [Ca2+]i of between 1 and 10 /M is set by the electrochemical forces acting on the Na+-Ca2+ exchange (Fig. 10). The removal of Ca2+ from the sarcoplasm will be relatively ineffective until aia has also fallen and this depends largely on the activity of the Na+ pump. This means that the Na+ pump is not only important in opposing the rise in a' a during Ca2+ depletion but also in limiting the Ca2+ loading on Ca2+ repletion (as suggested by Fig. 9). The responses of myocytes, lightly loaded with BAPTA, to Ca2+ repletion show that hypercontraction may not be accompanied by damage to the cell membrane because such cells retain their action and resting potentials. This is inconsistent with the proposal that the cell membrane is damaged by the hypercontraction as proposed previous (Grinwald & Nayler, 1982; Ganote et al. 1983). It furthermore suggests that the various consequences of a prolonged period of elevated [Ca2+]i (contracture, depletion of energy-rich phosphates, mitochondrial damage, activation of intracellular phospholipases and proteases) may be activated at different levels of free intracellular Ca2+. This work was supported by grants from the Wellcome Trust and the British Heart Foundation. 21-2

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