83

Journal of Physiology (1992), 447, pp. 83-102 With 7 figures Printed in Great Britain

CYTOSOLIC CALCIUM AND MYOFILAMENTS IN SINGLE RAT CARDIAC MYOCYTES ACHIEVE A DYNAMIC EQUILIBRIUM DURING TWITCH RELAXATION

BY H. A. SPURGEON, W. H. DUBELL, M. D. STERN, S. J. SOLLOTT, B. D. ZIMAN*, H. S. SILVERMAN, M. C. CAPOGROSSI, A. TALO AND E. G. LAKATTA From the Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health and * Cardiology Division, Johns Hopkins Medical Institutions, Baltimore, MD 21224, USA (Received 4 December 1990) SUMMARY

1. Single isolated rat cardiac myocytes were loaded with either the pentapotassium salt form or the acetoxymethyl ester (AM) form of the calcium-sensitive fluorescent probe, Indo-1. The relationship of the Indo-1 fluorescence transient, an index of the change in cytosolic calcium [Ca2+]i concentration, to the simultaneously measured cell length during the electrically stimulated twitch originating from slack length at 23 'C was evaluated. It was demonstrated that even if the Ca2l dissociation rate from Indo-1 was assumed to be as slow as 10 s-1, the descending limb ('relaxation phase') of the Indo-1 fluorescence transient induced by excitation under these conditions is in equilibrium with the [Ca21]i transient. Additionally, the extent of Indo-1 loading employed did not substantially alter the twitch characteristics. 2. A unique relationship between the fluorescence transient and cell length was observed during relaxation of contractions that varied in amplitude. This was manifest as a common trajectory in the cell length vs. [Ca2+]i phase-plane diagrams beginning at the time of cell relengthening. The common trajectory could also be demonstrated in Indo-1 AM-loaded cells. The Indo-1 fluorescence-length relation defined by this common trajectory is steeper than that described by the relation of peak contraction amplitude and peak fluorescence during the twitch contractions. 3. The trajectory of the [Ca2+]i-length relation elicited via an abrupt, rapid, brief (200 ms) pulse of caffeine directly onto the cell surface or by 'tetanization' of cells in the presence of ryanodine is identical to the common [Ca2+]i-length trajectory formed by electrically stimulated contractions of different magnitudes. As the [Ca2+]i and length transients induced by caffeine application or during tetanization in the presence of ryanodine develop with a much slower time course than those elicited by electrical stimulation, the common trajectory is not fortuitous, i.e. it cannot be attributed to equivalent rate-limiting steps for the decrease of [Ca2+]i and cell relengthening. 4. The [Ca2+]i-length relation defined by the common trajectory shifts appropriately in response to perturbations that have previously been demonstrated to MS 8978

84

H. A. SPURGEON AND OTHERS

alter the steady-state myofilament Ca2" sensitivity in skinned cardiac fibres. Specifically, the trajectory shifts leftward in response to an acute increase in pH or following the addition of novel myofilament calcium-sensitizing thiadiazinone derivatives; a rightward shift occurs in response to an acute reduction in pH or following the addition of butanedione monoxime. Each of these perturbations markedly shifted the trajectory in the absence of changes in the amplitude of the [Ca2+]i transient. 5. We conclude that the [Ca2+]i-cell length trajectory during the relaxation phase of the twitch contraction in single cardiac myocytes defines a quasi-equilibrium of cytosolic [Ca2+] myofilament Ca2+ binding and mechanical force and thus cell length. The occurrence of this trajectory indicates that the [Ca2+]i decay is a factor that governs relaxation of twitch contraction. Additionally, the position of this trajectory reflects the relative myofilament response to Ca2+. INTRODUCTION

Two general modes of amplitude modulation of the cardiac contraction are (1) a variation in the number of Ca2+ ions made available to bind to the myofilaments during the transient increase in cytosolic calcium [Ca2+]i concentration following excitation and a variation in the extent of myofilament displacement (shortening) or force production in response to a given [Ca2+]i transient (Endoh & Blinks, 1988). A direct assessment of the steady-state myofilament response to calcium has been obtained in preparations in which the membranes have been removed or rendered permeable to ions and in which [Ca2+]i has been clamped at varying levels (Ray & England, 1976; Fabiato & Fabiato, 1978; McClellan & Winegrad, 1978; Kentish, 1986). In intact cardiac cells or muscle a determination of the extent to which a change in the twitch contraction amplitude is due to an altered myofilament Ca2+ response requires the simultaneous assessment of contraction and the [Ca21]i transient. In this regard, studies using the chemiluminescent protein, aequorin, have shown that many perturbations that alter the strength of the cardiac contraction, e.g. receptor agonists via second messenger systems (Allen & Kurihara, 1980; Endoh & Blinks, 1988; Mclvor, Orchard & Lakatta, 1988), or changes in pH or novel cardiotonic drugs (Allen & Orchard, 1983; Endoh, Yanagisawa, Taira & Blinks, 1986) alter both the [Ca2+]i transient and the myofilament Ca2+ response. These studies have largely used measurements of peak aequorin luminescence and force, or the maximum rate of force change, to assess the myofilament response to Ca2 . However, an accurate assessment of the myofilament Ca2+ response requires that Ca2+ and contraction be measured under equilibrium conditions, i.e. at a time when both the level of [Ca2+]i and myofilament Ca2+ sensitivity determine myofilament Ca2+ binding. It has been argued that equilibrium conditions are not achieved during the twitch contraction at the times when peak [Ca2+]i is measured and that the relationship between peak aequorin luminescence and contraction underestimates the myofilament response to Ca2+ (Yue, Marban & Wier, 1986; Yue, 1987; Endoh & Blinks, 1988). This problem is exaggerated during perturbations that accelerate Ca2+ removal from the cytosol and thus accelerate the time course of the [Ca2+]i transient (Endoh & Blinks, 1988; Mclvor et al. 1988). An additional problem with assessment of the myofilament-Ca2+ response in multicellular intact cardiac muscle is that

CARDIAC MYOFILAMENT-Ca2+ RESPONSE AND RELAXATION 85 substantial heterogeneity occurs in the time course of shortening and force development among varying segments of this preparation (Huntsman, Joseph, Oiye & Nichols, 1979). Finally, external loading factors present in bulk tissue not only modulate but can often obscure the [Ca2±]i-myofilament relation during relaxation (Brutsaert & Sys, 1989; Sollott & Lakatta, 1989). In individual isolated cardiac cells a direct assessment of the relationship between [Ca2+]i and cell length, and thus myofilament length, can be made in the absence of external mechanical loading and intercellular heterogeneity. This preparation can be loaded with fluorescent probes sensitive to levels of Ca2+ at or near the diastolic level (Grynkiewicz, Poenie & Tsien, 1985). A method to simultaneously measure contraction, [Ca2+]i, as reported by the fluorescent Ca21 probe, Indo- 1, and membrane potential or current in individual cardiac cells has been reported recently (Spurgeon, Stern, Baartz, Raffaeli, Hansford, Talo, Lakatta & Capogrossi, 1990). Using this method in the present study we examined the relationship between Indo-1 fluorescence and contraction, particularly at later times during the twitch, i.e. at a time when [Ca21]i is approaching its baseline value and when equilibrium conditions between [Ca2+]i and the myofilaments might indeed be achieved. Our results show that during relaxation of twitch contractions that originate from slack length and vary in amplitude, a unique relation exists between cell length and Indo-1 fluorescence. This is manifest as a common trajectory in the cell length [Ca2+]i or Indo- 1 fluorescence phase-plane diagrams. This trajectory is steeper than that described by the peak contraction amplitude verus peak [Ca2+]i. The [Ca2+]i-length relation during the relaxation portion of electrically stimulated twitches is the same as that due to increases in [Ca2+]i and reductions in cell length elicited via the abrupt and transient application of caffeine or by 'tetanization' of the cell in the presence of ryanodine; it shifts appropriately in response to perturbations that have previously been demonstrated to alter the steady-state myofilament Ca2+ sensitivity in skinned cardiac fibres. Thus, the [Ca2+]i-cell length trajectory during the relaxation phase of the twitch contraction in single cardiac myocytes appears to define a quasi-equilibrium of cytosolic [Ca2+], myofilament Ca2+ binding, myofilament force, and thus cell length. The relative position of the trajectory reflects the relative myofilament Ca2+ response; a shift in the position of the trajectory reflects a shift in the myofilament Ca2+ response. The occurrence of the common trajectory also indicates that, under the present conditions, the decay of [Ca2+]i is a factor that determines relaxation. METHODS

Cell isolation procedure Ventricular myocytes were enzymatically dissociated as previously described (Spurgeon et al. 1990). Briefly, 2- to 4-month-old male Wistar rats from the Gerontology Research Center colony were decapitated, the hearts quickly removed and retrogradely perfused with 50 ml of a nominally Ca2`-free bicarbonate buffer at 36 +1 C, and continuously gassed with 95 % oxygen and 5 % CO2 to keep the pH at 7-40 + 0-05. The perfusate was then switched to a similar solution to which collagenase and CaCl2 had been added to achieve a final concentration of 160 units ml-' and 50 /LM, respectively. After approximately 20 min of perfusion with this medium the ventricles were cut off, and single cardiac myocytes were mechanically disaggregated and resuspended in a bicarbonate buffer with 10 mM-bathing [Ca2+]. Some cells were batch loaded with the free acid (FA) form of Indo- 1 during mechanical disaggregation (after collagenase-protease digestion) in a small volume

86

H. A. SPURGEON AND OTHERS

of HEPES-buffered solution containing 1 mM-Indo-I FA (pentapotassium salt) and 250 ,uM-Ca(CI2 and resuspended as above.

kSzimultaneous measurement of length and Indo-1 fluorescence Cell length and Indo- 1 fluorescence were measured simultaneously as recently described (Spurgeon et al. 1990). Cell length was simultaneously monitored using red light (650-750 nm) to form a bright-field image of the cell which was projected onto a photodiode array with a 3 ms scan time. Single myocytes bathed in HEPES-buffered medium were loaded with the ester derivative (AMI form) of the fluorescent Ca2" probe Indo- 1. Fluorescence was excited by epi-illumination with 10us flashes of 350+5 nm light. Paired photomultipliers collected Indo-1 emission by simultaneously measuring spectral windows of 391-434 and 477-507 nm (410 and 490 nm channels respectively) selected by bandpass interference filters. The ratio of Indo-1 emission at the two wavelengths was calculated using a pair of fast integrator sample-and-hold circuits under the control of a VAX 11/730 computer and was taken as a measure of [Ca2J]i. None of the drugs used in the present study, i.e. caffeine, butanedione monoxime (BDM) or EMD 53998, affected the 410:490 ratio of Indo-1 fluorescence. Both the loading of the Ca2" probe into the cells and the experiments were done at 23 'C. Another subset of myocytes was loaded directly with Indo-1 FA through low-resistance patch-type electrodes. The microelectrode-filling solution consisted of (in mM): KCl, 120; NaCl, 10; HEPES, 20; MgCl2, 5 with 100 1aM-Indo-I FA (pentapotassium salt), pH adjusted to 7 2 with KOH. Microelectrodes (1-3 MQ) were pulled from glass capillary tubes containing an internal filament (no. 30-32-0, FHC, Inc., Brunswick, ME, USA) on a BB-CH MIechanex two-stage microelectrode puller. Following the establishment of a gigaohm seal between the microelectrode and the cell membrane, recordings of Indo-1 fluorescence intensity at 410 and 490 nm were taken every 2 min until the background fluorescence had decreased to a steady level. The seal was then broken and the Indo-1 allowed to diffuse into the cytosol. Cytosolic calcium transients could be recorded within 5 min of breaking the seal. The cells were stimulated with 100 ms voltage clamp pulses from a holding potential of -40 mV to a step potential of 0 mV. Corrected fluorescence transients were generated by subtracting the background fluorescence at each wavelength, after correction by an algorithm that takes into account the intensity fluctuations of the xenon strobe lamp used for fluorescence excitation. In the case of batch-loaded Indo-1 FA cells, background substraction values were computed from the mean fluorescence at each wavelength of a large number of experimentally matched, non-loaded cells prepared from the same hearts. The cytosolic calcium concentration was calculated from an 'in vivo' calibration curve (Spurgeon et al. 1990). RESUILTS

The first goal of the present studies was to examine the relationship between the [Ca2+]i transients and contractions that vary in amplitude. In rat cardiac cells this situation occurs during the contraction staircase following stimulation from rest (Spurgeon et al. 1990). Figure IA illustrates the [Ca2+]i and contraction in a single rat ventricular myocyte field stimulated regularly from rest. The figure shows that the negative staircase in contraction is paralleled by a negative staircase in [Ca2+]i. In order to examine the time course of the [Ca2+]i transient relative to that of the contraction, the twitch and [Ca2+]i transient from the initial beat are displayed at higher sensitivity in Fig. 1B. The cytosolic calcium concentration increases prior to contraction and peaks about the time of the maximum rate of cell shortening, then decays rapidly while shortening continues to its peak value. Cell relengthening (relaxation) is accompanied by a further, slow decline in [Ca2+]i to the diastolic level. Figure 1 C shows a plot of the phase-plane diagram of cell length and [Ca2+]i (,UM) during the twitch contractions shown in Fig. 1A. Arrows on each trajectory represent 50 ms isochrones. Within each contraction the phase-plane diagram of cell length and [Ca2+]i forms a counter-clockwise loop (shortening proceeds upwards). Thus, during cell relengthening the cell length at a given [Ca2+]i is less than that

CARDIAC MYOFILAMEANT-Ca2+ RESPONxSE AND RELAXATIO.N

87

during the shortening phase of contraction. This suggests that an equilibrium between the myofilaments and [Ca2+]i is not achieved during the shortening phase of contraction. Note, however, that during the relaxation phase, the cell length and [Ca21]i transients (which differ in their individual peak amplitudes) converge to a

A

B

088

102

~~~~~~~~~[0-98]

i

E

E

CCD

cD 100

102

E~~~~~~~ V)

0-11

[Ca

2](P)0-88

15

158 6.(,M

6

pCa Fig. 1. A, simultaneously measured [Ca'+]i and cell length following stimulation from rest of a representative single rat myocyte that had been loaded with the free acid form of Indo-1 via the batch method. B, the initial transients in A are depicted with greater resolution. C, phase-plane trajectories of cell length-[Ca 2+]i for the transients in A (loops proceed in a counter-clockwise direction). Although the individual contractions and [Ca'+]i transients vary in magnitude a common trajectory occurs during relaxation. Arrows on each trajectory denote 50 ms isochrones. D, the dashed line depicts the common trajectory in C. The relation between peak contraction amplitude, measured as the end-systolic length (ESL) and pCa is denoted by *. The relation between ESL and fluorescence ratio measured at the time of ESL is denoted by *. Note that the latter relation, which defines the common trajectory in C, lies to the left of the former.

H. A. SPURGEON AND OTHERS

88 A

C

85

1.45

0)1 0

t99

-

E 0) C

0)

85 1I1-45 1

B

Fluorescence (410/490 nm)

1.4 a) ^ c

E

C C

1 1

99 -L s

08 0

84.7

500

ms

Fig. 2. A, simultaneously measured Indo-1 fluorescence ratio and twitch contractions in a rat myocyte stimulated from rest and loaded with Indo-1 AM. B, two events are depicted: (1) the initial twitch in A and its corresponding fluorescence transient following stimulation from rest, and (2) the fluorescence transient and contraction (bold traces) elicited by a rapid, brief (200 ms) pulse of caffeine (10 mm in pipette) applied directly onto the surface of the cell in A via the micropipette. C, phase-plane diagrams of length vs. fluorescence for the (1) initial electrically stimulated event in A, (2) the 14th event, (3) the steady-state (SS) electrically stimulated transients following rest, and (4) caffeine-induced transients (Caf), which is depicted with a bold line. Note that the phase-plane diagram of the slowly developing caffeine-induced transients does not exhibit the hysteresis observed in the phase-plane diagram of electrically stimulated transients and superimposes the late 'common trajectory' of the electrically stimulated transients.

CARDIAC MYOFILAMENT-Ca2+ RESPONSE AND RELAXATION 89 common trajectory (arrow). This common trajectory occurs during the [Ca2]i decline, at a time shortly after cell relengthening begins. This unique trajectory among contractions and [Ca2+]i transients that vary in peak amplitude appears to represent a quasi-equilibrium state of [Ca2+]i and myofilament Ca2+ binding. A common trajectory of [Ca2+]i and contraction was observed in all cells studied. Figure 1 D shows that the common trajectory, represented by the fluorescence at the end-systolic length, is shifted to the left of the relationship between the peak fluorescence achieved and the end-systolic length. Incubation of cells with the acetoxymethylester (AM) form of Indo- 1 is a convenient method to load the Indo-1 probe and permits experiments of longer duration than does loading with the free acid form of Indo-1. While we have shown that the AM method of loading precludes a strict calibration of [Ca2+]i from the Indo1 fluorescence (Spurgeon et al. 1990) it does facilitate the study of the relationship of fluorescent transients and twitch contractions in a large number of cells. Figure 2A shows the fluorescence transients and contractions during stimulation from rest in a rat cell loaded with Indo- 1 AM. As is the case for cells loaded with Indo- 1 FA, the phase-plane diagrams of the twitches and fluorescence transients of Indo-1 AMloaded cells conform to a common trajectory during the relaxation phase of the contraction (Fig. 2 C). To further test the notion that the common trajectory of [Ca2+]i and contraction during the relaxation phase of contraction does indeed indicate that a dynamic equilibrium is achieved during the twitch, we compared the trajectories of electrically stimulated twitches with those formed by slower increases and declines in [Ca2+]i and contraction, due to rapid addition and wash-out of caffeine (Stern, Silverman, Houser, Josephson, Capogrossi, Nichols, Lederer & Lakatta, 1988) or those elicited by 'tetanization' during rapid stimulation in the presence of ryanodine (Yue et al. 1986; Yue, 1987). In Figure 2B, a rapid, brief (200 ms) pulse of caffeine was applied via a micropipette (10 mM-caffeine in pipette) onto the surface of the same cell in Fig. 2A to induce sarcoplasmic reticulum (SR) Ca2+ release. Also depicted in Fig. 2B is the initial beat of the staircase in A. As compared to the electrically stimulated fluorescence and length transients, the caffeine-induced Ca2+ release and contraction rise and decay substantially more slowly. Time-to-peak Indo-1 fluorescence and contraction in the caffeine-induced transients was 401 and 410 ms respectively, vs. 47 and 185 ms respectively in the electrically stimulated transients. The phase-plane diagrams of both electrically stimulated beats and the caffeine-induced transients are superimposed in Fig. 2 C. Note that the phase-plane diagram of the caffeine-induced transients does not exhibit the marked hysteresis of the electrically stimulated transients. This suggests that the [Ca2+]i and myofilament Ca2+ binding are closer to equilibrium throughout both cell shortening and relengthening as compared to the electrically stimulated transient. Still, in the phase-plane diagram the trajectory of the caffeine-induced transients superimposes on that of the common late trajectory of the electrically stimulated transients. It is noteworthy that the caffeine-induced trajectory superimposes on that due to electrically stimulated transients all the way up to the onset of relaxation of the largest contraction. The overlap of the caffeineinduced trajectory and electrically induced late trajectory strongly supports the notion that during relaxation [Ca2+]i and myofilament Ca2+ binding reach a quasiequilibrium state.

H. A. SPURGEON AND OTHERS

90

Ryanodine depletes the SR Ca2" store (Hansford & Lakatta, 1987) and abolishes electrically stimulated twitches in resting cells. Rapid stimulation can partially and transiently reload the SR store, as the SR Ca2" pump is not directly affected by ryanodine (duBell, Spurgeon, Lakatta, & Lewartowski, 1989). In Fig. 3A rapid B

A

81 2

C:

Caffeine after

E

ryanodine Ryanodine

.2 -C ~

10 s _____________

10 Hz

104. 1

1.45 Fluorescence (410/490 nm)

Fig. 3. A, simultaneously measured Indo-t fluorescence and cell length in the same rat myocyte as in Fig. 5, but stimulated at 10 Hz in the presence of ryanodine (10 1aM). Caffeine (10 mm in pipette) is pulsed onto cell at arrow. B, the phase-plane diagram of the change in fluorescence and length during stimulation 10 Hz in panel A (ryanodine) is superimposed on the phase-plane of diagrams for the events in Fig. 5C. The trajectory during 'tetanization' during stimulation at 10 Hz in the presence of ryanodine superimposes that of the common trajectory in Fig. 5C.

electrical stimulation of the same cell used in Fig. 2 in the presence of ryanodine leads to a slowly developing, steady increase in Indo- 1 fluorescence and decrease in cell length. At the termination of electrical stimulation caffeine is pulsed onto the cell. This elicits a very slow increase in Indo- 1 fluorescence and a decrease in cell length which very slowly return to the baseline (pre-stimulation) levels following cessation of stimulation. Figure 3B shows the phase-plane trajectory of the ryanodine-caffeine application transient in A and those due to electrical stimulation and caffeine prior to ryanodine exposure. Note that the common trajectory during ryanodine exposure lies on the same trajectory as that during the relaxation of the electrically stimulated twitches prior to ryanodine. The data presented thus far suggest that during relaxation (cell relengthening) the myofilaments achieve a quasi-equilibrium with [Ca21]i. Thus, under a given condition, the length-fluorescence trajectory during relaxation ought to reflect the myofilament Ca21 sensitivity. If this were indeed the case, the trajectory should shift appropriately following perturbations that alter the steady myofilament response to

CARDIAC MYOFILAMENT-Ca2+ RESPONSE AND RELAXATION 91

Ca2+. This was examined by exposing cells to perturbations that are known to shift the steady-state myofilament Ca2+ response in skinned cardiac fibres. One of the actions of butanedione monoxime (BDM) is to reduce the steady-state myofilament response to Ca2+ (Li, Sperelakis, Ten Eick & Solaro, 1985). Figure 4A A

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Fig. 4. A, simultaneously measured Indo-1 fluorescence transients and cell length during Hz in control (Con) and following the addition of steady-state stimulation at 0o5 butanedione monoxime (BDM) to a representative rat myocyte. B, cell lengthfluorescence phase-plane trajectories of transients in A. C, autoscaled fluorescence

transients and twitches in control and after 6 mm-BDM in A.

shows Indo- 1 fluorescence transients and contractions in a cell prior to and during BDM exposure. In the presence of 6 mm-BDM the contraction is diminished in amplitude and reduced in duration while the fluorescence transient is not diminished but rather augmented in amplitude and duration, as might be predicted for myofilament Ca2+ desensitization (Endoh & Blinks, 1988). Resting cell length is also increased. Increasing the concentration of BDM to 10 mm nearly abolishes the twitch contraction. The fluorescence transient becomes reduced in magnitude compared to the lower BDM concentrations, suggesting that the higher concentration of BDM has additional effects on [Ca2+]i as recently shown using aequorin in ferret muscle (Blanchard, Smith, Allen & Alpert, 1990). Still, neither the magnitude nor duration of fluorescence transient in 10 mM-BDM is less than that prior to the drug addition. Figure 4B shows the phase-plane trajectories for the transients depicted in A. As predicted for a decrease in the myofilament Ca2+ response, the relaxation trajectories for BDM are shifted to the right. It might be expected that with the presence of a decrease in the myofilament Ca2' response relaxation would begin at a higher [Ca2+]i or sooner in time at a given [Ca2+]i. Figure 4B shows that the reduction of the myofilament Ca2+ response does permit cell relengthening at a higher Indo- 1

H. A. SPURGEON AND OTHERS

92

fluorescence ratio. In the time domain, this is manifest as an earlier onset of cell relengthening, as illustrated by the normalized transients prior to and during drug application (Fig. 4C). Changes in pH are known to alter the myofilament Ca2+ sensitivity of skinned muscle fibres (Fabiato & Fabiato, 1978). In the present study cells were exposed to A

C

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NH4Ca alkalosis

N

R

NH4CI alkalosis

90.7 0.87 1.35 Fluorescence (410/490 nm)

250 ms

Fig. 5. A, simultaneous recordings of Indo-1 fluorescence transients and contractions during steady-state stimulation of O-5 Hz in control (Con) during the alkalotic phase of NH4Cl addition and during the acidification phase of NH4C1 removal. B, cell length-fluorescence phase-plane trajectories of the transients in A. C, autoscaled fluorescence transients and twitches for events in A: R, removal; C, control.

NH4Cl to alter cytosolic pH. This manoeuvre, which has been well characterized previously in cardiac muscle (Bountra & Vaughan-Jones, 1989), initially causes an intracellular alkalosis which is gradually and partially compensated during continued NH4Cl exposure. Upon NH4Cl wash-out, a transient acidosis occurs prior to return to a control pH level. In preliminary experiments using the pH-sensitive fluorescent indicator Snarf- 1 in single rat myocytes (Blank, Silverman, Chung, Stern, Hansford, Lakatta & Capogrossi, 1990) it was shown that a 3 min exposure of rat myocytes to NH4Cl causes an initial alkalotic shift of 0-21 pH units and an acid overshoot of 0-07 pH units upon NH4Cl wash-out (H. S. Silverman, personal communication). Figure 5A shows the Indo-1 transient and twitch contractions in a representative rat myocyte prior to, during and following a 3 min exposure to NH4Cl. During the alkalotic phase following acute NH4Cl exposure the contraction amplitude increases in the absence of a change in the fluorescence transient. During the acidosis upon NH4Cl wash-out the contraction amplitude is depressed with a slight upward shift in the fluorescence transient. The length-fluorescence phase-plane diagrams for control, acute NH4Cl addition and NH4Cl removal are depicted in Fig. 5B. Note that the

CARDIAC MYOFILAMENT-Ca2+ RESPONSE AND RELAXATION 93 trajectory during relaxation is shifted leftward and rightward of control for the NH4Cl alkalotic and acidotic phases respectively. Note also in B that the shift in the myofilament Ca2' response during alkalotic and acidotic phases causes cell relengthening to begin at lower and higher Indo- 1 fluorescence ratios respectively. In A

B 119.

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~~~~EMD

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Fluorescence (410/490 nm) Fig. 6. A, simultaneously measured Indo-1 fluorescence transients and contractions in a representative rat myocyte stimulated at 0 5 Hz prior to and following addition of the calcium sensitizing drug, EMD 53998, to the bath. B, cell length-fluorescence phase-plane trajectories for transients in A. C, autoscaled fluorescence transients and twitches for events in A.

the time domain (Fig. 5C) this corresponds to a later onset of relaxation during the alkalosis and an earlier onset of relaxation during acidosis compared to control. Myofilament Ca2+ sensitivity is enhanced by thiadiazinone derivatives (Solaro & Riiegg, 1982; Ferroni, Hano, Ventura, Lakatta, Klockow, Spurgeon & Capogrossi, 1991). Figure 6A shows that EMD 53998, a novel thiadiazinone derivative (kindly supplied by E. Merck, Darmstadt, Germany) increases the twitch amplitude without increasing the amplitude of the Indo-1 fluorescence transient. This substance also reduces the resting cell length. Figure 6B shows that the phase-plane diagram of length-Indo- 1 fluorescence is shifted upward and to the left by EMD 53998, consistent with its effect to markedly shift the force-pCa relation leftward in skinned cardiac fibres (I. Lues, Merck Pharmaceuticals, Darmstadt, Germany, personal communication). The figure also shows that cell relengthening begins at a lower fluorescence level in the presence of the drug than in control. This is manifest in the time domain (Fig. 6C) as a retardation of the onset of relaxation in the presence of the drug. The results presented thus far are interpreted to indicate that during the twitch contraction in single rat ventricular myocytes in the absence of external mechanical

H. A. SPURGEON AND OTHERS

94

loading, [Ca21]i, as indexed by Indo-1 fluorescence, and the myofilaments achieve a quasi-equilibrium state during relaxation. However, the fluorescent probe Indo-1 is a Ca2+ buffer and it might be argued that the extent to which cells are loaded with the probe may artifactually affect the [Ca21]i transient and contraction. In order to achieve a signal-to-noise ratio sufficient to study transients of individual twitches TABLE 1. Effect of Indo- 1 AM loading (20-30 /M) on action potential and contraction (means+ S.E.M.) in rested twitch in single rat ventricular myocytes Indo-1 AM-loaded Non-loaded cells cells (n= 9) (n= 8) P Twitch amplitude n.s. 14-7+2-3 16-2+2-0 (percentage resting cell length) n.s. 193-9+32-1 214-5+35-5 Velocity of shortening

(,um s-')

Time-to-peak shortening (ms) Half-relaxation time (ms) Resting membrane potential (mV) 90% action potential repolarization (ms) 95% action potential repolarization (ms)

276+9-8

318+17-0

004

436+ 19-3 -67-1 + 1-5

514+34-3 -72-1 +2-5

n.s. n.s.

177 +32-3

164+47-2

n.s.

247 +35-3

246+47-6

n.s.

without averaging, cells in the present study have been loaded with an approximate concentration of 30-50 /tM-Indo- 1 (Spurgeon et al. 1990). The effect of this loading on the twitch contraction under conditions used in the present study was determined by comparing twitch amplitude and duration in cells with Indo-1 loading to those not loaded with the probe. The transmembrane action potential (TAP) characteristics were also measured in Indo-1-loaded and non-loaded cells, as the slow repolarization of the action potential in rat cells is modulated by the decay in [Ca21]i which produces a decaying inward current via Na+{-Ca2+ exchange and/or a non-specific cation channel flux (Mitchell, Powell, Terrar & Twist, 1984; duBell, Boyett, Spurgeon, Talo, Stern & Lakatta, 1991). Table 1 shows the data for twitch and TAP characteristics of the initial excitation following rest, i.e. the initial beat in the staircase protocols depicted in Figs 1-6. Except for time-to-peak shortening, which shows a modest reduction in Indo-1 AM-loaded cells, neither twitch amplitude nor duration differs significantly from that of non-Indo-1-loaded cells. Additionally, neither resting membrane potential nor the action potential duration are altered. Table 2 compares the TAP and twitch characteristics of the last beat of the train during 0 5 Hz stimulation, which approximates the steady-state value during stimulations at this rate, to those first beats following rest for the same cells in Table 1. There are no major significant differences between Indo-1-loaded and non-loaded cells, indicating that the staircase kinetics are not markedly altered by the degree of Indo- 1 loading achieved under the conditions of the present study. As the twitch and action potential characteristics under the conditions employed in the present study are essentially unaltered by Indo- 1 loading it may be concluded

CARDIAC I _OFILAMENT-a2+ RESPONVSE AN,D RELAXATION 95 that the quasi-equilibrium achieved between [Ca2+]i and myofilaments during twitch relaxation is not likely to be an artifact of the presence of Indo-1 within the cell. However, the interpretation of the common trajectory requires the assumption that the Indo-1 fluorescence ratio reflects the instantaneous behaviour of [Ca2+]i during the declining ('relaxation') phase of the transient. The extent to which this is valid TABLE 2. Effect of Indo- 1 AM loading on action potential and contraction staircase in single rat ventricular myocytes upon stimulation from rest Percentage decrease Indo- 1 AM-loaded Non-loaded cells P cells (n = 6) (n = 5) (beat 9 vs. beat I) Twitch amplitude 72-5 + 5-1 56-2 + 8-1 n.s. 71 3 +4-5 Velocity of shortening 42-6 + 14 1 n.s. Time-to-peak shortening 301 +44 21-3+8-6 n.s. Half-relaxation time 125+92 26-2+52 n.s. Resting membrane 0-50+0-17 0-36+0-6 n.s. potential Time to 90% 320+11-1 30-5+10-7 n.s. repolarization Time to 95% 493+4-9 42-3+10-6 n.s. repolarization

depends on the rate at which calcium concentration changes with time in relation to the kinetic rate constants of calcium binding and release by Indo-1. The measured dissociation rate of calcium-Indo- 1 in free aqueous solution is 130 s-1 (Jackson, Timmerman, Bagshaw & Ashley, 1987) which is quite rapid relative to the time scale of the twitch. However, it has been observed that the effective dissociation rate for the analogous calcium indicator, Fura-2, is reduced 5- to 8-fold in the intracellular environment in skeletal muscle fibres (Baylor & Hollingworth, 1988; Klein, Simon, Sziics & Schneider, 1988). This leads to serious errors in estimating the time course of the calcium transient in skeletal muscle using Fura-2, and similar effects might occur with Indo-1 in cardiac muscles. However, the cardiac muscle twitch, especially at room temperature, is very much slower than that in skeletal muscle, and the duration of the calcium 'relaxation' phase is considerably longer than the release phase. Therefore, it is unlikely that significant error is made by assuming that Indo1 is in equilibrium with calcium during the relaxation phase. In order to make certain that this assumption is valid, we estimated the magnitude of the possible 'kinetic' correction to the Indo-1 ratio, in the following manner. We assume that Indo-1 may exist either free (f) or bound (b) to calcium, according to the following reaction scheme:

Kf[Ca2+][Indo- 1] [Indo-I :Ca]

Indo-1 +Ca2+

Kblndo- 1: Ca This reaction scheme gives a differential equation for the fraction x of Indo- 1 bound to calcium:

dxldt Kf [Cal+] (I x) KbX, =

(1)

H. A. SPURGEON AND OTHERS

96

which may be solved for [Ca2+]: [Ca2+] = (dx/dt +Kbx)/(Kf(1-x)). If we assume dx/dt = 0 (i.e. equilibrium), then this reduces to

[Ca2+] =Kdxl (I -x), A

(2)

(3)

B 2.4

1.7

E a)

0)

C~~~~~~~~~~~~~~~~C+ 0) (0 "a 0

1.2

0.2 /

.

250 ms Fig. 7. Measured Indo-1 ratio transient (bold trace) following a single stimulation of a

rested rat cell injected with the free acid form of Indo- 1 via a patch pipette and a 'kinetically corrected ratio' obtained by assuming that the intracellular Indo-1-[Ca2+] dissociation rate is only 10 s-' (see text for details). As seen in the figure, the kinetic correction is negligible during the relaxation phase.

where Kd = Kb/Kr is the equilibrium dissociation constant. The bound fraction x can be expressed in terms of the fluorescence ratio R = F410/F490 by using the specific fluorescence (F) intensities of the bound and free forms of Indo- 1 in the two wavelength bands, and if this bound fraction x(R) is used in eqn (3), we obtain a calibration equation valid at equilibrium, expressing [Ca2+] in terms ofR (the final form of this relationship has been given by Grynkiewicz et al., 1985). In order to obtain a calibration including kinetic effects, we should instead insert x(R) into eqn (2). Alternatively, we can obtain a kinetically corrected ratio Rc, which is defined as that ratio which, if used with the equilibrium calibration, would give the true calcium concentration obtained using the true ratio with the kinetic calibration. An expression for the kinetic correction can be obtained by (a) inserting x(R) into eqn (2), remembering that R is a function of time, (b) putting the resulting true calcium concentration on the left of eqn (3) and solving for the apparent bound fraction x which would exist if the true calcium concentration were in equilibrium with Indo1, and then (c) calculating the fluorescence ratio which would be associated with the apparent bound fraction. After considerable algebra, this results in Rc = R - (/.(Rmax -R)dR/dt)/(Kb(Rmin -fR max) - fdR/dt + (,-1 )Kb R), (4) where Rmin and Rmax are the fluorescence ratios at zero and saturating [Ca2+]

CARDIAC MYOFILAMENT Ca2+ RESPONSE AND RELAXATION 97 respectively and , is the ratio of 490 nm fluorescence at saturating and zero [Ca2+]. The corrected ratio can then be used with the usual equilibrium calibration formula to give the true calcium concentration. In order to determine the possible magnitude of the kinetic correction, we have used eqn (4) to correct ratio data obtained from cells loaded with the free acid form of Indo- 1 via a patch pipette. Figure 7 shows a typical Indo- 1 ratio tracing from such a cell, superimposed on the corrected ratio obtained from eqn (4) assuming that the intracellular Indo-1-Ca2 dissociation rate Kb is only 10 s-5, which would be more than a twelvefold reduction from the rate found in free solution. As seen in the figure, the kinetic correction is negligible during the relaxation phase. DISCUSSION

The present study used single cardiac myocytes contracting from slack length which avoids the complications of heterogeneity and external loading present in intact cardiac muscle. The absence of external restoring forces and the lower temperature (23 °C) result in a relatively slow relaxation of electrically stimulated twitches in these cells even in the absence of Indo-1 loading (Krueger, Forletti & Wittenberg, 1980; Spurgeon et al. 1990). It has been shown previously by measurements of contraction prior to and after Indo-1 AM loading of the same cell that the decay of the fluorescence transient parallels the cell relengthening (Spurgeon et al. 1990). The extent to which Indo-1 fluorescence decay accurately reports the decay in the [Ca2+] transient remains an issue. This relates to whether the rate of Ca2+ dissociation from Indo-1 is sufficiently rapid to report relative to the true decay of [Ca2+]1. While direct measurements in solution report the rate of Ca2+ dissociation from fluorescent probes to be rapid (Jackson et al. 1987), estimates in intact skeletal muscle have been interpreted to indicate that it is slow (Baylor & Hollingworth, 1988; Klein et al. 1988). The present results show that in intact cardiac cells, under the conditions of the present study, that even when the slowest (an order of magnitude less) measured dissociation rate of Ca2+-Fura 2 measured in skeletal muscle (Klein et al. 1988) is assumed for Indo-1, the measured Indo-1 fluorescence transients require essentially no correction in the decay phase. Thus, the quasiequilibrium observed between length and Indo-1 fluorescence during the relaxation phase of the twitch cannot be due to an artifactually slow decay of the Indo- 1 transient due to a slow Ca2+-Indo-1 dissociation rate. These considerations lead us to believe that the off-rate of Ca2+ from Indo- 1 is sufficiently rapid for the fluorescence emission ratio to accurately track [Ca2+]i during relaxation. In the present study the extent of Indo-1 AM loading, estimated to be 30-50 fM (Spurgeon et al. 1990), provided a sufficient signal-to-noise ratio to measure individual transients without averaging but did not substantially alter the twitch amplitude, relaxation time, staircase kinetics or the Ca2+-sensitive, slow repolarization phase of the action potential. It has previously been shown that concentrations of Fura-2 of about this magnitude do not alter contraction in single barnacle cells (Jackson et al. 1987). These results suggest that in cells relatively lightly loaded with fluorescent probes, the [Ca2+]i transient following excitation under the conditions of the present experiments is not markedly buffered. 4

PHY 447

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H. A. SPURGEON AND OTHERS

While the chemiluminescent protein aequorin has been employed in a single study to measure the [Ca2"]i transient in single cardiac cells (Cobbold & Bourne, 1984), no direct comparison can be made with the present study because the time course of the change in [Ca2+]i was not presented nor was the contraction measured. However, in single barnacle cells loaded with aequorin, it has been shown that an elevation of aequorin light, and thus of free Ca2", persists during the entire phase of relaxation (Ashley, Potter, Strang, Godber, Walton & Griffiths, 1985). Additionally, the estimated [Ca2+]i during twitch relaxation, measured by refined techniques for aequorin light collection and deconvolution of the light signal in single frog skeletal muscle fibres, does not return to baseline prior to the completion of force relaxation (Cannell, 1986). However, a direct comparison of the [Ca2+]i-relaxation relation in these single muscle fibres with the cardiac cells in the present study is precluded by the presence of parvalbumin in the former cell type. In intact cardiac muscle the aequorin signal peaks early during the twitch, i.e. at about the time of peak dT/dt (the derivative of tension with respect to time) (Allen & Kurihara, 1980; Yue, 1987), just as it does in single barnacle cells (Ashley et al. 1985). In the present study in rat cells, peak Indo- 1 fluorescence also occurs early during the twitch contraction, at about the time at which the peak shortening velocity is achieved. In intact cardiac muscle the timing of the aequorin decay relative to twitch relaxation has been found to be variable among different studies (Allen & Kurihara, 1980; Allen & Kurihara, 1982; Hess & Wier, 1984). In some cases the light returns to baseline prior to the onset of muscle relaxation while in others it reaches its baseline during relaxation but well before its completion. This variation in the relationship between relaxation and the aequorin light decay among studies probably results in part from (1) differences in the extent to which preparations have been loaded with aqueorin, i.e. differences in the signal-to-noise ratio, (2) differences in external loading due to varying degrees of stretch applied to the muscle and (3) heterogeneities among muscle segments, particularly during relaxation (Huntsman et al. 1979), which precludes a strict comparison of the decay of any Ca2+ indicator to that of contraction in bulk muscle preparations. Additionally, as the relationship between aequorin light and [Ca2+]i is non-linear, small but important changes in free Ca2+ concentration occurring at values close to the resting free [Ca2+]i can be overlooked if time of the light transient is not deconvoluted at high sensitivity (Ashley et al. 1985). This manoeuvre has not routinely been applied to the tail of the aequorin transient in studies of intact cardiac muscle. However, in single barnacle cells, a slow tail of aequorin is observed and corresponds to the terminal relaxation when the aequorin light signal has been deconvoluted and expressed as [Ca2+], the aequorin transient shifts to the right, i.e. becomes prolonged (Ashley et al. 1985). Under certain conditions in intact cardiac muscle a 'slow tail' of the aequorin transient also parallels relaxation (Morgan & Morgan, 1984; Wier & Hess, 1984). In the single rat cardiac cells loaded with Indo-1 in the present study, as with single barnacle cells or intact cardiac muscle injected with aequorin (Ashley et al. 1985; Yue, 1987) it is apparent that [Ca2+]i does not equilibrate with myofilaments at the time at which peak [Ca21]i is achieved. This is evidenced in the present study by the hysteresis between the contraction and relaxation limbs of the phase-plane diagram for a given electrically stimulated twitch contraction. However, the

CARDIAC MY'OFILA1ENThCa2+ RESPONSE AND RELAXATION

99

common trajectory of cell length and [Ca2+]i during the slow portion of [Ca2+]i decay associated with large and small contractions is interpreted to indicate that at this time a quasi-equilibrium between [Ca2"] and myofilaments is achieved. This interpretation is strengthened by the observation that phase-plane diagrams of the slowly increasing [Ca2+]i and slowly developing contraction in response to caffeine pulses or tetanic stimulation in the presence of ryanodine superimpose on the common trajectory during relaxation of the electrically stimulated twitches. In these experiments the brief exposure (milliseconds) to caffeine probably precluded its effect to shift the myofilament Ca21 sensitivity, as this has been found to be time dependent (Konishi, Kurihara & Sakai, 1984) and concentration dependent (O'Neill, Donoso & Eisner, 1990). Finally, our results show that the common [Ca2+]i-length trajectory during relaxation shifts appropriately with perturbations known to alter the steady state [Ca2+]i-contraction relationship in skinned cardiac fibres (Fabiato & Fabiato, 1978; Solaro & Riiegg, 1982; Li et al. 1985). Thus it seems that during the relaxation phase of the [Ca 2+]i transient, myofilament Ca2+ binding achieves an equilibrium with [Ca2+]i, and the myofilament Ca21 sensitivity, myofilament force, and the [Ca2+]i determine cell relengthening in the non-externally loaded cardiac cell. The common [Ca2+]i-length trajectory suggests that intracellular restoring forces, which vary in magnitude among contractions of varying amplitudes, and play a role in determining the extent of myofilament shortening, are not exclusively rate limiting the relengthening of these cells. Rather, the establishment of an equilibrium between myofilament Ca2+ binding, force and [Ca2+]i probably governs relaxation in these cells. It might also be argued that mechanical restoring forces, though of different peak magnitude in contractions of different amplitude, achieve a unique value during the common trajectory of length and [Ca2+]i, and thus, the restoring force also retains a regulatory role in governing relaxation. A case for [Ca2+]i regulation of slow relaxation has also been made in chick embryonic cells (Clusin, 1981; Lee & Clusin, 1987) and detergent-treated rat and cat cells (Brutsaert, Claes & DeClerek, 1978). In the present study cells contracted and relaxed in the absence of the influence of external mechanical loading. We have observed that during auxotonic contractions in stretched myocytes, relaxation is accelerated in the absence of a change in the decay of the fluorescence transient (Sollott & Lakatta, 1989). Thus, external loading can modulate or override the Ca2' dependence of relaxation and may obscure the myofilament response to Ca2+ as well. Figure 1 C also shows that a common trajectory occurs early, i.e. during the initial 25 ms following excitation (arrows on each loop indicate 50 ms isochrones). It would be expected that at any given myofilament Ca2+ sensitivity this initial part of the trajectory would be common to all beats in a staircase, while this early trajectory would also shift as the myofilament Ca2+ sensitivity shifts; however, Ca2+ binding to troponin-C (TnC) has not reached equilibrium at this time and a delay occurs between TnC binding and contraction. Thus, the 'early' common trajectory is of no interest to the goals of the study. Note also in Fig. 2 that the 'early' trajectory does not occur when the rate of increase of [Ca2+]i and cell shortening are markedly reduced, i.e. in response to a rapid and brief application of caffeine directly onto the surface of the myocyte. The figure shows that while the [Ca2+]i transient and 4-2

too

H. A. SPURGEON AND OTHERS

contraction elicited by the caffeine application and those initiated by electrical stimulation have a common 'late trajectory', an early common trajectory between caffeine and electrically stimulated events does not occur. As the maximum contractile response to Ca2" was not defined in the present study, the shifts in the common trajectory are not interpreted as representing myofilament Ca2" sensitivity, but rather referred to as changes in the myofilament Ca 2+ response (Endoh & Blinks, 1988). It is not certain whether the maximum of the contraction-pCa relationship in single cells can be defined using fluorescent probes which saturate at a [Ca2+] of 10 /tM (Grynkiewicz et al. 1985; Spurgeon et al. 1990). However, given that the in situ force-pCa relationship derived by tetanization in the presence of ryanodine (Yue et al. 1986), in contrast to the case in skinned cardiac cell fragments (Fabiato, 1981), shows saturation at a [Ca2+]i between 1 and 2 /IM, it is possible that the maximum of the contraction-pCa relationship can be determined under the conditions of the present study. Additional studies in this regard are required. Also, it needs to be emphasized that force was not measured in the present studies and strict comparisons of shortening and force-Ca2+ relations are unwarranted at the present time. Still, the idea that the common [Ca2+]i-length trajector does reflect a [Ca2+]i-myofilament equilibrium will permit its usefulness to assess mechanisms of contraction amplitude modulation via modification of the myofilament Ca2+ response in intact isolated cells. REFERENCES

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