Simultaneous and potential

measurement of Ca2+, contraction, in cardiac myocytes

HAROLD A. SPURGEON, MICHAEL D. STERN, GUENTER BAARTZ, STEFANO RAFFAELI, RICHARD G. HANSFORD, ANTTI TALO, EDWARD G. LAKATTA, AND MAURIZIO C. CAPOGROSSI Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland 21224

SPURGEON, HAROLD A., MICHAEL D. STERN, GUENTER BAARTZ,~TEFANO RAFFAELI,RICHARD G. HANSFORD, ANTTI TALO,EDWARD G.LAKATTA,AND MAURIZIO C.CAPOGROSSI. Simultaneous measurement of Ca2’ contraction and potential in cardiac myocytes. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H574-H586, 1990.-A system is described that can simultane( [ Ca2+]i), cell length, ously record cytosolic Ca2+ concentration and either membrane potential or current in single cardiac myocytes loaded with the fluorescent Ca2+ indicator indo-I. Fluorescence is excited by epi-illumination with 3%ps flashes of 350 I~I 5 nm light from a xenon arc. Indo-l fluorescence is measured simultaneously in spectral windows of 391-434 nm and 457-507 nm, and the ratio of indo-l emission in the two bands is computed as a measure of [Ca2+]i for each flash. With cells loaded with the permeant acetoxymethyl ester of indo-1, quantitation of [Ca2+]; is not precise, owing to subcellular compartmentation of indo-I; however, the instrument would allow full quantitation if indo-l free acid was introduced by microinjection. Simultaneously, cell length is measured on-line from the bright-field image of the cell. Because fluorescence collection is time gated during the brief flash, and red light (650-750 nm) is used for the bright-field image, cell length and [Ca2+]i measurements are obtained simultaneously without cross talk. Membrane potential or current can be recorded simultaneously with indofluorescence and cell length via standard patch-clamping techniques. excitation-contraction coupling; cytosolic cent calcium ion indicators; indo-l

calcium ions; fluores-

CHANGES IN MEMBRANE POTENTIAL, cytosolic Ca2+concentration ( [Ca2+]i), and sarcomere length occur in myocardial tissue with each cardiac cycle. A fundamental understanding of how the heart functions would derive from the simultaneous measurement of these parameters. However, until recently this has not been readily possible either in multicellular cardiac preparations or in single cardiac cells. The recent development of the second generation of the fluorescent Ca2+ indicators, indo-l and fura- (15), and of preparations of dissociated myocardial cells that appear to be valid physiological models has represented a significant advance in measurements of [Ca”‘]; in single cardiac myocytes. Here we describe an apparatus that, without requiring signal averaging and with a 3-ms time resolution, allows simultaneous monitoring of [Ca2+];, cell length, and membrane potential or currents in a single cardiac myocyte with

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$1.50 Copyright

simultaneous visualization of the cell on a video monitor. When indo-l is loaded into myocytes by the pentaacetoxymethyl (indo-l/AM), as was done in the examples shown in this paper, there is substantial fluorescence because of indo-l, hydrolyzed and trapped in mitochondria (see below), which precludes absolute quantitation of the calcium transient. This problem would be circumvented if indo-l free acid were microinjected into the cell, as has been done for fura- (3, 5), in which case calcium measurement by the instrument would be fully quantitative. METHODS

AND RESULTS

Apparatus design.The optical system used for the simultaneous measurement of [Ca2+];, cell length, and membrane potential or currents in single cardiac myocytes is a modified Zeiss fluorescence microscope (model IM-35; Fig. 1). Measurement of indo-l fluorescence. The measurement of [Ca2+]i uses the fluorescent Ca2+probe indo-1. A xenon arc (Point Source Lamp, model 35S, Chadwick-Helmuth Electronics, El Monte, CA) powered by a modified stroboscope (model 236, Chadwick-Helmuth Electronics) is used as the source of illumination for epifluorescence, producing a 3.8~ps light pulse with an energy of -0.36 J/ flash at a repetition rate of up to 333 Hz. Subsequent to the experiments outlined in this paper, we replaced the light source with a faster system (FX193 lamp, FY714 light pack, and PS450 supply, EG&G, Salem, MA) that is cheaper, more stable, more compact, and better isolated with respect to electrical noise generation.. In addition, the EG&G unit is capable of 1-ms repetition rates at an intensity equal to the Chadwick-Helmuth unit at 3ms rates. An interference filter (Oriel, Stratford, CT) selects a wavelength of 350 t 5 nm to excite indo-1, and a 395-nm long-pass dichroic mirror (Zeiss) directs the exciting light to a X100, 1.3 NA UV fluorglycerin-immersion objective (Nikon). After excitation at 350 nm, the Ca2+-bound and -free forms of this indicator emit fluorescence peaking at -410 and 485 nm, respectively (15). Fluorescent light is collected from the whole microscope field, which contains a single myocyte. The cell is simultaneously illuminated with red (650750 nm) light through the normal bright-field illumination optics of the microscope. The bright-field and fluo-

0 1990 the American

Physiological

Society

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FIG. 1. A: diagram of apparatus used for simultaneous determination of 410-to-490 nm ratio of indo-l fluorescence, cell length, and membrane potential or current in single cardiac myocytes (see text for details). B: dual integrator for processing of photomultiplier signals. Gated integration with high-frequency response is accomplished by using sample/hold amplifiers to track integral. Analog switches are used to shift integrator signal input between ground reference and light signals, integrating evoked fluorescent light for typically 25 ps after each excitation lamp flash.

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rescence images are separated by a 600-nm short-pass dichroic mirror (custom designed by Andover, Lawrence, MA to operate at a 22.5” angle of incidence) inserted in place of a mirror normally used to direct the microscope image to a camera port. The fluorescent light is subsequently split by a 465-nm dichroic mirror, and customdesigned rectangular band-pass interference filters (Andover) select wavelength bands of 391-434 nm (“410-nm channel”) and 457-507 nm (“490-nm channel”). The ratio of these provides an estimate of the bound-to-free ratio of indo-1, which minimizes quantum (shot) noise error by optimizing the trade off between increased light collection (with broader filters) and avoidance of the overlap between the bound and free spectra (with narrower filters). Plane convex lenses (Rolyn Optical, Covina, CA) of 50-mm focal length converge the beam toward the sensitive portion of two photomultipliers (EM1 9893B/350, Thorn EM1 Gencom, Fairfield, NJ). The photocurrents from the two fluorescence channels are integrated during a time-gated window of 25 pus after the flash by an integrator-sample and hold circuit (Fig. 1B) in which great care was taken to assure thermal and differential gain tracking between both processing channels, which is essential because of the high gains employed. Overall errors between channels were t1.5 mV between channels across the entire range of *lo V (3 parts in 20,000 tracking error) with long-term DC drift of 3 mV in 6 mo. The signals evoked by each flash were individually digitized and stored after each flash. The entire acquisition process is under control of a VAX ll/ 730 computer with LPA-11 Lab interface. The ratio of the fluorescence intensities in the two channels is computed off-line, providing a calcium estimate for each flash. The time resolution of individual calcium measurements is limited only by the calcium-binding kinetics of the dye (18). Because the fluorescence signal is acquired only during the brief period when it is very intense, cross talk from the bright-field image, the average intensity of which is very much larger, is entirely eliminated. On the other hand, the brevity of the strobe flash reduces the average intensity of the ultraviolet exciting light to the minimum compatible with statistical significance of the individual calcium estimates, minimizing photochemical damage to the cell and the dye. Similar levels of average intensity could be utilized by an efficient continuous illumination system, but the signals would need to be filtered or averaged over a time equal to the interflash interval, reducing the time-precision of the calcium estimate. Calibration of apparatus response to changes in calcium ion concentration ([Ca2+/) as measured with indo-l (free acid). Indo-l (free acid) was added to a solution comprising (in mM): 140 NaCl, 5.4 KCl, 20 N-2-hydroxyethylpiperazine-N’ -2-ethanesulfonic acid (HEPES), 1 NaH2P02, and 1 MgSO* of pH 7.20 to give a concentration of 2.5 PM. Mixtures of CaC12and ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraaceticacid (EGTA), carefully adjusted to pH 7.2, were added to aliquots of the medium described above to give 2 mM chelating agent and known values of free-ionized [Ca”‘]. To stabilize the

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[Ca”‘] at more than 1 PM, dibromo-1,2-bis(2-aminophenoxyethane-N,N,N’,N’tetraacetic acid was substituted for EGTA, with a stability constant taken to be 1.23 X 10m6M. For EGTA, the stability constant was taken from Burgess et al. (4). Calculated values of free [Ca”‘] of these buffers were found to lie on a linear standard curve established using a Ca2+-selective electrode (World Precision Instruments, New Haven, CT) down to values of 10B7M Ca2+. A 200-~1 aliquot of each solution was placed in the microscope chamber, and the light emitted at each of the wavelengths (410 and 490 nm) was measured and corrected for the autofluorescence of the microscope optics. The measurements obtained on the microscope were found to be linear functions of the corresponding fluorescence intensities measured from the same solutions using a Perkin-Elmer LS-5 spectrofluorimeter. The values of the 410-to-490 nm fluorescence ratio obtained on the microscope, plotted as a function of free [Ca”‘] are shown as the asterisks in Fig. 2. The solid curve is the best least-squares fit to these points by the function (ax + b)/(x + c), which would be the expected shape of the calibration curve if the binding of Ca2’ to indo-l follows first-order kinetics. Figure 2 also presents calibration efforts in intact myocytes loaded with the membrane-permeant ester of indo-l, as discussed below. Myocyte isolation procedure. Single cardiac myocytes were isolated from the ventricle of rats according to a technique previously described (7). Briefly, the heart was retrogradely perfused with a low-Ca2+, collagenase-containing, bicarbonate buffer (36 t 1°C; pH 7.4 & 0.05), and the perfusion was terminated when the tissue became soft (25-30 min). The left ventricle was then mechanically dissociated, and myocytes were resuspended in a bicarbonate-buffered solution with 1.0 mM extracellular calcium concentration ([Ca”‘],) and stored at 37°C in a

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FIG. 2. Calibration of indo-l fluorescence ratio (410/490 nm) obtained on microscope system. * Obtained for indo-l free acid solution with calcium buffered by ethylene glycol-bis(P-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) or di-bromo 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). The solid line is best fit to the asterisks. Rectangles are in vivo calibrations in intact, deenergized indo-l/AM loaded cells with calcium, pH, and membrane potential equilibrated by ionophores (see text). Dotted line shows calculated values of fluorescence ratio as a function of cytosolic calcium ion concentration [Ca2+];, assuming that 51% of dye is sequestered in mitochondria at a fixed [Ca”‘] of 66 nM. Inset shows same curve on an expanded horizontal scale.

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CO2 incubator until used. Loading of myocytes with indo-I/AM. Indo-l/AM was loaded into cell suspensions in vials or into single cells on the microscope stage. In the latter method, an aliquot of 0.2 ml of cell suspension in a HEPES-buffered medium with 1 mM [Ca2+10is added to a similar medium in the experimental chamber, and 20 min is allowed for cell attachment to the cover slip in the absence of flow. Indol/AM is then loaded into cells by a recently described method (26): briefly, 35 ~1 of a solution containing 50 ~1 of 1 mM indo-l/AM in dimethylsulfoxide (Sigma Chemical, St. Louis, MO), 2.5 ~1 of 25% wt/wt Pluronic F-127 (BASF Wyandotte, Wyandotte, MI) in dimethylsulfoxide, and 75 ~1 of fetal bovine serum (Sigma) are added to the experimental chamber and mixed using a gentle air stream. After a period of exposure to the indicator, which varies between 30 s and 5 min depending on the degree of loading desired, the superfusion is restarted to wash the indo-l/AM from the chamber. In the bulk method of loading cells in vials, a proportionally higher volume of a similar solution of indo-l/AM is used to load 2 ml of a cell suspension, at room temperature, in a plastic scintillation vial (final concentration of indo-l/AM, 25 ,uM). After l-5 min, myocytes are gently centrifuged for 30 s, resuspended in a HEPES-buffered medium with 1 mM [Ca2+j0, and stored at room temperature until used. Regardless of the method used‘9loading of indo-l /AM into single cardiac cells is always done at 23°C and all experiments are also conducted at a similar temperature because a more rapid loss of the indicator from the myocytes occurs at higher temperatures. Decrease of indo-l fluorescence over 30 min was 2.4 t 1.1% (means t SE, n = 6) at 24°C and 21.3 & 3.2% (n = 10) at 37OC. The more rapid loss of dye at the higher temperature may represent extrusion of the free acid from the cell by an active anion transporter. Direct determination of Ca2+-sensitive fluorescence of lysates of loaded cell suspensions (Perkin-Elmer spectrofluorimeter, excitation 333 nm, emission 410 nm) by comparison with the free acid form of indo-l indicated intracellular concentrations in the range 30-42 PM, when cell volume was assumed to be 1.5 pl/mg protein. Determination of CaL+sensitivity of products of indo-ll AM hydrolysis within cell. It is conceivable that fluorescent products of partial hydrolysis of the indo-l/AM might be generated within the cell or that the dissociation constant (&) of the dye for Ca2+or that the fluorescence spectra of the intracellular probe might differ from the values determined in aqueous solution (18). To investigate, and if possible obviate, these problems, we sought to calibrate the probe in vivo by equilibrating Ca2+ concentrations across the cell membrane by a modification of a method previously described (21). This was done as follows. After fluorescence transients and contractile responses in normal physiological media were investigated, the cell was depolarized and energy depleted by superfusing a solution comprising 132 mM KCI, 10 mM K-HEPES, 1 mM MgSO*, 2 PM rotenone, 2 PM carbonyl cyanide-ptrifluoromethoxy-phenylhydrazone (FCCP), and 10 ng/ ml valinomycin (pH 7.2). Subsequently, the cell was

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equilibrated with 5.5 PM, 1 PM, 500 nM, 200 nM, and Cl0 nM free Ca2+ by successive exposure to portions of this medium, with the addition of 20 PM 4-Br-A23187 and appropriate Ca2+/EGTA buffers (2 mM EGTA). For 5.5 ,uM Ca2+, N-hydroxyethyl ethylenediaminetriacetic acid (HEDTA) was used instead of EGTA. To generate the lowest possible value of [Ca2+];, 2 mM EGTA was used, followed by superfusion with medium containing 0.1 mM BAPTA/AM. In each case, equilibration was judged to be complete when the 410-to-490 nm ratio became constant; this was complete within 5 min at the higher values of Ca2+,but was slower in response to Ca2+free EGTA. In the latter case, the addition of BAPTA/ AM generated a further decline in the 410-to-490 nm ratio. The results of the in vivo calibration are shown as the rectangles in Fig. 2. They fall tolerably well along the curve given by the in vitro calibration, considering the uncertainties in attempting to abolish Ca2+, pH, K, and electrical potential gradients across the membrane(s) of an intact cell. We interpret this as evidence that most of the indo-l in the cell is in the Ca2+-sensitive penta-ionic state and that contamination by fluorescent products of incomplete hydrolysis is not significant in rat cardiac myocytes under the conditions employed. Evidence for compartmentalization of indo-l within the cell. Application of the calibration curve in Fig. 2 to the fluorescence signals obtained during electrically stimulated twitches of intact cells yields values for peak cytosolic free Ca2+ considerably lower than the values generally accepted, even when the indo-l loading is kept to levels at which the contraction is not altered by the calcium-buffering effect of the dye (see below). Although we have shown above that this is not caused by the presence of calcium-insensitive metabolites of indo-l/ AM, it could be because of partial sequestration of the indo-l within intracellular compartments where the rapid variation of calcium during the twitch does not occur. Under the steady-state equilibration conditions employed for the in vivo calibration, such compartmentalized dye would be visible. The compartment under the greatest suspicion is the mitochondria, which occupies more than 40% of the cell by volume (17). To detect compartmentalization of dye, we measured the releasability of indo-l from suspensions of myocytes by 2 ,uM digitonin, which is expected to disrupt the cholesterol-rich sarcolemma, and Triton X-100, which disrupts all intracellular membranes. Cells were loaded with indo-l/AM for 5 or 10 min and washed and incubated at 23°C for 1 h. Fluorescence was monitored at 400 nm, with excitation at 350 nm. Digitonin was added, and the cells were rapidly centrifuged (Eppendorf Microfuge, 30 s) at 1, 2, 3, 5, or 10 min. Indo-l in the supernatant was measured as 400 nm fluorescence quenchable by 0.2 mM Mn. The pellet was dissolved in 200 ~1 of 0.5% Triton X-100, diluted to 2 ml in medium containing 1 mM Ca”+, and assayed for Mn2+-quenchable fluorescence. All fractions were assayed for the enzymes citrate synthase and lactic dehydrogenase as markers for the mitochondrial matrix and cytosolic compartments, respectively.

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The results of this procedure showed that indo-l release by digitonin was complete within 3 min. Digitonin released 82% of the cytosolic enzyme L-lactate dehydrogenase, whereas 98% of the mitochondrial citrate synthase remained with the pellet. Correcting for the 18% of the cytosolic compartment not released by the low concentration of digitonin employed, we found that 51 t 3% of the Mn2+ (and presumably Ca2+)-sensitive indo-l was in noncytosolic compartments. No significant amount of Mn2’-insensitive indo-l-related fluorescence was detected. These results are compatible with the hypothesis that the lipid-permeant indo- l/AM distributes uniformly within all cellular compartments and is subsequently completely hydrolyzed. The lion’s share of the noncytosolic dye would be expected to be in the mitochondria. Studies of isolated mitochondria directly loaded with indo-l show that, for cytosolic Ca2+ 300 pm/s is impossible because the video system saturates, whereas the CCD signal continues to show incremental increases. Thus compared with the video system the photodiode system has superior speed and sensitivity in following the rapid excursions in cell length that can occur in cardiac myocytes, whereas video edge tracking has the advantage that it can reanalyze archival data stored on videotape.

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FIG. 4. A: representative example of same twitch measured simultaneously with video analyzer and with photodiode without filtering or signal averaging in a rat cardiac myocyte not loaded with a Ca2+ indicator (T = 23C, [Ca”‘], = 2 mM). A 40-ms delay in video relative to real-time diode signal between beginning of cell shortening as measured by two systems is observed (see text). B: shifting tracings from A by 40 ms shows that velocity components of cell shortening are not recovered by simply compensating for video delay. C: velocities of cell shortening as measured simultaneously with video analyzer and photodiode. Same myocyte, not loaded with a Ca2+ indicator, was studied under different inotropic conditions obtained by varying [Ca”‘], from 0.25 to 5 mM. Each point represents velocity of shortening for same twitch simultaneously measured with dimension analyzer and diode array. As change in length over time (dl/dt) increases, difference between 2 systems becomes more pronounced, with video analyzer underestimating velocity of cell shortening.

Ratio method vs. single wavelength determination. One of the advantages of a probe such as indo-l is a shift in emission wavelength on binding Ca? Thus changes in [Ca2+]i can be expressed as changes in ratio of fluorescence at wavelengths that are optimal for bound and unbound forms rather than as a change in the fluorescence intensity at a single wavelength. Changes in the emitted light unrelated to changes in [Ca”‘], which can occur because of variation in the intensity of exciting light source, uneven illumination of the optical field, motion artifacts, or a decrease in the intracellular con-

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centration of the indicator secondary to loss through the sarcolemma of photobleaching, are effectively compensated. Figure 5A shows the unfiltered, unaveraged fluorescence transients and twitches in a rat myocyte stimulated from rest. Fluorescence intensity is depicted both at 410 nm, and as the 410-to-490 nm ratio. The ratio shows a better signal-to-noise ratio than either of the two individual wavelengths because of the cancellation of fluctuations in the flashlamp intensity. The indo-l fluorescence intensity is typically 3-10 times the background autofluorescence of cell and microscope optics. The software provides for subtraction of autofluorescence before computation of the ratio; however, optimum cancellation of lamp intensity noise is obtained if this is not done. Because the autofluorescence is small (Fig. 5C), and, fortuitously, has a wavelength ratio similar to that of indo-l at resting [Ca2+];, it introduces little error and is not important when absolute calibration of the indo-l signal is not attempted. Although individual (unaveraged) unfiltered transients can be easily resolved in this apparatus, Gaussian filtering before computation to reduce the random variations can improve the quality of light transients without blunting their characteristic features or shifting temporal relationships (Fig. 5B). Figure 5 also shows the cell contraction and illustrates that the negative staircase in twitch amplitude that occurs when rat cells are stimulated from rest (7, 12, 16) still occurs in the presence of indo-1. Clearly the negative contraction staircase is caused by a negative staircase in the [ Ca2+]; transients that elicit contractions. Whereas the [Ca2+]i transients derived by the ratio method are relatively free from distortion caused by motion artifact, fluorescence measured at each individual wavelength can be significantly altered. The effect of cell motion within an optical field unevenly illuminated by the exciting light source is shown in Fig. 6. After an electrically stimulated twitch the myocyte was purposely displaced within the optical field (arrows) leading to a decrease in the intensity of the emitted light both at 410 and 490 nm; however, the 410-to-490 nm ratio was not affected by this perturbation. Thus cell motion, especially if it occurs within an unevenly illuminated optical field, produces an artifact at the individual wavelengths that is well corrected by the ratio method. It is known that changes in the intracellular concentration of indo-l can occur either because of loss of the dye from the cell or photobleaching. However, these two factors should equally affect the two individual wavelengths of emitted fluorescent light and, if autofluorescence is subtracted, have no influence on their ratio. We assessed the effect of time alone on the intensity of the emitted light at the two individual wavelengths and their ratio by continuously superfusing myocytes for 1 h with sampling of indo-l fluorescence restricted to 20 s at the beginning and at the end of that period; in other experiments we followed a similar protocol but illuminated and sampled the cell every 10 min for 20 s. Under both conditions the loss of light at 410 and 490 nm was small (Table 1), and this decrease was slightly greater when the cells were illuminated during the course of the ex-

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107. 5. Fluorescence transients at 410 nm, 490 nm, and as the 410-to-490 nm ratio and contractions from an indol-loaded rat myocyte field stimulated from rest at 0.2 Hz in 1 mM [Ca”‘],. 410-to-490 mn ratio of indofluorescence has a better signal-to-noise ratio than either of two individual wavelengths because of cancellation of lamp intensity noise. Same data are shown before (A) and after (B) Gaussian filtration with a 3-db corner frequency of 10 Hz. This algorithm removes bulk of shot noise without introducing inordinate phase shifts. C shows absolute levels of fluorescence in 2 wavelength bands for microscope autofluorescence alone (A), an unloaded cell (C), and an indo-lloaded cell during a twitch (T). FIG.

periment, probably because of photobleaching. However, for both groups, the ratio of the two wavelengths remained constant over time. Effect of indo-l/AM loading on contractile properties of cardiac ceLI. Additional experiments were designed to determine the effect of loading with indo-l/AM on the contractile properties of individual myocytes, and twitches were obtained before and after exposure to indoI/AM. During washout of the indicator the cells consistently showed bursts of either spontaneous synchronous contractions or contractile waves (6, 7) and potentiation of electrically stimulated twitches that resolved spontaneously within l-5 min when myocytes became again

quiescent unless electrically stimulated (results not shown). A representative example of the effect of a “moderately heavy” (5 min) indo-l/AM loading on myocyte contractility and on the 410-to-490 nm ratio of indo-l fluorescence is shown in Fig. 7A. After loading, the 410-to-490 nm ratio of indo-l fluorescence shows a poor signal-tonoise ratio that improves with time and is accompanied by a progressive decrease in the diastolic ratio and a higher peak systolic ratio during the twitch. An increase in twitch amplitude occurs in the immediate postloading period, but with time the contraction becomes smaller, has a longer time to peak, and a slower terminal phase

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6. Effect of cell motion within an unevenly illuminated field on indo-l fluorescence. From top to bottom, tracings represent emittedindo-l fluorescence at 410 nm, 490 nm, the 410-to-490 nm ratio, and cell length. Myocyte was electrically stimulated to twitch, then moved within optical field (arrows), and finally returned to its original position. During electrically stimulated twitch, intensities of emitted light at 410 and 490 nm move in opposite directions; in contrast mechanical movement of unstimulated myocyte produces same directional changes at both wavelengths and this motion artifact is canceled in 410-to-490 nm ratio ([ Ca2+lo was 1 mM). FIG.

1. Effect of time and intermittent on indo-l fluorescence TABLE

Group

410, nm 490, nm 410/490,

nm

92.1t8.3 93.8t7.2 99.3t0.8

A

excitation Group

B

81.8k2.2 82.4t3.2

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Effect of time (60 min) alone (group A; n = 4) and intermittent excitation at 350 nm (group B; n = 5) on emitted indo-l fluorescence at 410 nm, 490 nm, and their ratio (see text). Values are means t SE and are expressed as % control (see text).

of relaxation likely related to increased buffering of [Ca2+]; by the indicator. These time-dependent changes in the emitted light and contraction may represent continuing de-esterification of the AM derivative of indo-1. Both the changes in contractility and indo-l fluorescence reach steady state within 30-60 min after washout of the indicator. Figure 7B shows, in another cell, that the effect of indo-l/AM loading on twitch amplitude can be compensated by raising [Ca”‘],, which enhances twitch shortening and the associated fluorescence transient; still, the relaxation phase of the twitch is prolonged. Note that as -[Ca”‘], is increased from 1 to 3 to 5 mM there is also an increase in the 410-to-490 nm ratio of indo-l fluorescence at rest; when [Ca”‘], is reduced back to 1 mM the resting indo-l ratio declines (not shown). It is apparent that loading of an adult cardiac myocyte with the Ca”+ indicator can significantly decrease the amplitude of the twitch and prolong its relaxation. The robustness of the indo-l fluorescence ratio transients after electrical stim-

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MYOCYTES

ulation in Figs. 5 and 6 suggest that less loading may still produce sufficient indo- 1 fluorescence but less twitch buffering. Indeed, a shorter exposure (30 s before the start of washout) to the AM form of the indicator, as shown in Fig. 8, was sufficient to give a fluorescence transient with a good signal-to-noise ratio and only a minor decrease in the amplitude of the twitch without affecting the time course of contraction. A slow terminal phase of relaxation of the mechanically unloaded myocyte contracting from slack length is usually observed in single adult cardiac cells even in the absence of any additional intracellular-Ca2’ buffer (cf. contraction in Fig. 4A and control contraction before indo-l loading in Fig. 8). This slow phase of relaxation, which is present in the cell before indo-l loading, appears unchanged after loading with indo-l/AM (Fig. 8, A and B) and is associated with slow decrease in the indo-l fluorescence ratio (Fig. 8C). The consistency of the latter observation suggests that small changes in [Ca2+]; that occur throughout the diastol .ic interval and modulate diastolic properties of cardiac m uscle can . be monitored by our system. An additional example of the utility of this system in this regard is observed in Fig. 9, that contrasts ,&adrenergic stimulation with an increase in [Ca2+10. Although both increase the amplitude of the [Ca2+]; transient, isoproterenol causes its duration to decrease, but, in particular, markedly accelerates the relaxation of the terminal phase of the [Ca2+]; transient. Similar changes are observed in the twitch amplitude and duration. As previously described (28) in cardiac myocytes loaded with the Ca2+-probe fura-2, a small decrease in resting [Ca2+]i, shown here by indo-l fluorescence, also occurs after isoproterenol. It is noteworthy that for similar peak fluorescence transients in 3 mM [Ca”‘], or isoproterenol with 1 mM [Ca”‘], (Fig. 9B) there is a smaller contraction in isoproterenol; and comparable results have been obtained with papillary muscle injected with aequorin (11). This is compatible with previous studies that showed an effect of ,&adrenergic stimulation to phosphorylate troponin-I (27) and decrease of myofilament sensitivity to Ca2+ (22) but could also be caused by the abbreviation of the calcium transient in isoproterenol. Simultaneous measurement of membrane potential, [Ca2+]i transient, and contraction. When conventional patch-pipette techniques are used, simultaneous measurement of the membrane potential or currents can be added to those of indo-l fluorescence and contraction. The apparatus in Fig. 1 is equipped with a high-impedante amplifier (Axopatch-IA patch clamp; Axon Instruments, Burlingame, CA) to measure membrane potential or currents. The 600-A pulse of the xenon arc generates a substantial amount of radio frequency noise; by very careful shielding of the lamp and its power supply, it was possible to avoid interference with the pica-ampere headstage amplifier. An example of simultaneously measured action potential, [Ca2+]i transient and contraction in a rat myocyte studied in the current-clamp mode is given in Fig. 1OA. This, to our best understanding, represents the first demonstration of simultaneous measurement of all three

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FIG. 7. Effect of 5-min loading with indo-l/AM on contractility and on 410to-490 nm ratio of indo-l fluorescence in 2 representative myocytes loaded on stage of microscope (see METHODS). Each myocyte was continuously field stimulated at 0.2 Hz. A: bottom tracings represent rested-state twitch in control and at different times during 1 h after loading with indo-l/AM. Top tracings represent 41O-to-490 nm ratio of indo-l fluorescence for associated contractions. [Ca”‘], was 1 mM. B: 1 h after exposure to indicator myocyte shows a decrease in twitch amplitude and a prolongation in its time course. An increase in [Ca”‘], enhances contraction and associated fluorescence transients, as well as resting fluorescence ratio.

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Downloaded from www.physiology.org/journal/ajpheart at Karolinska Institutet University Library (130.237.122.245) on February 12, 2019.

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MYOCYTES FIG. 9. Effect of P-adrenergic stimulation or an increase in [Ca”‘], on amplitude and time course of twitch. A: isoproterenol (0.5 PM), in addition to increasing amplitude of [Ca2+]; transient and of twitch, shortens their time course, in particular late phase of relaxation, and lowers diastolic indo-l fluorescence ratio (1 mM [ Ca”‘],). B: in a different myocyte, effects like those described in A were present when [Ca2+]; transients of similar magnitude were obtained during stimulation either in 3 mM [Ca”‘], or in 1 mM [Ca”‘], with 0.5 PM isoproterenol. For similar [Ca2+]i transients a smaller contraction occurs during p-adrenergic stimulation than in control, suggesting a decreased myofilament responsiveness to Ca2+ in isoproterenol. C: traces in isoproterenol from B and controls from A and B were normalized to same peak amplitude to emphasize effect of P-adrenergic stimulation on time to peak and on early and late phases of relaxation.

FIG. 10. A: transmembrane action potential, fluorescence transient, and length changes during a rested-state contraction in a myocyte loaded with indol/AM for 5 min and bathed in 1.0 mM [Ca”‘].. Cell length was monitored via photodiode array. B: Membrane current, fluorescence transient, and twitch in response to a 200-ms voltage step from -45 to 0 mV in an indo-l-loaded rat myocyte ([Ca”‘], was 1 mM). C: Ca2’ currents, [ Ca2’]i, and contractions monitored by video system in a representative rat myocyte studied under voltage-clamp conditions. Cell was heavily Ca2+ loaded by prior 5-s pulses in 3 mM [Ca”‘],. After last pulse, spontaneous SR Ca2’ release occurred that produced cell shortening and a small inward current.

parameters in an individual adult cardiac cell. Under these experimental conditions the decay of the latter part of the fluorescence transient, terminal repolarization of membrane potential, and elongation of cell length appear to parallel each other. With the apparatus operating in the voltage-clamp mode (Fig. 1OB) simultaneous measurement of Ca2+ current, [Ca2+]; transient, and contraction can be made. With the system described it is also possible to monitor the 410-to-490 nm ratio of indo-l fluorescence, cell length, and membrane current during spontaneous SR Ca2+ release, that has been reported to occur in a variety of circumstances that lead to augmentation of cell Ca2+ (7-9). This type of Ca2+ release produces a small depolarization (7, 8) caused by an inward current and causes a propagated contractile wave with the cell (6-9). A representative example is shown in Fig. IOC, in a cell bathed in 3 mM [ Ca2+10 and heavily Ca2+ loaded by prior

pulses of long duration. The electrically stimulated phasic SR Ca2+ release is relatively homogeneous and causes a twitch, whereas the subsequent more localized spontaneous SR Ca2+ release causes a spontaneous contractile wave and a small inward current. Figure 10 also demonstrates that on repolarization from a long-lasting clamp, a long inward tail current parallels the decay of [Ca2+];. This decaying inward current on repolarization has recently been rigorously studied and is thought to reflect Na-Ca exchange current (1, 14, 23, 24). Figure 1lA shows the effect of long depolarizations to different membrane potentials on the 410-to-490 nm ratio of indo-l fluorescence and cell length in a representative myocyte loaded with the AM form of the indicator. Figure 1lB depicts the relationship between the maximal amplitude of cell shortening and the ratio of indo-l fluorescence during the first second of each pulse and each of the subsequent 4 s for the same cell in Fig.

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that can occur when [Ca’+]; remains elevated up to 5 s, are not of such magnitude to change the relationship between cell length and indo-l fluorescence obtained during the first second of the pulse. These data suggest that the compartmentalization of the indicator into noncytosolic spaces does not interfere with the assessment of Ca2+-myofilament interaction obtained by plotting cell shortening vs. the change in [Ca2+]; obtained during a twitch.

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FIG. 11. Effect of 5-s depolarizations from a holding potential of -50 mV to various potentials up to +lO mV on relationship between 410/490 nm ratio of indo-l fluorescence and cell length at different times during voltage clamp in a representative rat myocyte. To obtain a wide range of amplitudes for both signals [Ca”‘], was varied between 3 and 0 mM: a) +5 mv, [Ca”+], = 3 mM; b) -30 mV, [Ca”‘], = 3 mM; c) -35 mV, [Ca2+], = 3 mM; d) -40 mV, [Ca”‘], = 3 mM; e) 0 mV, 2 min in Ca2+-free medium; and f) 0 mV, 3 min in Ca2+-free medium. A: tracings were superimposed to show range of cell shortening and ratio of indo-l fluorescence that was possible to achieve. After each pulse a slow decay occurred in both signals and sufficient time was allowed for a complete return to control values before repeating the depolarization. B: cell shortening and ratio of indo-l fluorescence during first second of each pulse (a) and during each of 4 subsequent seconds (0). Note that all points fall on same line which suggests that during 5 s of depolarization there was no change in contribution of indo-l fluorescence from noncytosolic compartments (see text).

1lA. If mitochondrial loading with Ca2+ occurred with long depolarizations, this would lead to an increase in the 410-to-490 nm ratio of indo-l fluorescence not paralleled by further cell shortening and thus change the relationship between these two parameters obtained during the first second of the pulse. The main information to be derived from this figure is that all points fall on the same line, which suggests that changes in [Ca”‘] in noncytosolic compartments, such as the mitochondria,

Although other investigators have used indo-l in either adult myocytes (10) or in embryonic cardiac cell aggregates (19, 25), there are no descriptions of a system that allows the simultaneous measurements of the 410-to-490 nm ratio of indo-l fluorescence, cell length, and membrane potential or currents, using unaveraged values and with a fast time resolution and limited exposure to UV light, in single cardiac myocytes. The Ca2+ indicator, indo-1, allows the emitted fluorescence at 410 and 490 nm to be expressed as a ratio, overcoming the problems caused by changes in the emitted light unrelated to changes in [Ca2+];, that may arise from fluctuations in the intensity of the exciting light source (Fig. 5A), bleaching or loss of the indicator from the cell (Table 1), and uneven illumination of the optical field or motion artifact (Fig. 6). Both prior reports (10, 25) and the present one have observed that loading cardiac cells with fluorescent-Ca2+ probes can alter the contraction. There are transient and long-term effects in this regard. In monolayer cultures of spontaneously contracting chick embryo ventricular cells loaded with indo-l/AM, a transient decrease in twitch amplitude and prolongation of its duration with a return of these parameters to control values within 30 min after washout of the residual AM, has been observed (25). Under the conditions we employed after a 5-min loading with indo-l/AM and subsequent washout of excess dye in the bath, there is a progressive decrease in extent of cell shortening accompanied by an increase in signal-to-noise ratio of the fluorescence transient, possibly caused by continuing de-esterification of the dye. Both changes in contractility and [Ca2+]; reach steady state within 30-60 min, with a persistent rather than transient decrease in twitch amplitude and prolongation of its time course relative to control before dye loading. However, an increase in [Ca”‘], could restore twitch amplitude to control values (Fig. 7B). An important characteristic of the present method is that indo-l/AM-induced changes in the contractile properties of the myocyte could be largely prevented by lighter loading with the probe (Fig. 8). In these lightly indo-lloaded single adult cardiac myocytes, we have observed a late phase of relaxation in cell length that is unchanged from that measured before indo-l loading and is paralleled by a slow decrease in [Ca2+];. Thus under these conditions it appears unlikely that the slow phase of relaxation represents an artifact caused by buffering of [Ca2+]; by the indicator. Rather, the results of Figs. 8 and 10, A and B, suggest that [Ca2+]; does continuously and slowly fall, during early diastole at least, and that this

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H586

CA’+,

CONTRACTION,

AND

MEMBRANE

change is associated with a slow repolarization of the membrane potential. Similar observations on cell length and/or diastolic [Ca”‘]; have been made in spontaneously beating clusters of chick embryo ventricular cells (2, 19) and in rabbit hearts (20) with indo-1. With the present technique it is possible to show (Fig. 9) that both the early rapid decline and the late decay of the [Ca2+]; transient can be modulated by P-adrenergic stimulation, which is thought to enhance the removal of Ca2+ from the cytosol (13, 29). The preliminary results presented with our instrument indicate the usefulness of the simultaneous measurement of indo-l fluorescence, current, and contraction, despite the uncertainties in calibration occasioned by compartmentation in cardiac myocytes loaded with the AM ester. It is possible that alterations in the loading protocol might reduce this problem. Alternatively, direct loading of the free acid of indo-l by pipette should make possible an absolute calibration of the cytosolic calcium measurement, which will further enhance the value of this method in studies of cardiac excitation-contraction coupling. The authors gratefully acknowledge Maurice Zimmerman for numerous contributions to the mechanical fabrication of the apparatus. Address for reprint requests: M. C. Capogrossi, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute of Aging, NIH, 4940 Eastern Ave., Baltimore, MD 21224. Received

19 September

1988; accepted

in final

form

4 October

1989.

REFERENCES 1. BARCENAS-RUIZ, L., D. J. BEUCKELMANN, AND W. G. WIER. Sodium-calcium exchange in heart: membrane currents and change in [Ca2+];. Science Wash. DC. 238: 1720-1722, 1987. 2. BARRY, W. H. Is ischemic contracture preceded by a rise in free calcium? In: Diastolic Relaxation of the Heart, edited by W. Grossman and B. H. Lorell. Boston, MA: Nijhoff, 1987. 3. BEUCKELMANN, D. J., AND W. G. WIER. Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. J. Physiol. Lond. 405: 233-255, 1988. 4. BURGESS, G. M., J. S. MCKINNEY, A. FABIATO, B. A. LESLIE, AND J. W. PUTNEY, JR. Calcium pools in saponin-permeabilized guinea pig hepatocytes. J. Biol. Chem. 258: 15336-15345, 1983. 5. CANNELL, M. B., J. R. BERLIN, AND W. J. LEDERER. Effect of membrane potential changes on the calcium transient in single rat cardiac muscle cells. Science Wash. DC 238: 1419-1423, 1987. 6. CAPOGROSSI, M. C., S. HOUSER, A. BAHINSKI, AND E. G. LAKATTA. Synchronous occurrence of spontaneous localized calcium release from the sarcoplasmic reticulum generates action potential in rat cardiac ventricular myocytes at normal resting membrane potential. Circ. Res. 61: 498-503, 1987. CAPOGROSSI, M. C., A. A. KORT, H. A. SPURGEON, AND E. G. LAKATTA. Single adult rabbit and rat cardiac myocytes retain the Ca2+- and species-dependent systolic and diastolic properties of intact muscle. J. Gen. Physiol. 88: 589-613, 1986. CAPOGROSSI, M. C., AND E. G. LAKATTA. Frequency modulation and synchronization of spontaneous oscillations in cardiac cells. Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H412-H418, 1985. CAPOGROSSI, M. C., M. D. STERN, H. A. SPURGEON, AND E. G. LAKATTA. Spontaneous Ca2’ release from the sarcoplasmic reticulum limits Ca2+-dependent twitch potential in individual cardiac myocytes: a mechanism for maximum inotropy in the myocardium. J. Gen. Physiol. 91: 133-155, 1988.

POTENTIAL

IN

CARDIAC

MYOCYTES

10. DUBELL, W. H., C. PHILIPS, S. R. HOUSER. A technique for measuring cytosolic free Ca2+ with indo-l in feline myocytes. In: Biology of Isolated Adult Cardiac Myocytes, edited by W. A. Clark, R. S. Decker, and T. K. Borg. New York: Elsevier, 1987. 11. ENDOH, M., AND J. R. BLINKS. Actions of sympathomimetic amines on the Ca2+ transient and contractions of rabbit myocardium: reciprocal changes in myofibrillar responsiveness to Ca2+ mediated through CY- and ,&adrenoceptors. Circ. Res. 62: 247-265, 1988. 12. FABIATO, A. Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calciuminduced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle. J. Gen. Physiol. 78: 457-497, 1981. 13. FABIATO, A., AND F. FABIATO. Relaxing and inotropic effects of cyclic AMP on skinned cardiac cells. Nature Lond. 253: 556-558, 1975. 14. FEDIDA, D., D. NOBLE, Y. SHIMONI, AND A. J. SPINDLER. Inward current related to contraction in guinea-pig ventricular myocytes. J. Physiol. Lond. 385: 565-589, 1987. 15. GRYNKIEWICS, G., M. POENIE, AND R. Y. TSIEN. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. BioZ. Chem. 260: 3440-3450,1986. 16. HAWORTH, R. A., P. GRIFFIN, B. SALEH, A. B. GOKNUR, AND H. A. BERKOFF. Contractile function of isolated young and adult rat heart cells. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): Hl484H1491,1987. 17. HERBENER, G. M., R. H. SWIGART, AND C. A. LANG. Morphometric comparison of the mitochondria populations of normal and hypertrophic heart. Lab. Inuest. 28: 96-103, 1973. 18. JACKSON, A. P., M. P. TIMMERMAN, 6. R. BAGSHAW, AND C. C. ASHLEY. The kinetics of calcium binding to furaand indo-1. FEBS Lett. 216: 35-39, 1987. 19. LEE, H.-C., AND W. T. CLUSIN. Cytosolic calcium staircase in cultured myocardial cells. Circ. Res. 61: 934-939, 1987. 20. LEE, H.-C., N. SMITH, R. MOHABIR, AND W. T. CLUSIN. Cytosolic calcium transients from the beating mammalian heart. Proc. Natl. Acad. Sci. USA 84: 7793-7797, 1987. 21. LI, Q., R. A. ALTSCHULD, AND B. T. STOKES. Quantitation of intracellular free calcium in single adult cardiomyocytes by furafluorescence microscopy: calibration of furaratios. Biochem. Biophys. Res. Commun. 147: 120-126, 1987. 22. MCCLELLAN, G. B., AND S. WINEGRAD. Cyclic nucleotide regulation of the contractile proteins in mammalian cardiac muscle. J. Gen. Physiol. 75: 283-295, 1980. 23. MITCHELL, M. R., T. POWELL, D. A. TARRAR, AND V. W. TWIST. Electrical activity and contraction in cells isolated from rat and guinea-pig ventricular muscle: a comparative study. J. Physiol. Lond. 391: 527-544,1987. 24. MITCHELL, M. R., T. POWELL, D. A. TARRAR, AND V. W. TWIST. Calcium-activated inward current and contraction in rat and guinea-pig ventricular myocytes. J. Physiol. Lond. 391: 545-560, 1987. 25. PEETERS, G. A., V. HLADY, J. H. B. BRIDGE, AND W. H. BARRY. Simultaneous measurement of calcium transients and motion in cultured heart cells. Am. J. Physiol. 253 (Heart Circ. Physiol. 22): Hl400-Hl408,1987. 26. POENIE, M., J. ALDERTON, R. STEINHARDT, AND R. TSIEN. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science Wash. DC 233: 886-889, 1986. 27. RAY, K. P., AND P. J. ENGLAND. Phosphorylation of the inhibitory subunit of troponin and its effect on the calcium dependence of cardiac myofibril adenosine triphosphatase. FEBS Lett. 70: 11-16, 1976. 28. SHEU, S.-S., V. S. SHARMA, AND M. KORTH. Voltage-dependent effects of isoproterenol on cytosolic Ca concentration in rat heart. Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H697-H703, 1987. 29. TADA, M., M. A. KIRCHBERGER, D. I. REPKE, AND A. M. KATZ. The stimulation of calcium transport in cardiac sarcoplasmic reticulum by adenosine 3’:5’-monophosphate-dependent protein kinase. J. BioZ. Chem. 249: 6174-6180, 1974.

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