Basis for late rise in fura 2 R signal reporting [Ca2+]i during relaxation in intact rat ventricular trabeculae

Yandong Jiang, Michael F. Patterson, David L. Morgan, and Fred J. Julian

Department of Anesthesia Research Laboratories, Brigham and Women's Hospital, Boston, Massachusetts 02115

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Intact rat ventricular trabeculae were injected with the salt form of fura 2, and the fura 2 ratio signal (R) was used to report intracellular Ca2+ concentration ([Ca2+]i). The fixed end relaxation phase of a twitch is associated with a slowing of the decay of the R signal, or even a reversal, to form a distinct bump, indicating a transient rise in [Ca2+]i. The bump is most prominent at 30°C, and motion artifact is not its cause. Increasing doses of 2,3-butanedione monoxime caused progressive attenuation of the twitch and bump. Increasing the bathing Ca2+ concentration potentiated the twitch and enhanced the bump. Imposed muscle shortening during relaxation caused a much quicker force decline, and this led to the appearance of a much more prominent associated bump. The amplitude of the bump depends on the amplitude of twitch force and the rate of relaxation. These findings can be explained, as in skeletal muscle, by making cross-bridge attachment and Ca2+ binding to troponin C strongly cooperative; therefore, the bump during fast relaxation is produced by a reversal of this cooperativity, leading to rapid dissociation of Ca2+ from troponin C into the myoplasm.

fluorescence; intracellular calcium; 2,3-butanedione monoxime; bathing calcium; ramp shortening

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IT HAS LONG BEEN appreciated in skeletal muscle research that the relaxation phase of a tetanus often shows two distinct phases (10, 14). After cessation of stimulation, there is a slow phase of force decline soon followed by a fast phase. It has been shown that the transition between these two phases coincides with the onset of increased sarcomere shortening in parts of a skeletal muscle with lengthening of other regions, typically near the ends, so that overall the muscle remains at a fixed length (8, 14). Later, it was shown that the transition in relaxation rate coincides with the onset of a small reduction in the rate of decline of the aequorin luminescence signal, which suggests a reduction in the rate of decline of myoplasmic free Ca2+ concentration ([Ca2+]i) (5).

A possible hypothesis to explain this phenomenon involves cooperative binding of myosin to actin and that of Ca2+ to troponin C (TnC). This explanation postulates that the onset of rapid relaxation indicates an acceleration of cross-bridge detachment rate and turnover (14) and that this leads to a reduction in the binding constant of TnC for Ca2+ (27). Thus, similar to previous discussions (2, 11, 13, 25), during accelerated relaxation, the affinity of TnC for Ca2+ decreases rapidly, causing the rate of release of Ca2+ to increase, leading to a decrease in the rate of decline of [Ca2+]i. This hypothesis has been recently described in detail and modeled for skeletal muscle (21).

It is also possible in cardiac muscle that extra Ca2+ is released from TnC as a consequence of reversal during relaxation of the strongly positive cooperative interactions between Ca2+ binding to TnC and myosin attachment to actin. This idea would be in agreement with that presented in a well-known review (1) in which quick releases of muscles (both cardiac and skeletal) imposed during the later stages of a twitch produce "bumps" in [Ca2+]i attributed to release of Ca2+ from myofibrils as a consequence of reduced Ca2+ affinity to TnC. This is further supported by work in cardiac muscle indicating that applied quick length changes during relaxation result in the appearance of extra Ca2+ in the myoplasm, whose most likely source is Ca2+ bound to TnC (19). In addition, experiments using cardiac trabeculae similar to those used in this study in which sudden length decreases were applied during relaxation produced bumps in the fura 2 [Ca2+]i signal that were interpreted as reflecting changes in the Ca2+ affinity of TnC (4). It was later confirmed that such changes in [Ca2+]i in similar trabeculae subjected to quick release could be observed (7).

However, in intact skeletal muscle, contraction is extremely cooperative, as revealed by the very steep force-[Ca2+]i relation (21), and this strong cooperativity is very similar in intact cardiac muscle (9). The reversal process of the cooperativity during naturally occurring twitch relaxation in skeletal muscle produces a bump (21), so the same phenomenon should be observed in cardiac muscle. Otherwise, there would have to be some fundamental difference between the basic contractile system in skeletal muscle and in cardiac muscle. Here, we present evidence indicating the presence of a consistent late slowing, or even reversal (bump), in the decline of the fura 2 ratio (R) signal reporting [Ca2+]i during ordinary fixed end twitches in cardiac muscle, i.e., in the absence of applied external length perturbations, which is an important distinction when comparing our findings to those described above. We also present evidence indicating the bump signals a true rise in [Ca2+]i, thus eliminating, for example, motion artifact as a significant factor. Finally, we test the hypothesis that the extra Ca2+, which leads to a bump, is liberated from TnC during reversal of the strong cooperativity between Ca2+ binding and cross-bridge detachment during fixed end relaxation. Preliminary findings have been presented to the Biophysical Society (23, 24).

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

A recent paper (16) presents many important details of the methods used in this work. The following condensed version provides essential information either not provided in the above paper or of particular importance here.

Dissection procedure. Male Lewis-Brown-Norway rats (150-175 g) were used in these experiments (Harlan, Indianapolis, IN). Hearts were quickly removed and placed in a cold modified Krebs solution (MKS). The hearts were then perfused retrograde through the aorta using a Langendorff apparatus for 10 min at room temperature with the same MKS containing (in mM) 108 NaCl, 6 KCl, 1.2 MgCl2, 2 CaCl2, 24 NaHCO3, 10 sodium pyruvate, and 4 glucose as well as 5 U/1,000 ml insulin and were bubbled with 95% O2-5% CO2, final pH 7.4. Finally, the hearts were flushed with dissection solution, which is the standard MKS plus 15 mM KCl (final KCl concentration = 21 mM).

Trabeculae similar to those described previously were isolated from right ventricles (6, 22). These trabeculae (length ~2.5-4 mm, width ~0.1-0.2 mm, thickness ~0.05 mm) were attached at one end to a force transducer (model 400A, Cambridge Technology), and the other end was tied to a stainless steel wire connected to a micropositioner. In an important step in the mounting procedure, cyanoacrylate tissue adhesive (Histoacryl blau, B. Braun, Melsungen, Germany) was used to fix firmly the two ends of the preparation to the force transducer wire and the micropositioner wire. We have previously shown that this method of attachment of a papillary muscle very much decreases extra end compliance as demonstrated by flash photography and use of markers and spot-follower apparatus (18). Consequently, internal movement associated with extra compliance at the ends of the preparation is minimized, thereby ensuring a contraction as close to isometric as possible.

Bath setup and electrical stimulation. The preparation was placed into a temperature-regulated chamber (8 mm × 6 mm × 5 mm) milled into a Lucite plate. The chamber had a glass coverslip floor (thickness 0.12 mm). The Lucite plate with its chamber was positioned on the stage of an inverted microscope (Nikon Diaphot 300) fitted with a fluorescence system [Photon Technology International (PTI) Deltascan 4000]. Preparations were superfused at a rate of 12 ml/min with MKS. The MKS solutions were vigorously bubbled with 95% O2-5% CO2. The temperature in the bath was maintained at 30 ± 0.2°C unless otherwise stated. The preparations were stimulated at 0.5 Hz with 5-ms pulses via platinum plate electrodes in the bath connected to a stimulator (Grass S88). The stimulus strength was adjusted to 1.5 times threshold, and tension was continuously monitored on both a chart recorder (Hewlett Packard 7133A) and one channel of a digital oscilloscope (Nicolet 4094B). The optimal tension response with respect to length was obtained. Resting sarcomere length resulting in maximum tension upon stimulation was always in the range of 2.18-2.30 µm. These values were measured from hard copy striation patterns sampled from the video image (using a Nikon ×40 objective) with a UP1200 Mavigraph color video printer (Sony).

Fluorescence system. The PTI Deltascan 4000 system was used in these experiments, and a 75-W xenon lamp was the source for excitation light. The light passed through a scanning monochromator (entrance and exit slit widths set to 10 nm) that was programmed to select wavelengths of 345 and 380 nm for excitation of fura 2. Light exiting the monochromator was carried by a quartz fiber-optic bundle to a filter cassette coupled to the microscope objective. Upon entering the cassette, the light was first short-pass filtered (<470 nm) to remove the residual 510-nm light before reflecting off a dichroic mirror (400 nm long pass) en route to the objective, an Olympus ×10 (Dapo UV/340; numerical aperture 0.4). Fluorescence from the preparation passed back through the objective and dichroic mirror to the side port of the microscope. Here, a second dichroic mirror passed the light to a photometer with a rectangular region-of-interest adjustment, and this light was then sent to a photomultiplier using the analog mode. Just before reaching the photomultiplier tube, light signals were passed through emission filters that transmitted wavelengths of 510 ± 20 nm. The voltage output from the analog photomultiplier was sent to a low-pass analog filter (Stanford Research Systems, model SR650) with a 500-Hz cutoff and then to a four-channel digital oscilloscope (Nicolet 4094B), sampling at a frequency of 2 kHz. A dual disk recorder (Nicolet XF-44/2) was used to record to floppy disks the tension and fluorescence signals. The fluorescence system was controlled by PTI software ("Oscar") running on an IBM-compatible 486 computer (American Megatrends) used to control shutter openings, monochromator settings, and other automated data acquisition programs.

Dye-loading procedures. Before the trabeculae were loaded with fura 2, autofluorescence (emission at 510 nm) was measured at a pacing rate of 0.5 Hz and 30°C. Preparations were then loaded by iontophoretic injection of fura 2 dye by a method already described (4), in MKS with extracellular Ca2+ concentration ([Ca2+]o) of 0.5 mM at 22°C with no stimulation. Micropipettes were pulled on a Flaming/Brown micropipette puller (model P87, Sutter Instrument), and tips were filled with 1 µl of 2 mM fura 2 (pentapotassium salt, Molecular Probes, Eugene, OR). The pipettes were then back-filled with a 150 mM potassium acetate solution (pH 7.3) and connected via a micropipette holder to the headstage of a WPI electrometer module (model S-7071A). Tip resistances of 200-300 MOmega were typical in micropipettes filled with the fura 2 solution before impalement of single myocytes, and stable membrane potentials of -40 to -60 mV were required for successful dye loading. After impalement, injection of the dye into the myocyte was achieved by passing a negative current of 3-5 nA. After loading, the preparation was superfused again with the standard MKS, the temperature was increased to 30°C from room temperature, and the pacing was resumed at 0.5 Hz. About 40 min were allowed to let the dye diffuse, yielding a complete uniformity of fluorescence. Fura 2 was loaded to levels such that the added fluorescence was about three times the autofluorescence observed at 358 nm (the isosbestic point for fura 2) before loading.

Application of 2,3-butanedione monoxime and nonstandard [Ca2+]o. The application of 2,3-butanedione monoxime (BDM) was done by direct addition of BDM (Aldrich Chemical) into the standard MKS in the treatment bottle, yielding final concentrations of 2, 5, or 10 mM. When nonstandard [Ca2+]o was required, the standard MKS (2 mM Ca2+) was replaced by an identical solution except that the Ca2+ concentrations were 0.5, 1, or 4 mM.

Servo control. A servo-controlled ramp release of length was applied at a predetermined point in time. In this case, the end of the trabecula opposite the transducer end was tied and glued in the same way to a stainless steel wire extending from the shaft of a servo motor. Control and timing signals could then be applied to the command unit controlling the servo motor to produce a length ramp of adjustable slope and amplitude. The amplitude of the release was determined using flash photography to relate ramp size to applied voltage. The apparatus and techniques for using servo control in the study of muscle function have been previously described (17).

Data collection and analysis. The fluorescence and twitch force records presented here are all averages of nine sweeps. The autofluorescence (a) values at excitation wavelengths 345 nm (F345a) and 380 nm (F380a) were interleaved in time so that a sweep at 345 nm was followed by one at 380 nm and so on until a total of 18 sweeps was recorded. These were then averaged in two groups of nine. In this way, a noise reduction by a factor of three was achieved. The fluorescence values after dye loading were collected in the same way as those for the autofluorescence, yielding total (t) fluorescence at the two excitation wavelengths (F345t and F380t). The F345a and F380a were then subtracted from the F345t and F380t, respectively, yielding the dye-related fluorescence F345 and F380. The R signal was formed by dividing F345 by F380. This made it possible to use the R signal to greatly minimize the effect of motion artifact on fura 2 reporting of [Ca2+]i. The R signals presented in the results are all formed from fluorescence signals that are the average of nine sweeps. Fluorescence and twitch force were analyzed using Vu-Point 3 data analysis software (Maxwell Laboratories). The R signal was transformed to [Ca2+]i by using the equation [Ca2+]i = Kd · beta  · (R - Rmin)/(Rmax - R). The values of dissociation constant (Kd) · beta , Rmax, and Rmin were obtained from a recent paper (16).

Controls. Varying temperature, [Ca2+]o, and adding BDM caused autofluorescence changes at both excitation wavelengths of 345 and 380 nm, which were maximally ~10% (without added fura 2). The impact of these changes was minimized by addition of fura 2 fluorescence to exceed the initial autofluorescence by a factor of about three. The actual impact of the change in autofluorescence on the R value is, therefore, no more than ~3%. This shifts the R signals up or down slightly, but it does not affect the shape and the size of the bump. Shortening and restretching a relaxed muscle during a ramp also caused autofluorescence and fura 2 fluorescence at excitation wavelengths 345 and 380 nm to increase and decrease, respectively, by ~5%. This impact is also greatly minimized by forming R. Compared with the prominence of the bump, the R signal fluctuation caused by a ramp is insignificant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The effects of temperature on [Ca2+]i and the prominence of the bump together with twitch force from a typical experiment (n = 3) are shown in Fig. 1, A and B. For the traces at 30°C, this leads to a [Ca2+]i clearly displaying a prominent bump during relaxation. This has been a consistent observation in our experiments. In contrast, the traces in Fig. 1, A and B, at 21°C, show a larger twitch force with both a slower rise and a much prolonged decay phase. The associated [Ca2+]i shows a very small sign of a late slowing in its decay rate. At 37°C, the twitch force is smaller with a much quicker rise and more rapid decay. However, there is a clear late slowing in the rate of decay of the [Ca2+]i, but less prominent than at 30°C. The choice of 30°C, therefore, seems to optimize conditions for best observing a bump.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Intracellular Ca2+ concentration ([Ca2+]i) and forces in twitches at different temperatures. Pacing frequency was 0.5 Hz, and bathing Ca2+ concentration was 2 mM. A: [Ca2+]i. B: corresponding twitch forces. Numbers (21, 30, and 37) above each trace in each panel indicate temperature (°C) at which traces were recorded. Twitch forces were scaled with respect to amplitude of 30°C twitch. Note in A that most prominent bump occurs at 30°C. In B, amplitudes and shapes of twitches differ markedly, whereas upstrokes and peak amplitudes of associated [Ca2+]i, in A, are similar.

Even at 30°C, the change in the rate of late decay of the R signal is variable. The R and force signals from twitches of three different trabeculae are shown in Fig. 2. The R signal is used instead of [Ca2+]i because the onset time of the bump is most important, not the peak amplitude. In all panels of Fig. 2, the R signal rises in response to electrical stimulation much more quickly than does twitch force and reaches a peak much sooner than does the twitch force. Then, after reaching the peak, R falls initially rapidly, and this is followed by a slower decay phase in the R signal during relaxation. In almost all trabeculae we have studied, the decay phase of the R signal contains a significant portion deviating away from a smooth decay during the later part. The deviation in the R signal occurs invariably after the R signal has decayed by at least 50%, whereas the force decays by <50%. Three cases are shown in Fig. 2. In Fig. 2A, the deviation is most obvious, leading to a reversal of the decay phase and the appearance of a bump in the R signal. In Fig. 2C, the opposite extreme is shown, with a deviation much less obvious than in Fig. 2A. In Fig. 2B, our most typical result is shown, and in this most often observed case, the deviation is about halfway between those shown in Fig. 2, A and C. It is obvious that the deviation is connected to the relaxation phase of contraction. The most apparent correlation between prominence of the deviation and the relaxation phase is in the rapidity with which the force returns to baseline. If, as in Fig. 2A, the force quickly returns to baseline, there will be a large deviation with a reversal in the decay phase. At the other extreme, as in Fig. 2C, a slowly decaying force signal leads to only a small deviation with no sign of a phase reversal. Most commonly, as in Fig. 2B, the force decays at an intermediate rate, and this leads to an obvious deviation with little sign of a reversal phase.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Fura 2 R signals (noisy traces) and twitch forces (smooth traces) from 3 different trabeculae at 30°C. Preparations were paced at frequency 0.5 Hz, and bathing Ca2+ concentration was 2 mM. All records have had their baselines set to zero followed by normalization with respect to maximum values. Results are shown in A-C with prominence of late slowing, or even a rise, in R signal being in order A > B > C. In each panel, a smooth curve was fit to points within decay segments of R signal flanking bump, as indicated by arrows, using a double exponential transform from SigmaPlot. R signals were filtered to remove noise components to make clear the close approximation of fitted curves. Also, in each panel, a vertical dashed line has been added to indicate time point of departure of decay of R from smooth course predicted by fitted curves. Note that in order A, B, C, vertical dashed lines shift to right as relaxation and decay of R signal become slower.

The slowing of the decay, or even a rise, in the [Ca2+]i is linked to the extent of force generation. A typical experiment done using BDM is shown in Fig. 3, where BDM is used to inhibit active force generation, thus diminishing twitch amplitude and reducing the prominence of the bump. These experiments were repeated in five trabeculae. The expectation here is that BDM, in increasing doses, will markedly diminish the number of attached cross bridges (3, 20), and this will influence the prominence of the bump. The force traces in Fig. 3, A-D, show that BDM reduced the twitch peak progressively with increasing BDM concentration. This is clearly accompanied, as shown in Fig. 3, E-H, by a progressive decline in the prominence of the bump. At the highest dose of BDM used, the [Ca2+]i declines smoothly with no sign of a slowing or bump. Note that BDM did not significantly affect the amplitude of the [Ca2+]i, nor did it influence the early declining phase of [Ca2+]i during relaxation. The BDM effects were essentially completely reversible, since control [Ca2+]i and twitch responses were nearly identical before and after application of BDM.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3.   [Ca2+]i and twitch forces from a rat ventricular trabecula treated with various doses of 2,3-butanedione monoxime (BDM). Pacing frequency was 0.5 Hz, and bathing Ca2+ was 2 mM. Temperature was 30°C. A-D: force traces. E-H: corresponding [Ca2+]i. Number in each panel (0, 2, 5, 10) indicates BDM concentration (in mM). In A and E, [Ca2+]i trace and force trace are average of initial control and posttreatment control after washing BDM. BDM effect was nearly completely reversible. Bump areas of [Ca2+]i signals were calculated as follows: data points from 2 segments of decay phase of [Ca2+]i flanking bump were fit with a modified single exponential equation from SigmaPlot, in a way similar to that described in legend for Fig. 2. Fitted curve was subtracted from Ca2+ concentration curve to yield difference. Finally, area beneath each curve was obtained using area transform in SigmaPlot. For each BDM concentration, [Ca2+]i signals and corresponding twitch forces were recorded after twitch peak reached its steady-state value. Twitch forces have been scaled with respect to averaged control value.

The correlation between the slowing in the fall of the [Ca2+]i, or its reversal, and the magnitude of the twitch force was further examined by variation in the bathing Ca2+ concentration, [Ca2+]o, which very much influenced twitch force amplitude. Typical results are shown in Fig. 4, and the experiments were repeated in four different trabeculae. The twitch force results in Fig. 4B show that increasing [Ca2+]o produced an obviously positive correlation between [Ca2+]o and twitch peak amplitude. In addition, the shape of the twitch force is modified in that increasing [Ca2+]o leads not only to an increase in force amplitude, but also to an abbreviation with a quicker decline of the twitch force. As is apparent in Fig. 4A, the twitch force variations produced by altering [Ca2+]o are accompanied by large changes in the associated [Ca2+]i. At the highest [Ca2+]o used, there is an obvious bump in the associated [Ca2+]i, and this gradually disappears as the [Ca2+]o is reduced. These results support the hypothesis that augmented force generation and, therefore, number of attached cross bridges, is connected to the occurrence of an enhanced late slowing, or even rise, in the [Ca2+]i. The variation in [Ca2+]o effects were completely reversible.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   [Ca2+]i and twitch forces from a trabecula treated with various concentrations of bathing Ca2+ concentration ([Ca2+]o). Pacing frequency was 0.5 Hz with temperature of 30°C. A: [Ca2+]i signals. B: corresponding twitch forces. In each panel, numbers above each trace indicate appropriate [Ca2+]o in mM. For each [Ca2+]o, [Ca2+]i and corresponding twitch forces were recorded after twitch peak reached its steady-state value. Twitch forces have been scaled with respect to normal control value for [Ca2+]o, 2 mM. Note, in A, that as [Ca2+]o increases, there is a progressive accentuation in prominence of late slowing in [Ca2+]i. There is also an obvious increase in amplitude of [Ca2+]i with increasing [Ca2+]o, and relaxed state level is shifted slightly upward at highest [Ca2+]o. This is accompanied, in B, by a progressive increase in twitch amplitude and a quickening in relaxation phase.

It is important to know whether there is a close correlation between the bump area, defined by the area between the falling part of the [Ca2+]i curve and a curve fit to the descending [Ca2+]i curve containing points flanking the bump, but not including the bump, and the peak amplitude of the corresponding twitch force. The way curves are fit is shown in Fig. 3. The relation between these two variables generated either by varying BDM concentration, from experiments similar to those shown in Fig. 3, or by varying [Ca2+]o, from experiments similar to those shown in Fig. 4, is shown in Fig. 5. The linear regression fit for all of the data shown in Fig. 5 is also included in the figure. In both cases, as shown by the linear regression, the bump area falls nearly linearly with peak amplitude of the twitch force, and the intercept on the abscissa is near zero. This means, with either treatment, that the bump area is proportional to the peak amplitude of the twitch force and that when the twitch force becomes very small the bump disappears, or is not detectable.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 5.   Plots of relative bump area as functions of corresponding relative peak amplitude of twitch forces with variation of either [Ca2+]o or BDM concentration. Data for BDM experiments are shown as solid triangles (means ± SE, n = 5), where average control, without BDM, value for both twitch amplitude and bump area was set to 1. Twitch force amplitude and bump areas obtained with BDM present (2, 5, and 10 mM) are given relative to those for controls. Data for varying [Ca2+]o are given as solid circles (means ± SE, n = 4). Average for peak twitch force amplitude and bump area for 4 mM [Ca2+]o was set to 1 and was used to rescale other values obtained for 2, 1, and 0.5 mM [Ca2+]o. Bump areas were defined as described for BDM experiments in legend for Fig. 3. Solid line is a linear regression (y = -0.1767 + 1.15x, r2 = 0.964) used to fit all data points obtained by both varying [Ca2+]o and varying BDM concentration.

The longitudinal motion in all these trabeculae was too small to be quantified accurately enough to correlate with the deviations and bumps in the R signals, or [Ca2+]i. For this reason, twitches in which an overall shortening was imposed during relaxation were studied. These data were obtained by applying a ramp length shortening to isolated trabeculae at a point during relaxation just before bumps were observed in the control twitches. Typical results are shown in Fig. 6. Figure 6A shows the raw fluorescence signals, Fig. 6B the R signal, and Fig. 6C the force during a twitch contraction with ramp release. The raw fluorescence signals in Fig. 6A show the expected deviations in opposite directions coinciding with late slowing in the R signal. The corresponding bump is apparent in Fig. 6B. These experiments were repeated in five different trabeculae; four were done at 30°C and one was done at 21°C.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Raw fluorescence at excitation wavelengths of 345 and 380 nm, R signal, and corresponding twitch force with an imposed ramp. Pacing frequency was 0.5 Hz, temperature was 30°C, and bathing [Ca2+]o was 2 mM. Muscle length was 3.5 mm, and relaxed average sarcomere length was 2.2 µm. Ramp amplitude was ~5% of muscle length, and slope was 4 muscle lengths/s. Average sarcomere length at end of ramp was 2.1 µm, so average sarcomere length decrease was 0.1 µm. A: raw fluorescence trace for 345 nm was shifted up by 0.1 units, and that for 380 nm was shifted down by 0.1 units to avoid initial crossover. R signal formed from fluorescence traces in A is shown in B, and corresponding force is shown in C. In each panel, time course of change in muscle length produced by ramp has been added. A downward deflection of this record indicates shortening, whereas an upward one is lengthening. It is evident that marked change in slope of force relaxation produced by ramp length decrease (C) is associated with a prominent bump in R signal. Note in A at reversal of ramp (length increase back to initial length) that large changes occur in both raw fluorescence signals. However, in B, R signal shows no sign of any change during same time period, indicating complete motion artifact cancellation.

In Fig. 7, the same ramp record on an expanded time scale is shown, together with a control record from the same muscle taken just before obtaining the record shown in Fig. 6. Figure 7A shows the R signal, and Fig. 7B shows the forces. In Fig. 7, A and B, the ramp length record has been added for clarification. There can be no doubt that the much more rapidly occurring relaxation in the force record produced by the applied ramp length decrease led to a much more prominent bump in the R signal shown in Fig. 7A and that this bump is closely linked in time with the shortening. Compared with the bump in the twitch without ramp, the bump in the twitch with ramp is of greater amplitude and narrower width. Difference curves of [Ca2+]i are shown in Fig. 7C to reveal the extra Ca2+ released in the bumps during relaxation with ramp and without ramp (control). The areas under these curves are very similar. This indicates that the bump in the [Ca2+]i is linked to the applied ramp shortening.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   R signals and corresponding twitch force with and without imposed ramp. R signal and force with imposed ramp were from Fig. 6, and corresponding controls for R signal and force were obtained from same preparation. A: R signals. B: corresponding twitch forces. In both panels, time course of applied ramp is shown for clarity. Control force with a much more rounded decay phase is associated with a gentle rise and fall of bump in R signal. However, when a ramp is applied to make very much steeper force decay (B), associated R record (A) shows a bump with markedly increased size and narrower width whose onset is close to that of ramp. R signals from A were transformed into [Ca2+]i as described in METHODS. From transformed curves, bump areas were calculated as described in legend for Fig. 3. Area under control curve was 91% of that under ramp curve.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

An important first question concerns our consistent observation of a prominent slowing in the rate of decay, or even a reversal, of the R signal or [Ca2+]i, which seems to be in contrast to the findings of other workers in the field. This difference in frequency of observation of some kind of deviation from smooth decay in the R signal or [Ca2+]i would become less puzzling if an adequate explanation could be found. We believe the difference can be mostly explained as a result of our choice of experimental temperature, and the results shown in Fig. 1 substantiate this belief. Our standard experimental temperature is 30°C, chosen as a compromise between body and room temperatures. Because others have worked at room temperature, this could explain why the bump is often not apparent even though the same preparation loaded with fura 2 salt was used (4). It should also be noted in Fig. 1 that the upstrokes and peak amplitudes of the [Ca2+]i are quite similar and that they differ most in their decay phases. In addition, in Fig. 1, it can be seen that the twitches vary markedly in height and duration, thus supporting the view that in cardiac muscle also it is a parameter more similar to [Ca2+]i half-width, rather than peak amplitude, which determines the amplitude and shape of the resultant twitch, as in skeletal muscle (15, 26). The prominence of the slowing, or rise, is subject to some variation, as shown in Fig. 2, and this seems mainly to be because of variation in individual trabecular relaxation rates.

The bump always occurs during twitch force relaxation, so it might be caused by motion artifact. This seems unlikely for the following reasons. The microscopic field for fluorescence collection covered ~90% of the total length of the preparation. This made fluorescence changes during a twitch due to longitudinal motion negligible. The microscopic field also enclosed about two times the width of the preparation. This made it possible to make sure the preparation moved only within this field, which was confirmed using a video camera before data collection began. Movements of the preparation during a twitch could be out of the focal plane, and this could influence F345 and F380. However, use of a ×10 objective, which has a large depth of focus, minimized effects due to motion vertical to the focal plane. In addition, the R signal, not a single- wavelength fluorescence signal, was used, and this ratioing process greatly reduces the effects of motion on the raw F345 and F380 signals. This is supported by results shown in Fig. 6, where it can be seen that the F345 and F380 move in opposite directions during both the early upstroke and the bump in the R signal in a twitch. In addition, in Fig. 6A, the reversal of the ramp release produces large changes in fluorescence at both F345 and F380 of similar size, time course, and polarity, i.e., decreases in this case, whereas the corresponding R signal, in Fig. 6B, shows a smooth decay without any sign of these motion-induced changes. This shows how motion-induced changes in raw fluorescence at both excitation wavelengths are canceled by using the R signal. The reversal of the ramp release occurs essentially in the relaxed state, so the changes in the raw fluorescence signals due to motion during the reduction of muscle length by the imposed ramp should be very nearly opposite to those shown for reextension. This serves as a control for the ramp release when the muscle is relaxed. Furthermore, if the late slowing in the bump is because of random motion artifact, it should, at least in some cases, occur in the opposite direction, which we have never observed. In conclusion, the bump is unlikely to be a result of motion artifact.

A pertinent next question concerns the origin of the extra amount of Ca2+ during relaxation. It has been proposed that the late slowing in the R signal (or its transformation into [Ca2+]i) may be because of a secondary release of Ca2+ from the sarcoplasmic reticulum (SR) (4), and evidence is cited to support this idea (22). We believe that this is unlikely for the following reasons. The deviations in the decay phase of the R signals or [Ca2+]i we observed occurred before the force returned to baseline, i.e., they were very different in time course from those thought to be associated with a secondary release of Ca2+ from the SR in after-contractions (22). The bump depends on twitch force amplitude, as shown in Figs. 3 and 4. Altering twitch force amplitudes with either BDM or variation of [Ca2+]o leads to corresponding changes in prominence of the bump, as well as showing a nearly linear relation of bump area with peak twitch amplitude, as shown in Fig. 5. In addition, BDM does not alter the times to peak, the early decays, and relaxed levels of the [Ca2+]i, as shown in Fig. 3. This implies that SR Ca2+ release and uptake, and even SR Ca2+ content, is not markedly influenced by BDM. The disappearance of the bump with BDM treatment is tightly linked to the reduction of force generation, rather than to SR function. There seems to be no strong reason to link force decline during relaxation to a secondary Ca2+ release from the SR. Abolishing SR function seems to enhance, not reduce, the bump (7). The use of a twitch force potentiator, EMD-53998, caused a delayed relaxation accompanied by a delayed occurrence of a bump (7). Because this experiment was done in trabeculae with disabled SR function, the delayed bump must be linked to delayed relaxation and not caused by secondary release of Ca2+ from the SR. For all of these reasons, it seems unlikely that the bump is caused by a secondary release of Ca2+ from the SR.

We propose here that the late slowing in the decline of the R signal, or even a rise, in cardiac muscle is caused by the same mechanism thought to occur in skeletal muscle. As mentioned in the introduction, this would be mainly because of the reversal during relaxation of the strongly cooperative relation between cross-bridge attachment and Ca2+ binding to TnC. If this hypothesis is true, the following conditions should be linked to the prominence of the bump.

The prominence of the bump should be proportional to that of the twitch force, since the cooperativity is dependent on the number of attached cross bridges. This idea is supported by the results from the BDM experiment shown in Fig. 3. With increasing doses of BDM, the amplitude of the twitch force and the prominence of the bump gradually decreased. With 10 mM BDM, the peak twitch force was reduced by ~80%, and this was associated with disappearance of the bump. The doses of BDM did not alter the peak amplitudes of [Ca2+]i, as shown in Fig. 3 and also by others (3). Also, in Fig. 3, the time course of the [Ca2+]i was essentially not affected. The only significant change in [Ca2+]i is the gradual disappearance of the bump. This indicates that at these doses BDM acts specifically to diminish twitch force and abolish the bump, while not influencing other characteristics of the [Ca2+]i, thus not affecting SR function. BDM is known to work primarily downstream at the cross-bridge level and reversibly inhibit active force generation (3, 20). It is also known that BDM does not alter the affinity of Ca2+ for TnC in the dose ranges used here (12); therefore, the extent of Ca2+ occupancy of TnC should not be changed if the [Ca2+]i signal remains mostly unaffected. This indicates that BDM's action to abolish the bump is not because of a reduction of Ca2+ binding to TnC. The bump is abolished by BDM as a result of its reducing the number of attached cross bridges, thus reducing cooperativity between attached cross bridges and Ca2+ affinity for TnC. During relaxation, reduced cooperativity leads to a less rapid Ca2+ release from TnC, thus producing a less prominent bump. This supports the idea that it is TnC binding sites for Ca2+ that serve as the most likely source for the extra Ca2+ forming a bump.

Another point arguing in favor of the hypothesis that the extra Ca2+ comes from TnC are the results obtained from altering [Ca2+]o as shown in Fig. 4. Changing [Ca2+]o from 2 mM to either lower or higher concentrations results in corresponding decreases or increases, respectively, in the amplitudes of both the Ca2+ transients and twitch forces. The larger twitches were associated with more attached cross bridges, and this led to stronger cooperativity between myosin binding to actin and Ca2+ binding to TnC. In contrast to the BDM effect, which decreases the number of attached cross bridges without altering the amount of Ca2+ bound to TnC, the inotropic effect of altering [Ca2+]o is because of changing the amount of Ca2+ bound to TnC, which in turn influences the number of attached cross bridges. Increasing [Ca2+]o produced larger twitch forces and more prominent bumps, whereas decreasing [Ca2+]o produced opposite effects. This result also supports our hypothesis that the larger twitches liberated more Ca2+ into the myoplasm, thus causing the progressively more pronounced bumps. This hypothesis is further supported by the near-linear correlation shown in Fig. 5, obtained from all of the data from the varying [Ca2+]o and BDM experiments, between area under the bump and peak twitch force.

The amplitude of the bump should be proportional to the rate of the relaxation because the appearance of a bump is affected by both the rate of the Ca2+ released from TnC into the myoplasm and the rate of Ca2+ removal from the myoplasm. The prominence of a bump should be associated with a difference between these two rates. If the extra Ca2+ is released from TnC during relaxation, and reverse cooperativity between detaching cross bridges and Ca2+ affinity of TnC exists, then the rate of cross-bridge detachment and the rate of Ca2+ dissociation from TnC should be linked. An increase in relaxation rate should lead to a more synchronized Ca2+ dissociation from TnC resulting in a more prominent bump. This can be seen in Fig. 2, which shows three different trabeculae each with a different relaxation rate. It seems apparent from Fig. 2 that the faster the relaxation, the more prominent is the bump. An increase in the rate of cross-bridge detachment results in an increase in the rate of Ca2+ liberation from TnC into the myoplasm, and this should lead to a more prominent bump, assuming the rate of Ca2+ removal from the myoplasm is unchanged. Using an applied ramp greatly speeds up the relaxation rate, which should not change the rate of Ca2+ removal from the myoplasm, leading to a bump with much larger amplitude and smaller width compared with that without ramp (Fig. 7, A and C). This indicates that the rate of relaxation is an important factor influencing the prominence of the bump. This may also explain the effect of temperature on the prominence of the bump, since at 30°C, the difference between the release and uptake rates of Ca2+ into and from the myoplasm may be maximal.

In our work, the bump always occurs during the relaxation phase of a twitch contraction, not after force returns to baseline to produce an after-contraction as a result of secondary Ca2+ release from the SR (22). The earlier relaxation begins, the earlier the bump occurs, as shown in Fig. 2. Imposed muscle shortening, as shown in Figs. 6 and 7, leads to an advance in time of the relaxation phase, and this is accompanied by a time advance in the onset of the bump. This clearly contrasts with the twitch without ramp and its corresponding bump, as shown in Fig. 7. As shown in Fig. 7C, the areas under the difference curves of the bump with ramp and the bump without ramp are nearly identical, even though the two bumps are much different in shape and time course. This indicates that the imposed ramp makes the bump peak earlier as a result of synchronized Ca2+ release from TnC, whereas the total amount of extra Ca2+ released is about the same as in the bump without ramp. In addition, there is no sign of a second bump in the bump with ramp occurring at the same time as in the bump without ramp. If the bump without ramp is because of Ca2+ not from TnC, then in the bump with ramp there should be a corresponding secondary bump appearing after the ramp-induced bump. This strongly argues in favor of the idea that bumps are produced by Ca2+ released from TnC during relaxation, and not by Ca2+ that comes from the SR or elsewhere.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of General Medical Sciences Grant GM-48078 (to F. J. Julian).

    FOOTNOTES

Address for reprint requests: F. J. Julian, Dept. of Anesthesia Research Laboratories, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.

Received 10 September 1997; accepted in final form 30 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Allen, D. G., and J. C. Kentish. The cellular basis of the length-tension relation in cardiac muscle. J. Mol. Cell. Cardiol. 17: 821-840, 1985[Medline].

2.   Allen, D. G., and S. Kurihara. The effects of muscle length on intracellular calcium transients in mammalian cardiac muscle. J. Physiol. (Lond.) 327: 79-94, 1982[Abstract].

3.   Backx, P. H., W. Gao, M. D. Azan-Backx, and E. Marban. Mechanism of force inhibition by 2,3-butanedione monoxime in rat cardiac muscle: roles of [Ca2+]i and cross-bridge kinetics. J. Physiol. (Lond.) 476: 487-500, 1994[Abstract].

4.   Backx, P. H., and H. E. D. J. ter Keurs. Fluorescent properties of rat cardiac trabeculae microinjected with fura 2 salt. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H1098-H1010, 1993[Abstract/Free Full Text].

5.   Cannell, M. B. Effect of tetanus duration on the free calcium during the relaxation of frog skeletal muscle fibres. J. Physiol. (Lond.) 376: 203-218, 1986[Abstract].

6.   De Tombe, P. P., and H. E. D. J. ter Keurs. Force and velocity of sarcomere shortening in trabeculae from rat heart: effects of temperature. Circ. Res. 66: 1239-1254, 1990[Abstract].

7.   Dobrunz, L. E., P. H. Backx, and D. T. Yue. Steady-state [Ca2+]i-force relationship in twitching cardiac muscle: direct evidence for modulation by isoproterenol and EMD 53998. Biophys. J. 69: 189-201, 1995[Abstract].

8.   Edman, K. A. P., and F. W. Flitney. Laser diffraction studies of sarcomere dynamics during isometric relaxation in isolated muscle fibres of the frog. J. Physiol. (Lond.) 329: 1-20, 1982[Medline].

9.   Gao, W. D., P. H. Backx, M. Azan-Backx, and E. Marban. Myofilament Ca2+ sensitivity in intact versus skinned rat ventricular muscle. Circ. Res. 74: 408-415, 1994[Abstract].

10.   Gillis, J. M. Relaxation of vertebrate skeletal muscle. A synthesis of the biochemical and physiological approaches. Biochim. Biophys. Acta 811: 97-145, 1985[Medline].

11.   Guth, K., and J. D. Potter. Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers. J. Biol. Chem. 262: 13627-13653, 1987[Abstract/Free Full Text].

12.   Gwathmey, J. K., R. J. Hajjar, and R. J. Solaro. Contractile deactivation and uncoupling of cross bridges. Circ. Res. 69: 1280-1292, 1991[Abstract].

13.   Hannon, J. D., D. A. Martyn, and A. M. Gordon. Effects of cycling and rigor cross-bridges on the conformation of cardiac troponin C. Circ. Res. 71: 984-991, 1992[Abstract].

14.   Huxley, A. F., and R. M. Simmons. Mechanical transients and the origin of muscular force. In: Cold Spring Harbor Symposia on Quantitative Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1972, p. 669-680.

15.   Jiang, Y., J. D. Johnson, and J. A. Rall. Parvalbumin relaxes frog skeletal muscle when the sarcoplasmic reticulum Ca-ATPase is inhibited. Am. J. Physiol. 270 (Cell Physiol. 39): C411-C417, 1996[Abstract/Free Full Text].

16.   Jiang, Y., and F. J. Julian. Pacing rate, halothane, and 2,3-butanedione monoxime affect fura 2 reporting of [Ca2+]i in intact rat trabeculae. Am. J. Physiol. 273 (Cell Physiol. 42): C2046-C2056, 1997[Abstract/Free Full Text].

17.   Julian, F. J., and D. L. Morgan. Intersarcomere dynamics during fixed-end tetanic contractions of frog muscle fibres. J. Physiol. (Lond.) 293: 365-378, 1979[Abstract].

18.   Julian, F. J., D. L. Morgan, R. L. Moss, M. Gonzalez, and P. Dwivedi. Myocyte growth without physiological impairment in gradually induced rat cardiac hypertrophy. Circ. Res. 49: 1300-1310, 1981[Abstract].

19.   Kurihara, S., and K. Komukai. Tension-dependent changes of the intracellular Ca2+ transients in ferret ventricular muscles. J. Physiol. (Lond.) 489: 617-625, 1995[Abstract].

20.   Li, T., N. Sperelakis, R. E. Teneick, and R. J. Solaro. Effects of diacetyl monoxime on cardiac excitation-contraction coupling. J. Pharmacol. Exp. Ther. 232: 688-695, 1985[Abstract].

21.   Morgan, D. L., D. R. Claflin, and F. J. Julian. The relationship between tension and slowly varying intracellular calcium concentration in intact frog skeletal muscle. J. Physiol. (Lond.) 500: 177-192, 1997[Abstract].

22.   Mulder, B. J. M., P. P. de Tombe, and H. E. D. J. ter Keurs. Spontaneous and propagated contractions in rat cardiac trabeculae. J. Gen. Physiol. 93: 943-961, 1989[Abstract].

23.   Patterson, M. F., Y. Jiang, and F. J. Julian. The slowing of the rate of decrease of the intracellular fura 2 ratio (R) signal during twitch relaxation in rat trabeculae (Abstract). Biophys. J. 72: A166, 1997.

24.   Patterson, M. F., and F. J. Julian. Measurement of intracellular calcium transients (ICTs) in rat trabeculae with iontophoretically injected fura 2 dye (Abstract). Biophys. J. 70: A54, 1996.

25.   Ridgway, E. B., and A. M. Gordon. Muscle calcium transient. Effect of post-stimulus length changes in single fibers. J. Gen. Physiol. 83: 75-103, 1984[Abstract].

26.   Sun, Y.-B., F. Lou, and K. A. P. Edman. The relationship between the intracellular Ca2+ transient and the isometric twitch force in frog muscle fibres. Exp. Physiol. 81: 711-724, 1996[Abstract].

27.   Weber, A., and J. M. Murray. Molecular control mechanisms in muscle contraction. Physiol. Rev. 53: 612-673, 1973[Free Full Text].


AJP Cell Physiol 274(5):C1273-C1282
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society