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Unloaded shortening increases peak of Ca²⁺ transients but accelerates their decay in rat single cardiac myocytes

¹Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655; ²Institute of Environmental Studies, Graduate School of Frontier Sciences, University of Tokyo, Tokyo 113-0033; ³Department of Physiology, Dental School, Tsurumi University, Yokohama 230-0063; ⁴Cardiovascular Division, Toranomon Hospital, Tokyo 105-0001; ⁵Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, and ⁶Recognition and Formation, Precursory Research for Embryonic Science and Technology, Tsukuba 305-8568; and ⁷Department of Physiology, Teikyo University, Tokyo 173-8605, Japan

Submitted 7 January 2003 ; accepted in final form 17 April 2003

	ABSTRACT

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It is of paramount importance to investigate the relation between the time-dependent change in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (Ca²⁺ transients) and the mechanical activity of isolated single myocytes to understand the regulatory mechanisms of heart function. However, because of technical difficulties in performing mechanical measurements with single myocytes, the simultaneous recording of Ca²⁺ transients and mechanical activity has mainly been performed with multicellular cardiac preparations that give conflicting results concerning Ca²⁺ transients during isometric twitches and during twitches with unloaded shortening. In the present study, we coupled intracellular Ca²⁺ measurement optics with a force measurement system using carbon fibers to examine the relation between Ca²⁺ transients and the mechanical activity of rat single ventricular myocytes over a wide range of load. To minimize the possible load dependence of sarcoplasmic reticulum Ca²⁺ loading, contraction mode was switched at every twitch from unloaded shortening to isometric contraction. During a twitch with unloaded shortening, the Ca²⁺ transients exhibited a higher peak and a higher rate of decay than transients during an isometric twitch. Similarly, when we changed the contraction mode in every pair of twitches, Ca²⁺ transients were dependent only on the mode of contraction. Mechanical uncoupling with 2,3-butanedione monoxime abolished this dependence on the mode of contraction. Our results suggest that Ca²⁺ transients reflect the affinity of troponin C for Ca²⁺, which is influenced by the change in strain on the thin filament but not by the length change per se.

calcium ion transients; load dependence; carbon fibers

The pumping action of the heart depends on rhythmic twitches of cardiac muscle in which both the length and the tension of the component myocytes are changing continuously, raising the possibility that the Ca²⁺ transients and the mechanical activity in each myocyte may affect each other throughout each cardiac cycle. Therefore, investigation of the relation between Ca²⁺ transients and mechanical activity in isolated single myocytes is of paramount importance to understanding the function of the heart. However, because of the technical difficulties in firmly clamping both ends of a myocyte without damaging it, simultaneous recording of Ca²⁺ transients and mechanical activity has been made mainly with multicellular cardiac preparations, with conflicting results (13, 14, 16). The diversity of the results may arise, at least in part, from a nonuniform distribution of Ca²⁺ indicators as well as from the nonuniform mechanical conditions of the component myocytes. Although an attempt was made to evaluate the effect of mechanical loading in single guinea pig myocytes (23), those authors could not find any difference in Ca²⁺ transients, probably because of the limited range in mechanical loading condition applied in that study.

Recently, we developed (24) an experimental method to measure the contractile function of a single cardiac myocyte under a wide range of loading conditions. In this study, by coupling [Ca²⁺]_i measurement optics with this system, we compared the time course of Ca²⁺ transients in isometric twitches and in twitches with unloaded shortening of single cardiac myocytes. We show that the Ca²⁺ transients during a twitch with unloaded shortening show a higher peak and a higher rate of decay than those during an isometric twitch.

	MATERIALS AND METHODS

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Myocyte isolation and loading with indo 1-AM. All studies were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Hearts were removed from adult male Wistar rats (200–300 g) under pentobarbital anesthesia (50 mg/kg), and the aorta was cannulated so as to perfuse the heart consecutively with the following solutions: 1) 1.8 mM Ca²⁺ HEPES-Tyrode solution (in mM: 137 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 0.33 NaH₂PO₄, 5 HEPES, and 5 glucose, pH 7.4 adjusted by NaOH at 37°C) for 5 min; 2) Ca²⁺-free HEPES-Tyrode solution for 4 min; and 3) Ca²⁺-free HEPES-Tyrode solution containing 0.2 mg/ml collagenase (Nitta Zerachin, Osaka, Japan) and 0.04 mg/ml protease (type XIV, Sigma) for 4 min. The left ventricle was then cut into small pieces and gently stirred in 0.2 mM Ca²⁺ HEPES-Tyrode solution for 10 min. After the solution was sieved through a 220-µm nylon mesh to remove undigested ventricular tissues, ventricular myocytes were collected by centrifugation of the cell suspension. To load indo 1-AM, myocytes were resuspended and incubated in 1.8 mM Ca²⁺ HEPES-Tyrode solution containing 5 µM indo 1-AM (Wako, Japan) and 0.02% Pluronic F127 (Sigma) as a dispersing agent for 60 min. Finally, the myocytes were washed to remove excess indo 1 and transferred to the experimental chamber mounted on the stage of an inverted microscope (IMT-2, Olympus, Tokyo, Japan; objective x40, numerical aperture 0.75). All solutions used were kept at 37°C with a thermoelectric device (Thermoplate, TOKAI HIT).

Myocyte mounting. As shown in Fig. 1A, both ends of a rod-shaped myocyte were firmly held with a pair of carbon fibers, each connected to a micromanipulator (24). One fiber was thick and rigid (RF; diameter 30–40 µm, compliance 0.015–0.02 m/N) and served as the mechanical "ground," and the other thin, compliant fiber (CF; diameter 5–7 µm, compliance 5.5–7.5 m/N) was used to record length changes of the preparation. The compliance of the carbon fibers was calibrated with a glass microneedle whose compliance had been determined against an actual weight (25). These fibers were made from a mixture of fine graphite granules (1–5 µm in diameter) and resin oligomer and were shaped into rods by thrusting through a thin hole in a block of sapphire. By this procedure, the graphite granules, rich in charged residues, were lined up along the surface of the carbon fiber to increase its surface charge, thus enabling its firm attachment to the myocyte. The myocyte held between the carbon fibers (RF and CF) was stretched to a sarcomere length of 2.1 µm and was field stimulated at 0.5 Hz to produce a series of twitches.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Diagram of the force-length and calcium measurement system for a single cardiac myocyte. A: bright-field image (>550 nm) of the compliant fiber (CF) was projected onto the photodiode array (S3903; Hamamatsu Photonics), and its output was used as feedback signal to the driver circuit of the piezoelectric translator (PT; P-841.40; Physik Instrumente). During the shortening mode, the PT followed the movement of the fiber induced by active contraction (active softening of the system stiffness) to make the load virtually zero. Command signal was generated by a personal computer (PC). RF, rigid fiber. B: intracellular Ca2+ concentration (405 nm to 495 nm) ratio during length change applied to the quiescent cardiac myocyte. Amplitude and velocity of the applied sinusoid were adjusted to those of the actual contraction.

Two different modes of twitch by feedback system. The image of the CF was projected onto a 256-element photodiode array (S3903; Hamamatsu Photonics) to give the myocyte length signal, which was fed, after appropriate amplification, to the feedback circuit driving a piezoelectric translator (PT; P-841.40, Physik Instrumente). The PT was mechanically connected to the CF via a rigid glass rod in such a way as to reduce the displacement of the CF caused by the myocyte. Thus, by changing the loop gain of the feedback system, we could change the effective compliance of the CF. If the gain was large enough, the compliance of the CF was made very close to zero, the myocyte contracted virtually isometrically, and the feedback signal fed to the PT served as the myocyte isometric force signal. On the other hand, even if the gain was set at zero the bending of the CF by myocyte shortening produced a certain load because of the stiffness of the CF. To produce unloaded myocyte shortening, therefore, we applied a command signal generated in the software of a personal computer (LabVIEW 6i; National Instruments) to the feedback system in such a way that the resulting PT movement produced CF movement that followed myocyte shortening without any deflection of the CF. With this softening of the CF, we could make the myocyte shorten under a virtually unloaded condition. In this shortening mode, the myocyte shortened by 15%, corresponding to a decrease in sarcomere length from 2.1 to 1.8 µm.

Recording of Ca²⁺ transients. The basic principle used in the fluorescence measurement system is described elsewhere (21). Light from a high-pressure Hg arc lamp was passed through a band-pass filter (Omega Optical) to obtain excitation light at 338 nm. The two peaks of indo 1 fluorescence emission (405 and 495 nm) were separated by a dichroic mirror and band-pass filter system (W-View optics; Hamamatsu Photonics) and were projected onto a pair of photomultipliers (H5783; Hamamatsu Photonics). The ratio of the photomultiplier output (405 nm-to-495 nm ratio) served as a measure of [Ca²⁺]_i (Fig. 1A). To enhance the signal-to-noise ratio, we applied symmetrical moving averaging (no. of points = 15, corner frequency = 29.5 Hz) to the raw data. One end of the myocyte attached to the CF was also illuminated by light from a halogen arc lamp (high pass filtered >550 nm) so as to project its image onto the photodiode array. To ascertain whether the 405 nm-to-495 nm ratio was influenced by the myocyte movement, sinusoidal length changes similar in amplitude and velocity to those of unloaded myocyte shortening were applied to one end of an unstimulated myocyte. As shown in Fig. 1B, the 405 nm-to- 495 nm ratio remained unchanged by the applied length changes, indicating that it is not affected by the myocyte movement per se.

All experiments were performed at 37°C, and the data were digitized at 1 kHz and recorded by an analog-to-digital converter (Power Lab; ADInstruments) and a personal computer. For comparing the means of two groups, Student's t-test was used.

	RESULTS

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Figure 2A shows typical records of the length and force changes and the Ca²⁺ transients during a series of twitches of a single ventricular myocyte. The two modes of twitch ("isometric" and "unloaded shortening") were switched from one to the other after each twitch. The Ca²⁺ transients during unloaded shortening twitches showed a higher peak amplitude and a higher rate of decay than those during isometric twitches. The peak amplitude of the Ca²⁺ transients (405 nm-to-495 nm ratio) was 1.46 ± 0.18 (mean ± SD; n = 30) during unloaded shortening twitches and 0.90 ± 0.12 (n = 30) during isometric twitches, i.e., they were 1.6 times higher in the former than in the latter (P < 0.01). We did not find a significant difference in diastolic level of the 405 nm-to-495 nm ratio between the two modes of contractions (0.42 ± 0.15 unloaded vs. 0.44 ± 0.14 isometric). Meanwhile, the time constant of decay of the Ca²⁺ transients, estimated by an exponential fit, was 132 ± 53 ms (n = 30) during unloaded shortening twitches and 352 ± 169 ms (n = 30) during isometric twitches, i.e., they were significantly shorter in the former than in the latter (P < 0.05). The averaged two types of Ca²⁺ transients obtained from the same myocyte are compared in Fig. 2B. Although the initial peak of the unloaded shortening Ca²⁺ transients is higher than that of the isometric transients, the two transients cross each other in the early decay period, so that the isometric Ca²⁺ transients are above the unloaded shortening Ca²⁺ transients for the rest of the decay period.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Ca2+ transients during the two modes of contraction. A: from top, change in cell length, developed force, Ca2+ transient (405 nm-to-495 nm ratio), and pacing signal. B: Ca2+ transients averaged for isometric (gray) and shortening (black) contractions are superimposed to show the difference in time course.

In rat ventricular myocytes, the Ca²⁺ for activation of contraction is mostly released from the sarcoplasmic reticulum (SR), which takes up and stores the Ca²⁺ released during the preceding twitch (3). Consequently, the change in either the release from, or uptake by, the SR during the preceding twitch would alter the Ca²⁺ loading of the SR to influence the Ca²⁺ transients during the following twitch. To exclude the possibility that the two different Ca²⁺ transients are associated with a different time course of Ca²⁺ release from the SR depending on the mode of preceding twitch, the mode of twitch was switched after each pair of twitches. If the mode of twitch affects the Ca²⁺ transients during the subsequent twitch, the Ca²⁺ transients in each twitch pair of the same mode should differ significantly from each other. As shown in Fig. 3A, however, we did not find any appreciable change in time course of the Ca²⁺ transients in two successive twitches of the same mode, indicating that the amount of Ca²⁺ release from the SR is constant during the alternative mode-switching protocol used in the present study.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Ca2+ transients under different conditions. A: difference in Ca2+ transients when the contraction mode was switched after each pair of twitches. From top, change in cell length, developed force, Ca2+ transient (405 nm-to-495 nm ratio), and pacing signal. B: Ca2+ transient in the presence of 2,3-butanedione monoxime. Force signal is omitted in B because it was virtually zero.

To examine whether the shortening movement per se affects the time course of Ca²⁺ transients, we also recorded the Ca²⁺ transients in a myocyte in which 5 mM 2,3-butanedione monoxime (BDM) (8) suppressed its mechanical activity. As shown in Fig. 3B, the addition of BDM decreased the peak amplitude and prolonged the time constant of decay of the Ca²⁺ transients in both modes of contraction. However, the overall time courses of Ca²⁺ transients were not significantly influenced by the applied shortening movement similar in time course to that in unloaded shortening twitch [peak amplitude: 0.65 ± 0.04 isometric vs. 0.67 ± 0.06 shortening, n = 6, not significant (NS); time constant: 415 ± 42 ms isometric vs. 402 ± 89 ms shortening, n = 6, NS]. These results were in agreement with the report by Kurihara et al. (15) using ferret ventricular muscle and can be taken to indicate that the strain on the thin filament caused by strong cross-bridge formation plays a significant role in determining the amplitude and time course of the Ca²⁺ transient.

	DISCUSSION

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Mechanical property of a single cardiac myocyte. Physiological investigations of the function of cardiac muscle have been made mostly with multicellular preparations such as those of ventricular trabeculae or papillary muscle because of the technical difficulties in performing experiments with isolated single myocytes, which are much smaller in size than single skeletal muscle fibers. The development of techniques to perform experiments with single myocytes under controlled mechanical conditions has been sought for many years to obtain definite results without the complications arising in multicellular preparations (5, 6, 9, 18, 19). Among these techniques the carbon fiber technique is superior to others in terms of its ease of application and causes minimal damage to the sarcolemma (9), but its high system compliance allowed us to evaluate the mechanical property of myocyte only at low-load conditions. In the previous study (24), we could overcome this problem with a specially developed carbon fiber and feedback enhancement of the system stiffness. In the present study, we added an optical system to record the Ca²⁺ transients of a myocyte simultaneously with its mechanical response.

Effect of load and length change on Ca²⁺ transients. So far, conflicting results have been reported about the effect of load and length change on Ca²⁺ transients. In cat papillary muscle, the Ca²⁺ transient measured with aequorin declines faster during an isometric twitch than in a twitch with unloaded shortening, although their peaks do not differ significantly (13, 16). On the other hand, in rat ventricular trabeculae the Ca²⁺ transients measured with fura 2 show a higher peak in a twitch with shortening than during an isometric twitch, with no difference in their time course (14). To eliminate the influence of possible nonuniformity in distributions of load and Ca²⁺ indicators in these multicellular preparations, White et al. (23) studied the effect of mechanical loading in single guinea pig ventricular myocytes loaded with fura 2 but found no difference in Ca²⁺ transients between isotonic (unloaded) and auxotonic (mechanically loaded) contractions, thus showing a clear contrast to the present results. Although they used a carbon fiber technique similar to that of the present study, attention should be paid to the range of applied load. First, under auxotonic contractions the maximum force was 300 nN in their study (23), significantly different from ours, in which myocytes developed 1 µN under isometric conditions with feedback. Second, in their isotonic mode (unloaded condition) myocytes were laid on the bottom of the experimental chamber and made to contract 7 µm (5% of the resting cell length). Because such unattached cells often adhere to the surface to create external load (6), we tried to minimize the load by lifting up the myocytes with carbon fibers and making the fiber follow the shortening (active softening of the system). As a result, the myocytes shortened 15% of the resting cell length, thus allowing us to study the two extremes in contraction mode (isometric and unloaded). This difference in the range of load could account for the discrepant results between the two studies. In some cases, however, the shortening distance of the cell reached 2 µm under high load. If we could have achieved truly isometric condition, these differences in the time course of Ca²⁺ transient could be more pronounced because the strain on the thin filament is greater under such a condition.

Possible mechanisms. The Ca²⁺ transients in cardiac muscle are determined not only by the Ca²⁺ influx across the sarcolemma and the Ca²⁺ release from the SR but also by the Ca²⁺-buffering action of TnC on the thin filament (4). By adopting protocols changing the mode of contraction after each twitch or after a pair of twitches (Figs. 2A and 3A), we could minimize the influence of load-dependent changes in transsarcolemmal Ca²⁺ flux and/or SR Ca²⁺ loading. Our results can be taken to indicate that the difference in the time course of Ca²⁺ transients between the two modes of twitch may largely result from the change in Ca²⁺ affinity of TnC, which is dependent on the difference in mechanical condition between the two modes of twitch.

It has been suggested that the Ca²⁺ affinity of TnC on the thin filament increases with increasing strain on the thin filament, which is produced either by stretching or by isometric force generation of the myocyte (10). Studies using ⁴⁵Ca²⁺ demonstrated that Ca²⁺ binding to TnC is associated with activation (20) and that the inhibition of cycling cross bridges by vanadate or extensive muscle shortening decreases Ca²⁺ binding in cardiac muscle preparations (12). By this reasoning, the Ca²⁺ affinity of TnC is higher during an isometric twitch than during an unloaded shortening twitch. The resulting increase in amount of Ca²⁺ bound to TnC thus reduces [Ca²⁺]_i and the peak height of isometric Ca²⁺ transients compared with unloaded shortening Ca²⁺ transients (Fig. 2B). On the other hand, the slower decay rate of isometric Ca²⁺ transients compared with that of unloaded shortening Ca²⁺ transients may be explained as being due to a slower Ca²⁺ detachment from TnC after an isometric twitch.

However, we, of course, cannot exclude other mechanisms responsible for the load dependence of Ca²⁺ transients. It has been shown that acute changes in Ca²⁺ current (trigger Ca²⁺) can induce a drastic change in fractional SR Ca²⁺ release (2). Thus acute changes in loading condition have the potential to change SR Ca²⁺ release by stimulating the stretch-activated channel current (17). Also, the direct effect of loading condition on SR Ca²⁺ release and/or uptake should be considered. To investigate the relative contributions of these factors, further studies are required.

In summary, we have successfully measured Ca²⁺ transients and mechanical response simultaneously in single cardiac myocytes over a wide range of loading conditions to show clear load dependence of Ca²⁺ transients at the cellular level. Further studies are needed to fill the gap between the present results and those reported in multicellular preparations.

	DISCLOSURES

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This work was supported by grants from the Vehicle Racing Commemorative Foundation, the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, and the Ministry of Education, Culture, Sports, and Technology of Japan (13670692).

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. Sugiura, Inst. of Environmental Studies, Graduate School of Frontier Sciences, Univ. of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: sugiura{at}k.u-tokyo.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

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