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Characteristics of Sarcomere Shortening in Single Frog Atrial Cardiac Cells during Lightly Loaded Contractions

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Characteristics of Sarcomere Shortening in

Single Frog Atrial Cardiac Cells during

Lightly Loaded Contractions

MERRILL TARR, JOHN W. TRANK, AND PAUL LEIFFER

SUMMARY We studied sarcomere performance in single isolated intact cardiac cells using techniques that allow direct measurement of sarcomere length and force. This investigation dealt primarily with sarcomere performance during twitch contractions under lightly loaded conditions. In such contrac-tions, there was a significant portion of the contraction in which sarcomere shortening occurred at constant velocity over a significant range of sarcomere lengths. The constant velocity phase of shortening was followed by a phase of shortening in which sarcomere velocity decreased markedly. Both the velocity and extent of sarcomere shortening depended on the stimulus parameters used to excite the cell. With threshold stimulation, sarcomere velocities during the constant velocity phase of shortening ranged from 1 to 5.5 /im/sec in different cells and significant slowing of sarcomere shortening began at sarcomere lengths of 1.8-2.0 /im. In contrast, when cells were stimulated with a long duration stimulus (200 msec) of large current strength, sarcomere velocities during the constant velocity phase ranged from 6 to 12 /tm/sec, and significant slowing did not occur until a sarcomere length of about 1.6 lira was reached. The threshold stimulus strength-stimulus duration relationship was determined on the single cell, and it was found to be of the type expected for a cell having an intact excitable membrane capable of generating an action potential when depolarized to a fixed voltage threshold. The data presented in this paper give direct evidence that the lightly loaded cardiac sarcomere has a velocity of shortening which depends on the level of contractile activation but is independent of sarcomere length at sarcomere lengths greater than about 1.6 pm. Circ Res 48: 189-200, 1981

THERE have been few direct determinations of the relationships between sarcomere length and devel-oped force in cardiac muscle. Elegant experiments have been performed to determine these relation-ships in single cardiac cells in which the cell mem-brane has been removed, and these experiments on skinned cardiac cells have given information about the relationships between sarcomere length and

From the Department of Physiology, University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kansas.

Supported by U.S. Public Health Service Grant HL 18943 and a Kansas Heart Association Fellowship to Paul Leiffer.

Address for reprints: Dr. Merrill Tarr, Department of Physiology, University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kansas 66103.

Original manuscript received October 26, 1979; accepted for publica-tion September 9, 1980.

developed force at various levels of contractile ac-tivation (Fabiato and Fabiato, 1976,1978). However it is difficult to make an extrapolation from these data to predict quantitatively sarcomere perform-ance during a twitch contraction in the intact tissue. Attempts to measure sarcomere performance di-rectly during twitch contractions in intact tissue have been limited due to the general difficulty of directly measuring sarcomere lengths in intact liv-ing cardiac tissue. Recently, the laser diffraction technique has been applied to very thin bundles of intact cardiac tissue and it as been possible with this technique to measure directly the performance of a large group of sarcomeres during twitch con-tractions (Nassar et al., 1974; Krueger and Pollack, 1975). However, interpretation of data derived from

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the intact tissue suffers from uncertainty about the distribution of forces within the tissue (Manring et al., 1977).

Ideally, one would like to determine sarcomere performance in a very simple cardiac preparation in which the sarcomere length can be measured directly and in which the distribution of forces is relatively well defined. Single cardiac cells perhaps can be used for this purpose and attempts at such investigations presently are being made by our-selves and other investigators (Tarr et al., 1979; Brady et al., 1979).

The present paper presents results of an investi-gation of sarcomere shortening in single frog atrial cells during twitch contractions in which the sar-comeres within the cell developed relatively small forces (i.e., lightly loaded contractions). Sarcomere performance at light loads is of interest for several reasons. First, there are at present few data avail-able in which the time course of sarcomere shorten-ing in cardiac muscle has been measured directly under these conditions, and the data available relate only to rat ventricular tissue (Pollack and Krueger, 1976; Krueger and Wittenberg, 1978). The majority of data, on a variety of cardiac tissues, relates velocity of tissue shortening to tissue length (Brut-saert et al., 1971; Henderson and Brut(Brut-saert, 1974). The interpretation of these data with regards to the sarcomere velocity-sarcomere length relationship suffers from inhomogeneities in sarcomere lengths which exist in the tissue, as well as from the lack of any definitive relationship between changes in over-all tissue length and changes in sarcomere length within the intact tissue (Winegrad, 1974; Gay and Johnson, 1967; Nassar et al., 1974; Pollack and Hunstman, 1974; Krueger and Pollack, 1975; Julian and Sollins, 1975). Second, the sarcomere velocity approaches the so-called unloaded or maximum velocity (Vmax) as the external load approaches zero

provided internal loads are negligible. There has been considerable controversy and interest as to whether or not Vmax depends on sarcomere length

(Sonnenblick, 1962; Pollack, 1970). Third, there appears to be considerable difference between the length dependence of shortening velocity in cardiac muscle compared to that in skeletal muscle. Studies that have been done in skeletal muscle demonstrate that the lightly loaded skeletal muscle sarcomere is capable of shortening at constant and high velocity down to sarcomere lengths as short as 1.6-1.7 fim (Gordon et al., 1966; Costantin and Taylor, 1973). In contrast, the data presently available on sarco-mere or tissue shortening in intact cardiac tissue indicate that the velocity of shortening decreases markedly at sarcomere lengths below 1.9-2.0 /urn (Brutsaert et al., 1971; Henderson and Brutsaert, 1974; Pollack and Krueger, 1976).

The results presented in this paper demonstrate

that during lightly loaded twitch contractions the sarcomeres in the single cardiac cell shorten at constant velocity over a significant range of

sarco-mere lengths. Furthermore, the data demonstrate that under some conditions the cardiac sarcomere shortens at constant and high velocity down to sarcomere lengths as short as 1.6 /nm before any significant slowing occurs. Both the velocity and extent of sarcomere shortening were dependent on the stimulus parameters used to excite the cell, a finding which indicates that the level of contractile activation in the single cardiac cell can be altered by the stimulus conditions. Data are also presented that demonstrate that the isolated cardiac cell has a stimulus strength-stimulus duration relationship similar to the type expected for a cell having an intact excitable membrane capable of generating an action potential dependent on the fast inward so-dium current.

Methods

Preparation of Isolated Frog Atrial Cells Isolated single frog atrial cardiac cells generally were prepared by trypsin-collagenase dispersion of intact frog (Rana catesbeiana) atrial tissue, as de-scribed previously (Tarr and Trank, 1976). How-ever, recently we have modified the cell isolation procedure in the following ways. First, trypsin (0.5 mg/ml) alone is adequate to disperse the cells from the tissue. Second, the cells are harvested without centrifugation merely by removing the suspension of dispersed cells from the remaining undigested tissue by means of a pipette. A few drops of the suspended cells then are placed directly into a cul-ture dish containing 2.5 ml of Ringer's solution having the following composition: NaCl =111 mM, KC1 = 5.4 mM, CaCl2 = 1.8 mM, 10 mM

Tris(Hydroxymethyl)aminomethane, and glucose = 4 mM. The pH of the Ringer solution was adjusted to 7.3 at 25°C by adding 12.4 N HC1.

Preparation and Calibration of Cantilever Force Beams

The methods for preparation and calibration of the cantilevered glass force beams have been pre-sented previously (Tarr et al., 1979).

Determination of Sarcomere Length and Developed Force

A Reichert Bio vert 111 microscope was used to view the cell, the sarcomere pattern within the cell, and the position of the force beam to which the cell was attached (see below). To obtain relatively high magnification, a Zeiss 40 x long-working distance (1.5 mm) objective having a numerical aperature of 0.6 was used; this objective is equivalent to a 62.5 X objective when placed on the Biovert microscope, since the Biovert has a 250-mm tube length rather than the conventional 160-mm tube length. The microscope condenser also had a numerical apera-ture of 0.6 and it had a working distance of about 20 mm. A beam splitter was placed in the optical path to allow simultaneous viewing by two TV

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cameras. Each TV camera was focused on the real image produced by the microscope objective; lenses of different focal lengths were used to provide dif-ferent magnifications. The total length resolution was limited by the microscope objective to approx-imately 0.5 /im.

To measure the force developed by the cell dur-ing a twitch contraction, the cell was attached to a poly -L -lysine- coated cantilevered force beam by simply touching the force beam to the cell surface near one end of the cell; the end of the cell was not wrapped around the beam as was the case in our previous work (Tarr et al., 1979). The other end of the cell then was sucked into the end of a fluid-filled pipette under controlled vacuum. The suction pipette was filled with Ringer's solution containing bovine albumin (1 mg/ml). The albumin reduced the tendency for the cell to stick to the inside surface of the pipette and thereby reduced the drag forces on the cell when the cell moved through the pipette opening during a contraction. This fluid-filled pipette also served as a stimulating electrode. Figure 1 gives an example of a typical TV image of the preparation obtained from the TV micro-scope system. The upper half of the TV image displays the edge of the force beam to which the cell was attached and the sarcomere pattern as viewed with one TV camera. The lower half of the TV image displays the force beam, the cell, and the suction pipette as viewed by the other TV camera. An adjustable electronic reticle composed of verti-cal lines was added to the composite video to pro-vide a direct and continuous dimensional reference for the TV display; the reticle line spacing was calibrated by comparison with the spacing of a stage

FIGURE 1 Monitor display obtained from the closed

circuit TV-microscope system. The upper part of the TV screen shows the sarcomere pattern and force beam at the magnification used for the determination of the sarcomere length and developed force; the force beam is barely visible at the far left edge of the picture. The lower part of the TV screen shows the force beam, cell and stimulus pipette at low magnification. The vertical reti-cle spacings are 5 fim (upper) and 22 fim (lower).

micrometer displayed through the TV microscope system.

A permanent retrievable store of the TV display was recorded on a video tape recorder (Sony AV 3600). Synchronized stroboscopic illumination was used to "freeze" the motion of the sarcomeres and force beam at time intervals of 16.67 msec. Each TV frame was identified uniquely by a six-digit real-time clock and a two-digit frame count. Electrical stimulation of the cell was provided by passing constant current between the suction pipette (an-ode) and a Ag/AgCl electrode (cath(an-ode) placed in the bathing medium. The stimulus was applied at the rate of 0.2 Hz, and the application of the stim-ulus to the cell was identified by means of a video display stimulus marker which generated a single vertical line at the left margin of the TV display. The onset and termination of this mark was coin-cident with the onset and termination of the stim-ulus. Both the stimulus marker and the digital frame identification were recorded on the video tape, along with the image of the preparation, and these allowed precise analysis of the timing of the cells mechanical events with respect to the electri-cal stimulus.

Analysis of the video-taped data was done using the stop frame capability (pause mode) of the video tape recorder in combination with a double TV cursor (Tarr et al., 1979). For sarcomere length determinations, the length occupied by 10 sarco-meres (upper half of TV display) was measured with the TV cursors and an average sarcomere length was calculated. The total length resolution of the microscope optics (0.5 fim) limits the preci-sion of an average sarcomere dimenpreci-sion determined from a group of 10 sarcomeres to about 0.05 /j.m (0.5 /mi/10). Applying the 0.5-jum length resolution to the measurement of the displacement of the force beam gives a force resolution on the order of 2.5 nN for a force beam having a compliance of 0.2 /im/nN, and 1.0 nN for a force beam having a compliance of 0.5 jum/nN. The position of the force beam prior to sucking the cell into the suction pipette was taken as the zero force position. The force in the cell at any given time during a twitch contraction (i.e., developed force) was determined from the displace-ment of the force beam relative to its position prior to the initiation of the contraction. The total force in the cell was the sum of the resting force plus developed force.

In some experiments the performance of a cell segment containing many sarcomeres (e.g., 30-60) was investigated by measuring the distance be-tween the point of cell attachment to the force beam and a prominent marker (e.g., cell nucleus) on the cell surface. The average sarcomere length within the cell segment at any time during the contraction was computed as the ratio of the seg-ment length to the number of sarcomeres in the segment. The number of sarcomeres in the cell segment was assumed to be equal to the ratio of the

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resting cell segment length to the resting average sarcomere length computed from a group of 10 sarcomeres.

In some experiments, a second noncompliant poly-L-lysine glass beam was attached to the cell surface by simply touching the beam to the cell at a point close to the opening of the stimulus pipette. The cell then was stimulated and allowed to develop force and shorten in an auxotonic fashion. The forces devloped by the cell during auxotonic twitch contractions then were compared to those devel-oped by the same cell during the lightly loaded twitch contraction under identical conditions of stimulus strength and stimulus duration. In this manner it was possible to determine if the forces developed by the cell during the lightly loaded contraction were relatively small compared to the force-generating capability of the cells.

Results

The Lightly Loaded Condition

The lightly loaded condition was accomplished by attaching one end of the cell to a flexible canti-levered force beam and drawing the other end of the cell through the opening of a fluid-filled pipette with controlled suction. Under these conditions the initial force on the cell was the resting force required to set the initial sarcomere length, and the forces developed by the cell during contraction were those forces developed by the cell as the unattached end of the cell moved through the opening of the suction pipette during the contraction. Since the unat-tached end of the cell was relatively free to move during the contraction, the forces developed by the cell during these contractions were relatively small. To determine whether these forces were indeed small with respect to the force-generating capability

of the cell, the forces developed by a given cell during auxotonic twitch contractions were com-pared to those developed during lightly loaded twitch contractions under identical conditions of stimulus strength and stimulus duration. It was not possible to determine auxotonic forces on all cells that were investigated under lightly loaded condi-tions. In some cases the cell either did not survive the attachment of the second beam, or it developed sufficient force during the auxotonic contraction to pull free of its attachment to one of the beams. It was possible, however, to obtain partial data on nine different cells under a variety of conditions of initial sarcomere length and stimulus conditions. The data obtained from these cells are summarized in Table 1. It is apparent that the peak total force developed by the cells during the lightly loaded contractions (PFL) was variable and increased as the initial sarcomere length increased. The peak total force developed by the cell during auxotonic twitch contractions (PFA) also was variable and depended on the stimulus parameters used to excite the cell. The ratio PFLrPFA was variable and ranged from a low of 0.03 to a high of 0.27. However, when the initial sarcomere length was 2.6 jum or less (i.e., when the resting force on the cell was small) and the cell was stimulated with a long duration stimulus (100-200 msec) of large amplitude (0.7 /XA or greater), the ratio of PFL:PFA was always less than 0.10. Thus, under these conditions it seems reasonable to conclude that the forces developed by the cell under the so-called lightly loaded conditions were probably less than 10% of the force-generating capabilities of the sarcomeres. The so-called lightly loaded contractions represent conditions where the external load on the cell during the contraction was approximately that of the preload required to set the initial sarcomere length.

TABLE 1 Resting Forces and Peak Total Forces during Lightly Loaded and

Auxotonic Twitch Contractions

Experiment no. 1 3-1-79 2 4-16-79 2 4-17-79 1 4-24-79 1 5-21-79 2 5-22-79 1 5-31-79 2 6-1-79 4 6-1-79 I (MA) 0.2 0.7 1.0 0.7 0.7 0.4 1.0 0.7 0.7 1.0 1.0 0.9 0.9 0.8 0.8 0.8 0.8 D (msec) 100 100 100 100 20 50 100 10 100 10 200 10 200 10 100 10 100 SL ((im) 2.5 2.5 2.5 2.7 2.6 2.6 2.6 2.5 2.5 2.2 2.2 2.8 2.8 2.8 2.8 2.4 2.4 RF (nNl 0.9 0.9 0.9 21.6 13.3 6.9 6.9 3.1 3.1 2.5 2.5 7.0 7.0 11.1 11.1 2.2 2.2 PFL (nN| 4.4 4.6 5.6 32.2 20.2 10.2 9.4 8.8 8.3 3.4 6.4 18.6 16.2 19.4 21.6 6.4 6.7 PFA (n,\) 55.9 75.5 98.0 127.1 120.5 73.9 186.4 78.6 94.1 106.4 160.0 132.0 196.4 70.9 129.4 116.4 176.4 PFL:PFA 0.08 0.06 0.06 0.25 0.17 0.14 0.05 0.11 0.09 0.03 0.04 0.14 0.08 0.27 0.17 0.05 0.04 I = stimulus current; D = stimulus duration; SL = initial sarcomere length; RF = resting force; PFL = peak total force (resting + developed) during lightly loaded contraction; PFA = peak total force during auxotonic contraction..

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Characteristics of the Lightly Loaded Contraction

Typical examples of the time course of sarcomere shortening and the forces developed (change in force from resting force) during lightly loaded twitch contractions in one cell (Experiment no. 4 6-1-79, Table 1) are shown in Figure 2. Contractions elicited by constant current stimulation (0.8 /iA) at two different stimulus durations (10 and 100 msec) are shown. This figure demonstrates the following consistent findings concerning such lightly loaded contractions. First, there is a considerable portion of the contraction during which the sarcomeres shorten at constant velocity (within the measure-ment error of ±0.05 fim). Second, the velocity of sarcomere shortening during the constant velocity phase depends markedly on the stimulus param-eters used to excite the cell. In this example, the constant velocity for the 10-msec stimulus duration was 3.1 fim/sec compared to a velocity 9.7 jitm/sec for the 100-msec stimulus duration. Third, the con-stant velocity phase of shortening is followed by a phase of shortening in which sarcomere velocity decreases markedly. As will be demonstrated in other examples, the sarcomere length at which this decrease in velocity occurs also depends on the stimulus parameters used to excite the cell. Fourth, the force developed by the cell during the sarcomere shortening is not constant. The initial onset of sarcomere shortening is associated with a rapid rise

2.6 1 2 2 18 o 10 msec • 100 msec 250 5 0 0 UJ 4 O a. o 0 250 500 TIME (msec)

FIGURE 2 Sarcomere length vs. time (upper) and

de-veloped force vs. time (lower) during lightly loaded twitch contractions elicited by constant current (0.8 fiA) stimulation at two different stimulus durations (10 msec and 100 msec). Zero time is the onset of the stimulus.

in developed force and the constant velocity phase generally is associated with a decline in or only a slight change in developed force. In the example shown in Figure 2, the peak change in developed force is about 4.4 nN (100-msec stimulus) and the resting force in this cell was 2.2 nN (Table 1). Thus, the total maximum force developed by this cell during this contraction was 6.6 nN, assuming the sarcomeres supported the total external load (rest-ing force + developed force) as the sarcomeres shortened. (At sarcomere lengths below 2.2 ^m, the assumption that the sarcomere supports the total external load is valid, since the resting sarcomere lengths in these single cells at zero external load were generally in the range of 2.1-2.4 jiim.) In com-parison, the total force developed by this cell aux-otonically (PFA) when stimulated with a 100-msec duration stimulus was 176.4 nN. Thus, the ratio of PFL:PFA in this cell was 0.04, and it seems reason-able to conclude that the total force supported by the sarcomeres under the so-called lightly loaded contraction was indeed small in relation to the force-generating capabilities of the sarcomeres. The Effects of Stimulus Parameters

The data presented in Figure 2 demonstrate that the velocity of sarcomere shortening can depend on the stimulus parameters. A detailed investigation of the effects of the magnitude of stimulus current and stimulus duration on the time course of sarcomere shortening under lightly loaded conditions was done on five different cells. Figure 3 gives a typical ex-ample of the effects of sequentially applied stimulus currents of increasing magnitude (stimulus duration of 200 msec) on the sarcomere kinetics and force development during lightly loaded twitch contrac-tions. In this example, several effects of stimulus current are apparent. First, the delay between stim-ulation and the onset of shortening decreased as the magnitude of the stimulus current increased. Second, the velocity of sarcomere shortening during the constant velocity phase of shortening increased with an increase in stimulus current. Third, the sarcomere length at which a subsequent slowing of sarcomere velocity occurs (arrows) depended on the magnitude of the stimulus current. Fourth, the total extent of sarcomere shortening depended on the magnitude of stimulus current. The effects of stim-ulus strength on sarcomere shortening also de-pended on stimulus duration. At short stimulus durations (10-20 msec), the velocity and extent of sarcomere shortening were not affected by the stim-ulus current strength. Similarly, at low current strengths, the velocity and extent of sarcomere shortening were not dependent on stimulus dura-tion.

The effects of stimulus current strength and stim-ulus duration on sarcomere velocity during the con-stant velocity phase of shortening are summarized in Figure 4. With a 10-msec stimulus duration, the

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2 . 8 r pomps

5 0 0

250 500 TIME (msec)

FIGURE 3 Sarcomere length and developed force vs.

time during lightly loaded twitch contractions elicited by three different, stimulus current strengths at a con-stant stimulus duration of 200 msec. Resting force = 9

/IN.

velocity of sarcomere shortening for any given cell was not dependent on stimulus strength, provided the stimulus current was above threshold strength: however, a great deal of cell-to-cell variability oc-curred in the sarcomere velocity ranging from a low of about 1 fim/sec to a high of about 5.5 jum/sec.

With a stimulus duration of 100 msec, the velocity of shortening was more dependent on stimulus strength than with a 10-msec stimulus. For example, in one cell, the velocity of sarcomere shortening increased almost 5-fold (2.0-9.8 /im/sec). In con-trast, in another cell the velocity increased about two-fold from 1.4 to 3.2 fim/sec. With a 200-msec stimulus duration, the velocity of shortening be-came very dependent on stimulus strength, and all cells showed an increased velocity of shortening with an increase in stimulus strength.

Sarcomere Kinetics from Segment Length Measurements

To confirm that the observations with regard to the velocity and extent of sarcomere shortening

made on only 10 sarcomeres were representative of the properties of a significant number of sarcomeres within the cell, the properties of an increased num-ber of sarcomeres were investigated in a few cells where a prominent landmark on the cell surface allowed an accurate measurement of a cell segment length between the landmark and the point of at-tachment of the cell to the force beam (see Meth-ods). The average sarcomere length at any time during the contraction was computed from the ratio of the segment length to the number of sarcomeres in the segment. A typical example obtained from one cell is shown in Figure 5. In this case the cell segment length between the force beam and the landmark on the cell surface contained 31 sarco-meres with an average sarcomere length of 2.59 /im. The results of sarcomere performance computed from the cell segment measurements were similar to those obtained on only 10 sarcomeres. In the example shown in Figure 5, sarcomere shortening had a constant velocity phase, and at large stimulus strengths significant slowing of sarcomere velocity occurred only at sarcomere lengths below about 1.6

jum.

The Slow Velocity Phase of Sarcomere Shortening

In Figure 3 it is apparent that a phase of de-creased sarcomere velocity follows the constant ve-locity phase of sarcomere shortening. The sarco-mere length at which the slow velocity phase begins also depends on stimulus strength. For example, in Figure 3 at a current strength of 0.2 pA, the slow velocity phase of sarcomere shortening begins at a sarcomere length of 1.97 /j.m and at a total force of about 14 nN (resting force = 9 nN). In contrast, at a current strength of 0.9 /xA, the slow velocity phase begins at a sarcomere length of 1.54, also at a total

10 msec 100 msec 200 msec

10 E Q ._ 9 6L 8 2 CURRENT(pamps)

FIGURE 4 Summary of the effects of stimulus current

strength and stimulus duration on the velocity of sar-comere shortening. Data from five different cells are presented.

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2 . 8 r

i m p s

500

250 500 TIME (msec)

FIGURE 5 Sarcomere length and developed force vs.

time as computed from cell segment length determina-tions. Data obtained from three contractions elicited by stimulation with a 200-msec duration stimulus at three different stimulus strengths. Resting force = 38 NN.

force of about 14 nN. The dependence of the onset of the slow velocity phase on stimulus parameters suggests that the onset of the slow velocity phase depends more on the time course of contractile activation than on sarcomere length per se. In other words, the slow velocity phase begins when the level of contractile activation decreases to the point at which the total force supported by the sarco-meres is no longer small with respect to the force-generating capability of the sarcomeres. To test this hypothesis, the sarcomere performance was com-pared for given stimulus strengths and durations when the contractions were initiated from different initial sarcomere lengths and thus had different external loads. (An increase in sarcomere length increased the preload on the cell, thereby increasing the total force supported by the sarcomeres at sarcomere lengths less than about 2.2 /um.) The sarcomere length was increased by decreasing the pressure in the suction pipette and thereby stretch-ing the cell. In performstretch-ing these experiments, it was essential to keep the length of cell in the stimulus pipette constant by repositioning the suction

pi-pette, since we found that the level of contractile activation elicited by long duration stimulus (100-200 msec) could be altered by changing the length of cell in the stimulus pipette.

Figure 6 demonstrates the sarcomere perform-ance for three different stimulus strengths when the initial sarcomere length was 3.04 /tm (resting force = 45 nN). The data presented in this figure were obtained from the same cell, as were the data pre-sented in Figure 3; the data in Figure 6 and 3 are to be compared. In Figure 6, it is apparent the constant velocity phase of sarcomere shortening occurs over a large range of sarcomere length. For example, for the case of a stimulus current of 0.9 JUA, the constant velocity phase spans a sarcomere length range of about 2.9 /mi down to about 1.8 ftm. It is also apparent by comparing Figure 6 to Figure 3 that the onset of the slow velocity phase occurs at an increased sarcomere length and at an increased total load when the contraction begins from an initial sarcomere length of 3.04 fim. For example, for a stimulus of 0.9 juA the slow velocity phase

3.2 r pomps 12 f o r 250 5 0 0

3

CE O U 10 O 2 so 500 TIME (msec)

FIGURE 6 Sarcomere length and developed force vs.

time during lightly loaded contractions beginning from a long initial sarcomere length. Stimulus duration = 200 msec. Resting force = 45 AIN.

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begins at a sarcomere length of 1.8 /*m (total force = 60 nN) for the contraction beginning from the 3.04-jum length compared to 1.5 jum (total force = 14 nN) for the contraction beginning from the 2.61-/xm length. For a stimulus of 0.2 /xA, the slow veloc-ity phase begins at a length of 2.12 /im (total force = 70 nN) for the contraction initiated from the 3.04-jum length compared to 1.97 /im (total force = 14 nN) for the contraction initiated from the 2.61-jum length. These results would be consistent with the hypothesis that the primary determinant of the onset of the slow velocity phase of sarcomere shortening at sarcomere lengths greater than about 1.6 jum is the level of contractile activation relative to the total force supported by the sarcomeres, rather than sarcomere length per se.

The Threshold Stimulus Strength-Duration Relationship

In light of the finding that the velocity of sarco-mere shortening depended on stimulus strength and duration, we were curious as to whether or not the isolated single cardiac cell had a threshold strength-duration relationship similar to what would be ex-pected for an excitable cardiac tissue having an intact membrane. In other words, does the stimulus excite an action potential in these cells? To answer this question, the relationship between the magni-tude of stimulus current and stimulus duration which produced a contractile response having a sharp threshold was determined on 10 single cardiac cells. There was very little variability in the strength-duration relationships of these single cells, and the results obtained from five of the cells are shown in Figure 7. The figure plots the ratio of

6

-I / -I Rh

-16

30

STIMULUS DURATION (msec)

60

FIGURE 7 Ratio of threshold stimulus current (I) to

rheobase current (Im,) as a function of stimulus duration. Data from five representative cells are presented. The solid line describes the Lapicque-Hill equation (see text).

| 50 8 LiJ Of 0 6 | 04 O Control A TTX(80nM) o TTX(l60nM) 0 20 40 60 80 100 STIMULUS DURATION (msec)

FIGURE 8 Effect of tetrodotoxin (TTX) on the threshold

strength-duration relationship of one cell. Stimulus cur-rent is plotted as a function of stimulus duration. TTX was added to the bathing fluid to produce a final TTX concentration as indicated.

stimulus current (I) to rheohase current (IRh) as a

function of the stimulus duration. The solid line describes the Lapicque-Hill equation I/Iith = [1 —

exp (—t/r)]"1 having a time constant (T) of 15 msec.

The Lapicque-Hill equation (Lapicque, 1907; Hill, 1936) has been found to describe reasonably the strength-duration relationship of a number of excit-able tissues.

To determine whether the excitatory process elicited by the stimulus depended on the fast inward sodium current responsible for phase 0 of the car-diac action potential, the effect of tetrodotoxin on the stimulus strength-duration relationship was in-vestigated. Figure 8 gives typical results obtained from one cell. Tetrodotoxin caused a rapid shift in the strength-duration relationship such that it was no longer possible to excite the cell with short duration stimuli (10 msec or less). Also, the thresh-old stimulus current was increased at long duration stimuli by tetrodotoxin.

Discussion

At present, much of our knowledge concerning the effect of sarcomere length on the velocity and extent of sarcomere shortening at light or near zero external loads has been derived from studies on intact tissue in which the velocity of tissue shorten-ing has been related to tissue length. For example, Brutsaert et al. (1971) found in cat papillary muscle that the velocity of muscle shortening either at constant external preload or under so-called zero load clamp conditions decreased markedly at tissue lengths less than 88% of Lmax (Lma, is the tissue

length at maximum isometric tension). Similar re-sults have also been reported for frog cardiac muscle (Henderson and Brutsaert, 1974). These studies along with studies in tetanized cat papillary muscle (Ford and Forman, 1974), in which the tissue length

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dependence of the force-velocity relationship of car-diac muscle was determined under conditions of relatively constant activation, suggest that the un-loaded sarcomere velocity (i.e., Vmax)in cardiac

mus-cle is highly length dependent at sarcomere lengths below about 1.9 jum. The interpretation of these results with regard to the sarcomere velocity-sar-comere length relationship is uncertain, however, since the inference of sarcomere length from overall tissue length is uncertain. For example, Winegrad (1974) has shown that connective tissue within the intact tissue renders the relationship between over-all muscle length and sarcomere length unpredict-able, and many investigators have found significant disproportionate changes in overall tissue length relative to changes in sarcomere length within the tissue (Gay and Johnson, 1967; Nassar et al., 1974; Pollack and Hunstman, 1974; Kruegar and Pollack, 1975; Julian and Sollins, 1975). Furthermore, in the intact tissue, significant nonuniformities of sarco-mere length occur, and it is therefore difficult to define the dependence of various parameters of contractile performance on sarcomere length from tissue length measurements alone.

We know of only two studies in cardiac muscle in which the time course of sarcomere shortening at light or near zero external loads has been measured directly, and these studies have been limited to rat ventricular tissue. Pollack and Krueger (1976) de-termined the time course of sarcomere shortening in thin bundles of rat papillary muscle by using the laser diffraction technique to measure sarcomere length. They found that the velocity of sarcomere shortening under very lightly loaded conditions de-creased markedly at sarcomere lengths below 2.0 (im. For example, the velocity at a sarcomere length of 1.8 jiim was only about 30% of that at a sarcomere length of 2.2 jum. Recently, Krueger and Wittenberg (1978) used the laser diffraction technique to mea-sure sarcomere kinetics during twitch contractions in isolated single rat ventricular cells. In a prelimi-nary report they stated that in unattached cells (i.e., external load of zero) the resting sarcomere length ranged from 1.9 to 2.0 jum and that during the twitch contraction the initial shortening veloc-ity was nearly the maximal velocveloc-ity. However, the relationship between sarcomere velocity and sar-comere length was not given. Thus, the data pres-ently available, in which direct measurments of sarcomere kinetics during lightly loaded contrac-tions have been made, appear to support the con-clusions inferred from tissue velocity-tissue length measurements that the unloaded sarcomere veloc-ity is highly length dependent at sarcomere lengths below 1.9-2.0 jum.

Our results on sarcomere shortening in single frog atrial cells during lightly loaded contractions show many similarities to those obtained from car-diac preparations where the external load was clamped very close to zero. First, the velocities of

shortening are very similar. Henderson and Brut-saert (1974) found the peak velocity in zero load clamped frog ventricular tissue to be in the range of 1-2 initial muscle lengths/sec. If we assume the initial sarcomere length in the intact tissue was 2.3

jam, then an unloaded velocity of shortening of 1-2

initial muscle lengths/sec would give a sarcomere velocity of 2.3-4.6 /nm/sec. These values are in the range of sarcomere velocities which we obtained on the single cell with short duration stimulation (Fig 4). The sarcomere velocities of 10-12 /im/sec re-ported by Pollack and Krueger (1976) and Krueger and Wittenberg (1978) in rat ventricular tissue are similar to the sarcomere velocities of 6-12 jum/sec that we obtained when the single cell was stimu-lated with long duration stimuli of large current strength. Second, similar to other cardiac prepara-tions, significant sarcomere slowing occurred at a sarcomere length of 1.8-2.0 /xm when the single cell was stimulated with a threshold stimulus. Thus, our results concerning sarcomere performance in the intact single cardiac cell are in some respects similar to those deduced from the performance of intact cardiac tissue during lightly loaded contrac-tions. However, there is one difference. The data presented in this paper demonstrate for the first time that, under some conditions, the cardiac sar-comere can shorten at constant and high velocity down to a sarcomere length of about 1.6 ^m before any significant slowing occurs.

The finding of a constant velocity of sarcomere shortening under conditions in which the external forces were relatively small tempts one to conclude that Vmax is independent of sarcomere length and

time over the sarcomere length range in which the constant velocity occurred. However, this conclu-sion might be erroneous. It is possible that time-and length-dependent variations in Vmax, isometric

force (Po), and internal loads could interact in such

a manner as to produce a constant velocity of shortening over a significant range of sarcomere lengths. The constant velocity phase cannot be explained entirely by the changes in external load that occurred during the lightly loaded contrac-tions, since constant velocities of shortening were obtained under conditions of nearly constant exter-nal loads (Fig. 2) and under conditions of increasing load (Figs. 3 and 6), as well as under conditions of decreasing load (Fig. 5). Unfortunately, it is beyond the state of the art to obtain the information re-quired to give a definitive answer as to how the length-independence of the lightly loaded sarco-mere velocity actually occurred, since this would require information as to the length and time de-pendency of Po and internal loads. We must

em-phasize that the finding of a constant sarcomere velocity over a significant range of sarcomere lengths would be compatible with either of two hypotheses: (1) a time- and length-independent Vmax; and (2) a time- and length-dependent Vmax.

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The effect of stimulus parameters on the velocity and extent of sarcomere shortening that we found in the single cardiac cell are similar to what Costan-tin and Taylor (1973) found in voltage-clamped segments of skeletal muscle fibers in which the level of contractile activation was controlled by the mem-brane potential imposed on the segment. These investigators found that both the extent of sarco-mere shortening and the velocity of sarcosarco-mere shortening at a given sarcomere length could be graded by varying the magnitude of the depolarizing step applied to the cell membrane. For example, when a 100-msec depolarizing step of 32 mV was applied to the membrane, sarcomere shortening occurred from an initial length of 2.3 jum down to about 1.9 /urn, and a considerable slowing of sarco-mere velocity began at a sarcosarco-mere length of about 2.1 jtim. In contrast, when the membrane was de-polarized by 55 mV for 100 msec, sarcomere shortening occurred with constant and high velocity from an initial length of 2.1 jum down to about 1.6 (im. Shortening then continued with a markedly decreased velocity on down to a sarcomere length of about 1.3 /im. These results are similar to those we obtained when the single cardiac cell was stim-ulated with 100- to 200-msec current pulses of var-ious amplitudes (Fig. 3).

The finding that the extent of sarcomere shorten-ing durshorten-ing lightly loaded contractions depends on stimulus parameters indicates that the level of con-tractile activation in the single cardiac cell can be altered by the stimulus parameters used to excite the cell. For example, in Figure 3, the sarcomeres shortened to about 1.8 /urn when the cell was stim-ulated with 200-msec current pulse of 0.2 JXA com-pared to a shortening to 1.4 jum when the cell was stimulated with a 200-msec pulse of 0.9 juA. The forces developed by the sarcomeres were similar in both cases, and, therefore, the data demonstrate that the sarcomere was able to develop the same force at a shorter sarcomere length for the 0.9 /xA case than for the 0.2 juA case. Similarly, the peak forces developed during auxotonic twitch contrac-tions depended on the stimulus parameters used to excite the cell (Table 1). Thus, the ability of the sarcomeres in the single cardiac cell to develop force (i.e., P,,) can be altered by the stimulus param-eters used to excite the cell.

The dependence of the velocity of sarcomere shortening during lightly loaded contractions on stimulus parameters strongly suggests, although it does not prove, that Vmax in the single intact cardiac

cell depends on the level of contractile activation (i.e., the intracellular calcium level). A dependence of Vmax on contractile activation is to be expected in light of the following observations. It has been demonstrated that Vmax of glycerinated skeletal

muscle fibers depends on the level of intracellar calcium (Julian, 1971; Wise et al., 1971). Also, Fa-biato and FaFa-biato (1978) recently have computed Vmax from tension and sarcomere oscillations in

partially activated single-skinned dog cardiac cells and found that Vmax was highly sensitive to pCa. It

seems reasonable to conclude that the level of in-tracellular calcium during the twitch contraction in the single intact cardiac cell can be altered by the stimulus parameters used to excite the cell. The stimulus-dependent changes in intracellular cal-cium produce changes in Vmax and P,, which affect

both the velocity and extent of sarcomere shorten-ing.

Exactly how the stimulus parameters alter the intracellular calcium concentration is not clear at present. In this regard it is important to note that the data indicate that the single cell has an intact membrane capable of producing an action potential when electrically stimulated. This conclusion is sup-ported by the following observations. The contrac-tile response behaved in an all-or-none manner to a threshold stimulus, in that there appeared to be a very sharp stimulus threshold for contractile ac-tivation. The velocity and extent of sarcomere shortening were not dependent on the stimulus duration, provided a threshold current strength was used to excite the cell. Also, the velocity and extent of sarcomere shortening were not dependent on the stimulus current magnitude, provided the stimulus was of short duration (<50 msec). Identical con-tractile responses would be expected by stimulating either with suprathreshold currents of short dura-tion or with threshold currents of long duradura-tion if the cell produced an all-or-none action potential in response to these stimuli, since the magnitude and time course of contractile activation would be the same in both cases.

The conclusion that these single cardiac cells produce all-or-none action potentials is supported further by the nature of the threshold stimulus strength-stimulus duration relationship obtained from these single cells. First, the strength-duration relationship of these isolated cells was found to be reasonably described by the Lapicque-Hill equa-tion, an equation that adequately describes the strength-duration relationship of a variety of excit-able tissues under a variety of stimulus conditions. This relationship given by the equation I/LRH = [1 - exp (—t/r)]"1 describes the strength-duration

curve expected from a membrane having an equiv-alent circuit composed of a resistance (R) and ca-pacitance (C) in parallel which produces an action potential whenever the membrane is brought to a fixed voltage threshold. In this equation, T = RC and T equals the membrane time constant when the membrane is polarized uniformly (Noble and Stern, 1966; Fozzard and Schoenberg, 1972). In our exper-iments it is certain that the cell was not polarized uniformly, since the stimulus current entered the cell in the region of the cell in the stimulus pipette and exited the cell in the region between the stim-ulus pipette and the force beam. Nevertheless, the value of T obtained from the strength-duration re-lationship for the single cell was on the order of 15

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msec; a value equal to the membrane time constant of intact frog atrial tissue (Tarr and Sperelakis, 1964; Tarr and Trank, 1971). Second, the strength-duration relationship that we obtained on the single frog atrial cell is remarkably similar to the strength-duration relationship reported by Goto et al. (1971) for bundles of intact frog ventricular tissue. These investigators found that the strength-duration re-lationship of the fast inward sodium current had a chronaxie of 9.8 msec, a value remarkably close to a chronaxie of about 10 msec which would be esti-mated from the strength-duration relationship of the single cell (Fig. 7). Third, tetrodotoxin in a concentration known to block the fast sodium cur-rent in intact frog atrial tissue (Tarr, 1971) produced a shift in the strength-duration relationship of the single cell (Fig. 8), as would be expected if the excitatory response of the single cardiac cell de-pended on the fast inward sodium current. These observations suggest that the single frog atrial cells used in the present investigation had intact and functional excitable membranes which produced an action potential dependent on the fast inward so-dium current when the membrane was depolarized to the threshold potential.

Although the data strongly suggest that the sin-gle cell produced an action potential in response to the stimulus current, we cannot give a definitive answer as to how suprathreshold long-duration stimuli altered the level of intracellular calcium during the twitch contraction, since the relation-ships between applied current, action potential con-figuration, and excitation-contraction coupling are unknown. One possibility is that the stimulus-de-pendent contractile responses were mediated via the effects which the applied depolarizing current had on the plateau phase of the action potential. It is well established that depolarizing currents ap-plied during the plateau of the cardiac action poten-tial enhance contractile activation (Goto and McC. Brooks, 1970). Another possibility is that the stim-ulus current not only excited an action potential but also directly influenced the release of calcium from storage sites either on the cell membrane or from the sarcoplasmic reticulum within the cell. There also have been several reports that high current stimuli and long duration stimuli can elicit catecholamine release in intact tissue, thereby pro-ducing augmentation of the contractile response (Whalen, 1958; Furchgott et al., 1959; Brady et al., 1960). However, these catecholamine-induced ino-tropies developed after a considerable delay and increased with continued stimulation. In the single cell, the change in contractile activation with large stimulus currents was immediate; a change in stim-ulus current altered the contractile response on the first stimulus. Furthermore, preliminary experi-ments have demonstrated that graded contractile responses to long duration stimuli still exist in the presence of DL-propranolol (10~ to 10~ g/ml). Thus, the stimulus-dependent effects seen in the

single cell would not appear to be catecholamine-induced inotropies.

The data presented in this paper demonstrate several important findings regarding the properties of the isolated single cardiac cell. First, the isolated cell has a stimulus strength-stimulus duration re-lationship of the type expected for a cardiac cell having an intact excitable membrane capable of generating an action potential. Second, under lightly loaded conditions the sarcomeres shorten at constant and high velocity over a large range of sarcomere length, and this high velocity of shorten-ing can occur down to sarcomere lengths as short as 1.6 jum. Third, the level of contractile activation of the single cell can be altered by the stimulus parameters used to excite the cell; the level of contractile activation affects both the velocity and extent of sarcomere shortening during lightly loaded contractions.

References

Brady AJ, Abbot BC, Mommaerts WFH (1960) Inotropic effects of trains of impulses applied during the contraction of cardiac-muscle. J Gen Physiol 44: 415-432

Brady AJ, Tan ST, Ricchiuti NV (1979) Contractile force mea-sured in unskinned isolated adult rat heart fibers. Nature 282: 728-729

Brutsaert DL, Claes VA, Sonnenblick EH (1971) Velocity of shortening of unloaded heart muscle and the length-tension relation. Circ Res 29: 63-75

Costantin LL, Taylor SR (1973) Graded activation in frog muscle fibers. J Gen Physiol 61: 424-443

Fabiato A, Fabiato F (1976) Dependence of calcium release, tension generation and restoring forces on sarcomere length in skinned cardiac cells. Eur J Cardiol 4 (suppl): 13-27 Fabiato A, Fabiato F (1978) Myofilament-generated tension

oscillations during partial calcium activation and activation dependence of the sarcomere length-tension relation of skinned cardiac cells. J Gen Physiol 72: 667-699

Ford LE, Forman R (1974) Tetanized cardiac muscle. Ciba Found Symp (new series) 24: 137-150

Fozzard HA, Shoenberg M (1972) Strength-duration curves in cardiac Purkinje fibres: Effects of liminal length and charge distribution. J Physiol (Lond) 226: 593-618

Furchgott RF, de Gubareff T, Grossman A (1959) Release of autonomic mediators in cardiac tissue by suprathreshold stim-ulation. Science 129: 328-329

Gay WA, Johnson EA (1967) An anatomical evaluation of the myocardial length-tension diagram. Circ Res 21: 33-43 Gordon AM, Huxley AF, Julian FJ (1966) The variation in

isometric tension with sarcomere length in vertebrate muscle fibres. J Physiol (Lond) 184: 170-192

Goto M, McC. Brooks C (1970) Positive and negative inotropic action of polarizing current on the frog ventricle. Am J Physiol 218: 1038-1045

Goto M, Kimoto Y, Kato Y (1971) A study on the excitation-contraction coupling of the bullfrog ventricle with voltage clamp technique. Jap J Physiol 21: 159-173

Henderson AH, Brutsaert DL (1974) Force-velocity-length re-lationship in heart muscle: Lack of time-independence during twitch contractions of frog ventricle strips with caffeine. Pflue-gers Arch 348: 59-64

Hill AV (1936) Excitation and accommodation in nerve. Proc R Soc Lond [Biol] 119: 305-355

Julian FJ (1971) The effect of calcium on the force-velocity relation of briefly glycerinated frog muscle fibres. J Physiol (Lond) 218: 117-145

Julian FJ, Sollins MR (1975) Sarcomere length-tension relations in living rat papillary muscle. Circ Res 37: 299-308

Krueger JW, Pollack GH (1975) Myocardial sarcomere dynamics

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during isometric contraction. J Physiol (Lond) 251: 627-643 Krueger JW, Wittenberg BA (1978) Dynamics of myofilament

sliding in single intact cardiac muscle cells (abstr). Circulation 58: (suppl II): 33

Lapicque L (1907) Recherches quantitative sur l'excitation elec-triquer des nerfs traitee comme une polarisation. J Physiol (Paris) 9: 620-635

Manring A, Nassar R, Johnson EA (1977) Light diffraction of cardiac muscle: An analysis of sarcomere shortening and mus-cle tension. J Mol Cell Cardiol 9: 441-459

Nassar R, Manring A, -Johnson EA (1974) Light diffraction of cardiac muscle: Sarcomere motion during contraction. Ciba Found Symp (new series) 24: 57-82

Noble D, Stern RB (1966) The threshold conditions for initiation of action potentials by excitable cells. J Physiol (Lond) 187: 129-162

Pollack GH (1970) Maximum velocity as an index of contractil-ity. Circ Res 26: 111-127

Pollack GH, Hunstman LL (1974) Sarcomere length-active force relations in living mammalian cardiac muscle. Am J Physiol 227: 383-389

Pollack GH, Krueger JW (1976) Sarcomere dynamics in intact

cardiac muscle. Eur J Cardiol 4 (suppl): 53-65

Sonnenblick EH (1962) Force velocity relations in mammalian heart muscle. Am J Physiol 202: 931-939

Tarr M (1971) Two inward currents in frog atrial muscle. J Gen Physiol 58: 523-543

Tarr M, Sperelakis N (1964) Weak electrotonic interaction be-tween contiguous cardiac cells. Am J Physiol 207: 691-700 Tarr M, Trank J (1971) Equivalent circuit of frog atrial tissue as

determined by voltage clamp-unclamp experiments. J Gen Physiol 58: 511-522

Tarr M, Trank JW (1976) Preparation of isolated single cardiac cells from adult frog atrial tissue. Experientia 32: 338-339 Tarr M, Trank JW, Leiffer P, Shepherd N (1979) Sarcomere

length-resting tension relation in single frog atrial cardiac cells. Circ Res 45: 554-559

Whalen WJ (1958) Apparent exception to the "all or none" law in cardiac muscle. Science 127: 468-469

Winegrad S (1974) Resting sarcomere length-tension relation in living frog heart. J Gen Physiol 64: 343-355

Wise RM, Rondinone JF, Briggs FN (1971) Effect of calcium on force-velocity characteristics of glvcerinated skeletal muscle. Am J Physiol 221: 973-979

Evidence That the Velocity of Sarcomere

Shortening in Single Frog Atrial Cardiac

Cells is Load Dependent

MERRILL TARR, JOHN W. TRANK, PAUL LEIFFER, AND NEAL SHEPHERD

SUMMARY Recent experiments using laser diffraction techniques to determine the time course and extent of sarcomere shortening in thin bundles of cardiac tissue have given results which suggest that the velocity of sarcomere shortening in cardiac muscle is independent of the developed force (Nassar et a]., 1974; Krueger and Pollack, 1975). However, the anatomical complexity of the intact tissue precludes a definitive interpretation of the data, since the exact relationship between the force being borne by the total tissue to the force being borne by any observed group of sarcomeres is uncertain. The single frog atrial cell provides a simple cardiac preparation in which the relationship between sarcomere velocity and sarcomere force is well defined, since these cells are only 1-2 myofibrils wide. The purpose of the present investigation was to determine if sarcomere velocity in the single frog atrial cell is dependent on force by measuring the time course of sarcomere shortening in single cells under conditions in which the cell developed markedly different forces. The results presented in this paper give direct evidence that the velocity of sarcomere shortening in the single cardiac cell depends on the force being developed by the sarcomeres. Thus, cardiac sarcomeres have a type of force-velocity relationship, although the exact nature of this relationship could not be determined in these experi-ments. Circ Res 48: 200-206, 1981

IN preliminary experiments on single frog atrial cells we found that the rate of force development during auxotonic twitch contractions was relatively constant for a significant portion of the rising phase

From the Department of Physiology, University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kansas, and Department of Physiology, Duke University Medical Center. Dur-ham, North Carolina.

Supported by U.S. Public Health Service Grant HL 18943. Kansas Heart Association Fellowship to Paul Leiffer, and a North Carolina Heart Association Grant-in-Aid to Dr. Neal Shepherd.

Address for reprints: Dr. Merrill Tarr, Department of Physiology, University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kansas 66103.

Received January 4, 1980; accepted for publication August 7, 1980.

of force development. Since the rate of force devel-opment in these experiments was directly propor-tional to the average sarcomere shortening velocity within the cell, the data indicated that the velocity of sarcomere shortening remained relatively con-stant even though force was increasing dramati-cally. Other investigators have found similar results in intact cardiac tissue (Nassar et al., 1974; Krueger and Pollack, 1975). However, the interpretation of the data derived from intact tissue is complicated by uncertainties with regard to the distribution of forces within the tissue (see Manring et al., 1977); i. e., the relationship between the force being borne

Figure

Figure 1 gives an example of a typical TV image of the preparation obtained from the TV  micro-scope system
TABLE  1 Resting Forces and Peak Total Forces during Lightly Loaded and Auxotonic Twitch Contractions
FIGURE  2 Sarcomere length vs. time (upper) and de- de-veloped force vs. time (lower) during lightly loaded twitch contractions elicited by constant current (0.8 fiA) stimulation at two different stimulus durations (10 msec and 100 msec)
FIGURE  4 Summary of the effects of stimulus current strength and stimulus duration on the velocity of  sar-comere shortening
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References

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