and the Department of Biological Chemistry, University of California-Los Angeles School of Medicine, Los Angeles,. California 90024. Received for publication ...
Pages 367-374 Vol. 3, No. 5, 1983 Printed in U.S.A.
0196-4763/83/0305-0367$00.00/0
CYTOMETRY Copyright 0by the Society for Analytical Cytology
A Video-Commter for the Chronotrotic and Inotropic Miasurements of the Beacing of Cultured Heart Cells' Isaac Harary, Garn Wallace and Greg Bristol The Laboratory of Biomedical and Environmental Sciences and the Molecular Biology Institute, University of California, and the Department of Biological Chemistry, University of California-Los Angeles School of Medicine, Los Angeles, California 90024 Received for publication July 19, 1982; accepted November 12, 1982
A video-computer system has been developed to measure the chronotropy and inotropy of cultured heart cells. The motion of the cell is followed by recording of the changes of light intensity from the edge of a beating cell. With this system the velocity of contraction and relaxation, the time to peak contraction, relaxation time and the displacement of the cell in culture can be measured for the first time. Also, the rate of beating can be measured beyond the limits set by visual counting. With the application of this technique to the cultured heart cell sys-
For the last 20 years cultured embryonic (4) and postnatal (9) heart cells have proved very useful for the investigation of the biochemistry, pharmacology, electrophysiology and cellular biology of the heart (8).The beating rate can be monitored easily in this model system and studies have been conducted on the effects of drugs and hormones (10, 11, 20), on the role of ions (5, 19) and on intermediates such as cyclic AMP (13). Unfortunately, the force of contraction has proved more difficult to measure because of the small size and fragility of the heart cells. However, other parameters associated with inotropy can be measured. Inotropy is a general clinical term which refers to various properties related to useful aspects of the force of contraction. Qualitative estimates have been made of inotropic changes and several attempts to achieve a more quantitative estimation by photoelectric monitoring of single beating cells have been reported (2, 15). Traces of the beating were made with this method and the chronotropic response was measured from changes in the beating rate and the inotropic response from changes in amplitude derived from changes in the intensity of light. The amplitude alone cannot be equated with the force of contraction. More importantly, since the amplitude of contraction is measured by shifts in the
' This work was supported by Department of Energy Contract EY76-C-03-0012. 367
tem we have found that norepinephrine increases the velocity of relaxation thus reducing the ratio of contraction velocity to relaxation velocity, and reduces the twitch time. Increased external calcium, on the other hand, has little effect on either the velocity of contraction or relaxation but, like norepinephrine, decreases the twitch time. Key terms: Video-computer, inotropic and chronotropic measurements, cultured rat heart cells
light from the edge of a moving cell, many factors, such as the surface topography of the cell could contribute to the results and introduce artifacts in the actual amplitude. We have developed a digitized video-microscope computer system in which the velocity and time of contraction and the rate of beating are also measured by recording changes in light intensity from the edge of a beating heart cell. The earlier system estimated amplitude from the change in light intensity alone. In our system the estimation of inotropic changes is made from a direct measurement of the displacement of the cell and the rate of change in light intensity. These parameters are used to calculate the velocity of contraction. In addition, with our system, the velocity of relaxation, the time to peak contraction, the relaxation and the twitch time can be derived. The computer system provides many other advantages. The beating rate can be measured beyond the limits set by visual counting. Statistics can be gathered on the regularity of the beating periods, changing rates and velocity. In addition, the information can be gathered, organized, processed through programmed calculations and stored for future use. The program gives an accurate beating rate in 6 to 10 sec, an estimation of the rate of contraction in 30 sec, and a measure of the displacement of the cell in 20 sec. In this paper we describe the system and its operation and
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demonstrate its usefulness by an examination of the effect of calcium a n d norepinephrine on rat heart cells in culture.
Materials and Methods Cultured heart cells: The cultured heart cells were prepared from newborn rat hearts (10, 12). In all of the experiments reported here the cells were allowed to grow to confluence and the beating was recorded from cells in 4- to 5-day-old cultures. The medium used was CMRL with 5% horse and 5% fetal calf serum. The details of the medium and procedures have been described elsewhere (12). Video-computer system: A model BU-13 Unitron inverted microscope (Unitron Instruments Inc., Plainview, NY) with Zeiss phase objectives and condenser rings (Carl Zeiss Inc., Thornwood, NY) was fitted with a McBain Instruments (Burbank, CA) C-mount lens adapter to which a model WV-1300 Panasonic TV camera was mounted (Fig. 1). The microscope stage was enclosed in plexiglass and its tejmperature was maintained a t 37°C with a resistance heater controlled by a model 74 YSI thermistor controller (Yellow Springs Instrument Co., Yellow Springs, OH). The video signal from the camera passes through a VW-400 Panasonic Monitor which displays the actual image and into a Video-Digitizer board installed in a Vector MX microcomputer (Vector Graphic, Inc., Westlake Village, CA). From the microcomputer the digitized image is passed to and displayed on the graphics monitor. The console monitor displays the commands entered into the keyboard and the data output from the computer. Besides the console keyboard and display the computer’s accessories include a High Resolution Graphics board (Vector Graphic) and its monitor, which can display 16 levels of gray a t 120
I
x 128 resolution or black and white a t 240 x 256 resolution, a Malibu printer with graphic dot plotting (Malibu Electronics Corp., Westlake Village, CA), a North Star Floating Point board (Northstar Computers Inc., Berkeley, CA), and a Q-T calendar clock (QT Computer System, Lawndale, CA). The operating system is based on the Micropolis Disk Operating System (MDOS). It occupies the low end of memory up to 7800 H. Data and picture buffers take up the next 6800 H. The system is controlled through an interactive executive structure which requests commands and parameters from the console. The video-computer system relies on a program which digitizes the light intensity from the edge of a single beating heart cell or from a cell in a confluent sheet of cells. The moving edge fluctuates in light intensity as the light gradient moves back and forth over the scan lines from the camera. Signals from the pixels which exhibit fluctuating light intensity are graphically displayed on a video screen as brightness versus time. In this way the changes in the digital light intensities are translated into measures of motion and the movement is traced in a wave pattern which is related to the change in the position of the edge. Using the video-computer system described we have developed programs to measure the beating rate and velocity of contraction in the cultured heart cell system. Beating rate measurements: The rate program measures the beating rate of the heart cells of a single area. With a locator (Fig. 2A) procedure a suitable area is chosen and changing light intensities for each video frame are noted and collected (Fig. 2B). The changes in brightness in the maximum pixel resulting from cell movement are graphically displayed on a video screen (Fig. 3A). The collection period is divided into 1to 40 segments and upper and lower limits are
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FIG. 1. Diagram of system. The microscope is an inverted Unitron with Zeiss optics. Its stage is enclosed in plexiglass and temperature is regulated with a YSI thermistor controller. The Panisonic camera is mounted on the side with a McBain adapter. The monitor and displays are Panisonic monitors. The Paper Tiger dot-matrix printer can also do graphics. T h e accessories listed under VECTOR MX communicate directly with S-100 bus of the microcomputer and are mounted inside the cabinet. (See text for functions)
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isotonic and isometric contractions should not be significant factors in determining the relative effect on a single cell since the mix should remain the same in the experimental cell although it can vary from cell to cell. Our video-computer system is a noninvasive procedure which deals with the capacity of the cell to move upon contraction. It does not measure force directly but has been programmed to measure several inotropic changes such as the velocity of contraction and relaxation, the contraction, relaxation and total twitch time, and the displacement or distance moved by the cell. Two basic measurements are made: the rate of change in light intensity resulting from cellular movement, and the actual displacement of the cell. From these measurements the other parameters are calculated. The rate of change in light intensity is derived from the variations in light intensity. The amplitude of contraction cannot be derived from the amplitude of the change in the light intensity, since the measurement made is an average of the light intensities in different pixels. Despite the fact that the light intensity changes because of movement of the bright area in and out of the measured pixels, the extent of the displacement of the cell is not the only factor in the light change. The rate of change in intensity must depend upon
FIG. 2. A, camera image digitized. The graphics display board generates 128 x 120 pixels a t 16 gray levels. The dashed box defines the moving edge of a heart cell. The size and position of the box are controlled by the operator. B, quarter-second box scans. This sequence of digitized images is from the box-enclosed cell edge shown in Figure 2A. The pictures are generated as a continuous sequence of columns a t a rate of 60 Hz/sec which accounts for the distortion of the moving shape. Normally this sort of strip display is not used. Instead each image replaces its predecessor in the box, a process which demonstrates the cell movement. set and marked as horizontal lines on the graph. The course of the intensity curve as it crosses above and below these lines triggers the marking of a “beat” as a tic above the trace (Fig. 3B). The beating rate is expressed as an average rate determined by an analysis of periods in which the beating is regular (within an error range). If the error range is relaxed to 100%all measured beats are included in the calculation of the beating rate. Thus, an average statistical or an actual beats-per-minute count can be derived. A record of times and beating rates a t preset intervals can be generated and graphed to demonstrate the change in rate over the whole time period. The effect of isoproterenol on the beating rate is shown in Figure 4. The beating rate was measured every 15 sec and plotted against time. Measurement of the velocity of contraction: The force of contraction in the whole heart or isolated cardiac muscle can be measured directly by following the course of tension development. In an isotonic contraction the force can be estimated by the determination of the velocity of contraction and the magnitude of shortening since these parameters are proportional to the force of contraction. In addition, the time to peak tension, relaxation time and twitch duration can, in some instances, be correlated with the force of contraction (15). In the cell culture system the cells are attached on one side to the culture dish and are free to move only on the unattached surface. The contraction results in a partial shortening and shift of the cytoplasm. Thus, the contraction of the heart cell is an undetermined mixture of isotonic and isometric contraction, and each cell varies. Therefore, to be meaningful all measurements of changes with experimental manipulation must be compared to the basal measurements of that cell. Since the same cell is used for the control and experimental measurement we are relying on a relative value. T h e actual mix of
FIG. 3. A , trace of motion. The light intensity from one point on the camera image is digitized in each frame and plotted on the graphics display in a dot mode in a positive direction. Each beat is represented by a positive peak. This picture covers 4 sec of data. B, recognition and marking of beats. Detail of motion trace with beats marked. Here the peaks are negative and move downwards. The horizontal lines are the computer’s discrimination levels. Only those peaks which cross the line are counted. The vertical tic marks show where a beat is recognized.
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minutes FIG. 4. Beating rate over time. To heart cells with uniform beating rate isoproterenol (final media concentration = M ) is added a t 1.5 min. A graphic record of the beats per min over an 8 min period is generated and printed out. Arrow shows time of addition. both the velocity and extent of displacement. Therefore, the displacement is a necessary value and is measured separately (see section on “Displacement measurement” in this article). We derive the velocity of contraction from the measurement of the maximum displacement o f a cell image in micrometers (D,.~) and the rate of change in light intensity ( I )which results from the movement of the cell.-This change is a function of time ( Z ( t ) ) .From the measurement of Z ( t )we can find the maximal change in light intensity of Z max. The fractional ( f ) change in light intensity with time is
- - - f ( t ) . This is a shape function with values from 0 to 1. The position change of the cell D ( t ) is directly proportional to Z ( t )because the light is recorded from pixels in linear sections of an intensity gradient. Thus D ( t ) and I ( t ) have the same shape:
D ( t )- Z(t)- f ( t ) Dmax Zmax Therefore, the position change of the cell can be determined from Z(t)
this equation D ( t ) = D,,, x f ( t )or D ( t ) = D,,, X-.
I,,,
The derivative
of this function is the velocity of movement of the cell.
dD(t)dZ( t ) / d t V ( t )=-DmaxXdt Zmax The maximum velocity of contraction is thus derived by multiplying the maximum of the cell movement by the maximum rate of change in light intensity resulting from the movement of the cell. D,,, has the dimensions of distance and dz(t”dt has the dimension of l / T . ~
Zmax
Thus V ( t )has the dimensions of distance/time. The measurement of the rate of change in light intensity is based on the analyses of a composite contraction which is the average of a large number of single contractions. It begins like a beating rate measurement by positioning a square. Maximum change pixels tend to be on the cell edges that are moving perpendicularly to the line of the edge where the intensity gradient is steepest (Fig. 2B). The pixels are located and synchronized, trial curves from these pixels are collected (Fig. 5A) and the curves are summed to a master curve with a positive direction (Fig. 5B). Acceptable peaks from the test sequence are averaged to produce a reference peak. All acceptable peaks in the data are positioned so that they have the least absolute differences
from the reference. They are then summed and averaged to produce a representative contraction peak which is displayed as a normalized average curve (NAC) (Fig. 6A). From NAC a new curve of sequential differences which approximates the first derivative can be calculated and displayed on the graphics display. The curve is the rate of change in light intensity (RL) (Fig. 6 B ) .T o derive the actual velocity of contraction we need to have both the rate of change in light intensity and the maximum distance (displacement) moved by the cell. The acceleration curve (ACC) (Pig. 6C) is the second derivative of NAC. The peak values are calculated and displayed. They are used to calculate the various inotropic changes described. Time measurements: The time to peak contraction (TPC) can be most easily seen from the contraction curve which displays a single contraction over a time period measured in camera frames. However, the actual time can be derived most accurately by use of the acceleration and rate curves which are derived from the contraction curve. The first positive peak of the acceleration curve represents the beginning of the rise in the ascending limb of the position curve where the acceleration ip a t a maximum. The negative peak of the acceleration curve represents the peak of contraction. Thus the time between these two peaks represents the time to reach the peak of contraction (Fig. 6 C ) . The time of the peak of contraction can also be derived from the rate curve. The zero point between the positive and negative peaks represents the peak of contraction where the rate of change is zero. This is useful when the negative peak of the acceleration curve is not smooth enough to read the exact time. The relaxation time (RT) can also be calculated from the acceleration or rate curves. The beginning of the descent of the contraction curve corresponds to the negative peak of acceleration or to the zero Doint of the velocitv curve. The end of the contraction corresDonds to the second positive peak of acceleration. Thus the time between the negative peak of the acceleration curve, or the zero point of the velocity curve, and the second positive peak of the acceleration curve, represents the relaxation time. The total twitch time is the sum of the time to peak contraction and the relaxation time. It is equivalent to the time between the first and second positive peaks of the acceleration curve. In summary, NAC (Fig. 6 A ) displays the averaged light curve. RL displays its first derivative and ACC displays its second derivative (Fig. 6C). RL and ACC also list out the local maxima and minima and zero crossings from which the pertinent times can be obtained. At low beating rates the data tends to be obscured by background noise which consists of electronic noise from the camera and Brownian movement. Increase of the signal-to-noise ratio has diminished the contribution of electronic noise permitting significant measurements. Displacement measurement: Because of inherent artifacts in the use of light the actual amplitude of contraction of the cell cannot be derived from the changes in light intensity alone. The height of the averaged peak has three sources of variation unrelated to actual motion: intensity of illumination, focusing of the image, and number of pixels summed. We are not able to control these variables, therefore, we have no way of knowing the actual position change of the part of the cell we observe. Also, different parts of a cell shorten to different extents during a contraction cycle. In order to eliminate this variation, the averaged peak is normalized to a maximum of 200. We trade off loss of information about peak height for standardization of shape. The trace of the average peak is only proportional to distance moved, and therefore its derivative is only proportional to velocity of contraction. If the amplitude of motion of a cell does not change during an experiment, any changes in the derivative curve will reflect actual changes in velocity of contraction. However, changes in amplitude of contraction will not necessarily show up in the derivative curve. The velocity of contraction has therefore been derived from both the rate of change in light intensity and from a determination of the extent of cellular movement. (See section on Measurement of the Velocity of Contraction for derivation of the relationship.) The cell displacement is derived from an independent measurement. A displacement routine ( D )has been developed to collect light values from the square area and to construct two pictures in memory: I
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FIG. 5. A, trial curves from edge pixels. T h e intensities of nine high amplitude pixels along a n edge were measured simultaneously for 4 sec. Note that they are well synchronized but their peaks point down. This data is used to produce the algebraic transformation for each pixel’s signal so that the sum will be a maximum and a peak up (Fig. 5 B ) . The transformation is made by multiplication by a sign and by a baseline subtraction. B, summed signal. Signals from several pixels on a cell edge are recorded in each frame. The digitized values are transformed, added and multiplied by a scale factor to fit the screen height. The result is continuously displayed and stored. Since the collection time is several times 4 sec, old data is wiped from the screen as new data is added. T h e ordinate gives the raw sum values before scaling. a “moved” picture representing the object a t the peak of contraction, and a large “base” picture representing the object and surroundings a t rest. Using a minimum convolution algorithm the routine calculates the magnitude of the vector displacement between the two pictures. The velocity of contraction (CV) is derived from the velocity of change in light intensity multiplied by the distance moved.
cantly. However, the ratio of CV to RV normalized the data and subtracted the wide variation in the basal velocities of the cells thereby reducing the RSD to 9.4. For example, in section B of Table 1 the RSD of CV and RV were 41% and 60% while the ratio of CV/RV was 17%. Given the limitations of the biological system it seems the most reliable parameters Results measured are the twitch time and ratio of velocities, except in Measurement of cultured rat heart cells: In order for those cases where an inotropic change occurs which is larger the measurements described to be useful it is necessary to than approximately 15% of the basal value. The most imporknow the degree of variation in the biological system that was tant factor that emerges from this study is that to be meaningused. We measured the variation of the parameters described ful all measurements must be made, before and after experiin the same beating cell, in different beating cells in the same mental manipulations, on the same cell. Variations from cell dish and in cells in different dishes (Table 1).As can be seen, to cell or from dish to dish are too large to allow meaningful the measurement with the largest variation is the displace- comparative measurements. Effect of calcium on CV and R V and twitch time: ment. In section A, (CV) and relaxation velocity (RV), which are derived from calculations using the D measurement, give There have been mixed reports on the effect of extracellular a relative standard deviation (RSD) of approximately 17%. calcium (Ca,) on the inotropic action in cardiac muscle. Both Thus, only changes above this value will be measured signifi- positive (3, 17, 18) and negative (1) inotropic actions have
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73
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Frames
Frames
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FIG.6. A, normalized average curve (NAC); average of 60 peaks. The abcissa is marked in frames. Each peak recorded is positioned by minimum difference from a reference peak and summed. The sum is scaled to a maximum of 200 and displayed. B, the rate of change in light intensity (RL). This curve is produced from Figure 6A by successive differences. It corresponds to a velocity curve of the change of light intensity. C, second derivative of motion. The curve is produced from Figure 6B by successive differences. It corresponds to an acceleration curve. There is some high frequency noise. Table 1 Variation in Contraction of Cultured Heart Cells
A" % SD
Bb % SD
C' % SD
Beating Rate (B/min)
Displacement
RL'
RL-
cv
RV
Ratio of Velocities
64.9 9.0 61.8 1.9 64.90 5.9
Pm 5.77 11.7 4.38 43.1 5.2 22.7
72.1 5.7 66.5 6.8 67.4 6.8
49.0 10.3 46.4 16.3 47.8 19.1
114.04 16.2 78.98 40.9 95.95 25.1
77.39 17.2 58.32 60. 70.17 42.5
1.48 9.4 1.47 17. 1.44 15.3
Twitch Time (Set)
0. I60 12.7 0.179 16.8 0.181 12.6
All cells measured are from the same primary culture. RL' and RL- are the first derivatives of the NAC curve and express the velocity of change in light intensity during contraction and relaxation. CV, contraction velocity; RV, relaxation velocity; velocity = D X VL X 0.687 p/sec, where D equals distance of cell movement in pixels (1 pixel = 2.52 pm). Average of ten readings on the same cell. Average of readings on ten different cells in the same dish. 'Average of readings on ten different cells in ten different dishes.
been reported. We decided to determine the effect of a low level (0.2 mM) and normal serum level (1.3 mM) of Ca, on the inotropic properties of cultured rat heart cells in order to determine how these cells react. Sixteen separate plates of
confluent cultured cells were used and paired measurements were made on a cell in each plate under 0.2 mM and 1.3 mM Ca, (Table 2). In this experiment the velocity of contraction and relaxation did not change significantly nor did the ratio of
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Table 2 Effect of Calcium on the Znotropic Properties of Rat Heart Cells Displacement
cv
RL'
RL
54.0 60.4 9.9 12.
