High Resolution On-line Analysis of Cardiac Action Potentials Using a Motorola 68000 Microcomputer

ROBERT KING r JOYCE CATLOW r MICHAEL GWILT, AND ALAN HIGGINS

We have developed a microcomputer system for the instantaneous on-line anal- ysis of cardiac action potentials (APs) based on a Motorola 68000 microprocessor and 50kHz A/D converter. This system can accurately analyze APs from all parts of the heart, including the extremely fast upstrokes of APs from Purkinje fibers. Facilities for tabulation of results, production of hard-copy plots of APs, pooling of experimental data, and statistical analyses are available off-line. The routine use of this system in our laboratory has greatly increased the accuracy and throughput of these time-consuming experiments.

Key Words: Cardiac electrophysiology; Action potentials; On-line analysis; Microcomputer

INTRODUCTION The recording of intracellular cardiac action potentials (APs) plays an essential

part in the investigation of drugs that act on the myocardium. These experiments, which typically measure several parameters from a number of action potentials (APs), generate large amounts of raw data.

The most basic method of analysis relies on measuring AP parameters by hand from hard copies. Recent publications, e.g., Binah et al. (1987) and Sawada et al. (1987), suggest that this tedious and time-consuming method remains widely used. A number of computerized methods of AP analysis have now been described (Millar and Vaughan Williams, 1982; Gray and Freeman, 1986; Fusi et al., 1984 Elharrar and Lovelace, 1979). All these methods, while representing a major advance on hand measurement, contain drawbacks, e.g., limited sampling rates, off-line analysis, and limited (8-bit) resolution.

We have developed a microcomputer system that features full on-line analysis of cardiac APs at 50 kHz sampling rate and 12-bit resolution, which can analyze APs from all parts of the heart with instantaneous visualization of the analyzed AP. Tab- ulation of results, hard copies of APs, and statistical analyses are all available using a specially written suite of programs.

A preliminary account of an earlier version of this system has appeared elsewhere (Catlow et al., 1986).

From the Department of Discovery Biology, Pfizer Central Research, Sandwich, Kent, UK. Address reprint requests to: Dr. M. Gwilt, Department of Discovery Biology, Pfizer Central Research,

Sandwich, Kent CT13 9NJ, U.K. Received December 1987; revised and accepted March 1988.

151 Journal of Pharmacological Methods 20, 151-160 (1988) 0160-5402/88/$3.50 © 1988 Elsevier Science Pubhshing Co., Inc., 52 Vanderbllt Avenue, New York, NY 10017

152 R. King et al.

Methods

Electrophysiological Recording

APs were recorded from atrium, ventricle, Purkinje fibers, or sinoatrial node using standard microelectrode techniques. Signals were amplified using an Axoclamp 2A and viewed on an oscilloscope connected in parallel with the computer.

Hardware

The main features of the hardware are as follows: In our system, two microcom- puters are used, a satellite terminal and mainframe, both of which are based on the Motorola 68000 microprocessor. The satellite terminal contains a 50 kHz A/D con- verter (Eltec 68K), 128K of memory and a graphics processor. This machine, which performs all of the on-line analysis, is connected to the mainframe via a port selector.

The mainframe, located in a central computer room, contains 875K memory and is linked to Winchester storage disks (64 M capacity), a plotter (Gould Colorwriter), and a line printer. This machine handles program and data storage, statistical anal- yses, and hard-copy production on behalf of 64 laboratory-based satellites. How- ever, the programs described here could be readily adapted to run on a single stand- alone microcomputer.

$1

CALIBRATION-J ~

PULSE GENERATOR OUTPUT

I I Ine×t

I I

PU LS E

~.~ STIMULUS ARTEFACT

POTENTIAk~ fl

FIGURE 1. Timing of pulses to control data acquisition. One cycle of control pulses is shown. Pulse S, initiates A/D conversion and also triggers the oscilloscope. 52 triggers a 60 mV, 10 ms calibration pulse (only used at the start of an experiment). $3 triggers the stimulator to elicit APs. The 5153 interval is set to a convenient value (usually 100 ms).

On-line Analysis of Cardiac Action Potentials 153

v

l ERO &CALIBRATE I

t ENTER EXPERIMENT TITLE

'9 II REQUEST OPTION

Y Z ' . . . . . . . . ~ N

DIGITISE DATA ] CONTAINING NEXT AP

T

I I T I C"EO O"ST'MOLOSA" EFAC I

AND LOCATE AP IN DATA T

I CALCULATE AP PARAMETERS [

T DISPLAY PARAMETERS& AP ON VDU I

R LAS AP?~ , N

WITH

I TRANSMIT DATA TO

MAINFRAME

ENTER HEADING FROM KEYBOARD

Y y

%, EXIT~ • V

FIGURE 2.

PRINT RESULTS & PLOT HARD COPIES J

Flow diagram of AP analysis programs.

Software

Programs (written in Assembler) are loaded into the satellite terminal from the mainframe. Experimental and programming events are timed externally by pulses from a Devices Digitimer pulse generator, as shown in Figure 1. The general layout of programs outlining keyboard commands, etc. is shown in Figure 2.

The calibration procedure is the same for all programs. First, the computer re- quires a calibration of OmV, obtained by adjusting the Axoclamp output to OmV and supplying a keyboard command to initiate digitization of baseline points. A 60 mV calibration pulse, supplied by the microelectrode amplifier, is used as a voltage calibration. All records are referred to these calibrations for the remainder of the experiment. A title for the experiment can then be entered via the keyboard.

154 R. King et al.

Analysis of APs from Ventricular Muscle, Atrial Muscle, and Conducting Tissue

Following a keyboard command, the next "Sl" pulse (see Figure 1) initiates ND conversion of one second of data containing an AP. This data is then analysed as shown in Figure 3. Data is twice filtered digitally using a simple "bel l" filter, so that:

Xt = 1/4.X,-~ + 1/2.Xi + 1/4.X~÷~

where X~ = value of ith data point X, after filtering. The resting potential (RP) is defined as the average 6f the first four points of the

record. The AP is located by searching for data 6 mV above baseline (any deflection lasting less than 10 ms is identified as stimulus artefact and ignored), and its upstroke is differentiated by calculating the potential gradient between successive groups of three digitized points to yield the maximum rate of depolarization (MRD). The AP peak is found as the highest point of the record (excluding stimulus artefact), and the RP is subtracted from it to yield the AP amplitude (APA). Finally, the times taken from the point of the MRD to where the AP repolarises by 20%, 50%, and 90% (APD20, APDs0, and APDg0 respectively) are found.

These calculated values are displayed on the VDU alongside a digitized display of the AP and an expanded view of its uptake so that the digitized record can be visually checked for contamination by artefacts. In particular, the expanded view of the AP upstroke allows inspection of the stimulus artefact to ensure that it does not distort the upstroke itself. Also displayed are a list of the available keyboard commands and the number of APs analyzed since the current heading was entered. If required, a second command transmits the calculated parameters to the main- frame for storage and subsequent printing. An alternative command stores the re- sults, but also transmits digitzed data for a hard copy plot (see Figure 4); for ease of transmission, only every 16th point is sent to the mainframe. A further command allows entry of headings into the results.

A variant of this program has proved necessary for the analysis of calcium de- pendent "slow" APs, which have MRDs one tenth or less those of normal ventricular muscle (see, e.g., Molyvadas and Sperelakis, 1982). Differentiation of these up- strokes at 50 kHz (20 microsec differentiation interval) resulted in overestimates of MRD, presumably due to interference by baseline noise. This was overcome by extending the differentiation interval to 4 ms. Now, rates of rise as low as 1 Vs -1 (verified using a ramp generator) can be differentiated accurately. Also in this pro- gram, the time limit for the detection of the stimulus artefact has been increased to 20 ms to account for the higher stimulation intensities required for these experiments.

Analysis of APs from Sinoatrial Node (SAN)

APs from the sinoatrial node (SAN) arise spontaneously and do not require stim- ulation, so that A/D conversion cannot be synchronized with APs by means of elec- tronic timing pulses. Instead, the program has to identify the main features of the AP itself. As SAN APs are very different from other APs, the general form is shown schematically in Figure 5 to aid visualization.

155

i ? IKEYBOARD COMMAND I

t CONTROL

ISTORE IN MEMORY OF SATELLITE TERMINAL I

I'BELL" FILTER TWICE I ?

CALCULATE AVERAGE VALUE OF FIRST I FOUR POINTS OF RECORD ( =RP) I

? _ I FIND LARGEST POTENTIAL GRADIENT BETWEEN

N ~ THREE SUCCESSIVE POINTS IN REMAINING I PART OF RECORD

I AOOUST SEARCH I ~ ~~~'~'~'~r"=#~'~T I START TO AVOID I I" ~ . . . . . . . ~n~,~"~' . . . . . . / I ARTEFACT I J, ~ . . . . . . /

+ , IDENTIFIED I T Y

AS ~ ' |IDENTIFIED AS ACl'ION POTENTIAL I I ARTEFACT ...l I Y

ILARGEST GRADIENT - MRDI ?

SUBTRACT RP FROM AP PEAK

SEARCH FORWARDS FROM AP PEAK. FIND FIRST POINT AT WHICH AP HAS REPOLARISED BY 20I OF ITS AMPLITUDE ( - AP~o ).

REPEAT FOR 501 ~ 90g REPOLARISATION (APD 50 G APD 90

Y I DISPLAY G STORE RESULT.S I

T t DISPLAY ~ RETURN TO MAIN PROGRAM I

'INVALID DATA' ON VDU

FIGURE 3. Detailed procedure for analysis of action potentials from fast conducting tissues. For further details of control pulse and bell filter, see Fig. 1 and text. Abbreviations; RP = resting potential; MRD = maximum rate of depolarization; APA = action potential ampli- tude; APD = action potential duration.

156

_A 6o

0

-J -J S= Z

~ - - 6 0

I"-

- - 120

GUINEA-PIG P A P I L L A R Y MUSCLE

RESTING dV/dt AMPLITUDE T20 T50 Tgo -84 296 114 117 196 227

I o ,oo 2o0 ~ ,~o .~o .Go 7~o .Go .Go

s 6o

_> 0 =,

z

T-

z - 6 0

-120

RESTING dV/dt AMPLITUDE T20 T50 Tgo - 9 0 576 121 3 195 308

I I I I I I I I 0 100 2 0 0 300 4 0 0 5 0 0 6 0 0 700 S00 9 0 0

c__ 50

3°

z

~--so

-100

MAX. DIA. SLOW TO MAX. MEAN APD POT POT DEP POT DEP REP 15 41 4 3 . 6 5 216.98 -35.78 7 62 0.72 109 7

I I I I I I I I 0 100 2 0 0 300 4 0 0 5 0 0 6 0 0 7 0 0 800 9 0 0

TIME IN MILLISECONDS

On-line Analysis of Cardiac Action Potentials 157

maximum I upstroke /(" velocity

maximum diastolic potential

0mV

~, CYCLE LENGTH ~ I

~" " - I I

~ i ~ " ~ i ~ A PD I pi!ntial ~ " ~ ~ ~ T a ! e - o f f

Phase IV depolarisation

FIGURE 5. Main features of sinoatrial node AP. A schematic representation of an SAN re- cording is shown with important points for analysis (see text).

First, the cycle to be analyzed is defined. The highest positive maximum differ- ential in the buffer is found, which is very likely to be the MRD of an AP upstroke. The MRD of sinus node APs is much greater than the rate of phase IV depolarization; therefore, the first two maximum positive differentials in the buffer are also likely to represent AP upstrokes. Thus, the cycle to be analyzed is defined as lying between the peaks following these points. The most positive point in the cycle is used to calculate the AP overshoot, and the 2nd maximum positive differential referred to above is stored as the MRD.

The maximum diastolic potential is found as the most negative value of the cycle. The data is searched backwards from the point of MRD (already calculated) until the value of the instantaneous differential falls by 75%--the potential at this point

FIGURE 4. Hard copy plots of single APs. Examples of APs are shown from (A) guinea pig papillary muscle, (B) dog Purklnje fibre and (C) rabbit SA node. AP parameters in (A) and (B) are (left to right): Resting potential (mV), maximum rate of depolarization (Vs-1), AP am- plitude (mV) and action potential durations at 20% (T20), 50% (TS0), and 90% (Tg0) repolar- ization. Parameters in (C) are (left to right) maximum potential, i.e., overshoot (mV), max- imum diastolic potential (mV), rate of phase IV depolarization mVs -1, take-off potential (mV), maximum rate of depolarization (Vs-l), mean rate of repolarization (Vs-1), and action po- tential duration (ms).

158

7/5/85 RESTING dV/dt AMPLITUDE T20 T50 T90

-92 230 136 73 172 203 -90 268 128 70 169 202 -83 209 122 57 166 197 -78 214 116 61 166 199 -85 233 121 71 169 199

* -87 231 129 61 164 197 -92 273 134 86 167 197 -89 266 128 78 170 199 -97 216 131 67 165 198 -95 215 125 59 163 197

D-SOTALOL 1E-06M -85 332 129 57 161 197 -89 260 115 54 170 199 -78 238 115 57 168 200 -94 264 115 57 158 197 -77 256 112 43 162 196 -88 263 125 55 153 193 -84 278 123 65 163 195 -85 202 120 62 166 199 -79 226 117 48 164 200

* -83 335 120 50 162 199 1E-05M

* -90 282 130 61 177 214 -91 311 126 63 171 218 -93 254 133 77 180 217 -99 265 137 69 182 217 -81 253 113 68 185 221 --89 263 126 69 181 220 -96 249 127 82 189 223 -90 253 121 78 191 223 -94 271 128 66 185 222 -90 183 114 56 189 223

1E-04M -87 232 123 69 209 258 -92 311 122 56 198 256 -89 248 120 61 201 257 -98 297 126 70 209 254 -89 257 119 58 198 251 -87 213 124 43 182 242 -91 276 127 50 190 245 -82 217 118 58 195 243 -91 238 120 54 196 242

*-91 224 122 61 202 246

FIGURE 6. Hard copy output of results from a single experiment. All of the results in this table were analyzed on line. Each AP is from a separate microelectrode impalement. APs that were plotted out are marked by an asterisk. AP parameters displayed are as shown in Fig. 4. Note the effects of D-sotalol on action potential duration (see Carmeliet, 1985).

On-line Analysis of Cardiac Action Potentials 159

is the "take-off potential." The slope of the middle 50% of the record between the maximum diastolic and take-off potentials is the rate of phase IV (pacemaker) de- polarization. Action potential duration is measured between the beginning of the cycle and the maximum diastolic potential. Finally, the mean rate of repolarization is calculated as overshoot minus maximum diastolic potential divided by action potential duration.

As before, these parameters are displayed alongside the digitized record. Vertical cursors are displayed at the peaks defining the cycle, the maximum diastolic po- tential, and the take-off potential to ensure that the AP has been analyzed correctly. Calculated AP parameters and data for hard copies (see Figure 4) are passed to the mainframe following acceptance of APs, as before.

Data Storage and Statistics

During an experiment, the data generated is held in the memory of the mainframe. At the end of the experiment hard copies are plotted and results both printed out and stored on Winchester disk. All files on disk are identified by a file number so that files can be recalled by separate analysis programs, their data pooled, and standard statistical tests carried out off-line.

RESULTS

Figure 6 shows a table of results from a single experiment. Headings were inserted during the course of the experiment, as described above. Asterisks at the left-hand margin denote those APs that were plotted out. Such hard copy plots of APs from ventricular muscle, Purkinje fiber, and sinoatrial node are shown in Figure 4.

DISCUSSION

Our main aim in developing this system was to increase the efficiency of these time-consuming experiments and to increase the accuracy of measurement. Also, instantaneous analysis allows rapid estimation of the quality of an impalement and onset of drug effects, allowing better experimental control. A typical experiment, as illustrated in Figure 6, generates 240 separate results. The time taken to analyze these by hand, as in Binah et al. (1987) or Sawada et al. (1987) or even by an off- line computer system (Millar and Vaughan Williams, 1982; Gray and Freeman, 1986) might approach the time taken in performing the experiment itself. Clearly, any correctly functioning computer-based analysis must be more accurate than hand measurement due to its superior objectivity and its voltage and time resolution. In particular, the upstrokes of APs in Purkinje fibres are very fast, of the order of 500- 800 Vs -1. Computer modelling studies of action potentials (McAIlister et al., 1975) indicate that the rate of depolarization during the upstroke is far from uniform and that the MRD is maintained for only a very small time interval. Therefore, faster sampling rates should provide better estimates of MRD in Purkinje fibers.

The flexibility of our system is evident in its ability to analyze APs from all parts of the heart in addition to storing data and performing statistical analyses. The system, being built from readily available and relatively inexpensive components

160 R. King et al.

can be easily modified to fulfil other experimental requirements. Indeed, this system is also used to control and analyze a wide variety of other in vitro and in vivo experiments. In particular, the DIA facility of the A/D converter can be used to supply command pulses for the on-line control of experiments.

REFERENCES

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Carmeliet E (1985) Electrophysiological and voltage clamp analysis of the effects of sotalol on isolated cardiac muscle and Purkinje fibres. J Pharrnacol Exp Ther 232: 817-825.

Catlow CJ, Gwilt M, Higgins AJ, King RC (1986) Real time analysis of intracellular cardiac action po- tentials using a Motorola 68000. Br J Pharmacol 88:422P.

Elharrar V, Lovelace DE (1979) On-line analysis of intracellular electrophysiological data using a mi- crocomputer. Am J Physio1237: H400-H408.

Fusi F, Piazzesi G, Amerini S, Mugelli A, Livi S (1984) A low-cost microcomputer system for automated

analysis of intracellular cardiac action potentials. J Pharmacol Methods 11:61-66.

Gray P, Freeman S (1986) A computerised approach to the collection and analysis of cardiac action potentials. J Pharmacol Methods 15:347-357.

McAllister RE, Noble D and Tsien RW (1975) Re- construction of the electrical activity of cardiac Purkinje fibres. J Physio1251 :I-59.

Millar JS, Vaughan Williams EM (1982) Differential actions on rabbit nodal, atrial, Purkinje cell and ventricular potentials of melperone, a bradycar- diac agent delaying repolarisation: Effects of hy- poxia. Br J Pharmacol 75:109-121.

Sawada K, Shoji T, Igarashi T, Hiraoka M (1987). Voltage- and rate-dependent depression of V'max of action potentials by a new antiarrhythmic agent, E-0747, in swine cardiac Purkinje fibres. J Cardiovasc Pharmacol 9:51-56.