Software Package. Rolf Thieleczek. Distributed by SCCE UG Essen*, Germany

Transcription

1 Cardiac Action Potential Analysis (CAPA) Software Package by Rolf Thieleczek Distributed by SCCE UG Essen*, Germany *Science Consulting Cardiac Cellular Electrophysiology UG (haftungsbeschränkt), Bargmannstraße 27, Essen, Germany C A P A, p 1 17

2 Cardiac Action Potential Analysis (CAPA) Software Package Author: Rolf Thieleczek Release: vers Created Using: Used MATLAB Toolboxes: MATLAB Release 8.5 (R2015a) Curve Fitting, Statistics, Compiler Third party MATLAB files: Filename Copyright X collection (ExcelCOM) Yvan Lengwiler, 2015 exporttopptx Stefan Slonevskiy, export_fig Oliver J. Woodford, Yair M. Altman, 2014 uipickfiles Douglas M. Schwarz, 2007 peakfinder Nathanael C. Yoder, 2013 legendflex Kelly Kearney, 2015 figtitle Chad Greene, 2013 inputsdlg Takeshi Ikuma, 2015 Please pay attention to the provided license information. Description: The CAPA software package has been developed for automatically detecting and analyzing spontaneous or evoked Action Potentials (AP) in membrane potential recordings of cultured or isolated cardiomyocytes or multicellular cardiac muscle preparations. AP in a recording are detected and analyzed automatically in a user-independent manner. No user input will be needed except loading of the data files and setting of frame conditions for the analysis. The user is set free to judgment of the results obtained by CAPA. The software is provided as standalone MATLAB application including the MATLAB Compiler Runtime (MCR) that is needed to run the application. C A P A, p 2 17

3 In order to be able to use CAPA, your computer must be running under a 64-bit version of MS Windows (Windows 7 or newer). You first have to install MCR on your computer by executing the provided capainstaller_mcr.exe as administrator (see CAPA_installation.pdf for details). The package includes sample recordings of evoked (sample_evap.txt) # and spontaneous AP (sample_spap.txt). Call these data to see the figures that are created in the progress of analysis. These figures and the analysis results will automatically be saved to PowerPoint and Excel files, respectively. Their names will be composed of a given user-id, the data filename, and the current date and time. # generously provided by: # Dept. of Pharmacology and Toxicology, Dresden University of Technology, Germany and Pharmacology, UMG, Göttingen, Germany Dept. of Files Included in the Package: These files are stored in a folder named CAPA***package. Please copy this folder to the directory C:\TEMP of your hard disk and install the software as described in CAPA_Installation.pdf. C A P A, p 3 17

4 Further Products Required: Microsoft PowerPoint 2007 (or newer) Microsoft Excel 2007 (or newer) Both have to be installed on your computer in order to create pptx and xlsx result-files and enabling direct data transfer to them in the progress of analysis. CAPA Trial Version: A Trial version of CAPA is available on request from the contact address below. The Trial Version is fully functional except that saving of your analysis results to Excel is limited and usage is restricted to a certain period of time. Contact: If you have any questions please contact Prof. Dr. Erich Wettwer scce_ug@online.de or Disclaimer: The CAPA software package is provided 'as is' without any endorsement made and without warranty of any kind, either expressed or implied. No guarantees for the accuracy of the codes, output information and figures are made. CAPA codes and outputs can only be used at your own discretion and risk and with agreement that you will be solely responsible for any damage and that the authors and their affiliate institutions accept no responsibility for errors or omissions in CAPA codes, outputs, figures, and documentation. In no event shall the authors, developers or their affiliate institutions be liable to you or any third parties for any special, direct, indirect or consequential damages and financial risks of any kind, or any damages whatsoever, resulting from, arising out of or in connection with the use of CAPA. The user of CAPA agrees that the codes and algorithms are subject to change without notice. C A P A, p 4 17

5 Main Features of CAPA Start CAPA by double clicking the capa icon created on your desktop during installation. Please be patient, the startup process may take a few moments. An entrance screen like the one below will be shown for a couple of seconds (you can skip over it). A File Selection Dialogue allows selection and loading of input data provided as ASCCI txt-file. If this dialogue is not visible completely on screen, double click C A P A, p 5 17

6 on a vacant region of this figure. You can load several data files which will be analyzed file by file in one run. If you do so please make sure they are containing the same type of data (e.g. all showing a distinct resting potential). To all of them the same analysis conditions selected in an Input Dialogue (see below) will be applied. Please put your data files in a folder C:\CAPA which is the default location CAPA uses for data file loading and saving of result files. The data must be provided as 2-column matrix with time given in seconds and voltage in mv. The decimal separator must be set to POINT and a thousands separator must not be set. The loaded data are displayed in an initial figure together with the Input Dialogue shown below. It enables setting of a user-id and offers determination the AP thresholds in the recording by linear regression to either the resting potential over the whole range or to the terminal part of the pre-upstroke phase of each AP. In the latter case the length of the section for linear fitting can be chosen as fraction of the peak-to-peak distance. The first method should be used if a distinct resting potential is visible in the recording whereas the second one is always applicable. If your recording shows features like a plateau and groove in the shoulder of the AP you may check the corresponding box in order to determine them. Check the last box if you want to suppress the output on screen of figures created during the course of analysis. C A P A, p 6 17

7 If a linear regression to the resting potential has been chosen, the mean resting potential of the recording will be determined together with the drift occurring during the recording. In case of threshold determination to individual AP, the Rate of Diastolic Depolarization will be determined from the chosen terminal part of each peakto-peak interval. The analysis will run without any further user input needed. In the progress of analysis Figures will be created and directly saved to a PowerPoint file. Obtained analysis results will also be saved automatically to an Excel file. Both of these result files will get the same stem name composed of the given user- ID, the data filename, and the current date and time. They will be saved to the data file directory C:\CAPA CAPA. In the following some figures and data obtained with the included sample data files are presented. The next figure shows a sample recording of spontaneous AP with clearly discernible diastolic depolarization phases. C A P A, p 7 17

8 The rate of diastolic depolarization (DDR) and the threshold will be determined automatically for each individual AP of the trace as is exemplified below for the second of the three AP shown above. The DDR is given by the slope of the linear regression to the terminal part of the peak-to-peak interval (here chosen to be 30% of the peak-to-peak distance). Data that are lying above the regression line (green) and are heading towards the AP peak are considered as belonging to the rising phase of the AP (red). The starting point of this rising phase is taken as initial estimate of the AP threshold. It will be checked wheter the AP upstroke after this threshold is continuously rising (defined here by dv/dt >= 0). In the presented example this condition is met. In case it is not, the initially determined threshold will be corrected according to the definition given above. C A P A, p 8 17

9 For these types of AP preceded by a diastolic depolarization phase the minimum diastolic potential (MDP) will also be obtained by non-linear fitting for each individual AP as exemplified below for the first two AP of the sample file. The peak potentials of the AP in a recording are determined generally independent of the method of threshold determination chosen. They are obtained by non-linear fits to the peak region of individual AP as shown again for the first two AP of the sample recording. For an explanation of the AP parameters please see the list at the end of the document. C A P A, p 9 17

10 The next sample recording shown involves electrical stimulation of cardiac cells which causes discernible stimulation artefacts. CAPA is able to detect them and thus avoiding that they disturb the AP analysis. This recording shows a resting potential of about -70 mv instead of distinct individual diastolic depolarization phases. Furthermore, the AP exhibit a shoulder phase after the spike with a groove like dip followed by a plateau. The thresholds of these types of AP are determined by a linear regression to the resting potential data over the whole recording as shown in the next figure. C A P A, p 10 17

11 The threshold points of the AP are projected on the regression fit line. The slope of the regression line reflects the drift in the resting potential (RP drift) during the recording. If this drift is small or absent the regression line can be regarded as a mean resting potential which is equal to the mean of the individual threshold values. Summary In case of threshold determination by linear regresssion to individual pre-ap phases, the rate of diastolic depolarization and the minimum diastolic potential will be writen to the Excel result file. When threshold determination by linear regresssion to the baseline of the whole recording is used, the mean resting potential +- SEM and the drift in resting potential +- SE is transferred to Excel instead. Please see the example Excel files shown further below. C A P A, p 11 17

12 If chosen in the input dialogue, the groove and plateau potentials and times will automatically be determined by a non-linear regression to the shoulder- phases of the AP as shown below. The groove reflects a local minimum and the plateau a local maximum in the shoulder of these AP. They are indicated in the recording as shown below together with further determined key parameters of the AP that define their shape. C A P A, p 12 17

13 The maximum rate of depolarization (MDR) of an AP is determined automatically by a non-linear fit to the peak region of the first derivative of the AP upstroke as indicated below for the last of the AP shown above. In this case a very small correction of the initially determined threshold (vertical black line) is made according to the definition of the rising phase of an AP given before. The MDR is obtained as the maximum of the non-linear fit to the first derivative of the membrane potential behind the corrected threshold (vertical magenta line). C A P A, p 13 17

14 Each detected AP will be documented in a single figure with some of its determined parameters as shown below for the second AP of both of the sample recordings. As characteristic feature the AP duration at 90% repolarization is indicated by the red bar. AP with Distinct Diastolic Depolarization AP with Distinct Resting Potential C A P A, p 14 17

15 A superposition of all detected AP on their thresholds will reveal a change in shape of the AP during the recording. Finally, an average AP with STD and SEM will be created from the analyzed AP and the corresponding data will be available in the Excel result file. Superimposed and Average AP Bioengineered Human Myocytes Human Atrium C A P A, p 15 17

16 List of parameters that can be obtained automatically with CAPA from recordings like those presented before. Meaning of the Parameters: 1 ThrPot Threshold Potential 2 ThrTime Threshold Time 3 PP Peak Potential 4 MDP Minimumimum Diastolic Potential* 5 MDPt Time from threshold to MDP* 6 AMPthr AP Amplitude from ThrPot to PP 7 AMmdp AP Amplitude from MDP to PP* 8 MDR Maximum Depolarization Rate 9 DDR Diastolic Depolarization Rate* 10 DP Diastolic Potential* 11 AP20 Potential at 20% repolarization 12 AP50 Potential at 50% repolarization 13 AP90 Potential at 90% repolarization 14 APD20 AP Duration from ThrPot to AP20 15 APD50 AP Duration from ThrPot to AP50 16 APD90 AP Duration from ThrPot to AP90 * only for recordings with distinct diastolic depolarization An explanation of the determined parameters will also be given in the head of the Excel result file. C A P A, p 16 17

17 The determined parameters are automatically written to an Excel file with the applied methods of analysis being mentioned. Below you find cut out pieces of data that are transferred to Excel for the shown sample recordings. Excel Data Sheets Created During Analysis Analysis considering distinct Diastolic Depolarization Analysis considering distinct Resting Potential Please let us know if you need special parameters to be determined in your AP recordings. To ensure correct data transfer to Excel, make sure that inside Excel the decimal separator is set to POINT and the thousands separator is not set (empty space). Otherwise Excel will probably misinterpret the received data. C A P A, p 17 17