2.1. Setup and Tissue Preparation
Nearly all mapping systems consist of a number of sensitive detectors, from which the signal gets amplified, processed, displayed and then stored for further analysis.
2.1.1. vECG and Pacing Electrodes
vECG recordings associated with spontaneous sinus and paced rhythms, were obtained steadily throughout the experiment and stored for off-‐line analysis (Figure 5b). Signals were amplified and low-‐pass filtered at 200Hz using an electronic amplifier (HSE, Germany) and digitally sampled at 10 kHz with a 16-‐bit resolution using the NI USB-‐6216 BNC (National Instruments) digitizer and custom-‐made software using LabView (National Instruments). At the center of the left ventricular wall, a bipolar specialized cardiac pacing electrode (50-‐100 kOhm, FHC, USA) was gently placed, stimulating the preparations at a BCL of 100ms at 2.5 diastolic threshold, with 2ms impulse duration. Capture was confirmed both electronically and optically.
2.1.2. Murine Heart Isolation and Langendorff Perfusion
Mice were heparinized (40 units) and anesthetized using volatile 100% Isoflurane (Floren, Abbat-‐
Germany). Excised through a sternotomy, intact hearts were carefully cannulated, connected to a custom-‐made perfusion setup and retrogradely perfused at 2.5-‐3 mL.min-‐1 via the aorta with Tyrode solution, under quasiphysiological conditions. The temperature was set to 38±1°C, oxygen content (95%O2 and 95% CO2), pH to 7.4 and ionic concentration (in mmol.L-‐1): NaCl 130, NaHCO3 24, KCl 4, MgCl2 1, CaCl2 1.8, KH2PO4 1.2, C6H12O6 5.6, 1% albumin (Bovine Serum Albumin, Sigma) and insulin (Insuman Rapid, Sanofi-‐Aventis, 5IU/L). Hearts were immersed with constantly warm Tyrode solution, within a custom-‐made glass chamber that ensures adequate temperature throughout the experimental period, where the solution within serves as a conducting medium for in-‐vitro custom-‐
made Ag-‐AgCl ECG electrodes placed horizontally and parallel to the septum, at a distance of 1mm from the epicardial surface. Hearts with a spontaneous beating rate exceeding 450beats/min for more than 20min after connecting them to the perfusion setup were exclusively used for further measurements. The left ventricular free wall was projected with maximal cross-‐sectional diameter in the objective light path to ensure consistent mapping of the conduction spread, by maximizing the exposed stimulated area to the exciting monochromatic beam (Figure 5a).
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Setup and Tissue Preparation 53
2.1.3. Excitation-Contraction Decoupler and VSD Staining
Of the limitations of cardiac optical mapping of the heart are imaging artifacts, caused by successive contractions. Motion artifacts can strongly distort or obscure voltage optical measurements. Several strategies are adopted to overcome this obstacle, which could be mechanical in nature,
large time constants, are not suitable for mapping fast activities like the ones taking place in the murine heart, where the entire ventricular AP starts and terminates in ~40ms.
Fast dyes, on the other hand, do not rely on movement back and forth between the different compartments of the cardiac cell, but change their optical characteristics by changing conformation.
These dyes provide rapid absorbance and fluorescence responses to membrane potential (time constants in the order of one ms) and are capable of recording fast AP525. Fast dyes are divided into different categories based on their chemistry. Several mechanisms account for a given dye’s sensitivity, including changing its conformation, its orientation within the plasma membrane or its charge distribution (electrochromism) within the plasma membrane526. The rotation-‐dimer
Setup and Tissue Preparation 55
to membrane voltage changes by an electrochromic mechanism, i.e. by charge redistribution of the surface of the molecule527. For a comprehensive review on organic dyes, their chemistry, mechanisms and sensitivity in optical mapping, refer to the book chapter by Loew “Design and Use of Organic Voltage Sensitive Dyes” (2010)526.
2.1.4. 2D Optical Mapping Setup
After the heart rate reached steady state (typically 20min from initiation of retrograde perfusion), measurements were carried out after staining the heart with a voltage-‐reporter dye (Di-‐4-‐ANEPPS, Molecular Probes, Portland, OR), by injecting a 1mL bolus at 30μmol.L-‐1, into a 10mL compliance chamber proximal to the aorta (section 2.1.2). Concisely, excitation light from a 100W short-‐arc mercury lamp (HBO103W/2, Olympus, Germany) was collocated, using an upright macroscope (MVX10, Olympus) and made monochromatic using an interference filter (515±15nm) and dichromatic filter (570nm), then collected using a long-‐pass filter (>590nm) and projected onto a 100 100 pixel CMOS (Complementary Metal Oxide Semiconductor camera, Ultima-‐L, SciMedia) recording array (Figure 5b). Imaging with Di-‐4-‐ANEPPS was obtained at a frame rate of 2 kHz. To reveal the signal, background noise was subtracted from each frame. Imaging was carried out using a 0.63 objective (MVPLAPO 0.63X, NA 0.15; Olympus), at a zoom of 2.5 in order to provide the largest view of the left ventricular free wall with a pixel resolution of 100µm. Prior to any further analysis, the optical signal was modified in order to optimize the fluorescence change and increase the SNR. To study conduction spread at steady state pacing of 10Hz, preparations were paced using a bipolar electrode (Figure 5b, section2.1.1). The raw data collected from the camera were frame-‐
selected using custom-‐made open source software written in Java that was optimized for the mouse heart, constituting a platform input into another custom-‐made routine written in MATLAB (the Mathworks) for further analysis.
2.1.5. Animal Models Used in the Current Study and Drugs
Adult 12-‐16weeks male dystrophin-‐deficientlv B6Ros.CG-‐Dmd(mdx-‐5cv)/J micelvi (nlvii=17+7) along with their WT gender and age matched controls C57BL6/J (n=17+10); and 20-‐22 weeks old male heterozygous ∆KPQ micelviii (n=3+4) with their corresponding WT control (n=3+4) were selected for the following studies. To avoid any position induced changes in fiber orientation, to maximally expose the region of fluorescence and to provide a supposedly homogeneous area of conduction spread; we restricted the measurements to the LV free wall for all excised hearts. Hearts were stained with a Di-‐4-‐ANEPPS and activations were recorded as waves emanated from the tip of a bipolar electrode, placed at the centre of the LV free wall, under automated steady state pacing at BCL=100ms. The pharmacological intervention consisted of perfusing the cannulated hearts with the regular Tyrode solution (section 2.1.2) containing 1µM Flecainide (Flecainide Acetate, Sigma Aldrich) after the control (non-‐treated) measurement was recorded and showed an elliptical behavior. Measurements with Flecainide were taken at time points 0, 1, 3, 5, 7 and 10 min, to assure close monitoring of activation changes due to the drug. Unlike the experimental protocol used for the mdx and their WT counterpart that lasted up to 10min exposure to Flecainide, steady state was
(Biophys J (2006) 90:2938-‐2945), fluorescence in rabbit hearts loaded with Di-‐4-‐Anepps is optimal between 0.5-‐1mm below the epicardial surface.
lv Dystrophin-‐deficiency is described in the Introduction section 1.2.2.
lvi These mice will be referred to as mdx (for simplicity) throughout this work.
lvii n = number of experiments. Two major experimental studies were conducted in this thesis. n = x + y means the total number of animals used for both studies, where x indicates the number of animals used in the study that investigated the different analytical strategies in CV evaluation using optical mapping, and y indicates the number of animals used to conduct the heterogeneity study with Flecainide.
lviii The ΔKPQ mouse, harboring the mutation for Long QT syndrome 3, is described in the Introduction section 1.4.3.
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no longer achievable for the ∆KPQ beyond 5min of treatment (i.e. either entrainment at 10Hz frequency was no longer possible or consecutive APs showed considerable beat-‐to-‐beat variability which prevented temporal averaging). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University Medical Center Göttingen and by veterinarian state authority LAVES (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) in compliance with the humane care and use of laboratory animals.
wave front, segregating the depolarized tissue from the quiescent tissue; consequently boundaries at 0.5ms interval (corresponding to the sampling rate) were computed by a linear interpolation of