Endocardial role in arrhythmias induced by acute ventricular stretch and the involvement of Purkinje fibres, in isolated rat hearts

Figures

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  Fig. 1. Acute left ventricular stretch induces ectopics. Upper trace, schematic change in balloon volume, and change in pseudo-ECG in a Langendorff perfused, isolated rat heart in sinus rhythm. The red arrow, at approximately 4 s, indicates the onset of LV stretch by injection of 100 μL into a balloon placed in the LV lumen. Mechanically induced ectopics began during the increase in luminal volume, with a spontaneous return to sinus rhythm at approximately 11 s. Ectopic activations were identified by their timing and waveform compared to sinus configurations. Lower trace, expanded time scale of the pseudo-ECG trace highlighting a period of ectopic activations (between 5 and 8 s) and during the return to sinus rhythm (between 12 and 15 s). Ectopic activations had varied waveforms including negative and positive going initial vectors while regular P-R waveforms are visible in the sinus trace. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 2. Lugol attenuates mechanically induced ectopics A. Upper trace, schematic of change in balloon volume, lower trace, pseudo-ECG, from a Langendorff perfused isolated rat heart in sinus rhythm. The red arrows indicates stretch of the LV by injection of 100 μL into a balloon placed in the LV lumen. Records from the same heart before (Tyrode) and after (Lugol) irrigation of the LV lumen with 0.1 mL Lugol (applied over a 10 s period, then Tyrode's washed). Mechanically-activated ectopics were evoked in the Tyrode's traces, disrupting the pseudo-ECG. In this heart, there were no mechanically-activated ectopics with Lugol and the heart beat rhythmically throughout stretch. B&C. Number of ectopics and period of ectopics for individual stretches under Tyrode's to Lugol conditions (B) and Tyrode's to Tyrode's (C). Lugol caused a significant reduction in both parameters, Tyrode's had no effect on the number of ectopics, while the period was significantly increased (see text). Data from 16 stretches in B&C. Bars indicate P < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 3. Necrotic tissue is restricted to the endocardial surface in Lugol treated hearts. The left ventricular (LV) lumen of isolated Langendorff perfused rat hearts were exposed to Lugol (120 mM KI, 38.6 mM I2) solution for 10 s, followed by Tyrode's wash. Hearts were stained with TTC and fixed in 10% formalin prior to imaging. (A) LV, with incision through the free wall. White, necrotic, tissue is restricted to the endocardial surface to a depth of approx. 200 μm. Prominent, ablated PFs are visible e.g. below the green asterick and on the septal wall. (B) Presence of necrotic tissue on the LV endocardial surface contrasts with its absence in both the LV myocardium and in (C), the endocardial surface of the untreated RV free wall taken from the same heart. (A&B) Yellow asterick denotes myocardium of the LV free wall, green and blue astericks denotes ablated tissue on the luminal surface of LV free wall and LV septal wall respectively. Scale bars, A–C: 2 mm. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 4. Lugol treatment attenuates endocardial connexion 40. The left ventricular (LV) lumen of isolated Langendorff perfused rat hearts were exposed to either (A) Tyrode's or (B) Lugol (120 mM KI, 38.6 mM I2) solution for 10 s, followed by Tyrode's wash. Cells were identified with DAPI stain (blue) and Purkinje fibres identified by dense connexin-40 staining (Cx40; bright green). (A) Dense Cx40 staining in the LV luminal surface of hearts exposed to Tyrode's only. In A the LV lumen is occluded in part of the image but follows the line of dense Cx40 staining indicated by the white arrow. (B) Absence of dense Cx40 stain on the LV luminal surface (Lumen) exposed to Lugol solution. (C) Relative intensity of Cx40 fluorescent signal at the luminal surface and deep myocardium, based upon the pixel gray value measured using Image J (Fiji). Data presented as mean ± SEM with one-way ANOVA performed in SPSS (P < 0.05). Tissue was imaged with an Evos Cell Imaging System. Scale bars, 250 μm. Data from n = 3 Tyrode treated hearts and n = 3 Lugol treated hearts. Bars indicate P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of…

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  Fig. 5. TRPM4 is more abundant in rat Purkinje fibres than myocardium and a TRPM4 blocker attenuates stretch-induced ectopics A. Purkinje Fibres (PF) and myocardium (M) were excised from the same area of rat LV free wall. (A) Tissue was immunolabelled for TRPM4 with primary antibody rabbit anti-TRPM4 (1:100; PA5-77324, Invitrogen) and secondary antibody goat anti-rabbit (1:200, Alexa 594. Thermofisher Scientific). Tissue was mounted in Prolong Gold (Thermofisher Scientific) on a glass slide. Two-dimensional images were acquired using an FV1000 Olympus confocal microscope. Increased TRPM4 immunofluorescence was observed in PF compared to M. Scale bar 250 μm. B. The integrated density and mean gray value of TRPM4 label was measured from myocardium and PFs (n = 3 hearts) using Image J-(Fiji) and presented as the mean integrated density ± SEM. Statistical analysis of data was performed in GraphPad Prism 7.03, and differences were compared using paired t-tests. The bar above the columns indicates (P < 0.05). C. Number of ectopics and period of ectopics for individual stretches under Tyrode to 50 μM 9-Phenanthrol (9-Phen) conditions in intact Langendorff hearts. 9-Phen (a TRPM4 blocker) caused…

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  Fig. 6. Activation from sinus stimulation spreads faster than from endomyocardial stimulation A. Optical mapping of left ventricular (LV) epicardial activation in 2 hearts in response to sinus stimulation (upper images) or focal endomyocardial stimulation near the LV base (lower traces). Images show colour coded 3 ms contours. Images are 80 × 80 pixels and 17.5 mm squares. In response to sinus stimulation, which involves conduction through the Purkinje network, there is a rapid spread of activation. In contrast focal endomyocardial stimulation results in a slower spread of activation. B. The fraction of epicardial surface activated at the 6 ms contour (colour identified by the asterix in A) in 4 hearts was significantly greater in response to sinus stimulation compared with endomyocardial stimulation. Thus the activation pattern can inform about the stimulation source. Hearts identified by symbol shape, individual data points and mean ± SEM shown (Bars indicate P < 0.05, paired t-test). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 7. Stretch-induced ectopic activation mimics sinus and focal stimulation Pseudo-ECG (upper trace), optically mapped action potentials (middle trace) and epicardial activation maps, as described in Fig. 6 (lower images), in the left ventricle (LV) of a Langendorff rat heart. At the red arrow the LV is dilated from 50 μL to 150 μL by inflation of an indwelling balloon over a 2 s period, triggering ectopics. Activation maps from selected points (1–5) show sinus stimulations at 1&5 with typical rapid activation spread. Excitations 2&4 are ectopic but have activation patterns that closely mimic the sinus pattern, indicating the involvement of the Purkinje network. Excitation 3 is ectopic with activation spreading more slowly from a more basal site. Stretch-induced ectopics are multi-focal (see Discussion). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 8. Stretch-induced ectopic activations Epicardial activation in 6 rat left ventricles (as described in Fig. 6). For each heart an example of activation in response to sinus stimulation and stretch-induced ectopics are shown with the associated pseudo-ECG waveform. The amplitude of the sinus R-wave is given as a voltage reference. Sinus and ectopic type 1 activations spread rapidly while ectopic type 2 spread more slowly. Positive and negative going pseudo-ECG waveforms occur with both type of activation pattern. For each heart both ectopic types were taken from the same stretch (see Fig. 7). Varied activations within a single run of dilution-induced ectopics are common. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)