β-Adrenergic Receptor Stimulation and Alternans in the Border Zone of a Healed Infarct: An ex vivo Study and Computational Investigation of Arrhythmogenesis

Figures

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  FIGURE 1. Langendorff heart overview. (A) Schematic depiction of the positioning of stimulator and the mapped area (L/R, left/right; A/V, atrium/ventricle). (B) An example of the mapped field of view in an infarcted heart. (C) A schematic illustration of the pacing and stimulation protocol. AM, alternans mapping; +NE, norepinephrine (1 μmol/L) for 15 min; +Meto, metoprolol (10 μmol/L) for 15 min. After each AM instance, the heart was not stimulated for 2–3 min for the heart rate to stabilize before the following measurement took place.

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  FIGURE 2. MRI and staining of infarction. (A) An example of MRI data (systole and diastole in a sham and an infarcted heart, with red arrows indicating the position of the akinetic infarct). (B) An example of Masson’s trichrome staining of apical tissue section showing fibrotic scar formation (blue arrow).

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  FIGURE 3. Rapid pacing induces calcium alternans in sham and infarcted hearts. (A) Normalized calcium alternans magnitude of recordings in control condition against base cycle length for infarcted (n = 8) and sham hearts (n = 3). Asterisks represent statistical significance of the difference between sham and infarcted hearts at the level of 0.05 (∗); assessed using Mann–Whitney U test. (B–D) Sample smoothed calcium traces illustrating the values of normalized alternans magnitude.

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  FIGURE 4. Border zone vulnerability to alternans. (A) The ratio of alternans magnitude in border zone and non-infarcted zone of each recording, plotted as gray points against base cycle length (bcl). Data from a single heart are connected with a dashed line. In blue is given the median and 25 and 75 quantiles. Points above the red line manifest more alternans in border zone than in non-infarcted zone. Asterisks represent statistical significance of alternans in non-infarcted zone versus border zone within each heart at the level of 0.05 (∗); assessed using the Wilcoxon signed rank test. The black arrow in bottom left corner indicates a special trace discussed further in the text. (B) Duration of calcium transient at 90% calcium recovery at bcl 140 in infarcted hearts (NZ, non-infarcted zone; BZ, border zone) and in sham hearts; dashed lines connect data from the same heart. Asterisks represent statistical significance of difference between sham and infarcted hearts at the level of 0.01 (∗∗) or 0.05 (∗); the Wilcoxon signed rank test was used for comparing BZ and NZ, given the paired nature of data, while for the other two comparisons, Mann–Whitney U test was used. (C) An example of…

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  FIGURE 5. Effects of norepinephrine and subsequent β-blockade. (A) The effect of norepinephrine perfusion and subsequent β-blockade on normalized alternans magnitude at high pacing frequencies (each circle is an average of normalized alternans magnitudes of the given heart over bcls of 80, 85, and 90). Asterisks represent statistical significance at the level of 0.01 (∗∗), as assessed using the Wilcoxon signed rank test; n.s., not significant. (B) Sample alternans maps from a single recording under the three conditions (control, norepinephrine, and norepinephrine+metoprolol), with sample smoothed traces of the red-marked-pixels shown below. NAM, normalized alternans magnitude. (C) The effect of norepinephrine and subsequent β-blockade on CaTD90 (calcium transient duration, at 90% calcium recovery level) at pacing of 140 ms bcl (statistical significance assessed using the Wilcoxon signed rank test; ∗ corresponds to p < 0.05). (D) Correlation between CaTD90 from (A) and normalized alternans magnitude at fast pacing frequencies from (C), matched by heart and condition. See Supplementary Figure S5 for a separation of (A,C) by normal and border zone.

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  FIGURE 6. Pre-paced fiber at control condition and with simulated β-adrenergic receptor stimulation. (A) In the left half is a heat map of membrane potential of cells in the fiber over time, visualizing two action potentials manifesting alternans in the border zone. Every row of the image gives the membrane potential of a single cell in the fiber over time (the x-axis). The gray NZ/BZ/NZ box indicates the location of non-infarcted (NZ) and border (BZ) zones, with the numbers specifying the index of each cell in the fiber. The black rectangle labeled “C. block” indicates the segment of the fiber and the time of stimulation used in searching for the vulnerable window of conduction block. The black square labeled “PVC” shows the segment and time used in testing vulnerability to PVCs. To the right are examples of action potentials from NZ (cell index 220) and BZ (cell index 128), corresponding to single rows of the heat map. The black lines under action potentials specify the APD90. (B) An analogy of (A) with β-adrenergic receptor stimulation.

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  FIGURE 7. Alternans and conduction block. (A) Examples of three scenarios observed for S2 interval between 175 and 200 ms, with heat and elevation coding the membrane potential: no conduction of S2 stimulus beyond the stimulated segment of the fiber (left image), a conduction block in the BZ (central image), and full conduction through the fiber (right image). The bar under these three cases shows the S2 intervals in which these three conditions occurred. (B) Results of an identical protocol as in (A), but using a fiber pre-paced in the presence of β-adrenergic receptor stimulation and manifesting no alternans. No conduction block was observed and thus is not shown.