Proarrhythmic Electrical Remodeling by Noncardiomyocytes at Interfaces With Cardiomyocytes Under Oxidative Stress

Figures

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  Figure 1. Patterning cardiomyocyte–noncardiomyocyte interfaces. (A) Side-1 cell type for all monolayer groups was neonatal rat ventricular cardiomyocytes (NRVMs). Side-2 cell type was either NRVMs in homotypic interfaces (left panel) or noncardiomyocytes in heterotypic interfaces (right panel). (B) The protocol schedule allowed the formation of heterotypic coupling at the interface over 48 h prior to optical mapping of monolayers by day 7.

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  Figure 2. Divergent anisotropies of impulse propagation by cardiomyocytes vs. Cx43+ noncardiomyocytes. (A–H) Representative activation maps (6-ms isochrones) of the entire monolayer under baseline (left column) vs. H2O2 condition (right column) during a single spontaneous excitation wavefront emerging from side-1 NRVMs, reaching the interface, and with or without success crossing the interface to reach side 2. Note the longitudinal anisotropy of Cx43+ noncardiomyocytes (horizontal arrows in C,D) in contrast to the transverse anisotropy of NRVMs (vertical/near vertical arrows in A–D). To ease comparison of the two test conditions for the same monolayer (same row), both activation maps were constructed based on the longer activation duration of the entire monolayer, which was always that under H2O2 condition. Thus, the four monolayers were mapped based on four different activation-time ranges indicated by numbers next to the color scales. The two activation durations of the two sides in a monolayer were indicated next to the corresponding sides in the monolayer activation map. *ARVF main activation direction and activation durations (C,G) were measured using the 2D wave display method (see…

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  Figure 3. Cx43+ noncardiomyocytes and H2O2 impaired NRVM activation and slowed conduction. (A–H) Representative activation maps (6-ms isochrones) of side-1 NRVMs under baseline (left column) vs. H2O2 condition (right column) during a single spontaneous excitation wavefront sweeping across side 1 of the monolayer. To ease comparison of the two test conditions for the same side 1 (same row), both maps were constructed based on the longer of the two side-1 NRVM activation durations, which was always that under H2O2 condition. Thus, the four monolayers were mapped on four different activation-time ranges indicated by numbers next to the color scales. The activation duration of side 1 in each monolayer was indicated next to its corresponding activation map. Please refer to Supplementary Figure 2 for a summary of independent and synergistic prolongation of NRVM activation duration by Cx43+ noncardiomyocytes and H2O2. (I,J) Independent and synergistic slowing of NRVM conduction by Cx43+ noncardiomyocytes and H2O2. Number of monolayers: NRVM-NRVM, n = 9; NRVM-HeLa, n = 5; NRVM-ARVF, n = 11; NRVM-HeLaCx43, n = 8. Symbols: box plots with mean (+), median, first and third quartiles (box), minimum and…

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  Figure 4. Cx43+ noncardiomyocytes and H2O2 prolonged APD by impairing NRVM repolarization. (A–H) Representative APD80 maps of side-1 NRVMs under baseline (left column) vs. H2O2 condition (right column) during a single excitation wavefront sweeping across side 1 of the monolayer. To ease intergroup and intercondition comparisons, all maps were constructed based on the longest APD80 range of 159–500 ms, which was that of the side-1 NRVM monolayer in co-culture with HeLaCx43 cells under H2O2 condition. The mean of the APD80 distribution was indicated next to the corresponding APD80 map. (I,J) Means of five APD80 measurements from five successive excitation wavefronts corrected for the spontaneous NRVM beating rate by Bazett formula under baseline vs. H2O2 condition. Note the independent and synergistic prolongation of mean APD80,c by Cx43+ noncardiomyocytes and H2O2. Number of monolayers: NRVM-NRVM, n = 9; NRVM-HeLa, n = 5; NRVM-ARVF, n = 11; NRVM-HeLaCx43, n = 8. Symbols: box plots with mean (+), median, first and third quartiles (box), and minimum and maximum (whiskers). nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA with Tukey's post-hoc analysis for…

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  Figure 5. Cx43+ noncardiomyocytes and H2O2 were independently and synergistically arrhythmogenic. (A) Cx43+ noncardiomyocytes and H2O2 independently and synergistically increased the likelihood of successful induction of new arrhythmia triggers, and non-reentrant and reentrant arrhythmias. Number of monolayers: NRVM-NRVM, n = 9; NRVM-HeLa, n = 5; NRVM-ARVF, n = 11; NRVM-HeLaCx43, n = 8. (B,C) Representative optical voltage tracings from two different monolayers illustrate how both HeLaCx43 cells and H2O2 successfully induced the emergence of new tachy-bradyarrhythmia mediated by arrhythmia triggers (B), monomorphic “ventricular tachycardia,” and bradyarrhythmia mediated by variable conduction block (C). DAD/EAD, delayed/early afterdepolarization (D,E) NRVM activation maps (6-ms isochrones) and corresponding 2D wave displays from seven and six different locations within two different NRVM-ARVF monolayers exposed to H2O2. ARVFs and H2O2 synergistically induced the emergence of new EAD-mediated non-reentrant arrhythmia (5–4–3–2–1, D right panel) and a reentrant circuit (3–2–1–2′-3′-4, E right panel). NRVM, neonatal rat ventricular cardiomyocyte; ARVF, aged rat ventricular myofibroblast.