Phenanthrene impacts zebrafish cardiomyocyte excitability by inhibiting IKr and shortening action potential duration

Figures

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  Figure 1.. Phenanthrene (Phe) shortens APD in zebrafish ventricular cardiomyocytes. (A and B) Representative AP traces at 0.5 Hz exposed to 3 µM Phe (A) and 10 µM Phe (B; with and without 2 µM E-4031 [ERG channel blocker]) with recovery from inhibition (wash). (A) Inset: Molecular structure of phenanthrene. (C and D) Scatter plot showing mean ± SEM of APD50 and APD90 in the absence and presence of 3 µM Phe (C; *, P < 0.05; Wilcoxon test) and 10 µM Phe (D; *, P < 0.05; Wilcoxon test). (E and F) No significant change in resting membrane potential (RMP; E) and dV/dt was observed in presence of phenanthrene (F). All data represented as mean ± SEM (n = 4–9; N = 5).

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  Figure 2.. Effect of 10 µM phenanthrene (Phe) on L-type Ca2+ channel currents (ICaL) in ventricular zebrafish myocytes. (A) Representative current traces. (B) Scatter plot showing mean ± SEM. ICaL density elicited by depolarization to −10 mV (n = 10; N = 3; **, P < 0.01; Wilcoxon test) in absence (control; black) and presence of 10 µM phenanthrene (orange). Dotted line represents current trace of recovery from inhibition after 5 min washout. (C) Concentration–response curve of phenanthrene on ICaL, revealing an IC50 of 4.8 ± 0.8 µM (Hill slope, nH = 1.3 ± 0.2). (D) Peak ICaL density I-V plot (n = 6; N = 2; *, P < 0.05; two-way ANOVA with the Holm–Šídák post hoc analysis). (E) steady-state activation (G/Gmax) and inactivation (I/Imax) obtained using (inset) double-pulse protocol in the absence (control; black) and presence of 10 µM phenanthrene (orange; n = 6; N = 2). Act, activation; Inact, inactivation.

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  Figure S1.. Effect of phenanthrene (Phe) on Ba2+ currents (IBaL) of L-type Ca2+ channels. (A) Representative current traces of IBaL elicited by depolarization to −10 mV in the absence (control; black) and presence of 10 µM phenanthrene (orange). (B) Bar graph showing mean ± SEM percentage change in peak currents (I) and total charge (Q) of Ca2+ (n = 5–10; N = 2) and Ba2+ (n = 5; N = 3) in the absence and presence of 10 µM phenanthrene (*, P < 0.05; Mann–Whitney Test). (C and D) Representative traces of cumulative charge transfer as integral of time for calcium currents (C) and barium currents (D) in the absence and presence of 10 µM phenanthrene.

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  Figure 3.. Phenanthrene (Phe) inhibits the native zfERG channel (IKr) and increases rate of channel deactivation. (A) Representative IKr peak tail current trace recorded in absence (black) and presence of 3 µM phenanthrene (red; inset) elicited by voltage protocol. (B) Concentration–response curve of phenanthrene on peak IKr tails, revealing an IC50 of 3.3 ± 0.2 µM (Hill slope, nH = 1.8 ± 0.2). (C) Time-course plot showing a maximum of 69 ± 5% recovery from inhibition by 10 µM phenanthrene after 10 min of washout (n = 6; N = 2). (D) Representative IKr tails (normalized to peak) in the absence (black) and presence of 1 µM (blue) and 3 µM (red) phenanthrene, fitted to a standard double exponential curve (green), to evaluate rate of current decay. (E and F) Bar plot presenting time-constant values of fast- and slow-deactivating current components (τfast and τslow, respectively; E; P < 0.01) and proportion of amplitude of fast-deactivating current component (Af) as a fraction of total amplitude of fast- and slow-deactivating current component (Af + As) obtained from fitting (F) show significant decrease in τfast and corresponding increase in Af component with increasing concentration in…

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  Figure S2.. Effect of phenanthrene (Phe) on other potassium current (IK). (A) Representative current traces of net IK elicited by depolarization to +40 mV in the absence (control; black) and presence of 3 µM phenanthrene (red). External solution contained 2 µM E-4031 to block IKr. (B) Bar graph showing mean ± SEM percentage change in peak currents in the absence and presence of 3 µM (n = 5; N = 2) and 25 µM (n = 4; N = 2) phenanthrene.

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  Figure 4.. Phenanthrene (Phe) significantly shifts the voltage of repolarizing IKr during AP-like command by −6 mV. (A) Representative traces of ventricular AP-like command waveform protocol used to elicit shown IKr current trace. (B) Mean ± SEM percentage response of IKr exhibiting an inhibition of 24 ± 7% in the presence of 3 µM phenanthrene (red). Mean peak voltage during the repolarization phase is shown in the corresponding colored dashed line exhibiting a significant shift of ∼6 mV from −46.4 mV for control to −52.5 mV in the presence of 3 µM phenanthrene (n = 6; N = 2; *, P < 0.05; Wilcoxon test).

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  Figure 5.. Effect of phenanthrene (Phe) on response of IKr to premature stimulation. (A) Magnified view of the dotted area of (inset) paired ventricular AP-like command waveform protocol. (B–D) Representative trace of families of transient currents elicited corresponding to each depolarization in the absence (B; black) and presence (C) of 3 µM phenanthrene (red) and 2 µM E-4031 (D; blue). (E) Percentage response of peak IKr transients in the absence (black) and presence of 3 µM phenanthrene (red). Peak IKr transients for each cell under control conditions were normalized to the maximal current transient during the protocol. Peak IKr transients in the presence of 3 µM phenanthrene (red) are represented as percentage response relative to its corresponding percentage control transient. First, peak, and last transient obtained are labeled a, b, and c, respectively. All data represented as mean ± SEM (n = 7 or 8; N = 3).