Understanding the cardiac toxicity of the anthropogenic pollutant phenanthrene on the freshwater indicator species, the brown trout (Salmo trutta): From whole heart to cardiomyocytes

Abstract

Freshwater systems are faced with a myriad of stressors including geomorphological alterations, nutrient overloading and pollution. Previous studies in marine fish showed polyaromatic hydrocarbons (PAHs) to be cardiotoxic. However, the cardiotoxicity of anthropogenic pollutants in freshwater fishes is unclear and has not been examined across multiple levels of cardiac organization. Here we investigated the effect of phenanthrene (Phe), a pervasive anthropogenic pollutant on a sentinel freshwater species, the brown trout (Salmo trutta). We first examined the electrical activity of the whole heart and found prolongation (∼8.6%) of the QT interval (time between ventricular depolarization and repolarization) of the electrocardiogram (ECG) and prolongation (∼13.2%) of the monophasic action potential duration (MAPD) following ascending doses of Phe. At the tissue level, Phe significantly reduced trabecular force generation by ∼24% at concentration 15 μM and above, suggesting Phe reduces cellular calcium cycling. This finding was supported by florescent microscopy showing a reduction (∼39%) in the intracellular calcium transient amplitude following Phe exposure in isolated brown trout ventricular myocytes. Single-cell electrophysiology was used to reveal the mechanism underlying contractile and electrical dysfunction following Phe exposure. A Phe-dependent reduction (∼38%) in the L-type Ca²⁺ current accounts, at least in part, for the lowered Ca²⁺ transient and force production. Prolongation of the MAPD and QT interval was explained by a reduction (∼70%) in the repolarising delayed rectifier K⁺ current following Phe exposure. Taken together, our study shows a direct impact of Phe across multiple levels of cardiac organization in a key freshwater salmonid.

Keywords: AP; Cardiac action potential; ECG; Electrophysiology; Fish; PAHs; Poly aromatic hydrocarbons; Toxicology; brown trout.

Fig. 1

The impact of Phenanthrene (Phe) on the electrical properties of the brown trout heart. (A) Representative ECG traces from a heart contracting at 0.5 Hz showing QT interval prolongation in presence of increasing Phe concentration. (B) The corrected QT interval (QTc, see methods) under control conditions and upon exposure to 15 μM of Phe at 0.5 Hz, and 0.8 Hz . (C) Representative MAP traces from an isolated heart at 0.5 Hz showing QT interval prolongation in presence of Phe concentration at 15 μM and above. (D) Mean data showing MAP duration at 90% repolarization (MAPD₉₀) in control conditions and following exposure to 15 μM Phe. Individual hearts are shown by circles with bar showing mean ± S.E.M (n = 7–9 hearts). *p < 0.05, two-way ANOVA. MAP and QT_c data for all exposure concentrations and all frequencies are shown in Table S1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

The impact of Phenanthrene (Phe) on the contractile properties of the brown trout heart. Force of contraction was significantly reduced when ventricular strips were exposed to 15 μM Phe and stimulated at varying frequency (0.2–1.0 Hz). (A) Representative trace from a ventricular strip contracting at 0.2 Hz showing peak height which was then converted to force. Mean ± S.E.M force expressed as percentage, normalised to control force contracting at 0.2 Hz in (B) DMSO control exposed group and (C) 15 μM exposed group. The solid black line represents the basal force of contraction prior to exposure of the strips to either DMSO or 15 μM Phe (blue line) whereas the dotted black line shows adrenaline (1 μM) exposure increasing contractility, attenuating the effects of Phe. (n = 8–12) *p < 0.05, two-way ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Intracellular Ca²⁺ recording from ventricular myocytes of brown trout exposed to 5, 15 and 25 μM Phe incubation 1 h prior to recording. Phe significantly reduced intracellular Ca²⁺ transients. (A) Ca²⁺ transient signals recorded from myocytes contracting at 0.2 Hz. Black trace represent control condition (exposed to ≤ 0.1% DMSO ) while the red, blue and green line represent traces from cells exposed to 5, 15 and 25 μM Phe respectively. (B) Ca²⁺ transient mean amplitude (ΔF/F₀) in control and increasing Phe concentrations (red bar, 5 μM; blue bar 15 μM; red bar, 25 μM) (** p < 0.01, one-way ANOVA). (C) Bar graph showing significant effect by Phe on Ca²⁺ rise time (* p < 0.05, one-way ANOVA). (D) Bar graph showing non-significant reduction of decay of Ca²⁺ transient calculated as tau (ms). Values are mean ± S.E.M (N = 6, n = 69, control; 54, 5 μM; 67, 15 μM; and 62, 25 μM). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Phenanthrene (Phe) disrupts cardiac ion flux in isolated brown trout ventricular cardiomyocytes. (A) Effect of Phe on action potential duration (APD). (Ai) Representative traces of action potential elicited at 0.5 Hz in absence (Control) and presence of 10 μM Phe and (Aii) Mean action potential duration at 10% (APD₁₀), 50% (APD₅₀) and 90% (APD₉₀) in absence (Control) and presence of 10 μM and 30 μM Phe (n = 5 for each data set from min. two fish). (B) Effect of Phe on I_kr currents. (Bi) Representative traces of I_kr currents in absence (Control) and presence of 10 μM Phe; inset – voltage protocol used to elicit I_kr currents. (Bii) Bar graph of mean I_kr current density in absence (Control) and presence of 10 μM Phe (*** p=0.0002; n = 6 from min. two fish). (Biii) concentration-response curve yielded an the IC₅₀ value of 7.2 ± 0.6 μM (n = 6 for each data set from min. two fish). (C) Effect of Phe on I_CaL currents. (Ci) Representative trace of I_CaL currents in absence (Control) and presence of 30 μM Phe; inset – voltage protocol used to elicit I_CaL currents. (Cii) Mean I_CaL current density in absence (Control) and presence of 30 μM Phe (** p=0.001; n = 4 from two fish). Values are mean ± S.E.M (total n = 34 from total of 8 fishes).

Fig. 5

Summary of the effects of phenanthrene on fish cardiovascular system and the potential implications of exposure on fish species population.