Use of cardiac cell cultures from salmonids to measure the cardiotoxic effect of environmental pollutants

Abstract

Environmental stressors such as micro- and nanosized plastic particles (MNPs) or crude oil have a detrimental effect on aquatic animals; however, the impact upon the cardiovascular system of fish remains relatively under-researched. This study presents a novel approach for investigating the effect of crude oil and MNPs on the cardiac system of fish. We used salmonid larvae and cardiac cell cultures derived from hearts of salmonid fish and exposed them to environmental stressors. Following exposure to plastic particles or crude oil, the larvae exhibited some variation in contraction rate. In contrast, significant alterations in the contraction rate were observed in all cardiac cell cultures. The greatest differences between the control and treatment groups were observed in cardiac cell cultures derived from older brown trout. Following 7 days of exposure to MNPs or crude oil in Atlantic salmon larval hearts or cardiac cell cultures, there were only minor responses noted in mRNA expression of the selected marker genes. These findings show the use of a novel in vitro technique contributing to the existing body of knowledge on the impact of MNPs and crude oil on the cardiovascular system of salmonids and the associated risk.

Keywords: cell cultures; crude oil; heart; microplastic; nanoplastic.

FIGURE 1

Heart rate in Atlantic salmon (Panel P2), brown trout (Panel P3) and rainbow trout (Panel P4) larvae during exposure to microplastic particles (MP), nanoplastic particles (NP) and crude oil (CrO) at different concentrations. Non‐exposed larvae are treated as control (CTR). The overview (Panel P1) provides a definition of a heartbeat and an illustration of the method of counting it. A heartbeat is defined as the transition from a state of the heart in which the blood is absent (P1a) to a state in which the blood is present (P1b), and then back to a state of the heart in which the blood is absent. The red circles indicate the position and the state of the heart, which may be either bloodless (clear appearance of the heart) (P1a) or blood‐filled (dark appearance of the heart) (P1b). The number of heartbeats was counted manually with a hand counter (P1c). Results are presented as scatter plots of the number of heartbeats per 30 s with all data points and the mean shown. * Indicates statistically significant differences in cardiac contractions of exposed larvae compared to unexposed controls within the time point at p < .05. Statistical analysis was performed using two‐way ANOVA, with multiple comparison test performed using the Holm‐Sidak method.

FIGURE 2

Cardiac cell cultures from rainbow trout. (a) Immunocytochemistry for alpha‐sarcomeric actin (green), nuclei (red) residual autofluorescence (yellow). (b) Immunocytochemistry for tropomyosin (green), nuclei (red) residual autofluorescence (yellow). (c and d) frames from Video S1 showing the non‐contracted (c) and contracted culture (d), note the areas of contraction marked with white and black arrows and the reduction in distance (indicated by distance units—du) between the dead cell aggregates during contraction.

FIGURE 3

Number of contractions per 30 s in cardiac cell cultures from Atlantic salmon (Panel P2), brown trout (Panel P3) and rainbow trout (Panel P4) larvae and older brown trout (Panel P5) during exposure to microplastic particles (MP), nanoplastic particles (NP) and crude oil (CrO) at different concentrations. Non‐exposed cardiac cell cultures are treated as control (CTR). The overview (Panel P1) illustrates the definition of a contraction of the cardiac cell culture and the method of counting it. A contraction is defined as the transition from a resting, non‐contracting state of the cells (P1a) to contracting state of the cells (P1b), and then back to a resting, non‐contracting state of the cells. Contractions were counted by ImageJ tool Myocyter (P1c). Results are presented as scatter plots with all data points and the mean shown. * Indicates statistically significant differences in cardiac contractions of exposed cell cultures compared to unexposed controls within the time point at p < .05. Statistical analysis was performed using two‐way ANOVA with multiple comparison test using the Holm‐Sidak method.

FIGURE 4

Levels of mRNA encoding Atlantic salmon genes related to oxidative stress (a: Cat, b: Gpx1a, c: Gpx1b1, d: Gpx1b2, e: Sod2), pro‐inflammatory response (f: Il‐1b, g: Il‐6, h: Il‐8) and suppressors of cytokine signalling (i: Socs1, j: Socs3) after 7‐day exposure of Atlantic salmon larvae and Atlantic salmon heart cell cultures to different concentrations of micro plastic (MP), nano plastic (NP) particles or crude oil. Results are presented as scatter plots with all data points and the mean shown. Data are presented as normalized copy numbers. * indicates statistically significant differences in expression levels of exposed cardiac cells and exposed hearts of Atlantic salmon larvae compared to unexposed controls at p < .05. Statistical analysis was performed using one‐way ANOVA. Where data were not normally distributed, Kruskal‐Wallis one‐way ANOVA on ranks was used.