Cardiac responses to elevated seawater temperature in Atlantic salmon

Abstract

Background: Atlantic salmon aquaculture operations in the Northern hemisphere experience large seasonal fluctuations in seawater temperature. With summer temperatures often peaking around 18-20°C there is growing concern about the effects on fish health and performance. Since the heart has a major role in the physiological plasticity and acclimation to different thermal conditions in fish, we wanted to investigate how three and eight weeks exposure of adult Atlantic salmon to 19°C, previously shown to significantly reduce growth performance, affected expression of relevant genes and proteins in cardiac tissues under experimental conditions.

Results: Transcriptional responses in cardiac tissues after three and eight weeks exposure to 19°C (compared to thermal preference, 14°C) were analyzed with cDNA microarrays and validated by expression analysis of selected genes and proteins using real-time qPCR and immunofluorescence microscopy. Up-regulation of heat shock proteins and cell signaling genes may indicate involvement of the unfolded protein response in long-term acclimation to elevated temperature. Increased immunofluorescence staining of inducible nitric oxide synthase in spongy and compact myocardium as well as increased staining of vascular endothelial growth factor in epicardium could reflect induced vascularization and vasodilation, possibly related to increased oxygen demand. Increased staining of collagen I in the compact myocardium of 19°C fish may be indicative of a remodeling of connective tissue with long-term warm acclimation. Finally, higher abundance of transcripts for genes involved in innate cellular immunity and lower abundance of transcripts for humoral immune components implied altered immune competence in response to elevated temperature.

Conclusions: Long-term exposure of Atlantic salmon to 19°C resulted in cardiac gene and protein expression changes indicating that the unfolded protein response, vascularization, remodeling of connective tissue and altered innate immune responses were part of the cardiac acclimation or response to elevated temperature.

Figure 1

Cardiac expression of selected genes in fish reared under normal and elevated temperature. Relative mRNA transcription levels of A)heat shock protein 70, HSP70; B)carnitine palmitoyltransferase 1, CPT1; C)peroxisome proliferator-activated receptor (PPAR)γ coactivator 1α, PGC1α; D)α-hemoglobin; E) T cell antigen CD8 alpha in fish reared at normal (14°C, filled circles) and elevated (19°C, open circles) temperature for 21 and 56 days. Data are mean log₂ expression ratio ± SEM relative to the average of normalized controls (14°C, 21 days), real-time qPCR. Statistical differences (adjusted p-value < 0.05; pairwise t-tests of the four groups, N = 9) are indicated between temperatures (21 days: a*, 56 days: b*) and time points for 19°C fish (c*).

Figure 2

Inducible nitric oxide synthase (iNOS) expression in cardiac tissues of fish reared under normal and elevated temperature. Immunofluorescence staining of iNOS (red color) and DAPI nuclear counterstain (white color) in cardiac tissues of fish reared for 56 days at normal temperature (14°C, upper panels) and high temperature (19°C, lower panels). At 14°C iNOS is expressed at low levels and in a few cells in the compact (A) and spongy (B) myocardium. At 19°C iNOS is expressed in a higher number of cells in the compact myocardium (C), and strong staining of individual myocytes is observed in the spongy myocardium (D). Panels A-D show one representative micrograph of three sections examined per fish from a total of three fish per temperature group. The 40 μm scale bar in panel A applies to all panels in the figure. E: Average number of positive cells ± SEM in spongy myocardium, calculated using a larger field of view (25× objective).

Figure 3

Vascular endothelial growth factor (VEGF) expression in cardiac tissues of fish reared under normal and elevated temperature. Immunofluorescence staining of VEGF (red color) in cardiac tissues of fish reared for 56 days at control temperature (14°C, A) and elevated temperature (19°C, B). A: VEGF positive cells (arrows) are mainly located around already existing epicardial vasculature at 14°C. B: VEGF positive cells (arrows) are evenly distributed along the entire epicardium at 19°C. Panels A-B show one representative micrograph of three sections examined per fish from a total of three fish per temperature group, with one representative region per group shown at higher magnification (inset). The 400 μm scale bar in panel A applies to both panels in the figure. C: Average number of positive cells per mm epicardium ± SEM, calculated using a larger field of view (25× objective).

Figure 4

Collagen I expression in cardiac tissues of fish reared at normal and elevated temperature. Immunofluorescence staining of collagen I (red color) and DAPI nuclear counterstain (white color) in cardiac tissues of fish reared for 56 days at 14°C (left panels, A, C, E) and 19°C (right panels, B, D, F). A: Collagen I is abundant in the epicardium and vasculature (arrow) at 14°C. B: Increased signal intensity is observed at 19°C. C: At 14°C collagen I is expressed in cell clusters in the compact (c) but not in the spongy (s) myocardium. D: At 19°C collagen I is strongly expressed in larger structures resembling connective tissue of the compact (c) but not in the spongy (s) myocardium. E-F: Cells in the spongy myocardium show weak staining at both temperatures. Fluorescence intensities are shown as LUT (Look-Up Table) images in panels A-D (inset). Panels show one representative micrograph of three sections examined per fish from a total of three fish per temperature group. The 50 μm scale bar in panel A applies to all panels in the figure.