Cardiac responses to elevated seawater temperature in Atlantic salmon

Environmental temperature has been termed the master abiotic factor which controls and limits all biochemical, physiological and life history activities in teleost fishes [1]. The thermal optimum for different species have been extensively studied, representing the temperature where the difference between routine and maximum metabolic rates is greatest; i.e. the aerobic scope is at its maximum [2]. For Atlantic salmon (Salmo salar L.), optimum temperature for growth in sea has been found to occur at 13-15°C [3], with upper critical temperatures around 22°C [4]. In response to natural temperature fluctuations outside of the thermal tolerance window, fish respond by behavioral, biochemical and physiological modifications in order to maintain cellular homeostasis and physiological performance [5, 6]. As the key organ supplying oxygen and fuels to the circulatory system for energy production, the heart has a major role in the physiological plasticity and acclimation to different thermal conditions in fish, showing alterations in cardiorespiratory performance, myocardial morphology and expression and phosphorylation of structural genes and proteins [7–10]. The occurrence of a thermal optimum (T_opt) for cardiovascular function is reflected by different salmonid species having different T_opt for maximum oxygen uptake, aerobic scope and critical swimming speed [11, 12]. At temperatures below and above T_opt the scope for aerobic metabolism will decline until a critical temperature (T_crit) is reached, where no aerobic activity can be performed besides routine metabolism [1]. In salmonids, the decreased aerobic scope observed with increasing temperatures above T_opt is associated with a limited oxygen supply suggested to be caused by a failure in maximum cardiac output to increase above T_opt[13]. Acclimation to high temperatures has been associated with cardiac remodeling of tissue composition and morphology [10], which is assumed to compensate for the decreased power-generating ability [14]. The nitric oxide synthase (NOS) system is another important inter- and intracellular regulator of cardiac function and oxygen supply in fish [15], and in long-term warm acclimated eel (Anguilla anguilla) inhibition of NO production significantly reduced the Frank-Starling response [16]. Another interesting yet poorly understood aspect of cardiac responses to temperature increase in fish is the effects on hematological and immunological responses, which may have a significant impact on the health and disease performance of Atlantic salmon in aquaculture, since heart is a target organ for several harmful viral pathogens [17, 18].

In Atlantic salmon aquaculture in the Northern hemisphere, fish are exposed to large seasonal fluctuations in seawater temperature. Peak summer temperatures around 18-20°C are regularly experienced at production sites in the western and southern regions of Norway, causing concerns regarding the possible negative impact on productivity, fish performance and welfare. We recently reported that long-term exposure (56 days, simulating a warm water period in aquaculture) of adult (~2 kg) Atlantic salmon to 19°C under controlled conditions significantly reduced growth performance when compared to fish reared at 14°C, a difference driven by a 50% reduction in feed intake [19]. The objective of the present study was to investigate effects of such temperature increase on molecular responses in cardiac tissues from the same experimental fish. To achieve this, cDNA microarray screening and single gene expression validation with real-time qPCR were employed to evaluate transcriptional changes after 21 days (simulating a short warm water period) and 56 days (simulating a long warm water period) thermal acclimation. In addition, expression of selected proteins of interest were analysed with immunofluorescence microscopy in cardiac tissues after long-term thermal acclimation.

Seasonal fluctuations in seawater temperature are naturally occurring in the aquaculture of Atlantic salmon. Particularly in the summer months, western and southern regions of Norway experience temperature increments above thermal preference (15°C [3, 29, 30]) peaking around 18-20°C. In a recent study we observed significantly reduced feed intake, growth performance and endogenous energy storage in large (~2 kg) Atlantic salmon after long-term exposure to 19°C under controlled conditions [19]. This poor performance was linked to suppressed endocrine appetite regulation, leading to a negative energy homeostasis with depleted lipid stores in muscle and whole carcass. Based on these findings and the same experimental fish, the current study aimed to further understand how the chronic temperature elevation affected molecular processes at the levels of gene and protein expression in the heart, as a key organ for thermal plasticity and acclimation in salmonids [31, 32].

Among genes that were strongly up-regulated with elevated temperature, it was not surprising to find several chaperones and other genes involved in the unfolded protein response. This included three heat shock proteins (HSPs), which are among the most studied proteins in the general response to a variety of stressors and perturbations in mammals and fish [33]. Although their use as suitable indicators of stressed states in fish has been disputed [34], synthesis of HSPs to maintain proper folding/refolding of proteins has been shown highly expensive for the organism [35]. In spite of a severely compromised energy homeostasis in the experimental fish, the sustained induced transcription of HSPs over time could indicate a need for chaperone activity and the unfolded protein response during cardiac acclimation to the elevated temperature.

The nitric oxide synthase system is an important inter- and intracellular regulator of mechanical performance and oxygen supply in the fish heart [15], and iNOS is exclusively expressed in ventricular cardiomyocytes under basal condition and after LPS stimulation [36]. Although the physiological and pathophysiological regulation of the NOS/NO system is very complex and only partly understood in fish, studies have shown that NOS influences cardiac inotropy (i.e. cardiac output) of salmon [37] and the red-blooded icefish T. bernacchii[36], which was also sensitive to acute temperature change [16]. Our results showing increased immunofluorescence staining of iNOS in compact and spongy myocardium may be a further indication of an involvement in the thermal acclimation or response to increased temperature. NO has also been shown to induce vasodilation and reduce coronary resistance under hypoxia in salmonids [38]. In this process, VEGF is another important regulator which promotes proliferation and migration of endothelial cells in the formation of new blood vessels under cardiac physiological and pathological conditions [39]. Whether the induced expression of VEGF and iNOS in our study reflected increased vascularization and/or vasodilation to compensate for increased oxygen demand with elevated temperature should be subject to further study. In this regard, the lower abundance of several transcripts for heme biosynthesis and ATP production/energy metabolism after short- and long-term exposure to elevated temperature could also indicate that oxygen transport/uptake and possibly aerobic metabolism was affected.

A fundamental mode for increasing oxygen carrying capacity in vertebrates is by increasing the cardiac output. In salmonids, several studies have suggested that a limitation at the level of the heart is the primary cause of limited oxygen supply with increasing temperature because maximum cardiac output fails to increase above thermal optimum [13]. Furthermore, acclimation to high temperatures has been associated with increased thickness of the compact myocardium, which is assumed to compensate for the decreased power-generating ability [14] or simply reflecting an increased activity level at higher temperatures [40]. A recent study with warm-acclimated rainbow trout also reported that the increased thickness of the compact layer was associated with reduced connective tissue, however this was only observed for male and not female fish [10]. Correspondingly, we observed lower abundance of collagen mRNA with high temperature, however effects of sex was not determined. On the contrary, immunostaining demonstrated increased deposition of collagen I in large bundles exclusively in the compact myocardium at high temperature. This discrepancy between protein and transcript levels of collagen could be methodology based, since gene expression was measured in total RNA extracted from the whole myocardium (both spongy and compact layers) whereas immunofluorescence detection of collagen showed increased staining with temperature only in the compact myocardium, which typically comprises 30-50% of the heart in athletic fishes [7]. In fact, Klaiman et al.[10] reported reduction in muscle bundle area in spongy myocardium with warm acclimation, thus lower collagen gene expression levels could simply reflect altered proportions of compartments with increased temperature. Pointing in the same direction, we also observed substantially lower transcript levels of several cytoskeleton genes including actin, myosin and troponin after short- and long-term acclimation. In view of the severely compromised growth performance and energy homeostasis of the fish in this study after long-term high temperature, implications of the compact myocardium increase in collagen I protein expression on cardiac function and possibly fibrosis should be further studied. Reduced mitochondrial capacity above thermal optimum has been demonstrated for fish [41]. Interestingly, we found significantly higher mRNA levels of CPT1 (after 56 days) and PGC1α (after 21 days) in fish kept at 19°C. It could be speculated whether this was a tissue response in order to balance the overall energetic status, a notion supported from reduced hepatic beta-oxidation in the same fish [19].

High temperature affected cardiac expression of important immune-related genes. The salmon heart is an immune-relevant organ in the sense that it represents a major replication site for several viral pathogens of high importance [17, 18]. Common for these diseases is the strong inflammation of the myocardium which coincides with a strong activation of a CD8 T cell response [18, 23, 42]. Consequently, given the temperature-induced gene expression of cellular immune components including CD8 alpha in the present study, it may be speculated if high seawater temperatures can further elevate myocardial inflammation during infection and thus represent a relevant risk factor affecting disease outcome. It has been proposed that low environmental temperature diminishes the humoral immune response, since primary antibody response has been found to be either dampened or retarded at lower temperatures [43, 44]. Here, we report that expression of some genes involved in the innate humoral immune response were down-regulated with high temperature. At the same time, a suite of genes representing cellular components were up-regulated, including some involved in B- and T-cell development. Our understanding of immunological effects of elevated temperatures in fish is far from complete, but studies have shown significant hematological modulation in response to thermal acclimation in salmonids [45–47]. A study with rainbow trout reported induced complement lytic activity and opsonization capacity with increased temperature in rainbow trout [48], which could agree with our finding of temperature-induced gene expression of complement C6, a part of the membrane attack complex. Another aspect relates to whether our observed regulation of cardiac innate immune response genes is ascribed to the temperature increase per se or (re)allocation of resources in view of the negative growth performance and energy homeostasis of the fish. This is a subject that should deserve attention in future research.

This study provides new knowledge on the molecular responses of cardiac tissues during long-term exposure to elevated seawater temperature reflective of the peak summer temperatures experienced in Atlantic salmon aquaculture. We report temperature-induced changes in expression of genes and proteins indicating that the unfolded protein response, vascularization and remodeling of connective tissue as well as altered innate immune responses were affected during cardiac acclimation to elevated temperature.