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    Topic review

    Hypoxic/Thermal Stress in Fish Heart

    Subjects: Physiology
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    Teleost fish are often regarded with interest for the remarkable ability of several species to tolerate even dramatic stresses, either internal or external, as in the case of fluctuations in O2 availability and temperature regimes. These events are naturally experienced by many fish species under different time scales, but they are now exacerbated by growing environmental changes. This further challenges the intrinsic ability of animals to cope with stress. The heart is crucial for the stress response, since a proper modulation of the cardiac function allows blood perfusion to the whole organism, particularly to respiratory organs and the brain. In cardiac cells, key signalling pathways are activated for maintaining molecular equilibrium, thus improving stress tolerance. In fish, the nitric oxide synthase (NOS)/nitric oxide (NO) system is fundamental for modulating the basal cardiac performance and is involved in the control of many adaptive responses to stress, including those related to variations in O2 and thermal regimes.

    1. Introduction

    Living organisms are constantly exposed to stress. After Seyle’s definition of stress as “the non-specific response of the body to any demand placed upon it” and the consequent declinations of the different degrees of responses from eustress to distress [1][2], decades of research have established that the ability to face stress-dependent challenges represents a basic mechanism to maintain organism homeostasis. Many factors shape the stress response, making the scenario highly complex. In the attempt to draw a framework in which networks and circuits involved in the crosstalk between the environment and the organisms are recapitulated, several stress-related concepts have been developed, such as stressome (the catalogue of genes and their products involved in maladaptive stress response) and stressotope (the adaptive background that includes the circuits of molecular mediators involved in the stress response, up to the population level) [3]. An important aspect in the context of the adaptive response is that multiple stresses often converge in challenging the organism, and this elicits a response that is the result of many events, occurring at a multilevel scale, from the whole organism to molecular signalling [3]. This is the case of related stresses, such as hypoxia and temperature that, particularly in water environments, move together. 
    An important body of the literature describes the mechanisms that, in aquatic species, sustain cardiac adaptation to environmental challenges, with attention not only to the upper and lower limits of this adaptation, but also to the molecular pathways that are recruited during the exposure to multiple challenges, an event that is becoming more and more frequent in damaged natural environments. A recent example is the cardiac transcriptomic response described in Fundulus grandis developing larvae to four combined stressors (O2 availability, temperature, salinity, and polycyclic aromatic hydrocarbons) [4]. It was found that each single stress, administered alone, affects the heart in terms of beat-to-beat hemodynamic and development. At the same time, stress combination potentiates the effects on the heart, either positively or negatively, by affecting canonical pathways involved in heart contractility, vasomotility, and cardiomyocyte proliferation. These pathways include the cardiac nitrergic system [4]. As demonstrated by many papers, in the heart, this system is at the crossroads of many stress-sensitive circuits [5][6][7][8][9][10]. It is under a modulation elicited by stress, and at the same time, it is crucial for shaping the stress response, either adaptive or maladaptive (Figure 1).
    Figure 1. Graphical representation of the review content. Under conditions of O2 limitations and temperature variations, the nitric oxide synthase (NOS)/nitric oxide (NO) system, by directly or indirectly interacting with critical kinases, e.g., AMP-activated protein kinase (AMPK), modulates molecular mechanisms that in the fish heart sustain stress tolerance and adaptation.

    2. The NOS/NO System and the Fish Heart

    NO is a gasotransmitter generated by the family of NOS isoenzymes, which include the constitutive endothelial (eNOS) and neuronal (nNOS) and the inducible (iNOS) isoforms. By using molecular O2 and NADPH as essential cofactors, these enzymes convert L-arginine into L-citrulline and NO.
    NO exerts its physiological effects either by soluble guanylate cyclase (sGC)-dependent mechanisms or reacting with hemes, thiols, or amines, forming iron-nitrosyl (FeNO), S-nitroso (SNO), and N-nitroso (NNO) compounds [11]. NO has a very short half-life. It is rapidly metabolized to nitrite in reaction with O2 [12] and is inactivated by oxidation to nitrate in reaction with oxygenated hemoglobin (Hb) and myoglobin (Mb). Under hypoxic and/or acidic conditions, nitrite may represent a reservoir of NO, since it can be reduced back to NO by a variety of non-enzymatic and enzymatic pathways. 
    Although non-univocal results can be found in the literature, it is currently recognized that, as in mammals, a functional NOS/NO system is present in the fish heart [13][14][15][16]
    Cardiac NOSs have been documented in eurythermal fish species (Thunnus thynnus thynnus [17]; Anguilla anguilla (A. anguilla) [17][18]; Carassius auratus (C. auratus) [8]), cold-adapted Antarctic teleosts (icefish Chaenocephalus aceratus (C. aceratus), hemoglobinless Chionodraco hamatus (C. hamatus), and red-blooded Trematomus bernacchii (T. bernacchii) [19][20]), and lungfish (Protopterus dolloi [21] and Protopterus annectens [7]).
    In steelhead trout (Oncorhynchus mykiss (O. mykiss)), exogenous NO enhances myocardial relaxation and this, in turn, influences isometric twitch duration and muscle contractility. This effect is related to contraction frequency, as the twitch duration is reduced by 25% at a frequency of 20 beats per min (bpm), but only by 5% at 80 bpm [22]. In eel [23], salmon [24], and goldfish [8], a tonic release of NO negatively modulates the basal mechanical performance of the heart. In addition, NO influences the Frank–Starling mechanism (salmon [24]; eel [25]; goldfish [8]), a fundamental cardiac trait that, in all vertebrates, allows the myocardium to increase stroke volume (SV), and consequent cardiac output (CO), in response to increased venous return (preload). As detected in the goldfish isolated and perfused heart, the sensitivity to filling pressure decreases when NO generation is prevented by NOS inhibition via L-NMMA or, downstream, when cGMP production is blocked by ODQ [8], thus involving the classic NO/cGMP-dependent cascade. As in the steelhead trout [22], in the eel, the nitrergic control of the Frank-Starling response is related to an enhanced relaxation, possibly due to calcium reuptake by SERCA2a pumps controlled by phospholamban S-nitrosylation [20]. Of note, NO produced in the heart can mediate actions distal from the site of production thanks to the storage into the blood as nitrite. In fish, compared to terrestrial animals, the nitrite pool can be enriched by uptaking nitrite from the environment across the respiratory surfaces [26]. In addition, as observed in the crucian carp (Carassius carassius (C. carassius)), the tissue nitrite pool may be further increased by the nitrate reductase activity generally mediated by xanthine oxidoreductase and Mb [27]. This results in an enhanced NO availability with consequent larger effects on the cardiovascular function. This mechanism is of particular importance during deep hypoxia and anoxia when, being NOS enzymes unable to produce NO because of the absence of O2, it contributes to cardioprotection [27][28].
    The role of NO2 in the cardiac nitrergic control has been studied in the eel A. anguilla, and in the icefish C. hamatus, two species characterized by extremely different ecophysiological traits [29]. In the eel, nitrite negatively influences the contractility of the isolated and perfused heart. This occurs by modulating the NOS activity and the cGMP/PKG pathway. Moreover, nitrite influences the Frank–Starling response through a mechanism which recruits the NO/cGMP/PKG pathway and requires protein S-nitrosylation [30]. Contrary to the eel, in the icefish nitrite induces a NOS-dependent increase in contractility, similar to the effect induced by NO. Icefish lacks Hb, a key protein in NO homeostasis, which is able to not only scavenge NO, but also generate it from NO2 (for references, see [29]). It has been proposed that, in the icefish, the reduction of NO2 to NO occurs through cardiac Mb that in this fish may represent th[31] predominant form of NO2 reductase [29].

    2.1. Hypoxia

    Water hypoxia (O2 levels: ±2.8mg L-1; [32] is a major limiting environmental factor. It is routinely experienced by aquatic animals, either chronically or on a diel or seasonal basis, and results from complex processes including mixing, air–water exchange, and fluctuations in the pattern of O2 production and consumption [33] [34]. In recent years, aquatic hypoxia is increasing due to anthropogenic influences and climate changes [35][36], with severe impact on individual organisms, communities, and ecosystems.

    In the heart, a general response to hypoxia is bradycardia, together with the depression of myocardial contractility, and the O2 consumption rate. These effects are particularly evident in hypoxia-intolerant species [37][38][39]. In contrast, hypoxia-tolerant species such as the crucian carp and the goldfish retain a normal cardiac function and even potentiate it by activating a complex molecular machinery that is only partially known [8][40][41].

    2.2. The NOS/NO System in the Fish Heart under Hypoxia

    A general effect of the activation of the NOS/NO system in the heart under hypoxia is the limitation of mitochondrial O2 consumption for the NO competitive binding with O2 to CytC oxidase [42]. When O2 availability is reduced, this contributes to preserving myocardial efficiency by enhancing the force generated per O2 consumed [43][44]. A comparison between the cardiac response to low O2 in hypoxia-intolerant vs. hypoxia-tolerant fish has been carried out by using ventricular strips from trout and goldfish [38]. In both species, NO generated by NOS activation inhibits respiration rates and contributes to improving myocardial efficiency. However, when NO is generated from nitrite conversion, different behaviors are observed in the two species. In fact, in trout but not in goldfish, myocardial O2 consumption is reduced without changes in force development. This is attributed to differences in oxygen affinity and then in the nitrite reductase capacity of myocardial Mb. With less O2 available, trout Mb may readily de-oxygenate, thus generating NO from nitrite, while the goldfish Mb, by remaining saturated with O2, is prevented by reducing nitrite [38]. In the steelhead trout, the role of the nitrergic system in the mechanical response of the heart to low O2 has been further investigated [22]. By exposing spongy ventricular strips from animals acclimated to low oxygen (PO2 = 8 kPa) to the NO donor (SNP), they observed that hypoxic acclimation scarcely influences the frequency-related NO-dependent effect on twitch duration and muscle contractility. The above studies suggest that, in trout, hypoxia exposure does not significantly influence the cardiac isometric contractility in response to NO. However, the authors do not exclude the possibility that the use of muscle strips may fail to reveal additional effects of NO that may be preserved in the whole heart preparation, closer to the in vivo situation [22][38]. In line with this, evidence obtained in goldfish, by using ex vivo isolated and perfused working heart preparations, a technique that prevents the constraints imposed by the use of limited parts of the organ, shows that the potentiated basal performance, typical of acute O2 limitation, is accompanied by an increased myocardial NOS expression [8]. The possibility that a more expressed enzyme generates a higher amount of NO and this in turn affects the myocardial performance is confirmed by the evidence that NO scavenging with PTIO, as well as NOS inhibition by L-NMMA, reduces the hypoxia-dependent increase of contractility. Moreover, under hypoxia, NOS inhibition by L-NMMA unchanges the Frank–Starling response of the goldfish heart. In contrast, a significant reduction of the myocardial sensitivity to stretch is observed if NO is removed from the tissue by PTIO and if sGC is inhibited by ODQ. This is of relevance, since it indicates that the effects elicited by NO involve the cGMP cascade but are NOS-independent, thus requiring other routes for NO generation [45].

    In the hypoxic goldfish heart, the molecular events of downstream NO generation exclude the involvement of the cGMP-dependent signalling [45]. Non cGMP-dependent pathways represent an important route for NO to control its molecular targets. These pathways are mainly represented by protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine (Cys) residues [46]. A significant reduction in the degree of S-nitrosylated proteins has been reported in the hypoxic goldfish heart with respect to the normoxic counterpart. In mammals, the significance of a dysregulated protein S-nitrosylation is correlated with both cardiac disorders [47] and with protective mechanisms against the development of myocardial dysfunction under stress [48]. Proteins encountering denitrosylation in the hypoxic goldfish heart and the related functional significance have not yet been identified. However, it is reasonable to hypothesize that this event, by activating still undefined protective programs, contributes to preserving myocardial function when challenged by hypoxia [45].

    Under hypoxia, NO may determine protein nitration. Data obtained in the hypoxic goldfish heart suggest the presence of hypoxia-induced nitration, since an increased expression of Nox2, the catalytic subunit of NADPH oxidase [45], and 3-nitrotyrosine [49] has been reported. If further data confirm the occurrence of nitration, putative targets must be identified. Based on the available information, some proteins can be hypothesized. One is the SERCA2a pump, the integral membrane protein controlling cardiac Ca2+ homeostasis by actively transporting the ion into the sarcoplasmic reticulum. It is susceptible to nitrosative and oxidative modifications for the presence of several cysteine and tyrosine residues [50][51]. The structural proximity to mitochondria exposes SERCA2a pumps to reactive O2/nitrogen species generated as by-products of the oxidative phosphorylation [52]. The nitrotyrosine modification of SERCA2a has been observed in several pathophysiological conditions, and nitrated SERCA2a is utilized as a cardiac marker of nitrative stress [50]. Although direct evidence on SERCA2a pumps nitration in fish is not available, the significant reduction of the hypoxia-induced time-course increase of the goldfish heart performance observed under conditions of SERCA2a inhibition [45] points to SERCA2a as a putative target of nitration in the hypoxic heart.
    In general, it appears that, in the goldfish heart, NO activates a protective program that sustains the performance under hypoxic challenge. Consistent with this, it was found that NO positively modulates cardiac sarcolemmal KATP channels, a response that, like the KATP-dependent protection observed in the ischemic mammalian myocardium [53], may contribute to the cardiac hypoxia tolerance of this species [5]. In addition, the hypoxic goldfish heart also shows an enhanced expression of the hypoxia inducible factor   (HIF-1α) [8]. In mammals, HIF1α/NO interaction is involved in hypoxia-elicited cardio-protective responses. Under hypoxia, HIF-1α activates genes critical for cell survival, including NOS [54]; at the same time, high NO concentrations (>1 µM) stabilize HIF-1α, leading to an increase in the dimeric form of protein which, by binding HREs sites, and enhances NOS gene expression and thus NO generation [55].

    2.3. Temperature

    Aquatic ectotherms depend on the thermal milieu to regulate their metabolic rate. Apart from species living in extremely stable environments, many fish routinely face temperature fluctuations associated not only with ontogenetic and/or seasonal changes, but also with diurnal changes especially in shallow water bodies. Nevertheless, their phenotypic plasticity (developmental or reversible acclimation) allows compensation by altering tolerance limits for optimizing the performance under changed temperature regimes [56]. While eurythermal fish, naturally subjected to large temperature changes, develop acclimation strategies for preserving their fitness, stenothermal species show specific evolutionary adaptations at the expense of reduced plasticity.
    In many eurytherm fish, temperature changes importantly influence the cardiac function that requires to be modulated to ensure an adequate CO. In addition, the upper thermal tolerance is partly determined by the capacity of the heart to ensure an adequate systemic O2 delivery [57]. This occurs by changing the heart rate (HR) more than the stroke volume [58][59]. When temperature acutely rises, the HR increases before declining at temperatures preceding the critical thermal maximum [58][59] and this compromises the cardiac function [57]. On the other hand, when the temperature drops, bradycardia occurs [60], and this is associated with an increased diastolic duration to maintain CO by increasing filling time and with little modifications of the systolic duration [61].

    Different from eurythermal species, stenotherm fish scarcely tolerate thermal challenges. This is the case of Antarctic teleost Channichthyidae that live in the extremely stable, frigid, and highly oxygenated Antarctic waters [62][63]. Some of them are unique among adult vertebrates, since they lack hemoglobin (Hb; [64]) and, in some species, also Mb [65]. This is compensated by extensive cardiocirculatory remodelling such as hypervolemia, low blood viscosity, large capillaries, cardiomegaly, and high blood flow with low systemic pressure and systemic resistance ([62] and references therein). 

    2.4. The NOS/NO System in the Fish Heart under Temperature Challenges

    In fish, the NOS/NO system plays a role in the regulation of the cardiac function in species adapted to both temperate and extreme thermal regimes, as well as in animals differently tolerant to thermal stress.In the eurythermal eel A. anguilla, the NOS/NO-dependent modulation of the Frank–Starling response [23][25] is impaired by temperature changes [66]. The positive modulation elicited by the intracardiac NO release on the Frank–Starling response disappears when animals are acutely exposed at temperatures lower orhigher than the acclimation one, both in the case of spring- and winter-like (acclimationtemperature: 20 °C and 10 °C, respectively) conditions. These effects are paralleled by reduced expression levels of NOS and pAkt, suggesting that the NO production via the Akt/NOS axis is temperature-dependent [66].
    Another example is provided by salmonids. In the eurythermal Atlantic salmon, longterm exposure to temperature enhancement is accompanied by an increased expression of iNOS in both compact and spongy ventricular myocardium, indicative of an enhanced NO production. At the same time, also VEGF expression increases [67]. This is interesting, since the two effects, if considered together, may call for a potentiated blood supply to the myocardium obtained by increasing vascularization (via VEGF) and/or by dilating the vessels (via NO). In fact, in salmonids, NO is known to induce vasodilation and reduce coronary resistance [13], thus contributing to compensating for the increased O2 demand under elevated temperature. The relationship between temperature variations and the cardiac nitrergic control may be of great importance in fish living under extreme temperatures. Unfortunately, this aspect remains unexplored, although the information so far available indicates that the NOS/NO system plays a role in the modulation of the basal cardiac performance of these animals. In Antarctic teleosts, functional NOSs are present in the heart of the hemoglobinless C. aceratus and C. hamatus and the red blooded T. bernacchii. An eNOS-like enzyme is mainly present in the lacunae of the spongy ventricle, while iNOS is basally expressed in the cytoplasm of myocardiocytes [19][20]. Despite the similar distribution, physio-pharmacological studies show that NO differently affects the contractility of the three species [20]. In fact, endogenous NO (L-arginine administration) reduces contractility in T. bernacchii, contrary to the stimulatory effect observed in the two icefish species. In addition, while in C. hamatus the NO-induced effects are cGMP-dependent, in T. bernacchii and C. aceratus these effects are cGMP-independent. The authors suggest that, in the absence of respiratory pigments, the loss of NO-oxygenase activities associates with Hb/Mb and the consequent increased NO levels may account for the observed differences [20].

    Tropical lungfish represent a peculiar model organism, since during warm seasons they undergo aestivation, a metabolic adaptation associated with functional modifications in tissues and organs including heart, kidney, gills, lung, and skeletal muscle [7][21][68][69][70]. A very interesting aspect of the lungfish is the ability of the myocardium to ensure contractility during warm aestivation, maintaining an appropriate blood perfusion to the whole organism. The lungfish heart, as observed in Protopterus dolloi [21] and Protopterus annectens [7], expresses NOS enzymes, and this expression increases under aestivation [21]. It has been proposed that the consequent enhanced NO release preserves the heart by sustaining cardiac bioenergetics in the presence of metabolic depression and reduced myocardial O2 consumption [7][21][69]

    3. Upstream and Downstream the NOS/NO System: AMP-Activated Protein Kinase (AMPK) as a Candidate in Fish

    Despite the growing data confirming NO as a key mediator in the cardiac response of fish to O2 and temperature stress, further research is needed to discover the complete molecular networks that are orchestrated by this gasotransmitter. A point of attention is the role played by critical kinases that may contribute to shape the adaptive response to stress. Together with PKA, PKG, PI3K, Akt, and others, the AMPK represents an interesting candidate. High AMP levels, generated when ATP consumption exceeds production, activate the kinase, and this results in a general shift from anabolism to catabolism, a modulation of gene and protein expression, a post-translational modification of metabolic enzymes, and mitochondrial biogenesis [71][72]. The AMPK activity also relates with metabolic redox and oxidative equilibria, although conclusive cause-effect models are still ongoing. It was suggested that the kinase is either inactivated by oxidative stress [73] or stimulated by the exposure to H2O2 and NO [74]
    An aspect that needs to be highlighted is the relationship between the AMPK and the nitrergic system. The kinase is located upstream of NOS enzymes. In mammalian cultured cardiomyocytes, the AMPK activates eNOS and nNOS by phosphorylation, thus contributing to NO production [75]. At the same time, an AMPK-dependent pathway activates Akt, thus indirectly modulating that in cardiomyocytes is involved in eNOS phosphorylation [76].
    In the heart, the relationship between AMPK and the nitrergic system is of importance, particularly under stress. The AMPK is activated under ischemia-reperfusion, increased workload, and glucose uptake impairment, all conditions calling for a reduced O2 and ATP, and a perturbation of the oxidative/nitrosative equilibrium [77][78][79]. Notably, the AMPK itself is regulated by NO that acts as an endogenous activator of the kinase. Under normal condition, NO stimulates the AMPK via an sGC/cGMP/Ca2+/CaMKβ pathway, while under stress the kinase is activated by the overproduced peroxynitrite via a PKC/LKB1 cascade [74]. The significance of this positive feedback between the AMPK and the nitrergic system, as well as the mechanisms of reciprocal control, is still an issue of active debate in mammals.
    So far, the biological role of the enzyme has been explored in several teleost species in relation to different challenges. Data in the hypoxia/anoxia-resistant crucian carp and goldfish [80][81] suggest that the kinase is sensitive to O2 reduction in a tissues-specific manner. In the goldfish exposed to 12 h of severe hypoxia, the AMPK is activated in the liver and presumably not in other tissues including heart, brain, and gills [81]. It is unclear whether the absence of enzyme activation is one of the physiological compensatory mechanisms that prevent energy decline under low O2, such as regional blood shunts and/or activations of alternative metabolic routes able to sustain cell bioenergetics [41]. At the same time, the available data may be influenced by the experimental context. For example, in goldfish, a short time of exposure and a partial O2 availability may be not enough to induce enzyme activation [81]. Consistent with this, in the crucian carp, a prolonged exposure (up to 7 days) to severe anoxia, but not hypoxia, is accompanied by an increased activation (i.e., phosphorylation) of the AMPK in the heart. This suggests that the kinase may be quiescent until complete anoxia is achieved [82]. In addition, this event is reversible, since the levels of the phosphorylated enzyme return to pre-anoxic levels after reoxygenation, suggesting an adaptive role [81]. This may be advantageous for hypoxia-tolerant species, since it allows increasing hypoxia tolerance before the AMPK-mediated metabolic adjustments is recruited. In this way, protein synthesis and other AMPK downregulated anabolic pathways continue to function under hypoxia along with the ability to preferentially shunt blood flow to at-risk organs (e.g., brain and heart) [82].
    In line with the role as a multistressor-dependent kinase, in fish, the AMPK function is related to temperature challenges, although the role of the kinase is still unclear. In salmonids (O. mykiss and O. kisutch), the AMPK phosphorylation correlates with optimal temperature for the aerobic scope, measured in terms of the maximum HR [83]. Recent observations on the heart of the olive flounder, Paralichthys olivaceus, show that cold stress activates the AMPK together with its upstream modulators (LKB1 and CAMKK) and downstream targets (SITR1, FOXO1A, and TFAM), and this positively affects the fish adaptive response to cold [84]. However, Nilsson and collaborators reported no changes in the AMPK phosphorylation in the heart of crucian carp acclimated to temperatures (4, 10, and 20 °C), which are naturally experienced by the species, suggesting that the kinase remains quiescent within the adaptive thermal range of the animal [85].
    To the best of our knowledge, the interplay between the AMPK and the nitrergic system in the fish heart in relation to stress, either single or multiple, has received scarce, or even null, attention. However, the data above illustrated clearly indicate that both are present and active in the fish heart and are involved in the adaptive response to O2 and temperature changes. The possibility that the extreme cardiac flexibility of several fish species, as in the case of cyprinids, may take advantage of signalling pathways that converge on both the NOS/NO system and the AMPK is intriguing and deserves attention.

    The entry is from 10.3390/antiox10091401


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