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Damage to the human heart is an irreparable process that results in a permanent impairment in cardiac function. There are, however, a number of vertebrate species including zebrafish (Danio rerio) that can regenerate their hearts following significant injury. In contrast to these regenerative species, mammals are known to have high levels of thyroid hormones, which has been proposed to play a role in this difference in regenerative capacity. However, the mechanisms through which thyroid hormones effect heart regeneration are not fully understood. Here, zebrafish were exposed to exogenous triiodothyronine (T 3 ) for two weeks and then their hearts were damaged through cryoinjury to investigate the effect of thyroid hormones on ECM remodeling and the components of the immune response during heart regeneration. Additionally, cardiac fibroblasts derived from trout, another species of fish known to display cardiac regenerative capacity, were exposed to T 3 in vitro to analyze any direct effects of T 3 on collagen deposition. It was found that cryoinjury induction results in an increase in myocardial stiffness, but this response was muted in T 3 exposed zebrafish. The measurement of relevant marker gene transcripts suggests that T 3 exposure reduces the recruitment of macrophages to the damaged zebrafish heart immediately following injury but had no effect on the regulation of collagen deposition by cultured trout fibroblasts. These results suggest that T 3 effects both the immune response and ECM remodeling in zebrafish following cardiac injury., Competing Interests: Declaration of competing interest I do not know the purpose of this declaration as I could not find relevant information on the journal website. However, the authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

The ability of the vertebrate heart to remodel enables the cardiac phenotype to be responsive to changes in physiological conditions and aerobic demand. Examples include exercise-induced cardiac hypertrophy, and the significant remodeling of the trout heart during thermal acclimation. Such changes are thought to occur in response to a change in hemodynamic load (i.e. the forces that the heart must work against to circulate blood). Variations in hemodynamic load are caused by either a volume overload (high volume of blood returning to the heart, impairing contraction) or a pressure overload (elevated afterload pressure that the heart must contract against). The changes observed in the heart during remodeling are regulated by multiple cellular signaling pathways. The cardiac response to these regulatory mechanisms occurs across levels of biological organization, affecting cardiac morphology, tissue composition and contractile function. Importantly, prolonged exposure to pressure overload can cause a physiological response - that improves function - to transition to a pathological response that causes loss of function. This Review explores the role of changes in hemodynamic load in regulating the remodeling response, and considers the cellular signals responsible for regulating remodeling, incorporating knowledge gained from studying biomedical models and comparative animal models. We specifically focus on the renin-angiotensin system, and the role of nitric oxide, oxygen free radicals and transforming growth factor beta. Through this approach, we highlight the strong conservation of the regulatory pathways of cardiac remodeling, and the specific conditions within endotherms that may be conducive to the development of pathological phenotypes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

A decrease in environmental temperature represents a challenge to the cardiovascular system of ectotherms. To gain insight into the cellular changes that occur during cold exposure and cold acclimation we characterized the cardiac phosphoproteome and proteome of zebrafish following 24 h or 1 week exposure to 20°C from 27°C; or at multiple points during 6 weeks of acclimation to 20°C from 27°C. Our results indicate that cold exposure causes an increase in mitogen-activated protein kinase signalling, the activation of stretch-sensitive pathways, cellular remodelling via ubiquitin-dependent pathways and changes to the phosphorylation state of proteins that regulate myofilament structure and function including desmin and troponin T. Cold acclimation (2-6 weeks) led to a decrease in multiple components of the electron transport chain through time, but an increase in proteins for lipid transport, lipid metabolism, the incorporation of polyunsaturated fatty acids into membranes and protein turnover. For example, there was an increase in the levels of apolipoprotein C, prostaglandin reductase-3 and surfeit locus protein 4, involved in lipid transport, lipid metabolism and lipid membrane remodelling. Gill opercular movements suggest that oxygen utilization during cold acclimation is reduced. Neither the amount of food consumed relative to body mass nor body condition was affected by acclimation. These results suggest that while oxygen uptake was reduced, energy homeostasis was maintained. This study highlights that the response of zebrafish to a decrease in temperature is dynamic through time and that investment in the proteomic response increases with the duration of exposure., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Pacific hagfish (Eptatretus stoutii) are an ancient agnathan vertebrate known to be anoxia tolerant. To study their metabolic organization and the role of the mitochondria in anoxia tolerance we developed a novel protocol to measure mitochondrial function in permeabilized cardiomyocytes and how this is affected by one hour of anoxia followed by reoxygenation. When measured at 10 °C the mitochondria had a respiration rate of 2.1 ± 0.1pmol/s/mg WW during OXPHOS with saturating concentrations of glutamate, malate, and succinate. This is comparatively low compared to other ectothermic species. The functional characteristics of the mitochondria were quantified with mitochondrial control ratios. These demonstrated that proton leak contributed to just under 50% of the oxygen flux, with the remainder going towards ATP phosphorylation. Finally, when the preparations were exposed to an anoxia-reoxygenation protocol there was no difference in respiration compared to that of a heart sample from the same animal maintained under normoxia for the same time. When Complex I alone or Complex I and II were stimulated following one hour of anoxia there was no decline in oxygen flux observed. However, if Complex II was activated alone there was a significant decline in respiration. This decrease was however also observed in the mitochondria maintained in normoxia for one hour. In conclusion, Pacific hagfish cardiac mitochondria demonstrated a low rate of oxygen consumption, a loosely coupled electron transfer system, and a resistance to one hour of anoxia., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2024. Published by Elsevier Inc.)

Cold acclimation of zebrafish causes changes to the structure and composition of the heart. However, little is known of the consequences of these changes on heart function or if these changes are reversible with rewarming back to the initial temperature. In the current study, zebrafish were acclimated from 27℃ to 20°C, then after 17 weeks, a subset of fish were rewarmed to 27°C and held at that temperature for 7 weeks. The length of this trial, 23 weeks, was chosen to mimic seasonal changes in temperature. Cardiac function was measured in each group at 27°C and 20°C using high frequency ultrasound. It was found that cold acclimation caused a decrease in ventricular cross-sectional area, compact myocardial thickness, and total muscle area. There was also a decrease in end-diastolic area with cold acclimation that reversed upon rewarming to control temperatures. Rewarming caused an increase in the thickness of the compact myocardium, total muscle area, and end-diastolic area back to control levels. This is the first experiment to demonstrate that cardiac remodeling, induced by cold acclimation, is reversible upon re-acclimation to control temperature (27°C). Finally, body condition measurements reveal that fish that had been cold-acclimated and then reacclimated to 27°C, were in poorer condition than the fish that remained at 20°C as well as the control fish at week 23. This suggests that the physiological responses to the multiple changes in temperature had a significant energetic cost to the animal. SUMMARY STATEMENT: The decrease in cardiac muscle density, compact myocardium thickness and diastolic area in zebrafish caused by cold acclimation, was reversed with rewarming to control temperatures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Millions of liters of diluted bitumen (dilbit), a crude oil product from Canada's oil sands region, is transported through critical Pacific salmon habitat each day. While the toxicity of the water-soluble fraction of dilbit (WSFd) to early life-stages of salmon is known, quantitative data on life-stage differences in sensitivity to WSFd is missing. To fill this knowledge gap, we exposed two juvenile life-stages of coho salmon (O. kisutch) in parallel to very low (parts per billion), environmentally-relevant concentrations of WSFd for acute (48 h) and sub-chronic (4 wk) durations. The relative sensitivities of the two life-stages (fry and parr) were assessed by comparing the timing and magnitude of biological responses using common organismal and molecular endpoints of crude oil exposure. A significant reduction in body condition occurred in both fry and parr after 4 wk exposure to WSFd. Both life-stages also experienced a concentration-dependent decrease in time-to-loss-of-equilibrium during a hypoxia challenge test at both 48 h and 4 wk of exposure. Although organismal responses were similar, molecular responses were distinct between life-stages. In general, unexposed fry had higher baseline values of hepatic phase I biotransformation indicators than unexposed parr, but induction of EROD activity and cyp1a mRNA expression in response to WSFd exposure was greater in parr than in fry. Neither gst nor hsp70 mRNA expression, markers of phase II biotransformation and cell stress, respectively, were reliably altered by WSFd exposure in either life-stage. Taken together, results of this study do not support differential sensitivities of coho fry and parr to WSFd. All the same, the potential for ontogenic differences in the expression and induction of phase I biotransformation need to be considered because age does matter for these endpoints if they are used as bioindicators of exposure in post-spill impact assessments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

An understanding of the risks associated with diluted bitumen (dilbit) transport through Pacific salmon habitat necessitates the identification and quantification of hazards posed to early life stages. Sockeye from the embryo to juvenile stage (8 months old) were exposed to four concentrations of the water-soluble fraction of Cold Lake dilbit (summer blend; concentrations of 0, 13.7, 34.7, and 124.5 μg/L total polycyclic aromatic compounds). Significant mortality (up to 18% over controls) only occurred in the embryo to swim-up fry stage. Impaired growth was seen in the alevin, swim-up, and juvenile stages (maximum reduction 15% in mass but not fork length). Reductions in both critical (maximum 24% reductions) and burst (maximum 47% reductions) swimming speed in swim-up fry and juveniles were seen. Alterations in energy substrate reserves (reductions in soluble protein and glycogen content, elevations in whole-body lipid and triglyceride levels) at all stages may underlie the effects seen in swimming and growth. Dilbit exposure induced a preexercise physiological stress response that affected the recovery of postexercise biochemistry (cortisol, glycogen, lactate, triglyceride concentrations). The transcript abundance of the cytochrome P450 1A gene (cyp1a) was quantified in alevin head regions (containing the heart) and in the hearts of swim-up fry and juveniles and showed a concentration-dependent increase in the expression of cyp1a at all life stages. Environ Toxicol Chem 2022;41:1937-1949. © 2022 SETAC., (© 2022 SETAC.)

The passive mechanical properties of the vertebrate heart are controlled in part by the composition of the extracellular matrix (ECM). Changes in the ECM, caused by increased blood pressure, injury or disease can affect the capacity of the heart to fill with blood during diastole. In mammalian species, cardiac fibrosis caused by an increase in collagen in the ECM, leads to a loss of heart function and these changes in composition are considered to be permanent. Recent work has demonstrated that the cardiac ventricle of some fish species have the capacity to both increase and decrease collagen content in response to thermal acclimation. It is thought that these changes in collagen content help maintain ventricle function over seasonal changes in environmental temperatures. This current work reviews the cellular mechanisms responsible for regulating collagen deposition in the mammalian heart and proposes a cellular pathway by which a change in temperature can affect the collagen content of the fish ventricle through mechanotransduction. This work specifically focuses on the role of transforming growth factor β1, MAPK signaling pathways, and biomechanical stretch in regulating collagen content in the fish ventricle. It is hoped that this work increases the appreciation of the use of comparative models to gain insight into phenomenon with biomedical relevance., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Rainbow trout (Oncorhynchus mykiss) live in environments where water temperatures range between 4 °C and 20 °C. Laboratory studies demonstrate that cold and warm acclimations of male trout can have oppositional effects on cardiac hypertrophy and the collagen content of the heart. The cellular mechanisms behind temperature-induced cardiac remodelling are unclear, as is why this response differs between male and female fish. Studies with cultured trout cardiac fibroblasts suggests that collagen deposition is regulated, at least in part, by mitogen-activated protein kinase (MAPK) cell signalling pathways. We, therefore, hypothesized that temperature-dependent cardiac remodelling is regulated by these signalling pathways. To test this, male and female trout were acclimated to 18 °C (warm) in the summer and to 4 °C (cold) in the winter and the activation of MAPK pathways in the hearts were characterized and compared to that of control fish maintained at 12 °C. In addition, cardiac collagen content, cardiac morphology and the expression of gene transcripts for matrix metalloproteinases (MMP) -9, MMP-2, tissue inhibitor of matrix metalloproteinases and collagen 1α were characterized. p38 MAPK phosphorylation increased in the hearts of female fish with cold acclimation and the phosphorylation of extracellular signal-regulated kinase increased in the hearts of male fish with warm acclimation. However, there was no effect of thermal acclimation on cardiac morphology or collagen content in either male or female fish. These results indicate that thermal acclimation has transient and sex-specific effects on the phosphorylation of MAPKs but also how variable the response of the trout heart is to thermal acclimation., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Western painted turtles (Chrysemys picta bellii) are the most anoxia-tolerant tetrapod. Survival time improves at low temperature and during ontogeny, such that adults acclimated to 3°C survive far longer without oxygen than either warm-acclimated adults or cold-acclimated hatchlings. As protein synthesis is rapidly suppressed to save energy at the onset of anoxia exposure, this study tested the hypothesis that cold acclimation would evoke preparatory changes in protein expression to support enhanced anoxia survival in adult but not hatchling turtles. To test this, adult and hatchling turtles were acclimated to either 20°C (warm) or 3°C (cold) for 5 weeks, and then the heart ventricles were collected for quantitative proteomic analysis. The relative abundance of 1316 identified proteins was compared between temperatures and developmental stages. The effect of cold acclimation on the cardiac proteome was only evident in the context of an interaction with life stage, suggesting that ontogenic differences in anoxia tolerance may be predicated on successful maturation of the heart. The main differences between the hatchling and adult cardiac proteomes reflect an increase in metabolic scope with age that included more myoglobin and increased investment in both aerobic and anaerobic energy pathways. Mitochondrial structure and function were key targets of the life stage- and temperature-induced changes to the cardiac proteome, including reduced Complex II proteins in cold-acclimated adults that may help down-regulate the electron transport system and avoid succinate accumulation during anoxia. Therefore, targeted cold-induced changes to the cardiac proteome may be a contributing mechanism for stage-specific anoxia tolerance in turtles., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Long-term cocaine use is associated with a variety of neural and behavioral deficits that impact daily function. This study was conducted to examine the effects of chronic cocaine self-administration on resting-state functional connectivity of the dorsal anterior cingulate (dACC) and putamen-two brain regions involved in cognitive function and motoric behavior-identified in a whole brain analysis. Six adult male squirrel monkeys self-administered cocaine (0.32 mg/kg/inj) over 140 sessions. Six additional monkeys that had not received any drug treatment for ~1.5 years served as drug-free controls. Resting-state fMRI imaging sessions at 9.4 Tesla were conducted under isoflurane anesthesia. Functional connectivity maps were derived using seed regions placed in the left dACC or putamen. Results show that cocaine maintained robust self-administration with an average total intake of 367 mg/kg (range: 299-424 mg/kg). In the cocaine group, functional connectivity between the dACC seed and regions primarily involved in motoric behavior was weaker, whereas connectivity between the dACC seed and areas implicated in reward and cognitive processing was stronger. In the putamen seed, weaker widespread connectivity was found between the putamen and other motor regions as well as with prefrontal areas that regulate higher-order executive function; stronger connectivity was found with reward-related regions. dACC connectivity was associated with total cocaine intake. These data indicate that functional connectivity between regions involved in motor, reward, and cognitive processing differed between subjects with recent histories of cocaine self-administration and controls; in dACC, connectivity appears to be related to cumulative cocaine dosage during chronic exposure.

The development of anoxia within tissues represents a significant challenge to most animals because of the decreased capacity for aerobic ATP production, the associated loss of essential cellular functions and the potential for detrimental tissue oxidation upon reoxygenation. Despite these challenges, there are many animals from multiple phyla that routinely experience anoxia and can fully recover. In this Review, we integrate knowledge gained from studies of anoxia-tolerant species across many animal taxa . We primarily focus on strategies used to reduce energy requirements, minimize the consequences of anaerobic ATP production and reduce the adverse effects of reactive oxygen species, which are responsible for tissue damage with reoxygenation. We aim to identify common strategies, as well as novel solutions, to the challenges of anoxia exposure. This Review chronologically examines the challenges faced by animals as they enter anoxia, as they attempt to maintain physiological function during prolonged anoxic exposure and, finally, as they emerge from anoxia. The capacity of animals to survive anoxia is also considered in relation to the increasing prevalence of anoxic zones within marine and freshwater environments, and the need to understand what limits survival., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Petrogenic chemicals are common and widespread contaminants in the aquatic environment. In Canada, increased extraction of bitumen from the oil sands and transport of the major crude oil export product, diluted bitumen (dilbit), amplifies the risk of a spill and contamination of Canadian waterways. Fish exposed to sublethal concentrations of crude oil can experience a variety of adverse physiological effects including osmoregulatory dysfunction. As regulation of water and ion balance is crucial during the seawater transition of anadromous fish, the hypothesis that dilbit impairs seawater acclimation in Atlantic salmon smolts (a fish at risk of exposure in Canada) was tested. Smolts were exposed for 24 d to the water-soluble fraction of dilbit in freshwater, and then transferred directly to seawater or allowed a 1 wk depuration period in uncontaminated freshwater prior to seawater transfer. The seawater acclimation response was quantified at 1 and 7 d post-transfer using established hematological, tissue, and molecular endpoints including gill Na + /K + -ATPase gene expression (nka). All smolts, irrespective of dilbit exposure, increased serum Na + concentrations and osmolality within 1 d of seawater transfer. The recovery of these parameters to freshwater values by 7 d post-transfer was likely driven by the increased expression and activity of Na + /K + -ATPase in the gill. Histopathological changes in the gill were not observed; however, CYP1A-like immunoreactivity was detected in the pillar cells of gill lamellae of fish exposed to 67.9 μg/L PAC. Concentration-specific changes in kidney expression of a transmembrane water channel, aquaporin 3, occurred during seawater acclimation, but were resolved with 1 wk of depuration and were not associated with histopathological changes. In conclusion, apart from a robust CYP response in the gill, dilbit exposure did not greatly impact common measures of seawater acclimation, suggesting that significant osmoregulatory dysfunction is unlikely to occur if Atlantic salmon smolts are exposed sub-chronically to dilbit., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Canada's oil sands industry continues to expand and the volume of diluted bitumen (dilbit) transported across North America is increasing, adding to spill risk and environmental contamination. Dilbit exposure is known to cause adverse effects in fish, but linking molecular and cellular changes with ecologically-relevant individual performance metrics is needed to better understand the potential consequences of a dilbit spill into the aquatic environment. Therefore, this study examined the effects of dilbit exposure on subcellular responses in cardiac and skeletal muscle in relation to swimming performance in a migratory fish species at risk of exposure, Atlantic salmon. Smolts were exposed subchronically to environmentally relevant concentrations of the water-soluble fraction of dilbit (WSFd) for 24 d, and then a subset of exposed fish underwent a depuration period of 7 or 14 d, for a total of 3 experimental time points. At each time point, repeat swimming performance was assessed using sequential critical swimming speed tests (U crit ) separated by a 24 h rest period, and then several tissues were collected to determine biotransformation enzyme activation, energetic responses, and gene expression changes. U crit was unaffected in fish exposed to 67.9 μg/L total initial polycyclic aromatic compounds (PAC), but fish showed a decreased reliance on lipid metabolism for adenosine triphosphate (ATP) in the heart that was maintained through 7 d depuration. In contrast, U crit increased in fish exposed to 9.65 μg/L PAC, corresponding to an increased reliance on anaerobic metabolic pathways in cardiac and red skeletal muscle, with partial recovery after 7 d depuration. As expected, at both concentrations WSFd hepatic cyp 1A-mediated biotransformation reactions increased, as measured by EROD activity, which remained elevated for 7 d but not after 14 d depuration. Transcript abundance of cyp1a was also increased in muscle tissue and recovered by 14 d depuration. The expression of other stress-related genes increased in white muscle of dilbit-exposed fish, but were largely unchanged in cardiac and red muscle. The transcriptional profile of cardiac tissue was compared to that of sockeye salmon similarly exposed to WSFd in a previous experiment, and is provided in supplemental text. Combined, these results demonstrate that dilbit exposure alters gene expression and enzyme activities related to xenobiotic exposure, cellular stress, and muscle energetics in juvenile Atlantic salmon without impairing swimming performance, and that most of these changes are recoverable within 14 d depuration., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We recently described lasting changes in the cardiac proteome of American alligators (Alligator mississippiensis) reared under hypoxic conditions, that resemble what embryos encounter in natural nests. While these changes were consistent with functional differences in cardiac performance induced by developmental hypoxia, the magnitude of this response was dwarfed by a much greater effect of development alone (76% of the total differentially abundant proteins). This means that substantial differences in relative steady-state protein expression occur in the hearts of alligators as they mature from egg-bound embryos to 2-year-old juveniles, and this developmental program is largely resistant to variation in nest conditions. We therefore performed functional enrichment analysis of the 412 DA proteins that were altered by development but not hypoxia, to gain insight into the mechanisms of cardiac maturation in this ectotherm. We found that the cardiac proteome of alligators at 90% of embryonic development retained a considerable capacity for transcription and translation, suggesting the heart was still primarily invested in growth even as the animal approached hatching. By contrast, the cardiac proteome of 2-year-old juveniles was weighted towards structural and energetic processes typical of a working heart. We discuss our results in the context of differences in cardiac development between ectothermic and endothermic oviparous vertebrates, and argue that the robust developmental program of the alligator heart reflects a slow-paced ontogeny, unburdened by the requirement to support the elevated peripheral oxygen demand typical of endothermic animals from a young age.

The form and function of the rainbow trout heart can remodel in response to various stressors including changes in environmental temperature and anemia. Previous studies have hypothesized that changes in biomechanical forces experienced by the trout myocardium as result of such physiological stressors could play a role in triggering the remodeling response. However, there has been no work examining the influence of biomechanical forces on the trout myocardium or of the cellular signals that would translate such a stimuli into a biological response. In this study, we test the hypothesis that the application of biomechanical forces to trout cardiac fibroblasts activate the cell signaling pathways associated with cardiac remodeling. This was done by cyclically stretching cardiac fibroblasts to 10% equibiaxial deformation at 0.33 Hz and quantifying the activation of the p38-JNK-ERK mitogen activated protein kinase (MAPK) pathway. After 20 min, p38 MAPK phosphorylation was elevated by 4.2-fold compared to control cells ( P <0.05) and after 24 h of stretch, p38 MAPK phosphorylation remained elevated and extracellular-regulated kinase 1/2 was phosphorylated by 2.4-fold compared to control ( P <0.05). Together, these results indicate that mechanotransductive pathways are active in cardiac fibroblasts, and lead to the activation of cell signaling pathways involved in cardiac remodeling., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Warm acclimation of rainbow trout can cause a decrease in the collagen content of the heart. This ability to remove cardiac collagen is particularly interesting considering that collagen deposition in the mammalian heart, following an injury, is permanent. We hypothesized that collagen removal can be facilitated by microRNA-29b (miR-29b), a highly conserved, small, non-coding RNA, as a reduction in this microRNA has been reported during the development of fibrosis in the mammalian heart. We also used a bioinformatics approach to investigate the binding potential of miR-29b to the seed sequences of vertebrate collagen isoforms. Cultured trout cardiac fibroblasts were transfected with zebrafish mature miR-29b mimic for 7 days with re-transfection occurring after 3 days. Transfection induced a 17.8-fold increase in miR-29b transcript abundance ( P <0.05) as well as a 54% decrease in the transcript levels of the col1a3 collagen isoform, compared with non-transfected controls ( P <0.05). Western blotting demonstrated that the level of collagen type I protein was 85% lower in cells transfected with miR-29b than in control cells ( P <0.05). Finally, bioinformatic analysis suggested that the predicted 3'-UTR of rainbow trout col1a3 has a comparatively higher binding affinity for miR-29b than the 3'-UTR of col1a1 Together, these results suggest that miR-29b is a highly conserved regulator of collagen type I protein in vertebrates and that this microRNA decreases collagen in the trout heart by targeting col1a3 ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Deer mice, Peromyscusmaniculatus, live at high altitudes where limited O 2 represents a challenge to maintaining oxygen delivery to tissues. Previous work has demonstrated that hypoxia acclimation of deer mice and low altitude white-footed mice (P. leucopus) increases the force generating capacity of the diaphragm. The mechanism behind this improved contractile function is not known. Within myocytes, the myofilament plays a critical role in setting the rate and level of force production, and its ability to generate force can change in response to changes in physiological conditions. In the current study, we examined how chronic hypobaric hypoxia exposure of deer mice and white-footed mice influences the Ca 2+ activation of force generation by skinned diaphragmatic myofilaments, and the phosphorylation of myofilament proteins. Results demonstrate that myofilament force production, and the Ca 2+ sensitivity of force generation, were not impacted by acclimation to hypobaric hypoxia, and did not differ between preparations from the two species. The cooperativity of the force-pCa relationship, and the maximal rate of force generation were also the same in the preparations from both species, and not impacted by acclimation. Finally, the relative phosphorylation of TnT, and MLC was lower in deer mice than white-footed mice, but was not affected by acclimation. These results indicate that species differences in diaphragm function, and the increase in force production with hypoxia acclimation, are not due to differences, or changes, in myofilament function. However, it appears that diaphragmatic myofilament function in these species is not affected by chronic hypobaric hypoxia exposure.

Hypoxic exposure during development can have a profound influence on offspring physiology, including cardiac dysfunction, yet many reptile embryos naturally experience periods of hypoxia in buried nests. American alligators experimentally exposed to developmental hypoxia demonstrate morphological and functional changes to the heart that persist into later life stages; however, the molecular bases of these changes remain unknown. We tested if targeted and persistent changes in steady-state protein expression underlie this hypoxic heart phenotype, using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Alligator eggs were reared under normoxia or 10% hypoxia, then either sampled (embryo) or returned to normoxia for 2 years (juvenile). Three salient findings emerge from the integrated analysis of the 145 differentially expressed proteins in hypoxia-reared animals: (1) significant protein-protein interaction networks were identified only in up-regulated proteins, indicating that the effects of developmental hypoxia are stimulatory and directed; (2) the up-regulated proteins substantially enriched processes related to protein turnover, cellular organization, and metabolic pathways, supporting increased resource allocation towards building and maintaining a higher functioning heart; and (3) the juvenile cardiac proteome retained many of the signature changes observed in embryonic hearts, supporting long-term reprogramming of cardiac myocytes induced by hypoxia during critical periods of development.

Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia and their systemic hearts continue to work throughout the exposure. Recent work demonstrates that glycogen stores are utilized in the E. stoutii heart during anoxia but that these are not sufficient to support the measured rate of ATP production. One metabolic fuel that could supplement glycogen during anoxia is glycerol. This substrate can be derived from lipid stores, stored in the heart, or delivered via the blood. The purpose of this study was to determine the effect of glycerol on the contractile function of the excised E. stoutii heart during anoxia exposure. When excised hearts, perfused with metabolite free saline (mf-saline), were exposed to anoxia for 12 h, there was no difference in heart rate, pressure generation (max-dP), rate of contraction (max-dP/dt sys ), or rate of relaxation (max-dP/dt dia ) compared to hearts perfused with mf-saline in normoxia. However, hearts perfused with saline containing glycerol (gly-saline) in anoxia had higher max-dP, max-dP/dt sys , and max-dP/dt dia than hearts perfused with mf-saline in anoxia. Tissue levels of glycerol increased when hearts were perfused with gly-saline in normoxia, but not when perfused with gly-saline in anoxia. Anoxia exposure did not affect the activities of triglyceride lipase, glycerol kinase, or glycerol-3-phosphate dehydrogenase. This study suggests that glycerol stimulates cardiac function in the hagfish but that it is not derived from stored lipids. How glycerol may stimulate contraction is not known. This could be as an energy substrate, as an allosteric factor, or a combination of the two.

The collagen content of the rainbow trout heart increases in response to cold acclimation and decreases with acclimation to warm temperatures. This ability to remodel the myocardial extracellular matrix (ECM) makes these fish useful models to study the cellular pathways involved in collagen regulation in the vertebrate heart. Remodelling of the ECM in the mammalian heart is regulated, in part, by myofibroblasts which arise from pre-existing fibroblasts in response to transforming growth factor-β1 (TGF-β1). We have previously demonstrated that treatment of cultured rainbow trout cardiac fibroblasts with human TGF-β1 causes an increase in collagen production. Here, we showed that repetitive treatment of rainbow trout cardiac fibroblasts with a physiologically relevant concentration of human recombinant TGF-β1 results in a ∼29-fold increase in phosphorylated small mothers against decapentaplegic 2 (pSmad2); a 2.9-fold increase in vinculin protein, a 1.2-fold increase in cellular size and a 3-fold increase in filamentous actin (F-actin). These are common markers of the transition of fibroblasts to myofibroblasts. Cells treated with TGF-β1 also had highly organized cytoskeletal α-smooth muscle actin, as well as increased transcript abundances of mmp-9 , timp-2 and col1a1 Furthermore, using gelatin zymography, we demonstrated that TGF-β1 treatment causes a 5.3-fold increase in gelatinase activity. Together, these results suggest that trout cardiac fibroblasts have the capacity to differentiate into myofibroblasts and that this cell type can increase extracellular collagen turnover via gelatinase activity. Cardiac myofibroblasts are, therefore, likely involved in the remodelling of the cardiac ECM in the trout heart during thermal acclimation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Zebrafish is rapidly becoming a key model organism for studying a variety of biological processes from molecules to organisms. Interactions involving actin, a contractile protein and part of the cytoskeleton, are regulated by actin binding proteins in the majority of physiological processes in eukaryotic cells. To understand the contribution of actin proteins to the physiological processes of zebrafish, it is important to identify the diverse isoforms of actin encoded by its genome; however, significant sequence identity complicates isoform assignments. Through a combination of human-directed sequence and functional analysis, we have assigned and performed localization of actc1c, a previously undesignated cardiac actin gene, and propose an updated assignment of α-actin protein isoform identities in zebrafish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The early life stages of Pacific salmon are at risk of environmental exposure to diluted bitumen (dilbit) as Canada's oil sands industry continues to expand. The toxicity and latent effects of dilbit exposure were assessed in sockeye salmon (Oncorhynchus nerka) exposed to water-soluble fractions of dilbit (WSFd) from fertilization to the swim-up stage, and then reared in clean water for 8 months. Mortality was significantly higher in WSFd-exposed embryos, with cumulative mortality up to 4.6-fold higher in exposed relative to unexposed embryos. The sublethal effects of WSFd exposure included transcriptional up-regulation of cyp1a, a concentration-dependent delay in the onset and progression of hatching, as well as increased prevalence of developmental deformities at total polycyclic aromatic hydrocarbon (TPAH) concentrations ≥35 μg L -1 . Growth and body composition were negatively affected by WSFd exposure, including a concentration-specific decrease in soluble protein concentration and increases in total body lipid and triglyceride concentrations. Mortality continued during the first 2 months after transferring fish to clean water, reaching 53% in fish exposed to 100 μg L -1 TPAH; but there was no latent impact on swimming performance, heart mass, or heart morphology in surviving fish after 8 months. A latent effect of WSFd exposure on brain morphology was observed, with fish exposed to 4 μg L -1 TPAH having significantly larger brains compared to other treatment groups after 8 months in clean water. This study provides comprehensive data on the acute, sub-chronic, and latent impacts of dilbit exposure in early life stage sockeye, information that is critical for a proper risk analysis of the impact of a dilbit spill on this socioeconomically important fish species., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hagfishes use their defensive slime to ward off gill-breathing predators. Slime gland refilling is a surprisingly slow process, and previous research has shown that the composition of the slime exudate changes significantly during refilling, which likely has consequences for the functionality of the slime. This study set out to expand our understanding of slime gland refilling by examining the cellular processes involved in refilling of the glands, as well as determining where in the gland the main slime cells - the gland thread cells and gland mucous cells - arise. Slime glands were electro-stimulated to exhaust their slime stores, left to refill for set periods of time, and harvested for histological and immunohistochemical examination. Whole slime glands, gland thread cell morphometrics and slime cell proportions were examined over the refilling cycle. Slime glands decreased significantly in size after exhaustion, but steadily increased in size over refilling. Gland thread cells were the limiting factor in slime gland refilling, taking longer to replenish and mature than gland mucous cells. Newly produced gland thread cells underwent most of their growth near the edge of the gland, and larger cells were found farthest from the edge of the gland. Immunohistochemical analysis also revealed proliferating cells only within the epithelial lining of the slime gland, suggesting that new slime cells originate from undifferentiated cells lining the gland. Our results provide an in-depth look at the cellular dynamics of slime gland refilling in Pacific hagfish, and provide a model for how slime glands refill at the cellular level., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Hagfishes are known for their unique defensive slime, which they use to ward off gill-breathing predators. Although much is known about the slime cells (gland thread cells and gland mucous cells), little is known about how long slime gland refilling takes, or how slime composition changes with refilling or repeated stimulation of the same gland. Slime glands can be individually electrostimulated to release slime, and this technique was used to measure slime gland refilling times for Atlantic and Pacific hagfish. The amount of exudate produced, the composition of the exudate and the morphometrics of slime cells were analyzed during refilling, and as a function of stimulation number when full glands were stimulated in rapid succession. Complete refilling of slime glands for both species took 3-4 weeks, with Pacific hagfish achieving faster absolute rates of exudate recovery than Atlantic hagfish. We found significant changes in the composition of the exudate and in the morphometrics of slime cells from Pacific hagfish during refilling. Over successive stimulations of full Pacific hagfish glands, multiple boluses of exudate were released, with exudate composition, but not thread cell morphometrics, changing significantly. Finally, histological examination of slime glands revealed slime cells retained in glands after exhaustion. Discrepancies in the volume of cells released suggest that mechanisms other than contraction of the gland musculature alone may be involved in exudate ejection. Our results provide a first look at the process and timing of slime gland refilling in hagfishes, and raise new questions about how refilling is achieved at the cellular level., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The present study examined how the expression of enhanced green fluorescent protein (eGFP) and human cardiac actin (ACTC) in zebrafish Danio rerio influences embryonic heart rate (R H ) and the swim performance and metabolic rate of adult fish. Experiments with the adults involved determining the critical swimming speed (U crit , the highest speed sustainable and measure of aerobic capacity) while measuring oxygen consumption. Two different transgenic D. rerio lines were examined: one expressed eGFP in the heart (tg(cmlc:egfp)), while the second expressed ACTC in the heart and eGFP throughout the body (tg(cmlc:actc,ba:egfp)). It was found that R H was significantly lower in the tg(cmlc:actc,ba:egfp) embryos 4 days post-fertilization compared to wild-type (WT) and tg(cmlc:egfp). The swim experiments demonstrated that there was no significant difference in U crit between the transgenic lines and the wild-type fish, but metabolic rate and cost of transport (oxygen used to travel a set distance) was nearly two-fold higher in the tg(cmlc:actc,ba:egfp) fish compared to WT at their respective U crit . These results suggest that the expression of ACTC in the D. rerio heart and the expression of eGFP throughout the animal, alters cardiac function in the embryo and reduces the aerobic efficiency of the animal at high levels of activity., (© 2017 The Fisheries Society of the British Isles.)

Background: The prevalence of major depression in those with HIV/AIDS is substantially higher than in the general population. Mechanisms underlying this comorbidity are poorly understood. HIV-transactivator of transcription (Tat) protein, produced and excreted by HIV, could be involved. We determined whether conditional Tat protein expression in mice is sufficient to induce depression-like behaviors and oxidative stress. Further, as oxidative stress is associated with depression, we determined whether decreasing or increasing oxidative stress by administering methylsulfonylmethane (MSM) or diethylmaleate (DEM), respectively, altered depression-like behavior., Methods: GT-tg bigenic mice received intraperitoneal saline or doxycycline (Dox, 25-100 mg/kg/day) to induce Tat expression. G-tg mice, which do not express Tat protein, also received Dox. Depression-like behavior was assessed with the tail suspension test (TST) and the two-bottle saccharin/water consumption task. Reactive oxygen/nitrogen species (ROS/RNS) were assessed ex vivo. Medial frontal cortex (MFC) oxidative stress and temperature were measured in vivo with 9.4-Tesla proton magnetic resonance spectroscopy (MRS)., Results: Tat expression increased TST immobility time in an exposure-dependent manner and reduced saccharin consumption. MSM decreased immobility time while DEM increased it in saline-treated GT-tg mice. Tat and MSM behavioral effects persisted for 28 days. Tat and DEM increased while MSM decreased ROS/RNS levels. Tat expression increased MFC glutathione levels and temperature., Conclusions: Tat expression induced rapid and enduring depression-like behaviors and oxidative stress. Increasing/decreasing oxidative stress increased/decreased, respectively, depression-like behavior. Thus, Tat produced by HIV may contribute to the high depression prevalence among those with HIV. Further, mitigation of oxidative stress could reduce depression severity.

Objective: Cerebral photobiomodulation (PBM) improves mood and cognition. Cerebral metabolic enhancement is a mechanism proposed to underlie PBM effects. No PBM studies to date have applied phosphorus magnetic resonance spectroscopy ( 31 P MRS), which can be used to assess metabolic intermediates such as phosphocreatine (PCr) and adenosine triphosphate, the latter of which is elevated by PBM. Accordingly, we used 9.4 Tesla 31 P MRS to characterize effects of single and repeat cerebral PBM treatments on metabolism. PBM was delivered to healthy adult beagles in the form of transcranial laser treatment (TLT) at a wavelength of 808 nm, which passes safely through the skull and activates cytochrome C oxidase, a mitochondrial respiratory chain enzyme., Methods: Isoflurane-anesthetized subjects (n = 4) underwent a baseline 31 P MRS scan followed by TLT applied sequentially for 2 min each to anterior and posterior cranium midline locations, to irradiate the dorsal cortex. Subjects then underwent 31 P MRS scans for 2 h to assess acute TLT effects. After 2 weeks of repeat TLT (3 times/week), subjects were scanned again with 31 P MRS to characterize effects of repeat TLT., Results: TLT did not induce acute 31 P MRS changes over the course of 2 h in either scan session. However, after repeat TLT, the baseline PCr/β-nucleoside triphosphate ratio was higher than the scan 1 baseline (p < 0.0001), an effect attributable to increased PCr level (p < 0.0001)., Conclusions: Our findings are consistent with reports that bioenergetic effects of PBM can take several hours to evolve. Thus, in vivo 31 P MRS may be useful for characterizing bioenergetic effects of PBM in brain and other tissues.

Cold acclimation of rainbow trout, Oncorhynchus mykiss , causes collagen to increase within the extracellular matrix (ECM) of the myocardium, while warm acclimation has the opposite effect. The mechanism responsible for this remodelling response is not known. In mammals, transforming growth factor beta-1 (TGF-β1) stimulates collagen deposition within the myocardial ECM. Therefore, we hypothesized that TGF-β1 regulates trout myocardial ECM turnover and predicted that TGF-β1 would induce collagen deposition in cultured rainbow trout cardiac fibroblasts. We found that treatment of trout cardiac fibroblasts with 15 ng ml -1 human recombinant TGF-β1 caused an increase in total collagen at 48 and 72 h and an increase in collagen type I protein after 7 days. We also found that TGF-β1 treatment caused an increase in the transcript abundance of tissue inhibitor of metalloproteinase 2 ( timp-2 ) and matrix metalloproteinase 9 ( mmp-9 ) at 24 h. Cells treated with TGF-β1 also had lower levels of the gene transcript for mmp-2 after 48 h and higher levels of the gene transcript for collagen type I α1 ( col1a1 ) after 72 h. These changes in gene expression suggest that the increase in collagen deposition is due to a decrease in the activity of matrix metalloproteinases and an increase in collagen synthesis. Together, these results indicate that TGF-β1 is a regulator of ECM composition in cultured trout cardiac fibroblasts and suggest that this cytokine may play a role in regulating collagen content in the trout heart during thermal acclimation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Pipelines carrying diluted bitumen (dilbit) from Canada's oil sands traverse North America, including the freshwater habitat of Pacific salmon, posing a risk of environmental release and aquatic exposure. Swimming performance is impacted in juvenile sockeye (Oncorhynchus nerka) exposed to dilbit; therefore biomarkers of dilbit exposure will be valuable for monitoring at-risk salmon stocks. This study characterized changes in the serum proteome of sockeye exposed to a sub-lethal and environmentally relevant concentration of dilbit using isobaric tags for relative and absolute quantitation (iTRAQ), and included a range of experimental conditions to permit identification of biomarkers that are robust across time (1 and 4wk) and exercise level (at rest and following a swim test). Over 500 proteins were identified and quantified in sockeye serum, with dilbit exposure significantly altering the abundance of 24 proteins irrespective of time and exercise, including proteins associated with immune and inflammatory responses, coagulation, and iron homeostasis. An increase in creatine kinase (CK) activity in serum of dilbit-exposed salmon confirmed the higher CK protein abundance measured using iTRAQ. The combination of 4wk dilbit exposure and a swim test had a greater effect on the serum proteome than either treatment alone, including a marked increase in tissue leakage proteins, suggesting that aerobic exercise exacerbates the serum proteome response to dilbit, and the increased cellular damage could impede exercise recovery. This study provides a foundation for the development of bio-monitoring tools for salmon stock assessments, and offers new insights into the sub-lethal toxicity of crude oil exposure in fish., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The changes in the cardiac proteome of rainbow trout (Oncorhynchus mykiss) were quantified during the early phases (4, 7, and 14d) of a typical exercise-training regime to provide a comprehensive overview of the cellular changes responsible for developing a trained heart phenotype. Enhanced somatic growth during the 14d experiment was paralleled by cardiac growth to maintain relative ventricular mass. This was reflected in the cardiac proteome by the increased abundance of contractile proteins and cellular integrity proteins as early as Day 4, including a pronounced and sustained increase in blood vessel epicardial substance - an intercellular adhesion protein expressed in the vertebrate heart. An unexpected finding was that proteins involved in energy pathways, including glycolysis, β-oxidation, the TCA cycle, and the electron transport chain, were generally present at lower levels relative to Day 0 levels, suggesting a reduced investment in the maintenance of energy production pathways. However, as the fish demonstrated somatic and cardiac growth during the exercise-training program, this change did not appear to influence cardiac function. The in-depth analysis of temporal changes in the cardiac proteome of trout during the early stages of exercise training reveals novel insights into cardiac remodelling in an important model species., Biological Significance: Rainbow trout hearts have a remarkable ability for molecular, structural, and functional plasticity, and the inherent athleticism of these fish makes them ideal models for studies in comparative exercise physiology. Indeed, several decades of research using exercise-trained trout has shown both conserved and unique aspects of cardiac plasticity induced by a sustained increase in the workload of the heart. Despite a strong appreciation for the outcome of exercise training, however, the temporal events that generate this phenotype are not known. This study interrogates the early stages of exercise training using in-depth proteomic analysis to understand the molecular pathways of cardiac remodelling. Two major and novel findings emerge: (1) structural remodelling is initiated very early in training, as evidenced by a general increase in proteins associated with muscle contraction and integrity at Day 4, and (2) the abundance of proteins directly involved in energy production are decreased during 14d of exercise training, which contrasts the general acceptance of an exercise-induced increase in aerobic capacity of muscle, and suggests that regulation of energy pathways occurs at a different biological level than protein abundance., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Diaphragmatic weakness is a feature of heart failure (HF) associated with dyspnea and exertional fatigue. Most studies have focused on advanced stages of HF, leaving the cause unresolved. The long-standing theory is that pulmonary edema imposes a mechanical stress, resulting in diaphragmatic remodeling, but stable HF patients rarely exhibit pulmonary edema. We investigated how diaphragmatic weakness develops in two mouse models of pressure overload-induced HF. As in HF patients, both models had increased eupneic respiratory pressures and ventilatory drive. Despite the absence of pulmonary edema, diaphragmatic strength progressively declined during pressure overload; this decline correlated with a reduction in diaphragm cross-sectional area and preceded evidence of muscle weakness. We uncovered a functional codependence between angiotensin II and β-adrenergic (β-ADR) signaling, which increased ventilatory drive. Chronic overdrive was associated with increased PERK (double-stranded RNA-activated protein kinase R-like ER kinase) expression and phosphorylation of EIF2α (eukaryotic translation initiation factor 2α), which inhibits protein synthesis. Inhibition of β-ADR signaling after application of pressure overload normalized diaphragm strength, Perk expression, EIF2α phosphorylation, and diaphragmatic cross-sectional area. Only drugs that were able to penetrate the blood-brain barrier were effective in treating ventilatory overdrive and preventing diaphragmatic atrophy. These data provide insight into why similar drugs have different benefits on mortality and symptomatology, despite comparable cardiovascular effects., (Copyright © 2017, American Association for the Advancement of Science.)

This data article presents the first description of the rainbow trout cardiac ventricle at the level of the proteome, with more than 700 proteins identified and quantified using isobaric tags for relative and absolute quantitation (iTRAQ) and LC-MS/MS. The abundances of these proteins were compared across 4 durations of moderate exercise training (0, 4, 7, and 14 d), and a total of 107 proteins were differentially abundant during the course of the training program. The differentially abundant proteins are presented here grouped by functional classification. In the research article associated with this data [1], the temporal changes in the cardiac proteome are discussed in the context of cardiac remodelling and development of a trained heart phenotype.

Diluted bitumen (dilbit; the product of oil sands extraction) is transported through freshwater ecosystems critical to Pacific salmon. This is concerning, because crude oil disrupts cardiac development, morphology, and function in embryonic fish, and cardiac impairment in salmon can have major consequences on migratory success and fitness. The sensitivity of early life-stage salmon to dilbit and its specific cardiotoxic effects are unknown. Sockeye salmon parr were exposed to environmentally relevant concentrations of the water-soluble fraction (WSF) of dilbit for 1 wk and 4 wk, followed by an examination of molecular, morphological, and organismal endpoints related to cardiotoxicity. We show that parr are sensitive to WSF of dilbit, with total polycyclic aromatic hydrocarbon (PAH) concentrations of 3.5 µg/L sufficient to induce a liver biomarker of PAH exposure, and total PAH of 16.4 µg/L and 66.7 µg/L inducing PAH biomarkers in the heart. Furthermore, WSF of dilbit induces concentration-dependent cardiac remodeling coincident with performance effects: fish exposed to 66.7 µg/L total PAH have relatively fewer myocytes and more collagen in the compact myocardium and impaired swimming performance at 4 wk, whereas the opposite changes occur in fish exposed to 3.5 µg/L total PAH. The results demonstrate cardiac sensitivity to dilbit exposure that could directly impact sockeye migratory success. Environ Toxicol Chem 2017;36:354-360. © 2016 SETAC., (© 2016 SETAC.)

Thermal acclimation causes the heart of some fish species to undergo significant remodelling. This includes changes in electrical activity, energy utilization and structural properties at the gross and molecular level of organization. The purpose of this Review is to summarize the current state of knowledge of temperature-induced structural remodelling in the fish ventricle across different levels of biological organization, and to examine how such changes result in the modification of the functional properties of the heart. The structural remodelling response is thought to be responsible for changes in cardiac stiffness, the Ca 2+ sensitivity of force generation and the rate of force generation by the heart. Such changes to both active and passive properties help to compensate for the loss of cardiac function caused by a decrease in physiological temperature. Hence, temperature-induced cardiac remodelling is common in fish that remain active following seasonal decreases in temperature. This Review is organized around the ventricular phases of the cardiac cycle - specifically diastolic filling, isovolumic pressure generation and ejection - so that the consequences of remodelling can be fully described. We also compare the thermal acclimation-associated modifications of the fish ventricle with those seen in the mammalian ventricle in response to cardiac pathologies and exercise. Finally, we consider how the plasticity of the fish heart may be relevant to survival in a climate change context, where seasonal temperature changes could become more extreme and variable., Competing Interests: The authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The Ca 2+ affinity of cardiac troponin C (cTnC) from rainbow trout is significantly greater than that of cTnC from mammalian species. This high affinity is thought to enable cardiac function in trout at low physiological temperatures and is due to residues Asn 2 , Ile 28 , Gln 29 , and Asp 30 (Gillis et al., 2005, Physiol Genomics, 22, 1-7). Interestingly, the cTnC of the African clawed frog Xenopus laevis (frog cTnC) contains Gln 29 and Asp 30 but the residues at positions 2 and 28 are those found in all mammalian cTnC isoforms (Asp 2 and Val 28 ). The purpose of this study was to determine the Ca 2+ affinity of frog cTnC, and to determine how these three protein orthologs influence the function of complete troponin complexes. Measurements of Ca 2+ affinity and the rate of Ca 2+ dissociation from the cTnC isoforms and cTn complexes were made by monitoring the fluorescence of anilinonapthalenesulfote iodoacetamide (IAANS) engineered into the cTnC isoforms to report changes in protein conformation. The results demonstrate that the Ca 2+ affinity of frog cTnC is greater than that of trout cTnC and human cTnC. We also found that replacing human cTnC with frog cTnC in a mammalian cTn complex increased the Ca 2+ affinity of the complex by 5-fold, which is also greater than complexes containing trout cTnC. Together these results suggest that frog cTnC has the potential to increase the Ca 2+ sensitivity of force generation by the mammalian heart., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dyspnea and reduced exercise capacity, caused, in part, by respiratory muscle dysfunction, are common symptoms in patients with heart failure (HF). However, the etiology of diaphragmatic dysfunction has not been identified. To investigate the effects of HF on diaphragmatic function, models of HF were surgically induced in CD-1 mice by transverse aortic constriction (TAC) and acute myocardial infarction (AMI), respectively. Assessment of myocardial function, isolated diaphragmatic strip function, myofilament force-pCa relationship, and phosphorylation status of myofilament proteins was performed at either 2 or 18 wk postsurgery. Echocardiography and invasive hemodynamics revealed development of HF by 18 wk postsurgery in both models. In vitro diaphragmatic force production was preserved in all groups while morphometric analysis revealed diaphragmatic atrophy and fibrosis in 18 wk TAC and AMI groups. Isometric force-pCa measurements of myofilament preparations revealed reduced Ca(2+) sensitivity of force generation and force generation at half-maximum and maximum Ca(2+) activation in 18 wk TAC. The rate of force redevelopment (ktr) was reduced in all HF groups at high levels of Ca(2+) activation. Finally, there were significant changes in the myofilament phosphorylation status of the 18 wk TAC group. This includes a decrease in the phosphorylation of troponin T, desmin, myosin light chain (MLC) 1, and MLC 2 as well as a shift in myosin isoforms. These results indicate that there are multiple changes in diaphragmatic myofilament function, which are specific to the type and stage of HF and occur before overt impairment of in vitro force production., (Copyright © 2016 the American Physiological Society.)

Background: Obsessive compulsive disorder (OCD) is a debilitating condition with lifetime prevalence of 1-3%. OCD typically arises in youth but delays in diagnosis impede optimal treatment and developmental studies of the disorder. Research using genetically modified rodents may provide models of etiology that enable earlier detection and intervention. The SAPAP3 knockout (KO) transgenic mouse was developed as an animal model of OCD and related disorders (OCRD). KO mice exhibit compulsive self-grooming behavior analogous to behaviors found in people with OCRD. Striatal hyperactivity has been reported in these mice and in humans with OCD., Methods: Striatal and medial frontal cortex 9.4 Tesla proton spectra were acquired from young adult SAPAP3 KO and wild-type control mice to determine whether KO mice have metabolic and neurochemical abnormalities., Results: Young adult KO mice had lower striatal lactate ( P =0.006) and glutathione ( P =0.039) levels. Among all mice, striatal lactate and glutathione levels were associated (R=0.73, P =0.007). We found no group differences in medial frontal cortex metabolites. At the age range studied, only 1 of 8 KO mice had skin lesions indicative of severe compulsive grooming., Conclusion: Young adult SAPAP3 KO mice have striatal but not medial frontal cortex MRS abnormalities that may reflect striatal hypermetabolism accompanied by oxidative stress. These abnormalities typically preceded the onset of severe compulsive grooming. Our findings are consistent with striatal hypermetabolism in OCD. Together, these results suggest that striatal MRS measures of lactate or glutathione might be useful biomarkers for early detection of risk for developing compulsive behavior disorders.

Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia at 10°C. Such anoxia tolerance demands the mobilization of anaerobic fuels and the removal of metabolic wastes--processes that require a functional heart. The purpose of this study was to measure the metabolic response of the excised, cannulated hagfish heart to anoxia using direct calorimetry. These experiments were coupled with measurements of cardiac pH and metabolite concentrations, at multiple time points, to monitor acid-base balance and anaerobic ATP production. We also exposed hagfish to anoxia to compare the in vitro responses of the excised hearts with the in vivo responses. The calorimetry results revealed a significant reduction in the rate of metabolic heat production over the first hour of anoxia exposure, and a recovery over the subsequent 6 h. This response is likely attributable to a rapid anoxia-induced depression of aerobic ATP-production pathways followed by an upregulation of anaerobic ATP-production pathways such that the ATP production rate was restored to that measured in normoxia. Glycogen-depletion measurements suggest that metabolic processes were initially supported by glycolysis but that an alternative fuel source was used to support the sustained rates of ATP production. The maintenance of intracellular pH during anoxia indicates a remarkable ability of the myocytes to buffer/regulate protons and thus protect cardiac function. Altogether, these results illustrate that the low metabolic demand of the hagfish heart allows for near-routine levels of cardiac metabolism to be supported anaerobically. This is probably a significant contributor to the hagfish's exceptional anoxia tolerance., (© 2015. Published by The Company of Biologists Ltd.)

Adolescents are highly vulnerable to addiction and are four times more likely to become addicted at first exposure than at any other age. The dopamine D1 receptor, which is typically overexpressed in the normal adolescent prefrontal cortex, is involved in drug cue responses and is associated with relapse in animal models. In human drug addicts, imaging methods have detected increased activation in response to drug cues in reward- and habit-associated brain regions. These same methods can be applied more quantitatively to rodent models. Here, changes in neuronal activation in response to cocaine-conditioned cues were observed using functional magnetic resonance imaging in juvenile rats that were made to over-express either D1 receptors or green fluorescent protein by viral-mediated transduction. Reduced activation was observed in the amygdala and dopamine cell body regions in the low cue-preferring/control juvenile rats in response to cocaine cues. In contrast, increased activation was observed in the dorsal striatum, nucleus accumbens, prefrontal cortex, and dopamine cell bodies in high cue-preferring/D1 juveniles. The increase in cue salience that is mediated by increased D1 receptor density, rather than excessive cocaine experience, appears to underlie the transition from aversion to reward in cue-induced neural response and may form the basis for habit-forming vulnerability.

Background: Decreased availability of the N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine is thought to promote NMDAR hypofunction and contribute to the pathophysiology of schizophrenia, including neuroanatomical abnormalities, such as cortical atrophy and ventricular enlargement, and neurochemical abnormalities, such as aberrant glutamate and γ-aminobutyric acid (GABA) signaling. It is thought that these abnormalities directly relate to the negative symptoms and cognitive impairments that are hallmarks of the disorder. Because of the genetic complexity of schizophrenia, animal models of the disorder are extremely valuable for the study of genetically predisposing factors. Our laboratory developed a transgenic mouse model lacking serine racemase (SR), the synthetic enzyme of d-serine, polymorphisms of which are associated with schizophrenia. Null mutants (SR-/-) exhibit NMDAR hypofunction and cognitive impairments. We used 9.4 T magnetic resonance imaging (MRI) and proton spectroscopy (MRS) to compare in vivo brain structure and neurochemistry in wildtype (WT) and SR-/- mice., Methods: Mice were anesthetized with isoflurane for MRI and MRS scans., Results: Compared to WT controls, SR-/- mice exhibited 23% larger ventricular volumes (p<0.05). Additionally, in a medial frontal cortex voxel (15 μl), SR-/- mice exhibited significantly higher glutamate/water (12%, t=1.83, p<0.05) and GABA/water (72%, t=4.10, p<0.001) ratios., Conclusions: Collectively, these data demonstrate in vivo neuroanatomical and neurochemical abnormalities in the SR-/- mouse comparable to those previously reported in humans with schizophrenia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Reducing temperature below the optimum of most vertebrate hearts impairs contractility and reduces organ function. However, a number of fish species, including the rainbow trout, can seasonally acclimate to low temperature. Such ability requires modification of physiological systems to compensate for the thermodynamic effects of temperature on biological processes. The current study tested the hypothesis that rainbow trout compensate for the direct effect of cold temperature by increasing cardiac contractility during cold acclimation. We examined cardiac contractility, following thermal acclimation (4, 11 and 17°C), by measuring the Ca(2+) sensitivity of force generation by chemically skinned cardiac trabeculae as well as ventricular pressure generation using a modified Langendorff preparation. We demonstrate, for the first time, that the Ca(2+) sensitivity of force generation was significantly higher in cardiac trabeculae from 4°C-acclimated trout compared with those acclimated to 11 or 17°C, and that this functional change occurred in parallel with a decrease in the level of cardiac troponin T phosphorylation. In addition, we show that the magnitude and rate of ventricular pressure generation was greater in hearts from trout acclimated to 4°C compared with those from animals acclimated to 11 or 17°C. Taken together, these results suggest that enhanced myofilament function, caused by modification of existing contractile proteins, is at least partially responsible for the observed increase in pressure generation after acclimation to 4°C. In addition, by examining the phenotypic plasticity of a comparative model we have identified a strategy, used in vivo, by which the force-generating capacity of cardiac muscle can be increased., (© 2014. Published by The Company of Biologists Ltd.)

Xenon (Xe) is a noble gas that has been developed for use in people as an inhalational anesthestic and a diagnostic imaging agent. Xe inhibits glutamatergic N-methyl-D-aspartate (NMDA) receptors involved in learning and memory and can affect synaptic plasticity in the amygdala and hippocampus, two brain areas known to play a role in fear conditioning models of post-traumatic stress disorder (PTSD). Because glutamate receptors also have been shown to play a role in fear memory reconsolidation--a state in which recalled memories become susceptible to modification--we examined whether Xe administered after fear memory reactivation could affect subsequent expression of fear-like behavior (freezing) in rats. Male Sprague-Dawley rats were trained for contextual and cued fear conditioning and the effects of inhaled Xe (25%, 1 hr) on fear memory reconsolidation were tested using conditioned freezing measured days or weeks after reactivation/Xe administration. Xe administration immediately after fear memory reactivation significantly reduced conditioned freezing when tested 48 h, 96 h or 18 d after reactivation/Xe administration. Xe did not affect freezing when treatment was delayed until 2 h after reactivation or when administered in the absence of fear memory reactivation. These data suggest that Xe substantially and persistently inhibits memory reconsolidation in a reactivation and time-dependent manner, that it could be used as a new research tool to characterize reconsolidation and other memory processes, and that it could be developed to treat people with PTSD and other disorders related to emotional memory.

Thermal acclimation can alter cardiac function and morphology in a number of fish species, but little is known about the regulation of these changes. The purpose of the present study was to determine how cold acclimation affects zebrafish (Danio rerio) cardiac morphology, collagen composition and connective tissue regulation. Heart volume, the thickness of the compact myocardium, collagen content and collagen fiber composition were compared between control (27°C) and cold-acclimated (20°C) zebrafish using serially sectioned hearts stained with Picrosirius Red. Collagen content and fiber composition of the pericardial membrane were also examined. Cold acclimation did not affect the volume of the contracted heart; however, there was a significant decrease in the thickness of the compact myocardium. There was also a decrease in the collagen content of the compact myocardium and in the amount of thick collagen fibers throughout the heart. Cold-acclimated zebrafish also increased expression of the gene transcript for matrix metalloproteinase 2, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 2 and collagen Type I α1. We propose that the reduction in the thickness of the compact myocardium as well as the change in collagen content may help to maintain the compliance of the ventricle as temperatures decrease. Together, these results clearly demonstrate that the zebrafish heart undergoes significant remodeling in response to cold acclimation., (© 2014. Published by The Company of Biologists Ltd.)

Hagfish slime threads were recently established as a promising biomimetic model for efforts to produce ecofriendly alternatives to petroleum polymers. Initial attempts to make fibers from solubilized slime thread proteins fell short of achieving the outstanding mechanics of native slime threads. Here we tested the hypothesis that the high strength and toughness of slime threads arise from the ability of constituent intermediate filaments to undergo a stress-induced α-to-β transition. To do this, we made fibers from human vimentin proteins that were first allowed to self-assemble into 10 nm intermediate filaments. Fibers made from assembled vimentin hydrogels underwent an α-to-β transition when strained and exhibited improved mechanical performance. Our data demonstrate that it is possible to make materials from intermediate filament hydrogels and that mimicking the secondary structure of native hagfish slime threads using intermediate filament self-assembly is a promising strategy for improving the mechanical performance of biomimetic protein materials.

Hypoxia exposure during embryonic development of rainbow trout causes developmental delay and bradycardia and alters the ontogeny of cardiac regulatory control mechanisms. The purpose of this study was to characterize how hypoxia exposure from the day of fertilization until stage 34 (57 d postfertilization) affects the aerobic fitness and growth of the hatched fish at multiple stages. In addition, we characterized the expression of gene transcripts for seven troponin I (TnI) isoforms to examine the effect of hypoxia treatment on cardiac muscle development. Results demonstrate that the critical swimming speed of the hypoxia-exposed fish was significantly less than that of the control group at stage 35 and the fry stage. Growth was reduced in the hypoxia-treated fish between stages 35 and 37, as was the relative lipid content at stage 37. Finally, six TnI isoforms were found in all hearts. One of these isoforms, RTcTnI, decreased in abundance between stage 35 and the fry stage, but hypoxia-exposed fish had higher levels than did controls at the fry stage. The abundance of AScTnI2 was significantly lower in hypoxia-exposed fry fish than in controls. These results indicate that chronic hypoxia exposure during embryonic development has long-term consequences on aerobic fitness, growth, and cardiac gene expression following hatch.

Obsessive compulsive disorder (OCD) is a debilitating condition, the etiology of which is poorly understood, in part because it often remains undiagnosed/untreated for a decade or more. Characterizing the etiology of compulsive disorders in animal models may facilitate earlier diagnosis and intervention. Doberman pinschers have a high prevalence of an analogous behavioral disorder termed canine compulsive disorder (CCD), which in many cases responds to treatments used for OCD. Thus, studies of CCD may help elucidate the etiology of compulsive disorders. We compared brain structure in Dobermans with CCD (N=8) and unaffected controls (N=8) to determine whether CCD is associated with structural abnormalities comparable to those reported in humans with OCD. We obtained 3 Tesla magnetic resonance structural and diffusion images from anesthetized Dobermans and subjected images to segmentation, voxel based morphometry, and diffusion tensor analyses. CCD dogs exhibited higher total brain and gray matter volumes and lower dorsal anterior cingulate cortex and right anterior insula gray matter densities. CCD dogs also had higher fractional anisotropy in the splenium of the corpus callosum, the degree of which correlated with the severity of the behavioral phenotype. Together, these findings suggest that CCD is associated with structural abnormalities paralleling those identified in humans with OCD. Accordingly, the CCD model, which has a number of advantages over other animal models of OCD, may assist in establishing the neuroanatomical basis for and etiology of compulsive disorders, which could lead to earlier diagnosis of and new treatments for humans and animals with these disorders., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Functional MRI (fMRI) has emerged as a powerful technique for assessing neural effects of psychoactive drugs and other stimuli. Several experimental approaches have been developed to use fMRI in anesthetized and awake animal subjects, each of which has its advantages and complexities. We sought to assess whether one particular method to scan alert postanesthetized animals can be used to assess fMRI effects of opioid agonists. To date, the use of fMRI as a method to compare pharmacological effects of opioid drugs has been limited. Such studies are important because mu and kappa opioid receptor agonists produce distinct profiles of behavioral effects related both to clinically desirable endpoints (e.g., analgesia) and to undesirable effects (e.g., abuse potential). This study sought to determine whether we could use our fMRI approach to compare acute effects of behaviorally equipotent (3.2 μg/kg) intravenous doses of fentanyl and U69,593 (doses that do not affect cardiorespiratory parameters). Scans were acquired in alert male cynomolgus macaques acclimated to undergo fMRI scans under restraint, absent excessive stress hormone increases. These opioid agonists activated bilateral striatal and nucleus accumbens regions of interest. At the dose tested, U69,593 induced greater left nucleus accumbens BOLD activation than fentanyl, while fentanyl activated left dorsal caudate nucleus more than U69,593. Our results suggest that our fMRI approach could be informative for comparing effects of opioid agonists., (PsycINFO Database Record (c) 2013 APA, all rights reserved)

The classic myelin basic protein (MBP) family of central nervous system (CNS) myelin arises from transcription start site 3 of the Golli (gene of oligodendrocyte lineage) complex and comprises splice isoforms ranging in nominal molecular mass from 14 kDa to (full-length) 21.5 kDa. We have determined here a number of distinct functional differences between the major 18.5-kDa and minor 21.5-kDa isoforms of classic MBP with respect to oligodendrocyte (OLG) proliferation. We have found that, in contrast to 18.5-kDa MBP, 21.5-kDa MBP increases proliferation of early developmental immortalized N19-OLGs by elevating the levels of phosphorylated ERK1/2 and Akt1 kinases and of ribosomal protein S6. Coculture of N2a neuronal cells with N19-OLGs transfected with the 21.5-kDa isoform (or conditioned medium from), but not the 18.5-kDa isoform, caused the N2a cells to have increased neurite outgrowth and process branching complexity. These roles were dependent on subcellular localization of 21.5-kDa MBP to the nucleus and on the exon II-encoded segment, suggesting that the nuclear localization of early minor isoforms of MBP may play a crucial role in regulating and/or initiating myelin and neuronal development in the mammalian CNS., (Copyright © 2012 Wiley Periodicals, Inc.)

Hagfish slime threads, which make up the fibrous component of the defensive slime of hagfishes, consist primarily of proteins from the intermediate filament family of proteins and possess impressive mechanical properties that make them attractive biomimetic models. To investigate whether solubilized intermediate filament proteins can be used to make high-performance, environmentally sustainable materials, we cast thin films on the surface of electrolyte buffers using solubilized hagfish slime thread proteins. The films were drawn into fibers, and the tensile properties were measured. Fiber mechanics depended on casting conditions and postspinning processing. Postsecondary drawing resulted in fibers with improved material properties similar to those of regenerated silk fibers. Structural analyses of the fibers revealed increased molecular alignment resulting from the second draw, but no increase in crystallinity. Our findings show promise for intermediate filament proteins as an alternative source for the design and production of high performance protein-based fibers.