Cardiac remodeling caused by cold acclimation is reversible with rewarming in zebrafish (Danio rerio).

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.

Age matters: Comparing life-stage responses to diluted bitumen exposure in coho salmon (Oncorhynchus kisutch).

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.

Diluted Bitumen Affects Multiple Physiological Systems in Sockeye Salmon (Oncorhynchus nerka) Embryo to Juvenile Life Stages.

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.

Regulation of collagen deposition in the trout heart during thermal acclimation.

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 ß1, 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.

Cold acclimation induces life stage-specific responses in the cardiac proteome of western painted turtles (Chrysemys picta bellii): implications for anoxia tolerance.

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.

Surviving anoxia: the maintenance of energy production and tissue integrity during anoxia and reoxygenation.

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.

Effects of diluted bitumen exposure on Atlantic salmon smolts: Molecular and metabolic responses in relation to swimming performance.

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 (Ucrit) separated by a 24 h rest period, and then several tissues were collected to determine biotransformation enzyme activation, energetic responses, and gene expression changes. Ucrit 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, Ucrit 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.

Effects of diluted bitumen exposure and recovery on the seawater acclimation response of Atlantic salmon smolts.

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.

Growing up gator: a proteomic perspective on cardiac maturation in an oviparous reptile, the American alligator (Alligator mississippiensis).

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.

Short-term cyclical stretch phosphorylates p38 and ERK1/2 MAPKs in cultured fibroblasts from the hearts of rainbow trout, Oncorhynchus mykiss.

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.

microRNA-29b knocks down collagen type I production in cultured rainbow trout (Oncorhynchus mykiss) cardiac fibroblasts.

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.

Characterizing the influence of chronic hypobaric hypoxia on diaphragmatic myofilament contractile function and phosphorylation in high-altitude deer mice and low-altitude white-footed mice.

Deer mice, Peromyscusmaniculatus, live at high altitudes where limited O2 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 Ca2+ activation of force generation by skinned diaphragmatic myofilaments, and the phosphorylation of myofilament proteins. Results demonstrate that myofilament force production, and the Ca2+ 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.

Hypoxia-induced reprogramming of the cardiac phenotype in American alligators (Alligator mississippiensis) revealed by quantitative proteomics.

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.

Contractile function of the excised hagfish heart during anoxia exposure.

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/dtsys), or rate of relaxation (max-dP/dtdia) 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/dtsys, and max-dP/dtdia 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.

Transforming growth factor-ß1 induces differentiation of rainbow trout (Oncorhynchus mykiss) cardiac fibroblasts into myofibroblasts.

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-ß1 (TGF-ß1). We have previously demonstrated that treatment of cultured rainbow trout cardiac fibroblasts with human TGF-ß1 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-ß1 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-ß1 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-ß1 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.

Developmental and latent effects of diluted bitumen exposure on early life stages of sockeye salmon (Oncorhynchus nerka).

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.

Cellular mechanisms of slime gland refilling in Pacific hagfish (Eptatretus stoutii).

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.

Identification of the actc1c cardiac actin gene in zebrafish.

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.

Emptying and refilling of slime glands in Atlantic (Myxine glutinosa) and Pacific (Eptatretus stoutii) hagfishes.

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.

Transforming growth factor beta-1 (TGF-ß1) stimulates collagen synthesis in cultured rainbow trout cardiac fibroblasts.

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-ß1) stimulates collagen deposition within the myocardial ECM. Therefore, we hypothesized that TGF-ß1 regulates trout myocardial ECM turnover and predicted that TGF-ß1 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-ß1 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-ß1 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-ß1 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-ß1 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.