Short communication: Baroreflex function in embryonic emus (Dromiceius novaehollandiae).

The baroreflex involves cardiovascular homeostatic mechanisms that buffer the system against acute deviations in arterial blood pressure. It is comprised of the cardiac limb which involves adjustments in heart rate and the peripheral limb which involves adjustments in vascular resistance. This negative feedback loop mechanism has been investigated in numerous species of adult vertebrates, however our understanding of the maturation and functional importance of the reflex in developing animals remains poorly understood. In egglaying species, our knowledge of this mechanism is limited to the domestic chicken embryo and the embryonic alligator. While each of these species possess a cardiac baroreflex prior to hatching, they differ in the timing when it becomes functional, with the embryonic chicken possessing the reflex at 90% of incubation, while the alligator possesses the reflex at 70% of incubation. In an effort to determine if bird species might share similar patterns of active baroreflex function, we studied embryonic emus (Dromiceius novaehollandiae). However, we hypothesized that emus would possess a pattern of baroreflex function similar to that of the American alligator given the emu embryo possesses functional vagal tone at 70% of incubation, possibly indicating a more mature collection of cardiovascular control mechanism than those found in embryonic chickens. Our findings illustrate that emu embryos possess a hypotensive baroreflex at 90% of incubation. Therefore, our data fail to support our original hypothesis. While only two species of birds have been studied in this context, it could indicate that baroreflex function is not essential for cardiovascular homeostasis in birds for the majority of in ovo development.

Cardiovascular function during early development is suppressed by cinnamon flavored, nicotine-free, electronic cigarette vapor.

OBJECTIVES: Vaping products continue to remain popular among teens and young adults despite an overall lack of research regarding their potential health effects. While much research focuses on respiratory effects associated with electronic cigarette use, their effects on other systems, including embryonic cardiovascular function and development due to maternal use during pregnancy, also needs to be evaluated. Here, we assessed the impact of nicotine-free, cinnamon and chocolate flavored, electronic cigarette vapor on cardiovascular function during early development by exposing wild-type zebrafish embryos to electronic cigarette vapor. METHODS: Vapor was produced from a second-generation style vape pen and was incorporated into dechlorinated water at 0.6, 12, and 25 puffs/L, where one puff equals 55 ml of vapor. Vapor infused water was distributed among flasks to which zebrafish embryos were added. Exposures lasted for 24 hours and cardiovascular videos were recorded. Videos were analyzed and end systolic volume, end diastolic volume, stroke volume, heart rate, cardiac output, red blood cell density, and arterial and venous blood vessel diameters were measured. RESULTS: Here, it was found that embryonic exposure to nicotine free, cinnamon, and not chocolate, flavored electronic cigarette vapor at 25 puffs/L significantly decreased all cardiovascular parameters measured, with the exception of blood vessel diameter. No significant effect on any measured parameter was observed at 0.6 or 12 puffs/L with either flavor. CONCLUSION: These results indicate that cinnamon flavored electronic cigarette vapor can affect cardiovascular function during early development, even in the absence of nicotine, particularly at elevated exposure concentrations.

Vascular parameters continue to decrease post-exposure with simultaneous, but not individual exposure to BPA and hypoxia in zebrafish larvae.

How fish respond to hypoxia, a common stressor, can be altered by simultaneous exposure to pollutants like bisphenol A (BPA), a plasticizer. BPA is cardiotoxic and interferes with the hypoxia inducible factor pathway (HIF-1α), therefore disrupting the hypoxic response. Co-exposure to hypoxia and BPA also causes severe bradycardia and reduced cardiac output in zebrafish larvae. The purpose of this work was to determine how the cardiovascular effects of co-exposure vary with BPA concentration and persist beyond exposure. Zebrafish embryos were exposed to 0, 0.01, 0.1, 1, and 100 µg/L of BPA during normoxia (>6.0 mg/L O2) and hypoxia (2.0 ±â€¯0.5 mg/L O2) between 1 h post fertilization (hpf) and late hatching (72-96 hpf). Heart rate, cardiac output, and red blood cell (RBC) velocity were determined through video microscopy and digital motion analysis at late hatching and 10 days post fertilization (dpf), several days post exposure. In comparison to the hypoxic control, RBC velocity was 25% lower with 0.01 µg/L BPA and hypoxia at late hatching. At 10 dpf, the difference in RBC velocity between these treatments doubled, despite several days of recovery. This coincided with a 24% thinner outer diameter for caudal vein but no effect on cardiac or developmental parameters. Statistical interactions between BPA and oxygen concentration were found for arterial RBC velocity at both ages. Because the co-occurrence of both stressors is extremely common, it would be beneficial to understand how BPA and hypoxia interact to affect cardiovascular function during and after exposure.

Hypoxia exacerbates the cardiotoxic effect of the polycyclic aromatic hydrocarbon, phenanthrene in Danio rerio.

The Deepwater Horizon oil spill of 2010 released a mixture of polycyclic aromatic hydrocarbons (PAHs) into the Gulf of Mexico presenting a complex exposure regime for native species. Concurrently, the Gulf has experienced an increase in hypoxic events due to agricultural runoff from the Mississippi River outflow. This combination presents a unique physiological challenge to native species and a challenge for researchers. The purpose of this study was to determine how the cardiotoxic PAH, phenanthrene interacts with hypoxia to affect the cardiovascular system of larval zebrafish (Danio rerio). We exposed zebrafish larvae to 0, 1, 100, and 1000 µg/L of phenanthrene in combination with normoxia and hypoxia. At late hatching, video of hearts and vessels were used to measure heart rate (ƒH), stroke volume (SV), cardiac output (Q), red blood cell velocity, and caudal vessel diameter. We found that the highest concentration of phenanthrene caused a 58, 80, and 84% decrease in ƒH, Q, and arterial red blood cell velocity in normoxia and an 88, 98, and 99% decrease in hypoxia, respectively. Co-exposed larvae also experienced higher rates of edema and lordosis in addition to a 33% increase in mortality rate with co-exposure to hypoxia at the 1000 µg/L concentration of phenanthrene. At 12 dpf, baseline swimming behavior was similar between treatments indicating partial recovery from embryonic exposure. This study shows that phenanthrene decreases cardiac parameters, most significantly heart rate and that this effect is exacerbated by simultaneous exposure to hypoxia.

Bisphenol A alters the cardiovascular response to hypoxia in Danio rerio embryos.

The purpose of this study was to determine if the cardiovascular response to hypoxia was altered by the presence of bisphenol A (BPA) in Danio rerio embryos. It was expected that BPA exposure would affect cardiovascular parameters during hypoxia more than normoxia due to an interaction between BPA and the hypoxia-inducible factor (HIF-1α) pathway. We demonstrate that BPA exposure has a minimal effect during normoxia but can severely affect the cardiovascular system during a hypoxic event. Cardiovascular response was measured in vivo using video microscopy and digital motion analysis. RBC density increased 35% in hypoxia alone but decreased 48% with addition of 0.25mg/L BPA. Tissue vascularization (% coverage) was unaffected by hypoxia alone but decreased 37% with addition of 0.25mg/L BPA. The diameter and RBC velocity of arteries were more sensitive than veins to BPA exposure during both normoxia and hypoxia. Arterial RBC velocity decreased 42% during normoxia and 52% during hypoxia with 1mg/L BPA. This decrease in velocity may in part be due to the 86% decrease in heart rate (ƒH) observed during co-exposure to hypoxia and 5mg/L BPA. While stroke volume (SV) was unaffected by treatment, cardiac output (Q) decreased by 69% with co-exposure. ƒH and Q were not affected by BPA exposure during normoxia. Development ultimately slowed by 146% and mortality rates were 95% during hypoxia when exposed to 5mg/L BPA. Our results show for the first time that BPA exposure alters the cardiovascular system during hypoxia more so than normoxia.

Leptin expression affects metabolic rate in zebrafish embryos (D. rerio).

We used antisense morpholino oligonucleotide technology to knockdown leptin-(A) gene expression in developing zebrafish embryos and measured its effects on metabolic rate and cardiovascular function. Using two indicators of metabolic rate, oxygen consumption was significantly lower in leptin morphants early in development [<48 hours post-fertilization (hpf)], while acid production was significantly lower in morphants later in development (>48 hpf). Oxygen utilization rates in <48 hpf embryos and acid production in 72 hpf embryos could be rescued to that of wildtype embryos by recombinant leptin coinjected with antisense morpholino. Leptin is established to influence metabolic rate in mammals, and these data suggest leptin signaling also influences metabolic rate in fishes.

The influence of ontogenetic dietary fluctuations on zebrafish size and swimming performance.

Phenotypic flexibility is critical in determining fitness. As conditions change during ontogeny, continued responsiveness is necessary to meet the demands of the environment. Studies have shown that subsequent ontogenetic periods of development can interact with one another and shape developmental outcomes. The role genetic variation within populations plays in shaping these outcomes remains unclear. Four full-sib families of zebrafish Danio rerio were raised under for dietary regimes: high food rations for 60 days (HH), low food rations for 60 days (LL), high food rations for 30 days followed by low food rations for 30 (HL), and low food rations for 30 days followed by high food rations for 30 (LH). While the low ration diet significantly reduced body length at 30 days, diet was no longer a significant factor at day 60. Only family level variation influenced body length. Furthermore, there was significant family level variation in the manner in which swimming performance responded to fluctuating dietary conditions. Some families increased swimming performance in response to dietary change, while others did not. These results suggest that plastic responsiveness to subsequent environmental changes can be trait specific and vary significantly within populations.

Guided inquiry lab exercises in development and oxygen consumption using zebrafish.

Zebrafish have become a model organism in many areas of research and are now being used with more frequency in the classroom to teach important biological concepts. The two guided inquiry exercises in this article are each aimed at a different level of instruction, but each can be modified to fit the needs of many high school or college-level courses. The "Zebrafish Development and Environment" exercise teaches high school students about zebrafish development by presenting a series of embryos at different ages. Without access to visual references, students are asked to rank developing zebrafish by age and explain their choices. The students also learn about the heart and circulatory system and the effects of temperature on physiological processes. The second exercise, "Oxygen Consumption," is a 2-week laboratory designed for introductory college biology majors and involves the concept of oxygen consumption as a predictor of metabolic rate. During the first week of lab, students are introduced to the concept and learn how to measure oxygen consumption in zebrafish. In the second week, they perform an instructor-approved experiment of their own design, analyze the results using statistics, and write a report.

Cadherin2 (N-cadherin) plays an essential role in zebrafish cardiovascular development.

BACKGROUND: Cadherins are cell surface adhesion molecules that play important roles in development of vertebrate tissues and organs. We studied cadherin2 expression in developing zebrafish heart using in situ hybridization and immunocytochemical methods, and we found that cadherin2 was strongly expressed by the myocardium of the embryonic zebrafish. To gain insight into cadherin2 role in the formation and function of the heart, we analyzed cardiac differentiation and performance in a cadherin2 mutant, glass onion (glo). RESULTS: We found that the cadherin2 mutant had enlarged pericardial cavity, disorganized atrium and ventricle, and reduced expression of a ventricular specific marker vmhc. Individual myocardiocytes in the glo mutant embryos became round shaped and loosely aggregated. In vivo measurements of cardiac performance revealed that the mutant heart had significantly reduced heart rate, stroke volume and cardiac output compared to control embryos. Formation of the embryonic vascular system in the glo mutants was also affected. CONCLUSION: Our results suggest that cadherin2 plays an essential role in zebrafish cardiovascular development. Although the exact mechanisms remain unknown as to the formation of the enlarged pericardium and reduced peripheral blood flow, it is clear that myocardiocyte differentiation and physiological cardiovascular performance is impaired when cadherin2 function is disrupted.

Cardiovascular system in larval zebrafish responds to developmental hypoxia in a family specific manner.

BACKGROUND: Genetic and environmental variation are both known to influence development. Evolution of a developmental response that is optimized to the environment (adaptive plasticity) requires the existence of genetic variation for that developmental response. In complex traits composed of integrated sets of subsidiary traits, the adaptive process may be slowed by the existence of multiple possible integrated responses. This study tests for family (sibship) specific differences in plastic response to hypoxia in an integrated set of cardiovascular traits in zebrafish. RESULTS: Cardiac output, which is the integrated product of several subsidiary traits, varied highly significantly between families, and families differed significantly in the degree and direction of response to developmental oxygen level. The cardiac output response to oxygen environment was entirely family specific with no significant overall trend due to oxygen level. Constituent physiological variables that contribute to cardiac output all showed significant family specific response to hypoxia. Traits that were not directly related to cardiac output, such as arterial and venous diameter, and red blood cell velocities did not respond to hypoxia in a family specific manner. CONCLUSION: Zebrafish families vary in their plastic response to hypoxia. Genetic variation in plastic response to hypoxia may therefore provide the basic ingredient for adaptation to a variable environment. Considerable variation in the degree of familial response to hypoxia exists between different cardiovascular traits that may contribute to cardiac output. It is possible that the integration of several subsidiary traits into cardiac output allows the maintenance of genetic variance in cardiac response.

A three-dimensional functional assessment of heart and vessel development in the larva of the zebrafish (Danio rerio).

There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart's irregular shape and its significant change in morphology during the first 6 d of development. By use of a three-dimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross-sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%-600% increase in cardiac output (from 10 to 50-60 nL min(-1)) occurs on days 5 and 6 postfertilization in Danio rerio. Cross-sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between-individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross-sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three-dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O2 supply to internal tissues.

Ontogeny of cardiovascular control in zebrafish (Danio rerio): effects of developmental environment.

The goal of this symposium paper was to identify and quantify developmental plasticity in the onset of cardiovascular responses in the zebrafish. Developmental plasticity was induced by altering the developmental environment in one of three ways: (1) by developing zebrafish in a constant current of 5 body lengths per second, (2) by developing zebrafish at a colder temperature (20 degrees C), and (3) by developing zebrafish in severe hypoxia (DO=0.8 mg/L). Early morphological development was significantly affected by each of the treatment environments with hypoxia slowing development the most and producing the highest variation in measurements. Development in constant water current did not significantly affect the timing onset of cardiovascular responses to the pharmacological agents applied. Development at 20 degrees C significantly delayed the onset of all cardiovascular responses measured by 2-3 days. Development in hypoxia, however, not only delayed onset of all cardiovascular responses, but also shifted the onset relative to the developmental program. Hypoxia clearly has a profound affect on the onset of cardiovascular regulation and it will take many more studies to elucidate the mechanisms by which hypoxia is having its effect. Furthermore, long term studies are also needed to assess whether the plasticity measured in this study is adaptive in the evolutionary sense.

Maturation of cardiovascular control mechanisms in the embryonic emu (Dromiceius novaehollandiae).

Our understanding of avian embryonic cardiovascular regulation has been based on studies in chickens. The present study was undertaken to determine if the patterns established in chickens are generally applicable to the emu, a ratite bird species. We studied cardiovascular physiology over the interval from 60% to 90% of the emu's 50-day incubation period. During this period, embryonic emus exhibit a slight fall in resting heart rate (from 171 beats min(-1) to 154 beats min(-1)) and a doubling of mean arterial pressure (from 1.2 kPa to 2.6 kPa). Exposures to 15% or 10% O(2) initially decreased heart rate during the first period of emu incubation studied [60% of incubation (60%I)] but increased heart rate in the 90%I group. Arterial pressure responded to hypoxia with an initial depression (-1.6 kPa) at 60%I and 70%I but showed no response during the later periods of incubation (80%I and 90%I). In addition, tonic stimulation of both cholinergic and adrenergic (alpha and beta) receptors was present on heart rate at 70%I, with the cholinergic and beta-adrenergic tone increasing in strength by 90%I. Arterial pressure was dependent on a constant beta-adrenergic and constant alpha-adrenergic tone from 60%I to 90%I. A comparison with embryonic white leghorn chickens over a similar window of incubation revealed that emus and white leghorn chickens both possess an adrenergic tone on heart rate and pressure but that only emus possess a cholinergic tone on heart rate. Collectively, these data indicate that the maturation of cardiovascular control systems differs between white leghorn chickens and emus, inviting investigation of additional avian species to determine other patterns.