Casey A. Mueller

Comparative cardiovascular physiology: future trends, opportunities and challenges

The inaugural Kjell Johansen Lecture in the Zoophysiology Department of Aarhus University (Aarhus, Denmark) afforded the opportunity for a focused workshop comprising comparative cardiovascular physiologists to ponder some of the key unanswered questions in the field. Discussions were centred around three themes. The first considered function of the vertebrate heart in its various forms in extant vertebrates, with particular focus on the role of intracardiac shunts, the trabecular (‘spongy’) nature of the ventricle in many vertebrates, coronary blood supply and the building plan of the heart as revealed by molecular approaches. The second theme involved the key unanswered questions in the control of the cardiovascular system, emphasizing autonomic control, hypoxic vasoconstriction and developmental plasticity in cardiovascular control. The final theme involved poorly understood aspects of the interaction of the cardiovascular system with the lymphatic, renal and digestive systems. Having posed key questions around these three themes, it is increasingly clear that an abundance of new analytical tools and approaches will allow us to learn much about vertebrate cardiovascular systems in the coming years.

Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings

Critical windows are periods of developmental susceptibility when the phenotype of an embryonic, juvenile or adult animal may be vulnerable to environmental fluctuations. Temperature has pervasive effects on poikilotherm physiology, and embryos are especially vulnerable to temperature shifts. To identify critical windows, we incubated whitefish embryos at control temperatures of 2°C, 5°C, or 8°C, and shifted treatments among temperatures at the end of gastrulation or organogenesis. Heart rate (fH) and oxygen consumption ( [Formula: see text] ) were measured across embryonic development, and [Formula: see text] was measured in 1-day old hatchlings. Thermal shifts, up or down, from initial incubation temperatures caused persistent changes in fH and [Formula: see text] compared to control embryos measured at the same temperature (2°C, 5°C, or 8°C). Most prominently, when embryos were measured at organogenesis, shifting incubation temperature after gastrulation significantly lowered [Formula: see text] or fH. Incubation at 2°C or 5°C through gastrulation significantly lowered [Formula: see text] (42% decrease) and fH (20% decrease) at 8°C, incubation at 2°C significantly lowered [Formula: see text] (40% decrease) and fH (30% decrease) at 5°C, and incubation at 5°C and 8°C significantly lowered [Formula: see text] at 2°C (27% decrease). Through the latter half of development, [Formula: see text] and fH in embryos were not different from control values for thermally shifted treatments. However, in hatchlings measured at 2°C, [Formula: see text] was higher in groups incubated at 5°C or 8°C through organogenesis, compared to 2°C controls (43 or 65% increase, respectively). Collectively, these data suggest that embryonic development through organogenesis represents a critical window of embryonic and hatchling phenotypic plasticity. This study presents an experimental design that identified thermally sensitive periods for fish embryos.

The Regulation Index: A New Method for Assessing the Relationship between Oxygen Consumption and Environmental Oxygen

Critical oxygen pressure (PC) is used in respiratory physiology to measure the response to hypoxia. PC defines the partial pressure of oxygen (Po2) at which an oxygen regulator switches to a conformer. However, not all animals show such clear patterns in oxygen consumption rate (), and there are many methods for determining PC. This study assesses two methods that determine regulatory ability and four that calculate PC. A new method, the regulation index (RI), assigns to an animal a relative measure of regulatory ability by calculating the area under the versus Po2 curve that is greater than a linear trend. The six methods are applied to developmental data of two amphibians, Pseudophryne bibronii and Crinia georgiana. The four methods used to determine PC produced similar results but failed to identify the increase in regulation on hatching in C. georgiana or the greater regulation in larval C. georgiana compared with P. bibronii. Of the two methods that evaluated regulation, only the RI satisfactorily represented the entire range of Po2. The RI is advantageous because it has clearly defined limits and does not constrain data to fit any single pattern. The RI can be used in concert with PC, which can be easily calculated during the RI analysis, to provide a clearer definition of the response to environmental Po2.

Developmental Critical Windows and Sensitive Periods as Three-Dimensional Constructs in Time and Space*

A critical window (sensitive period) represents a period during development when an organism’s phenotype is responsive to intrinsic or extrinsic (environmental) factors. Such windows represent a form of developmental phenotypic plasticity and result from the interaction between genotype and environment. Critical windows have typically been defined as comprising discrete periods in development with a distinct starting time and end time, as identified by experiments following an on and an off protocol. Yet in reality, periods of responsiveness during development are likely more ambiguous that depicted. Our goal is to extend the concept of the developmental critical window by introducing a three-dimensional construct in which time during development, dose of the stressor applied, and the resultant phenotypic modification can be utilized to more realistically define a critical window. Using the example of survival of the brine shrimp (Artemia franciscana) during exposure to different salinity levels during development, we illustrate that it is not just stressor dose or exposure time but the interaction of these two factors that results in the measured phenotypic change, which itself may vary within a critical window. We additionally discuss a systems approach to critical windows, in which the components of a developing system—whether they be molecular, physiological, or morphological—may show differing responses with respect to time and dose. Thus, the plasticity of each component may contribute to a broader overall system response.

Embryonic development of lake whitefish Coregonus clupeaformis: a staging series, analysis of growth and effects of fixation

A reference staging series of 18 morphological stages of laboratory reared lake whitefish Coregonus clupeaformis is provided. The developmental processes of blastulation, gastrulation, neurulation as well as development of the eye, circulatory system, chromatophores and mouth are included and accompanied by detailed descriptions and live imaging. Quantitative measurements of embryo size and mass were taken at each developmental stage. Eggs were 3·19 ± 0·16 mm (mean ± s.d.) in diameter at fertilization and embryos reached a total length (LT ) of 14·25 ± 0·41 mm at hatch. Separated yolk and embryo dry mass were 0·25 ± 0·08 mg and 1·39 ± 0·17 mg, respectively, at hatch. The effects of two common preservatives (formalin and ethanol) were examined throughout development and post hatch. Embryo LT significantly decreased following fixation at all points in development. A correction factor to estimate live LT from corresponding fixed LT was determined as live LT = (fixed LT )(1·025) . Eye diameter and yolk area measurements significantly increased in fixed compared with live embryos up to 85-90% development for both measurements. The described developmental stages can be generalized to teleost species, and is particularly relevant for the study of coregonid development due to additionally shared developmental characteristics. The results of this study and staging series are therefore applicable across various research streams encompassing numerous species that require accurate staging of embryos and descriptions of morphological development.

The Physiology of the Avian Embryo

Abstract The freshly laid avian egg contains most of the materials needed for embryonic growth and development, but lacks the oxygen and heat needed for successful development. Microscopic pores in the eggshell allow O2 to diffuse into the egg from the environment and water vapor and CO2 produced by the embryo to diffuse out. The adult bird has a key role in incubation, providing not only the heat necessary for embryonic development but also controlling the microclimate of the egg. In the poultry industry and for research purposes, the adult bird can be conveniently replaced by an incubator. The majority of research on avian incubation is undertaken using artificially incubated chicken (Gallus gallus domesticus) eggs. Thus, in this chapter, the chicken embryo is used to elucidate the development of physiological function during avian incubation, supplemented by additional species when data are available. Developmental physiology of the gas exchange, acid–base, cardiovascular, osmoregulatory and thermoregulatory systems are examined. The optimal conditions for artificial incubation are outlined and embryonic responses to incubation extremes are described.

Dynamics of metabolic compensation and hematological changes in chicken (Gallus gallus) embryos exposed to hypercapnia with varying oxygen

In day 15 chicken embryos, we determined the time course responses of acid-base balance and hematological respiratory variables during 24h exposure to 15, 20, 40 or 90% O(2), in the presence of 5% CO(2). Hypercapnic respiratory acidosis was initially (2h) only slightly (∼20%) compensated by metabolic alkalosis in normoxic/hyperoxic embryos. After 6h, respiratory acidosis was partially (∼40-50%) compensated not only in normoxic/hyperoxic embryos, but also in hypoxic embryos. However, partial metabolic compensation in 15% O(2) could not be preserved after 24h. Preservation of metabolic compensation required oxygen concentration ([O(2)]) above 20%, but the magnitude of partial metabolic compensation was unrelated to [O(2)]. Hematocrit (Hct), together with mean corpuscular volume (MCV), markedly increased in hypercapnic hypoxia, and was maintained at 24h due to a subsequent increase in red blood cell concentration ([RBC]). In contrast, Hct, together with MCV, decreased in hypercapnic normoxia/hyperoxia accompanied by a subsequent decrease in [RBC] at 24h. Regulation of variables takes place similarly irrespective of environmental [O(2)] above 20%, matching acid-base regulation.

The actions of the renin-angiotensin system on cardiovascular and osmoregulatory function in embryonic chickens (Gallus gallus domesticus).

Using embryonic chickens (Gallus gallus domesticus), we examined the role of the renin-angiotensin system (RAS) in cardiovascular and osmotic homeostasis through chronic captopril, an angiotensin-converting enzyme (ACE) inhibitor. Captopril (5 mg kg⁻¹ embryo wet mass) or saline (control) was delivered via the egg air cell daily from embryonic day 5-18. Mean arterial pressure (MAP), heart rate (ƒ(H)), fluid osmolality and ion concentration, and embryonic and organ masses were measured on day 19. Exogenous angiotensin I (ANG I) injection did not change MAP or ƒ(H) in captopril-treated embryos, confirming ACE inhibition. Captopril-treated embryos were significantly hypotensive, with MAP 15% lower than controls, which we attributed to the loss of vasoconstrictive ANG II action. Exogenous ANG II induced a relatively greater hypertensive response in captopril-treated embryos compared to controls. Changes in response to ANG II following pre-treatment with phentolamine (α-adrenergic antagonist) indicated a portion of the ANG II response was due to circulating catecholamines in captopril-treated embryos. An increase in MAP and ƒ(H) in response to hexamethonium indicated vagal tone was also increased in the absence of ACE activity. Captopril-treated embryos had lower osmolality, lower Na⁺ and higher K⁺ concentration in the blood, indicating osmoregulatory changes. Larger kidney mass in captopril-treated embryos suggests disrupting the RAS may stimulate kidney growth by decreasing resistance at the efferent arteriole and increasing the fraction of cardiac output to the kidneys. This study suggests that the RAS, most likely through ANG II action, influences the development of the cardiovascular and osmoregulatory systems.

The Importance of Perivitelline Fluid Convection to Oxygen Uptake of Pseudophryne bibronii Eggs

The ciliated epithelium of amphibian embryos produces a current within the perivitelline fluid of the egg that is important in the convective transfer of oxygen to the embryo’s surface. The effects of convection on oxygen uptake and the immediate oxygen environment of the embryo were investigated in Pseudophryne bibronii. Gelatin was injected into the eggs, setting the perivitelline fluid and preventing convective flow. Oxygen consumption rate () and the oxygen partial pressure (Po2) of the perivitelline fluid were measured in eggs with and without this treatment. decreased in eggs without convection at Gosner stages 17–19 under normoxia. The lack of convection also shifted embryos from regulators to conformers as environmental Po2 decreased. A strong Po2 gradient formed within the eggs when convection was absent, demonstrating that the loss of convection is equivalent to decreasing the inner radius of the capsule, an important factor in gas exchange, by 25%. also declined in stage 26–27 embryos without cilia-driven convection, although not to the extent of younger stages, because of muscular movements and a greater skin surface area in direct contact with the inner capsule wall. This study demonstrates the importance of convective flow within the perivitelline fluid to gas exchange. Convection is especially important in the middle of embryonic development, when the perivitelline space has formed, creating a barrier to gas exchange, but the embryos have yet to develop muscular movements or have a large surface area exposed directly to the jelly capsule.

Hypercapnic thresholds for embryonic acid–base metabolic compensation and hematological regulation during CO2 challenges in layer and broiler chicken strains

Time specific acid-base metabolic compensation and responses of hematological respiratory variables were measured in day 15 layer (Hyline) and broiler (Cornish Rock) chicken embryos during acute hypercapnic challenges (3, 6, 10 and 20% CO2). Control acid-base status and hematology differed between two strains. Broiler embryos were relatively respiratory acidotic and had higher hematocrit (Hct) and hemoglobin concentration. The partial metabolic compensation for respiratory acidosis produced by ≤ 10% CO2 exposures occurred in proportion to CO2 concentrations in both strains, but metabolic compensation for 20% CO2 respiratory acidosis was depressed at 2, 6 and 24h, particularly in broiler embryos. Exposure to ≤ 10% CO2 induced the same hematological responses across CO2 concentrations; i.e., Hct and mean corpuscular volume (MCV) increased while RBC concentration remained unchanged. In response to 20% CO2 exposure, Hct and MCV increased dramatically in both stains. Consequently, altered acid-base and hematology responses to 20% CO2 exposure compared to ≤ 10% CO2 suggest that the hypercapnic threshold to compensation for acidosis and regulation of hematology is >10% CO2.