Introduction

The renin angiotensin system (RAS) is a vital humoral system contributing to cardiovascular and renal function in adult vertebrates. In adults, renin released by the kidneys cleaves angiotensinogen to produce angiotensin I (ANG I). ANG I is cleaved by angiotensin-converting enzyme (ACE) to produce angiotensin II (ANG II). ANG II has direct effects on the vasculature causing vasoconstriction (Bottari et al. 1993; Jӧhren et al. 2004), and has many secondary effects, including the release of catecholamines, aldosterone, and arginine vasotocin, which impact cardiovascular function (Farrell et al. 2001; Dendorfer et al. 2002; Harrison-Bernard 2009). While the role of ANG II in adult cardiovascular function has been investigated in multiple adult vertebrates (e.g., Nishimura et al. 1982; Brown et al. 1983; Brooks and Reid 1986; Garner et al. 1987; Matsumura et al. 1989; Lumbers et al. 1992; Esteves et al. 2012), its role across development and the maturation of its cardiovascular regulatory ability has been investigated in only two vertebrate groups.

To date, our knowledge of the role of ANG II during development comes from studies of fetal rats, fetal sheep, and embryonic chickens (Siegel and Fisher 1980; Robillard and Nakamura 1988; Crossley II et al. 2010; Mueller et al. 2013, 2014). ANG II contributes to cardiovascular control in all species studied to date; however, there are unique features to its mode of action between these species. For example, in embryonic chickens, ANG II significantly contributes to the maintenance of baseline mean arterial pressure (MAP) and attenuates short-term baroreflex control (Mueller et al. 2013, 2014). In contrast, in fetal sheep, ANG II does not alter the set point or sensitivity of the baroreflex, but does have a stronger influence in newborn sheep (Segar et al. 1994). Such interspecific differences may result from a higher level of circulating ANG II in the plasma of embryonic chickens compared to fetal mammals (Crossley II et al. 2010). The extent to which ANG II contributes to developmental cardiovascular function in other vertebrate groups, such as reptiles, remains to be studied. In ovo reptilian studies may provide insight into the role of the RAS during the evolution of vertebrates, including the important transition to endothermy.

The American alligator (Alligator mississippiensis) is a model species used to examine the development of the reptilian cardiovascular system (Crossley II and Burggren 2009). In embryonic alligators, MAP increases from 60 to 90% of development, while heart rate (fH) stays relatively unchanged (Crossley II and Altimiras 2005). Circulating catecholamines are the primary source of an adrenergic tone on the system in the final third of embryonic development (Eme et al. 2011a), while cholinergic tone is absent (Eme et al. 2011a). Furthermore, embryonic alligators first show baroreflex control of fH at 70% of development that is exclusively a response to hypertensive stimulus (Crossley II et al. 2003a, b, c). The function of the RAS system in the regulation of cardiovascular function in embryonic alligators has been investigated with exogenous injection of ANG II and they respond in a similar manner to embryonic chickens (Tate et al. 2012). The hypertension, and corresponding transient bradycardia, in response to ANG II injection increases from 70 to 90% of development, suggesting an increasing role of ANG II in maintaining baseline pressure during embryonic development. However, the developmental importance of ANG II to baseline cardiovascular function, and how it may interact with short-term regulation such as barostatic reflexes, in alligator embryos is unknown.

We investigated the effects of ACE inhibition on cardiovascular development to further understand the role of ANG II in the cardiovascular function of embryonic American alligators. We chronically treated embryos with captopril, an angiotensin-converting enzyme (ACE) inhibitor that prevents conversion of ANG I into the vasoactive ANG II. We hypothesized that chronic treatment with captopril would disrupt the RAS, resulting in hypotensive embryos. We predicted the chronic hypotensive response would be dose-dependent with a stronger response in the high-dose (~ 450 mg kg embryo−1) treatment compared to the low-dose (~ 5 mg kg embryo−1) treatment group. We examined the response to injections of exogenous ANG I and ANG II in the low-dose treatment group. The hypertensive response to ANG II is partly due to catecholamine release in chicken embryos treated with captopril (Mueller et al. 2014), and thus, we predicted that embryos in the low-dose treatment group would show a reduced intensity in response to acute ANG II following α-adrenergic blockade. We also explored the possible interactions between ANG II and cardiac baroreflex function. Alligator embryos have a hypertensive, but no hypotensive, baroreflex at 90% of incubation (Crossley II et al. 2003a, b, c). We predicted that chronic disruption of the RAS would result in the baroreflex set point being shifted to a lower pressure, i.e., a left shifted baroreflex curve. However, given the relative immaturity of the alligator baroreflex compared to the chicken at 90% of development, we did not predict as strong a change in the sensitivity of the baroreflex as observed previously in chickens following captopril treatment (Mueller et al. 2013).

Materials and methods

Alligator egg incubation

Seven clutches of American alligator eggs were obtained from the Rockefeller Wildlife Refuge in Grand Chenier, LA, USA, and transported to the University of North Texas. Two eggs from each clutch were staged to determine each clutches’ post-laying time, as previously described [72 days total incubation period at 30 °C; (Ferguson 1985; Crossley II and; Altimiras 2005; Eme et al. 2011a)]. Eggs were buried to the midpoint of the egg in a bed of vermiculite mixed in a 1:1 ratio with water and incubated at 30 °C in a walk-in Percival environmental room (model IR-912L5; Percival Scientific, Perry, IA) in plastic containers (2.5-L Ziploc Container, SC Johnson, Racine, WI, USA). Water content of the vermiculite was maintained by weighing the box two or three times weekly and adding water to keep the mass constant, as previously described (Eme et al. 2011b; Tate et al. 2016).

Egg boxes were then sealed inside large Ziplock® bags that had two holes that allowed parallel inflow and outflow of air. Air was passed through a H2O bubbler to ensure adequate water saturation within the bag (~ 9.0 L min−1). Gas composition within the bag was monitored with an oxygen analyzer (S-3AII, Ametek Applied Electrochemistry, IL, USA) connected to a PowerLab® data recording system connected to a computer running LabChart Pro® software (v 7 ADInstruments, CO, USA), and data recorded at 40 Hz. All studies were approved by UNT IACUC #11-007. Monitoring gas composition allowed for certainty that oxygen levels were ~ 20% throughout incubation as embryos grew.

Chronic captopril injection protocols for low- and high-dose treatment groups

Four experimental treatment groups were used, chronic high-dose captopril injection embryos (N = 12; 450 ± 50 mg kg embryo−1), chronic low-dose captopril injection embryos (N = 7; 5 ± 0.2-mg kg embryo−1), saline only injection embryos (N = 6), and control no injection embryos (N = 3). Captopril and saline injection eggs were cleaned and had a small area of the outer, calcified eggshell removed (0.5-cm diameter) using a 0.3-cm-diameter drill bit and a Dremel®, leaving the underlying eggshell and chorioallantoic membrane intact. Each ‘hole’ was in the ‘dorsal’ midline of the egg, adjacent to, but not directly on top of the embryo. Alligator embryos remain stationary during incubation, and, therefore, have a ‘top’ and ‘bottom’ side; the early stage embryo attaches and develops on the top/dorsal side (Ferguson 1985). The hole was covered with 100% clear silicone (DAP Auto/Marine sealant; DAP Products Inc., Baltimore, MD, USA). Embryos received captopril or saline injections every other day throughout the duration of the experiment, from 50 to 90% of embryonic development (~ day 35–65 post-laying).

Chronic high- and low-dosage injection concentrations were based on embryo mass from Deeming and Fergusson (1989), Crossley II and Altimiras (2005), and Crossley II et al. (unpublished). The chronic high dosage represented a supramaximal dose intended to provide complete inhibition of angiotensin-converting enzyme (ACE) to demonstrate ‘proof of principle’ in using captopril. The chronic low dose was similar to that previously used in a study of embryonic chickens (Mueller et al. 2013), and 10× higher than a previously reported study of juvenile American alligators (Silldorff and Stephens 1992 used 0.5 ± 0.2 mg kg embryo−1). This dose was chosen to ensure blockade of ACE while reducing the mortality seen in the supramaximal dose. Embryo mass was estimated for each estimated day post-laying by generating a regression of body mass and days post-laying using a log-based best fit line: [body mass = 0.0000552 g × (days post-laying)3.2196]. As the embryo grew larger from 50 to 90% of development, eggs were candled and care was taken to avoid disturbing the embryo with the needle.

Embryos in both low- and high-dose chronic captopril treatments received injections of captopril diluted in saline (warmed to 30 °C) using a 1 cc syringe and a sterile 25-gauge needle into the egg, so the CAM was bathed in solution. The silicone covering on the egg was wiped with 70% ethanol prior to and following each injection. In the high-dose experiment, 30 mg mL−1 of captopril was delivered at the following individual injection volumes during embryonic windows: 50–60% of development 100 µL, 60–70% 200 µL, 70–80% 300 µL, and 80–90% 400 µL. Approximately 3.6 mL of fluid was given to each embryo over the course of the high-dose experiment. In the low-dose captopril experiment, injection of captopril was performed using a glass Hamilton syringe and a 25-gauge needle. The glass syringe was wiped with ethanol between injections. Injections of 1 or 5 mg mL−1 were delivered at the following individual injection volumes during embryonic windows: 50–60% of development 28–46 µL, 60–70% 16–62 µL, 70–80% 17–25 µL, and 80–90% 26–38 µL. Approximately 0.6 mL of fluid was given to each embryo over the course of the experiment. Saline only treatment embryos mirrored the low-dose captopril treatment schedule, with 0.6 mL of fluid given to each embryo over the course of the experiment, and control embryos received no chronic injections whatsoever.

Embryonic surgery and pharmacological injection protocols

Measurements of embryonic alligators were conducted at 90% of embryonic development/incubation, which reflected in ovo developmental stages 27 or 28 (Ferguson 1985; Eme et al. 2011a, c). Surgery occurred on the day following final captopril or saline injection. Eggs were removed from their incubators and candled to determine embryo orientation and location of an accessible tertiary chorioallantoic membrane (CAM) artery. Eggs were placed in a temperature-controlled surgical chamber (30 °C) and a portion of the eggshell removed under a dissection microscope (Leica MZ6; Leica Microsystems). A tertiary CAM artery was isolated for arterial pressure monitoring and drug injection, and an occlusive catheter was inserted into the artery under a dissection microscope using heat-pulled, heparinized, and saline-filled PE 50 tubing, as previously described (Crossley II and Altimiras 2005; Eme et al. 2011a, b). The catheter was attached to a pressure transducer 1–3 cm above the egg (ADInstruments model MLT0699, Colorado Springs, CO, USA) via saline-filled PE 50 tubing, connected to an Octabridge amplifier (ADInstruments, Colorado Springs, CO, USA), and the pressure signal acquired (40 Hz). Pressure transducers were calibrated prior to each measurement period with a vertical column of saline, and heart rate was determined with a software tachograph calculated based on the arterial pressure pulse.

Following catheterization, embryos were transferred to a six-chamber (730-mL chamber volume, with one embryo per chamber placed on cotton), water-jacketed, stainless steel experimental apparatus and allowed to recover for at least 60 min. Temperature (30 °C) was maintained throughout the apparatus’ chambers by recirculating water (30 °C) from a constant temperature circulator (VWR 1165; VWR International, LLC, West Chester, PA, USA). Each chamber in the apparatus had a stainless steel lid, with three small holes that allowed for the catheter line (1 × 5 mm3) and airlines (2 × 3 mm3) to enter the chamber. Air was continuously pumped into each chamber at ~ 0.2 L·min−1 connected to a 2-m heating coil lining the chambers. Following a 60-min recovery, all embryos received a control injection of heparinized saline equivalent to total volume of each drug injection plus saline flush to document any responses from injection alone. Individual total injection volumes did not surpass 5% of total blood volume (Tate et al. 2012). All injections were administered through a T connector in the arterial catheter line, and all drug injection concentrations were based on previously published pharmacological studies of alligator embryos (Silldorff and Stephens 1992; Crossley II et al. 2003a, b, c; Crossley II and; Altimiras 2005; Eme et al. 2011a, c; Tate et al. 2012).

In the high-dose captopril experiment, all embryos were instrumented to measure baseline arterial pressure (MAP, kPa) and heart rate (fH, beats min−1) only, with baseline levels taken for 5–10-min periods at least 60 min after catheterization. The control, saline, and low-dose captopril treatments were instrumented to measure baseline MAP and fH; and in addition, each of these treatments was subjected to a pharmacological protocol. The protocol included angiotensin I (ANG I) injection (2000 ng kg−1) to assess the success of chronic ACE inhibition by captopril and angiotensin II (ANG II) injection (2000 ng kg−1) to examine the pressor response after ACE inhibition. Both ANG I and ANG II were custom-synthesized crocodilian peptides (synthesized by Conlon). Baroreflex function was then assessed by examining reflexive fH responses to pharmacological manipulation by a series of doses of sodium nitroprusside (SNP; 25–100 µg kg−1; Sigma-Aldrich, Saint Louis, MO, USA), a vasodilator that acts via the release of nitric oxide, and phenylephrine (25–100 µg kg−1; Sigma-Aldrich), a vasoconstrictor, and α1-adrenoreceptor agonist. The α-adrenergic contribution of the ANG II pressor response was examined by blockade with the α-adrenoreceptor antagonist, phentolamine (3 mg kg−1; Sigma-Aldrich), followed by a second dose of ANG II (2000 ng kg−1). Each 50 µL drug injection was flushed with 50 µL of saline to ensure the drug had entered the CAM artery. ANG I and II dosages were based on previously published data on chicken and alligator embryos (CrossleyII et al. 2010; Tate et al. 2012; Mueller et al. 2014). This pharmacological protocol is described below:

  1. 1.

    Response to control saline injection volume. Saline injection of a volume equal to total drug injection plus flush volumes (100 µL) was given 60 min after catheterization. Changes in MAP and fH were based on mean values taken over 5–10-min periods preceding and following control injection.

  2. 2.

    Angiotensin I (ANG I, 2000 ng kg−1). Given 30 min after control saline. Changes in MAP and fH were based on mean values taken over 5-min period preceding injection and 30 s–1-min period immediately after injection.

  3. 3.

    Angiotensin II (ANG II, 2000 ng kg−1). Given 60 min after ANG I. Changes in MAP and fH were based on mean values taken over 5-min period preceding injection and 30 s–1-min period immediately after injection.

  4. 4.

    Sodium nitroprusside (SNP, 25 µg kg−1). Given 60 min after ANGII. Changes in MAP and fH were based on mean values taken over 5-min period preceding injection and 30 s–1-min period immediately after injection.

  5. 5.

    SNP (50 µg kg−1). Administered and measurements taken as for 4, above.

  6. 6.

    SNP (100 µg kg−1). Administered and measurements taken as 4, above.

  7. 7.

    Phenylephrine (25 µg kg−1). Administered and measurements taken as 4, above.

  8. 8.

    Phenylephrine (50 µg kg−1). Administered and measurements taken as 4, above.

  9. 9.

    Phenylephrine (100 µg kg−1). Administered and measurements taken as 4, above.

  10. 10.

    Phentolamine (3 mg kg−1). Given 60 min after last phenylephrine dose. Changes in MAP and fH were based on mean values taken over 5-min period preceding injection and 5-min period 30 min after injection.

  11. 11.

    ANG II (2000 ng kg−1). Given approximately 30 min after phentolamine when MAP and fH were stabilized. Changes in MAP and fH were based on mean values taken over 5-min period preceding injection and 30-s–1 min period immediately after injection.

Following completion of each trial, embryos were euthanized with an overdose of isoflurane (Isoflo; Abbott Laboratories, North Chicago, IL, USA), and egg mass and wet embryo, yolk, heart, lung, liver, and kidney masses determined ± 0.001 g using an analytical balance (Mettler Toledo XS204; Columbus, OH, USA). Absolute blood pressure was corrected for a pressure transducer’s distance above the egg by adding the distance from the transducer to the catheter (cm) to recorded pressure (kPa).

Statistical analyses

All data met the parametric assumptions of normality and equal variances. Egg mass, embryo mass, yolk mass, organ mass, mass-specific organ mass, and baseline MAP and fH levels were compared between control, saline, low-dose captopril and high-dose captopril treatments with separate one-way analysis of variance (ANOVA), and a post hoc Tukey’s HSD test. In control, saline and low-dose captopril embryos only, the pre-injection and post-injection MAP or fH after the injections 1, 2, 3, 10, and 11 were compared between treatments with separate two-way repeated-measures ANOVAs and post hoc Tukey’s HSD. For comparisons of the percentage changes in MAP or fH following drug injections, a one-way ANOVA and post hoc Tukey’s HSD were used (Eme et al. 2011a, c). For responses to SNP and phenylephrine, a two-way repeated-measure ANOVA was used to assess differences between treatments across doses for each drug.

To assess baroreflex function, minimum/maximum MAP values after each drug injection were plotted against the corresponding fH for each individual embryo. Only instances of an inverse relationship between MAP and fH were included in analyses. Corresponding fH and MAP were analyzed for each embryo using a four-variable sigmoidal logistic function (Reid 1996):

$${f_{\text{H}}}{\text{~=~}}\frac{{\left( {{\text{max}} - {\text{~min}}} \right)}}{{\left( {{\text{1~+~(MAP/}}C{)^B}} \right)}}+{\text{min,}}$$

where max and min are the maximum and minimum fH (beats min−1), respectively, attained by the baroreflex, B is the maximum slope of the line coefficient (beats min−1 kPa−1), and C is the MAP (kPa) when fH is at the midpoint of its range. The best fit was determined using the quasi-Newton iterative method in the nonlinear estimation module of Statistica 10 (StatSoft, Tulsa, OK, USA). The gain (G50) of the baroreflex when MAP = C was calculated as follows:

$${G_{{\text{50}}}}={\text{ABS}}\left( {\frac{{ - B\left( {{\text{max}} - {\text{min}}} \right)}}{{4C}}} \right).$$

For comparison between treatments, the gain was normalized as a percentage change in fH per unit change in MAP (G; % kPa−1), and adapted to the four-variable sigmoid model (Berger et al. 1980; Altimiras et al. 1998):

$$G=\frac{{100B}}{{{\text{min}}}}.$$

Differences in baroreflex parameters were tested with a two-way ANOVA with Tukey’s HSD, with treatment and baroreflex method as the main effects. Data presented as mean ± SEM and differences were accepted as statistically significant at α = 0.05.

Results

Survival and baseline MAP and f H for high-dose captopril, low-dose captopril, saline, and control embryos

All control, saline injection, and low-dose captopril treatment embryos survived until 90% of development. In comparison, 24% of embryos survived high-dose captopril treatment. Baseline MAP was significantly affected by treatment (P < 0.001, one-way ANOVA, Table 1). MAP of control and saline embryos were not significantly different. However, MAP of the low-dose captopril embryos was significantly reduced (by 41%) compared to saline embryos. MAP of the high-dose captopril embryos was significantly lower than all other treatments, reduced by 72% compared to control embryos, 74% compared to saline embryos, and 56% compared to the low-dose captopril embryos (Table 1). Baseline fH of the high-dose captopril-treated embryos was significantly lower than control, saline, and low-dose captopril-treated embryos (P < 0.001, one-way ANOVA, Table 1).

Table 1 Baseline mean arterial pressure (MAP, kPa) and heart rate (fH, beats min−1) of control, saline injection, low-dose captopril injection, and high-dose captopril injection embryos
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ANG I and ANGI II responses for low-dose captopril, saline, and control embryos

There was no change in MAP or fH in any treatment following control injections of saline equivalent to the drug injection volume (100 µL) (P = 0.941, two-way RM ANOVA). MAP was significantly affected by the interaction between treatment and before and after ANG I injection (P = 0.003, two-way RM ANOVA). Despite low-dose captopril embryos having a slightly lower MAP, the three treatments did not differ significantly immediately prior to the ANG I injection. Following administration of ANG I, MAP of the control and saline embryos was significantly higher than the low-dose captopril embryos. However, ANG I did not cause a significant hypertensive response in any treatment (Tukey’s HSD, Fig. 1a). fH was not significantly affected by ANG I injection (P = 0.072), treatment (P = 0.983), or the interaction (P = 0.342, Fig. 1b).

Fig. 1
figure 1

Pre- and post-injection values for mean arterial pressure (a MAP, kPa) and heart rate (a fH, beats min−1) in response to angiotensin I (ANG I, 2000 ng kg−1), and MAP (c) and fH (d) in response to angiotensin II (ANG II, 2000 ng kg−1) in control, saline injection, and low-dose captopril injection embryos. Data presented as mean ± SEM. Pre: before injection, post: ~1 min after injection. Different letters in A indicate significant differences (P < 0.05) due to a significant interaction between treatment and pre- and post-injection values (two-way RM ANOVA, Tukey’s HSD). Different letters in B and D indicate significant differences (P < 0.05) between pre- and post-injection values for all treatments combined, which did not differ (two-way RM ANOVA, Tukey’s HSD). N as follows for all bars shown for each treatment: control = 3, saline = 6, low-dose captopril = 7

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There was no treatment effect (P = 0.052) or significant interaction between treatment and MAP before and after injection of ANG II (P = 0.936, two-way RM ANOVA). However, for all treatments combined, ANG II injection caused a significant hypertensive response (before ANG II: 1.55 ± 0.18 kPa, after ANG II: 2.61 ± 0.24 kPa, P = 0.001, two-way RM ANOVA, Fig. 1c). Average MAP values increased by 80 ± 12% in control, 57 ± 6% in saline embryos, and 90 ± 9% in low-dose captopril embryos. The % increase in the low-dose captopril-treated embryos was significantly greater compared to saline, but not control embryos (P = 0.036, one-way ANOVA, Tukey’s HSD). fH was not affected by treatment (P = 0.572, two-way RM ANOVA) or the interaction of treatment and before and after ANG II injection (P = 0.888, two-way RM ANOVA). However, ANG II caused a significant bradycardia when all treatments were combined (before ANG II: 80 ± 1 beats min−1, after ANG II: 51 ± 4 beats min−1, P < 0.001, two-way RM ANOVA, Fig. 1d). fH decreased by 37% in control embryos, 34% in saline embryos and 38% in low-dose captopril embryos. The % decrease in fH did not differ between treatments (P = 0.884, one-way ANOVA).

Adrenergic contribution to ANG II response for low-dose captopril, saline, and control embryos

There was no significant interaction between treatment and MAP before and after injection of the α-adrenergic receptor blocker phentolamine (P = 0.482, two-way RM ANOVA). Phentolamine caused a significant hypotension (P < 0.001, two-way RM ANOVA, Table 2). There was a treatment effect on MAP before and after phentolamine combined, with low-dose captopril embryos significantly lower than saline embryos (control: 1.14 ± 0.19 kPa, saline: 1.21 ± 0.14 kPa, low-dose captopril: 0.91 ± 0.08 kPa, P = 0.018, two-way RM ANOVA). Phentolamine had no significant effect on fH in any treatment (P = 0.304, two-way RM ANOVA) and there was no treatment effect on fH before or after injection (P = 0.826, two-way RM ANOVA, Table 2).

Table 2 Mean arterial pressure (MAP, kPa) and heart rate (fH, beats min−1) before and after injection of phentolamine in control, saline injection, and low-dose captopril injection embryos
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There was no significant interaction between treatment and MAP before and after injection of ANG II before or after phentolamine (P = 0.166, two-way RM ANOVA). A treatment effect was also absent in the MAP response for ANG II before or after phentolamine (P = 0.541, two-way RM ANOVA). Therefore, with all treatments combined, the absolute increase in MAP (ΔMAP) induced by ANG II was depressed following phentolamine administration (P < 0.0001, two-way RM ANOVA, Fig. 2a). The intensity of the ANG II hypertensive response was unchanged in control (before block: 80 ± 12%, after block: 60 ± 16%, P = 0.374), saline (before block: 57 ± 6%, after block: 69 ± 3%, P = 0.128), and low-dose captopril embryos (before block: 90 ± 9%, after block: 78 ± 9%, P = 0.378).

Fig. 2
figure 2

Absolute change in mean arterial pressure (a ΔMAP, kPa) and heart rate (b ΔfH, beats min−1) in response to angiotensin II (ANG II) before and after α-adrenergic blockade with phentolamine (3 mg kg−1) in control, saline injection, and low-dose captopril injection embryos. Data presented as mean ± SEM. Different letters indicate significant differences between pre- and post-phentolamine responses for all treatments combined (two-way RM ANOVA). N as follows for all bars shown for each treatment: control = 3, saline = 6, low-dose captopril = 7

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There was no significant interaction between treatment and fH before and after injection of ANG II before or after phentolamine (P = 0.849, two-way RM ANOVA). A treatment effect was also absent in the fH response for ANG II before or after phentolamine (P = 0.556, two-way RM ANOVA). Therefore, with all treatments combined, there was a significant decrease in the amplitude of the bradycardic response to ANG II following phentolamine administration (P = 0.007, two-way RM ANOVA, Fig. 2b). The intensity of the ANG II bradycardic response was unchanged in control (before block: − 37 ± 13%, after block: − 23 ± 13%, P = 0.508) and saline embryos (before block: − 33 ± 10%, after block: − 28 ± 6%, P = 0.690). However, the intensity significantly decreased following phentolamine treatment in low-dose captopril embryos (before block: − 38 ± 4%, after block: − 18 ± 3%, P = 0.002).

Baroreflex for low-dose captopril, saline, and control embryos

Despite a slight decrease in MAP in response to the 100 µg kg−1 dose of SNP in control and saline embryos, the hypotension was not significant at this or lower doses in any treatments (P = 0.232, two-way ANOVA, Table 3). For all doses combined, low-dose captopril embryos had a lower MAP compared to control and saline embryos (P = 0.006, two-way ANOVA). SNP did not induce a reciprocal increase in fH in any embryos tested, confirming the previous findings that alligators lack a hypotensive baroreflex response at 90% of incubation (Crossley II et al. 2003a, b, c).

Table 3 Mean arterial pressure (MAP, kPa) and corresponding heart rate (fH, beats min−1) before (baseline) and following doses of sodium nitroprusside (SNP) and phenylephrine in control, saline, and low-dose captopril embryos
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Phenylephrine induced a hypertensive response in all treatments. For all treatments combined, baseline MAP was significantly lower compared to MAP following all doses of phenylephrine, which did not differ from each other (P < 0.001, two-way ANOVA, Table 3). For doses combined, captopril embryos had lower MAP compared to control and saline embryos (P < 0.001, two-way ANOVA). Increased MAP due to phenylephrine induced a reciprocal fH decrease in 94% of the injections given, indicative of a hypertensive baroreflex response.

Baroreflex parameters determined for each embryo were used to calculate treatment averages (Table 4) and overall baroreflex curve estimates (Fig. 3a). Despite a trend for the low-dose captopril embryos to have a left shifted baroreflex curve, no baroreflex parameters, including C, which represents the curve midpoint, were significantly different between treatments (P > 0.05, one-way ANOVA, Table 3). In addition, a single curve was fitted to each treatment using all embryo data points combined, which resulted in lower B, gain, and normalized gain (Table 4; Fig. 3b).

Table 4 Baroreflex parameters in control, saline injection, and low-dose captopril injection embryos
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Fig. 3
figure 3

Baroreflex curve estimation for the relationship between mean arterial pressure (MAP, kPa) and heart rate (fH, beats min−1) in control, saline injection, and low-dose captopril injection embryos. a Curves represent a four-variable sigmoidal logistic function (see “Materials and methods” for details) determined from calculated mean baroreflex parameters for each treatment (see Table 4). b Curves represent a four-variable sigmoidal logistic function fitted to all data points for each treatment (see Table 4 for parameter values). Data presented as mean ± SEM for the maximal response in MAP and fH after injection of phenylephrine. Data points with asterisks indicate resting values. N for each treatment: control = 3, saline = 6, low-dose captopril = 7

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Body and organ masses for high-dose captopril, low-dose captopril, saline, and control embryos

Egg mass did not differ significantly between treatments (P = 0.458, one-way ANOVA, Table 5). Embryo mass of high-dose captopril embryos was significantly lower compared to control embryos (P = 0.005, one-way ANOVA). Accordingly, yolk wet mass was higher in high-dose captopril injection embryos compared to all other treatments (P < 0.001, one-way ANOVA). High-dose captopril embryos had smaller hearts than control and saline treatments (P < 0.001, one-way ANOVA). However, mass-specific heart mass did not differ between treatments (P = 0.904, one-way ANOVA). High-dose captopril embryos had significantly reduced lung and liver mass compared to all other treatments (P = 0.003 and < 0.001, respectively, one-way ANOVA). The treatment effect was absent for mass-specific lung mass (P = 0.122, one-way ANOVA). However, high-dose captopril embryos had a significantly reduced mass-specific liver mass compared to low-dose captopril embryos (P = 0.004, one-way ANOVA). Kidney mass did not differ between treatments (P = 0.655, one-way ANOVA), but mass-specific kidney mass was greater in the high-dose captopril treatment compared to other treatments (P < 0.001, one-way ANOVA).

Table 5 Egg mass (g), embryo mass (g), yolk mass (g), organ masses (g), and mass-specific organ masses (g kg−1) for control, saline injection, low-dose captopril injection (5 mg kg embryo−1), and high-dose captopril injection (450 mg kg embryo−1) embryos
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Discussion

ANG II exerts a significant influence on cardiovascular function in adult vertebrates, and there is growing evidence that it plays a role in cardiovascular homeostasis during development of endotherms (Segar et al. 1994; Mueller et al. 2014). However, a few studies have investigated the role of ANG II in the structural and functional development of the cardiovascular system in ectothermic vertebrates. This is particularly relevant in reptiles, because those species studied to date lack cardiovascular regulatory mechanisms that typify adult vertebrates, a phenotype that may shift the dependence to humoral regulatory systems such as the RAS (Crossley II et al. 2003a, b; Eme et al. 2011a). Unfortunately, the current available ANG II antagonists, that are functional in adults, are ineffective during development of both avian (Crossley II et al. 2010) and crocodilian species studied to date (Crossley personal communication). Therefore, we elected to utilize captopril, a known ACE inhibitor, to infer the role of ANG II in alligator embryonic development. Here, we demonstrate that ANG II has an essential, but not a fully matured, role in cardiovascular regulation during alligator embryonic development. By chronically treating embryonic alligators with captopril, an ACE inhibitor, we explored how ANG II contributes to developmental cardiovascular function and regulation. Overall, ANG II contributes to resting MAP and may influence resting fH. However, we found that ACE inhibition does not appear to significantly adjust short-term baroreflex function. These findings highlight that while ANG II appears to be of importance during the latter part of alligator development, as in endotherms, the modality in which it contributes to cardiovascular regulation appears to be taxa specific.

Baseline MAP and f H

Both low- and high-dose chronic captopril treatment created an altered cardiovascular phenotype, with a dose-dependent effect on both MAP and fH (Table 1). The low-dose captopril treatment created embryos with lowered MAP but no change in fH. These results supported our hypothesis and are similar to chicken embryos, in which a moderate captopril dose reduced MAP but did not alter fH (Mueller et al. 2014). However, the decrease in MAP in the low-dose captopril treatment compared to the saline treatment was a stronger hypotensive response (41% decrease) than measured in chicken embryos (15% decrease) following a similar dosage (Mueller et al. 2014) and in fetal sheep also treated with captopril (6–13%) (Robillard et al. 1983; Lumbers et al. 1992). The strong hypertensive response suggests that ANG II plays a larger role in baseline MAP in alligator embryos than in chicken embryos or fetal sheep. Furthermore, a significant bradycardia occurred along with the large decrease in MAP in the high-dose captopril treatment. Thus, this treatment caused a significant slowdown in overall cardiovascular function. The additional decrease in MAP following the high-dose captopril treatment may suggest that this treatment was highly stressful to the embryos, as evidenced by this experiment producing 76% mortality.

ANG I and ANGI II responses

ANG I did not induce a hypertensive or bradycardic response in any of the embryos (Fig. 1a, b). While the pressor response that occurs following ANG I injection is usually not as strong as when exogenous ANG II is injected, a hypertensive response usually does occur in the presence of a functional RAS (Nishimura et al. 1982; Mueller et al. 2014). This could be due to an insufficiently high dosage. It should be noted that our ANG I dosage (2000 ng kg−1) was similar to previously reported to elicit a strong response in day 19 chicken embryos (Mueller et al. 2014). However, the ANG I used in this studied was a synthesized crocodilian peptide, and thus different from the ANG I used in the chicken study, and a higher dosage may be required. We can assume that alligator embryos do have a functional ACE, as chronic captopril treatment results in embryos that were hypotensive (Table 1). While further investigations are needed it is conceivable that ACE may be a limiting factor to ANG II production in embryonic alligators, or another factor involved in cardiovascular regulation may be interacting with the RAS to reduce the effect of ANG I injection.

Injection of exogenous ANG II caused a significant hypertension (Fig. 1c), and this response was consistent with a previous study in embryonic alligators (Tate et al. 2012). While the somewhat low MAP of the low-dose captopril embryos persisted, these embryos had a stronger response to ANG II, particularly compared to the saline embryos (90% increase compared to 57%). A similar response was observed following ANG II administration in chicken embryos and likely indicates that the absence of endogenous ANG II created embryos more sensitive to exogenous ANG II (Mueller et al. 2014). All treatments showed a similar bradycardic response to ANG II injection (Fig. 1d). In our previous study, captopril-treated chicken embryos did not show this response, instead responding with a brief tachycardia (Mueller et al. 2014). The altered response in chicken embryos treated with captopril was attributed to altered baroreflex sensitivity (Mueller et al. 2013), which is unchanged in low-dose captopril alligator embryos (see below).

Adrenergic contribution to ANG II response

The hypotensive response to α-adrenergic blocker phentolamine is consistent with a previous study in embryonic alligators (Table 2) (Eme et al. 2011a). The similar hypotensive response between treatments indicates that captopril treatment had no influence on basal α-adrenergic tone. The previous studies in embryonic chickens, alligators, and common snapping turtles (Chelydra serpentina) have also indicated that either ACE inhibition or hypoxia-induced hypotension did not change α-adrenergic tone (Eme et al. 2011a, 2013; Crossley II and; Altimiras 2012; Mueller et al. 2014). This study yet again confirms the lack of plasticity in this cardiovascular trait for reptilian embryos.

The absolute change in MAP in response to ANG II decreased in all treatments following α-adrenergic blockade (Fig. 2). Attenuation of the ANG II pressor response was similar between treatments. Furthermore, the intensity of the response to ANG II did not change before and after phentolamine, which indicates that the ANG II pressor response is without a α-adrenergic stimulation component. This contradicts our hypothesis that embryos treated with captopril would show a catecholamine contribution in the ANG II response and is different from the observations in adult alligators (Silldorff and Stephens 1992). However, the intensity of the corresponding bradycardia decreased in the low-dose captopril embryos following phentolamine administration (Fig. 2). This may suggest that there is some interaction between α-adrenergic blockade and the heart rate response to exogenous ANG II.

Baroreflex

Despite a significant hypotension following chronic captopril treatment, baroreflex parameters were not altered in low-dose captopril embryos compared to control and saline (Table 4). However, even a cursory examination of the baroreflex curves generated (Fig. 3) shows that the low-dose captopril embryos have a left shifted curve, indicative of a resetting to a lower MAP. The generation of baroreflex curves for each individual embryo allowed generation of mean and error values for baroreflex parameters and thus statistical analysis between the three treatment groups. However, Fig. 3A shows how the curves generated from these mean values are steep in their mid section with abrupt inflection points near the minimum and maximum fH. The steepness of the curves may be due to the low sample size for MAP values and corresponding fH values for each embryo, which created high B, gain, and normalized gain values.

By fitting baroreflex curves to the average data points generated for all embryos in each treatment, which allows curve fitting to a larger number of data points, the data are more accurately described (Fig. 3b). These curves have a less steep midsection around the MAP midpoint and less abrupt inflections at the minimum and maximum fH. While statistical analyses cannot be performed on the baroreflex parameters generated in this manner, there may be some evidence that the B, gain, and normalized gain values for the low-dose captopril-treated embryos are lower compared to control and saline embryos. This is the opposite to our previous findings in chicken embryos, which showed significantly higher values for these three parameters compared to control embryos, which indicates a more sensitive baroreflex (Mueller et al. 2013). The previous studies report mixed findings regarding the relationship between ANG II and baroreflex sensitivity. Disruption of the RAS in some adult organisms left shifted the baroreflex curve without changing the sensitivity (Hatton et al. 1981; Brooks and Reid 1986; Düsing et al. 1987; Matsumura et al. 1989), while others showed both a shift in the curve set point and an increase in sensitivity, i.e., a higher gain (Lee and Lumbers 1981; Guo and Abboud 1984; Garner et al. 1987). Whether ACE inhibition influences baroreflex sensitivity in an organism likely depends on the role of ANG II in the system, which may be partly dependent on endogenous levels of ANG II (Mueller et al. 2013). It may be argued that a higher level of ANG II, such as in chicken embryos, probably indicates an increased role of the vasoactive agent in short-term baroreflex function. Assessment of circulating ANG II levels in embryonic alligators would provide much needed data as to the relationship between ANG II and baroreflex sensitivity during development.

Masses

The high-dose captopril treatment impacted embryonic growth, as demonstrated by reduced embryo, heart, lung, and liver masses (Table 5). ACE inhibition during development has also been associated with a reduction in body mass in neonatal rats (Friberg et al. 1994). When corrected for embryo mass, the liver was significantly smaller and the kidneys significantly larger in the high-dose captopril treatment. In contrast to the current study, kidney growth was impaired following ANG II receptor antagonism in newborn rats and ACE inhibition during development of frog tadpoles (Tufro-McReddie et al. 1995). However, our previous study in chicken embryos showed an increase in dry kidney mass following captopril treatment similar to the low-dose captopril treatment of this study (Mueller et al. 2014). The larger kidney mass in bird and reptile embryos may be a result of renal abnormalities that have previously been observed in human and rat fetuses, such as overproduction of glomeruli, hyperplasia of the juxtaglomerular apparatus, tubular and interstitial inflammation, and thickening of arterial and arteriolar walls (Friberg et al. 1994; Martinovic et al. 2001; Daïkha-Dahmane et al. 2006), which reflects the role of the RAS in kidney growth and development.

Conclusion

This study is the first to examine the role of ANG II in cardiovascular function of a reptile embryo. Using chronic treatment with captopril, we demonstrate that ANG II contributes significantly to baseline pressure in embryonic alligators. This study provides evolutionary context for the RAS, demonstrating that it is important during the early development of both living groups of archosaurs (crocodilians and birds). While RAS has been shown to be important in adult reptiles, its functional importance in ovo in developing alligators suggests ancient origins for the RAS system, and that this system was possibly important during the time of the evolutionary transition to endothermy in birds and at the dawn of the age of mammals. We detected a dose-dependent reduction in MAP, despite evidence that the low-dose captopril treatment used was adequate to cause complete ACE blockade. These results suggest other secondary effects of the ACE inhibition that also contribute to the alteration in MAP. Interestingly, it does not appear that ANG II’s significant contribution to baseline pressure occurs through changes in short-term baroreflex control. This was evident in the lack of a change in baroreflex sensitivity with ACE inhibition. The previous studies, both in adult and developing vertebrates, demonstrate differing results for the interaction between ANG II and baroreflex control (Mueller et al. 2013 and references therein). This study further emphasizes that the role of ANG II in cardiovascular regulation is variable both between vertebrate groups and at different developmental stages. Yet, ANG II does appear to play some role in all species examined thus far, and will remain a useful target for exploring how the cardiovascular system functions during development and beyond.