ABSTRACT
Carnivorous reptiles exhibit an intense metabolic increment during digestion, which is accompanied by several cardiovascular adjustments responsible for meeting the physiological demands of the gastrointestinal system. Postprandial tachycardia, a well-documented phenomenon in these animals, is mediated by the withdrawal of vagal tone associated with the chronotropic effects of non-adrenergic and non-cholinergic (NANC) factors. However, herbivorous reptiles exhibit a modest metabolic increment during digestion and there is no information about postprandial cardiovascular adjustments. Considering the significant impact of feeding characteristics on physiological responses, we investigated cardiovascular and metabolic responses, as well as the neurohumoral mechanisms of cardiac control, in the herbivorous lizard Iguana iguana during digestion. We measured oxygen consumption rate (O2), heart rate (fH), mean arterial blood pressure (MAP), myocardial activity, cardiac autonomic tone, fH/MAP variability and baroreflex efficiency in both fasting and digesting animals before and after parasympathetic blockade with atropine followed by double autonomic blockade with atropine and propranolol. Our results revealed that the peak of O2 in iguanas was reached 24 h after feeding, accompanied by an increase in myocardial activity and a subtle tachycardia mediated exclusively by a reduction in cardiac parasympathetic activity. This represents the first reported case of postprandial tachycardia in digesting reptiles without the involvement of NANC factors. Furthermore, this withdrawal of vagal stimulation during digestion may reduce the regulatory range for short-term fH adjustments, subsequently intensifying the blood pressure variability as a consequence of limiting baroreflex efficiency.
INTRODUCTION
All vertebrates exhibit a significant post-feeding metabolic increment to supply the energetic demands of the digestive process, a cascade of events called specific dynamic action (SDA) characterized by food intake, physicochemical breakdown of macromolecules, and subsequent absorption and assimilation of nutrients (Andrade et al., 2005; Secor, 2009). Naturally, such an increase in metabolic rate demands several adjustments in many physiological systems to be sustained, for which cardiovascular adjustments are often required (Andrade et al., 2005; McCue, 2006; Wang et al., 2005; Secor, 2009).
The increase in heart rate (fH), cardiac output, myocardial activity, cardiac pumping capacity/contraction force and eventually mean arterial blood pressure (MAP), accompanied by a decrease in systemic vascular resistance, ensure a greater blood flow to the gastrointestinal system during digestion, supplying its high metabolic demands and facilitating the transportation of nutrients from the intestinal lumen to the bloodstream (Kågstrom et al., 1998; Axelsson et al., 2000; Secor, 2009; Secor and White, 2010; Slay et al., 2014; Enok et al., 2016; da Silva Vasconcelos et al., 2020). Such cardiovascular adjustments can be mediated by the autonomic nervous system through the secretion by nerve endings on target tissues or circulating catecholamines (Altimiras, 1999; Secor et al., 2000; Wang et al., 2001; Secor and White, 2010), and/or by non-adrenergic non-cholinergic (NANC) factors which can also be nerve-secreted or humoral molecules (Rigel, 1988; Wang et al., 2001; Skovgaard et al., 2009, 2017; Enok et al., 2012; Claësson et al., 2015; da Silva Braga et al., 2016; Guagnoni et al., 2021). The adrenergic and cholinergic neurotransmitters secreted by the autonomic nervous system neurons on target tissues are the leading modulators of short-term cardiovascular adjustments (Altimiras, 1999; Secor et al., 2000; Wang et al., 2001), whilst NANC factors released into the bloodstream are the main modulators of medium and long-term responses, although nerve-secreted NANC factors may also be capable of producing short-term cardiovascular alterations (Nilsson, 1983; Altimiras, 1999; Li et al., 2006; Enok et al., 2016; Joyce and Wang, 2020).
In most ectothermic vertebrates, specifically non-crocodilian, the well-known postprandial tachycardia that occurs during the SDA response is primarily determined by the withdrawal of vagal tone in combination with circulating NANC factors – except in the fish Dicentrarchus labrax, in which NANC factors are not involved (Iversen et al., 2010; Claësson et al., 2015; Wang et al., 2001; Skovgaard et al., 2009; Enok et al., 2012; Guagnoni et al., 2021). This withdrawal of vagal stimulation may limit the regulatory range for short-term fH adjustments without compromising the capacity for compensatory fH responses to hypotension via baroreflex (Guagnoni et al., 2021; Wang et al., 2021). Crocodilians, in contrast, show a postprandial tachycardia triggered by sympathetic stimulation along with NANC factors (da Silva Braga et al., 2016), a modulation similar to that observed in mammals (Canis lupus, Rattus norvegicus and Homo sapiens), in which the postprandial tachycardia and the digestion-associated thermogenesis may be related to a general increase in adrenergic activity and to the effects of NANC factors (Rothwell et al., 1982; Young et al., 1982; Acheson et al., 1983; DeFronzo et al., 1984; Diamond and LeBlanc, 1987; Astrup et al., 1989; Kelbæk et al., 1989; Baron, 1994).
The importance of NANC factors in the postprandial cardiovascular response has been extensively investigated, and their involvement in cardiac control during SDA has been reported in almost all vertebrates studied to date (Wang et al., 2001; Waaler et al., 2006; Enok et al., 2012; Claësson et al., 2015; Slay et al., 2014; da Silva Braga et al., 2016; Guagnoni et al., 2021). It is speculated that the tonic stimulation of fH during digestion by a circulating NANC factors provides a tachycardia to accommodate the postprandial rise in venous return (Enok et al., 2016; Joyce and Wang, 2020; Wang et al., 2021). However, the characterization of these factors and why they are not present during digestion in some animals remains unclear, although some studies have identified certain NANC molecules with positive cardiac chronotropic effects acting during digestion, such as intestinal polypeptides, insulin and histamine (Rigel, 1988; Baron, 1994; Skovgaard et al., 2009; Burggren et al., 2014).
The magnitude of postprandial metabolic responses and, consequently, digestion-related cardiovascular adjustments depends on many variables, including the physicochemical characteristics of the food, feeding intervals and the amount of food consumed (Secor and Phillips, 1997; Secor, 2001; Iglesias et al., 2003; Klein et al., 2006; Secor, 2009). These topics have been studied extensively in vertebrates, particularly those related to digestion-associated tachycardia, but the information currently available is restricted to specific groups (Andrade et al., 2005; da Silva Braga et al., 2016). Most studies on this topic have focused on carnivorous snakes, which exhibit very intense postprandial physiological responses as a result of their low standard metabolic rate (SMR) and the relatively large meals they consume in infrequent intervals, resulting in metabolic increments of up to 600% (Andrade et al., 2005; Wang et al., 2006; Secor, 2009). Studies of digestion within reptile groups with different dietary patterns, such as herbivorous lizards, remain not fully understood – a crucial lack of knowledge for the mechanistic understanding of the factors that influence postprandial responses and for making comparisons between the effects of animal-based and plant-based diets.
In this context, our study aimed to investigate the postprandial cardiovascular and metabolic responses, as well as the neurohumoral mechanisms of cardiac control, in a frequently feeding herbivorous lizard. We hypothesized that, although lizards exhibit a less pronounced increase in metabolic rate after feeding compared with carnivorous reptiles relative to their SMR, there are still significant demands for cardiovascular and metabolic adjustments in these animals, particularly because the processing of plant material takes longer than the digestion of animal tissues. While the broad regulation of digestive performance has been observed for infrequently feeding snakes, it remains to be seen whether this apparent adaptive response is characteristic of other frequently feeding reptiles. Using the lizard Iguana iguana as an experimental model, we analyzed the changes in cardiovascular and metabolic variables during digestion, before and after autonomic cardiac blockade using specific antagonists to the cholinergic and adrenergic effectors on the heart. We also analyzed the animals' fH and MAP variability combined with baroreflex assessment using the sequence method, characterizing a new approach for the study of the autonomic control of cardiovascular adjustments to digestion in ectotherms. Iguana iguana, an arboreal species with a wide distribution across the Americas, exhibits a plant-based diet and consumes smaller daily food portions compared with carnivorous lizards and snakes, making it an appropriate experimental model to contrast previous findings in carnivorous reptiles.
- BEI
baroreflex effectiveness index
- BPV
blood pressure variability
- fH
heart rate
- Gnorm
spontaneous baroreflex gain normalized
- Gsp
spontaneous baroreflex gain
- HRV
heart rate variability
- MAP
mean arterial blood pressure
- NANC
non-adrenergic non-cholinergic
- Pa
arterial pressure
- PS
systolic pressure
- PI
pulse interval
- RPP
rate–pressure product
- SDA
specific dynamic action
- SMR
standard metabolic rate
- O2
oxygen consumption
MATERIALS AND METHODS
Experimental animals
Twenty-four adult I. iguana (Linnaeus 1758) specimens of both sexes were obtained from a scientific breeding center (Jacarezário of the São Paulo State University, Campus of Rio Claro, São Paulo, Brazil) and transported to the Zoophysiology Laboratory of the São Paulo State University, Campus of São José do Rio Preto, São Paulo, Brazil. The lizards weighed 3.6±0.5 kg and were individually housed in 540 l plastic boxes at 25.0±1.0°C (mean±s.e.m.) and under natural photoperiod. The animals were divided into two main experimental groups, which were initially fasted for 96 h. One of these groups remained fasted throughout the instrumentation and experimental trials (fasting group), whereas the other was voluntarily fed (preserving the cephalic phase of digestion) with mixed chopped vegetables (collard greens, cabbage, kabocha squash, carrots, apple and banana; ∼3.5% of the animals' body mass) immediately prior to instrumentation (digesting group). All experimental trials were carried out during the spring and summer seasons. The experiments were approved by the São Paulo State University Ethics Committee for Animal Research (UNESP/IBILCE/CEUA, Case No. 200/2019), and performed in accordance with all of the regulations and ethical guidelines in Brazil.
Oxygen consumption
The postprandial oxygen consumption profile of I. iguana (O2; ml O2 h−1) was described and illustrated by a comparison of the SMR (determined as the minimumO2 measured in the fasting group; N=5) and the oxygen consumption after meal ingestion in the digesting group (N=6) (Secor and Phillips, 1997; Secor, 2009). Using an automated system of intermittently closed respirometry similar to that described by Cruz-Neto and Abe (1994), each lizard was placed in hermetically sealed respirometric chambers of an appropriate volume for their size (45 l). The automated respirometer was programmed for cycles of 30 min to flush the respirometric chamber with fresh air (volume 1.5–2.0 l min−1), followed by 30 min whereby the air contained in the chambers was just recirculated through an oxygen analyzer (Witrox, Loligo Systems Inc.). Oxygen content inside the respirometric chamber (never below 80%) was monitored with the software Witrox View 1.0 (Loligo Systems Inc.) and was used to calculate O2 every 60 min by the depletion of O2 concentration in the chamber for 3 days (all values were corrected for standard temperature and pressure).
Animal instrumentation and cardiovascular measurements
Before the surgical procedure, each of 14 lizards was sedated through an inhalation mask perfused with isoflurane (3%) and oxygen (97%) until complete loss of righting reflexes. The trachea was then intubated for direct ventilation with isoflurane (1%) and oxygen (99%) at 4 breaths min−1 with a tidal volume of 20 ml kg−1, manually maintained using an anesthesia gas blender coupled to a breathing balloon and a chronometer (Colibri Inhalatory Anaesthesia Apparatus, Brasmed Veterinary Products, Paulínia, São Paulo, Brazil) (Mosley, 2005; Armelin et al., 2019; Filogonio et al., 2019). A local anesthetic (lidocaine 2%, 2 mg kg−1) was injected before a 3 cm longitudinal incision was made in the left thigh (Filogonio et al., 2019). A PE50 catheter filled with heparinized saline (100 IU ml−1, 0.9% NaCl) was occlusively inserted into the animal's femoral artery and fixed with an internal suture and cyanoacrylate glue. All procedures were performed under sterile conditions and took ∼45 min. After surgery, animals were transferred to a temperature-controlled chamber (25.1±0.2°C) in a silent room for 24 h recovery – this enabled the experiments to be performed entirely during the lizards’ maximum metabolic response (Fig. 1).
Following this recovery period, the femoral catheter was connected to a pressure transducer (Pressure 139 Transducer SS13L, BIOPAC Systems Incorporated, Goleta, CA, USA) calibrated against a static water column before measurements were obtained using a BIOPAC MP36 data acquisition system (BIOPAC Systems Incorporated) for uninterrupted acquisition arterial pressure (PA; kPa) at 1000 Hz. fH (beats min−1) was derived from the PA signal pulse and MAP (kPa) was calculated from the area under the pressure/time curve divided by the cardiac cycle time using LabChart® software (LabChart v.7.0, ADInstruments). Rate–pressure product (RPP; as a measure of myocardial activity; kPa min−1) was calculated as the product of fH and systolic pressure (PS; kPa) (Slay et al., 2014; Zena et al., 2016).
Experimental protocol
After verifying the PA signal stability, the cardiovascular variables were recorded for 30 min. Next, saline solution (0.9%) was administered in untreated fasting animals via the femoral catheter to verify the possible influence of drug injections on the cardiovascular variables. For this, the largest volume of drug administered was used as a reference for the volume of saline solution for each animal, and data were recorded for 30 min. After the acquisition of cardiovascular variables from untreated animals, the muscarinic cholinergic antagonist atropine (2.5 mg kg−1; Troiano et al., 2018) was intra-arterially administered and allowed to exert its effects for 1 h, after which the cardiovascular variables were recorded for another 30 min. Afterward, the β-adrenergic antagonist propranolol (3.5 mg kg−1; Sartori et al., 2015; Troiano et al., 2018) was administered via the femoral catheter, allowed to take effect for 1 h, and then the animals' variables were recorded for 30 min under the double autonomic block of the heart. Finally, to verify whether autonomic blockade was successful, intra-arterial injection of acetylcholine (0.3 ml kg−1; 200 μg ml−1 of saline solution) and adrenaline (epinephrine; 0.3 ml kg−1; 200 μg ml−1 of saline solution) was performed in fasting animals to investigate its effects on the cardiovascular variables under double autonomic blockade (Wang et al., 2001; Troiano et al., 2018; Armelin et al., 2019).
Calculation of adrenergic and cholinergic tone
Spectral analysis of beat-to-beat oscillations
To assess the sympathetic and parasympathetic cardiac modulation of the fH and PA fluctuations in a complementary approach to the cardiac autonomic tone, their fH and MAP variability were measured using power spectral analysis (Altimiras et al., 1997; Sanches et al., 2019). For this, raw PA signal traces composed of 256 cardiac cycles were extracted from the final moment of each data collection period (i.e. under untreated conditions and after the administration of autonomic antagonists for both groups) (Armelin et al., 2019; Duran et al., 2020). These signal portions were used to generate PI [heart rate variability (HRV); reciprocal to fH] and MAP tachograms in BIOPAC Student Lab Pro v3.7 software, which were converted to text files (*.txt) for processing in CardioSeries v2.4 software (custom software available at www.danielpenteado.com). The beat-to-beat series of each tachogram was resampled with data points every 333 ms via cubic spline interpolation (3 Hz). Next, the interpolated series were divided into half-overlapping segments of 256 points. A Hanning window was applied to attenuate side effects, and the spectra were calculated for all of the segments with a fast Fourier transformation and integrated into a single spectrum (Campbell et al., 2006; Armelin et al., 2019). Finally, the power spectral bands were calculated based on the location of the low-frequency (HRVLF; blood pressure variability, BPVLF) and high-frequency (HRVHF; BPVHF) peaks.
Analysis of the baroreflex
The lizards' baroreflex was evaluated through the sequence method using the software configuration previously standardized for reptiles proposed by Filogonio et al. (2019). Segments of PA signal composed of a continuous series of 600 cardiac cycles from the final moment of each data collection period of both groups (i.e. untreated condition and after the administration of autonomic antagonists) were extracted using BIOPAC Student Lab Pro v.3.7 software, which was converted into PI and PS tachograms and exported to text files (*.txt) to be analyzed in the CardioSeries v.2.4 software. The software was configured to detect sequences with three or more consecutive cardiac cycles at the time that the mean of PS changed progressively and was associated with gradual changes in PI. After sequence detection, directly proportional changes in PI were searched by the software utilizing delay 1, and the linear regression between PS and PI for each baroreflex sequence was calculated to acquire the correlation and slope coefficients. To avoid bias from regulatory mechanisms other than the baroreflex (Ide and Hoffmann, 2002; Armelin et al., 2021) that may trigger cardiovascular changes and generate stochastic baroreflex sequences, we included only sequences with a correlation coefficient of ≥0.8 in the analysis (Armelin et al., 2021).
Statistics
Initially, Shapiro–Wilk normality test was performed on all of the data, and the parametricity was confirmed. Regarding the postprandial metabolic response data, every O2 value obtained from the digesting group over the 3 days of trials was compared with SMR using a two-tailed unpaired t-test. Then, the cardiovascular data (fH, MAP, RPP, HRVLF, HRVHF, BPVLF, BPVHF, Gsp, Gnorm, BEI and the number of detected baroreflex sequences) were compared within and between groups and treatments using a two-way ANOVA for repeated measurements followed by a Holm–Šídák post hoc test. For the evaluation of changes in cardiac autonomic tone (adrenergic and cholinergic) induced by digestion, a one-way ANOVA followed by a Holm–Šídák post hoc test was performed. Possible differences existing between cardiovascular variables (fH and MAP) acquired before and after intra-arterial saline solution administration in fasting animals were assessed using a two-tailed unpaired t-test. Likewise, a two-tailed unpaired t-test was applied to cardiovascular variables (fH and MAP) acquired before and after intra-arterial administration of adrenaline and acetylcholine in fasting animals under double blockade treatment to verify that the autonomic heart block was effective. For all of the tests, the null hypothesis was rejected when P≤0.05. The statistical analyses were carried out using GraphPad Prism 9.0 commercial software (GraphPad Software Inc.). The Results section presents statistics details of multiple comparison tests between groups and treatments while Tables S1–S3 provide further information about the statistical tests. All values are expressed as means±s.e.m.
RESULTS
Following 4 days of fasting, the iguanas exhibited an SMR of 33.15±4.82 ml O2 h−1. Immediately after feeding, the first O2 measured at 0 h was significantly higher than SMR (P=0.046); this returned close to SMR levels after 6 h (Fig. 1; P=0.082). Then, the O2 values increased quickly and were significantly elevated within 18–24 h, peaking at 24 h with a O2 of 75.15±12.03 ml O2 h−1 (∼2.26-fold SMR; P=0.014). Thereafter, O2 declined at a fast pace and returned within 30–36 h to levels not significantly greater than those of fasting (56.15±9.35 to 38.31±8.57 ml O2 h−1, respectively; P=0.632).
Untreated digesting animals demonstrated a fH and RPP (Fig. 2A,C) significantly higher than those fasting animals under the same condition (P=0.001 and P=0.020, respectively), but MAP remained unchanged (Fig. 2B; P=0.534). However, there was no significant difference in fH and RPP between the fasting and digesting groups under double autonomic blockade of the heart (P=0.263 and P=0.764, respectively). Specifically, fasting untreated animals exhibited a fH of 29.12±2.56 beats min−1, MAP of 8.96±0.33 kPa and RPP of 320.82±28.37 kPa min−1. After muscarinic cholinergic blockade with atropine, fH increased to 41.15±2.29 beats min−1 (P<0.0001), MAP was reduced to 7.80±0.28 kPa (P=0.049) and RPP did not change (361.68±22.23 kPa min−1, P=0.301). MAP remained unchanged (7.37±0.49 kPa, P=0.381) after double autonomic blockade with atropine and propranolol, whereas fH (23.75±1.22 beats min−1, P<0.0001) and RPP (211.31±15.52 kPa min−1, P=0.002) were reduced. In contrast, digesting untreated animals showed a fH of 42.76±3.45 beats min−1, MAP of 9.70±0.74 kPa and RPP of 485.05±63.55 kPa min−1. After atropine treatment, fH increased to 48.11±3.13 beats min−1 (P=0.017), but the MAP (8.86±0.75 kPa, P=0.097) and RPP (482.86±63.24 kPa min−1, P=0.955) did not change in these animals. The establishment of the double autonomic blockade caused a drastic decrease in fH (27.72±1.31 beats min−1, P<0.0001), MAP (6.99±0.21 kPa, P=0.001) and RPP (228.87±14.91 kPa min−1, P<0.0001).
Regarding the animals' cardiac autonomic control (Fig. 3), digestion did not induce changes in adrenergic tone (which was 42.03±1.65% in the fasting group and 41.54±2.80% in the digesting group; P=0.924) but was associated with a reduction in cholinergic tone from 26.27±5.17% to 8.84±3.83% (P=0.009), respectively. Moreover, the HRV and BPV analysis also revealed an impact of digestion on the animals' fH and MAP oscillation patterns. The spectra that describe those oscillation patterns were found to be below 0.30 Hz with a major low-frequency peak located between 0.00 and 0.10 Hz and a minor high-frequency peak above 0.10 Hz (Figs 4A,C and 5A,C). The digestion process induced a drastic decrease in HRV spectral amplitude (∼2.5 times) accompanied by a reduction in HRVLF spectral power (Fig. 4B,D; P=0.027), while the BPV spectral amplitude and BPVLF spectral power were increased (P=0.014). Specifically, fasting untreated animals showed a HRVLF of 1429.25±633.49 ms2 (Fig. 4B) and BPVLF of 0.0079±0.0013 kPa2 (Fig. 5B). The administration of atropine induced a drastic reduction in HRVLF to 67.35±22.06 ms2 (Fig. 4B; P=0.0001) but did not change BPVLF (0.0036±0.0010 kPa2; Fig. 5B; P=0.842). After double autonomic blockade, HRVLF (113.79±25.86 ms2; Fig. 4B; P=0.997) and BPVLF (0.0029±0.0006 kPa2; Fig. 5B; P=0.998) remained unchanged. In contrast, digesting untreated animals showed a HRVLF of 575.68±300.24 ms2 which did not change after parasympathetic blockade with atropine (142.90±31.16 ms2, P=0.809) and double autonomic blockade (105.58±35.54 ms2, P=0.999) (Fig. 4D), but BPVLF was consistently reduced after these treatments (from 0.0181±0.0052 kPa2 to 0.0101±0.0052 kPa2 and 0.0068±0.0014 kPa2, respectively), without statistical significance (P=0.085 and 0.735, respectively) (Fig. 5D). No changes were observed in the HRVHF and BPVHF between the two groups under all pharmacological treatments (P=0.998).
We observed that digestion changed GSP (Fig. 6A; P=0.001), Gnorm (Fig. 6B; P=0.001) and the total number of baroreflex sequences detected (Fig. 6D; P=0.043), but did not affect BEI (Fig. 6C; P=0.151). Specifically, fasting untreated animals exhibited GSP and Gnorm of 1055.00±139.70 ms kPa−1 and 51.46±3.94%, which were drastically reduced after atropine injection (393.40±84.37 ms kPa−1 and 18.15±2.59%, respectively; P=0.0001) and remained stable with the establishment of the double autonomic blockade (446.10±98.06 ms kPa−1, P=0.660; and 23.50±5.88%, P=0.573, respectively). In contrast, GSP (439.10±142.30 ms kPa−1, P=0.001) and Gnorm (26.40±5.21%, P=0.001) of digesting untreated animals were lower compared with the fasting group in the same treatment, but they did not change after the parasympathetic blockade with atropine (325.40±102.30 ms kPa−1, P=0.572; and 20.83±5.83%, P=0.573, respectively) and after the double autonomic blockade (275.00±76.37 ms kPa−1, P=0.673; and 17.42±2.67%, P=0.573, respectively). No changes were observed in BEI and the number of baroreflex sequences detected between the two groups under all pharmacological treatments (P>0.100).
Finally, the intra-arterial administration of saline (0.9% NaCl) in untreated fasting animals did not elicit significant changes in fH and MAP (Table S4). Moreover, the intra-arterial administration of adrenaline (0.3 ml kg−1; 200 μg ml−1 of saline solution) and acetylcholine (0.3 ml kg−1; 200 μg ml−1 of saline solution) in double-blocked animals treated with atropine and propranolol (Tables S5 and S6) also did not change those variables.
DISCUSSION
Following 4 days of fasting, the iguanas exhibited a SMR of 33.15±4.82 ml O2 h−1 (Fig. 1), which was lower than that reported by Maxwell et al. (2003). This difference could be attributed to the contrasts in the experimental design, including factors such as animal maturity, captivity duration, body mass and the temperature to which the animals were exposed. All the animals used in our study were adults (∼3.6 kg) obtained from a long-term captive facility and maintained at 25.0°C±1.0 throughout the experiments. In contrast, Maxwell et al. (2003) assessed the SMR of a wide mass range of iguanas, from 16 g to 3.6 kg, including adults and juveniles kept under different captive conditions and for different periods of time (i.e. long-term captive, captive-hatched juveniles, wild-caught and feral lizards). Their study was conducted at 30°C, which combined with the other factors mentioned above could justify the increased SMR compared with our findings.
The fH and MAP values (Fig. 2A,B) observed for fasting I. iguana were similar to those reported in previous studies under the same experimental conditions (Moberly, 1968; Hohnke, 1975; Chinnadurai et al., 2010). Driven by the dominance of sympathetic tonus on basal cardiac function (Fig. 3), the high values of MAP and RPP (Fig. 2B,C) observed were similar or even higher than those of other species of Squamata with a functional separation of the pulmonary and systemic circulation – probably associated with the orthostatic tolerance of this arboreal species, which improves the blood flow against gravity during orthostatic stress and minimizes the effects of postural changes on tissue perfusion during climbing (Burggren and Johansen, 1982; Lillywhite, 1993, 1996; Seymour et al., 1993; Troiano et al., 2018; Armelin et al., 2019; Enok et al., 2012; Jensen et al., 2010, 2014; Slay et al., 2014).
Immediately after feeding, the first O2 measured at 0 h was significantly higher than SMR because of handling stress, but returned close to SMR levels after 6 h (Fig. 1). Then, O2 values increased quickly and were significantly elevated within 18–24 h, peaking at 24 h, with O2 120% greater than SMR values. This period to reach O2 peak during digestion was quite similar to the lizards Angolosaurus skoogi and Pogona vitticeps with a plant-based diet (Clarke and Nicolson, 1994; Buddemeyer et al., 2019 preprint) and carnivorous lizards Varanus albigularis and Varanus exanthematicus (Secor and Phillips, 1997; Hicks et al., 2000; Hartzler et al., 2006). However, the magnitude of the metabolic response in I. iguana was smaller than that of these carnivorous lizards, which can reach up O2 to 800% of the SMR, but higher when compared with that of the plant-based diet lizards A. skoogi (50% and 80% greater than SMR at 30°C after a 7% and 11% body mass meal; and 100% greater than SMR at 23°C after a 7% body mass meal) and P. vitticeps (67% greater than SMR at 26–29°C after a 5% body mass meal) studied by Clarke and Nicolson (1994) and Buddemeyer et al. (2019). Finally, O2 values returned at a fast pace close to SMR values within 30–36 h (Fig. 1; 56.15±9.35 to 38.31±8.57 ml O2 h−1, respectively).
The postprandial metabolic increment triggers enteric vasodilation and, consequently, requires a series of cardiovascular adjustments to supply the metabolic demands (Enok et al., 2013). In I. iguana, digestion triggers a postprandial tachycardia of ∼45% (Fig. 2A) similar to that in Chelydra serpentina (Wearing et al., 2017), but quite small compared with that of carnivorous ectotherms that feed on large prey. The renowned rise in oxygen consumption during digestion in carnivorous reptiles is attended by an equally impressive elevation of cardiac output that is supplied mainly by a significant increase in fH between 66% and 238% (Stinner and Ely, 1993; Hicks et al., 2000; Wang et al., 2001; Skovgaard et al., 2009; Slay et al., 2014; da Silva Braga et al., 2016).
Administration of atropine resulted in an increase of fH in both fasting and digesting animals, but while the former showed an increase of ∼41%, the latter had a small increment of ∼14% (Fig. 2A). However, fH was also decreased ∼44% in both groups after the establishment of double autonomic blockade with propranolol (Fig. 2A), decreasing directly the myocardial activity (Fig. 2C). These results are evidence that the parasympathetic nervous system plays the main role in the postprandial tachycardia control in I. iguana, which is consistent with the data on cardiac autonomic tone, which exhibited a reduction of ∼69% in cardiac cholinergic tone during digestion, whilst cardiac adrenergic tone remained unchanged (Fig. 3). Also, the fact of the intrinsic fH obtained during double autonomic blockade did not differ between groups indicates no contribution of NANC factors in the determination of postprandial tachycardia in this species, the first case recorded in digesting reptiles to date.
Except for the high adrenergic tone on the heart supporting the high blood pressure and myocardial activity (Fig. 2B,C), our results for the postprandial tachycardia control in I. iguana are exclusively similar to those of the fish D. labrax, in which this fH adjustment is mediated by a withdrawal of vagal tone (Fig. 3) with no influence of NANC factor chronotropic effects (Iversen et al., 2010). They differ from the results reported for other fishes, amphibians and non-crocodilian reptiles, in which this adjustment is mediated by a reduction of the cholinergic tone associated with the positive chronotropic effects of NANC factors (Wang et al., 2001; Skovgaard et al., 2009; Enok et al., 2012; Claësson et al., 2015; Guagnoni et al., 2021), and also from those reported for crocodilians and mammals that presented a postprandial tachycardia mediated by an increase in cardiac adrenergic tone associated with NANC factors (Rothwell et al., 1982; Young et al., 1982; Acheson et al., 1983; DeFronzo et al., 1984; Diamond and LeBlanc, 1987; Astrup et al., 1989; Kelbæk et al., 1989; Baron, 1994; da Silva Braga et al., 2016). Therefore, determining an evolutionary pattern for the involvement of NANC factors in postprandial cardiac stimulation in vertebrates is not possible. This emphasizes the importance of understanding why these factors are present in cardiac control in some animals and not in others, including experimental models that represent the diversity of ecological and physiological factors such as diet.
According to our HRV analyses, I. iguana exhibit fH oscillations of parasympathetic origin only, as seen by the fact that HRVLF and HRVHF were drastically reduced after atropine administration in fasting animals (Figs. 4A,B). The fact that HRVLF and HRVHF remained unchanged after the double autonomic blockade of the heart (Figs. 4A,B) in both experimental groups indicates an absence of cardiac sympathetic nervous activity in the beat-to-beat regulation of the heart, different from the majority of tetrapods (De Vera et al., 2012; da Silva Braga et al., 2016; Lopes et al., 2017; Zena et al., 2017; Armelin et al., 2019; Sanches et al., 2019). The results above mentioned suggest that the autonomic adrenergic cardiac tone (Fig. 3) is derived from circulating catecholamines and also indicate that nerve-secreted NANC factors are not involved in the cardiac control of this species. Lastly, the lower HRVLF observed in digesting untreated animals compared with fasting untreated animals is a consequence of the reduction in cardiac sympathetic tone triggered by digestion, which could lead to a reduction in the regulatory range for short-term fH adjustments – similar to that observed by Guagnoni et al. (2021) during digestion in O. niloticus.
The I. iguana baroreflex is initiated by baroreceptors in the wall of the truncus arteriosus (Bagshaw, 1985), and the efferent short-term regulation of fH is provided by a parasympathetic control – as evidenced by a marked decrease in HRV, Gsp, and Gnorm in unfed animals after muscarinic blockade with atropine and a subsequent lack of change in these variables after double blockade (Figs 4A and 6A,B). Moreover, it is important to emphasize that the BEI and the number of baroreflex sequences detected did not change after the double cardiac autonomic blockade, because atropine and propranolol are competitive antagonists and therefore may not prevent discrete beat-to-beat changes in fH mediated by the barostatic reflex, which can generate baroreflex sequences (Lee et al., 2002; Rang et al., 2016; Armelin et al., 2021). The reduction of Gsp and Gnorm in unfed animals after atropine and propranolol injection corroborates this hypothesis, as it is evidence that the responsiveness of the sinus node to baroreflex-mediated changes in cardiac autonomic activity is impaired by these drugs (Stauss et al., 2006; Laude et al., 2008a,b; Armelin et al., 2021).
Finally, digestion triggers gastrointestinal hyperemia induced by enteric vasodilation and intensifies blood pressure fluctuations, but short-term autonomic reflexes such as the baroreflex modulate the fH and peripheral resistance to restore MAP within the limits of normality (Bagshaw, 1985; Brzezinski, 1990; Chapleau, 2012; Mancia and Mark, 1983). Consequently, as observed in digesting iguanas, the limitation of the autonomic regulatory range of short-term fH adjustments (Figs 3 and 4C,D) is directly related to the impairment of baroreflex effectiveness (Fig. 6A,B,D) and might have contributed to increasing oscillations of arterial blood pressure (Fig. 5C), resulting in higher values of BPVLF compared with those observed in fasting untreated animals (Fig. 5B,D). However, the response curve for barostatic fH regulation could be shifted upward, allowing for the maintenance of the cardiac limb in baroreflex regulation and the capacity for compensatory fH responses to hypotension although the gain has been significantly reduced – as observed by Wang et al. (2021) in digesting Boa constrictor.
In conclusion, our study shows that I. iguana exhibits a higher metabolic response compared with other herbivorous lizards, but the time to reach O2 peak was quite similar (Clarke and Nicolson, 1994; Buddemeyer et al., 2019 preprint). The increased myocardial activity is associated with a subtle postprandial tachycardia mediated exclusively by a reduction in vagal tone. In contrast to other reptiles, in iguanas, the absence of NANC factors in the postprandial cardiac regulatory pattern highlights the need for future studies to extend beyond the cardiac effects of various regulatory peptides, also focusing on identifying these peptides and exploring the reasons for their presence in postprandial cardiac control in some animals and their absence in others, as observed in the case of D. labrax (Iversen et al., 2010) and I. iguana. We also suggest that digestion limits the autonomic regulatory range for short-term fH adjustments in I. iguana. This impairment may compromise the effectiveness of the baroreflex by reducing the responsiveness to baroreflex-mediated changes in arterial pressure. However, as reported by Wang et al. (2021) in digesting B. constrictor, it is possible that it could be compensated for without negative consequences through an upward shift of the baroreflex regulatory response curve. Therefore, we ascertain that the analysis of HRV and BPV, combined with baroreflex assessment using the sequence method, has broadened and enriched our study, providing a more comprehensive approach to understanding short-term autonomic control of postprandial cardiovascular adjustments in ectothermic vertebrates.
Acknowledgements
The authors would like to thank the INCT-FISC professors for their comments and suggestions on the present study and members of the Florindo Laboratory for assistance with animal care. We are deeply indebted to André Luiz da Cruz for providing both equipment and thoughtful discussions, as well Augusto Shinya Abe for providing some specimens of I. iguana.
Footnotes
Author contributions
Conceptualization: I.N.G.; Methodology: I.N.G., V.A.A., V.H.d.S.B.; Formal analysis: I.N.G.; Investigation: I.N.G., V.A.A., V.H.d.S.B., D.A.M., L.H.F.; Resources: D.A.M., L.H.F.; Data curation: I.N.G.; Writing - original draft: I.N.G.; Writing - review & editing: I.N.G., V.A.A., V.H.d.S.B., D.A.M., L.H.F.; Visualization: I.N.G., D.A.M., L.H.F.; Supervision: D.A.M., L.H.F.; Project administration: L.H.F.; Funding acquisition: D.A.M., L.H.F.
Funding
This study was supported by the Brazilian National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq) and the São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP), through the Brazilian National Institute of Science and Technology in Comparative Physiology (INCT-FISC; Proc. Number 2008/57712-4). I.N.G. also received a master's research fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Proc. Number 88887.342350/2019-00).
Data availability
Data are available from the Open Science Framework at https://osf.io/ymzv7/.
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References
Competing interests
The authors declare no competing or financial interests.