The eastern pygmy possum (Cercartetus nanus) is a small marsupial that can express spontaneous short bouts of torpor, as well as multi-day bouts of deep hibernation. To examine heart rate (fH) control at various stages of torpor in a marsupial hibernator, and to see whether fH variability differs from that of deep placental hibernators, we used radiotelemetry to measure ECG and body temperature (Tb) while measuring the rate of O2 consumption and ventilation. fH and O2 consumption rate during euthermia were at a minimum (321±34 beats min−1, 0.705±0.048 ml O2 g−1 h−1) at an ambient temperature (Ta) of 31°C. fH had an inverse linear relationship with Ta to a maximum of 630±19 beats min−1 at a Ta of 20°C. During entry into torpor at a Ta of 20°C, fH slowed primarily as a result of episodic periods of cardiac activity where electrical activity of the heart occurred in groups of 3 or 4 heart beats. When Tb was stable at 24°C in these torpor bouts, the episodic nature of fH had disappeared (i.e. no asystoles) with a rate of 34±3 beats min−1. For multi-day bouts of deep torpor, Ta was lowered to 6.6±0.8°C. During these deep bouts of torpor, Tb reached a minimum of 8.0±1.0°C, with a minimum fH of 8 beats min−1 and a minimum O2 consumption rate of 0.029±0.07 ml O2 g−1 h−1. Shivering bouts occurred in deep torpor about every 8 min, during which ventilation occurred, and fH was elevated to 40 beats min−1. The duration of the QRS complex increased from 12 ms during euthermia to 69 ms at a Tb of 8°C. These findings demonstrate the dynamic functioning range of fH to be about 600 beats min−1 (∼80-fold), one of the largest known ranges in mammals. Our study shows that despite a separation of ∼160 million years, the control and function of the cardiac system seems indistinguishable in marsupial and placental hibernating mammals.
The physiological processes that are engaged during bouts of torpor in mammalian hibernation are numerous and complex. These processes include, but are certainly not limited to, abandonment of euthermic thermoregulation (Heller, 1983), apnea which can last seconds to minutes (Lyman, 1965; Milsom and Jackson, 2011), and active suppression of metabolic rate (Geiser, 2004). Further, shared cardiovascular changes in animals that use torpor include bradycardia (Zosky and Larcombe, 2003; Swoap and Gutilla, 2009; Morhardt, 1970; Harris and Milsom, 1995) mediated by the autonomic nervous system (ANS) and peripheral vasoconstriction (Swoap and Gutilla, 2009; Osborne et al., 2005) mediated by the sympathetic nervous system (SNS). Other indicators of altered autonomic function during a bout of torpor include SNS activation of white adipose tissue (Swoap and Weinshenker, 2008) with the resultant drop in leptin and elevation in free fatty acid release, and withdrawal of SNS activity from brown fat (Cannon and Nedergard, 2004) in those organisms that have this heat-generating organ.
Heart rate (fH) during torpor has been examined in several placental mammals such as the 13-lined ground squirrel, the marmot, the black bear and long-eared bats (Tøien et al., 2011; Currie et al., 2014; Eagles et al., 1988; Hampton et al., 2010). Placental mammals show three distinguishing features of fH during hibernation (Milsom et al., 1999). First, all display low fH (between ∼8 and 12 beats min−1) during bouts of deep torpor. Second, fH slows during entrance into torpor in part as a result of asystoles, or skipped beats. Third, fH during deep torpor is elevated periodically, and that elevation is associated with ventilation (ventilatory tachycardia). Ventilatory tachycardia is also seen in deep torpor in a marsupial (Cercartetus concinnus: Zosky and Larcombe, 2003); however, little is known about the variability in fH during entrance into torpor in a marsupial hibernator. Further, in animals such as mice and Djungarian hamsters that utilize exclusively daily torpor during which the minimum body temperature (Tb) is around 20°C and the length of the torpor bout is measured in hours and not days or weeks, the minimum fH is much higher, around 70 beats min−1 (Hudson and Scott, 1979; Swoap and Gutilla, 2009). An increase in fH variability during entrance into torpor is also seen in these mammals that use daily torpor (Mertens et al., 2008; Vicent et al., 2017).
Marsupials diverged from placental mammals about 160 million years ago (Luo et al., 2011) and are interesting from an evolutionary point of view because they have lost functional brown fat (Oelkrug et al., 2015), which is widely considered crucial for thermogenesis during rewarming from hibernation. The objectives of the current experiment therefore were to measure the fH and fH variability characteristics in the marsupial eastern pygmy possum (Cercartetus nanus) at different depths of torpor to examine whether cardiac function especially during deep torpor shows any marsupial idiosyncrasies related to their different form of thermogenesis. This arboreal mammal is found in southeastern Australia and, from an experimental point of view, has an advantage because it enters torpor at a wide range of ambient temperature (Ta), resulting in shallow and short bouts of torpor at high Ta, as well as deep and multi-day bouts of torpor at low Ta (Geiser, 1993; Bartholomew and Hudson, 1962). Because the eastern pygmy possum can undergo both shallow brief torpor and deep hibernation, this organism allows for the examination of fH control in both of these situations. The eastern pygmy possum can hibernate without food for up to an entire year, longer than known for any other species, and also expresses some of the longest torpor bouts measured to date in the laboratory of up to 1 month (Geiser, 2007). This attribute of varied torpor bout duration and depth allows for examination of the full dynamic range of fH control in a mammal.
MATERIALS AND METHODS
Five eastern pygmy possums, Cercartetus nanus (Desmarest 1818), were caught using nest boxes near Dorrigo, NSW, Australia (30°22′S, 152°34′E). The mean minimum monthly Ta in Dorrigo is 4.4–15.0°C and the mean maximum Ta is 14.4 –24.1°C. The highest recorded maximum Ta is 36.3°C and the lowest recorded minimum Ta is −3.5°C. The animals were housed singly in cages with sawdust bedding and nest boxes containing bedding material on a 12 h:12 h light:dark schedule at a Ta of approximately 22°C. Animals were fed apples, walnuts, sunflower seeds, rolled oats and a mixture of high protein baby cereal, honey, a vitamin supplement, boiled eggs or protein powder and pureed fruit. Food was not provided ad libitum because pygmy possums tend to become obese in captivity. Water was freely available. Throughout the experiments, body mass ranged from 25 to 44 g (mean 35 g). All experiments were approved by the University of New England Animal Ethics Committee and were performed in accordance with the guidelines described by the National Health and Medical Research Council.
Each animal was implanted with an electrocardiogram (ECG) telemeter that (1) detects electrical signals across the heart, (2) measures Tb and (3) measures locomotor activity (ETA-F10, Data Sciences International, St Paul, MN, USA). For implantation, animals were anesthetized using 5% isoflurane in oxygen gas and maintained with 2.5–3% isoflurane for the duration of the implantation procedure. The telemeter was implanted in the abdominal cavity and ECG leads were placed subcutaneously, approximating a lead II configuration, and held in place by sutures used to close the body wall. Wound clips (7 mm size reflex clips, Fine Science Tools, Foster City, CA, USA) were used to close the abdominal incision. During the post-operative recovery period, animals were housed individually at 30°C in cages placed half atop a heating pad. The pygmy possums were allowed to recover for 10 days before any experimentation began.
Telemeter data (ECG recordings and Tb) were monitored for a period of 10 s, once per minute, using receivers beneath the home cage (RPC-1, Data Sciences International). After approximately 4 weeks of sampling, each possum was placed individually into a 0.5 l metabolic chamber at an initial flow rate of ∼300 ml air min−1 without food and water. These measurements began in the morning and were conducted during the daytime, the period of rest of pygmy possums. Air from the chamber was dried and directed into a Sable FC-1B oxygen analyzer for determination of oxygen content. Flow rate was measured with a mass flowmeter (Omega FMA-5606, Stamford, CT, USA). The Ta of the chamber, measured to the nearest 0.1°C with a calibrated thermocouple inserted ∼1 cm into the respirometry chamber, was initially set to 25°C to measure fH and oxygen consumption of euthermic possums. After 3 h, the Ta of the chamber was raised to approximately 32°C, and lowered again to 25°C at a rate of 1.6°C h−1 to measure fH and O2 consumption as a function of Ta and to determine basal values. The possum was moved back to its home cage after completion of the Ta ramp in the metabolic chamber. To examine deeper torpor bouts, the possums were each placed again in the metabolic chamber in the late afternoon and left there overnight with the Ta of the chamber set initially to 13–14°C. After the possum had entered torpor in the morning, as assessed in real time by the Tb of the animal, the air flow rate was lowered to approximately 150 ml min−1 to increase the O2 differential, and the Ta was lowered by 2–3°C approximately every hour to a minimum of 5–8°C. Sampling from the telemeters was continuous in this condition. Once the possum aroused, it was placed back into its home cage at 22°C. To assess the link between ventilation and fH control during torpor, the possums were placed in the metabolic chamber once again and held at 15°C overnight in the absence of water and food. A pressure transducer (MPX2010, Motorola, Denver, CO, USA) was used to monitor chamber pressure, from which ventilation frequency was determined (Cooper and Withers, 2010). For fH, fH variability and ECG noise analysis, raw data files of ECG recordings from the telemeters were imported into Ponemah Physiology Platform software (Data Sciences International). Noise detection was enabled and waveforms were analyzed.
All results are reported as means±s.e.m. Statistical analyses were performed in SPSS 15.0 (IBM Corp., Armonk, NY, USA). Repeated measures ANOVAs were performed and, when significance was shown, were followed with a post hoc Tukey test. P<0.05 was considered statistically significant.
When pygmy possums were housed in their cages in a room maintained at a Ta of 22±1°C and Tb and ECG tracings were monitored, three of the five possums periodically entered a torpor bout despite the presence of food and water (Fig. 1). At this Ta and on days when the possums did not enter torpor, the average 24 h fH for the group was 623±17 beats min−1 (n=5), with an average Tb over the same time frame of 35.5±0.4°C. On those days that the possums entered torpor spontaneously (Fig. 1), fH fell significantly to a minimum of 34±3 beats min−1 (n=3) with a minimum Tb of 24.1±0.1°C. The relationship between fH and Tb in the animals that entered torpor was complex (Fig. 1). fH significantly dropped before the onset of the reduction in Tb. This was followed by a more gradual decrease in fH as Tb declined. fH increased dramatically during the arousal period, which typically took less than 60 min.
Over 10 h, the Ta of the metabolic chamber housing an implanted pygmy possum was ramped from 25°C to 31–32°C and back down to 25°C (Fig. 2A). Fig. 2E,F shows typical ECG tracings from a pygmy possum at a Ta of 25 and 31°C. From these ECG tracings, both fH and noise from the ECG tracing were quantified together with the rate of O2 consumption (V̇O2). A typical response to the Ta ramp in V̇O2, fH and noise on the ECG tracing is shown in Fig. 2B–D. The calculated lower critical temperature of the thermoneutral zone from V̇O2 measurements was 30.0±0.3°C, similar to that found earlier for this same species (Song et al., 1997). The lowest V̇O2 in the euthermic possum was 0.705±0.048 ml O2 g−1 h−1 (n=5) at a Ta of 31.0±0.2°C and a Tb of 35.4±0.3°C. Similarly, the minimum fH at a Ta of 31°C was 294±31 beats min−1, and the noise in the ECG tracing dropped to 5.1±1.0% of the noise value at a Ta of 25°C.
When housed individually in a metabolic chamber at 13–14°C without food and water overnight for monitoring Tb, ECG tracings and V̇O2, all possums entered a bout of torpor within 24 h of residence in the metabolic chamber. Before the bout of torpor (i.e. euthermic, with a Tb of 35.5±0.3°C), the average fH of the possums was 624±11 beats min−1, not significantly different from the fH when housed at 22°C while not in the metabolic chamber. The average V̇O2 of the non-torpid pygmy possums at this Ta of 13–14°C was 3.76±0.29 ml O2 g−1 h−1. Once the possums entered torpor, Tb dropped to approximately 15°C (14.9±0.4°C), which was 1.3±0.3°C above Ta. The minimum V̇O2 at this Ta fell to 0.110±0.021 ml O2 g−1 h−1, and the fH dropped to 25±2 beats min−1 (n=5). When the Ta of the chamber was then lowered to 5–8°C over a period of several hours (see Fig. 3), Tb fell to an average of 8.0±1.0°C, which was 1.2±0.4°C above Ta. The V̇O2 at this Ta was 0.029±0.007 ml O2 g−1 h−1 and the minimum fH dropped to 9±1 beats min−1 (n=5). The lowest fH sustained in any possum during periods of a stable fH was 8 beats min−1 in a possum with a Tb of 6.0°C. Attempts to lower the Ta below 5°C evoked arousals in the possums (data not shown).
ECGs were examined during three phases of these deep torpor bouts shown in Fig. 3: pre-torpor, entrance into torpor, deep torpor. Before the possums entered torpor, fH was fast and had little variability (see ECG tracing in Fig. 4A and the accompanying Poincare plot). During entrance into torpor (Fig. 4B), the ECG displayed skipped beats, with the heart beats occurring episodically in sets of 3 or 4. The variability of fH is easily seen on the accompanying Poincare plot. During deep torpor, a consistent pattern was observed as shown in Fig. 4C,D. The fH was slow and steady for minutes at a time, followed by a slowly increasing fH, then a burst of ECG noise and an associated elevation of fH, typically to about 40 beats min−1. This elevation lasted 20–30 s, at which point the fH of the possum returned to a slow and steady level of 8–12 beats min−1. The QRS complex was also examined at three Tb throughout these deep bouts of torpor. The duration of the QRS complex within the ECG changed as a function of Tb. As Fig. 5 shows, the duration of the QRS increased 5.8-fold at a Tb of 8°C (69±9 ms) relative to the QRS duration in euthermia (12±1 ms). During arousal, fH was quickly obscured by the noise on the ECG tracing, presumably a result of the massive shivering that occurs during emergence from torpor in this species.
To examine whether the short bouts of tachycardia during deep torpor (Fig. 4D) were related to ventilation patterns, the implanted possums were placed in a metabolic cage with a pressure transducer for determination of breathing activities with simultaneous acquisition of Tb and fH at a Ta of 14°C. A typical tracing during euthermia is shown in Fig. 6A. During deep torpor, periods of apnea were clearly discernible between bouts of breathing. A typical bout of breathing during torpor is shown in Fig. 6B, with the region between 120 and 180 s shown on an expanded scale in Fig. 6C. The bouts of increased fH during deep bouts of torpor in the pygmy possum occurred as the animal began to breathe. fH slowed again during the subsequent apnea bout.
Mammalian fH can be tremendously flexible, varying greatly throughout the day, even on a beat-to-beat basis. This flexibility in fH is of particular interest in hibernators, where fH falls as low as a few beats per minute in a deep bout of torpor. The maximum fH for the eastern pygmy possum appears to be about 625 beats min−1, similar to an earlier estimate (Bartholomew and Hudson, 1962). The fH was very responsive to Ta above 25°C, falling to 325 beats min−1 at thermoneutrality at a Ta of 31°C, as has been shown before (Bartholomew and Hudson, 1962). This same relationship between fH and Ta exists in rats and mice. Importantly, the slope of the fH/Ta relationship is much less steep in rats and mice (8 and 15 beats min−1 °C−1, respectively) than it is with the possum (∼50 beats min−1 °C−1) between 25 and 31°C (Swoap et al., 2004). However, at Ta below 22°C when the possums were euthermic, the fH stayed unchanged, measured herein at 624 and 623 beats min−1 at 23 and 15°C, respectively. This lack of change of fH at cooler temperatures has been seen previously, including a measured fH of euthermic possums of 600–650 beats min−1 at a Ta of 5°C (Bartholomew and Hudson, 1962). This suggests that fH is at its maximum at 22°C, and that the SNS has a much greater influence than the parasympathetic nervous system (PNS) over the possums' heart period at Ta below 22°C. It is unclear whether cardiac output continues to increase as Ta drops through elevation in stroke volume in response to the elevated metabolic demand at cooler temperatures.
All animals that use torpor, either hibernation or daily torpor, exhibit a large drop in fH during entrance into torpor. This is true for eastern pygmy possum as well (present study and Bartholomew and Hudson, 1962). However, the extent to which fH falls differs between animals that use daily torpor and hibernators. With the lower absolute Tb in hibernators, it is not surprising that the absolute minimum fH in hibernators is substantially lower (5–10 beats min−1) compared with the minimum fH (70–150 beats min−1) in animals that use daily torpor (Milsom et al., 1999; Morhardt, 1970; Swoap and Gutilla, 2009; Zosky, 2002). However, even for any given Tb that both groups of animals can achieve, the hibernators have a substantially lower fH. For example, during entrance into torpor, the eastern pygmy possum had a fH of ∼35 beats min−1 at 24°C, similar to that seen in placental hibernators, woodchucks, hedgehogs and ground squirrels (Lyman, 1958; Harris and Milsom, 1995). This fH for hibernators at a Tb of 24°C is only a small fraction (20–45%) of what is observed in animals that use daily torpor at the same Tb, including placental mammals (the mouse and Djungarian hamster) and in the fat-tailed dunnart, a marsupial (Swoap and Gutilla, 2009; Mertens et al., 2008; Zosky, 2002), supporting the view that hibernators and daily heterotherms differ not only ecologically but also functionally (Ruf and Geiser, 2015).
The relationship between Tb and fH showed marked hysteresis (Fig. 1), suggesting that the control of fH during entrance into torpor and emergence from torpor are the result of different phenomena. Hysteresis loops are also observed with fH and Tb in the mouse (Swoap and Gutilla, 2009; Morhardt, 1970), and when examining metabolic rate as a function of Tb in dunnarts (Geiser et al., 2014), and in the QT interval of the ECG as a function of Tb in the Djungarian hamster (Mertens et al., 2008). Interestingly, the breadth of the fH/Tb loop is much greater in the eastern pygmy possum than in other hibernators as well as in animals that use daily torpor. For example, at a Tb of 30°C, the difference in fH between entrance and exit from torpor is about 400 beats min−1 in the eastern pygmy possum. For hibernators, such as the ground squirrel, and in animals that use daily torpor, such as the mouse and Djungarian hamster, fH breadth is approximately 200–275 beats min−1 (Lyman, 1965; Swoap and Gutilla, 2009; Mertens et al., 2008).
The variability of fH during entrance into torpor is common to all mammals that enter torpor (Milsom et al., 1999), and the eastern pygmy possum is no different (Fig. 4). There appear to be at least two sources of fH variability during torpor and entrance into torpor that are both linked to the ANS. First, the appearance of asystoles (skipped beats) during entrance into torpor (Fig. 4) is seen in many hibernators and is a consequence of elevated PNS activity during entrance into torpor (see Milsom et al., 1999, for a review). While we did not perform any pharmacological experiments in the current study, administration of atropine, a muscarinic receptor antagonist, into the closely related western pygmy possum during torpor significantly elevates fH and eliminates asystoles (Zosky and Larcombe, 2003). The second source of variability in fH (Fig. 4) occurred during the periods between bouts of apnea in deep torpor in the eastern pygmy possum. fH was elevated from 8 to 12 beats min−1 during apnea to approximately 40 beats min−1 during ventilation in deep torpor (Fig. 6). This ventilation-associated tachycardia is also seen in several other hibernating species, such as bears and ground squirrels, and is indicative of altered ANS activity (Dawe and Morrison, 1955; Tøien et al., 2011; Milsom et al., 1999; Harris and Milsom, 1995). Indeed, atropine administration during torpor in the western pygmy possum eliminates ventilation-associated tachycardia (Zosky and Larcombe, 2003). The low fH of 8 beats min−1 we measured here is in contrast with a previous study where this same species had a fH of 28 beats min−1 during torpor (Bartholomew and Hudson, 1962). While it is difficult to know the source of the difference between these studies, Bartholomew and Hudson (1962) were surprised at the relatively high torpid fH and concluded the animals were likely disturbed when instrumented during the bout of torpor. Notably, we used radiotelemetry here with indwelling lines that minimize disturbance of the possums.
We have shown here that the eastern pygmy possum has an enormous dynamic range in fH of 600 beats min−1, from 625 to 8 beats min−1 (an 80-fold difference), to match the metabolic flexibility (greater than 100-fold) in torpor and euthermia. The animal's Tb during torpor plays a role in this flexibility, which can be seen by the Q10 of the QRS duration of approximately 2 (Fig. 5), similar to that seen in the Djungarian hamster (Mertens et al., 2008). Two other much smaller mammals have been reported to have a greater dynamic range in fH, the Etruscan shrew (Fons et al., 1997) and the Gould's long-eared bat (Currie et al., 2014). The 2.4 g shrew has a dynamic range of 900 beats min−1 (euthermic rate of 1000 beats min−1 and torpid rate of 100 beats min−1) whereas the 9 g bat has a range of 800 beats min−1 (euthermic rate of 800 beats min−1 and torpid of 8 beats min−1). The rapid 600 beats min−1 increase in fH (and likely cardiac output) during recovery from torpor in the pygmy possum is probably important for meeting O2 demand for metabolic rate elevation and heat production, such as shivering in skeletal muscle, during rewarming as has been suggested for the shrew (Fons et al., 1997). To sum, despite the lack of functional brown fat, fH control in this marsupial hibernator throughout a bout of deep torpor appears indistinguishable from that of a placental hibernator in terms of depth, appearance of skipped beats and ventilation-associated tachycardia. However, there appears to be a large difference in minimum fH between hibernators and mammals that use daily torpor, even when measured at the same Tb.
We thank Christine Cooper and Phil Withers for the loan and instructions for the use of the pressure transducer.
Funding was provided for this study from a grant to F.G. from the Australian Research Council.
Conceptualization: S.S., F.G.; Methodology: S.S., F.G.; Formal analysis: S.S., F.G.; Investigation: G.K., F.G.; Resources: S.S., F.G.; Data curation: S.S.; Writing - original draft: S.S.; Writing - review & editing: S.S., G.K., F.G.; Supervision: G.K., F.G.; Project administration: F.G.; Funding acquisition: F.G.
The authors declare no competing or financial interests.