RESEARCH ARTICLE

Heart rate dynamics in a marsupial hibernatorSteven J. Swoap1,2,*, Gerhard Körtner2 and Fritz Geiser2

ABSTRACTThe eastern pygmy possum (Cercartetus nanus) is a small marsupialthat can express spontaneous short bouts of torpor, as well as multi-day bouts of deep hibernation. To examine heart rate (fH) control atvarious stages of torpor in a marsupial hibernator, and to seewhetherfH variability differs from that of deep placental hibernators, we usedradiotelemetry to measure ECG and body temperature (Tb) whilemeasuring the rate of O2 consumption and ventilation. fH and O2consumption rate during euthermia were at a minimum (321±34 beats min−1, 0.705±0.048 ml O2 g−1 h−1) at an ambienttemperature (Ta) of 31°C. fH had an inverse linear relationship withTa to a maximum of 630±19 beats min−1 at a Ta of 20°C. During entryinto torpor at a Ta of 20°C, fH slowed primarily as a result of episodicperiods of cardiac activity where electrical activity of the heartoccurred in groups of 3 or 4 heart beats. When Tb was stable at 24°Cin 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 ofdeep torpor, Ta was lowered to 6.6±0.8°C. During these deep boutsof torpor, Tb reached a minimum of 8.0±1.0°C, with a minimum fH of8 beats min−1 and a minimum O2 consumption rate of 0.029±0.07 mlO2 g−1 h−1. Shivering bouts occurred in deep torpor about every8 min, during which ventilation occurred, and fH was elevated to40 beats min−1. The duration of the QRS complex increased from12 ms during euthermia to 69 ms at a Tb of 8°C. These findingsdemonstrate the dynamic functioning range of fH to be about600 beats min−1 (∼80-fold), one of the largest known ranges inmammals. Our study shows that despite a separation of ∼160 millionyears, the control and function of the cardiac system seemsindistinguishable in marsupial and placental hibernating mammals.

KEY WORDS: Torpor, Hibernation, Cardiac, ECG, Metabolism

INTRODUCTIONThe physiological processes that are engaged during bouts oftorpor 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; Milsomand Jackson, 2011), and active suppression of metabolic rate(Geiser, 2004). Further, shared cardiovascular changes in animalsthat 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) andperipheral 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 oftorpor include SNS activation of white adipose tissue (Swoap andWeinshenker, 2008) with the resultant drop in leptin and elevationin free fatty acid release, and withdrawal of SNS activity frombrown fat (Cannon and Nedergard, 2004) in those organisms thathave this heat-generating organ.

Heart rate (fH) during torpor has been examined in severalplacental mammals such as the 13-lined ground squirrel, themarmot, 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 duringhibernation (Milsom et al., 1999). First, all display low fH (between∼8 and 12 beats min−1) during bouts of deep torpor. Second, fHslows during entrance into torpor in part as a result of asystoles, orskipped beats. Third, fH during deep torpor is elevated periodically,and that elevation is associated with ventilation (ventilatorytachycardia). Ventilatory tachycardia is also seen in deep torpor ina marsupial (Cercartetus concinnus: Zosky and Larcombe, 2003);however, little is known about the variability in fH during entranceinto torpor in a marsupial hibernator. Further, in animals such asmice and Djungarian hamsters that utilize exclusively daily torporduring which the minimum body temperature (Tb) is around 20°Cand the length of the torpor bout is measured in hours and not daysor weeks, the minimum fH is much higher, around 70 beats min

−1

(Hudson and Scott, 1979; Swoap and Gutilla, 2009). An increasein fH variability during entrance into torpor is also seen in thesemammals that use daily torpor (Mertens et al., 2008; Vicentet al., 2017).

Marsupials diverged from placental mammals about 160 millionyears ago (Luo et al., 2011) and are interesting from an evolutionarypoint of view because they have lost functional brown fat (Oelkruget al., 2015), which is widely considered crucial for thermogenesisduring rewarming from hibernation. The objectives of the currentexperiment therefore were to measure the fH and fH variabilitycharacteristics in the marsupial eastern pygmy possum (Cercartetusnanus) at different depths of torpor to examine whether cardiacfunction especially during deep torpor shows any marsupialidiosyncrasies related to their different form of thermogenesis.This arboreal mammal is found in southeastern Australia and, froman experimental point of view, has an advantage because it enterstorpor at a wide range of ambient temperature (Ta), resulting inshallow and short bouts of torpor at high Ta, as well as deep andmulti-day bouts of torpor at low Ta (Geiser, 1993; Bartholomew andHudson, 1962). Because the eastern pygmy possum can undergoboth shallow brief torpor and deep hibernation, this organism allowsfor the examination of fH control in both of these situations. Theeastern pygmy possum can hibernate without food for up to anentire year, longer than known for any other species, and alsoexpresses some of the longest torpor bouts measured to date in thelaboratory of up to 1 month (Geiser, 2007). This attribute of variedtorpor bout duration and depth allows for examination of the fulldynamic range of fH control in a mammal.Received 2 January 2017; Accepted 30 May 2017

1Department of Biology, Williams College, Williamstown, MA 01267, USA. 2Centrefor Behavioural and Physiological Ecology, Zoology, University of New England,Armidale, NSW 2351, Australia.

*Author for correspondence (sswoap@williams.edu)

S.J.S., 0000-0002-4651-2488

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mailto:sswoap@williams.eduhttp://orcid.org/0000-0002-4651-2488

MATERIALS AND METHODSAnimalsFive 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 recordedmaximum Ta is 36.3°C and the lowest recorded minimum Ta is−3.5°C. The animals were housed singly in cages with sawdustbedding and nest boxes containing bedding material on a 12 h:12 hlight:dark schedule at a Ta of approximately 22°C. Animals were fedapples, walnuts, sunflower seeds, rolled oats and a mixture of highprotein baby cereal, honey, a vitamin supplement, boiled eggs orprotein powder and pureed fruit. Food was not provided ad libitumbecause pygmy possums tend to become obese in captivity. Waterwas freely available. Throughout the experiments, body massranged from 25 to 44 g (mean 35 g). All experiments were approvedby the University of New England Animal Ethics Committee andwere performed in accordance with the guidelines described by theNational Health and Medical Research Council.

Radiotelemeter implantationEach 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, DataSciences International, St Paul, MN, USA). For implantation,animals were anesthetized using 5% isoflurane in oxygen gas andmaintained with 2.5–3% isoflurane for the duration of theimplantation procedure. The telemeter was implanted in theabdominal cavity and ECG leads were placed subcutaneously,approximating a lead II configuration, and held in place by suturesused 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 aheating pad. The pygmy possums were allowed to recover for10 days before any experimentation began.

Telemeter data (ECG recordings and Tb) were monitored for aperiod of 10 s, once per minute, using receivers beneath the homecage (RPC-1, Data Sciences International). After approximately4weeks of sampling, each possumwas placed individually into a 0.5 lmetabolic chamber at an initial flow rate of ∼300 ml air min−1without food and water. These measurements began in the morningand were conducted during the daytime, the period of rest of pygmypossums. Air from the chamber was dried and directed into a SableFC-1B oxygen analyzer for determination of oxygen content. Flowrate was measured with a mass flowmeter (Omega FMA-5606,Stamford, CT, USA). The Ta of the chamber, measured to the nearest0.1°C with a calibrated thermocouple inserted ∼1 cm into therespirometry chamber, was initially set to 25°C to measure fH andoxygen consumption of euthermic possums. After 3 h, the Ta of thechamber was raised to approximately 32°C, and lowered again to25°C at a rate of 1.6°C h−1 to measure fH and O2 consumption as afunction of Ta and to determine basal values. The possumwasmovedback to its home cage after completion of theTa ramp in themetabolicchamber. To examine deeper torpor bouts, the possums were eachplaced again in the metabolic chamber in the late afternoon and leftthere overnight with the Ta of the chamber set initially to 13–14°C.After the possum had entered torpor in the morning, as assessed inreal time by the Tb of the animal, the air flow rate was lowered toapproximately 150 ml min−1 to increase the O2 differential, and theTa was lowered by 2–3°C approximately every hour to a minimum of5–8°C. Sampling from the telemeters was continuous in thiscondition. Once the possum aroused, it was placed back into itshome cage at 22°C. To assess the link between ventilation and fH

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Fig. 1. Typical heart rate ( fH) and body temperature (Tb) tracings of a pigmy possum housed at 22°C. This pygmy possum entered a spontaneous bout oftorpor (food ad libitum) on one day (solid line) and remained euthermic (dashed line) on another (top). The dark phase is the first 12 h of the 24 h period, and ismarked by a black bar. These typical tracings show a minimum Tb during torpor of 24°C and a minimum fH of 35 beats min−1. The resulting hysteresis curveplotting fH as a function of Tb is also shown (bottom) for the spontaneous bout of torpor (solid line), with arrows indicating directionality of entry into and arousalfrom torpor.

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control during torpor, the possums were placed in the metabolicchamber once again and held at 15°C overnight in the absence ofwater and food.Apressure transducer (MPX2010,Motorola,Denver,CO, USA) was used to monitor chamber pressure, from whichventilation frequency was determined (Cooper and Withers, 2010).For fH, fH variability and ECG noise analysis, raw data files of ECGrecordings from the telemeters were imported into PonemahPhysiology Platform software (Data Sciences International). Noisedetection was enabled and waveforms were analyzed.

Data analysisAll results are reported as means±s.e.m. Statistical analyses wereperformed in SPSS 15.0 (IBM Corp., Armonk, NY, USA).Repeated measures ANOVAs were performed and, whensignificance was shown, were followed with a post hoc Tukeytest. P

and V̇O2, all possums entered a bout of torpor within 24 h ofresidence 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 possumswas 624±11 beats min−1, not significantly different from the fHwhen housed at 22°C while not in the metabolic chamber. Theaverage V̇O2 of the non-torpid pygmy possums at this Ta of 13–14°Cwas 3.76±0.29 ml O2 g

−1 h−1. Once the possums entered torpor, Tbdropped to approximately 15°C (14.9±0.4°C), which was 1.3±0.3°Cabove Ta. The minimum V̇O2 at this Ta fell to 0.110±0.021 mlO2 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 ofseveral hours (see Fig. 3), Tb fell to an average of 8.0±1.0°C, whichwas 1.2±0.4°C above Ta. The V̇O2 at this Ta was 0.029±0.007 mlO2 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 fHwas 8 beats min−1 in a possumwith a Tb of 6.0°C. Attempts to lowerthe 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 littlevariability (see ECG tracing in Fig. 4A and the accompanyingPoincare plot). During entrance into torpor (Fig. 4B), the ECGdisplayed skipped beats, with the heart beats occurring episodicallyin sets of 3 or 4. The variability of fH is easily seen on theaccompanying Poincare plot. During deep torpor, a consistentpattern was observed as shown in Fig. 4C,D. The fH was slow andsteady for minutes at a time, followed by a slowly increasing fH, thena burst of ECG noise and an associated elevation of fH, typically toabout 40 beats min−1. This elevation lasted 20–30 s, at which pointthe 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 QRScomplex within the ECG changed as a function of Tb. As Fig. 5shows, 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 ECGtracing, presumably a result of the massive shivering that occursduring emergence from torpor in this species.

To examine whether the short bouts of tachycardia during deeptorpor (Fig. 4D) were related to ventilation patterns, the implantedpossums were placed in a metabolic cage with a pressure transducerfor determination of breathing activities with simultaneousacquisition of Tb and fH at a Ta of 14°C. A typical tracing duringeuthermia is shown in Fig. 6A. During deep torpor, periods of apneawere clearly discernible between bouts of breathing. A typical boutof breathing during torpor is shown in Fig. 6B, with the regionbetween 120 and 180 s shown on an expanded scale in Fig. 6C. Thebouts of increased fH during deep bouts of torpor in the pygmypossum occurred as the animal began to breathe. fH slowed againduring the subsequent apnea bout.

DISCUSSIONMammalian fH can be tremendously flexible, varying greatlythroughout the day, even on a beat-to-beat basis. This flexibility infH is of particular interest in hibernators, where fH falls as low as afew beats per minute in a deep bout of torpor. The maximum fH forthe eastern pygmy possum appears to be about 625 beats min−1,similar to an earlier estimate (Bartholomew and Hudson, 1962). ThefH 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 betweenfH and Ta exists in rats and mice. Importantly, the slope of the fH/Ta

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Fig. 3. Typical tracings of pygmy possum rate ofoxygen consumption (V̇O2), fH and Tb duringprolonged torpor bouts. Possums were housedin a metabolic chamber set to a Ta of 13–14°C.Once the possum went into torpor, the Ta waslowered to 5°C over a 4 h period. After 22 h at thistemperature, the Ta was then slowly raised. Thepossum spontaneously aroused from torpor whenthe Ta reached 19°C. V̇O2, fH and Tb weresimultaneously measured throughout the entirerun. See Results for quantification of thesevariables.

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relationship is much less steep in rats and mice (8 and15 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 coolertemperatures has been seen previously, including a measured fH ofeuthermic possums of 600–650 beats min−1 at a Ta of 5°C

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Fig. 4. Typical ECG tracings and fH variability during deep torpor in the pygmy possum for different sections of the torpor bout. To the right ofeach ECG tracing is a Poincare plot, which plots any interbeat interval (IBIn) with the following IBI (IBIn+1). The ECG tracing and accompanying Poincare plotare shown (A) before the bout of torpor, (B) during descent into torpor and (C) during deep torpor. In addition, a longer time scale during deep torpor isshown (D) to illustrate the bursts of fH that occurred in this pygmy possum about every 8 min during deep torpor. The Poincare plots contain ∼65,000,30,000 and 35,000 data points for A–C, respectively.

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(Bartholomew and Hudson, 1962). This suggests that fH is at itsmaximum at 22°C, and that the SNS has a much greater influencethan the parasympathetic nervous system (PNS) over the possums’heart period at Ta below 22°C. It is unclear whether cardiac outputcontinues to increase as Ta drops through elevation in stroke volumein response to the elevated metabolic demand at coolertemperatures.All animals that use torpor, either hibernation or daily torpor,

exhibit a large drop in fH during entrance into torpor. This is true foreastern pygmy possum as well (present study and Bartholomew andHudson, 1962). However, the extent to which fH falls differsbetween animals that use daily torpor and hibernators. With thelower absolute Tb in hibernators, it is not surprising that the absoluteminimum 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 canachieve, 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 placentalhibernators, 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 observedin animals that use daily torpor at the same Tb, including placentalmammals (the mouse and Djungarian hamster) and in the fat-taileddunnart, a marsupial (Swoap and Gutilla, 2009; Mertens et al.,2008; Zosky, 2002), supporting the view that hibernators and dailyheterotherms differ not only ecologically but also functionally (Rufand Geiser, 2015).

The relationship between Tb and fH showed marked hysteresis(Fig. 1), suggesting that the control of fH during entrance into torporand 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 examiningmetabolic 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 theDjungarian hamster (Mertens et al., 2008). Interestingly, the breadthof the fH/Tb loop is much greater in the eastern pygmy possum thanin other hibernators as well as in animals that use daily torpor. Forexample, at a Tb of 30°C, the difference in fH between entrance andexit from torpor is about 400 beats min−1 in the eastern pygmypossum. For hibernators, such as the ground squirrel, and in animalsthat use daily torpor, such as the mouse and Djungarian hamster, fHbreadth is approximately 200–275 beats min−1 (Lyman, 1965;Swoap and Gutilla, 2009; Mertens et al., 2008).

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The variability of fH during entrance into torpor is common to allmammals that enter torpor (Milsom et al., 1999), and the easternpygmy possum is no different (Fig. 4). There appear to be at leasttwo sources of fH variability during torpor and entrance into torporthat are both linked to the ANS. First, the appearance of asystoles(skipped beats) during entrance into torpor (Fig. 4) is seen in manyhibernators and is a consequence of elevated PNS activity duringentrance into torpor (see Milsom et al., 1999, for a review). Whilewe did not perform any pharmacological experiments in the currentstudy, administration of atropine, a muscarinic receptor antagonist,into the closely related western pygmy possum during torporsignificantly elevates fH and eliminates asystoles (Zosky andLarcombe, 2003). The second source of variability in fH (Fig. 4)occurred during the periods between bouts of apnea in deep torporin the eastern pygmy possum. fH was elevated from 8 to12 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 hibernatingspecies, such as bears and ground squirrels, and is indicative ofaltered ANS activity (Dawe and Morrison, 1955; Tøien et al., 2011;Milsom et al., 1999; Harris and Milsom, 1995). Indeed, atropineadministration during torpor in the western pygmy possumeliminates ventilation-associated tachycardia (Zosky andLarcombe, 2003). The low fH of 8 beats min−1 we measured hereis in contrast with a previous study where this same species had a fHof 28 beats min−1 during torpor (Bartholomew and Hudson, 1962).While it is difficult to know the source of the difference betweenthese studies, Bartholomew and Hudson (1962) were surprised atthe relatively high torpid fH and concluded the animals were likelydisturbed when instrumented during the bout of torpor. Notably, weused radiotelemetry here with indwelling lines that minimizedisturbance 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 metabolicflexibility (greater than 100-fold) in torpor and euthermia. Theanimal’s Tb during torpor plays a role in this flexibility, which canbe 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 agreater 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 shrewhas a dynamic range of 900 beats min−1 (euthermic rate of1000 beats min−1 and torpid rate of 100 beats min−1) whereasthe 9 g bat has a range of 800 beats min−1 (euthermic rate of800 beats min−1 and torpid of 8 beats min−1). The rapid600 beats min−1 increase in fH (and likely cardiac output) duringrecovery from torpor in the pygmy possum is probably important formeeting O2 demand for metabolic rate elevation and heatproduction, such as shivering in skeletal muscle, duringrewarming as has been suggested for the shrew (Fons et al.,1997). To sum, despite the lack of functional brown fat, fH control inthis marsupial hibernator throughout a bout of deep torpor appearsindistinguishable from that of a placental hibernator in terms ofdepth, appearance of skipped beats and ventilation-associatedtachycardia. However, there appears to be a large difference inminimum fH between hibernators and mammals that use dailytorpor, even when measured at the same Tb.

AcknowledgementsWe thank Christine Cooper and Phil Withers for the loan and instructions for the useof the pressure transducer.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: 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 - originaldraft: S.S.; Writing - review & editing: S.S., G.K., F.G.; Supervision: G.K., F.G.;Project administration: F.G.; Funding acquisition: F.G.

FundingFunding was provided for this study from a grant to F.G. from the AustralianResearch Council.

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Cooper, C. E. and Withers, P. C. (2010). Comparative physiology of Australianquolls (Dasyurus; Marsupialia). J. Comp. Physiol. B 180, 857-868.

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