Cardiac Rhythms and Variation in Hibernating Arctic Ground Squirrels

Abstract

The dramatic decrease in heart rate (HR) during entrance into hibernation is not a mere response to the lowering of core body temperature $\left(T_{\text{b}}\right)$ but a highly regulated fall, as the decrease in HR precedes the drop in $T_{\text{b}}$. This regulated fall in HR is thought to be mediated by increased cardiac parasympathetic activity. Conversely, the sympathetic nervous system is thought to drive the increase of HR during arousal. Despite this general understanding, we lack temporal information on cardiac parasympathetic regulation throughout a complete hibernation bout. The goal of this study was to fill this gap in knowledge by using Arctic ground squirrels implanted with electrocardiogram/temperature telemetry transmitters. Short-term HR variability (root mean square of successive differences [RMSSD]), an indirect measure of cardiac parasympathetic regulation, was calculated in 11 Arctic ground squirrels. RMSSD, normalized as RMSSD/RR interval (RRI), increased fourfold during early entrance (from 0.2 ± 0.1 to 0.8 ± 0.2, P < 0.05). RMSSD/RRI peaked after HR dropped by over 90% and $T_{\text{b}}$ fell by 70%. Late entrance was delineated by a decline in RMSSD/RRI while $T_{\text{b}}$ continued to decrease. During arousal, HR started to increase 2 h before $T_{\text{b}}$, with a concurrent decrease in RMSSD/RRI to a new minimum. As $T_{\text{b}}$ increased to a maximum during interbout arousal, HR declined, and RMSSD/RRI increased. These data suggest that activation of the parasympathetic nervous system initiates and regulates the HR decrease during entrance into hibernation and that withdrawal of parasympathetic activation initiates arousal. We conclude that cardiac parasympathetic regulation persists throughout all phases of a hibernation bout—a feature of the autonomic nervous system’s regulation of hibernation that was not appreciated previously.

Introduction

Hibernation is a highly complex physiological process used by different species to adapt and survive difficult, and sometimes even extreme, conditions such as very low or high ambient temperatures where the availability of food or water is reduced and often insufficient (Lyman and Chatfield 1955). Essentially, hibernation lowers the energy demands required by organisms and provides a solution for the lack of resources, which can occur in very hot and dry areas such as Africa (defined as estivation; Storey and Storey 2012) or at high latitudes above the Arctic Circle. Hibernation encompasses numerous physiological processes, such as long apneic periods (Milsom and Jackson 2011; Sprenger and Milsom 2022), suppression of metabolic rate (Geiser 2004), and a decrease in core body temperature ($T_{\text{b}}$; Heller 1983). In addition to these modifications, a significant alteration in cardiac rhythm and heart rate (HR) occurs during different phases of hibernation (i.e., entrance, torpor, and arousal). Entrance into torpor corresponds with a decrease in metabolic rate and HR as well as a progressive decrease in the temperature regulation set point (followed by a lowering of $T_{\text{b}}$; Florant and Heller 1977; Heller 1979). Additionally, torpor is characterized by a drastic and prolonged reduction in metabolic rate, HR, and $T_{\text{b}}$. Finally, during the arousal stage, metabolic rate, HR, and $T_{\text{b}}$ increase to euthermic values.

HR during hibernation has been studied in various mammals, such as the thirteen-lined ground squirrel, the Syrian hamster, the marmot, the American black bear, and the eastern pygmy (Eagles et al. 1988; Hampton et al. 2010; Tøien et al. 2011; Horwitz et al. 2013; Swoap et al. 2017). All species show similar features in HR during the entrance phase that are consistent with increased parasympathetic input (Milsom et al. 1999). For example, during the entrance phase, asystoles and/or skipped heartbeats often occur with the slowing of HR (Lyman 1958). Moreover, blocking parasympathetic activity with atropine abolished skipped heartbeats and elevated overall HR, demonstrating the importance of parasympathetic activation during this phase (Zosky 2002; Zosky and Larcombe 2003). It is acknowledged that cardiac parasympathetic regulation continues to function during torpor, as evidenced by persistent respiratory sinus arrhythmias (RSAs; Harris and Milsom 1995; Laske et al. 2010; Swoap et al. 2017) and by baroreflex function measured with sequence methods (Horwitz et al. 2013).

Other studies support an essential role of the sympathetic nervous system during torpor. During the pre-entrance phase, several hibernators show peaks in metabolic rate, HR, and ventilation (Ortmann and Heldmaier 2000; Elvert and Heldmaier 2005; Hampton et al. 2010). Chemical sympathectomy with a 6-hydroxydopamine injection in Djungarian hamsters blocks this surge of metabolic activity and abolishes the onset of daily torpor for several days (Braulke and Heldmaier 2010). While these studies point to the importance of the autonomic nervous system (ANS) in hibernation, the temporal pattern of the parasympathetic contribution throughout entrance, torpor, and arousal is not fully known. The capability for noninvasive continuous monitoring is key to resolving this temporal pattern. In this regard, HR variability (HRV) is well accepted as a marker for cardiac autonomic function. Intrinsic HR is set by the sinoatrial node pacemaker cells, and the variability of that HR is dually regulated by the cardioinhibitory parasympathetic and cardioexcitatory sympathetic branches of the ANS. In time-domain measures, the short-term component of HRV (root mean square of successive differences [RMSSD]) is believed to be an indicator of the cardiac parasympathetic modulation of the heart (Task Force of the European Society of Cardiology the North American Society of Pacing Electrophysiology 1996).

Here, we build on advances in HRV interpretation to evaluate parasympathetic regulation of the heart during hibernation. The goal of this study was to identify parasympathetic involvement throughout different phases of a hibernation bout by using the Arctic ground squirrel (AGS; Urocitellus parryii) as the animal model.

Methods

Animal and Housing Conditions

Four adults (652 ± 61 g) and seven juveniles (448 ± 50 g) were used in this study. Animals were captured in early July near the Toolik Field Station, Alaska (68°38′N, 149°36′W), under permit from the Alaska Department of Fish and Game, and they were kept at the University of Alaska Fairbanks animal facility. All procedures were approved by the University of Alaska Fairbanks Institutional Animal Care and Use Committee in compliance with the Animal Welfare Act and in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.

After capture, all animals were held in a warm room (20°C, 16L∶8D cycle) until the insertion of surgical implants. After surgery, animals were returned to the warm room for postoperative recovery (∼10 d). When animals recovered (∼September), they were transferred into an isolated cold room with a winter-month setting (2°C, 4L∶20D cycle). Food (Mazuri laboratory chow, formula 5663) was removed after squirrels began to hibernate and throughout torpor and arousal to enhance the total time in hibernation. This was done considering that free-ranging AGSs do not eat during the hibernation season and that the presence of food shortens hibernation bouts. Water was freely available in the form of a gel-texture compound.

Surgery

Animals were implanted with electrocardiogram (ECG)/temperature telemetry transmitters (CTA-F40, 4.2 mL, 8 g; Data Sciences International, St. Paul, MN) in early to mid-August. All animals received antibiotics (sc. Baytril, 8.88 mg/kg) 12 h before surgery and an analgesic (sc. sustained-release buprenorphine, 1 mg/kg) immediately before the surgical procedure. Animals were anesthetized with isoflurane via a nose cone (5% in oxygen for induction, 2%–3% in oxygen for maintenance). The depth of anesthesia was maintained at such a level that there was no response to a toe pinch or any other surgical manipulation. The abdominal cavity was opened with a 2-cm ventral midline abdominal incision for the placement of the transmitter. The biopotential leads were tunneled subcutaneously and placed in a modified lead II position. A 1-cm right pectoral muscle area incision was made to secure the negative ECG lead, and a 1-cm left caudal rib region incision (approximately 2 cm to the left of the xiphoid process) was made to secure the positive ECG lead. After the procedure, animals received additional antibiotics (sc. Baytril, 8.88 mg/kg) every 12 h for 3 d.

Physiological Data Acquisition and Preprocessing

Physiological data were collected for 5 mo from the beginning of the hibernation season until spring. $T_{\text{b}}$ and ECG signals were recorded for 10 min every 40 min with a sampling frequency of 4,000 Hz with Ponemah (Data Sciences International). Data from one midseason hibernation bout (i.e., a full bout recorded between the months of December and January) from each animal were analyzed. Figure 1 shows $T_{\text{b}}$ data from one AGS with multiple hibernation bouts over the winter months. The hibernation bout between December and January was used for data analysis for this animal. The four phases of hibernation are defined as the following: (1) entrance (E in fig. 1) is from a drop of 0.5°C in $T_{\text{b}}$ to when $T_{\text{b}}$ becomes stable and the changes in $T_{\text{b}}$ are smaller than 0.5°C, (2) torpor (T in fig. 1) is from the end of entrance to the beginning of arousal when HR begins rising, (3) arousal (A in fig. 1) is from the end of torpor to when the changes in $T_{\text{b}}$ are less than 0.5°C, and (4) interbout arousal (IBA; fig. 1) is from the end of arousal to the beginning of the next entrance phase.

Figure 1.

Graph showing body temperature $\left(T_{\text{b}}\right)$ modification during the hibernation season in Arctic ground squirrels (19–055) and the subdivision of one bout in the following different phases: entrance (E), torpor (T), arousal (A), and interbout arousal (IBA). Data were recorded from September 2019 to February 2020 by utilizing an electrocardiogram/temperature telemetry transmitter.

HRV Analysis

R waves were initially marked with Ponemah and then manually checked to correct any misplaced marks. Artifacts caused by shivering (during arousal) or other interferences were excluded from analysis. Standard time-domain short-term HRV (RMSSD) was calculated for each 10-min recorded period by using the following equation:

$$\text{RMSSD}=\sqrt{\frac{{\sum }_{i=1}^{n-1}{\left({\text{RR}}_i-{\text{RR}}_{i+1}\right)}^2}{n-1}},$$

where $n$ is the number of RRI terms.

RMSSD reflects beat-to-beat variation in HR and is primarily under cardiac parasympathetic regulation (Kleiger et al. 1992). To account for the effect of HR on HRV measures, RMSSD was normalized to the RRI, resulting in the following modified parameter: RMSSD/RRI.

Statistical Analysis

All data are expressed as mean ± SEM. Data analysis was conducted in SPSS version 25.0 (IBM, Armonk, NY) or SigmaPlot (Systat Software, San Jose, CA). A one-way repeated-measures ANOVA was used to compare HR, $T_{\text{b}}$, and RMSSD/RRI across time, followed by Bonferroni post hoc tests when appropriate. A t-test was used for comparing entrance time versus arousal time.

Results

AGSs $(n=11)$ housed in a temperature-controlled chamber (ambient temperature = 2°C) entered hibernation at varying times. The first AGS started cooling at the beginning of September, and the last AGS entered torpor around the beginning of November. For the torpor bout between December and January, six AGSs had complete recordings from all four phases of hibernation, and five AGSs had only partial recordings because of technical difficulties (issues with the computer’s recording schedule and problems with the ECG trace). The entrance phase was about seven times longer than the arousal phase (entrance vs. arousal: 28.9 ± 1.7 h vs. 3.9 ± 0.01 h; t-test, P < 0.01), and the animals spent over 90% of the time in torpor (∼20 d for each torpor bout; table 1).

Table 1:

Duration of each phase of the hibernation bouts analyzed

	Entrance	Torpor	Arousal (period of increasing $T_{\text{b}}$)	Interbout arousal
n	11	9	9	9
Time (h):
Median	30	453	4	10
Range	21–35	304–529	3–4	7–12

Note. $T_{\text{b}}$ = body temperature.

ECG varies dramatically between stages of torpor (fig. S1). Figure 2 shows data collected from the six AGSs with complete recordings. HR during different phases of hibernation showed several alterations, followed by alterations in $T_{\text{b}}$. The relationship between HR and $T_{\text{b}}$ in the AGSs followed the wellknown hysteresis loop, which is explained by a suppression of thermogenesis with a subsequent decrease in metabolic rate during entrance. In the entrance phase, HR significantly dropped before the onset of the decrease in $T_{\text{b}}$, and the decrease in HR was more gradual after the animals initiated the cooling (fig. 2; Geiser 2021, p. 122). During torpor, HR and $T_{\text{b}}$ remained stable at their minimums. Similar to the entrance phase, HR increased before the rise in $T_{\text{b}}$ during the arousal phase, showing an overshoot at the end of this phase. Since the arousal phase was the shortest phase (table 1), there were fewer data points during this phase.

Figure 2.

Hysteresis plots of heart rate (HR) versus body temperature $\left(T_{\text{b}}\right)$. Data are from six Arctic ground squirrels with complete recordings of all four phases of a hibernation bout. Arrows indicate the directions of entrance into and arousal from torpor. HR significantly decreased before the onset of the reduction in $T_{\text{b}}$, followed by a more gradual decrease in HR as $T_{\text{b}}$ started falling. HR increased drastically over about 4 h during the arousal phase. Differences in the amount of data points during each phase are due to the length of the different phases. IBA = interbout arousal.

Figure 3 shows group data of all measurements over time from all 11 AGSs. HR dropped from ∼150 bpm at the end of IBA to ∼121 bpm at the onset of entrance (defined as a drop of 0.5°C in $T_{\text{b}}$, time zero of entrance in fig. 3). RMSSD, normalized as RMSSD/RRI, increased fourfold during early entrance (from 0.2 ± 0.1 to 0.8 ± 0.2; Bonferroni, P < 0.05), peaking at the time when HR dropped by over 90% (a level that was not significant during torpor; Bonferroni, P > 0.05). At which time, $T_{\text{b}}$ dropped by ∼70% (to 12°C ± 1°C), but it was still significantly higher (Bonferroni, P < 0.05) than $T_{\text{b}}$ during torpor (2.1°C±0.1°C). The second part of the entrance phase was delineated by a drop in RMSSD/RRI while $T_{\text{b}}$ decreased to its torpor minimum level. These data suggest that the primary driving factor for the drop in HR was the activation of the cardiac parasympathetic system and that the reduced sympathetic input during the second part of entrance may have contributed to maintaining HR at a lower level as the parasympathetic withdrawal occurred.

Figure 3.

The parasympathetic nervous system remains active throughout the torpor bout until its withdrawal precedes the initial increase in heart rate (HR). Group data $(n=11)$ of $T_{\text{b}}$, HR, and root mean square of successive differences (RMSSD)/RR interval (RRI) over time during entrance into and arousal from deep torpor are shown. Time zero represents the onset of entrance and arousal. The first part of the entrance consisted of an increase in RMSSD/RRI while HR dropped. RMSSD started to return to the level observed during interbout arousal (IBA) after HR had reached a torpor minimum level. A slow increase in HR before arousal was accompanied by a further decrease in RMSSD/RRI to the lowest level observed within the hibernation bout.

The onset of arousal was defined as an increase of 0.5°C in $T_{\text{b}}$ (time zero of arousal in fig. 3). Similar to the entrance phase, HR increased before detectable changes in $T_{\text{b}}$ and reached 87 ± 10 bpm at the onset of $T_{\text{b}}$ increase. HR started to increase 2 h before the onset of arousal, with a concurrent decrease in RMSSD/RRI to the lowest level. HR reached its peak of overshoot at 303 ± 12 bpm when $T_{\text{b}}$ was still at 24°C ± 2°C. As $T_{\text{b}}$ reached the IBA phase level, HR continued to decrease, and RMSSD/RRI increased slowly over time. These data suggest that cardiac parasympathetic withdrawal may contribute to the initial increase in HR and that the later dramatic increase in HR during arousal was likely mediated by sympathetic activation.

To better illustrate the temporal relationships among HR, HRV, and $T_{\text{b}}$, we looked for evidence of hysteresis between these parameters. To generate these curves, we plotted HR, HRV, and $T_{\text{b}}$ that were measured at the same time points from data shown in figure 3 against each other, as shown in figure 4. The hysteresis between HR and $T_{\text{b}}$ (fig. 4) showed the same pattern as seen in figure 2, confirming that the hibernation process is tightly regulated and highly conserved. Similarly, curves for RMSSD/RRI versus $T_{\text{b}}$ also showed a hysteresis loop (fig. 4), indicating that the biological processes are different during entrance versus arousal. Specifically, a higher RMSSD/RRI during entrance suggests a heightened parasympathetic control, and the decrease during torpor suggests the return of parasympathetic input from the heightened state. A minimum RMSSD/RRI was reached during arousal, suggesting a central inhibition of parasympathetic input and a slow removal of such inhibition during IBA. The relationship between RMSSD/RRI and HR also showed a hysteresis loop (fig. 4), demonstrating that the changes in RMSSD/RRI are not a function of changes in HR. Rather, it suggests a highly controlled temporal pattern of parasympathetic regulation throughout a hibernation bout.

Figure 4.

Different biological processes regulate phases of torpor. Hysteresis plots of heart rate (HR) versus body temperature $\left(T_{\text{b}}\right)$, root mean square of successive differences (RMSSD)/RR interval (RRI) versus $T_{\text{b}}$, and RMSSD/RRI versus HR are shown. Data are shown as the mean of all 11 animals. Standard error bars are omitted for clarity. Similar to HR versus $T_{\text{b}}$, curves for RMSSD/RRI versus $T_{\text{b}}$ showed a hysteresis loop, meaning that the biological processes are different between the different phases.

Discussion

For the first time, we showed the temporal pattern of parasympathetic activity throughout a complete hibernation bout, using beat-to-beat variation in RRIs (RMSSD) as a measure of cardiac parasympathetic regulation. Our data showed that the drop in HR during entrance was associated with a fourfold increase in RMSSD, suggesting that activation of the parasympathetic system initiates and regulates the decrease in HR during the entrance phase. RMSSD started to return to the level observed during IBA after HR had reached a torpor minimum level, suggesting that the low HR was achieved by a simultaneous decrease in the activities of both arms of the ANS during the latter part of entrance, as $T_{\text{b}}$ continued to decline to its torpor minimum level. In addition to reducing energy consumption, the decrease in the ANS activity allows the animal to reach a low HR during torpor by taking advantage of a ${\text{Q}}_{10}$ effect on cardiac pacemaker cells. Interestingly, the initial slow increase in HR before the onset of arousal (defined by an increase in $T_{\text{b}}$) was accompanied by a reduce in RMSSD to the lowest level, suggesting that parasympathetic withdrawal initiates the rise in HR as the first step of arousal. The rapid rise in HR during arousal was likely mediated by sympathoexcitation, with almost no opposing parasympathetic input.

Intrinsic HR is set by the sinoatrial node pacemakers, and the variability of that cardiac rhythm is dually regulated by the cardioinhibitory parasympathetic and cardioexcitatory sympathetic branches of the ANS (Yaniv et al. 2014). Thus, measures of HRV have been used as indicators of the ANS. Many factors, including RSA, physical activity, baroreflex, chemoreflex, cardiopulmonary reflex, somatic sensory inputs, visceral sensory inputs, and mental state, contribute to variations in HR via changes in the ANS. At rest during euthermia, RSA is a major source of variations in HR and is believed to be mediated primarily via the parasympathetic limb of the ANS. It is best represented by either calculating percentage of change in HR during a respiration cycle or using frequency domain analysis of RRIs. The high-frequency band of the power spectrum corresponds to the respiratory rate, and the power at this frequency band is used as an indicator of parasympathetic activity. One limitation of the frequency-domain HRV is that the frequency range changes with changes in respiratory rate.

The parasympathetic effects on HR are immediate because of its direct action on repolarizing channels and its fast neurotransmitter degradation. Conversely, the sympathetic effects on HR take several seconds because of the involvement of second messenger signaling and the slower removal of neurotransmitters. Thus, in the time-domain HRV, beat-to-beat changes in HR (short-term variability, RMSSD) are believed to be an indicator of the cardiac parasympathetic modulation of heart periods, while overall and longer-term HRVs (e.g., SD of normal-to-normal RRIs [SDNN], SD of all 5-min normal-to-normal RRI means [SDANN]) are under both cardiac parasympathetic and cardiac sympathetic regulation (Ernst 2017; Shaffer and Ginsberg 2017). This is further supported by studies showing that blocking the cardiac parasympathetic modulation with atropine significantly decreased both short-term and long-term HRV, while blocking the cardiac sympathetic modulation had significant effects only on overall long-term HRV but not on short-term HRV (Task Force of the European Society of Cardiology the North American Society of Pacing Electrophysiology 1996; Chen et al. 2008).

Our observation of a heightened parasympathetic input in the first part of the entrance phase, as suggested by a fourfold increase in short-term HRV, is consistent with previous studies demonstrating that parasympathetic activity is the dominant regulator that modulates and slows HR during this phase (Lyman and O’Brien 1963; Milsom et al. 1999). For example, the ground-breaking study by Lyman and O’Brien (1963) showed that blocking parasympathetic influence with atropine during the entrance phase abolished skipped heartbeats and asystoles and increased overall HR in the thirteen-lined ground squirrel. Our data further showed that RMSSD/RRI started to decrease when HR reached its torpor minimum level (fig. 3), suggesting that parasympathetic activation decreased once HR reached a minimum. Since HR remained low as RMSSD/RRI started to decrease, the data suggest that there may be a concurrent decrease in sympathetic activity, resulting in an overall lower ANS activity that could contribute to lower energy consumption. Using SDANN as a proxy of sympathetic activity, Evans et al. (2016) showed a dramatic drop in this HRV measure when a brown bear entered a den. While the reduced SDANN suggests reduced sympathetic activity and hence metabolic suppression, it is also consistent with lower cardiac sympathetic input. With simultaneous reduction of parasympathetic and sympathetic influences on HR, the ${\text{Q}}_{10}$ effect on HR may become important in maintaining low HR during torpor. Assuming that an AGS’s ${\text{Q}}_{10}$ effect on HR is similar to that found in rats and rabbits (∼2; Yamagishi and Sano 1967; Bolter and Atkinson 1988) and that the same ${\text{Q}}_{10}$ applies to the whole hibernation temperature range, we would expect HR during torpor to be ~17 bpm. Thus, while the ANS is important in the precise control of HR during the entrance and arousal phases (as evidenced by changes in HR before changes in $T_{\text{b}}$), AGSs may take advantage of ${\text{Q}}_{10}$ in the final phase of entrance leading to torpor, providing an additional way to conserve energy during torpor.

As observed in AGSs in our study, a number of studies also suggest that cardiac parasympathetic regulation continues to function during torpor, despite the extremely low $T_{\text{b}}$. For example, using RSA (percentage of HR change over respiration cycles) as an indicator of parasympathetic regulation, these studies demonstrated that RSA persisted during shallow daily torpor in dunnarts (Zosky 2002) and during deep hibernation in ground squirrels (Harris and Milsom 1995) and black bears (Laske et al. 2010). The RSA was abolished with atropine (Zosky 2002) or a cervical vagal block with lidocaine (Harris and Milsom 1995), suggesting a functioning parasympathetic system during torpor.

A somewhat surprising finding in our study is the drop in RMSSD/RRI before the onset of arousal. This drop in RMSSD/RRI suggests that a further reduction in cardiac parasympathetic activity supports the initial increase in HR before the rise in $T_{\text{b}}$. Similarly, using frequency-domain HRV analysis in the thirteen-lined ground squirrel, MacCannell et al. (2018) showed that the high-frequency band power (an indicator of parasympathetic regulation) was lower at the arousal threshold (compared to the entrance phase), suggesting a reduced parasympathetic activity at the onset of arousal. These data suggest that parasympathetic withdrawal mediates the initial increase in HR and that the increase in $T_{\text{b}}$ is supported by sympathetic activation because RMSSD was at its lowest level during this phase. In brown bears, Evans et al. (2016) showed that the sympathetic proxy (SDANN) increased after the rise in $T_{\text{b}}$. These data also support the interpretation that late sympathetic activation serves to finalize the restoration of euthermic metabolism (Evans et al. 2016).

RMSSD reflects the parasympathetic activity seen in RSA as well as in direct cardiac parasympathetic activity. We interpret changes in RMSSD as an increase in cardiac parasympathetic activity rather than as a consequence of altered respiration for several reasons. First, looking at the RMSSD/RRI hysteresis curve (fig. 4) during entrance, we do not see a drop in RMSSD/RRI when $T_{\text{b}}$ drops. Assuming that HRV is dependent on breathing (RSA), we should expect a lower HRV when $T_{\text{b}}$ reaches 10°C, considering that a hibernating AGS’s respiratory rate is <6 respirations per minute (Ma et al. 2005). However, we did not see a drop in RMSSD/RRI as $T_{\text{b}}$ declined and indeed saw a transient peak in HRV at $T_{\text{b}}$’s near 10°C. These data lead us to believe that even though RSA is a major contributor to HRV in euthermic animals, other regulatory mechanisms modulate HRV during entrance. We reason that even if HRV and RSA are associated with each other, they are not linked one to one. Eliminating RSA does not eliminate HRV. However, we cannot rule out the influence of RSA on RMSSD.

The current study has limitations. First, because of our software license limitation, we were able to perform only intermittent recordings (10 min of ECG were recorded every 40 min) to allow for recordings from multiple animals over the entire hibernation season (5 mo). Thus, we were unable to obtain other meaningful HRV indexes such as SDNN, a parameter representing both sympathetic and parasympathetic (plus humoral) factors. Second, dramatic changes in RRIs per se could change RMSSD. We do not believe our results reflect the simple change in HR seen over a hibernation bout because (1) we normalized RMSSD to the averaged RRI and (2) the RMSSD/RRI–HR relationship over a hibernation bout showed an open hysteresis loop, suggesting that the changes in RMSSD/RRI are not merely a function of changes in HR.

In conclusion, we use HRV as an estimate of parasympathetic activation to show that the parasympathetic nervous system remains functional throughout a torpor bout. As expected, we show an increase in parasympathetic nervous system activation during entrance into hibernation. The initial increase in activation then subsides without an increase in HR, suggesting that the decline in parasympathetic activity is accompanied by a presumed concomitant decrease in sympathetic nervous system activation that then preserves bradycardia throughout prolonged torpor. Just before arousal, parasympathetic activation is withdrawn. Withdrawal of parasympathetic activation removes the brakes on sympathetic activation required to stimulate thermogenesis and to warm the animal from the torpid state. Thus, during hibernation, vagal input remains low, although still present, until it is briefly withdrawn as the first sign of impending arousal. During the IBA phase, the parasympathetic activity gradually increases again to prepare the AGS for the next successive bout. Hence, autonomic regulation of cardiac function is maintained throughout all phases of hibernation. Although the temporal resolution of parasympathetic activation provided by HRV analysis is interpreted with the caveat that it has not been confirmed by pharmacological manipulation, the insights gained will inform further study of hibernation and efforts to produce synthetic torpor in species that do not normally hibernate.