Cardiac Adaptation and Cardioprotection Against Arrhythmias and Ischemia-Reperfusion Injury in Mammalian Hibernators

Abstract

Hibernation allows animals to enter an energy conserving state to survive severe drops in external temperatures and a shortage of food. It has been observed that the heart of mammalian hibernators exhibits intrinsic protection against ischemia-reperfusion (I/R) injury and cardiac arrhythmias in the winter whether they are hibernating or not. However, the molecular and ionic mechanisms for cardioprotection in mammalian hibernators remains elusive. Recent studies in woodchucks (Marmota Monax), the largest true hibernator, have suggested that cardiac adaptation occurs at different levels and mediates an intrinsic cardioprotection prior to/in the winter. The molecular/cellular remodeling in the winter (with or without hibernation) includes: 1) an upregulation of transcriptional factor, anti-apoptotic factor, nitric oxide synthase, protein kinase C-ε, and phosphatidylinositol-4,5-bisphosphate 3-kinase, 2) an upregulation of antioxidant enzymes (e.g. superoxide dismutase and catalase), 3) a reduction in the oxidation level of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and 4) alterations in the expression and activity of multiple ion channels/transporters. Therefore, the cardioprotection against I/R injury in the winter is most likely mediated by enhancement in signaling pathways that are shared by preconditioning, reduced cell apoptosis, and increased detoxification of reactive oxygen species (ROS). The resistance to cardiac arrhythmias and sudden cardiac death in the winter is closely associated with an upregulation of the antioxidant catalase and a downregulation of CaMKII activation. This remodeling of the heart is associated with a reduction in the incidence of afterdepolarizations and triggered activities. In this short review article, we will discuss the seasonal changes in gene and protein expression profiles as well as alterations in the function of key proteins that are associated with the occurrence of cardioprotection against myocardial damage from ischemic events and fatal arrhythmias in a mammalian hibernator. Understanding the intrinsic cardiac adaptive mechanisms that confer cardioprotection in hibernators may offer new strategies to protect non-hibernating animals, especially humans, from I/R injury and ischemia induced fatal cardiac arrhythmias.

1. Introduction-hibernation phenotype and cardiac protection

Hibernation is a seasonal physiological adaptation to periods of food shortage, low environmental temperature, and shortened daylight hours (detailed in review articles [11,18,2,3]). Hibernation allows animals to therefore enter an energy conserving state by eliminating the need to maintain a high body temperature and metabolic rate.

Throughout the winter (hibernation season), hibernators essentially undergo a series of long torpor bouts that are interrupted by brief euthermic interbout arousal periods. During torpor, hibernators enter a condition of low body temperature (as low as 2-10 ºC), suppressed metabolism (2-3% of the aroused condition), and extreme reductions in heart rate (3-10 beats/min). A growing body of evidence has suggested that the torpor-like state can be induced in non-hibernating animals by a single molecule or a combination of molecules [3,64]. Among the molecules thought to induce a torpor-like state are adenosine and 5-adenosine monophosphate (5′-AMP) both of which have attracted much attention. Adenosine is an inhibitory neuromodulator that is widely distributed in the central nervous system including the brainstem. Recent studies have reported that administration of the A1 receptor agonist cyclohexyladenosine into the lateral ventricle of the arctic ground squirrel induces hibernation which is similar to natural spontaneous hibernation [29,47]. In addition, 5′-AMP has also been shown to induce a torpor-like state in Syrian hamsters [19].

Natural hibernators undergo physiological and behavioral changes in order to survive seasonal limitations in nutrition and other environmental stressors such as low environmental temperatures. It is well known that severe hypothermia can lead to cardiac arrhythmias including ventricular tachycardia (VT) and ventricular fibrillation (VF) which can result in sudden cardiac death (SCD) in non-hibernators including humans [49,48]. However, numerous studies including ours have shown that the hearts of hibernating animals are resistant to cardiac arrhythmias induced by many interventions such as hypothermia, intracellular Ca²⁺ overload, and ischemia [30,31,67]. A recent study from our laboratory [67] has demonstrated that woodchucks which are true hibernators exhibit intrinsic cardioprotection against coronary artery occlusion (CAO)-induced arrhythmias and SCD in the winter. In addition, Yan et al. [62] have also reported that the hearts of woodchucks are highly protected against ischemia-reperfusion (I/R) injury in the winter whether they are hibernating or not. It seems likely that hibernating mammals undergo intrinsic seasonal adaptation processes (i.e. molecular and physiological changes) that allow them to prepare for winter hibernation and provide them with an ability to be protected against cardiac injury and arrhythmias. Hibernating animals may therefore serve as a powerful and instructive model for cardiovascular research on cardioprotection against I/R injury and sudden cardiac death.

In this article, we review recent progress in studies on seasonal remodeling of gene and protein expression, as well as alterations in the electrophysiological and Ca²⁺ handling properties that are associated with cardioprotection in mammalian hibernators.

2. Seasonal alterations in gene expression and protein synthesis in the heart

It is conceivable that the hibernation phenotype is regulated at the gene (genomic), protein expression and post translation modification levels. Multiple approaches such as genomics, trancriptomics, and proteomics have been used to investigate the molecular basis of hibernation [3]. Our present article focuses on seasonal changes in the pattern of gene and protein expression in the heart that are relevant to cardiac protection.

2.1. Molecules facilitating a metabolic switch from carbohydrate to lipid fuel

Upon entering into torpor, a switch from a carbohydrate-based metabolism to a lipid-based one occurs in hibernating animals so that stored fat serves as the primary source of ATP production [11]. Glucose oxidation rates are significantly lower in the hibernating heart compared to the non-hibernating heart [11,3,7]. During hibernation in the winter, an upregulation of pyruvate dehydrogenase kinase 4 (PDK4) expression has been detected in the heart of the thirteen-lined ground squirrel at both the mRNA and protein level [26,10,9]. PDK4 is a mitochondrial enzyme that blocks the conversion of pyruvate to Acetyl-CoA by pyruvate dehydrogenase (PDH). Therefore, upregulation of PDK4 reduces the supply of glycolytic intermediates entering the tricarboxylic acid (TCA) cycle in mitochondria making lipid hydrolysis the primary source of ATP synthesis. A recent study by Li et al. have analyzed cardiac proteomic changes by using isobaric tags for a relative and absolute quantification (iTRAQ) approach [36]. They have found that the lipid metabolic enzymes, acyl-coenzyme A synthetase medium-chain family member 5 (ACSM5) and mitochondrial acyl-coenzyme A thioesterase 9 (ACOT9) are upregulated during torpor in woodchucks compared to non-hibernating woodchucks in the summer. The upregulation of all of these molecules facilitate a metabolic switch from carbohydrate to lipid fuel. More detailed information can be found in the literature addressing this issue in relation to hibernation [11,3,7].

2.2. Ion channels and Ca²⁺ handling proteins

An earlier study by Yatani et al. has examined the changes of Ca²⁺ handling proteins during hibernation in woodchuck (Marmota monax) hearts [63]. The expression level of sarcoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a) has been found to be upregulated by 3-fold while phospholamban (PLB) has been reported to be downregulated by approximately 50% in hearts of hibernating (HB) woodchucks in the winter compared with non-hibernating (NHB) control woodchucks in the spring (March-April). The same pattern of change occurs at both the mRNA (using RT-PCR) and protein expression (using Western blot) levels. Thus, the ratio of SERCA2a to PLB is significantly increased during hibernation suggesting a higher activity of the SERCA 2a pump. The upregulation of SERCA2a and downregulation of PLB has also been confirmed at the gene level by Brauch et al. in the heart of hibernating ground squirrels [9]. The protein expression level of the L-type Ca²⁺ channel (LTCC) α_1C-subunit is also reduced (by 35 ± 8%) in hearts from hibernating woodchucks [63], while there are no changes in calsequestrin (CSQ) or Na/Ca exchanger 1 (NCX1) expression levels. In contrast, in hibernating thirteen-lined ground squirrels, Hampton et al. have reported an increase in the mRNA level of NCX1 [26]. Meanwhile, the mRNA levels of the ryanodine receptor 2 (RyR2) and Na/K-transporting ATPase subunit alpha-1 (AT1A1) are upregulated during hibernation in hibernating thirteen-lined ground squirrels [26]. These genes control ion transporters that are required for enhanced cardiac contraction and relaxation at low body temperatures. In the Siberian ground squirrel, upregulation of both Cx43 and Cx45 occurs during hibernation which may help maintain the regular cardiac electrical conduction pattern [23].

2.3. Antioxidant system

Besides the adaptive alternations in ion channels and Ca²⁺ handling proteins, changes in other molecules and signal transduction pathways during hibernation have also been reported. Specifically, since hibernators are exposed to oxidative stress during torpor-arousal cycles they appear to have adapted by developing a higher antioxidant system in the heart. For example, the following findings have been reported: 1) upregulation of the antioxidant catalase in woodchuck hearts during torpor [36,62] or without hibernation [67] in the winter. Higher levels of catalase have also been detected in the serum of Syrian hamsters during arousal [45], 2) increased expression/activity of superoxide dismutase (SOD) in the heart of woodchucks in the winter at cold environmental temperatures [62] and increased expression/activity of superoxide dismutase (SOD) in the heart of 13-lined ground squirrels [39] have also been reported and 3) upregulation of peroxiredoxins (Prx1-3) [40] and Heme oxygenase-1 (HO-1) [39] have been reported in hibernating ground squirrels.

2.4. Transcriptional factors

A iTRAQ analysis by Li et al. [36] has also detected the upregulation of two transcriptional factors, i.e. cAMP-response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT), in winter woodchucks in deep torpor compared to non-hibernating woodchucks in the summer. Further study by Yan et al. has revealed that the activity of CREB (p-CREB Ser133 level) is increased in hearts from hibernating woodchucks in the winter compared to hearts from woodchucks in the summer[62].

CREB is involved in multiple signaling pathways and plays a critical role in cell survival, proliferation, differentiation, adaptation, and apoptosis. CREB can be activated by several Ca²⁺ sensitive kinases such as CaMK, PKA, PKCs, Ras/Raf/MAPK, Akt/GSK3β, and p70S6K etc. through phosphorylation of CREB at Ser133 (p-CREB ser133) [15]. Among a diverse array of CREB-regulated genes are anti-apoptotic factors such as B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl) proteins which are upregulated in the heart of woodchucks in the winter. These findings suggest a potential cardioprotective mechanism mediated by reducing apoptosis via Bcl-2 and Bcl-xl upregulation [62]. In addition, CREB has also been shown to be a direct regulator that stimulates the expression of antioxidant genes including HO-1 [34], MnSOD (SOD2)[32], and catalase [55]. For example, phosphorylation of CREB at Ser133 by Akt promotes the detoxification of reactive oxygen species (ROS) by catalase, therefore protecting mitochondrial function under oxidative stress [55].

Morin et al. [39] have demonstrated regulation in the heart of hibernating 13-lined ground squirrels at both the mRNA and protein levels of nuclear factor erythroid 2–related factor 2 (Nrf2) transcription factor. Through binding to the antioxidant response element (ARE) in targeted gene promoters, Nrf2 promotes transcriptional activation of antioxidant genes such as catalase, SOD, Prx, and HO-1. Therefore, upregulation of Nrf2 might play an important role in cardioprotection against oxidative stress [17,12]. The protein levels of Nrf2 and its targets such as SOD and HO-1 are upregulated during hibernation following the same pattern as the torpor-arousal cycle. These data suggest an essential role for Nrf2 in the regulation of antioxidant defenses (i.e. ROS detoxification) during hibernation [39].

Both CREB and Nrf2 promote the expression of peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1α) which is a master regulator of antioxidant enzyme expression including Cu-ZnSOD (SOD1), SOD2, and glutathione peroxidase 1 (Gpx1) [27,54,4,46]. Taken together, elevated CREB and Nrf2 levels in woodchuck hearts in the winter may also play a protective role via upregulation of antioxidant genes and thereby detoxification of ROS. On the other hand, the transcriptional factor NFAT promotes the expression of a diverse array of ionic channels or transporters including SERCA [8].

2.5. Other molecules potentially relevant to cardioprotection

There are also changes in other genes and/or proteins in hibernating mammals. For instance, Li et al. have also revealed that the NO signaling, α-adrenergic, and protein kinase A signaling pathways are upregulated while the ER stress regulator 78 kDa glucose-regulated protein (GRP78) is downregulated in the heart of hibernating woodchucks [36]. General NOS activity and eNOS protein at both the total and phosphorylated levels are elevated in woodchuck myocardium in the winter, while there is no alteration in iNOS. Another study using a digital gene expression assay has revealed that the mRNA level of calmodulin (CaM), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and cytochrome c oxidase are downregulated in the thirteen-lined ground squirrel during hibernation [9].

Substantially decreased metabolic rates and protein synthesis rates (up to 3-fold lower) have been found in the liver, brain and heart tissue of thirteen-lined ground squirrels and arctic ground squirrels during hibernation (during both torpor and aroused sates) compared to the non-hibernation season [24]. Therefore, it is our thought that the molecular remodeling especially involving protein overexpression would likely start prior to the hibernation season. Detailed information regarding the exact phase(s) and trigger point(s) of genomic and proteomic changes are lacking, although tissue samples have been taken from different seasonal points in the torpor-arousal cycle in recent deep sequencing and proteomic studies [36,56].

3. Seasonal remodeling in cardiac electrophysiology and intracellular Ca²⁺ handling in mammalian hibernators

Cardiac electrophysiology and Ca²⁺ handling play an important role in the maintenance of heart function during hibernation. It has been identified that there are remarkable adaptive changes in the electrophysiological and Ca²⁺ handling properties in hibernating animals at the cellular level.

Unlike skeletal muscle, the heart of a hibernator must continue to contract even during torpor. During hibernation, the myocardium becomes hypertrophic to normalize wall stress in order to overcome increased vascular resistance most likely due to the increased viscosity of the blood and reduced vascular elasticity. The heart rate of a hibernator decreases dramatically (to 3-10 beats/min) during hibernation. As shown in ECG recordings from ground squirrels [14,41], the duration of the TP segment is prolonged by 40-70 times indicating suppression of SA node automaticity during hibernation. Meanwhile, the QRS complex duration and the P-R interval are both significantly increased in duration indicating a reduced electrical conduction.

3.1. Remodeling in the action potential and ionic currents

Yatani et al. [63] have made a systematic comparison between hibernating (HB) woodchucks and non-hibernating (NHB) control woodchucks. They reported : i) a shortened action potential duration (APD₅₀ and APD₉₀) in HB woodchuck cardiomyocytes vs. in NHB woodchuck cardiomyocytes; ii) decreased current density and faster inactivation of LTCC in HB woodchuck cardiomyocytes compared to NHB woodchuck cardiomyocytes consistent with the protein expression level of the 1C subunit of LTCC (see section 2.2). iii) no alterations in the density of the transient outward K⁺ current (I_to), inward rectifier K⁺ current (I_K1), or Na⁺/Ca²⁺ exchange current (I_NCX). Note the cardiomyocyte electrophysiology experiments were carried out at 22±2°C in the Yatani et al. study.

Lack of an action potential plateau and decreased LTCC current density has also been observed in hibernating ground squirrels [1,58]. Since the action potential morphology and duration are determined by the balance of depolarizing inward currents and repolarizing outward currents, the reduction of APD and the absence of the action potential plateau are likely due to reduced LTCC during hibernation.

3.2. Remodeling of intracellular Ca²⁺ handling and homeostasis

Intracellular Ca²⁺ homeostasis is maintained by a dynamic balance between Ca²⁺ entry and exclusion through the sarcolemma and between Ca²⁺ release from and re-uptake into organelles such as the sarcoplasmic reticulum (SR) and mitochondria. Numerous studies have observed a reduction in the LTCC current in cardiomyocytes from different hibernating mammals including woodchucks and the long-tailed ground squirrel (Citellus undulatus) [1,58,63]. While the decrease in LTCC current helps to prevent excessive Ca²⁺ entry across the cardiac cell sarcolemma, it also reduces the strength of the trigger for Ca²⁺-induced Ca²⁺ release (CICR). However, the Ca²⁺ transient amplitude is unchanged which can be explained by an enhanced gain in CICR. Up-regulation of the SERCA2a/PLB expression ratio and increased SERCA2a pump activity can increase SR Ca²⁺ uptake capacity thus increasing SR Ca²⁺ content [63]. In addition, the upregulation of RyR2 expression/activity further facilitates Ca²⁺ release from the SR. Therefore, an adaptation with increased stored Ca²⁺ in SR and an increased RyR Ca²⁺ release ability ensures a comparable [63] or even stronger contraction [58] in cardiomyocytes of hibernating animals.

It is important to point out that enhanced SERCA2a pump activity is able to sequester Ca²⁺ and maintain diastolic intracellular Ca²⁺ levels by re-uptaking Ca²⁺ into the SR Ca²⁺ store thereby maintaining a stable resting or diastolic level of intracellular Ca²⁺. This notion is supported by the observation of a lower incidence of Ca²⁺ sparks in woodchuck cardiomyocytes in the winter compared to the summer (Fig. 1) [61]. This finding also implies that the SR has less spontaneous leakage of Ca²⁺ in the winter. These adaptations in Ca²⁺ handling may mediate the mechanism(s) for protection against Ca²⁺ overload-induced injury and arrhythmias, since a reduced intracellular Ca²⁺ level (especially during diastole) reduces Na⁺- Ca²⁺ exchanger activity and thereby preventing the generation of early and delayed afterdepolarizations (EADs and DADs) and triggered activity.

Fig. 1. Incidence of Ca²⁺ sparks in ventricular myocytes from woodchucks in the winter vs. in the summer.

A: Representative line-scan images showing Ca²⁺ spark occurrence in ventricular myocytes isolated from woodchucks in the winter and in the summer at room temperature (~ 22°C). B: Summary of the frequency of Ca²⁺ sparks. The myocytes have fewer Ca²⁺ sparks in the winter than in the summer. ** p < 0.05 with unpaired Student’s t-test.

4. Protection against cardiac I/R injuries in woodchucks in the winter

An earlier study has reported that after monitoring creatine kinase leakage, the hearts of ground squirrels show significantly less I/R injury than rat hearts [25]. In agreement with this observation, a recent study by Yan et al. has assessed cardioprotection against I/R injury in woodchucks, a true hibernator [62]. While the ischemic areas at risk were similar among groups, myocardial infarction sizes were significantly smaller in woodchucks in the winter (whether hibernating or not) compared with those in the summer. These data indicate that woodchucks exhibit myocardial ischemic protection during the winter compared to woodchucks in the summer similar to what is elicited by ischemic preconditioning.

The underlying mechanisms for cardioprotection appear to involve multiple pathways such as endothelial nitric oxide synthase (eNOS) and NO synthesis, protein kinase C-ε (PKCε), and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) all of which are upregulated in the winter. The cardioprotective signal transduction pathways have been discussed in greater detail elsewhere [28]. Of interest is the increased expression of CREB and downstream gene targets including antioxidant enzymes (e.g. SOD, catalase, HO-1, Prx) and anti-apoptotic factors (e.g. Bcl-2, Bcl-xl ) in woodchuck hearts in the winter (with or without hibernation) [62] that has been reported. It has been well accepted that activated CREB increases the expression of Bcl-2 which inhibits caspase-3 activation and thus attenuating cardiomyocyte apoptosis. Therefore, the cardioprotection generated by woodchucks in the winter is most likely associated with a reduction in apoptosis by promoting Bcl-2 and Bcl-xl and increased detoxification of ROS. When CREB was overexpressed artificially via adenoviral CREB administration in the summer woodchuck heart, it induced cardioprotection similar to that observed in the woodchuck heart in the winter, supporting the postulation that CREB-activated transcriptional regulation is critical for cardioprotection in woodchucks in the winter.

5. Protection against cardiac arrhythmias in woodchucks in the winter

Sudden cardiac death (SCD) accounts for more than 300,000 deaths annually in the United States [51]. Most SCDs are related to severe arrhythmias such as VT and VF which often occur after myocardial ischemia [69]. While prevention/treatment of ischemia-induced arrhythmias and SCD remains a huge clinical challenge, the mechanisms for ischemia-induced arrhythmias and SCD are not well understood. Previous studies have observed that hearts of mammalian hibernators are resistant to cardiac arrhythmias induced by hypothermia and other proarrhythmic factors such as arrhythmia-inducing agents, Ca²⁺ overload, or electrical stimulation [31,30]. Although adaptive alternations in membrane ion channels, Ca²⁺ handling, connexin expression, and signaling molecules have been reported in the hearts of hibernating animals, the direct mechanistic link between adaptation of cellular and ionic properties and antiarrhythmic protection in hibernators is still unclear.

A recent study from our group has determined the underlying molecular and cellular mechanisms involved in the resistance against ischemia-induced arrhythmias in woodchucks [67]. We have demonstrated a seasonal difference in the susceptibility to arrhythmias after coronary artery occlusion (CAO) in woodchucks in the winter (Dec-Jan) vs. in the summer (June-July). As revealed by telemetric ECG, there was a significantly higher arrhythmia score in the incidence of VTs and VFs in woodchucks in the summer compared to the occurrence in the winter. Ventricular cardiomyocytes isolated from woodchucks in the winter were more resistant to H₂O₂-induced early afterdepolarizations (EADs) compared to cardiomyocytes isolated from woodchucks in the summer. The electrophysiology experiments in cardiomyocytes were carried out at 34-36°C in this study.

ROS including superoxide (O₂^·−), hydroxyl radical (HO^·), and hydrogen peroxide (H₂O₂) are derivatives of oxygen characterized by their high reactivity [44]. Under normal conditions, the myocardium reduces 95% of the oxygen to water via the mitochondrial electron transport chain leaving the remaining 5% to form ROS. However, under various pathological conditions (e.g. aging, heart failure, and I/R), ROS levels can become elevated and may predispose the heart to arrhythmias [35,52,43,50]. It has been reported that the generation of ROS is acutely potentiated by a shift to anaerobic metabolism during ischemia or ischemia/reperfusion [33,38,6]. Pathological concentrations of H₂O₂ causes early afterdepolarization (EAD), delayed afterdepolarization (DAD), and triggered activity (TA), and has been suggested as a potential proarrhythmic factor in rats, guinea pigs, and rabbits [59,53,60]. We have demonstrated that H₂O₂ induces EAD, DAD and TA via activation of CaMKII in rabbit ventricular myocytes [60]. CaMKII is a serine/threonine kinase that is ubiquitously expressed in various tissues including the heart. Autophosphorylation of threonine 286 in the presence of Ca²⁺ and CaM activation of CaMKII play essential roles in cardiac physiologic function [13,37]. CaMKII can also be activated by oxidation at M281/282 by ROS (e.g. via H₂O₂) [20]. Numerous studies including ours have demonstrated that excessive oxidation of CaMKII by ROS is associated with the generation of EADs, DADs and arrhythmias [60,65,66,68]. The potential mechanism(s) may be CaMKII-mediated activation of I_Ca,L [60] and/or activation of late I_Na [57].

We have further found that catalase expression in woodchuck hearts is significantly higher in the winter than in the summer. Catalase is widely expressed in the cytoplasm and efficiently detoxifies H₂O₂ into O₂ and H₂O. Consistent with the higher levels of catalase expression in the winter, the level of oxidized CaMKII (Ox-CaMKII) but not P-CaMKII is significantly lower in woodchuck hearts in the winter. The arrhythmia scores in woodchuck hearts in the summer are significantly reduced by overexpressing catalase (via adenoviral vectors) or inhibiting CaMKII activity (with KN-93). It is therefore postulated that natural adaptation by increasing antioxidative capacity (catalase expression) and reducing CaMKII activity can switch woodchucks from a “summer mode” to a “winter mode”, which confers a higher resistance against ROS-induced EADs and lethal arrhythmias during cardiac ischemia (Fig. 2). As discussed in section 3.1 and 3.2, the adaptations in action potential duration (shortening) and Ca²⁺ handling (reduced incidence of Ca²⁺ sparks) may also account for protection against ROS and Ca²⁺ overload-induced arrhythmias in the winter. Therefore, creation of a “winter mode” genotype resembling that in hibernating species might likely prove to be beneficial in preventing arrhythmias in patients suffering from myocardial ischemia (i.e. heart attacks). The profound protection conferred by catalase overexpression and/or CaMKII inhibition in this novel natural animal model may provide insights into new clinical directions for therapy for arrhythmias and SCD.

Figure 2. Mechanism for the resistance to ischemia-induced arrhythmias and sudden cardiac death (SCD) in woodchucks in the winter.

In “summer mode”, cardiac ischemia increases the level of ROS which oxidizes CaMKII and activates multiple ion channels thereby inducing EAD, DAD and triggered activity. Severe arrhythmias such as VF lead to SCD at a high rate (42%) in the summer. In “winter mode”, the woodchuck heart undergoes adaptation to reduce the level of ROS by upregulation of catalase. As a result, the CaMKII activity and ion channel activities are reduced so that woodchucks are more resistant to coronary artery occlusion-induced arrhythmia and SCD.

6. Conclusions and future research directions

Hibernating mammals represent a novel model for studying cardioprotection. Woodchucks (Marmota Monax) are the biggest true hibernating mammal thereby providing us with the opportunity to determine the molecular and cellular mechanism(s) mediating a reported resistance to cardiac I/R injury and arrhythmias. Recent studies in woodchucks by Lin et al. [62] and Zhao et al. [67] have revealed underlying mechanisms for intrinsic cardioprotection at different levels including transcriptional factors (e.g. CREB), anti-apoptotic factors (e.g. Bcl-1), antioxidant enzymes (e.g. catalase), protein kinases (e.g. CaMKII), and ion channels (Fig. 2 and Fig 3). Understanding the intrinsic cardiac adaptive mechanisms that confer cardioprotection in hibernators may suggest new strategies to protect non-hibernating mammals, especially humans from I/R injury and fatal cardiac arrhythmias induced by ischemia.

Figure 3. Schematic presentation of adaptation accounting for cardioprotection in woodchucks in the winter.

See main text for details.

The protection against I/R injury in woodchuck hearts in the winter is also likely mediated by a reduction in cell apoptosis (upregulation of anti-apoptotic factor Bcl-1) [62]. Ferroptosis has been recently revealed as a new form of non-apoptotic cell death caused by the accumulation of Fe-dependent lipid peroxidation [16]. The study of ferroptosis in the heart is only just emerging [5,22,21] and ferroptosis has been shown to be associated with cardiotoxicity induced by anti-cancer drugs and I/R injury. It will be important to determine whether there are novel anti-ferroptotic pathways that may explain the observed cardioprotection in woodchuck hearts. Future studies are warranted.

Besides small rodent hibernators such as ground squirrels and woodchucks, bears (e.g. genus Ursus) also hibernate for 4–6 months during the winter season without taking in any nutrition (see review article by Nelson and Robbins for comparisons between large and small hibernators [42]). However, their body temperatures only decline from approximately 37° to 33 °C while the mean heart rates slow to approximately 25 % of the active heart rate during the summer. While it has been shown that the QT intervals were shortened on ECG recordings in hibernating bears during the winter, the adaptive mechanisms of the electrophysiology and Ca²⁺ handling in bears are not well understood. Since bears likely demonstrate unique refined adaption with less hypothermia during hibernation, which may be more relevant to humans, it would be interesting to further investigate potential cardioprotection and underlying adaptation at the molecular/ionic cellular level in these large hibernators.