In the present intact rat study, we find that despite the presence of significant changes in cardiac electrophysiology induced by severe hypothermia and rewarming, no ventricular arrhythmias took place. All electrophysiological parameters were however normalised after rewarming. As predicted by the QRS/QTc ratio values, which remained higher than at normothermia throughout the protocol, we found that neither cooling, prolonged hypothermia, nor rewarming created substrate(s) for lethal ventricular arrhythmias in our rat model.
In humans, hypothermia-induced arrhythmias are common. Accordingly, it is interesting to assess risk for VF, considering the electrophysiological differential effects of hypothermia between humans and rats. The hypothermia-induced electrophysiological changes, in the presence of intact sinus rhythm, as observed in the present experiment, therefore gives valuable information of potential translational value. Background pathophysiologic mechanisms for hypothermia-induced VF and cardiac arrest in patients has been largely unknown. However, recent efforts with translational research into this topic have given possible groundbreaking results . In rabbit, both increased ventricular divergence  and heterogenic effect of hypothermia on transmural and longitudinal conduction  has been documented at 30 °C, with increased risk for developing VF. Cooling to 30 °C also enhanced epicardial APD dispersion, wavebreaks and re-entry, associated with increased vulnerability to pacing- induced VF . In pigs cooled to 30 °C, VF threshold is reduced by 72% . This is also found in dogs at 25 °C, where VF threshold was reduced compared to at 37 °C . Cardiac vulnerability, however, does seem to be promoted further by rewarming. In canine wedge preparations cooled to 26 °C, VF and VT occurred more frequently during rewarming than during cooling . In a similar model, hypothermia (32 °C) caused local re-excitation and development of polymorphic VT/VF . In two recent publications, we reported that the QRS/QTc ratio could be used as a highly predictable marker of VF threshold in rabbits [6, 7].
Ventricular ectopic activity is documented being increased in patients treated with therapeutic hypothermia , in addition to the occurrence of frequent non-sustained VT . However, most studies report that sustained ventricular arrhythmias are uncommon , with some exceptions as by Mirzoyev et al. who documented polymorphic VT in 11.7% of therapeutic hypothermia patients. Most cases with VT occurred at a core temperature around 34.7 °C, and defibrillation was necessary in most of these patients. Patients with polymorphic VT were hypokalaemic and had significantly prolonged QTc interval . Risk for VF is dependent of severity of hypothermia and pose a big challenge during rewarming. Of 19 accidental hypothermia patients admitted, with core temperatures between 17 °C – 29 °C, seven were in VF, while two presented with asystole . In a Japanese study of 60 patients, no patients with a core temperature above 26 °C developed VF . Recently we published a report indicating that the QRS/QTc interval could be used as a biomarker of risk for hypothermia-induced VF in such patients .
In the present study, we show that rats are largely resistant to ventricular arrhythmias induced by hypothermia. Still, rats are vulnerable to pacing- induced arrhythmias and several studies of normothermic cardiopulmonary resuscitation are carried out in well-established rat models of VF [22, 23]. The relative different electrophysiological effects when cooling rats, compared to hypothermic patients and animal species that have similar electrophysiology to humans, are therefore of interest. In the present study on rats, the QRS/QTc ratio increased steadily during cooling, remained stable during hypothermia, and was normalised during rewarming. Interestingly, when assessing human ECG-data (Fig. 4) , QRS/QTc values were similar to those in rats at normothermia and at severe hypothermia (< 24 °C). At these temperatures the QRS/QTc ratios in humans were found to be higher than 0.2, while initial cooling is associated with a reduction of QRS/QTc values during moderate and higher temperatures of severe hypothermia (32 − 24 °C). Exposure to such core temperatures is highly associated with occurance of ventricular arrhythmias in hypothermic patients . This is similar to the relation between temperature and the QRS/QTc ratio values in rabbit hearts, where low QRS/QTc values observed during moderate hypothermia correlated highly with increased VF-risk (VF threshold). In contrast, QRS/QTc values in rat never fell below 0.2 and no ventricular arrhythmias were recorded during the present experiments.
In a recent study, we found that moderate hypothermia presents a “vulnerable window” for hypothermia-induced VF and cardiac arrest in rabbit hearts. This pro-arrhythmic state was related to slowed cardiac conduction and repolarisation, whilst ventricular/ transmural activation remained relatively unaffected, producing an acquired long-QT syndrome . As transmural conduction is gap-junction dependent, we tested the gap-junction uncoupler Heptanol as an anti-arrhythmic measure. After administration, risk for VF was normalised and equal to in normothermic hearts .
Hypothermia-induced QT-prolongation is however not species dependent  or directly associated with increased risk for VF, as shown in the present rat model, where animals were resistant to hypothermia-induced ventricular arrhythmia despite a five-fold increase in QT-time after cooling to 15 °C. An explanation for this species-dependent difference could be the relative transmural distance across the ventricular wall, compared to cardiomyocyte size. Despite small heart size and short transmural distance in rats, compared to both humans and rabbits, cardiomyocyte size and volume is equal . Thus, in rats, transmural conduction is less dependent on gap junction activity than in rabbits and humans. The temperature dependent effect on ventricular/ transmural activation could therefore be less significant in rats than larger animals. Our data supports this theory, as hypothermia fails to induce a relative shortening of ventricular activation compared to repolarisation, observed through increasing QRS/QTc values during cooling. This is different from rabbit and human data (Fig. 4) . Accordingly, we speculate that the limited dependence on gap junction activity for transmural conduction is the underlying mechanism for resistance to hypothermia-induced VF in rats, compared to other species like rabbit and hypothermic patients [4, 6, 7].
According to our present and previous findings, identifying pro-arrhythmic activity during hypothermia depends on the ability to detect a heterogenic effect on ventricular/ transmural activation, relative to cardiac conduction and repolarisation [4, 6, 7]. Therefore it is not possible to predict the vulnerable window by assessing QT-time alone. Risk for hypothermia-induced arrhythmias rather seems to be associated with an exaggerated shortening of depolarisation timings (QRS) relative to repolarisation time corrected for RR-interval (QTc), namely QRS/QTc. From the present and previous translational studies [4, 6, 7], we propose that 0.2 defines the upper limit of the vulnerable window, as measured by QRS/QTc.