In line with existing literature29,30, we found that HRV time- and frequency domain measures derived from 24-h ECG significantly discriminated our cohorts of individuals with T1DM and polyneuropathy and age- and sex-matched healthy controls, indicating diabetes-induced dysautonomia. We further investigated the neurocardiac adaptability by investigating the electrocardiographically dynamic responses representing HRV measures before, during, and after exposure to tonic cold pain. Compared to healthy, we found convincingly adynamic responses in the T1DM group, evident as a delayed increase in SDNN and decreased parasympathetic regulation, contributing to a more chronometric heart rhythm. The findings reveal impaired neurocardiac adaptability to rapidly adjust to potential threats (here mimicked with cold pain exposure), causing physical or psychological alterations, and consequently, maintained homeostasis is mistrusted.
Table of Contents
Baseline characteristics
This study included 21 healthy and 48 individuals with T1DM and confirmed DSPN (decreased nerve conduction velocities and thermal pain tolerance), increased heart rate and systolic blood pressure, and 90% of had orthostatic hypotension, indicating both peripheral and severe autonomic nerve dysfunction. This was further supported by the mean clinical risk score QRISK3 of 20%, indicating a relatively high risk of cardiovascular events within the next 10 years. All participants in the T1DM group had diabetic retinopathy, and the number of participants with a UACR above > 30 mg/g (lower level of microalbuminuria) was larger in the T1DM group, reflecting the systemic presence of microvascular complications. Antihyperlipidemic and antihypertensive treatment is recommended in the diabetes treatment guidelines. This explains why a larger percentage of participants received these treatments in the T1DM group, reflected as lower levels of LDL than healthy.
Autonomic regulation reflected in HRV
The HRV measures are an emergent property assessing the neurocardiac adaptability, reflecting the heart-brainstem interactions and regulated through the non-linear regulated autonomic nervous system10. HRV describes the fluctuations in the time intervals between consecutive heartbeats. Mean RR is simply the time of the inter-beat interval. SDNN is the standard deviation of the RR-intervals and is considered the most robust parameter to quantify the inter-beat variability and neurocardiac adaptability, and it reflects both sympathetic and parasympathetic modulation in response to physiological influences38. RMSSD and HF are strongly believed to represent central parasympathetic regulation8, in contrast to the LF component of HRV, which reflects both sympathetic and parasympathetic activity. However, the utility of the LF component of HRV is largely debated39,40,41. The LF/HF ratio is believed to reflect the sympathovagal balance or sympathetic modulations8. According to Shaffer et al., a low LF/HF reflects parasympathetic dominance, whereas a high LF/HF reflects sympathetic dominance10. Finally, the indices have proven valuable in assessing individual risk stratification for cardiovascular events and the degree of neurocardiac dysautonomia6,42.
Our dynamic assessments provided the possibility to interpret the cold pain-induced comprehensive autonomic response, comparable to the assessment of orthostatic hemodynamics in response to, e.g., standing or tilting, representing initial, sustained, and recovery phases43. Compared to healthy, the diabetes group experienced less unpleasantness, but there was no difference in pain response to tonic cold pain or thermal heat stimulation. This rules out that peripheral neuropathies modulated the afferent upstream activation of the tonic pain experience, thereby biasing our HRV data. Consequently, as both groups were exposed to equipotent peripheral pain experiences, our findings of decreased parasympathetic response to tonic cold pain in the diabetic group are supported by impaired neurocardiac regulation, evident as a delayed increase in SDNN. Taken together, the adynamic HRV responses in T1DM to tonic pain indicate central dysautonomia, even when controlling for age and BMI.
Characteristics of HRV (ultra-short vs. long-term recordings)
We used ultra short-term HRV epochs (2 min) to assess the dynamic neurocardiac regulation. Such short epochs have less variability compared to 24-h recordings. Even though they are influenced by sinus arrhythmia, the baroreceptor reflex (negative feedback control of blood pressure), and rhythmic changes in vascular tone8,37, the 24-h recordings are further influenced by circadian rhythms, body core temperature, metabolism, sleep pattern, and the regulation of the renin-angiotensin system8,37, thereby providing the highest possible variability. The 24-h recordings are the gold standard for clinical HRV assessments because they are more accurate in predicting cardiovascular events than short recordings. Even though we use identical mathematical formulas to assess HRV from 24-h and short-term recordings, they represent complementary physiological measures. Simplistically, RMSSD and HF have been suggested to quantify the portion of parasympathetic regulation and used to estimate the sympathovagal balance8. Such indices are often used to assess individual risk stratification for cardiovascular events and the degree of neurocardiac dysautonomia42.
An example is the LF/HF ratio, which may describe the sympathovagal balance under controlled conditions44. However, the measure should be interpreted cautiously in situations where the neurocardiac regulation is altered in response to external situations, such as the cold pressor test, primarily because the total power (sum of the energy in VLF, LF, and HF bands) is highly variable and changes in response to external stimuli that challenges homeostasis10. In that perspective, the short-term data reflect vulnerability to underreport differences. Nonetheless, the recordings were analyzed as a series of 2-min epochs and revealed significant differences providing robust and complementary information on the neurocardiac adaptability to cold pain exposure.
Healthy controls vs. T1DM in 24-h HRV recordings
The 24-h recordings revealed impaired neurocardiac regulation, and the shown diminished RMSSD and HF support the diagnosis of cardiovascular autonomic neuropathy (CAN) characterized by severe sympathetic dominance and parasympathetic withdrawal. Even though HRV measures are known to be reduced in individuals with T1DM in comparison to healthy controls9,28,29,30,31,45, conflicting results exist, which may indicate co-activation or co-inhibition of the two tonically activated branches, allowing rapid physiological neurocardiac adaptation in response to the surroundings. Furthermore, attenuated HRV responses at rest, but not in response to cold pain, were shown in a cohort with 20 participants with type 2 diabetes compared to 10 healthy controls33. Consequently, LF/HF may not always index autonomic balance accurately7,10,32,33.
To our surprise, total power, VLF, LF, and HF components of the 24-h HRV measures were correlated to the mean QRISK3, supporting the relevance of assessing neurocardiac regulation with HRV measures. Interestingly, based on the ultra-short epochs, QRISK3 scores negatively correlated with SDNN, total power, VLF, LF, and HF between 2 and 6 min post-exposure, underlining that parasympathetic withdrawal and adynamic appearance of other HRV measures are associated with increased risk of having a cardiovascular event.
Complementary neurocardiac measures
The arterial baroreceptors buffer acute fluctuations in blood pressure during e.g., sympathetic dominance, deep breathing, or postural changes by transducing increased vascular distension into nervous electrical activity, which actuates parasympathetic activation and sympathetic inhibition44. Baroreceptor activity is activated through high-pressure (aortic arch) or both low- and high-pressure (carotid sinus) baroreceptors, triggering distinct pathways, the so-called cardio-vagal, the cardio-sympathetic or the vaso-sympathetic pathway46. We showed decreased CSB in T1DM, constituting the afferent branch of the cardiac-brainstem communication. Interestingly, we only found a tendency towards decreased CVT, constituting the efferent branch of the cardiac-brainstem communication, but previously it has been reported that CVT is reduced in T1DM34. Both findings are in accordance with the literature, plausibly explained by damage to central or peripheral (afferent and/or efferent) parts of the cardiac-brainstem circuit47.
Exposure to tonic cold pain
We found no group differences between healthy and T1DM in perceived pain, and hence, believe that the individuals were exposed to an equipotent painful stimulus, with a proportional autonomic response. Exposure to cold tonic pain is perceived with high inter-individual variability, which has classically been considered to activate the sympathetic response because it is supported by elevated adrenaline and noradrenaline levels, increased heart rate or blood pressure13,14,15,17,18,19,20,21,22,23,24. Nevertheless, novel data reveal that approximately 30% of healthy subjects respond to cold pain exposure with parasympathetic dominance48, suggesting a more complex response, where an individual physiological meaningful linear, and non-linear co-activation of the sympathetic and parasympathetic system is present.
HRV responses in healthy following to exposure to cold pain
We refined the existing methodologies and measured mean HRV responses in ultra-short epochs (2 min) before, during, and up to 14 min after cold tonic pain (2 min at 2 °C) to allow a more dynamic interpretation of the neurocardiac regulation in response to cold pain exposure. The experimental setting is comparable to, e.g., provocative tests such as treadmill tests, in which stressing the neurocardiac central regulatory system can reveal changes that otherwise may not be visible under resting conditions. In the healthy cohort, we show convincingly dynamic physiological adaptive capacity of the heart, evidenced by increased SDNN and RMSSD, supported by Sanchez-Gonzalez et al., who also found increased RMSSD in response to cold pain49. In contrast, Jarczewski et al. found a decrease in RMSSD14, and MacArtney et al. did not show changes in SDNN or RMSSD in response to the cold pressor test16. Differences in used methodologies and duration of the analyzed ECG epochs could explain these discrepancies. Example, Jarczewski et al. measured the mean HRV responses in a 10-min epoch following a similar cold pressor test (2 min at 0–4 °C), whereas MacArtney et al. measured mean HRV responses in a 5-min epoch during the cold pressor test (5 min, 5 °C)16,17. Furthermore, since especially parasympathetic modulation decreases with increasing age50, and as our cohort is approximately double the age as those in the Jarczewski et al. and MacArtney et al., the need for matched controls to reliably compare the HRV responses between our two groups is emphasized16,17.
The initial group level response to tonic cold pain showed an increase in total power and LF/HF ratio, indicating more LF (or less HF) power, plausibly revealing increased sympathetic response in response to cold pain exposure. The literature shows ambiguous results, some report increased LF25,49 and HF power25,49,51 within the first 2 min after hand immersion into ice water, whereas others describe decreased LF17 and HF52, which may be explained by individual responses, different methodologies, different central brainstem regulations (co-inhibition or reciprocal activation of the two branches), sympathetic ceiling, withdrawal, or enhanced compensatory parasympathetic tone.
HRV responses in T1DM with polyneuropathy following exposure to cold pain
Throughout the entire recording in T1DM, SDNN did not differ from the pre-test epoch indicating chronometrical heart rhythm, regardless of tonic cold pain exposure. This is supported by decreased mean RR and RMSSD in the initial response to cold pain exposure, indicating rapid sympathetic dominance, which interestingly is characterized by decreased total power, LF and LF/HF ratio decreased, and unchanged HF. At first sight, one could speculate that it solely represents sympathetic dominance, but it could also indicate that parasympathetic tone is at a minimum, and that is why it cannot be modulated more—even by a provocative test. Taken together, it indicates altered central neurocardiac regulation and the presence of cardiovascular autonomic neuropathy3,6. As a late sustained response in the recovery phase, sympathetic withdrawal or relatively enhanced parasympathetic activity was shown by increased RR intervals, but most evident is the presence of adynamic responses in SDNN, RMSSD, and total power, indicating non-adaptability of the neurocardiac regulation. To our knowledge, no previous studies have investigated such dynamic HRV response to tonic cold pain exposure in T1DM, unmasking the complexity of the concomitant sympathetic and parasympathetic autonomic regulation.
Differences in HRV responses between groups
In comparison to healthy, we found that time-domain (SDNN and RMSSD) and frequency-domain (total power, LF) responses to cold pain exposure were unphysiologically adynamic, leading to decreased cardiac adaptability, chronometrical HR, and ultimately diminished ability to counteract or adapt to threatening external factors. Interestingly, in response to tonic cold pain exposure, both T1DM and healthy controls experienced a rapid decrease in the LF/HF ratio; however, total power was only diminished in the T1DM group. As the parasympathetic response was unchanged, this finding indicates differences in sympathetic responses, which were less powerful and less adaptable in the T1DM group. This is further supported by a cross-sectional study conducted in a cohort of 52 participants with diabetes and 15 years of diabetes duration, where no alterations in HR and blood pressure were shown in response to cold pain exposure7. Taken together both branches show impaired responses. The parasympathetic response is adynamic and unadaptable, but the sympathetic response can still, but to a lesser degree than normal, be modulated.
Strengths and limitations
This is the first time that the dynamic HRV response to tonic cold pain has been investigated in a cohort of individuals with T1DM and polyneuropathy and compared with an age- and sex-matched cohort of healthy controls. Unsurprisingly, we showed convincing differences in the HRV measures based on 24-h recordings, supporting dysautonomia in the T1DM group. Furthermore, we showed robust group differences in the capacity of adapting the neurocardiac regulation in responses to tonic cold pain exposure. However, the first obvious limitation is that the participants with T1DM have long-term diabetes and severe verified polyneuropathy and the majority have orthostatic hypotension, indicative of severe CAN. Thus, our findings support clinical findings, but unfortunately, cardiovascular autonomic reflex tests were not carried out to diagnose cardiovascular autonomic neuropathy, hampering the ability to stratify the cohort according to the CAN stage. Secondly, 2 min epochs are ultra-short, making them vulnerable to physiological noise such as sinus arrhythmia, respiration patterns, coughing, certain movements, or stress response caused by the cold pressor test. However, data was consistently calculated within meaningful predefined intervals, and HRV analyses were conducted by the same person by use of validated software, minimizing the inter- and intra-observer differences. Thirdly, including the Poincaré plot could have revealed non-linear dynamics of consecutive RR intervals and thus provided additional information on the heart rate variability changes, but this was not included in the study. Fourthly, it has been shown in healthy that the cold pressor test causes high inter-individual variability, reflected in primarily parasympathetic and sympathetic responses, and thus, group comparisons yield a source of error. Nevertheless, in comparison to healthy, we showed that the parasympathetic response in T1DM was convincingly adynamic, and the magnitude of the adaptability of the sympathetic response was decreased. Lastly, it could have strengthened the interpretation of the neurocardiac regulation if novel parameters of sympathetic and parasympathetic activation, such as periodic dynamic depolarization of the T-wave and deceleration capacity, were used, but these were not available in this study.
