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. 2013 Sep 5;6(3):583–589. doi: 10.3400/avd.cr.13-00064

Cardiac Sympathetic Activity Assessed by Heart Rate Variability Indicates Myocardial Ischemia on Cold Exposure in Diabetes

Keita Sasaki 1,✉, Shonosuke Matsushita 2,✉, Fujio Sato 2,✉, Chiho Tokunaga 2,✉, Kazuyuki Hyodo 3,✉, Yuzuru Sakakibara 2,✉
PMCID: PMC3793179  PMID: 24130613

Abstract

Measurement of heart rate variability (HRV) is a non-invasive technique that can be used to investigate functioning of the autonomic nervous system, especially the balance between sympathetic and vagal activities. It is reported that dilatation of coronary microcirculation by augmentation of sympathetic nerve activity (SNA) caused by cold exposure was impaired in diabetes. The question of whether or not SNA in HRV could respond to coronary ischemia was evaluated by cold exposure in diabetic rats. It was found that diabetes with weight loss significantly increased SNA both in baseline and cold exposure, compared with control. A correspondence was also found with coronary ischemia. It can be concluded that measurement of HRV may provide useful information regarding the coronary risk of cold exposure in diabetes. (*English translation of J Jpn Coll Angiol 2012; 52: 295-301)

Keywords: heart rate variability (HRV), sympathetic nerve activity (SNA), diabetes, rat, cold exposure

Introduction

The world’s diabetic population in 2010 was 285 million, and has recently been increasing. Diabetes is known to markedly raise morbidity and mortality rates from cardiovascular disease,1) the risk factors of which include obesity, smoking, stress, old age, and cold stimulation.2,3) The measurement of the coronary blood flow by cardiac catheterization and positron emission tomography (PET) has suggested that cold stimulation induces contraction of the cardiac microvasculature by sympathetic nerve activity, but the induction of coronary vasoconstriction by cold exposure has not been directly confirmed.4,5)

We experimentally measured heart rate variability (HRV) by plain electrocardiogram (ECG) as an index of the cardiac sympathetic nerve function, and evaluated whether or not it allows the prediction of the risk of myocardial ischemia on cold exposure, which may be exacerbated in diabetes.6)

Methods

Preparation of diabetic rats and evaluation of aging

Diabetes was induced in male Wistar rats (5 weeks old) by administering streptozotocin (STZ, 65 mg/kg once, i.p.).7) The animals that showed a blood glucose level of 400 mg/dl or higher 20 weeks after STZ administration were regarded as a Diabetes Mellitus (DM) group8) and compared with controls cared for over the same period without STZ administration (n = 6 for each group).

This experiment was carried out with approval by the Institutional Review Board of the Laboratory Animal Resource Center, University of Tsukuba, Japan.

Protocol

PowerLab (ADInstruments, Sydney, Australia) was used for analysis. Electrocardiography was performed by immobilizing the limbs of rats in a supine position under isoflurane inhalation anesthesia (concentration: 2.5%). Needle electrodes were inserted subcutaneously at the positions of lead II (right shoulder: negative electrode, left costal margin: positive electrode, right abdominal region: indifferent electrode). In addition, a needle-shaped temperature sensor was inserted near the left femoral artery. A cooling spray was applied to the shaved left lower leg from a distance of 10 cm. The HRV index, ST changes, left ventricular ejection fraction (EF), and lower leg tissue temperature were recorded before and 30 seconds, 1, 3, 5, 10, and 15 minutes after spraying.

HRV analysis method, other methods

Method for RR analysis

HRV was analyzed with Chart Ver.5.5.6 (ADInstruments, HRV Ver.1.1) based on 500 consecutive pulses recorded from lead II of ECG.9) The parts of the ECG records showing arrhythmia were excluded from analysis. The intervals of the first derivations of R waves were used for RR frequency analysis. After high-speed Fourier transformation, the frequency (Hz, X-axis) and power (ms2, Y-axis) were plotted (Fig. 1).

Fig. 1.

Fig. 1

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Method of HRV. An interval of 500 beats without any ectopic beats was obtained from lead II of the surface ECG, and was transformed into first derivatives. The fast Fourier transform (FFT) of the RR interval was performed and expressed as a power-spectrum analysis of HRV. The horizontal axis represents frequency (Hz) and the vertical axis represents the power of the frequency band (ms2). HRV: heart rate variability; ECG: electrocardiogram.

Definition of frequency bands

Since the method for HRV evaluation has not been established, we defined the frequency bands as indices of the autonomic activity based on our own experiments.

Sympatholytic propranolol (0.2 mg/kg) was administered intraperitoneally10) to the rats of the control group (n = 6) under pentobarbital anesthesia (50 mg/kg, i.p.), and electrocardiograms recorded before and 1 and 5 minutes after the administration were analyzed for HRV. The power of the frequency spectrum decreased significantly 1 and 5 minutes after (0.14 ± 0.09 and 0.26 ± 0.12 ms2, respectively; p <0.005 for both) compared with before (0.52 ± 0.19 ms2) the administration in a frequency band of 0.04–0.67 Hz.11) Therefore, we judged that the power in this range is useful as an index of the sympathetic nerve activity (SNA) and defined this frequency band as low frequency (LF), and defined the power of the LF divided by the power of a LF + high frequency (HF) band (0.04–1.5 Hz) (LF/(LF + HF)) as an index of SNA. The frequency band of the parasympathetic nerve activity (PNA) was assumed to be 1.0–1.5 Hz according to previous reports,11,12) and HF/(LF + HF) was used as an index of PNA. Eventually, we defined the frequency bands of autonomic activities as 0.04 Hz <LF <0.67 Hz and 1.0 Hz <HF <1.5 Hz.

Definition of ST changes on ECG

ST changes were measured in ECG records with a stable baseline. An ST elevation or depression of 0.05 mV or greater was defined as an indication of ischemia.

Evaluation of the cardiac function by echocardiography

Echocardiography was performed using Vevo2100 (VISUAL SONICS, Toronto, Ontario, Canada) for rats and mice. The left ventricular internal dimension in systole and diastole (LVIDs and LVIDd, respectively) were measured in the M-mode before and after cold stimulation, and the left ventricular EF was calculated.

Synchrotron radiation coronary angiography

Synchrotron radiation coronary angiography was performed at the Synchrotron Facility of the High Energy Accelerator Research Organization (Tsukuba, Ibaraki, Japan). A cannula was inserted through the right carotid artery and advanced to the Valsalva sinus. Coronary angiography was performed by injecting 1 ml of a contrast medium at 1 ml/sec into the Valsalva sinus. The irradiation time was 30 msec (n = 4).

Statistical procedures

All data are expressed as the mean ± 1 SD (SD: standard deviation). The paired Student’s t-test was used for comparisons within the same group, and the unpaired Student’s t-test was performed for comparisons between the groups. The level of significance was p <0.05.

Results

Body weight and blood glucose level

The body weight of the rats was 757 ± 12 g in the control group and 367 ± 18 g in the DM group, and the body weight gain was significantly suppressed in the DM compared with the control group (p <0.00001). The casual blood glucose level was 74 ± 25 mg/dl in the control group and 519 ± 70 mg/dl, exceeding 400 mg/dl, in the DM group.

Changes in the body temperature

In the control group, the left lower limb tissue temperature was 32.6 ± 0.8°C before cooling, decreased to 8.3 ± 3.2°C immediately after 5-second application of cooling spray, but recovered to 14.7 ± 3.6°C after 1 minute, 27.7 ± 1.5°C after 5 minutes, and 28.9 ± 1.5°C after 10 minutes. Similarly, in the DM group, it was 33.5 ± 1.0°C before cooling, decreased to 8.2 ± 3.7°C immediately after cooling, but recovered to 15.3 ± 3.1°C after 1 minute, 27.6 ± 1.9°C after 5 minutes, and 28.9 ± 1.5°C after 10 minutes. No significant difference was noted between the 2 groups.

Changes in SNA

Before cooling, SNA was about 7 times higher in the DM than control group (0.222 ± 0.136 and 0.034 ± 0.021, respectively; p <0.001). The difference widened further 1 (0.348 ± 0.145 and 0.027 ± 0.019, respectively; p <0.0005) and 5 (0.279 ± 0.188 and 0.028 ± 0.021, respectively; p <0.01) minutes after cooling.

In the control group, SNA was not changed (p = NS) (NS: not significant) 1 or 5 minutes after compared with before cooling. In the DM group, it was increased significantly after 1 minute (p <0.01) and was also increased after 5 minutes, although not significantly (p = NS) (Fig. 2).

Fig. 2.

Fig. 2

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Changes in sympathetic nerve activity (SNA). Changes of LF/(LF + HF) which is an index of SNA in the Control group and Diabetes Mellitus (DM) group, before and after cold exposure, are shown. The SNA value in DM group was larger than Control group at baseline (about 7 times, unpaired t test, p <0.01). One minute after cold exposure, SNA value of DM group increased, and became about 10 times larger than Control group (unpaired t test, p <0.0005). There was no significant difference in SNA before cold exposure compared to 1 minute after cold exposure in the Control group (paired t test, p = NS). In the DM group, SNA was significantly increased 1 minute after cold exposure (paired t test, p <0.01). LF: low frequency; HF: high frequency; NS: not significant

Changes in PNA

HF/(LF + HF), an index of PNA, showed a significant decrease 1 minute after cooling in the DM compared with the control group (0.59 ± 0.21 and 0.89 ± 0.12, respectively; p <0.05), but there was no significant difference between the 2 groups after 5 minutes.

ST changes

The difference in the ST level between before and after cooling was regarded as the “ST change”. It was significantly greater in the DM than control group. The ST change (mV) after 1 minute was 0.08 ± 0.04 in the DM group and 0.03 ± 0.03 in the control group (p <0.05), and that after 5 minutes was 0.13 ± 0.08 and 0.02 ± 0.03 (p <0.05), respectively. This suggested that the risk of the induction of myocardial ischemia by cold stimulation was higher in the DM group (Fig. 3).

Fig. 3.

Fig. 3

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Changes of ST segment in ECG. Changes between ST segments before and after cold exposure were evaluated in lead II in surface ECG. ST changes (ST elevation or ST depression) were significantly larger in the DM group than Control group in 1 and 5 minutes after cold exposure (p <0.05). ECG: electrocardiogram; DM: Diabetes Mellitus.

Changes in the left ventricular EF

The difference in between before and after cooling was compared by echocardiography. Before cooling, EF was significantly lower (p <0.001) in the DM (58 ± 7%) than control (79 ± 9%) group. However, it showed no significant change between before and after cooling in either group.

Synchrotron radiation coronary angiography

Synchrotron radiation coronary angiograms were compared between the control (n = 2) and DM (n = 2) groups. While the coronary artery was dilated in the control group 1 minute after cooling, it was contracted in the DM group (Fig. 4).

Fig. 4.

Fig. 4

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Image of synchrotron radiation coronary angiography. Synchrotron radiation coronary angiography was performed on rats in both Control group and DM group. The LAD (left anterior descending artery) is shown in the frontal position. Compared with 1 minute after cold exposure, the rat in the DM group showed coronary vasoconstriction, whereas the rat in the Control group showed vasodilatation. DM: Diabetes Mellitus.

Discussion

Cold stimulation is a risk factor for the exacerbation of cardiovascular disease.3) Cold stimulation is transmitted from the sensory nerves of the skin, relayed by the autonomic center, and then transmitted as sympathetic stimulation to the peripheral organs. Since coronary artery contraction is also associated with the sympathetic tone in diabetes, we thought that the effect of cold exposure on the heart could be evaluated simply if the cardiac SNA could be evaluated and quantified. HRV is considered to be an index useful for evaluation of the balance between sympathetic and parasympathetic activities and for noninvasive and simple assessment of the autonomic nerve functions.9) In diabetes, the parasympathetic nerve function has been reported to be suppressed, and the sympathetic nerve function to be enhanced in relative terms.13) Also, in the heart, the SNA was shown to be increased by cold exposure.14,15) The HRV evaluation method in humans has nearly been established.9) For these reasons, we also thought that the cardiac SNA represented by HRV would be useful as an index of cardiac ischemia on cold exposure in diabetes.

This study is significant in that it attempted to quantify the sympathetic nerve function under a hyperglycemic condition using HRV as an inexpensive, simple, and noninvasive method compared with the conventional measurement of the coronary blood flow using coronary angiography or PET,4,5) and to evaluate its association with ischemic changes of the cardiovascular system.

In this study, no increase in SNA was observed on cold exposure in the control group. However, SNA was about 7 times higher before cooling, and about 10 times higher 1 minute after cooling (p <0.01), in the DM than control group even under anesthesia. This suggests that exposure to cold has a marked effect on SNA in the DM group (Fig. 2).

The above results may be partially explained by the involvement of adiponectin, an adipocytokine released from fat cells. An elevation of the blood adiponectin level suppresses SNA through the suprachiasmatic nucleus of the hypothalamus.16) In this study, marked weight loss was observed in the DM compared with the control group (367 ± 18 and 757 ± 12 g, respectively; p <0.00001). A rapid loss of body weight reduces the blood adiponectin level,17) and cold stimulation further reduces it by increasing the sympathetic tone.18) Therefore, the blood adiponectin level is considered to have been markedly reduced in the DM group, and this may have markedly promoted the increase in SNA on cold simulation compared with the control group. This, however, needs further evaluation.

In this study, also, the ST segment of ECG showed significant changes (depression and elevation) after compared with before cold simulation. ST changes on ECG have been reported to indicate myocardial ischemia in rats as well as humans,19) and the results of this study suggest that cold stimulation is likely to have induced myocardial ischemia in diabetic rats.

Synchrotron radiation coronary angiography showed dilation of the coronary artery in the control group but contraction in the DM group after cold exposure. Prior et al. reported that contraction of the cardiac microvasculature was induced by an increase in SNA due to cold stimulation in patients with type 2 diabetes.4) In addition, excitation of the sympathetic nervous system increases the release of catecholamines such as noradrenaline into the circulation, and platelet aggregation is enhanced in diabetes.20) From these observations, cold stimulation is considered to increase the risk of ischemic heart disease in diabetes.

A characteristic of HRV is that it can numerically quantify the sympathetic tone by frequency analysis. Activation of the sympathetic nervous system in diabetes on cold stimulation can thus be confirmed quantitatively.

Since cold stimulation induced a marked increase in the sympathetic tone, large ST changes on ECG, and coronary artery constriction on coronary angiography in the DM group, excitation of the sympathetic nervous system due to cold stimulation is considered to promote coronary vasoconstriction and may even lead to myocardial ischemia in the presence of diabetes. Therefore, HRV, which quantitatively represents the sympathetic nerve function, is considered to serve as an index of the risk of coronary ischemia on cold stimulation in diabetics.

Conclusion

Cold stimulation of diabetic rats evidently amplified the cardiac SNA and induced ischemic changes on ECG. HRV may be a quantitative index of rapid changes in SNA on cold stimulation in diabetes.

Acknowledgments

This English manuscript was kindly proofread by Mr. Avi Landau.

Disclosure Statement

None of the authors has any conlicts of interest to disclose.

Footnotes

*

This article is English Translation of J Jpn Coll Angiol 2012; 52: 295-301.

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