Letters coronary heart disease in women. Arch Intern Med 2003; 163: 205–209. 6 van Mill JG, Hoogendijk WJ, Vogelzangs N, Van Dyck R, Penninx BW. Insomnia and sleep duration in a large cohort of patients with major depressive disorder and anxiety disorders. J Clin Psychiatry 2010; 71: 239–246. 7 Borel AL, Pepin JL, Nasse L, Baguet JP, Netter S, Benhamou PY. Short sleep duration measured by wrist actimetry is associated with deteriorated glycemic control in type 1 diabetes. Diabetes Care 2013; 36: 2902–2908.
DOI: 10.1111/dme.12423
Effect of peripheral cholinergic stimulation on autonomic modulation in Type 2 diabetes with autonomic neuropathy: a randomized controlled trial Diabet. Med. 31, 761–762 (2014) Cardiovascular autonomic neuropathy is a reversible, serious and frequent complication in diabetes mellitus [1], presenting with reduced vagal modulation to the sinus node and beat-to-beat heart rate variability [2]. Alterations in the adrenergic and/or muscarinic receptor functions with a reduction in acetylcholine release [3] may be involved in this pathogenic process. Pyridostigmine bromide, an acetyl cholinesterase inhibitor, produces cholinergic stimulation by preventing the hydrolysis of acetylcholine and increasing its availability in the synaptic cleft [4]. It increases vagal modulation in healthy subjects [5] and heart failure patients [6], but the effect on cardiovascular autonomic neuropathy is unknown. In this study, we tested the hypothesis that increasing acetylcholine availability in the synaptic cleft improves vagal modulation. Thirty-four outpatients with Type 2 diabetes with cardiovascular autonomic neuropathy, attending the Endocrine Division at Hospital de Clınicas de Porto Alegre, were randomized to pyridostigmine and placebo, according to the intervention randomly drawn from a concealed envelope (Table 1). Presence of cardiovascular autonomic neuropathy was confirmed by the autonomic tests standardized by Ewing et al. [7] and a baseline 24-h Holter monitoring to calculate the following heart rate variability indices: mean of normal R–R intervals; standard deviation of normal R–R intervals; and root– mean–square successive differences [8]. The protocol was approved by the ethical committee of the institution and informed consent was obtained from each individual. Heart rate variability was re-evaluated while patients were receiving pyridostigmine bromide 30 mg orally three times daily for 24 h, or placebo. Patients and researchers were blinded to the interventions. The analysed data are described as mean SD. Differences in clinical characteristics between groups were compared using Student’s t-test for continuous variables and Pearson’s v2-test. Repeated-measures ANOVA was used to evaluate the effect
ª 2014 The Authors. Diabetic Medicine ª 2014 Diabetes UK
DIABETICMedicine
of the interventions on heart rate variability and minimum significance level was set at 5%. Both groups had similar clinical characteristics regarding age, gender, BMI, diabetes duration, blood pressure, cardiovascular autonomic neuropathy severity and use of drugs. Our results showed that pyridostigmine had no acute effect on autonomic modulation evaluated by heart rate variability, with an effect size d < 0.2 (Table 1). Differently, in our previous study in patients with heart failure with severely depressed heart rate variability, the short administration of pyridostigmine during only 24 h promoted a clear increase in time domain indices [6]. As in cardiovascular autonomic neuropathy peripheral nerves are anatomically compromised, it was not expected that a short intervention could restore its integrity, but an increase in acetylcholine availability should improve vagal modulation. The release of acetylcholine at the neuroeffector junction of the heart is regulated by the central nervous system and peripherally influenced by muscarinic receptors and pre-synaptic autoreceptors found at the postganglionic parasympathetic fibres [9]. These autoreceptors are muscarinic receptors subtypes that, when activated by endogenous acetylcholine, inhibit the release of acetylcholine in the synaptic cleft, causing a negative feedback [3,9,10]. Although acetyl cholinesterase inhibition with pyridostigmine increases the amount of acetylcholine in the synaptic cleft, in patients with diabetes, as the autoreceptors are up-regulated, this increase in acetylcholine may be followed by a negative feedback that reduces further acetylcholine release, preventing an improvement in cardiac autonomic modulation. The development of drugs that could modulate these up-regulated autoreceptors possibly would help in cardiovascular autonomic neuropathy control. There are some potential limitations to our study that could explain the lack of pyridostigmine effect. The drug was administered for a short period of time and longer exposure could bring different results. In this study, we did not assess cholinesterase activity to assure that the pyridostigmine dose used could properly block serum cholinesterase activity. In a previous study of our group, we have demonstrated that the same dose used in this study promoted a 14% reduction in cholinesterase activity [5] and increased autonomic modulation. In this present study, 58% of the patients were classified as having severe cardiovascular autonomic neuropathy. It is possible that, in a less compromised sample, pyridostigmine could increase heart rate variability. Although this study enrolled a small number of patients, the sample size was based on previous studies of our group with healthy individuals [5] and patients with heart failure [6] in whom the same dose of pyridostigmine significantly increased heart rate variability. In conclusion, peripheral acetyl cholinesterase blockage with oral administration of pyridostigmine bromide does not improve autonomic modulation of patients with Type 2 diabetes with cardiovascular autonomic neuropathy.
761
DIABETICMedicine
Letters
Table 1 Heart rate variability measurements
Time domain 24 h (ms) Mean of normal R–R intervals Standard deviation of normal R–R intervals Root–mean–square successive differences
Pyridostigmine (n = 17)
Placebo (n = 17)
Baseline
Pyridostigmine
Baseline
Placebo
P-value
727.5 74.6 100.6 28.2 19.3 9.5
748.3 99.6 107.4 26 20.8 12.7
733.8 104.9 104 40.6 19.3 9.4
733.9 111.5 108.9 36 20.4 10
0.17 0.79 0.83
Funding sources
This study was supported by the Fundo de Incentivo a` Pesquisa do Hospital de Clı´nicas de Porto Alegre (FIPE) that provided all the necessary resources and the authors acknowledge for financial support.
Competing interests
None declared. A. D. Harthmann1, W. I. B. P. Souza1, E. L. Ferlin2 and R. S. Moraes3 1 Exercise Pathophysiology Research Laboratory and Cardiovascular Division, Hospital de Clınicas de Porto Alegre, 2Biomedical Engineering Division, Hospital de Clınicas de Porto Alegre and 3Cardiology Division, Hospital de Clınicas de Porto Alegre, Porto Alegre, Brazil
References 1 Gaede P, Vedel P, Parving HH, Pedersen O. Intensified multifactorial intervention in patients with type 2 diabetes mellitus and microalbuminuria: the Steno type 2 randomized study. Lancet 1999; 353: 617–622. 2 Moraes RS, Ferlin EL, Polanczyk CA, et al. Three-dimensional return map: a new tool for quantification of heart rate variability. Auton Neurosci 2000; 83: 90–99.
762
3 Oberhauser V, Schwertfeger E, Rutz T, Beyersdorf F, Rump LC. Acetylcholine release in human heart atrium. Influence of muscarinic autoreceptors, diabetes, and age. Circulation 2001; 103: 1638–1643. 4 Stoelting RK. Anticholinesterase drugs and cholinergic agonists. In: Craig Percy R, ed. Pharmacology and Physiology in Anesthesic Practice. 3rd edn. Philadelphia: Lippincott–Raven Publishers, 1999: 224–246. 5 N obrega ACL, Reis AF, Moraes RS, Bastos BG, Ferlin EL, Ribeiro JP. Enhancement of heart rate variability by cholinergic stimulation with pyridostigmine in healthy subjects. Clin Auton Res 2001; 11: 11–17. 6 Behling A, Moraes RS, Rohde LE, Ferlin EL, Nobrega AC, Ribeiro JP. Cholinergic stimulation with pyridostigmine reduces ventricular arrytmia and enhances heart rate variability in heart failure. Am Heart J 2003; 146: 494–500. 7 Ewing DJ, Martyn CN, Young RJ, Clarke BF. The value of cardiovascular autonomic function tests: 10 years experience in diabetes. Diabetes Care 1985; 8: 491–498. 8 Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Circulation 1996; 93: 1043–1065. 9 Wetzel GT, Brown JH. Presynaptic modulation of acetylcholine release from cardiac parasympathetic neurons. Am J Physiol 1985; 248: 33–39. 10 Coulson FR, Fryer AD. Muscarinic acetylcholine receptors and airway diseases. Pharmacol Ther 2003; 98: 59–69.
ª 2014 The Authors. Diabetic Medicine ª 2014 Diabetes UK
DOI: 10.1111/dme.12423
Effect of peripheral cholinergic stimulation on autonomic modulation in Type 2 diabetes with autonomic neuropathy: a randomized controlled trial Diabet. Med. 31, 761–762 (2014) Cardiovascular autonomic neuropathy is a reversible, serious and frequent complication in diabetes mellitus [1], presenting with reduced vagal modulation to the sinus node and beat-to-beat heart rate variability [2]. Alterations in the adrenergic and/or muscarinic receptor functions with a reduction in acetylcholine release [3] may be involved in this pathogenic process. Pyridostigmine bromide, an acetyl cholinesterase inhibitor, produces cholinergic stimulation by preventing the hydrolysis of acetylcholine and increasing its availability in the synaptic cleft [4]. It increases vagal modulation in healthy subjects [5] and heart failure patients [6], but the effect on cardiovascular autonomic neuropathy is unknown. In this study, we tested the hypothesis that increasing acetylcholine availability in the synaptic cleft improves vagal modulation. Thirty-four outpatients with Type 2 diabetes with cardiovascular autonomic neuropathy, attending the Endocrine Division at Hospital de Clınicas de Porto Alegre, were randomized to pyridostigmine and placebo, according to the intervention randomly drawn from a concealed envelope (Table 1). Presence of cardiovascular autonomic neuropathy was confirmed by the autonomic tests standardized by Ewing et al. [7] and a baseline 24-h Holter monitoring to calculate the following heart rate variability indices: mean of normal R–R intervals; standard deviation of normal R–R intervals; and root– mean–square successive differences [8]. The protocol was approved by the ethical committee of the institution and informed consent was obtained from each individual. Heart rate variability was re-evaluated while patients were receiving pyridostigmine bromide 30 mg orally three times daily for 24 h, or placebo. Patients and researchers were blinded to the interventions. The analysed data are described as mean SD. Differences in clinical characteristics between groups were compared using Student’s t-test for continuous variables and Pearson’s v2-test. Repeated-measures ANOVA was used to evaluate the effect
ª 2014 The Authors. Diabetic Medicine ª 2014 Diabetes UK
DIABETICMedicine
of the interventions on heart rate variability and minimum significance level was set at 5%. Both groups had similar clinical characteristics regarding age, gender, BMI, diabetes duration, blood pressure, cardiovascular autonomic neuropathy severity and use of drugs. Our results showed that pyridostigmine had no acute effect on autonomic modulation evaluated by heart rate variability, with an effect size d < 0.2 (Table 1). Differently, in our previous study in patients with heart failure with severely depressed heart rate variability, the short administration of pyridostigmine during only 24 h promoted a clear increase in time domain indices [6]. As in cardiovascular autonomic neuropathy peripheral nerves are anatomically compromised, it was not expected that a short intervention could restore its integrity, but an increase in acetylcholine availability should improve vagal modulation. The release of acetylcholine at the neuroeffector junction of the heart is regulated by the central nervous system and peripherally influenced by muscarinic receptors and pre-synaptic autoreceptors found at the postganglionic parasympathetic fibres [9]. These autoreceptors are muscarinic receptors subtypes that, when activated by endogenous acetylcholine, inhibit the release of acetylcholine in the synaptic cleft, causing a negative feedback [3,9,10]. Although acetyl cholinesterase inhibition with pyridostigmine increases the amount of acetylcholine in the synaptic cleft, in patients with diabetes, as the autoreceptors are up-regulated, this increase in acetylcholine may be followed by a negative feedback that reduces further acetylcholine release, preventing an improvement in cardiac autonomic modulation. The development of drugs that could modulate these up-regulated autoreceptors possibly would help in cardiovascular autonomic neuropathy control. There are some potential limitations to our study that could explain the lack of pyridostigmine effect. The drug was administered for a short period of time and longer exposure could bring different results. In this study, we did not assess cholinesterase activity to assure that the pyridostigmine dose used could properly block serum cholinesterase activity. In a previous study of our group, we have demonstrated that the same dose used in this study promoted a 14% reduction in cholinesterase activity [5] and increased autonomic modulation. In this present study, 58% of the patients were classified as having severe cardiovascular autonomic neuropathy. It is possible that, in a less compromised sample, pyridostigmine could increase heart rate variability. Although this study enrolled a small number of patients, the sample size was based on previous studies of our group with healthy individuals [5] and patients with heart failure [6] in whom the same dose of pyridostigmine significantly increased heart rate variability. In conclusion, peripheral acetyl cholinesterase blockage with oral administration of pyridostigmine bromide does not improve autonomic modulation of patients with Type 2 diabetes with cardiovascular autonomic neuropathy.
761
DIABETICMedicine
Letters
Table 1 Heart rate variability measurements
Time domain 24 h (ms) Mean of normal R–R intervals Standard deviation of normal R–R intervals Root–mean–square successive differences
Pyridostigmine (n = 17)
Placebo (n = 17)
Baseline
Pyridostigmine
Baseline
Placebo
P-value
727.5 74.6 100.6 28.2 19.3 9.5
748.3 99.6 107.4 26 20.8 12.7
733.8 104.9 104 40.6 19.3 9.4
733.9 111.5 108.9 36 20.4 10
0.17 0.79 0.83
Funding sources
This study was supported by the Fundo de Incentivo a` Pesquisa do Hospital de Clı´nicas de Porto Alegre (FIPE) that provided all the necessary resources and the authors acknowledge for financial support.
Competing interests
None declared. A. D. Harthmann1, W. I. B. P. Souza1, E. L. Ferlin2 and R. S. Moraes3 1 Exercise Pathophysiology Research Laboratory and Cardiovascular Division, Hospital de Clınicas de Porto Alegre, 2Biomedical Engineering Division, Hospital de Clınicas de Porto Alegre and 3Cardiology Division, Hospital de Clınicas de Porto Alegre, Porto Alegre, Brazil
References 1 Gaede P, Vedel P, Parving HH, Pedersen O. Intensified multifactorial intervention in patients with type 2 diabetes mellitus and microalbuminuria: the Steno type 2 randomized study. Lancet 1999; 353: 617–622. 2 Moraes RS, Ferlin EL, Polanczyk CA, et al. Three-dimensional return map: a new tool for quantification of heart rate variability. Auton Neurosci 2000; 83: 90–99.
762
3 Oberhauser V, Schwertfeger E, Rutz T, Beyersdorf F, Rump LC. Acetylcholine release in human heart atrium. Influence of muscarinic autoreceptors, diabetes, and age. Circulation 2001; 103: 1638–1643. 4 Stoelting RK. Anticholinesterase drugs and cholinergic agonists. In: Craig Percy R, ed. Pharmacology and Physiology in Anesthesic Practice. 3rd edn. Philadelphia: Lippincott–Raven Publishers, 1999: 224–246. 5 N obrega ACL, Reis AF, Moraes RS, Bastos BG, Ferlin EL, Ribeiro JP. Enhancement of heart rate variability by cholinergic stimulation with pyridostigmine in healthy subjects. Clin Auton Res 2001; 11: 11–17. 6 Behling A, Moraes RS, Rohde LE, Ferlin EL, Nobrega AC, Ribeiro JP. Cholinergic stimulation with pyridostigmine reduces ventricular arrytmia and enhances heart rate variability in heart failure. Am Heart J 2003; 146: 494–500. 7 Ewing DJ, Martyn CN, Young RJ, Clarke BF. The value of cardiovascular autonomic function tests: 10 years experience in diabetes. Diabetes Care 1985; 8: 491–498. 8 Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Circulation 1996; 93: 1043–1065. 9 Wetzel GT, Brown JH. Presynaptic modulation of acetylcholine release from cardiac parasympathetic neurons. Am J Physiol 1985; 248: 33–39. 10 Coulson FR, Fryer AD. Muscarinic acetylcholine receptors and airway diseases. Pharmacol Ther 2003; 98: 59–69.
ª 2014 The Authors. Diabetic Medicine ª 2014 Diabetes UK
