- Email: [email protected]
Heart rhythm disturbances in the neonatal alloxan-induced diabetic rat
Pathophysiology 18 (2011) 185–192
Heart rhythm disturbances in the neonatal alloxan-induced diabetic rat Frank Christopher Howarth a,∗ , Mohamed Shafiullah a , Ernest Adeghate a , Milos Ljubisavljevic a , Michael Jacobson b a b
Faculty of Medicine & Health Sciences, UAE University, United Arab Emirates Biomedical Engineering, Higher Colleges of Technology, United Arab Emirates Received 5 May 2010; accepted 9 October 2010
Abstract Diabetic patients show a higher incidence of cardiac arrhythmias, including ventricular fibrillation and sudden death. Their electrocardiograms may show several alterations from normal patterns, many of them related to the QT. Various diastolic and systolic abnormalities are frequently reported in diabetic patients, and the severity of the abnormalities depend on the patients’ age and the duration of diabetes. The aim of this experimental study has been to clarify the progressive effects on heart rhythm in neonatal alloxan (ALX) (induced at 5 days of age) diabetic male rats. Cardiac biopotential data were acquired in vivo with a biotelemetry system. After an overnight fast blood glucose in diabetic rats, compared to age-matched controls, was elevated before and at 60, 120 and 180 min after a glucose challenge at 2 and 8 months of age. Heart rate and heart rate variability were modestly reduced and QT interval modestly prolonged in diabetic rats, compared to controls, at 2, 6 and 8 months of age. There was also an age-dependent decline in heart rate and prolongation in QT interval. At 8 months heart rate was 296 ± 8 bpm in diabetic compared to 311 ± 10 bpm in controls and heart rate variability was 27 ± 3 bpm in diabetic rats compared to 32 ± 4 bpm in controls. Physical activity was significantly reduced in diabetic rats, compared to controls, at 6 and 8 months of age. Body temperature was modestly reduced in diabetic rats, compared to controls, at 2, 6 and 8 months. In conclusion, the neonatal ALX-induced diabetes mellitus was associated with disturbances in heart rate, heart rate variability, QT interval which in turn may be associated with changes in physical activity and body temperature. © 2010 Elsevier Ireland Ltd. All rights reserved. Keywords: Neonatal alloxan-induced diabetes mellitus; Heart rate; Heart rate variability; QT interval; Physical activity; Body temperature; Male rats
1. Introduction Cardiovascular disease is the major cause of morbidity and mortality in diabetic patients, and the hearts of diabetic patients are in a compromised condition [1,2]. Diabetic patients show a higher incidence of cardiac arrhythmias, including ventricular fibrillation and sudden death. Their electrocardiogram may show several alterations from normal patterns, many of them related to the QT interval [3–6]. Doppler imaging, echocardiography, radionuclide angiogra∗ Corresponding author at: Department of Physiology, Faculty of Medicine & Health Sciences, UAE University, P.O. Box 17666, Al Ain, United Arab Emirates. Tel.: +971 3 7137536; fax: +971 3 7671966. E-mail address: [email protected] (F.C. Howarth).
0928-4680/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2010.10.001
phy and other techniques have demonstrated a variety of diastolic and systolic dysfunctions in diabetic patients and the severity of the abnormalities depend on the patients’ age and the duration of diabetes. Hemodynamic abnormalities include reduced left ventricular ejection fraction, impaired myocardial velocity at early diastole, abnormal relaxation during the early filling phase, prolonged isovolumetric relaxation, lower peak systolic and early diastolic velocity, impaired diastolic relaxation and filling and reduced peak filling rate [7–18]. Structurally diabetic hearts may have increased interventricular septum thickness, left atrial diameter and left ventricular mass index [17]. The mechanism leading to these pathological alterations in cardiac tissue is not fully understood. Therefore the progressive effects of neonatal alloxan (ALX) induced diabetes mel-
186
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
Table 1 General characteristics of ALX-induced diabetes in male rats (at the age of 5 days) has been followed. 2 months
Body weight (g) Fasting blood glucose (mg/dl)
Control (n = 6)
ALX (n = 8)
Control (n = 6)
ALX (n = 8)
191.8 ± 10.6 60.7 ± 2.3
177.1 ± 6.2 83.6 ± 3.3**
427.2 ± 28.9 80.7 ± 2.2
369.0 ± 9.0 96.1 ± 3.7**
p < 0.01.
litus on heart rhythm have been investigated in male rats.
2. Materials and methods 2.1. Induction of diabetes mellitus Diabetes was induced in 5-day-old male Wistar rats by a single intraperitoneal injection of ALX (200 mg/kg body weight; Sigma, St Louis, MO, USA). The ALX was dissolved in a citrate buffer solution (0.1 mol/l citric acid, 0.1 mol/l sodium citrate; pH 4.5). Age-matched controls received citrate buffer alone. Body weight, blood glucose and glucose tolerance, following an intraperitoneal injection of glucose (2 g/kg bodyweight) after an overnight fast, were measured periodically. Blood glucose was measured in small samples of blood collected from a tail nick with a glucometer (One Touch Ultra, Lifescan Inc., USA). Ethical approval for the project was obtained from the local Faculty of Medicine & Health Sciences Ethics Committee for Animal Research.
rate was recorded hourly and computed from the 5 min average of all normal-to-normal beats as detect by the R-wave of the QRS complex. The heart rate variability was determined as measured heart rate values as the standard deviation of 24 consecutive heart beats. The QT interval was recorded as the average of the time spans as measured from the Qwave minimum to the end of T-wave for all normal beats of the 5 min electrocardiogram. The corrected QT interval was determined from the QT and heart rate of each normal beat of the 5 min electrocardiogram and computed using the Bazzet
a
**
* 500
**
400 ** 300 200 100
2.2. Biotelemetry system
**
0 0
50
100
150
200
Time (min)
b Blood glucose (mg/dl)
Heart biopotential, body temperature and physical activity were monitored using a biotelemetry system (Data Sciences Int., St Paul, MN, USA) according to previously described techniques [19,20]. The system comprised the transmitter devices (TA11CTA-F40, Data Sciences Int., USA), the receivers (RPC-1), a data exchange matrix (20CH) and a personal computer for system configuration, control, acquisition, and data storage. The transmitter devices were surgically implanted in 14 male Wistar rats at 2 months of age, under general anesthesia (sodium pentobarbitone, 45 mg/kg, intraperitoneal). At the time of surgery ALX treated rats weighed 177.1 ± 6.2 g and age-matched control rats weighed 191.8 ± 10.6 g. The devices were inserted into the peritoneal cavity and electrodes from the transmitter were arranged in Einthoven bipolar—lead II configuration with one electrode connected to the right foreleg and one to the left hind leg. After recovery from surgery transmitters were then switched on by activation of a magnetic switch, located in the transmitter device, with a permanent magnet brought into close proximity to the animal. Electrocardiographic, body temperature and physical activity data were acquired 5 min per hour, 24 h per day for the duration of the 6-month study. Heart rate, heart rate variability, QT interval, and corrected QT interval were obtained from the electrocardiographic data. The heart
ALX Control
700 600
Blood glucose (mg/dl)
**
8 months
ALX Control
700 600
**
*
**
500 400 300 200 ** 100 0 0
50
100
150
200
Time (min) Fig. 1. Glucose tolerance. Animals treated with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age. Animals were fasted overnight and then received intraperitonealy 2 g glucose per kg bodyweight. Blood glucose was measured at time zero and then 30, 60, 120 and 180 min after glucose injection. Glucose tolerance tests were performed at (a) 2 and (b) 8 months. Data are mean ± SEM, n = 6–8 rats, *p < 0.01, **p < 0.05.
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
a
187
400
Control
380
ALX
360
BPM
340 320 300
360
280
320
260
280 8
240 8
12
16
20
24
12
28
16
32
20
24
28
32
Age (Week)
b
350
Control ALX
340
BPM
330 320 310 300 290 2
6
8
Age (Month)
Fig. 2. Heart rate of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters were implanted at 2 months. Electrocardiogram data acquired 5 min per hour, 24 h per day: (a) continuous heart rate data and mean trends (inset) and (b) mean heart rate at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
correction, which is QT divided by the square root of heart rate [21,22]. 2.3. Statistics Data were analyzed using SPSS (PASW Statistics v18) and presented as mean ± SEM. Statistical analysis was performed using either Independent samples t-test or Student’s t-test, as appropriate. p-Values < 0.05 were considered significant.
3. Results 3.1. General characteristics of neonatal ALX-induced diabetes Male rats were treated with ALX (200 mg/kg bodyweight, intraperitoneal injection) at 5 days of age. Body weight and
fasting blood glucose are shown in Table 1. Body weight was less and fasting blood glucose was significantly (p < 0.01) elevated in ALX treated rats compared to controls at 2 and 8 months of age. The results of glucose tolerance test at 2 and 8 months of age are shown in Fig. 1. At 60, 120 and 180 min after intraperitoneal injection of glucose (2 g/kg bodyweight) blood glucose was significantly higher in ALX treated rats compared to age-matched controls. 3.2. Effects of neonatal ALX-induced diabetes on heart rate and heart rate variability The effects of ALX-induced diabetes on continuous heart rate (mean trend inset) are shown in Fig. 2a. Mean heart rate at selected times is shown in Fig. 2b. At 2, 6 and 8 months of age the heart rate in diabetic rats was lower compared to age-matched controls. There was also an age-dependent progressive decline in heart rate in diabetic rats and in controls over the same time period. At 8 months of age the heart
188
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
a
50
Control
45
ALX
40 35
BPM
30 25 20
50
15
30
10 10
5
8
12
16
20
24
28
32
0 8
12
16
20
24
28
32
Age (Week)
b
40
Control ALX
35
BPM
30
25
20
15
2
6
8
Age (Month)
Fig. 3. Heart rate variability of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age were followed and transmitters were implanted at 2 months. Electrocardiogram data were acquired 5 min per hour, 24 h per day: (a) continuous heart rate variability data and mean trends (inset) and (b) mean heart rate variability at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
rate was 296 ± 8 bpm (n = 8) in diabetic rats compared to 311 ± 10 bpm (n = 6) in controls (p > 0.05). The effects of ALX-induced diabetes on continuous heart rate variability (mean trend inset) are shown in Fig. 3a. Mean heart rate variability at selected times is shown in Fig. 3b. At 2, 6 and 8 months of age heart rate variability in diabetic rats was reduced compared to age-matched controls. At 8 months after ALX treatment heart rate variability was 27 ± 3 bpm in diabetic rats compared to 32 ± 4 bpm in controls (p > 0.05). 3.3. Effects of neonatal ALX-induced diabetes on QT interval The effects of ALX-induced diabetes on continuous QT interval (mean trend inset) are shown in Fig. 4a. Mean QT interval at selected times is shown in Fig. 4b. At 2, 6 and 8 months of age QT interval in diabetic rats was prolonged compared to age-matched controls and there was also an age-dependent prolongation of QT interval in diabetic rats
and in age-matched controls. At 8 months of age QT interval was 61 ± 2 ms in diabetic rats compared to 58 ± 3 ms in controls. The effects of ALX-induced diabetes on continuous corrected QT interval (mean trend inset) are shown in Fig. 5a. Mean corrected QT interval at selected time points is shown in Fig. 5b. Similar diabetes and age-dependent changes in QT interval were observed after correction. At 8 months of age corrected QT interval was 134 ± 4 ms in diabetic rats compared to 130 ± 7 ms in controls. 3.4. Effects of neonatal ALX-induced diabetes on physical activity The effects of ALX-induced diabetes on physical activity (mean trend inset) are shown in Fig. 6a. Mean physical activity at selected times is shown in Fig. 6b. Physical activity was lower at 2, 6 and 8 months of age, at 6 and 8 months the reduction in physical activity was significant (p < 0.05).
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
a
189
70
Control ALX
65
msec
60 55 65
50
60 55
45 40
50 8
8
12
16
20
24
12
16
20
24
28
28
32
32
Age (Week)
b
66
Control ALX
64 62
msec
60 58 56 54 52 2
6
8
Age (Month)
Fig. 4. QT interval of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters were implanted at 2 months. Electrocardiogram data were acquired 5 min per hour, 24 h per day: (a) continuous QT interval data and mean trends (inset) and (b) mean QT interval at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
3.5. Effects of neonatal ALX-induced diabetes on body temperature
temperature at selected times is shown in Fig. 7b. At 2, 6 and 8 months of age body temperature in diabetic rats was slightly reduced compared to age-matched controls. At 8 months body temperature was 37.5 ± 0.1 ◦ C in diabetic rats compared to 37.7 ± 0.1 ◦ C in controls (p > 0.05).
The effects of ALX-induced diabetes on body temperature (mean trend inset) are shown in Fig. 7a. Mean body
a
b 145
170
140
msec
msec
150
130
135 130
110
90
125
8
12
16
20
24
Age (Week)
28
32
120
2
6
8
Age (Month)
Fig. 5. Corrected QT intervals of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters implanted at 2 months. Electrocardiogram data were acquired 5 min per hour, 24 h per day: (a) continuous corrected QT interval data and mean trends (inset) and (b) mean corrected QT interval at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
190
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192 6.0
4
Count per minute
0
4.0
b
Control ALX
2
5.0
8
12
16
20
24
28
32
3.0 2.0 1.0 0.0
8
12
16
20
24
28
3.0
Control ALX
2.5
Counts per min
a
2.0
*
1.0 0.5 0.0
32
*
1.5
2
6
8
Age (Month)
Age (Week)
Fig. 6. Physical activity of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters were implanted at 2 months. Physical activity data were acquired 5 min per hour, 24 h per day: (a) continuous physical activity data and mean trends (inset) and (b) mean physical activity at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats, *p < 0.05.
in type 2 diabetes mellitus. Fasting blood glucose was elevated and blood glucose concentrations 60, 120 and 180 min after a glucose challenge were significantly elevated in diabetic rats compared to controls. Glucose intolerance has been
4. Discussion When alloxan was administered to neonatal rats the animals developed characteristics that are frequently observed
a
38.8
Control
38.6
ALX
Body Temperature (C)
38.4 38.2 38.0 37.8 37.6 37.4
38.2
37.2
37.7
37.0
37.2
36.8 36.6
b
8
8
12
16
12
20
24
28
16
32
20 Age (Week)
24
28
38.0
32
Control
37.9
ALX
Temperature (C)
37.8 37.7 37.6 37.5 37.4 37.3 37.2
2
6 Age (Month)
8
Fig. 7. Body temperature of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters implanted at 2 months. Body temperature data were acquired 5 min per hour, 24 h per day: (a) continuous body temperature data and mean trends (inset) and (b) mean physical activity at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
previously demonstrated in the ALX-induced type 2 diabetic rat [23]. Rats received ALX (200 mg/kg bodyweight, intraperitoneal injection) at 5 days of age. Biotelemetry transmitters were surgically implanted at 2 months of age when the rats were of suitable size to accommodate the devices. Electrocardiographic data was then recorded for 6 months. At 2, 6 and 8 months of age there was a small reduction in heart rate in diabetic rats compared to age-matched controls. In addition there was an age-dependent progressive decline in heart rate in both diabetic and control rats. Previous studies in ALX-induced diabetic rats have demonstrated reductions in spontaneous atrial beating rates suggesting that intrinsic defects may partly underlie ALX-induced bradycardia [24]. The reductions in heart rate may have occurred at any stage prior to implantation of the biotelemetry devices and may partly be attributed to a direct action of ALX on the heart. Previous studies have demonstrated that rate is reduced in spontaneously beating rat heart when the hearts are perfused with either streptozotocin (STZ), a widely used diabetogenic agent, or ALX [25,26]. In addition a previous in vivo study has demonstrated that heart rate falls rapidly following the administration of STZ and there was a partial recovery of heart rate during insulin treatment [27]. Previous unpublished in vivo experiments in our laboratory have demonstrated a rapid decline in heart rate following administration of ALX (120 mg/kg bodyweight), which can be completely reversed with insulin treatment. Heart rate variability is an index of sympathovagal modulation of heart function and the small reduction in heart rate variability in ALX treated rats compared to controls may be indicative of defective autonomic regulation of heart function [19,28,29]. The prolongation of QT interval is consistent with frequently reported findings in diabetic patients [3–6]. Previous in vivo biotelemetry studies in STZ treated rats have also demonstrated a prolongation of the QT interval, events between depolarization and repolarization of ventricular myocardium, and are consistent with reduction in heart rate and previous reports of prolonged action potentials in papillary preparations from ALX-induced diabetic rat [30]. Other factors which may influence heart rate include physical activity and body temperature. The reductions in body temperature in diabetic rats, compared to controls, were very small. However, changes in physical activity were significant. Previous studies have demonstrated reduced activity and body temperature in STZ-induced diabetic rats and treatment of the rats with daily insulin was able to partially normalize heart rate and body temperature but did not normalize physical activity [27]. In conclusion, the neonatal ALX-induced diabetes mellitus in male rats was associated with disturbances in heart rate, heart rate variability, QT interval which in turn may be partly associated with changes in physical activity and body temperature.
191
Acknowledgement This project was supported by an Individual Grant from the United Arab Emirates University, Al Ain, United Arab Emirates.
References [1] J. Julien, Cardiac complications in non-insulin-dependent diabetes mellitus, J. Diab. Complications 11 (1997) 123–130. [2] N.S. Dhalla, G.N. Pierce, I.R. Innes, R.E. Beamish, Pathogenesis of cardiac dysfunction in diabetes mellitus, Can. J. Cardiol. 1 (4) (1985) 263–281. [3] M. Veglio, A. Chinaglia, P. Cavallo-Perin, QT interval, cardiovascular risk factors and risk of death in diabetes, J. Endocrinol. Invest. 27 (2) (2004) 175–181. [4] K. Takebayashi, R. Sugita, K. Tayama, Y. Aso, Y. Takemura, T. Inukai, The connection between QT dispersion and autonomic neuropathy in patients with type 2 diabetes, Exp. Clin. Endocrinol. Diab. 111 (6) (2003) 351–357. [5] B.S. Rana, M.M. Band, S. Ogston, A.D. Morris, S.D. Pringle, A.D. Struthers, Relation of QT interval dispersion to the number of different cardiac abnormalities in diabetes mellitus, Am. J. Cardiol. 90 (5) (2002) 483–487. [6] M. Veglio, G. Bruno, M. Borra, G. Macchia, G. Bargero, N. D’Errico, et al., Prevalence of increased QT interval duration and dispersion in type 2 diabetic patients and its relationship with coronary heart disease: a population-based cohort, J. Int. Med. 251 (4) (2002) 317–324. [7] H. von Bibra, I.S. Thrainsdottir, A. Hansen, V. Dounis, K. Malmberg, L. Ryden, Tissue Doppler imaging for the detection and quantitation of myocardial dysfunction in patients with type 2 diabetes mellitus, Diab. Vasc. Dis. Res. 2 (1) (2005) 24–30. [8] P. Chareonthaitawee, P. Sorajja, N. Rajagopalan, T.D. Miller, D.O. Hodge, R.L. Frye, et al., Prevalence and prognosis of left ventricular systolic dysfunction in asymptomatic diabetic patients without known coronary artery disease referred for stress single-photon emission computed tomography and assessment of left ventricular function, Am. Heart J. 154 (3) (2007) 567–574. [9] J.E. Sharman, B.A. Haluska, Z.Y. Fang, J.B. Prins, T.H. Marwick, Association of arterial wave properties and diastolic dysfunction in patients with type 2 diabetes mellitus, Am. J. Cardiol. 99 (6) (2007) 844–848. [10] L. Markuszewski, T. Grycewicz, R. Pietruszynski, D. Michalkiewicz, N. Roszczyk, Glycosylated hemoglobin and left ventricular diastolic dysfunction in patients with type 2 diabetes mellitus, Pol. Merkur. Lekarski 21 (121) (2006) 8–11. [11] A. Loimaala, K. Groundstroem, S. Majahalme, A. Nenonen, I. Vuori, Impaired myocardial function in newly onset type 2 diabetes associates with arterial stiffness, Eur. J. Echocardiogr. 7 (5) (2006) 341–347. [12] E.E. Jweied, R.D. McKinney, L.A. Walker, I. Brodsky, A.S. Geha, M.G. Massad, et al., Depressed cardiac myofilament function in human diabetes mellitus, Am. J. Physiol. Heart Circ. Physiol. 289 (6) (2005) H2478–H2483. [13] G. Bajraktari, S. Qirko, A. Bakalli, N. Rexhepaj, S. Elizi, Reduced left ventricular diastolic function in asymptomatic patients with noninsulin-dependent diabetes mellitus, Med. Arch. 58 (6) (2004) 339–341. [14] J.K. Boyer, S. Thanigaraj, K.B. Schechtman, J.E. Perez, Prevalence of ventricular diastolic dysfunction in asymptomatic, normotensive patients with diabetes mellitus, Am. J. Cardiol. 93 (7) (2004) 870–875. [15] A.K. Annonu, A.A. Fattah, M.S. Mokhtar, S. Ghareeb, A. Elhendy, Left ventricular systolic and diastolic functional abnormalities in asymptomatic patients with non-insulin-dependent diabetes mellitus, J. Am. Soc. Echocardiogr. 14 (9) (2001) 885–891. [16] E. Astorri, P. Fiorina, G. Gavaruzzi, A. Astorri, G. Magnati, Left ventricular function in insulin-dependent and in non-insulin-dependent
192
[17]
[18]
[19]
[20]
[21] [22] [23]
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192 diabetic patients: radionuclide assessment, Cardiology 88 (2) (1997) 152–155. P. Di Bonito, S. Cuomo, N. Moio, G. Sibilio, D. Sabatini, S. Quattrin, et al., Diastolic dysfunction in patients with non-insulin-dependent diabetes mellitus of short duration, Diab. Med. 13 (4) (1996) 321–324. K. Hiramatsu, N. Ohara, S. Shigematsu, T. Aizawa, F. Ishihara, A. Niwa, et al., Left ventricular filling abnormalities in non-insulin-dependent diabetes mellitus and improvement by a short-term glycemic control, Am. J. Cardiol. 70 (13) (1992) 1185–1189. F.C. Howarth, M. Jacobson, M. Shafiullah, E. Adeghate, Long-term effects of streptozotocin-induced diabetes on the electrocardiogram, physical activity and body temperature in rats, Exp. Physiol. 90 (6) (2005) 827–835. F.C. Howarth, M. Jacobson, M. Shafiullah, E. Adeghate, Long-term effects of type 2 diabetes mellitus on heart rhythm in the Goto–Kakizaki rat, Exp. Physiol. 93 (3) (2008) 362–369. B. Hosmane, C. Locke, D. Morris, QT interval: correction for heart rate, J. Appl. Res. 6 (4) (2006) 288–299. F.C. Howarth, E. Adeghate, M. Jacobson, Heart rate and QT interval in streptozotocin-induced diabetic rat, J. Med. Sci. 2 (3) (2009) 108–118. M.C. Soares de Alencar, C. Ribeiro, G.G. de Araujo, M.B. de Araujo, F. Barros Manchado-Gobatto, F.A. Voltarelli, et al., Exercise training in the aerobic/anaerobic metabolic transition prevents glucose intolerance in alloxan-treated rats, BMC Endocr. Disord. 8 (11) (2008) 11.
[24] A.T. Ozcelikay, N. Yidizoglu-Ari, A. Ozuari, Y. Ozturk, V.M. Altan, The effect of vanadate on alloxan-diabetic rat atria, Diab. Res. Clin. Pract. 19 (3) (1993) 189–194. [25] F.C. Howarth, R. Al Sharhan, A. Al Hammadi, M.A. Qureshi, Effects of streptozotocin-induced diabetes on action potentials in the sinoatrial node compared with other regions of the rat heart, Mol. Cell Biochem. 300 (1–2) (2007) 39–46. [26] K.A. Salem, M. Kosanovic, A. Qureshi, M. Ljubisavljevic, F.C. Howarth, The direct effects of streptozotocin and alloxan on contractile function in rat heart, Pharmacol. Res. 59 (4) (2009) 235–241. [27] F.C. Howarth, M. Jacobson, M. Shafiullah, E. Adeghate, Effects of insulin treatment on heart rhythm, body temperature and physical activity in streptozotocin-induced diabetic rat, Clin. Exp. Pharmacol. Physiol. 33 (4) (2006) 327–331. [28] A.E. Aubert, D. Ramaekers, F. Beckers, R. Breem, C. Denef, W.F. Van de, et al., The analysis of heart rate variability in unrestrained rats. Validation of method and results, Comput. Methods Prog. Biomed. 60 (3) (1999) 197–213. [29] M. Bootsma, C.A. Swenne, H.H. Van Bolhuis, P.C. Chang, V.M. Cats, A.V. Bruschke, Heart rate and heart rate variability as indexes of sympathovagal balance, Am. J. Physiol. 266 (4 Pt 2) (1994) H1565–H1571. [30] M.P. Sauviat, D. Feuvray, Electrophysiological analysis of the sensitivity to calcium in ventricular muscle from alloxan diabetic rats, Basic Res. Cardiol. 81 (5) (1986) 489–496.
Heart rhythm disturbances in the neonatal alloxan-induced diabetic rat Frank Christopher Howarth a,∗ , Mohamed Shafiullah a , Ernest Adeghate a , Milos Ljubisavljevic a , Michael Jacobson b a b
Faculty of Medicine & Health Sciences, UAE University, United Arab Emirates Biomedical Engineering, Higher Colleges of Technology, United Arab Emirates Received 5 May 2010; accepted 9 October 2010
Abstract Diabetic patients show a higher incidence of cardiac arrhythmias, including ventricular fibrillation and sudden death. Their electrocardiograms may show several alterations from normal patterns, many of them related to the QT. Various diastolic and systolic abnormalities are frequently reported in diabetic patients, and the severity of the abnormalities depend on the patients’ age and the duration of diabetes. The aim of this experimental study has been to clarify the progressive effects on heart rhythm in neonatal alloxan (ALX) (induced at 5 days of age) diabetic male rats. Cardiac biopotential data were acquired in vivo with a biotelemetry system. After an overnight fast blood glucose in diabetic rats, compared to age-matched controls, was elevated before and at 60, 120 and 180 min after a glucose challenge at 2 and 8 months of age. Heart rate and heart rate variability were modestly reduced and QT interval modestly prolonged in diabetic rats, compared to controls, at 2, 6 and 8 months of age. There was also an age-dependent decline in heart rate and prolongation in QT interval. At 8 months heart rate was 296 ± 8 bpm in diabetic compared to 311 ± 10 bpm in controls and heart rate variability was 27 ± 3 bpm in diabetic rats compared to 32 ± 4 bpm in controls. Physical activity was significantly reduced in diabetic rats, compared to controls, at 6 and 8 months of age. Body temperature was modestly reduced in diabetic rats, compared to controls, at 2, 6 and 8 months. In conclusion, the neonatal ALX-induced diabetes mellitus was associated with disturbances in heart rate, heart rate variability, QT interval which in turn may be associated with changes in physical activity and body temperature. © 2010 Elsevier Ireland Ltd. All rights reserved. Keywords: Neonatal alloxan-induced diabetes mellitus; Heart rate; Heart rate variability; QT interval; Physical activity; Body temperature; Male rats
1. Introduction Cardiovascular disease is the major cause of morbidity and mortality in diabetic patients, and the hearts of diabetic patients are in a compromised condition [1,2]. Diabetic patients show a higher incidence of cardiac arrhythmias, including ventricular fibrillation and sudden death. Their electrocardiogram may show several alterations from normal patterns, many of them related to the QT interval [3–6]. Doppler imaging, echocardiography, radionuclide angiogra∗ Corresponding author at: Department of Physiology, Faculty of Medicine & Health Sciences, UAE University, P.O. Box 17666, Al Ain, United Arab Emirates. Tel.: +971 3 7137536; fax: +971 3 7671966. E-mail address: [email protected] (F.C. Howarth).
0928-4680/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.pathophys.2010.10.001
phy and other techniques have demonstrated a variety of diastolic and systolic dysfunctions in diabetic patients and the severity of the abnormalities depend on the patients’ age and the duration of diabetes. Hemodynamic abnormalities include reduced left ventricular ejection fraction, impaired myocardial velocity at early diastole, abnormal relaxation during the early filling phase, prolonged isovolumetric relaxation, lower peak systolic and early diastolic velocity, impaired diastolic relaxation and filling and reduced peak filling rate [7–18]. Structurally diabetic hearts may have increased interventricular septum thickness, left atrial diameter and left ventricular mass index [17]. The mechanism leading to these pathological alterations in cardiac tissue is not fully understood. Therefore the progressive effects of neonatal alloxan (ALX) induced diabetes mel-
186
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
Table 1 General characteristics of ALX-induced diabetes in male rats (at the age of 5 days) has been followed. 2 months
Body weight (g) Fasting blood glucose (mg/dl)
Control (n = 6)
ALX (n = 8)
Control (n = 6)
ALX (n = 8)
191.8 ± 10.6 60.7 ± 2.3
177.1 ± 6.2 83.6 ± 3.3**
427.2 ± 28.9 80.7 ± 2.2
369.0 ± 9.0 96.1 ± 3.7**
p < 0.01.
litus on heart rhythm have been investigated in male rats.
2. Materials and methods 2.1. Induction of diabetes mellitus Diabetes was induced in 5-day-old male Wistar rats by a single intraperitoneal injection of ALX (200 mg/kg body weight; Sigma, St Louis, MO, USA). The ALX was dissolved in a citrate buffer solution (0.1 mol/l citric acid, 0.1 mol/l sodium citrate; pH 4.5). Age-matched controls received citrate buffer alone. Body weight, blood glucose and glucose tolerance, following an intraperitoneal injection of glucose (2 g/kg bodyweight) after an overnight fast, were measured periodically. Blood glucose was measured in small samples of blood collected from a tail nick with a glucometer (One Touch Ultra, Lifescan Inc., USA). Ethical approval for the project was obtained from the local Faculty of Medicine & Health Sciences Ethics Committee for Animal Research.
rate was recorded hourly and computed from the 5 min average of all normal-to-normal beats as detect by the R-wave of the QRS complex. The heart rate variability was determined as measured heart rate values as the standard deviation of 24 consecutive heart beats. The QT interval was recorded as the average of the time spans as measured from the Qwave minimum to the end of T-wave for all normal beats of the 5 min electrocardiogram. The corrected QT interval was determined from the QT and heart rate of each normal beat of the 5 min electrocardiogram and computed using the Bazzet
a
**
* 500
**
400 ** 300 200 100
2.2. Biotelemetry system
**
0 0
50
100
150
200
Time (min)
b Blood glucose (mg/dl)
Heart biopotential, body temperature and physical activity were monitored using a biotelemetry system (Data Sciences Int., St Paul, MN, USA) according to previously described techniques [19,20]. The system comprised the transmitter devices (TA11CTA-F40, Data Sciences Int., USA), the receivers (RPC-1), a data exchange matrix (20CH) and a personal computer for system configuration, control, acquisition, and data storage. The transmitter devices were surgically implanted in 14 male Wistar rats at 2 months of age, under general anesthesia (sodium pentobarbitone, 45 mg/kg, intraperitoneal). At the time of surgery ALX treated rats weighed 177.1 ± 6.2 g and age-matched control rats weighed 191.8 ± 10.6 g. The devices were inserted into the peritoneal cavity and electrodes from the transmitter were arranged in Einthoven bipolar—lead II configuration with one electrode connected to the right foreleg and one to the left hind leg. After recovery from surgery transmitters were then switched on by activation of a magnetic switch, located in the transmitter device, with a permanent magnet brought into close proximity to the animal. Electrocardiographic, body temperature and physical activity data were acquired 5 min per hour, 24 h per day for the duration of the 6-month study. Heart rate, heart rate variability, QT interval, and corrected QT interval were obtained from the electrocardiographic data. The heart
ALX Control
700 600
Blood glucose (mg/dl)
**
8 months
ALX Control
700 600
**
*
**
500 400 300 200 ** 100 0 0
50
100
150
200
Time (min) Fig. 1. Glucose tolerance. Animals treated with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age. Animals were fasted overnight and then received intraperitonealy 2 g glucose per kg bodyweight. Blood glucose was measured at time zero and then 30, 60, 120 and 180 min after glucose injection. Glucose tolerance tests were performed at (a) 2 and (b) 8 months. Data are mean ± SEM, n = 6–8 rats, *p < 0.01, **p < 0.05.
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
a
187
400
Control
380
ALX
360
BPM
340 320 300
360
280
320
260
280 8
240 8
12
16
20
24
12
28
16
32
20
24
28
32
Age (Week)
b
350
Control ALX
340
BPM
330 320 310 300 290 2
6
8
Age (Month)
Fig. 2. Heart rate of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters were implanted at 2 months. Electrocardiogram data acquired 5 min per hour, 24 h per day: (a) continuous heart rate data and mean trends (inset) and (b) mean heart rate at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
correction, which is QT divided by the square root of heart rate [21,22]. 2.3. Statistics Data were analyzed using SPSS (PASW Statistics v18) and presented as mean ± SEM. Statistical analysis was performed using either Independent samples t-test or Student’s t-test, as appropriate. p-Values < 0.05 were considered significant.
3. Results 3.1. General characteristics of neonatal ALX-induced diabetes Male rats were treated with ALX (200 mg/kg bodyweight, intraperitoneal injection) at 5 days of age. Body weight and
fasting blood glucose are shown in Table 1. Body weight was less and fasting blood glucose was significantly (p < 0.01) elevated in ALX treated rats compared to controls at 2 and 8 months of age. The results of glucose tolerance test at 2 and 8 months of age are shown in Fig. 1. At 60, 120 and 180 min after intraperitoneal injection of glucose (2 g/kg bodyweight) blood glucose was significantly higher in ALX treated rats compared to age-matched controls. 3.2. Effects of neonatal ALX-induced diabetes on heart rate and heart rate variability The effects of ALX-induced diabetes on continuous heart rate (mean trend inset) are shown in Fig. 2a. Mean heart rate at selected times is shown in Fig. 2b. At 2, 6 and 8 months of age the heart rate in diabetic rats was lower compared to age-matched controls. There was also an age-dependent progressive decline in heart rate in diabetic rats and in controls over the same time period. At 8 months of age the heart
188
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
a
50
Control
45
ALX
40 35
BPM
30 25 20
50
15
30
10 10
5
8
12
16
20
24
28
32
0 8
12
16
20
24
28
32
Age (Week)
b
40
Control ALX
35
BPM
30
25
20
15
2
6
8
Age (Month)
Fig. 3. Heart rate variability of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age were followed and transmitters were implanted at 2 months. Electrocardiogram data were acquired 5 min per hour, 24 h per day: (a) continuous heart rate variability data and mean trends (inset) and (b) mean heart rate variability at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
rate was 296 ± 8 bpm (n = 8) in diabetic rats compared to 311 ± 10 bpm (n = 6) in controls (p > 0.05). The effects of ALX-induced diabetes on continuous heart rate variability (mean trend inset) are shown in Fig. 3a. Mean heart rate variability at selected times is shown in Fig. 3b. At 2, 6 and 8 months of age heart rate variability in diabetic rats was reduced compared to age-matched controls. At 8 months after ALX treatment heart rate variability was 27 ± 3 bpm in diabetic rats compared to 32 ± 4 bpm in controls (p > 0.05). 3.3. Effects of neonatal ALX-induced diabetes on QT interval The effects of ALX-induced diabetes on continuous QT interval (mean trend inset) are shown in Fig. 4a. Mean QT interval at selected times is shown in Fig. 4b. At 2, 6 and 8 months of age QT interval in diabetic rats was prolonged compared to age-matched controls and there was also an age-dependent prolongation of QT interval in diabetic rats
and in age-matched controls. At 8 months of age QT interval was 61 ± 2 ms in diabetic rats compared to 58 ± 3 ms in controls. The effects of ALX-induced diabetes on continuous corrected QT interval (mean trend inset) are shown in Fig. 5a. Mean corrected QT interval at selected time points is shown in Fig. 5b. Similar diabetes and age-dependent changes in QT interval were observed after correction. At 8 months of age corrected QT interval was 134 ± 4 ms in diabetic rats compared to 130 ± 7 ms in controls. 3.4. Effects of neonatal ALX-induced diabetes on physical activity The effects of ALX-induced diabetes on physical activity (mean trend inset) are shown in Fig. 6a. Mean physical activity at selected times is shown in Fig. 6b. Physical activity was lower at 2, 6 and 8 months of age, at 6 and 8 months the reduction in physical activity was significant (p < 0.05).
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
a
189
70
Control ALX
65
msec
60 55 65
50
60 55
45 40
50 8
8
12
16
20
24
12
16
20
24
28
28
32
32
Age (Week)
b
66
Control ALX
64 62
msec
60 58 56 54 52 2
6
8
Age (Month)
Fig. 4. QT interval of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters were implanted at 2 months. Electrocardiogram data were acquired 5 min per hour, 24 h per day: (a) continuous QT interval data and mean trends (inset) and (b) mean QT interval at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
3.5. Effects of neonatal ALX-induced diabetes on body temperature
temperature at selected times is shown in Fig. 7b. At 2, 6 and 8 months of age body temperature in diabetic rats was slightly reduced compared to age-matched controls. At 8 months body temperature was 37.5 ± 0.1 ◦ C in diabetic rats compared to 37.7 ± 0.1 ◦ C in controls (p > 0.05).
The effects of ALX-induced diabetes on body temperature (mean trend inset) are shown in Fig. 7a. Mean body
a
b 145
170
140
msec
msec
150
130
135 130
110
90
125
8
12
16
20
24
Age (Week)
28
32
120
2
6
8
Age (Month)
Fig. 5. Corrected QT intervals of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters implanted at 2 months. Electrocardiogram data were acquired 5 min per hour, 24 h per day: (a) continuous corrected QT interval data and mean trends (inset) and (b) mean corrected QT interval at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
190
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192 6.0
4
Count per minute
0
4.0
b
Control ALX
2
5.0
8
12
16
20
24
28
32
3.0 2.0 1.0 0.0
8
12
16
20
24
28
3.0
Control ALX
2.5
Counts per min
a
2.0
*
1.0 0.5 0.0
32
*
1.5
2
6
8
Age (Month)
Age (Week)
Fig. 6. Physical activity of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters were implanted at 2 months. Physical activity data were acquired 5 min per hour, 24 h per day: (a) continuous physical activity data and mean trends (inset) and (b) mean physical activity at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats, *p < 0.05.
in type 2 diabetes mellitus. Fasting blood glucose was elevated and blood glucose concentrations 60, 120 and 180 min after a glucose challenge were significantly elevated in diabetic rats compared to controls. Glucose intolerance has been
4. Discussion When alloxan was administered to neonatal rats the animals developed characteristics that are frequently observed
a
38.8
Control
38.6
ALX
Body Temperature (C)
38.4 38.2 38.0 37.8 37.6 37.4
38.2
37.2
37.7
37.0
37.2
36.8 36.6
b
8
8
12
16
12
20
24
28
16
32
20 Age (Week)
24
28
38.0
32
Control
37.9
ALX
Temperature (C)
37.8 37.7 37.6 37.5 37.4 37.3 37.2
2
6 Age (Month)
8
Fig. 7. Body temperature of male rats injected with alloxan (ALX; 200 mg/kg bodyweight, ip) at 5 days of age and transmitters implanted at 2 months. Body temperature data were acquired 5 min per hour, 24 h per day: (a) continuous body temperature data and mean trends (inset) and (b) mean physical activity at 2, 6 and 8 months of age. Data are mean ± SEM, n = 6–8 rats.
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192
previously demonstrated in the ALX-induced type 2 diabetic rat [23]. Rats received ALX (200 mg/kg bodyweight, intraperitoneal injection) at 5 days of age. Biotelemetry transmitters were surgically implanted at 2 months of age when the rats were of suitable size to accommodate the devices. Electrocardiographic data was then recorded for 6 months. At 2, 6 and 8 months of age there was a small reduction in heart rate in diabetic rats compared to age-matched controls. In addition there was an age-dependent progressive decline in heart rate in both diabetic and control rats. Previous studies in ALX-induced diabetic rats have demonstrated reductions in spontaneous atrial beating rates suggesting that intrinsic defects may partly underlie ALX-induced bradycardia [24]. The reductions in heart rate may have occurred at any stage prior to implantation of the biotelemetry devices and may partly be attributed to a direct action of ALX on the heart. Previous studies have demonstrated that rate is reduced in spontaneously beating rat heart when the hearts are perfused with either streptozotocin (STZ), a widely used diabetogenic agent, or ALX [25,26]. In addition a previous in vivo study has demonstrated that heart rate falls rapidly following the administration of STZ and there was a partial recovery of heart rate during insulin treatment [27]. Previous unpublished in vivo experiments in our laboratory have demonstrated a rapid decline in heart rate following administration of ALX (120 mg/kg bodyweight), which can be completely reversed with insulin treatment. Heart rate variability is an index of sympathovagal modulation of heart function and the small reduction in heart rate variability in ALX treated rats compared to controls may be indicative of defective autonomic regulation of heart function [19,28,29]. The prolongation of QT interval is consistent with frequently reported findings in diabetic patients [3–6]. Previous in vivo biotelemetry studies in STZ treated rats have also demonstrated a prolongation of the QT interval, events between depolarization and repolarization of ventricular myocardium, and are consistent with reduction in heart rate and previous reports of prolonged action potentials in papillary preparations from ALX-induced diabetic rat [30]. Other factors which may influence heart rate include physical activity and body temperature. The reductions in body temperature in diabetic rats, compared to controls, were very small. However, changes in physical activity were significant. Previous studies have demonstrated reduced activity and body temperature in STZ-induced diabetic rats and treatment of the rats with daily insulin was able to partially normalize heart rate and body temperature but did not normalize physical activity [27]. In conclusion, the neonatal ALX-induced diabetes mellitus in male rats was associated with disturbances in heart rate, heart rate variability, QT interval which in turn may be partly associated with changes in physical activity and body temperature.
191
Acknowledgement This project was supported by an Individual Grant from the United Arab Emirates University, Al Ain, United Arab Emirates.
References [1] J. Julien, Cardiac complications in non-insulin-dependent diabetes mellitus, J. Diab. Complications 11 (1997) 123–130. [2] N.S. Dhalla, G.N. Pierce, I.R. Innes, R.E. Beamish, Pathogenesis of cardiac dysfunction in diabetes mellitus, Can. J. Cardiol. 1 (4) (1985) 263–281. [3] M. Veglio, A. Chinaglia, P. Cavallo-Perin, QT interval, cardiovascular risk factors and risk of death in diabetes, J. Endocrinol. Invest. 27 (2) (2004) 175–181. [4] K. Takebayashi, R. Sugita, K. Tayama, Y. Aso, Y. Takemura, T. Inukai, The connection between QT dispersion and autonomic neuropathy in patients with type 2 diabetes, Exp. Clin. Endocrinol. Diab. 111 (6) (2003) 351–357. [5] B.S. Rana, M.M. Band, S. Ogston, A.D. Morris, S.D. Pringle, A.D. Struthers, Relation of QT interval dispersion to the number of different cardiac abnormalities in diabetes mellitus, Am. J. Cardiol. 90 (5) (2002) 483–487. [6] M. Veglio, G. Bruno, M. Borra, G. Macchia, G. Bargero, N. D’Errico, et al., Prevalence of increased QT interval duration and dispersion in type 2 diabetic patients and its relationship with coronary heart disease: a population-based cohort, J. Int. Med. 251 (4) (2002) 317–324. [7] H. von Bibra, I.S. Thrainsdottir, A. Hansen, V. Dounis, K. Malmberg, L. Ryden, Tissue Doppler imaging for the detection and quantitation of myocardial dysfunction in patients with type 2 diabetes mellitus, Diab. Vasc. Dis. Res. 2 (1) (2005) 24–30. [8] P. Chareonthaitawee, P. Sorajja, N. Rajagopalan, T.D. Miller, D.O. Hodge, R.L. Frye, et al., Prevalence and prognosis of left ventricular systolic dysfunction in asymptomatic diabetic patients without known coronary artery disease referred for stress single-photon emission computed tomography and assessment of left ventricular function, Am. Heart J. 154 (3) (2007) 567–574. [9] J.E. Sharman, B.A. Haluska, Z.Y. Fang, J.B. Prins, T.H. Marwick, Association of arterial wave properties and diastolic dysfunction in patients with type 2 diabetes mellitus, Am. J. Cardiol. 99 (6) (2007) 844–848. [10] L. Markuszewski, T. Grycewicz, R. Pietruszynski, D. Michalkiewicz, N. Roszczyk, Glycosylated hemoglobin and left ventricular diastolic dysfunction in patients with type 2 diabetes mellitus, Pol. Merkur. Lekarski 21 (121) (2006) 8–11. [11] A. Loimaala, K. Groundstroem, S. Majahalme, A. Nenonen, I. Vuori, Impaired myocardial function in newly onset type 2 diabetes associates with arterial stiffness, Eur. J. Echocardiogr. 7 (5) (2006) 341–347. [12] E.E. Jweied, R.D. McKinney, L.A. Walker, I. Brodsky, A.S. Geha, M.G. Massad, et al., Depressed cardiac myofilament function in human diabetes mellitus, Am. J. Physiol. Heart Circ. Physiol. 289 (6) (2005) H2478–H2483. [13] G. Bajraktari, S. Qirko, A. Bakalli, N. Rexhepaj, S. Elizi, Reduced left ventricular diastolic function in asymptomatic patients with noninsulin-dependent diabetes mellitus, Med. Arch. 58 (6) (2004) 339–341. [14] J.K. Boyer, S. Thanigaraj, K.B. Schechtman, J.E. Perez, Prevalence of ventricular diastolic dysfunction in asymptomatic, normotensive patients with diabetes mellitus, Am. J. Cardiol. 93 (7) (2004) 870–875. [15] A.K. Annonu, A.A. Fattah, M.S. Mokhtar, S. Ghareeb, A. Elhendy, Left ventricular systolic and diastolic functional abnormalities in asymptomatic patients with non-insulin-dependent diabetes mellitus, J. Am. Soc. Echocardiogr. 14 (9) (2001) 885–891. [16] E. Astorri, P. Fiorina, G. Gavaruzzi, A. Astorri, G. Magnati, Left ventricular function in insulin-dependent and in non-insulin-dependent
192
[17]
[18]
[19]
[20]
[21] [22] [23]
F.C. Howarth et al. / Pathophysiology 18 (2011) 185–192 diabetic patients: radionuclide assessment, Cardiology 88 (2) (1997) 152–155. P. Di Bonito, S. Cuomo, N. Moio, G. Sibilio, D. Sabatini, S. Quattrin, et al., Diastolic dysfunction in patients with non-insulin-dependent diabetes mellitus of short duration, Diab. Med. 13 (4) (1996) 321–324. K. Hiramatsu, N. Ohara, S. Shigematsu, T. Aizawa, F. Ishihara, A. Niwa, et al., Left ventricular filling abnormalities in non-insulin-dependent diabetes mellitus and improvement by a short-term glycemic control, Am. J. Cardiol. 70 (13) (1992) 1185–1189. F.C. Howarth, M. Jacobson, M. Shafiullah, E. Adeghate, Long-term effects of streptozotocin-induced diabetes on the electrocardiogram, physical activity and body temperature in rats, Exp. Physiol. 90 (6) (2005) 827–835. F.C. Howarth, M. Jacobson, M. Shafiullah, E. Adeghate, Long-term effects of type 2 diabetes mellitus on heart rhythm in the Goto–Kakizaki rat, Exp. Physiol. 93 (3) (2008) 362–369. B. Hosmane, C. Locke, D. Morris, QT interval: correction for heart rate, J. Appl. Res. 6 (4) (2006) 288–299. F.C. Howarth, E. Adeghate, M. Jacobson, Heart rate and QT interval in streptozotocin-induced diabetic rat, J. Med. Sci. 2 (3) (2009) 108–118. M.C. Soares de Alencar, C. Ribeiro, G.G. de Araujo, M.B. de Araujo, F. Barros Manchado-Gobatto, F.A. Voltarelli, et al., Exercise training in the aerobic/anaerobic metabolic transition prevents glucose intolerance in alloxan-treated rats, BMC Endocr. Disord. 8 (11) (2008) 11.
[24] A.T. Ozcelikay, N. Yidizoglu-Ari, A. Ozuari, Y. Ozturk, V.M. Altan, The effect of vanadate on alloxan-diabetic rat atria, Diab. Res. Clin. Pract. 19 (3) (1993) 189–194. [25] F.C. Howarth, R. Al Sharhan, A. Al Hammadi, M.A. Qureshi, Effects of streptozotocin-induced diabetes on action potentials in the sinoatrial node compared with other regions of the rat heart, Mol. Cell Biochem. 300 (1–2) (2007) 39–46. [26] K.A. Salem, M. Kosanovic, A. Qureshi, M. Ljubisavljevic, F.C. Howarth, The direct effects of streptozotocin and alloxan on contractile function in rat heart, Pharmacol. Res. 59 (4) (2009) 235–241. [27] F.C. Howarth, M. Jacobson, M. Shafiullah, E. Adeghate, Effects of insulin treatment on heart rhythm, body temperature and physical activity in streptozotocin-induced diabetic rat, Clin. Exp. Pharmacol. Physiol. 33 (4) (2006) 327–331. [28] A.E. Aubert, D. Ramaekers, F. Beckers, R. Breem, C. Denef, W.F. Van de, et al., The analysis of heart rate variability in unrestrained rats. Validation of method and results, Comput. Methods Prog. Biomed. 60 (3) (1999) 197–213. [29] M. Bootsma, C.A. Swenne, H.H. Van Bolhuis, P.C. Chang, V.M. Cats, A.V. Bruschke, Heart rate and heart rate variability as indexes of sympathovagal balance, Am. J. Physiol. 266 (4 Pt 2) (1994) H1565–H1571. [30] M.P. Sauviat, D. Feuvray, Electrophysiological analysis of the sensitivity to calcium in ventricular muscle from alloxan diabetic rats, Basic Res. Cardiol. 81 (5) (1986) 489–496.