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Altered cardiac automaticity and conduction in experimental diabetes mellitus
Journal
of Molecular
Altered
and Cellular
Cardiac
Cardiology
( 1980)
Automaticity Diabetes
12, 134 1- 135 1
and Conduction Mellitus*
JOCHEN SENGES, JOHANNES BRACHMANN, CHRISTIAN HASSLACHER, EBERHARD WEIHE Abteilung
Innere
AND
Medizin III (Kardiologie), Medizinische Anatomisches Znstitut III, 69 Heidelberg,
(Received
11 June
in Experimental
DIETER PELZER, WOLFGANG KUBLER
Universitiitsklinik Germany
1979, accepted in revised form
3
and
June1980)
J. SENGES, J. BRACHMANN, D. PELZER, CH. HASSLACHER, E. WEIHE AND W. K~~BLER. Altered Cardiac Automaticity and Conduction in Experimental Diabetes Mellitus. Journal of Molecular and Cellular Cardiology ( 1980) 12, 1341- 135 1. The electrophysiological and ultrastructural alterations that occur in chronic diabetes mellitus were studied in isolated, superfused atria including sinoatrial (SA) and atrioventricular (AV) nodes that were obtained from rabbits subjected to alloxan-diabetes for 100 days. Transmembrane action potentials were recorded simultaneously with the His bundle electrogram. The results were compared with corresponding values observed in atria from normal, non-diabetic rabbits. The abnormalities of automaticity and conduction found in atria of alloxan-diabetic rabbits included lower sinus rate, longer poststimulatory sinus-node recovery time, inhomogeneity of atria1 conduction and the occurrence of AV nodal block. Ultrastructural analysis of the diabetic preparations revealed intracellular accumulation of abundant glycogen and various mitochondrial abnormalities but a normal appearance of the small coronary vessels. It was concluded that chronic alloxandiabetes mellitus may directly alter the electrophysiological and ultrastructural properties of nodal and atria1 fibers in the absence of vascular damage. KEY
WORDS:
Alloxan-induced
diabetes;
Sinus
node;
Atrium;
Ultrastructure;
Brady-
cardias.
1. Introduction The present experiments were derived from a clinical observation obtained in patients with permanent cardiac pacemakers that diabetes mellitus may represent a risk factor facilitating the development of severe sinoatrial nodal dysfunction and of high degree atrioventricular block [3, 41. Diabetes appears to be associated with a markedly increased mortality rate from cardiac disease [8]. This has been traditionally attributed to secondary coronary artery disease but recent experimental and clinical observations favour the hypothesis of a diabetic cardiomyopathy independent of vascular alterations [S, IO]. Address for 69 Heidelberg, * This work Electrostimulation, 1978. 0022-2828/80/
reprints: Jochen Sengcs, M.D., Medizinische Universitatsklinik, Bergheimerstr., Germany. was presented in part at the International Symposium: Troubles du Rhythme et Toulouse 1977 and at the III. Internationales Arrhythmie-Symposium, Wien 12 1341+
11 $02.00/O
0
1980 Academic
Press Inc.
(London)
Limited
1342
J. SENGES
ET AL.
The effects of diabetes mellitus on myocardial function and metabolism have been extensively studied in man and animals [9, 101 but the electrophysiological alterations have not yet been described. The purpose of the present study was to examine various parameters of automaticity and conduction in atria1 preparations obtained from chronic alloxan-diabetic rabbits and to relate the electrophysiologic results to ultrastructural findings observed in the corresponding tissues including the small coronary vessels.
2. Materials
and Methods
Sixteen male New Zealand white rabbits were selected for the study. The animals were divided at random into two groups consisting of eight normal control and of eight alloxan-treated rabbits. All control and alloxan-treated animals received the same diet, consisting of commercial rabbit chow (Georg Plange Co., Soest, W. Germany) and were kept under conditions for an average of 100 f 15 days. Alloxan monohydrate (130 mg/kg, i.v.) was administered to rabbits that had been fasted for the previous 15 hours [IZ]. In the chronic control and in the diabetic rabbits, venous blood samples were taken in the fasted state at the onset of the study and at one month intervals thereafter for determination of plasma values for glucose, cholesterol, triglycerides and urea. Electrofihysiological
studies
For the terminal study, preparations consisting of sinoatrial node, crista terminalis, pectinate muscles of atria1 appendage, intraatrial septum, coronary sinus, atrioventricular node and His bundle were isolated and superfused with modified Tyrode’s solution at 36 & 0.5”C. The pH (7.4 f 0.03; n = 16), Pcoz (42 f 1.8 mmHg) and PO, (460 & 30 mmHg) remained unchanged throughout the experiment. A detailed description of the methods of dissection and mounting of the preparation, of the composition of the Tyrode’s solution and of the recording device has been previously given [14]. After a 45 min equilibration period, trnsmembrane action potentials were recorded simultaneously from two sites of the preparation, by means of floating micro-electrodes. An extracellular His bundle electrogram was recorded through a close bipolar electrode. Although intracellular action potentials were recorded in various areas of the sinoatrial and atrioventricular node and of the crista terminalis, the great variability of the transmembrane electrical parameters made it very difficult to compare analogous regions in different preparations. Therefore, no systematic evaluation of the action potential parameters was attempted. For measuring diqerent conduction time, the atrium was stimulated through bipolar electrodes placed on the right atria1 appendage at a constant cycle length of 330 ms. Retrograde sinoatrial conduction time was measured as the interval
AUTOMATICITY
AND
CONDUCTION
IN
DIABETES
1343
between atria1 depolarization and the upstroke of the SA nodal action potential. Intraatrial conduction was determined as the interval between the upstroke of the atria1 action potential recorded at the upper part of the crista terminalis and the extracellular atria1 electrogram near the AV node. Total AV nodal conduction time was determined as the interval between atria1 and His deflection in the extracellular His bundle electrogram. Sinus-node recovery time was measured following suppression of the sinoatrial nodal pacemaker activity by atria1 stimulation at 330 ms cycle length for 1 min. Corrected sinus-node recovery time was defined as the recovery interval in excess of the basis cycle. Electrophysiological-ultrastructural
correlation
After the electrophysiological investigation, the histologic and-electromicroscopic features of each non-diabetic and diabetic preparation were analyzed. Sinoatrial node and crista terminalis were fixed by immersion. The methodcfor fixation and preparation of the tissue samples has been recently described in detail [15]. The sinoatrial nodal and atria1 tissue samples were separated under a stereo microscope and embedded in Epon. Coloured semithin sections were analyzed by light microscopy and lead stained ultrathin sections by electron microscopy. In order to exclude ultrastructural alterations that might have occurred during the preceding in vitro procedures, electronmicroscopy was also performed in four additional fresh control preparations exhibiting no significant ultrastructural difference to non-diabetic preparations that had ‘been electrophysiologically studied. 3. Results The alloxan-diabetic rabbits were observed for an average of 100 k 15 days after alloxan administration and compared with the normal animals observed for a similar duration. Some metabolic data obtained in the diabetic group before alloxan and at the terminal study are summarized in Table 1. Hyperglycemia was first observed at the fourth day after alloxan and persisted throughout the study. At the terminal study, the most pronounced change was a six-fold increase in fasting plasma glucose. The plasma concentration for urea was moderately increased, triglycerides and cholesterol remained unchanged. The increase in body weight was not significant. Most of the diabetic animals were obviously ill at the terminal study. The metabolic data obtained from the control group before and after a similar period of the time demonstrated no significant alterations. Electrophysiological The sinus rate and various diabetic and control animals
studies
conduction times of isolated atria obtained from are summarized in Table 2. In diabetic preparations.
1344 TABLE
J. SENGES
ET AL.
1. Metabolic data and serum values obtained of rabbits before (control) 100 h 15 days after induction of alloxan-diabetes mellitus (n = 8) Body weight (kg)
Glucose (mg/lOO ml)
2.8 f 0.9
90 f. 20
3.4 * 0.7
550 * 200*
Cholesterol (mg/lOO ml)
Control
Triglycerides (mg/lOO ml)
and
Urea (mg/lOO ml)
state
135 * 65
100 f 80
‘32 f
12
200 & 130
60 f. 20*
Alloxan-diabetic-state
90 * 45
Figures are means * S.D. Significance P of the difference of means was determined by Student’s t test. * P < 0.05.
TABLE
n
2. Comparison of electrophysiological parameters in diabetic and non-diabetic preparations SR (beats/min)
CSRT (ms)
of automaticity
and conduction
CTSA
CTA
CTAH
(4
W
W
45 * 9
24 + 6
55*
39 & 14*
66 & 20 218 B*
Control
130 f
13
100 * 20
11
Diabetes
110 & 15*’
140 * 35*
345
13
Results were determined following a 1 h equilibration period. The conduction times were measured at 180/min atria1 stimulation rate. n = number of experiments; SR, sinus rate; CSRT, corrected sinus-node recovery time; CT, conduction time; B, block of conduction; SA, sinoatrial retrograde; A, intraatrial; AH, atrioventricular nodal. Figures are means * S.D. Significance P of the difference of means was determined by Student’s t test. * P < 0.05.
the sinoatrial nodal automaticity was significantly sinus rate and a prolonged poststimulatory sinus-node significant disturbances of the sinoatrial conduction differences in the antegrade sinoatrial conduction because of possible shifts in the site of the dominant sinoatrial, intraatrial and atrioventricular nodal atria1 stimulation rate (180/min) were measured. conduction was not significantly different in diabetic
depressed including a lower recovery time. Although no were observed in both groups, time are difficult to evaluate pacemaker. Therefore, only conduction time at constant The retrograde sinoatrial and control preparations.
AUTOMATICITY
AND
CONDUCTION
IN
1345
DIABETES
The intraatrial conduction time was significantly prolonged in the diabetic preparations associated with inhomogeneity of the atria1 excitability. Unidirectional entrance block within a small atria1 area of a diabetic preparation is demonstrated in Figure 1. Both upper and middle trace show atria1 action potentials recorded from an upper and a lower site of the crista terminalis, the lower trace is a His bundle electrogram. At spontaneous sinus rate, o&Q the lower atria1 cell showed regular action potentials exhibiting normal excitability. In contrast, the spread of atria1 excitation failed to conduct to the upper atria1 cell demonstrating a stable resting potential. However, when an extracellular stimulate electrode was moved close to the upper atria1 recording site, the
60 mV
i
FIGURE 1. Inhomogeneity of intraatrial conduction in a preparation obtained from an alloxandiabetic rabbit. Top and middle trace show intracellular records from two different atria1 fibers localized within the crista terminalis. Direct electrical stimulation at the recording site of the upper trace is preceded and following by spontaneous sinus rhythm.
fiber was found to be fully excitable exhibiting action potentials of moderately reduced amplitude. After termination of direct extracellular stimulation, entrance block occurred again as indicated by the stable resting potential of the upper fiber. Inhomogeneity of atria1 conduction was not observed in control preparations. The atrioventricular nodal conduction was completely blocked during the equilibration period in two diabetic preparations which was not observed in the control preparations. Typical recordings obtained from a diabetic preparation are shown in Figure 2 demonstrating complete AV nodal block associated with two independent rhythms. The upper trace was recorded from an upper right atria1 fiber, the middle trace shows action potentials from a His bundle fiber and the lower trace is a His bundle electrogram recording both lower right atria1 and
I346 His bundle depolarizations. atria1 rate associated with are initiated at a regular fibers.
J. SENGES
Complete independent but much
ET
AL.
AV nodal block is indicated by the rapid depolarizations of the His bundle which slower rate from His bundle pacemaker
IllI III I I/ I 1L111
100 mV
JJ t’ I/ ?+A f t
1
I
3s
FIGURE 2. Complete atrioventricular nodal block occurring preparation. The top trace shows action potentials from a lower is an intracellular record from a His bundle fiber and the bottom Note complete AV nodal block associated with two independent
Ultrastructural
spontaneously in a diabetic atria1 right atria1 fiber, the middle trace trace is a His bundle electrogram. rhythms.
studies
Histological and electromicroscopic analysis of the non-diabetic control preparations revealed a normal morphological appearance of the sinoatrial node and the crista terminalis. In preparations obtained from alloxan-diabetic rabbits intracellular accumulation of abundant glycogen was generally observed. Mitochondrial abnormalities consisted of both signs of increased activation and of degenerative changes including increase in size and number of the mitochondria, the appearance of huge mitochondria extending up to four to six sarcomere lengths and the occurrence of mitochondrial myelin figures and lysosomes (Figure 3). However, the small coronary vessels including the arterio-capillaryvenous (ACV) system (Figure 4), the cardiac nerve endings and the cell membranes exhibited no morphological alterations.
FIGURE 3. Electron micrographs of atria1 and SA nodal cells obtained from a diabetic preparation. (A) atria1 muscle cell with damaged and enlarged mitochondria (Mil, Mi2). Formation of myelin figures and lysosomes (l-4). AG, atria1 granules. x 18 000. (B) SA nodal cross sectional transitional cell demonstrating myelin figures but normal intercalated disc with desmosomes (d) and a gap junction (g). Glycogen (glyc.). x 21 900. (C) SA nodal typical P cells with myelin figure (arrow). Note sparsity of membrane specializations at the P cell borders (arrow heads). d, desmosomes. x 21 900.
FIGCRE
3.
FIGURE 4. Electron micrographs of small coronary vessels obtained from a diabetic preparation. (A) Longitudinal section of a large arteriole within the SA node showing a normal vascular wall. el, internal elastica. x4250. (B) smaller arteriole with normal vascular structure. mv, micropinocytotic vesicles. x 84 400.
AUTOMATICITY
AND
CONDUCTION
IN
DIABETES
1349
4. Discussion The presence of chronic diabetes mellitus at the conclusion of the study was verified by the persisting marked hyperglycemia. The severity of the diabetic conduction was indicated by the mild uremia that were attributed to diabetes since the nephrotoxic dose of alloxan has been demonstrated to exceed markedly the diabetogenic dose given in the present experiments [II]. The view that the electrophysiological abnormalities observed in the diabetic atria were primarily related to acquired diabetes rather than to administration of alloxan per se is supported by earlier results indicating that the cardiac abnormalities induced by chronic alloxan-diabetes could be prevented if the pancreatic effects of alloxan were inhibited [9]. Direct myocardial toxicity has not been reported even after large doses of alloxan [II]. The relevance of the alloxan-diabetes model to the human situation is corroborated by the great similarity of cardiac abnormalities observed in alloxan-diabetic dogs, spontaneous diabetic dogs and human diabetic [S]. Recent epidemiological observations in patients with cardiac pacemakers have indicated that diabetes may represent a risk factor facilitating the occurrence of severe bradycardias including sinoatrial nodal dysfunction and high degree atrioventricular conduction disturbances [3, 41. The possibility that chronic diabetes may affect the electrophysiological properties of the heart was confirmed and extended by the present experiments. Abnormalities of automaticity and conduction in isolated atria obtained from alloxan-diabetic rabbits as compared with non-diabetic control preparations included slower sinus rate, longer poststimulatory sinus-node recovery time, inhomogeneity of atria1 conduction and atrioventricular nodal block. The electrophysiological alterations were associated with ultrastructural abnormalities consisting of intracellular accumulation of abundant glycogen and various mitochondrial aberrations including degenerative changes but also signs of increased activation and the appearance of huge mitochondria. The pathophysiological mechanism of the electrophysiological alterations observed in alloxan-diabetic atria could not be determined because the transmembrane electrical activity was not systematically studied. However, it could be speculated that persisting hyperglycemia present in chronic diabetes might alter the metabolic conditions regulating the ion permeabilities of the fiber membrane. It is well known that the nodal electrical activity is predominantly related to the slow transmembrane inward current by Ca and/or Na ions [16]. Metabolic studies suggest that this transmembrane current is modulated by the glycolytic rate dependent on the extracellular glucose concentration [7, 131. An alternative hypothesis is derived from the biochemical observation that diabetes induced aberrations of the myocardial lipid metabolism involved in the structure of the fiber membrane [I, 91.
1350
J. SENGES
E7 AL.
Cardiac disease in the human diabetic is traditionally attributed to coronary artery disease. However, recent experimental and human autopsy studies support the view that chronic diabetes mellitus can alter myocardial composition and function independent of vascular effects ‘favouring the hypothesis of a diabetic cardiomyopathy [6, 91. The present histological and ultrastructural examination of the diabetic atria did not reveal any lesions of the coronary vessels including the arterio-capillary-venous system. Therefore, the observed electrophysiological and ultrastructural alterations may be primarily related to diabetes rather than to secondary coronary artery disease. The experimental results support the concept based on clinical observations suggesting that severe bradycardia may occur in diabetic patients due to metabolic alterations in the absence of coronary artery disease [4]. Acknowledgements
We are grateful to manuscript and to technical assistance. tion within the SFB
Professor Wolf-Georg Forssmann for critical review of the Mrs Katharina Marquard and Miss Christine Mtiller for This work was supported by the German Research Founda90 “Cardiovasculares System”. REFERENCES
1. DENTON, R. M. & RANDLE, P. J. Concentrations of glycerides and phospholipids in rat heart and gastrocnemicus muscles: Effect of alloxan-diabetes and perfusion. Biochemical .Journal 104, 4 16-422 ( 1967). 2. DORTIMER, A. C., SHENOY, P. N., SHIROFF, R. A., LEAMAN, D. M., BABB, J. D., LIEDTKE, A. J. & ZELIS, R. Diffuse coronary artery disease in diabetic patients. Fact or fiction ? Circulation 57, 133-l 36 ( 1978). 3. HASSLACHER, C., WAHL, P., THORSPECKEN, R. & N~SSEL, E. Risikofaktoren der Arteriosklerose bei Patienten mit schweren bradykarden Rhythmusstorungen. 4.
9.
zeitschrift K~~BLER,
ftir
Kardiologie 65, 890-896 ( 1976). & HASSLACHER, CH. Erregungsbildungsstorungen: Herzrhythmusstiirungen, H. Antoni, F. Bender, E. Gerlach
W.
metabolische & M. Schlepper aspekte. In Eds, pp. 141-147. Stuttgart-New York: Schattauer Verlag (1979). LAZAROW, A. & PALAY, S. L. Production and course of alloxan diabetes in the rat. Journal of Laboratory Clinical Medicine 31, 1004-1015 ( 1946). LEDET, T. Histological and histochemical changes in the coronary arteries of old diabetic patients. Diabetologia 4, 268-272 (1968). MCDONALD, T. F. & MACLEOD, D. P. Metabolism and the electrical activity of anoxic ventricular muscle. Journal of Physiology 229, 559-582 (1973). OSTRANDER, JR. L. D., FRANCIS, JR. T., HAYNER, N. S., KJELSBERG, M. 0. & EPSTEIN, F. H. Relationship of cardiovascular disease to hyperglycemia. Annals of Internal Medicine 62, 1188- 1198 ( 1965). REGAN, T. H., ETTINGER, P. O., KHAN, M. I., JESRANI, M. U., LYONS, M. M., OLDEWURTEL, H. A. & WEBER, M. Altered myocardial function and metabolism in chronic diabetes mellitus without ischemia in dogs. Circulation Research 35, 222-237 (1974).
AUTOMATICITY 10.
11. 12. 13.
14.
15. 16.
AND
CONDUCTION
IN DIABETES
1351
REGAN, T. J., AHMED, S. S., LEVINSON, G. E., OLDEWURTEL, H. A., AHMAD, M. R., HAIDER, B. & LYONS, M. M. Cardiomyopathy and regional scar in diabetes mellitus. Transactions of the Association of American Physicians 88, 217-221 (1975). RERUP, C. C. Drugs producing diabetes through damage of the insulin secreting cells. Pharmacology Review 22, 485-518 (1970). SCHEYNIUS, A. & TALJEDAL, I. B. On the mechanism of glucose protection against alloxan toxicity. Diabetologia 7, 252-255 (197 1). SENGES, J.: BRACHMANN, J., PELZER, D., KRAMER, B. & K~~BLER, W. Combined effects of glucose and hypoxia on cardiac automaticity and conduction. Journal of Molecular and Cellular Cardiology 12, 3 1 l-323 (1980). SENGES, J., MIZUTANI, T., PELZER, D., BRACHMANN, J., SONNHOF, U. & KOBLER, W. Effect of hypoxia on the sinoatrial node, atrium and atrioventricular node in the rabbit heart. Circulation Research 44, 856-863 (1979). WEIHE, E. & KALMBACH, P. Ultrastructure of capillaries in the conduction system of the heart in various mammals. Cell and Tissue Research 192, 7787 (1978). ZIPES, D. P., BESCH, H. R. & WATANABE, A. M. Role of the slow current in cardiac electrophysiology. Circulation 51, 761-766 (1975).
of Molecular
Altered
and Cellular
Cardiac
Cardiology
( 1980)
Automaticity Diabetes
12, 134 1- 135 1
and Conduction Mellitus*
JOCHEN SENGES, JOHANNES BRACHMANN, CHRISTIAN HASSLACHER, EBERHARD WEIHE Abteilung
Innere
AND
Medizin III (Kardiologie), Medizinische Anatomisches Znstitut III, 69 Heidelberg,
(Received
11 June
in Experimental
DIETER PELZER, WOLFGANG KUBLER
Universitiitsklinik Germany
1979, accepted in revised form
3
and
June1980)
J. SENGES, J. BRACHMANN, D. PELZER, CH. HASSLACHER, E. WEIHE AND W. K~~BLER. Altered Cardiac Automaticity and Conduction in Experimental Diabetes Mellitus. Journal of Molecular and Cellular Cardiology ( 1980) 12, 1341- 135 1. The electrophysiological and ultrastructural alterations that occur in chronic diabetes mellitus were studied in isolated, superfused atria including sinoatrial (SA) and atrioventricular (AV) nodes that were obtained from rabbits subjected to alloxan-diabetes for 100 days. Transmembrane action potentials were recorded simultaneously with the His bundle electrogram. The results were compared with corresponding values observed in atria from normal, non-diabetic rabbits. The abnormalities of automaticity and conduction found in atria of alloxan-diabetic rabbits included lower sinus rate, longer poststimulatory sinus-node recovery time, inhomogeneity of atria1 conduction and the occurrence of AV nodal block. Ultrastructural analysis of the diabetic preparations revealed intracellular accumulation of abundant glycogen and various mitochondrial abnormalities but a normal appearance of the small coronary vessels. It was concluded that chronic alloxandiabetes mellitus may directly alter the electrophysiological and ultrastructural properties of nodal and atria1 fibers in the absence of vascular damage. KEY
WORDS:
Alloxan-induced
diabetes;
Sinus
node;
Atrium;
Ultrastructure;
Brady-
cardias.
1. Introduction The present experiments were derived from a clinical observation obtained in patients with permanent cardiac pacemakers that diabetes mellitus may represent a risk factor facilitating the development of severe sinoatrial nodal dysfunction and of high degree atrioventricular block [3, 41. Diabetes appears to be associated with a markedly increased mortality rate from cardiac disease [8]. This has been traditionally attributed to secondary coronary artery disease but recent experimental and clinical observations favour the hypothesis of a diabetic cardiomyopathy independent of vascular alterations [S, IO]. Address for 69 Heidelberg, * This work Electrostimulation, 1978. 0022-2828/80/
reprints: Jochen Sengcs, M.D., Medizinische Universitatsklinik, Bergheimerstr., Germany. was presented in part at the International Symposium: Troubles du Rhythme et Toulouse 1977 and at the III. Internationales Arrhythmie-Symposium, Wien 12 1341+
11 $02.00/O
0
1980 Academic
Press Inc.
(London)
Limited
1342
J. SENGES
ET AL.
The effects of diabetes mellitus on myocardial function and metabolism have been extensively studied in man and animals [9, 101 but the electrophysiological alterations have not yet been described. The purpose of the present study was to examine various parameters of automaticity and conduction in atria1 preparations obtained from chronic alloxan-diabetic rabbits and to relate the electrophysiologic results to ultrastructural findings observed in the corresponding tissues including the small coronary vessels.
2. Materials
and Methods
Sixteen male New Zealand white rabbits were selected for the study. The animals were divided at random into two groups consisting of eight normal control and of eight alloxan-treated rabbits. All control and alloxan-treated animals received the same diet, consisting of commercial rabbit chow (Georg Plange Co., Soest, W. Germany) and were kept under conditions for an average of 100 f 15 days. Alloxan monohydrate (130 mg/kg, i.v.) was administered to rabbits that had been fasted for the previous 15 hours [IZ]. In the chronic control and in the diabetic rabbits, venous blood samples were taken in the fasted state at the onset of the study and at one month intervals thereafter for determination of plasma values for glucose, cholesterol, triglycerides and urea. Electrofihysiological
studies
For the terminal study, preparations consisting of sinoatrial node, crista terminalis, pectinate muscles of atria1 appendage, intraatrial septum, coronary sinus, atrioventricular node and His bundle were isolated and superfused with modified Tyrode’s solution at 36 & 0.5”C. The pH (7.4 f 0.03; n = 16), Pcoz (42 f 1.8 mmHg) and PO, (460 & 30 mmHg) remained unchanged throughout the experiment. A detailed description of the methods of dissection and mounting of the preparation, of the composition of the Tyrode’s solution and of the recording device has been previously given [14]. After a 45 min equilibration period, trnsmembrane action potentials were recorded simultaneously from two sites of the preparation, by means of floating micro-electrodes. An extracellular His bundle electrogram was recorded through a close bipolar electrode. Although intracellular action potentials were recorded in various areas of the sinoatrial and atrioventricular node and of the crista terminalis, the great variability of the transmembrane electrical parameters made it very difficult to compare analogous regions in different preparations. Therefore, no systematic evaluation of the action potential parameters was attempted. For measuring diqerent conduction time, the atrium was stimulated through bipolar electrodes placed on the right atria1 appendage at a constant cycle length of 330 ms. Retrograde sinoatrial conduction time was measured as the interval
AUTOMATICITY
AND
CONDUCTION
IN
DIABETES
1343
between atria1 depolarization and the upstroke of the SA nodal action potential. Intraatrial conduction was determined as the interval between the upstroke of the atria1 action potential recorded at the upper part of the crista terminalis and the extracellular atria1 electrogram near the AV node. Total AV nodal conduction time was determined as the interval between atria1 and His deflection in the extracellular His bundle electrogram. Sinus-node recovery time was measured following suppression of the sinoatrial nodal pacemaker activity by atria1 stimulation at 330 ms cycle length for 1 min. Corrected sinus-node recovery time was defined as the recovery interval in excess of the basis cycle. Electrophysiological-ultrastructural
correlation
After the electrophysiological investigation, the histologic and-electromicroscopic features of each non-diabetic and diabetic preparation were analyzed. Sinoatrial node and crista terminalis were fixed by immersion. The methodcfor fixation and preparation of the tissue samples has been recently described in detail [15]. The sinoatrial nodal and atria1 tissue samples were separated under a stereo microscope and embedded in Epon. Coloured semithin sections were analyzed by light microscopy and lead stained ultrathin sections by electron microscopy. In order to exclude ultrastructural alterations that might have occurred during the preceding in vitro procedures, electronmicroscopy was also performed in four additional fresh control preparations exhibiting no significant ultrastructural difference to non-diabetic preparations that had ‘been electrophysiologically studied. 3. Results The alloxan-diabetic rabbits were observed for an average of 100 k 15 days after alloxan administration and compared with the normal animals observed for a similar duration. Some metabolic data obtained in the diabetic group before alloxan and at the terminal study are summarized in Table 1. Hyperglycemia was first observed at the fourth day after alloxan and persisted throughout the study. At the terminal study, the most pronounced change was a six-fold increase in fasting plasma glucose. The plasma concentration for urea was moderately increased, triglycerides and cholesterol remained unchanged. The increase in body weight was not significant. Most of the diabetic animals were obviously ill at the terminal study. The metabolic data obtained from the control group before and after a similar period of the time demonstrated no significant alterations. Electrophysiological The sinus rate and various diabetic and control animals
studies
conduction times of isolated atria obtained from are summarized in Table 2. In diabetic preparations.
1344 TABLE
J. SENGES
ET AL.
1. Metabolic data and serum values obtained of rabbits before (control) 100 h 15 days after induction of alloxan-diabetes mellitus (n = 8) Body weight (kg)
Glucose (mg/lOO ml)
2.8 f 0.9
90 f. 20
3.4 * 0.7
550 * 200*
Cholesterol (mg/lOO ml)
Control
Triglycerides (mg/lOO ml)
and
Urea (mg/lOO ml)
state
135 * 65
100 f 80
‘32 f
12
200 & 130
60 f. 20*
Alloxan-diabetic-state
90 * 45
Figures are means * S.D. Significance P of the difference of means was determined by Student’s t test. * P < 0.05.
TABLE
n
2. Comparison of electrophysiological parameters in diabetic and non-diabetic preparations SR (beats/min)
CSRT (ms)
of automaticity
and conduction
CTSA
CTA
CTAH
(4
W
W
45 * 9
24 + 6
55*
39 & 14*
66 & 20 218 B*
Control
130 f
13
100 * 20
11
Diabetes
110 & 15*’
140 * 35*
345
13
Results were determined following a 1 h equilibration period. The conduction times were measured at 180/min atria1 stimulation rate. n = number of experiments; SR, sinus rate; CSRT, corrected sinus-node recovery time; CT, conduction time; B, block of conduction; SA, sinoatrial retrograde; A, intraatrial; AH, atrioventricular nodal. Figures are means * S.D. Significance P of the difference of means was determined by Student’s t test. * P < 0.05.
the sinoatrial nodal automaticity was significantly sinus rate and a prolonged poststimulatory sinus-node significant disturbances of the sinoatrial conduction differences in the antegrade sinoatrial conduction because of possible shifts in the site of the dominant sinoatrial, intraatrial and atrioventricular nodal atria1 stimulation rate (180/min) were measured. conduction was not significantly different in diabetic
depressed including a lower recovery time. Although no were observed in both groups, time are difficult to evaluate pacemaker. Therefore, only conduction time at constant The retrograde sinoatrial and control preparations.
AUTOMATICITY
AND
CONDUCTION
IN
1345
DIABETES
The intraatrial conduction time was significantly prolonged in the diabetic preparations associated with inhomogeneity of the atria1 excitability. Unidirectional entrance block within a small atria1 area of a diabetic preparation is demonstrated in Figure 1. Both upper and middle trace show atria1 action potentials recorded from an upper and a lower site of the crista terminalis, the lower trace is a His bundle electrogram. At spontaneous sinus rate, o&Q the lower atria1 cell showed regular action potentials exhibiting normal excitability. In contrast, the spread of atria1 excitation failed to conduct to the upper atria1 cell demonstrating a stable resting potential. However, when an extracellular stimulate electrode was moved close to the upper atria1 recording site, the
60 mV
i
FIGURE 1. Inhomogeneity of intraatrial conduction in a preparation obtained from an alloxandiabetic rabbit. Top and middle trace show intracellular records from two different atria1 fibers localized within the crista terminalis. Direct electrical stimulation at the recording site of the upper trace is preceded and following by spontaneous sinus rhythm.
fiber was found to be fully excitable exhibiting action potentials of moderately reduced amplitude. After termination of direct extracellular stimulation, entrance block occurred again as indicated by the stable resting potential of the upper fiber. Inhomogeneity of atria1 conduction was not observed in control preparations. The atrioventricular nodal conduction was completely blocked during the equilibration period in two diabetic preparations which was not observed in the control preparations. Typical recordings obtained from a diabetic preparation are shown in Figure 2 demonstrating complete AV nodal block associated with two independent rhythms. The upper trace was recorded from an upper right atria1 fiber, the middle trace shows action potentials from a His bundle fiber and the lower trace is a His bundle electrogram recording both lower right atria1 and
I346 His bundle depolarizations. atria1 rate associated with are initiated at a regular fibers.
J. SENGES
Complete independent but much
ET
AL.
AV nodal block is indicated by the rapid depolarizations of the His bundle which slower rate from His bundle pacemaker
IllI III I I/ I 1L111
100 mV
JJ t’ I/ ?+A f t
1
I
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FIGURE 2. Complete atrioventricular nodal block occurring preparation. The top trace shows action potentials from a lower is an intracellular record from a His bundle fiber and the bottom Note complete AV nodal block associated with two independent
Ultrastructural
spontaneously in a diabetic atria1 right atria1 fiber, the middle trace trace is a His bundle electrogram. rhythms.
studies
Histological and electromicroscopic analysis of the non-diabetic control preparations revealed a normal morphological appearance of the sinoatrial node and the crista terminalis. In preparations obtained from alloxan-diabetic rabbits intracellular accumulation of abundant glycogen was generally observed. Mitochondrial abnormalities consisted of both signs of increased activation and of degenerative changes including increase in size and number of the mitochondria, the appearance of huge mitochondria extending up to four to six sarcomere lengths and the occurrence of mitochondrial myelin figures and lysosomes (Figure 3). However, the small coronary vessels including the arterio-capillaryvenous (ACV) system (Figure 4), the cardiac nerve endings and the cell membranes exhibited no morphological alterations.
FIGURE 3. Electron micrographs of atria1 and SA nodal cells obtained from a diabetic preparation. (A) atria1 muscle cell with damaged and enlarged mitochondria (Mil, Mi2). Formation of myelin figures and lysosomes (l-4). AG, atria1 granules. x 18 000. (B) SA nodal cross sectional transitional cell demonstrating myelin figures but normal intercalated disc with desmosomes (d) and a gap junction (g). Glycogen (glyc.). x 21 900. (C) SA nodal typical P cells with myelin figure (arrow). Note sparsity of membrane specializations at the P cell borders (arrow heads). d, desmosomes. x 21 900.
FIGCRE
3.
FIGURE 4. Electron micrographs of small coronary vessels obtained from a diabetic preparation. (A) Longitudinal section of a large arteriole within the SA node showing a normal vascular wall. el, internal elastica. x4250. (B) smaller arteriole with normal vascular structure. mv, micropinocytotic vesicles. x 84 400.
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4. Discussion The presence of chronic diabetes mellitus at the conclusion of the study was verified by the persisting marked hyperglycemia. The severity of the diabetic conduction was indicated by the mild uremia that were attributed to diabetes since the nephrotoxic dose of alloxan has been demonstrated to exceed markedly the diabetogenic dose given in the present experiments [II]. The view that the electrophysiological abnormalities observed in the diabetic atria were primarily related to acquired diabetes rather than to administration of alloxan per se is supported by earlier results indicating that the cardiac abnormalities induced by chronic alloxan-diabetes could be prevented if the pancreatic effects of alloxan were inhibited [9]. Direct myocardial toxicity has not been reported even after large doses of alloxan [II]. The relevance of the alloxan-diabetes model to the human situation is corroborated by the great similarity of cardiac abnormalities observed in alloxan-diabetic dogs, spontaneous diabetic dogs and human diabetic [S]. Recent epidemiological observations in patients with cardiac pacemakers have indicated that diabetes may represent a risk factor facilitating the occurrence of severe bradycardias including sinoatrial nodal dysfunction and high degree atrioventricular conduction disturbances [3, 41. The possibility that chronic diabetes may affect the electrophysiological properties of the heart was confirmed and extended by the present experiments. Abnormalities of automaticity and conduction in isolated atria obtained from alloxan-diabetic rabbits as compared with non-diabetic control preparations included slower sinus rate, longer poststimulatory sinus-node recovery time, inhomogeneity of atria1 conduction and atrioventricular nodal block. The electrophysiological alterations were associated with ultrastructural abnormalities consisting of intracellular accumulation of abundant glycogen and various mitochondrial aberrations including degenerative changes but also signs of increased activation and the appearance of huge mitochondria. The pathophysiological mechanism of the electrophysiological alterations observed in alloxan-diabetic atria could not be determined because the transmembrane electrical activity was not systematically studied. However, it could be speculated that persisting hyperglycemia present in chronic diabetes might alter the metabolic conditions regulating the ion permeabilities of the fiber membrane. It is well known that the nodal electrical activity is predominantly related to the slow transmembrane inward current by Ca and/or Na ions [16]. Metabolic studies suggest that this transmembrane current is modulated by the glycolytic rate dependent on the extracellular glucose concentration [7, 131. An alternative hypothesis is derived from the biochemical observation that diabetes induced aberrations of the myocardial lipid metabolism involved in the structure of the fiber membrane [I, 91.
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E7 AL.
Cardiac disease in the human diabetic is traditionally attributed to coronary artery disease. However, recent experimental and human autopsy studies support the view that chronic diabetes mellitus can alter myocardial composition and function independent of vascular effects ‘favouring the hypothesis of a diabetic cardiomyopathy [6, 91. The present histological and ultrastructural examination of the diabetic atria did not reveal any lesions of the coronary vessels including the arterio-capillary-venous system. Therefore, the observed electrophysiological and ultrastructural alterations may be primarily related to diabetes rather than to secondary coronary artery disease. The experimental results support the concept based on clinical observations suggesting that severe bradycardia may occur in diabetic patients due to metabolic alterations in the absence of coronary artery disease [4]. Acknowledgements
We are grateful to manuscript and to technical assistance. tion within the SFB
Professor Wolf-Georg Forssmann for critical review of the Mrs Katharina Marquard and Miss Christine Mtiller for This work was supported by the German Research Founda90 “Cardiovasculares System”. REFERENCES
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