Structural Changes of Atrial Myocardium due to Sustained Atrial Fibrillation in the Goat
Background After cardioversion of sustained atrial fibrillation (AF), the electrical and contractile functions of the atria are impaired, and recurrences of AF frequently occur. Whether remodeling of the structure of atrial myocardium is the basis for this problem is not known.
Methods and Results Sustained AF was induced by electrical pacing in 13 goats instrumented long-term. The goats were killed after 9 to 23 weeks, and the atrial myocardium was examined by light and electron microscopy. The changes were quantified in left and right atrial free walls, appendages, trabeculae, the interatrial septum, and the bundle of Bachmann. A substantial proportion of the atrial myocytes (up to 92%) revealed marked changes in their cellular substructures, such as loss of myofibrils, accumulation of glycogen, changes in mitochondrial shape and size, fragmentation of sarcoplasmic reticulum, and dispersion of nuclear chromatin. These changes were accompanied by an increase in size of the myocytes (up to 195%). There were virtually no signs of cellular degeneration, and the interstitial space remained unaltered. The duration of sustained AF did not significantly affect the degree of myolytic cell changes.
Conclusions Sustained AF in goats leads to predominantly structural changes in the atrial myocytes similar to those seen in ventricular myocytes from chronic hibernating myocardium. These structural changes may explain the depressed contractile function of atrial myocardium after cardioversion. This goat model of AF offers a new approach to study the cascade of events leading to sustained AF and its maintenance.
Restoration of sinus rhythm after atrial fibrillation (AF) by either pharmacological or electrical cardioversion is often associated with a delayed recovery of atrial contractile function. The time needed for recovery appears to be related to the duration of AF before cardioversion.1 Immediately after cardioversion, the atrial contraction is usually weak, but its strength increases progressively during the initial weeks of sinus rhythm.1 2 3 It is not clear whether this problem is based on functional or on structural changes. Alterations in myocardial structure in patients with atrial arrhythmias were described by Mary-Rabine et al.4 In a group of patients with atrial arrhythmias of various causes, these authors observed a variety of structural changes in the atrial myocardium. In addition to degenerative changes, part of the myocytes showed changes in cellular substructures, such as loss of myofibrils, presence of glycogen granules, accumulation of sarcoplasmic reticulum–like material, and aggregates of mitochondria. The patients with chronic AF showed the most pronounced structural alterations. It was not clear, however, whether these structural abnormalities were caused by AF itself or by the concomitant underlying heart disease.
To study the structural changes in the atria resulting from chronic AF, we maintained AF in normal goats for a prolonged period of time. After 9 to 23 weeks of sustained AF, several areas of the right and left atria were examined by light and electron microscopy to investigate the structural alterations due to AF and to compare the degree of change at different atrial sites. This study is the first in which structural alterations are assessed both qualitatively and quantitatively in an animal model of prolonged sustained AF.
Twenty female goats weighing between 41 and 82 kg were used for this study. Thirteen goats were subjected to sustained AF, and 7 goats in sinus rhythm served as controls. Animal handling was carried out in accordance with the guidelines of the American Society of Physiology and approved by the Animal Investigation Committee of Maastricht University.
Chronic AF Model
The goat model of sustained AF was described in more detail by Wijffels et al.5 Briefly, the goats were instrumented long-term with multiple unipolar electrodes sutured to the epicardial surface of the free wall of the right and left atria and on the bundle of Bachmann. Approximately 2 to 3 weeks after surgery, the animals were connected to an automatic AF pacemaker, which continuously reinduced AF by automatic delivery of a 1-second burst of stimuli (50 Hz) as soon as sinus rhythm resumed. In the present study, 13 of the 20 goats were connected to the fibrillation pacemaker, and AF was maintained for 20 weeks (median value; range, 10 to 31 weeks) before the goats were killed. Initially, the episodes of electrically induced AF were short-lasting and self-terminated within a few seconds. As a result of the maintenance of AF, the duration of AF progressively increased, and AF usually became sustained (defined as duration of AF of >24 hours) after an average of ≈1 week. After AF had become sustained, it was maintained for an additional period of 9 to 23 weeks.
At the end of the period of sustained (9 to 23 weeks) AF, the goats were anesthetized with thiopental (10 to 15 mg/kg) and ventilated by halothane (1% to 2%) and a 1:2 mixture of O2 and N2O. The heart was quickly removed, and parts of the left and right atrial wall, appendages, trabecular muscles, the interatrial septum, and the bundle of Bachmann were cut into small blocks (4 mm3), fixed in 3% glutaraldehyde in 90 mmol/L potassium phosphate, buffered to a pH of 7.4, and kept for at least 24 hours at room temperature. After they were washed in the same buffer for another 24 hours, the tissue blocks were postfixed for 1 hour in 1% osmium tetroxide, buffered with 50 mmol/L veronal acetate, rapidly dehydrated through a graded ethanol series, and routinely embedded in epoxy resin.6 The atria of the 7 goats in sinus rhythm were prepared in the same way.
For electron microscopy, ultrathin sections were cut from each sample, counterstained with uranium acetate and lead citrate, and examined in a Philips CM100 electron microscope.
Quantification of Atrial Remodeling
Sections 2 μm thick, derived from various sites of the atria, were examined by light microscopy. To quantify the degree of myolysis and to identify cells with accumulation of glycogen, sections were stained with PAS and counterstained with toluidine blue.7 To quantify the extent of myolysis in the cardiomyocytes, at least two sections per atrial site were examined and at least 200 cells per section were analyzed. The extent of cell change was evaluated only in cells in which the nucleus was present in the plane of the section. Cells were scored by morphometry as mildly myolytic if myolysis involved 10% to 25% of the cytosol and as severely myolytic if >25% of the sarcomeres were absent.
To assess the amount of connective tissue in the myocardium, morphometry was carried out with the aid of a grid of vertical and horizontal lines providing 121 intersections (points). This technique was applied in a previous study.7 In accordance with the principles of morphometry, counting of the number of points overlying a certain structure results in quantitative determination of the volume of the structure under investigation in relation to the volume of the entire tissue under the square grid. The total number of points was defined as 100%, and the points counted in the connective tissue were expressed as a percentage of the entire tissue within the limits of the grid. The same procedure was repeated on different areas of the same section. Blood vessels and perivascular interstitial cells were excluded from the connective tissue quantification.
To assess whether sustained AF induced changes in the size of cardiomyocytes, the surface area of the cardiomyocytes was measured with a digital imaging system (Metasystems equipment with ISIS software). Area measurements were performed for three categories of cells: (1) normal cardiomyocytes (without myolysis), (2) moderately affected cells (between 10% and 25% myolysis), and (3) severely affected cells (>25% myolysis). The measurements were performed in left atria, right atria, and interatrial septa from 6 goats with sustained AF. The number of cells that were evaluated for each tissue sample varied between 262 and 388.
Data on myolytic cell changes and connective tissue changes were tested for statistical significance by means of the Wilcoxon-Mann-Whitney rank-sum test. The effect of duration of sustained AF on myolytic cell changes was evaluated with the Spearman rank correlation test. Exact two-tailed probability values were obtained with the StatXact program.8
Data on myocardial cell size measurements were transformed to logarithms, because the original measurements proved to be log-normally distributed. Subsequently, a mixed-effects ANOVA9 with restricted maximum likelihood variance estimation was carried out with the SAS system for statistical analysis. In this analysis, the classification of the myocytes (site, severity of alteration) served as fixed effect, and the animal for which the measurements were obtained was considered as a random effect. Mean values, adjusted for the number of measurements and overall level of each animal, and the corresponding 95% CIs for the mean were obtained by back-transformation of the least-squares estimates.
Values of P<.05 were considered to indicate statistical significance.
Structural Changes After Sustained AF
Atrial myocytes from goats in sinus rhythm showed compact sarcomeres occupying the entire cytoplasm (Fig 1a⇓). In contrast, atrial myocytes of goats in sustained AF were often depleted of contractile material and showed accumulation of glycogen (Fig 1b⇓). In these cells, myolysis started in the perinuclear area and often extended toward the plasma membrane. This loss of sarcomeres did not result in atrophic degeneration of the cardiomyocytes. On the contrary, the myocytes were moderately enlarged (see quantitative assessments below). The myolysis and glycogen accumulation were seen at all different atrial sites.
No obvious changes were seen in the content or distribution of the connective tissue in the atrial myocardium. The numbers of fibroblasts, endothelial cells, pericytes, and interstitial mesenchymal cells were normal. Inflammatory cell infiltrates were virtually absent.
At the ultrastructural level, atrial myocytes from goats in sinus rhythm showed a highly organized sarcomeric structure with rows of uniformly sized mitochondria in between (Figs 2a⇓ and 3a⇓). Atrial granules were mainly confined to the perinuclear area. A typical distribution of heterochromatin in the form of clusters at the nuclear membrane was present in all cardiomyocyte nuclei (Fig 3a⇓).
The atrial myocytes from goats in sustained AF had undergone typical alterations of a nondegenerative nature. The following characteristic changes were observed: (1) Contractile material was depleted (myolysis). The disappearance of sarcomeres was often limited to the vicinity of the nucleus but also frequently involved the entire cytosol, in which then only fragments of sarcomeres were present (Figs 2b⇑ and 3b⇑). In cells showing a moderate degree of myolysis, the peripherally located sarcomeres maintained their structural integrity (Fig 3c⇑). As a result of the myolysis, remnants of sarcomeres, especially clumped Z-band material, are often seen (Fig 3c⇑ inset). (2) Huge amounts of glycogen filled the myolytic space in almost all cells that underwent myolysis (Fig 3d⇑). (3) A network of disorganized membranes, probably altered profiles of sarcoplasmic reticulum, were present in myolytic areas (Fig 3c⇑). (4) Typical changes in size and shape of mitochondria were seen in areas depleted of sarcomeres: many mitochondria had become elongated, with their cristae oriented in a longitudinal fashion (inset Fig 3c⇑). In cross sections, such mitochondria appeared as small, donut-like structures (inset Fig 3c⇑). (5) Although the sarcolemma of the affected cells appeared to be normal, the short membrane invaginations present in small numbers in normal atrial myocytes were only occasionally seen in the myolytic myocytes. There were no noticeable changes in the ultrastructure of the intercalated disks. (6) A final characteristic alteration was seen in the nuclei: whereas in normal myocytes the heterochromatin is clustered in condensed aggregates near the nuclear membrane, the heterochromatin was now dispersed uniformly throughout the nucleoplasm (Fig 3b⇑). These nuclei resembled the interphase nuclei of myocytes during embryonic/fetal development. Typical degenerative changes, such as cytoplasmic vacuolization, cytoplasmic edema, mitochondrial swelling, membrane disruption, accumulation of secondary lysosomes, membrane whorls, and lipid droplets, were virtually absent.
Quantification of the Structural Changes
Morphometric assessment of the number of myocytes with myolysis or glycogen accumulation, the extent of myolysis in each cardiomyocyte, and the amount of connective tissue (interstitial space) was performed in 13 goats with sustained AF and in 7 goats in sinus rhythm. Changes in the size of the atrial myocytes were correlated with the extent of myolysis in 6 goats with chronic AF.
In all goats with sustained AF, a considerable but variable proportion of the atrial myocytes (up to 92%) was affected by myolysis. Cells were considered to be mildly affected when 10% to 25% of the sarcomeres had disappeared. Cells with >25% myolysis were considered to be severely affected. The results of the morphometric analysis are presented in Table 1⇓, together with the statistical analysis. The percentage of myolytic myocytes was significantly higher in all parts of fibrillating atria examined. As an example, during sinus rhythm, 7.4% of the myocytes from the left atria showed some degree of myolysis, and 51.8% of the cardiomyocytes in the left atria from goats with chronic AF showed mild or severe myolysis. Other atrial segments yielded comparable results. As for the severe myolytic changes, a statistically significant increase was seen in all atrial parts with the exception of the right appendage and septum. Although the level of significance was not reached in these two parts, the increase in number of affected cells was substantial.
The myolytic changes were accompanied by an increase in size of the myocytes. In Table 2⇓, the mean sizes of the atrial myocytes, together with the 95% CIs, are given for myocytes without myolysis and those with mild and with severe myolysis. The cell size was significantly different in the three groups (up to 195%), indicating that the degree of myolysis clearly correlated with an increase in size of the myocyte. The changes in size were not different between left and right atria and the interatrial septum.
The connective tissue did not undergo any quantitative modification by prolonged AF: no significant differences in the percentage of connective tissue were found between atria of goats in sinus rhythm and sustained AF. The descriptive statistics, together with the probability values (median, range) of the morphometric analysis, are given in Table 3⇓. The percentage of interstitial tissue did not differ between the various atrial sites.
The duration of sustained AF did not significantly affect the degree of myolytic cell changes in the left atrium (r=.094, P=.756), right atrium (r=−.147, P=.663), and interatrial septum (r=−.071, P=.832), as indicated by the Spearman rank correlation test.
The present ultrastructural study and morphometric assessment in atrial tissue from goats in sustained AF revealed profound structural changes in the atrial myocytes. The novelty of this structural approach lies in the fact that this is the first study in which quantitative and qualitative light- and electron-microscopic changes are reported in an animal model of sustained AF. Qualitative structural data hitherto reported on AF are limited to a study in dogs after long-term (6 weeks) rapid atrial pacing.10 This study showed that rapid pacing induced the formation of enlarged and disarrayed fibers, giant mitochondria, dilated sarcoplasmic reticulum, and enlarged nuclei. However, the typical changes found in our model, such as myolysis, glycogen accumulation, typical chromatin changes, and the presence of slender elongated mitochondria were not seen. The reason why these changes are at variance with changes reported in our goat model is not clear, but different duration of fibrillation and species differences might be responsible.
Depletion of contractile material and accumulation of glycogen were identified as essential characteristics in a substantial proportion of the atrial myocytes of the AF goats. Two remarkable features accompanied these changes: first, the cells did not show atrophy; on the contrary, they were enlarged. The increase in cell size appeared to correlate with the degree of myolysis. The myocytes from the fibrillating atria did not show any degenerative changes. Second, quantitative assessment failed to demonstrate any alterations in the extracellular matrix. The observed structural changes did not appear to be influenced by the durations of sustained AF (9 to 23 weeks).
The changes in atrial myocytes described above should be compared with the alterations reported for a variety of other pathophysiological conditions of the atrial myocardium, both in humans and in animals.11 12 13 In patients with atrial arrhythmias, Mary-Rabine and coworkers4 showed that part of the atrial myocytes displayed loss of myofilaments, presented aggregates of small mitochondria and of sarcoplasmic reticulum, clusters of accumulated glycogen, and frequently showed inclusions that were interpreted as “lysosomal degeneration.” These structural abnormalities appeared to be more pronounced when the underlying pathological condition was aggravated by sustained AF.
Patients with an atrial septal defect and atrial dilatation have been reported to show focal degenerative changes.11 Some of the cells were myolytic and showed alterations in the myofilaments, sarcoplasmic reticulum, mitochondria, and cytosol similar to those seen in our study. Similar changes have also been described in cardiomyopathic feline hearts with atrial arrhythmias and in dogs with mitral valve stenosis and intermittent atrial arrhythmias.12 13 These changes were characterized as “degenerative,” although degeneration involving the lysosomal compartment was absent.
In most of the above-mentioned studies, the term degeneration was used as a synonym for loss of myofilaments. In our opinion, cellular alterations that are not accompanied by lysosomal degradation should not be interpreted as degenerative, but rather should be described as dedifferentiated. Some features seen in atrial myocytes during sustained AF closely resemble those of myocytes during heart development. These are the paucity of contractile filaments and sarcoplasmic reticulum, glycogen storage, mitochondrial shape changes, and nuclear chromatin distribution.14 15 In view of their resemblance to structural features of myocytes during heart development, they are interpreted as phenotypic changes characteristic of less differentiated cells. This is further supported by the close resemblance of these cells to pacemaker cells in the sinus and AV node and the transitional cells of the atrial myocardium.16 17 Berger and Rona16 describe the extranodal transitional cardiocytes as follows: “Their sarcomeres appear poorly organized, very similar to those in embryonic cardiocytes, and give the impression that these cells are less well specialized for contraction than working cardiocytes.”
What Causes the Structural Changes of the Atrial Myocardium due to Sustained AF?
The possible causes underlying the structural modifications during prolonged AF are not well understood. Sarcomere depletion might occur as a result of prolonged absence or downregulation of contractile activity. Noncontractile or less contractile segments of the heart become subjected to increased passive stretch, which may induce ultrastructural changes. The passive stretch of noncontracting myocardium may be particularly important for the explanation of the increase in size of the cardiomyocytes.18 19 A role for normal contractile activity in maintaining a normal sarcomere structure is further supported by mechanical unloading and reloading experiments.20 21 On the basis of these experiments, it was concluded that sustained contractile activity of cardiac muscle appears to be essential for the maintenance of the contractile filaments.
A striking observation was that in most of the myolytic cells, glycogen accumulated at sites at which sarcomeres were depleted. The reason for this glycogen storage is not known, but it has been suggested that a lowered oxygen supply/demand ratio may play an important role by switching the energy metabolism from the use of fatty acids to the use of glucose.14 White and coworkers22 showed that immediately after induction of AF, oxygen consumption in the atrial tissue increases more than threefold, resulting in a marked reduction of the flow reserve during AF. Whether AF actually causes ischemia is at present unknown. Intracellular accumulation of glycogen may be either the result of a metabolic excess of glucose or the consequence of impaired catabolism of glycogen, due to a deficient phosphorylase activity, for instance.23 The cell volume is maintained despite the depletion of contractile material, and in fact, an increase in cell volume was observed. We believe that the glycogen accumulation, which correlated strongly with the degree of sarcomere depletion, is responsible for the increase in cell volume. Storage of large quantities of glycogen has also been found in fetal cardiac muscle,15 in a number of cardiac cells such as Purkinje fibers and atrioventricular node cells,24 and in transitional atrial cells16 in adult hearts.
Sustained AF as a Model for Chronic Hibernating Myocardium
Chronic hibernating myocardium, a condition that occurs in patients as a result of low-flow ischemia caused by stenosis of one or more coronary arteries,25 26 displays structural changes in ventricular myocardium similar to the changes in atrial myocardium due to sustained AF.7 27 Replacement of contractile elements by glycogen, disorganization of sarcoplasmic reticulum, the presence of numerous mini-mitochondria, and changes in nuclear shape all are features also found during ischemia-induced myocardial hibernation. The long time required for recovery of contractile function (weeks to months), as seen in some patients after restoration of blood flow to the hibernating segments, may be explained by the long time needed to reverse the structural alterations assigned as cellular dedifferentiation, a process presumed to need time to reverse.28 29
To date, no animal models are available in which the structural adaptations of the myocardium mimic those of chronic hibernating myocardium in humans. The animal model presented here may be the first to lend itself to the study of the events leading to such cellular adaptations. Although we realize that this is a model with structural adaptations in atrial and not in ventricular myocardium, we think it can provide valuable answers to a series of essential questions pertaining to chronic hibernating myocardium.
Possible Clinical Relevance
In patients with chronic AF, there is a correlation between the duration of AF and the time needed to recover atrial contractile function after cardioversion. In these patients, several weeks may be needed for the atrial contractile function to recover.1 2 3
The changes in subcellular structure of the atrial myocytes due to prolonged episodes of AF as described in this article may explain the lack of immediate recovery of contractile function in the atria after successful conversion to sinus rhythm. After regular synchronous triggering of contraction of the atria has been restored, it may take the cardiomyocytes several days to rebuild a normal amount of sarcomeres. In vitro studies have shown that myolysis can be induced in cultures of cardiomyocytes in which beating was arrested by verapamil. After the verapamil was removed, it took the cells 3 days to rebuild a normal contractile machinery and recover from the arrest.21
The considerable loss of contractile material resulting from sustained fibrillation might explain the prolonged contractile failure after conversion to sinus rhythm in patients.1 2 One important difference between structural changes seen in our model and those reported in patients with AF is that the increase in connective tissue as found in patient material was not reproduced in the goat.4 11 The absence of measurable changes in the interstitial space in goats with chronic AF may explain the relatively fast recovery of normal function, ie, within 2 weeks (M.A., unpublished observations). An extensive modulation of the interstitial space, as apparently occurs in patients, would contribute to increased stiffness of the myocardium and thus impairment of contractile function and hence could explain the very long delay in recovery of normal function after cardioversion.1 It is conceivable that full recovery might occur only when the interstitial tissue has returned to normal proportions.
Although this study clearly shows that AF leads to marked structural changes in the atrial myocytes, the mechanisms causing these structural alterations are not known. Our present study cannot answer the question of whether quiescence of the atrial myocardium, elevated stretch on the atrial wall, or ischemia is the trigger for the structural changes observed. Another limitation is that the goats were killed after 9 to 23 weeks of sustained AF, and therefore the effects of long duration (years) remain unknown. In particular, myocyte degeneration and fibrosis formation, as observed in human diseases, require more time to occur, just as a prolonged period of time may be necessary to reverse such changes. Indeed, an important issue that needs to be dealt with concerns the reversibility of the structural changes after cardioversion, ie, the redifferentiation potential of the dedifferentiated cardiomyocytes.
Many but not all features observed in human diseases can be reproduced in the goats with sustained AF. This might be due to the relatively short time period of AF or to the absence of concomitant conditions that are often present in patients. An advantage of this goat model, however, is that structural changes due to “lone” AF can be investigated, and in view of the uniformity of the cell changes, it lends itself well to study of the cascade of events leading to chronic AF and the pathophysiology behind its chronicity.
Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 11-14, 1996, and published in abstract form (Circulation. 1996;94[suppl I]:I-593).
- Received April 2, 1997.
- Revision received May 29, 1997.
- Accepted June 14, 1997.
- Copyright © 1997 by American Heart Association
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