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Published online before print October 26, 2005, 10.1148/radiol.2373041347
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(Radiology 2005;237:861-867.)
© RSNA, 2005


Cardiac Imaging

Intraatrial Repair of Transposition of the Great Arteries: Use of MR Imaging after Exercise to Evaluate Regional Systemic Right Ventricular Function1

Laurens F. Tops, MD, Arno A. W. Roest, MD, Hildo J. Lamb, PhD, Hubert W. Vliegen, MD, Willem A. Helbing, MD, Ernst E. van Der Wall, MD and Albert de Roos, MD

1 From the Departments of Radiology (L.F.T., A.A.W.R., H.J.L., A.d.R.), Pediatric Cardiology (A.A.W.R.), and Cardiology (H.W.V., E.E.v.d.W.), Leiden University Medical Center, Albinusdreef 2, C2-S, 2333 ZA Leiden, the Netherlands; and Department of Pediatric Cardiology, Erasmus Medical Center-Sophia's Children Hospital, Rotterdam, the Netherlands (W.A.H.). Received August 2, 2004; revision requested October 8; revision received December 16; accepted January 18, 2005. Address correspondence to A.d.R. (e-mail: a.de_roos{at}lumc.nl).


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
PURPOSE: To prospectively assess regional systemic right ventricular (RV) function at rest and in response to exercise by using magnetic resonance (MR) imaging in patients who have undergone surgical correction at the atrial level for transposition of the great arteries (TGA).

MATERIALS AND METHODS: Informed consent was obtained, and the medical review board approved this study. In 25 adult patients (mean age, 25.8 years ± 4.7 [standard deviation]; 13 men) who had undergone correction for TGA (23.4 years ± 4.9 after surgery) and 11 healthy volunteers (mean age, 27.4 years ± 2.7; six men), systemic ventricular function was assessed with MR imaging (turbo field echo-planar imaging) at rest and during supine bicycle exercise. Regional wall thickness and wall thickening of the systemic RV were assessed and compared with those of the left ventricle in healthy volunteers by two investigators working together. Regional wall parameters were calculated by using the three-dimensional centerline method. Independent-samples t test and paired-samples t test were used for statistical analysis.

RESULTS: Ejection fraction of the systemic RV did not increase after exercise (56% ± 8 at rest to 55% ± 7 after exercise, P = .196). Mean RV wall thickening was impaired in patients with TGA at all levels both at rest and in response to exercise (P < .05). Moreover, the free wall and the anterior wall of the systemic RV had a smaller end-systolic thickness and a diminished thickening at rest and after exercise compared with findings in the normal left ventricle (P < .05).

CONCLUSION: The systemic RV of patients after intraatrial correction for TGA reveals regional functional disturbances at rest and in response to exercise.

© RSNA, 2005


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Since the introduction of the Mustard or Senning procedure (atrial redirection procedure), the life expectancy of patients born with a transposition of the great arteries (TGA) has improved. Although nowadays most patients undergo the arterial switch procedure, there are still many patients who have undergone surgery at the atrial level (1). In these patients, the right ventricle (RV) is functioning as the systemic ventricle. Most of these patients are asymptomatic when performing daily activities, but exercise testing often reveals diminished global ventricular function (2,3). Changes in diastolic and systolic volumes, decreased RV ejection fraction, and lack of increase in stroke volume response during exercise have been reported (24).

RV dysfunction, atrial arrhythmias, and venous baffle obstruction are risk factors for late death (57), and atrial arrhythmias may be preceded by RV dysfunction in patients who have undergone intraatrial correction for TGA (5,6). Regional perfusion defects with concordant wall motion abnormalities have been demonstrated in the systemic RV of patients with TGA (8,9). These regional perfusion defects may play a role in the pathogenesis of impaired RV function (9).

Several imaging modalities, such as echocardiography (6,7) and angiography (3,10), are available for assessment of ventricular function in patients with TGA, both at rest or after pharmacologic stress (3). However, these methods are limited by the use of geometric assumptions and the presence of scar tissue (11). Magnetic resonance (MR) imaging has proved to be a valid method to measure global ventricular function in patients in whom TGA has been corrected (4). Assessment of global ventricular function after physical exercise in patients with TGA has become feasible with MR imaging techniques (12). Moreover, MR imaging can be used to analyze regional wall function in patients with ventricular dysfunction caused by ischemia (13,14).

Thus, the purpose of our study was to prospectively assess regional systemic RV function at rest and in response to exercise by using MR imaging in patients who have undergone intraatrial surgical correction of TGA.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Patients
Twenty-five patients (13 men and 12 women; mean age, 25.8 years ± 4.7 [standard deviation]) with TGA corrected at the atrial level at a mean age of 2.6 years ± 2.3 were examined, and the findings were compared with those in 11 healthy volunteers (six men and five women; mean age, 27.4 years ± 2.7) who served as controls. All patients with TGA had participated in a previous MR imaging study on cardiovascular response to physical exercise (12). All patients were scheduled to undergo cardiac MR imaging at rest for clinical follow-up of cardiovascular function. None of the patients had contraindications for exercise MR imaging, such as the presence of a pacemaker, arrhythmias, claustrophobia, or the inability to perform bicycle exercise. The healthy volunteers had normal findings at clinical examination, had normal electrocardiographic (ECG) findings at rest, were without a history of cardiovascular disease, had no physical complaints, and were similar for age, weight, and body surface area (Table 1). Eleven patients underwent correction with the Mustard procedure; and 14 patients, with the Senning procedure. Fifteen patients had additional pulmonary stenosis and/or ventricular septal defect, which is referred to as complex TGA. All patients had New York Heart Association class I or II congestive heart failure (ie, symptoms of dyspnea or angina occur with moderate or severe exertion), and all had regular cardiac rhythm (22 patients had sinus rhythm, and three patients had an atrioventricular junctional rhythm) during the examinations. Informed consent was obtained from all individuals, and the medical review board of our institution approved the study.


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TABLE 1. Characteristics of TGA Patients and of Healthy Subjects

 
Exercise Testing
Exercise testing was performed within 1 month before the MR imaging examination to determine the patients' maximal exercise level. Each patient performed a graded maximal exercise test while in the supine position on an MR-compatible ergometer (MR imaging cardiac ergometer; Lode, Groningen, the Netherlands) with continuous recording of ECG and arterial oxygen saturation (supervised by A.A.W.R., H.W.V., or W.A.H.). Heart rate and workload at peak exercise were assessed. The following parameters were measured during the exercise test by using a gas analyzer (Oxycon Record; Jaeger, Höchberg, Germany): total ventilation, breathing frequency, oxygen uptake (VO2), and carbon dioxide release (VCO2). The respiratory quotient (VCO2/VO2) at peak exercise was used as an indicator of maximum effort.

MR Imaging
MR imaging was performed with a 1.5-T imager (ACS-NT15; Philips Medical Systems, Best, the Netherlands) equipped with a Powertrak 6000 gradient system and a CPR-6 cardiac research software package (Philips Medical Systems). At rest and with exercise, 10 consecutive sections in the left ventricle (LV) short-axis orientation with a thickness of 10 mm and an intersection gap of 1 mm were used to cover the LV and the RV from apex to base (15).

The short-axis images were obtained with a turbo field echo-planar imaging sequence, a body coil, and prospective ECG triggering. Imaging parameters included a repetition time msec/echo time msec of 14/4.8, a 128 x 40 matrix, a field of view of 420 x 120 mm, a flip angle of 30°, and a temporal resolution of 31 msec. After each radiofrequency excitation pulse, five lines of data were acquired (echo-planar imaging factor, five), which was repeated twice (turbo factor, two), so 10 k-lines per heart phase were acquired. All k-lines of one short-axis section were obtained during four heartbeats. Two short-axis sections were acquired during a breath hold of eight heartbeats at end expiration.

Exercise was performed with an MR-compatible bicycle ergometer that was fitted to the tabletop and positioned in the imager. The exercise level for MR imaging was based on the workload, corresponding to 60% of the maximum oxygen uptake measured during the preceding maximal exercise test. After reaching a steady heart rate, the patients with TGA and healthy volunteers stopped cycling. During this short exercise break, all participants immediately performed a single breath-hold procedure, and image acquisition was performed within eight heartbeats. Pedersen et al (16) accurately assessed flow measurements by using a breath-hold procedure shortly after exercise. The exercise state was well represented within 11 heartbeats, with a mean decrease in heart rate of less than 14% immediately after exercise. Furthermore, a previous report (17) showed no significant differences in heart rate recovery shortly after exercise between patients with TGA and healthy control subjects. After acquisition of the images, the participants resumed cycling until steady heart rate was reached again. The breath-hold procedure was then repeated to acquire a total of 10 short-axis sections.

Data Analysis
Global ventricular function parameters and regional wall function parameters of the systemic RV in patients who had been treated for TGA were assessed and compared with those of the systemic ventricle of healthy volunteers. Therefore, the RV of patients with TGA was compared with the LV of healthy volunteers. The short-axis images were analyzed manually by two observers (L.F.T. and A.A.W.R., 2 and 6 years of experience with cardiac MR imaging, respectively) working together in consensus. By using the MASS software package (Medis, Leiden, the Netherlands), the ejection fraction, EF, of the systemic ventricle was assessed with the following equation: EF = (EDV – ESV)/EDV · 100, where EDV is end-diastolic volume and ESV is end-systolic volume.

Wall Thickness and Thickening
For assessment of the end-diastolic wall thickness, end-systolic wall thickness, and wall thickening (as percentage of end-diastolic wall thickness), the three-dimensional centerline method (18) was used. The systemic ventricle was first classified into sections covering the whole ventricle from apex to base. The highest level where the total heart muscle could be identified was included, so imaging of the ventricle resulted in four to six sections.

By using centerline cords, which are placed at equal distance perpendicular to the "centerline," regional wall thickness could accurately be assessed (18). The centerline cords divided the ventricular cross section into eight equal segments, starting at the junction of the ventricles (Fig 1). For the systemic RV of the patients with TGA, the first two segments cover the inferior wall region, the subsequent two segments cover the free wall, and the final four segments cover the anterior and septal wall regions (Fig 2). In the healthy volunteers, the LV cross section was divided in the same way: Starting at the junction of the ventricles, the cross section was divided into eight equal segments covering the anterior, septal, inferior, and free wall regions. Similar regions of the systemic ventricular cross sections could therefore be analyzed.



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Figure 1. Short-axis MR image in healthy 31-year-old female volunteer. Image was acquired by using a breath-hold turbo field echo-planar imaging technique (14/4.8, 30° flip angle, echo-planar imaging factor of five, turbo factor of two, 10-mm section thickness, 128 x 40 matrix, and 420 x 120-mm field of view). The centerline method divides the LV wall into eight equal segments, starting at the junction of the ventricles (+). Image was obtained at rest, at end systole.

 


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Figure 2a. Short-axis MR images in 35-year-old female patient with TGA who had undergone intraatrial correction. Images were obtained (a) with and (b) without centerline cords (turbo field echo-planar sequence, 14/4.8, 30° flip angle, echo-planar imaging factor of five, turbo factor of two, 10-mm section thickness, 128 x 40 matrix, and 420 x 120-mm field of view). The systemic RV has been divided by the centerline cords into eight segments, which cover the inferior wall, the free wall, the anterior wall, and the septal wall regions.

 


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Figure 2b. Short-axis MR images in 35-year-old female patient with TGA who had undergone intraatrial correction. Images were obtained (a) with and (b) without centerline cords (turbo field echo-planar sequence, 14/4.8, 30° flip angle, echo-planar imaging factor of five, turbo factor of two, 10-mm section thickness, 128 x 40 matrix, and 420 x 120-mm field of view). The systemic RV has been divided by the centerline cords into eight segments, which cover the inferior wall, the free wall, the anterior wall, and the septal wall regions.

 
Statistical Analysis
An independent-samples t test was used to compare characteristics of the patients with TGA with those of the healthy volunteers to detect any differences in parameters during maximal exercise testing and to detect differences in all the ventricular wall parameters. Furthermore, an independent-samples t test was used to detect any differences in characteristics and exercise capacity between patients with TGA who underwent the Mustard procedure and those who underwent the Senning procedure and between patients with TGA with simple transposition and those with complex transposition.

Differences in heart rate and ventricular wall parameters at rest and in response to exercise were analyzed by using a paired-samples t test. This was performed for both the patients with TGA and the healthy volunteers.

To assess the possible correlation between the different variables, we calculated the intraclass correlation coefficients for wall thickness and wall thickening of the different regions.

All statistical analyses were performed by using computer software (SPSS for Windows, version 10.0; SPSS, Chicago, Ill). Results are expressed as mean ± standard deviation when appropriate. P < .05 was considered to indicate a statistically significant difference.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Exercise Testing
At maximal exercise testing, all participants reached a respiratory quotient of greater than 1.0, indicating at least near-maximum effort during the graded exercise test (19). Mean oxygen uptake per kilogram of body weight was lower in the patients with TGA than in the healthy subjects (29 [mL/min] · kg–1± 8 vs 44 [mL/min] · kg–1 ± 6, P < .001). Furthermore, peak workload differed between the patients with TGA and the healthy subjects (154 W ± 36 vs 232 W ± 31, P < .001), and heart rate at peak exercise was lower in the patients with TGA (164 beats per minute ± 18 vs 177 beats per minute ± 9, P = .03) (Table 2). There were no differences in exercise capacity between patients with simple transposition and those with complex transposition or between patients with TGA corrected with the Mustard procedure and those with TGA corrected with the Senning procedure.


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TABLE 2. Results of Maximal Exercise Testing in TGA Patients and in Healthy Subjects

 
Systemic Ventricular Function
Global function of the systemic ventricles could be assessed in both groups at rest and in response to submaximal exercise. Heart rate increased significantly after exercise in both the patients with TGA and in the healthy subjects (Table 3). The systemic RV of the patients with TGA had a significant lower ejection fraction at rest compared with that in the healthy volunteers (56% ± 8 vs 68% ± 6, P < .001), and the RV ejection fraction did not improve in patients with TGA in response to exercise (56% ± 8 at rest vs 55% ± 7 after exercise; P = .196, nonsignificant).


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TABLE 3. Global Ventricular Indices for TGA Patients and Healthy Subjects at Rest and after Exercise

 
Apart from global function, mean wall thickness and mean wall thickening, as well as regional wall thickness and wall thickening, were analyzed. At rest, the systemic ventricle of the patients with TGA had a similar mean end-diastolic wall thickness at all levels compared with that of the control group (Table 3). On the other hand, at all levels the mean end-systolic wall thickness was significantly smaller and the mean wall thickening was significantly lower in the patients with TGA compared with those of healthy volunteers at rest (Table 3).

During exercise, the mean end-systolic wall thickness was smaller in patients with TGA than in healthy control subjects, but only at the basal level (9.3 mm ± 1.5 in patients with TGA vs 10.9 mm ± 1.7 in healthy subjects, P = .016) and the midventricular level (9.4 mm ± 1.9 in patients with TGA vs 11.6 mm ± 1.3 in healthy subjects, P = .003). Mean wall thickening in the control group tended to be higher with exercise, but only the midventricular level showed a significant increase (87% ± 29 at rest vs 117% ± 30 at exercise, P = .043). Mean wall thickening of the systemic RV in the patients with TGA showed a slight increase in response to exercise at all levels, but this finding was significantly (P < .05) smaller in comparison with the values in the control group at all levels (Table 3).

Parameters of regional wall function were assessed in four regions of the ventricle: the septal wall, the inferior wall, the free wall, and the anterior wall. No differences were detected in regional end-diastolic wall thickness between patients with TGA and healthy subjects, both at rest and in response to exercise. Regional end-systolic wall thickness and regional wall thickening were diminished in the patients with TGA in two specific regions. In patients with TGA at rest, the end-systolic free wall and the end-systolic anterior wall were both thinner at all levels compared with these regions in the ventricles of the healthy subjects (P < .05). At all levels, wall thickening of the free wall region and the anterior wall region was significantly less in the patients with TGA than in the healthy subjects at rest (Table 4).


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TABLE 4. Regional Ventricular Indices for TGA Patients and Healthy Subjects at Rest and after Exercise

 
In response to exercise, the regional end-systolic wall thickness of the free wall and anterior wall in the patients with TGA was smaller at basal and midventricular levels (P < .05). Regional wall thickening was also diminished in the free wall and the anterior wall at these two levels in the patients with TGA in response to exercise (Table 4).

The intraclass correlation coefficients of the wall thickness and wall thickening of the regions were approximately 0.30 on average, reflecting a modest positive correlation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In this study, regional ventricular function of the systemic ventricle in patients with TGA who underwent atrial correction and in healthy control subjects was assessed with MR imaging at rest and during supine bicycle exercise. Global cardiac function of the patients with TGA was relatively well preserved at rest, but ejection fraction of the systemic RV failed to increase in response to exercise. Mean end-systolic wall thickness and mean wall thickening were decreased in the patients with TGA at rest and in response to exercise. Furthermore, regional wall function parameters (end-systolic wall thickness and wall thickening) were impaired in the free wall region and anterior wall region in the patients with TGA, possibly reflecting regional myocardial ischemia.

MR Imaging
Several imaging modalities are available for assessment of cardiovascular function in patients with congenital heart disease. Most of them, however, are limited by the presence of scar tissue or the use of geometric assumptions (11,20). MR imaging has been validated for assessment of global ventricular parameters such as RV mass, end-diastolic and end-systolic volumes, and ejection fraction in patients with congenital heart disease (17,20).

Regional systemic ventricular function in healthy individuals can be assessed with several techniques, both at rest and with pharmacologic stress (21,22). In the present study, MR imaging was used to assess regional cardiac function, which has been validated in previous reports (23,24). MR imaging has become available for analysis of ventricular function after supine bicycle exercise (25). The observations in the present study on the response of global ventricular function to exercise in healthy individuals are in accordance with those in previous reports (22,25). Furthermore, the observations in the present study on regional ventricular function in healthy individuals are in accordance with the results of previous studies (14,23). Sandstede et al (23) indicated that functional cardiac parameters are age dependent. Values of ejection fraction, end-diastolic and end-systolic wall thickness, and wall thickening in our healthy volunteers were comparable to those in subjects in the same age group in the study by Sandstede et al (23). To our knowledge, the present study uses MR imaging for the first time to assess regional wall function in patients with atrial-corrected TGA at rest and after exercise.

Systemic Ventricular Function
In the present study, mean end-systolic wall thickness and mean wall thickening of the systemic RV in patients with TGA were diminished compared with those of the LV in healthy volunteers. These impairments of ventricular wall function may contribute to late RV dysfunction and may be caused by global ischemia.

With the use of positron emission tomography (PET), diminished coronary flow reserve in patients with a systemic RV has been observed (26,27). According to Hauser et al (27), this decreased myocardial blood flow may cause recurrent asymptomatic ischemia, which may lead to scar tissue and late ventricular dysfunction. In contrast, Hornung et al (28) believe that hypertrophy of the systemic RV is not a consequence but rather is the cause of ischemia of the systemic ventricle in patients with TGA. They found increased RV myocardial mass 24 years after atrial correction, which correlated inversely with RV ejection fraction; this suggests a relationship between RV mass and impaired systemic ventricular function (28).

Episodes of myocardial hypoxia before correction of the transposition or suboptimal conditions during the operation may cause the functional ventricular wall motion abnormalities found in patients with TGA (10,29). This has also been noted in patients with other congenital heart abnormalities (30). Niezen et al (30) found global diminished wall thickness of the systemic ventricular wall in patients who underwent correction for tetralogy of Fallot, which may represent global myocardial injury acquired preoperatively or during surgery. Similarly, we speculate that the ventricular wall function abnormalities found in our patients with TGA may reflect global ischemia.

Furthermore, the present study showed impaired regional wall function in the anterior wall and the free wall of the patients with TGA. This may be caused by regional perfusion abnormalities (or ischemic changes), as has been demonstrated in previous studies (8,9). Regional perfusion abnormalities in hypertrophied or ischemic ventricles result in impaired regional wall thickening, which can be accurately assessed with MR imaging (13). Using the centerline method in an MR imaging study, Holman et al (14) found normal end-diastolic wall thickness and lower percentage ventricular wall thickening in patients with dysfunctional myocardium caused by myocardial infarction.

Our observations on the location of the regional wall function abnormalities are in accordance with the results of previous studies (8,9). Severe perfusion defects were most frequently found in the anterior wall of the systemic RV of patients with TGA (9). These regional perfusion defects found in patients with TGA were associated with significantly lower RV ejection fraction and may be used as a predictor of systemic ventricular failure (8). In response to exercise, our patients with TGA showed no deterioration in regional wall function, probably because their exercise level was below maximum. Using myocardial perfusion single photon emission computed tomography (SPECT), Lubiszewska et al (8) noted significant perfusion defects in patients with TGA 10.0 years ± 2.9 after atrial correction. In contrast to our findings, their findings indicated that the extent of perfusion abnormalities became greater in response to exercise.

In another study (9), regional wall thickening abnormalities were detected in 90% of the patients with TGA at a mean age of 15.5 years. Wall motion abnormalities were closely matched with fixed perfusion defects apparent at rest and with dipyridamole-induced stress. Fixed perfusion defects are suggestive of infarction or scarring, which may cause RV dysfunction in patients with TGA (9). In patients with congenitally corrected TGA, Hornung et al (31) found reversible and fixed perfusion defects associated with reduced regional wall thickening. They suggest a relationship between fixed perfusion defects, caused by ischemia or infarction, and ventricular dysfunction in these patients with systemic RVs (31). Therefore, the diminished end-systolic wall thickness and wall thickening in the anterior wall and septal wall found in our patients with TGA may reflect regional perfusion defects and may play a role in RV dysfunction.

Limitations
Our study may be limited because we compared the systemic RV to the LV of healthy volunteers. Although different anatomy and geometry make a direct comparison difficult, Singh et al (26) used a similar method in a PET study. They found no differences in myocardial blood flow at rest between the systemic RV of patients with TGA and the LV of healthy volunteers. Furthermore, our study may be limited by the use of a single plane in the evaluation of the RV. However, this approach is in agreement with that of other studies (11,23,28).

For assessment of dynamic changes during exercise, a high temporal resolution is needed. As a consequence, a less than optimal spatial resolution was accepted in the present study for technical reasons. However, previous studies have shown the feasibility of the technique (17,25), and the observations on regional ventricular function of healthy individuals in the present study are in accordance with the results of previous studies (14,23). No comparison with other imaging modalities (PET, SPECT) was made in our study. Therefore, no data on myocardial perfusion can be provided. New MR techniques (first-pass contrast enhancement, delayed enhancement) may be helpful to assess both myocardial perfusion and possible scar tissue in patients with TGA in the future.

Finally, we found a modest positive correlation of the variables by calculating the intraclass correlation coefficients. Therefore, statistically one could not treat them as fully independent, as one variable could be seen as a possible covariate, confounder, or influence on other variables of interest.

With exercise MR imaging, it was possible to assess regional systemic RV function in patients long after correction for TGA. The abnormal end-systolic wall thickness and wall thickening found in our patients with TGA may reflect diffuse injury of the systemic RV. Regional wall function was impaired mainly in the anterior wall and the free wall of the patients with TGA. These regional abnormalities may reflect regional perfusion defects and ischemia superimposed on the diffuse injury. MR imaging, therefore, may be useful for monitoring systemic ventricular function in detail over time in patients who have undergone atrial correction for TGA.


    FOOTNOTES
 

Abbreviations: ECG = electrocardiography • LV = left ventricle • RV = right ventricle • TGA = transposition of the great arteries

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, E.E.v.d.W., A.d.R.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, L.F.T., A.d.R.; clinical studies, L.F.T., A.A.W.R., W.A.H.; statistical analysis, L.F.T., A.d.R.; and manuscript editing, L.F.T., H.W.V., A.d.R.


    References
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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