Diffuse Myocardial Fibrosis in Children After Heart Transplantations: A Magnetic Resonance T1 Mapping Study

Background It is unclear whether the myocardium undergoes accelerated fibrotic remodeling in children after heart transplantation (HTx). Methods In this prospective study, cardiac magnetic resonance (CMR) studies in 17 patients 1.3 years (median, range 0.03-12.6 years) after HTx (mean age, 9.8 ± 6.2 years; 8 girls) were compared to CMR studies in 9 healthy controls (mean age, 12.4 ± 2.4 years; 4 girls). T1 measurements were performed at a midventricular short axis slice before (ie, native T1 times) and after the application of 0.2 mmol/kg gadopentetate dimeglumine in the interventricular septum, left ventricular (LV) free wall and encompassing the entire LV myocardium. The tissue-blood partition coefficient (TBPC), reflecting the degree of diffuse myocardial fibrosis, was calculated as a function of the ratio of T1 change of myocardium compared to blood. Native T1 times and TBPC were correlated with echocardiographic parameters of diastolic function. Results Native T1 times were significantly higher in HTx patients compared to controls in all regions assessed (LV free wall 973 ± 42 vs 923 ± 12 ms; P < 0.005; interventricular septum 1003 ± 31 vs 974 ± 21 ms, P < 0.05; entire LV myocardium 987 ± 33 vs 951 ± 16 ms; P < 0.005) and correlated with LV E/e' as an echocardiographic marker of diastolic dysfunction (r = 0.54, P < 0.05). The TBPC was elevated in the LV free wall (0.45 ± 0.06 vs 0.40 ± 0.03, P < 0.005) and the entire LV myocardium (0.47 ± 0.06 vs 0.43 ± 0.03, P < 0.05). Conclusions Evidence of diffuse myocardial fibrosis and is already present in children after HTx. It appears to be associated with diastolic dysfunction.

Background. It is unclear whether the myocardium undergoes accelerated fibrotic remodeling in children after heart transplantation (HTx). Methods. In this prospective study, cardiac magnetic resonance (CMR) studies in 17 patients 1.3 years (median, range 0.03-12.6 years) after HTx (mean age, 9.8 ± 6.2 years; 8 girls) were compared to CMR studies in 9 healthy controls (mean age, 12.4 ± 2.4 years; 4 girls). T1 measurements were performed at a midventricular short axis slice before (ie, native T1 times) and after the application of 0.2 mmol/kg gadopentetate dimeglumine in the interventricular septum, left ventricular (LV) free wall and encompassing the entire LV myocardium. The tissue-blood partition coefficient (TBPC), reflecting the degree of diffuse myocardial fibrosis, was calculated as a function of the ratio of T1 change of myocardium compared to blood. Native T1 times and TBPC were correlated with echocardiographic parameters of diastolic function. Results. Native T1 times were significantly higher in HTx patients compared to controls in all regions assessed (LV free wall 973 ± 42 vs 923 ± 12 ms; P < 0.005; interventricular septum 1003 ± 31 vs 974 ± 21 ms, P < 0.05; entire LV myocardium 987 ± 33 vs 951 ± 16 ms; P < 0.005) and correlated with LV E/e' as an echocardiographic marker of diastolic dysfunction (r = 0.54, P < 0.05). The TBPC was elevated in the LV free wall (0.45 ± 0.06 vs 0.40 ± 0.03, P < 0.005) and the entire LV myocardium (0.47 ± 0.06 vs 0.43 ± 0.03, P < 0.05). Conclusions. Evidence of diffuse myocardial fibrosis and is already present in children after HTx. It appears to be associated with diastolic dysfunction. T he outcomes after pediatric heart transplantation (HTx) have improved steadily over the last two decades. [1][2][3] with the most recent data from the International Society for Heart and Lung Transplantation (ISHLT) Registry showing a 1-and 5-year survival of approximately 90% and 75%, respectively. 4 Despite these advances, mortality remains significant, and ventricular dysfunction is not uncommon in children after HTx. 5,6 Diffuse myocardial fibrosis is increasingly identified as an important mechanism of myocardial functional decline in a variety of cardiac conditions. An increased amount of fibrosis has been demonstrated by endomyocardial biopsy (EMBx) in adult HTx patients with cardiac allograft vasculopathy (CAV), irrespective of rejection. 7 However, it is unclear whether pediatric heart transplant recipients are also prone to developing diffuse myocardial fibrosis.
Histological examination, the gold standard for myocardial fibrosis quantification, requires EMBx which is invasive and not without risks. Furthermore, the myocardial tissue obtained through percutaneous biopsy may not always be representative of the entire myocardium. Tissue-blood partition coefficient (TBPC) and extracellular volume fraction as markers of diffuse myocardial fibrosis can be measured noninvasively by cardiac magnetic resonance (CMR) on the basis of native and postcontrast myocardial longitudinal relaxation (T1) times. 8,9 The technique has been validated extensively and used in a broad spectrum of congenital and acquired heart conditions in adults and children. [10][11][12][13][14] The aim of this study was (1) to quantify the degree of myocardial fibrosis in children after HTx in comparison with healthy controls and (2) to explore the impact of myocardial fibrosis on cardiac function.

Patients
This is a single-center, cross-sectional cohort study, with retrospective analysis of prospectively collected data. Following approval by the institutional research ethics board, children and adolescents after HTx who underwent an EMBx as per routine clinical protocol between April 2010 and March 2011 were invited to undergo a CMR scan on the same day. Written informed consent was obtained from parents and/ or patients, and assent from patients where appropriate. Biopsies of the right ventricular aspect of the interventricular septum (IVS) were performed as part of routine surveillance. All EMBx specimens were interpreted by experienced cardiac pathologists who were blinded to the CMR results. The EMBx were diagnosed according to the classification of the ISHLT for acute cellular rejection (ACR) 15 and antibodymediated rejection. 16 As myocardial edema due to inflammation can expand the extracellular volume and increase native T1 times, patients with grade 2R or higher degree of rejection on EMBx were excluded from the study. Patient charts were reviewed for age of the organ donor, ischemia time of the donor heart, immunosuppressive medications, and number and severity of all previous episodes of rejection since HTx. Where available, the reports of fluoroscopic angiography for signs of CAV in accordance with the ISHLT grading system were reviewed. 17 Routine 2 yearly screening by coronary angiography for CAV began between 2 and 5 years after transplantation, depending on age, size, and risk factors for development of CAV. Intravascular ultrasound is not performed routinely at the authors' institution.
Results of cardiopulmonary exercise testing performed within 6 months of CMR were extracted from the patients' charts.
Asymptomatic children and adolescents with normal echocardiograms and electrocardiograms who underwent a screening CMR for family history of arrhythmogenic right ventricular cardiomyopathy were recruited as CMR controls.
In all patients and CMR controls, N-terminal probrain natriuretic peptide (NT-proBNP) was analyzed (Modular Analytics, Roche Diagnostics, Laval, QC, Canada) on the day of CMR.
Echocardiographic control values were extracted from the hospital database of healthy volunteers and were matched for patients' age and sex.

Magnetic Resonance Image Acquisition
The CMR examinations were performed on a 1.5 Tesla scanner (Magnetom Avanto, Siemens AG Healthcare Sector, Erlangen, Germany) using a phased-array multichannel surface receiver coil. A modified Look-Locker inversion recovery prototype sequence 18 was used to measure native and postcontrast longitudinal relaxation T1 times of myocardium and blood. The sequence consisted of 2 inversion-recovery prepared electrocardiogram synchronized Look-Locker experiments with inversion pulses of 100 ms and 150 ms, respectively, as well as 3 and 5 single-shot images after these inversion pulses. Other sequence parameters were as follows: Repetition and echo times 2.53 ms and 1.08 ms, respectively; in-plane resolution, 1.7 Â 1.7 mm; slice-thickness, 8 mm; flip angle, 35 degrees. Images were acquired in diastole at a single midventricular short axis slice orientation before and at least 10 minutes after the application of 0.2 mmol/kg gadopentetate dimeglumine (Magnevist, Bayer, Leverkusen, Germany). Breathholds were used in cooperative patients; all other patients were scanned during free breathing.
A stack of multiphase short axis slices was acquired in the steady state free precession technique for left and right ventricular volumes, as described previously. 19 The presence of late gadolinium enhancement (LGE) was determined qualitatively on standard long axis (4-chamber, 2-chamber, and 3-chamber) and short-axis slices using phase-sensitive inversion-recovery acquisitions longer than 10 minutes after administration of the contrast agent (same contrast injection as described above).

Image Analysis
Longitudinal relaxation times (T1 times) were measured using commercially available software (CVI42, Circle Cardiovascular Imaging, Calgary, AB, Canada). Contours were drawn at the midventricular short axis slice in the IVS (representing segments 8 and 9 of the American Heart Association 17 segments left ventricular (LV) model 20 ), the LV free wall (representing segments 11 and 12 20 ), and the entire LV myocardium (representing segments 7-12, 20 Figure 1). To avoid partial volume effects with blood, contours included only the central two thirds of the myocardium. T1 times in the blood pool were measured in the LV cavity; care was taken not to include trabeculations or papillary muscle. T1 times as well as standard deviation and R2, as a measure of goodness of curve fit of the relaxation curves, were recorded.
The TBPC of gadopentetate dimeglumine (as measure of fibrosis, with higher values reflecting increased fibrosis) was calculated as a function of the ratio of T1 change of myocardium precontrast and postcontrast compared to blood according to Flacke et al 8 : Ventricular volumes were extracted from the cine short axis stack in end-diastole and end-systole in the routine clinical fashion using commercially available software (QMass, version 7.2; Medis, Leiden, The Netherlands). Ventricular volumes are reported both indexed to body surface area as is customary in pediatric medicine as well as nonindexed because of a potential size mismatch between the host and the graft organ. Ejection fractions of both ventricles were calculated. 19

Echocardiography
The clinical echocardiogram 21 closest to the date of the CMR was reanalyzed by 1 reader, as long as it occurred within 60 days of the CMR. The routine protocol in each patient included ventricular and myocardial dimensions by 2-dimensional imaging, mitral valve inflow, and pulmonary vein flow characteristics by pulsed wave Doppler, isovolumic relaxation time, systolic, and diastolic velocities of the mitral valve annulus and IVS by tissue Doppler, as well as assessment of basal, mid, and apical circumferential strain, longitudinal strain, and torsion of the LV, all by 2-dimensional speckle tracking.
Measurements in controls were available through the hospital database of healthy volunteers.
All images of patients and controls had been obtained with a Vivid 7 or 9 ultrasound system (General Electric Medical Systems, Milwaukee, WI).

Statistical Methods
Descriptive statistics of continuous data are presented as the mean value ± standard deviation (SD) or median with interquartile range as appropriate; categorical variables are reported as number and percentage. After normality tests, unpaired t tests were used to compare variables between patients and controls, and between groups of patients, where applicable. The association of variables was expressed using Pearson correlation coefficient (r). A P value less than 0.05 was considered significant.

RESULTS
The study cohort consisted of 17 patients (47% girls) who had undergone HTx at a median age of 8.4 years (range, 6 days-16 years), and 9 healthy controls. The CMR studies occurred at a median of 1.3 years after HTx (range, 9 days-12.6 years). At the time of CMR, no patient had clinically significant rejection (biopsy grade, 0 R; n = 9; biopsy grade, 1 R; n = 8). Patient characteristics are summarized in Table 1 and standard CMR parameters in Table 2. Neither the patient nor the organ age was significantly different from controls ( Table 1). The LGE imaging did not reveal focal scarring in any of the patients or controls. Coronary angiography was performed in 2 patients within 6 months of the CMR date and showed no CAV in either. Immunosuppressive medication at the time of CMR consisted of tacrolimus and mycophenolate mofetil (MMF) (n = 13), tacrolimus, MMF and prednisone (n = 2), tacrolimus monotherapy (n = 1), and sirolimus with MMF (n = 1). Additional medications used include statins (n = 9), amlodipine for arterial hypertension (n = 5), and proton pump inhibitors (n = 5).

T1 Mapping Results
The R 2 values of T1 relaxation curves varied between 0.993 and 1.0 (mean 0.999) for the native T1 relaxation curves and between 0.998 and 1.0 (mean 1.0) for the postcontrast T1 relaxation curves, indicating an acceptable fit of the T1 relaxation curves. Native T1 times before the application of gadopentetate dimeglumine were significantly higher in HTx patients compared to controls in all investigated areas of the LV ( Table 3). The TBPC was elevated in patients after HTx in the LV free wall and the entire LV myocardium as compared to controls (Table 3). Both the patient and the control group showed higher native T1 times in the IVS than in the LV free wall (Table 3, patients IVS 1003 ± 31 ms vs LV free wall 973 ± 42 ms, P < 0.05 and controls IVS 974 ± 21 ms vs LV free wall 923 ± 12 ms, P < 0.001) as well as a higher TBPC value in the IVS than in the LV free wall (Table 3, patients IVS 0.48 ± 0.07 vs LV free wall 0.45 ± 0.06, P < 0.05 and controls IVS 0.45 ± 0.03 vs LV free wall 0.40 ± 0.03, P < 0.005). Figure 2 illustrates the differences of native T1 times and TBPC values between patients and controls for the 3 myocardial regions assessed.
There was no correlation between time since HTx and native T1, postcontrast T1 or TBPC. The 2 patients with the shortest ischemia times (82 and 88 minutes, respectively) were among the ones with the shortest native T1 times and lowest TBPC. Two patients underwent CMR early after transplantation (within the first 3 months). As native T1 values could be elevated in these patients due to edema, 22 they were excluded for a subanalysis, which did not show differences compared to patients who were older than 3 months from transplantation  No correlation existed between CMR fibrosis markers and heart age at the time of CMR (see caption of Table 1 for assessment of heart age in HTx patients).
Overall, 8 patients (47%) had previously experienced at least 1 episode of ISHLT 2R ACR. Since their HTx, 4 patients had 1 episode, 3 patients had 2 episodes, and 1 patient had 8 episodes of ISHLT 2R ACR. All episodes of previous ACR were asymptomatic incidental biopsy findings with no echocardiographic changes. No differences in native T1 times and TBPC were identified between patients with and without previous 2R ACR. One patient had had an episode of antibodymediated rejection with hemodynamic compromise 2.8 years after transplantation (2.2 years before CMR), and had required extracorporeal membrane oxygenation and intensification of immunosuppression. The native T1 time in the IVS of this patient was the highest of all patients.
Patients with arterial hypertension, as defined by medical treatment, did not have different T1 times, TBPC, or echocardiographic parameters as compared to normotensive patients.
No association was found between T1 values or TBPC and N-terminal probrain natriuretic peptide in the patient group.
Sex differences were not assessed in the HTx group as the gender of patient and donor may be discrepant and the etiology of fibrosis, that is, local versus humoral is a topic of debate. 23 In the control group, native T1 times were higher in girls in the IVS and entire LV (IVS female 992 ± 15 vs male 959 ± 11, P < 0.01, entire LV female 966 ± 9 vs male 940 ± 11, P < 0.005). Female values for TBPC were also slightly higher (IVS female 0.47 ± 0.03 vs male 0.45 ± 0.02, P < 0.05, entire LV female 0.44 ± 0.02 vs male 0.41 ± 0.03, P = 0.06). The difference in age between female and male controls was not significant (13.1 ± 3.2 years vs 11.9 ± 1.9 years, P = 0.13).

Systolic and Diastolic Ventricular Function
Echocardiographic data as presented in Table 4 were available in a subgroup of patients and/or controls only. In the patients, the median time interval between echocardiography and CMR was 22 days (range, 0-46 days). Tissue Doppler parameters in early diastole were reduced, and circumferential strain parameters of the patients were higher as compared to controls ( Table 4). The ratio of early diastolic mitral valve inflow (MV E) and early diastolic myocardial velocities of the lateral mitral annulus (MV e'), LV E/e', was elevated in the HTx patients (Table 4), although this difference did not reach statistical significance. LV E/e' correlated with native T1 times (entire LV r = 0.54, P < 0.05). None of the other echocardiographic parameters as assessed by standard 2-dimensional imaging, pulsed-wave Doppler, pulsed-and    Data are expressed as mean and standard deviation except for time since HTx and NT-proBNP (reported in median and range). a Heart age at CMR in the patients group, that is, allograft age, was calculated as the sum of the donor age at demise (median, 10.0 years; range, 1 month-29 years) and the time since HTx at CMR. b Data were available in 16 of 17 patients. NT-proBNP, N-terminal probrain natriuretic peptide.

Characteristics of patients and healthy controls (CMR controls)
color-tissue Doppler or speckle tracking imaging techniques correlated with native T1 times or TBPC.

Cardiopulmonary Exercise Tolerance
Exercise testing results were available in 8 patients (Table 1) who underwent cardiopulmonary exercise test at the age of 13.7 ± 2.7 years, with a median time interval of 3 months from CMR (range, 1-4 months). There was no correlation of percent of predicted peak VO 2 or predicted peak workload and native T1 times or the TBPC.

DISCUSSION
Diffuse myocardial fibrosis is the presumed universal morphological substrate of adverse ventricular remodeling in patients with various types of heart disease. The CMR T1 mapping approach allows for a noninvasive estimation of the myocardial fibrosis content. 10,13,14,24,25 In a small number of adult patients after HTx (n = 9), postcontrast T1 times correlated with myocardial collagen content. 26 Prolonged native T1 times, that is, before contrast application, and increased TBPC values by CMR both indicate increased diffuse myocardial fibrosis, in the absence of other conditions that expand the extracellular space, such as edema. In the current study, native T1 values and TBPC as a measure of myocardial fibrosis were higher in children after HTx compared to healthy controls. The most significant differences in native T1 values and TBPC between HTx patients and controls were observed in the free wall of the LV ( Figure 2B), whereas values in the IVS overlapped between the 2 groups ( Figure 2C). The LV free wall is a predilection site of functional and structural changes in many disease processes, including Duchenne's muscular dystrophy or dilated cardiomyopathy. 27 It is tempting to speculate that the free wall is more susceptible to adverse fibrotic remodeling because of increased chronic stress in this region. The CMR indices of diffuse fibrosis in the IVS, on the other hand, were closer to the ones of healthy controls.
The hearts of the pediatric HTx patients examined in this study had normal echocardiographic dimensions and preserved ejection fractions. Several echocardiographic markers of diastolic function were abnormal, as has been reported previously. 6,28,29 Circumferential strain was elevated in the patients   as compared to controls. Whether this observation is merely a reflection of the higher heart rates in HTx patients, is unclear, but heart rate dependencies of tissue Doppler measurements are well known, including in children after HTx. 28,30,31 Native T1 times correlated with the LV E/e' ratio by echocardiography as a marker of diastolic dysfunction and surrogate of elevated LV filling pressures. This association is in line with the reported association between histological myocardial fibrosis and restrictive physiology in patients who underwent retransplantation. 32 No correlation of diffuse myocardial fibrosis markers was found with other markers of diastolic dysfunction. The transplanted hearts exhibited signs of diffuse fibrosis even though neither the transplanted patients nor their hearts were older than the controls. These findings suggest that mechanisms of fibrosis other than aging are at play. Possible etiologies of increased fibrosis in pediatric transplant recipients include factors related to the transplantation surgery, such as ischemia and ischemia-reperfusion injury, donorspecific antibodies, arterial hypertension, CAV, drug toxicity, 29,33 and previous episodes of rejection, although data about the association of rejection and fibrosis are conflicting. 34,35 The reason of fibrosis in the patients studied remains speculative, but could be related to ischemic injury, as interestingly, 2 patients with very short ischemia times had the lowest measures of fibrosis.

Results of echocardiography
CAV is a likely cause of fibrosis, as has been suggested in adults, 7 although the evidence remains anecdotal. Conceptually, the option of monitoring for end-organ damage-related signs of CAV noninvasively and without ionizing radiation is attractive, especially in infants and children. As a marker and possible determinant of myocardial dysfunction, diffuse myocardial fibrosis appears to have a role independent of circumscribed patchy scarring. 24,36 Of note, none of the patients in this study showed signs of localized scars by LGE. In both controls and HTx patients, native T1 times and TBPC were higher in the IVS than in the free wall of the left ventricle, suggesting a higher amount of collagen and connective tissue in the septum. This is in keeping with data from healthy adults, where regional differences and also higher amounts of connective tissue in the septum were measured, 8,37 although the topic is not undebated. 38 Female controls exhibited higher native T1 times and TBPCs both for the IVS and the entire LV than boys. This corroborates previous studies, at least up to the age of 45 years. 12 Further studies with serial CMR studies in a larger cohort of patients are needed to confirm and better understand the preliminary findings presented here. Changes in indices of fibrosis over time and the relationship with CAV remain to be explored. Animal and human studies have shown that myocardial fibrosis is reversible in some conditions. In this context CMR derived fibrosis indices may prove invaluable for the identification of patients who may benefit from antifibrotic treatment. 13 Limitations Several limitations of this pilot study must be mentioned: The cohort size was small, potentially obscuring associations between fibrosis and functional parameters, and only two patients underwent coronary angiography, prohibiting the examination of a possible relationship between CAV and CMR fibrosis markers. The controls for CMR and echocardiography consisted of different individuals and matching for age and sex was only feasible for echocardiography, but not for CMR controls. Only 1 short-axis slice was studied with T1 mapping CMR which may not be representative for the complete LV myocardium. For future studies, measurements in several slices reflecting all regions of the heart are recommended. It is recognized that native T1 and, to a lesser extent TBPC values, are elevated in acute ischemia, infarction and myocarditis (reflecting myocardial edema). 11 However, these causes are unlikely in the patients studied here, given the lack of clinical or histological evidence.

CONCLUSION
Cardiac magnetic resonance markers of diffuse myocardial fibrosis are present in children after HTx. They appear to be associated with measures of diastolic dysfunction by echocardiography.