The impact of water exchange on estimates of myocardial extracellular volume calculated using contrast enhanced T1 measurements: A preliminary analysis in patients with severe aortic stenosis

Guidelines recommend measuring myocardial extracellular volume (ECV) using T1‐mapping before and 10–30 min after contrast agent administration. Data are then analyzed using a linear model (LM), which assumes fast water exchange (WX) between the ECV and cardiomyocytes. We investigated whether limited WX influences ECV measurements in patients with severe aortic stenosis (AS).


INTRODUCTION
Society for Cardiovascular Magnetic Resonance (SCMR) guidelines recommend measuring myocardial extracellular volume (ECV) with T 1 maps taken before and 10-30 min after a 0.1 to 0.2 mmol/kg dose of contrast agent. 1 Subsequent analysis employs a linear model (LM) which assumes fast cell-interstitial (transcytolemmal) water exchange (WX), predicting a linear relationship between the change in relaxation rates, R 1 (=1/T 1 ), in myocardium and blood following contrast agent administration. 2,3A pre-clinical study 2 showed that when there was a large difference between intra-and extra-cellular T 1 following contrast administration, use of a LM can significantly underestimate ECV.The two-site exchange model (2SXM), 4 which accounts for WX, can be fitted to data acquired at a range of contrast agent concentrations in myocardial tissue and blood. 2 This provides estimates of both ECV (corrected for WX) and the residence time of water in the cell (τ ic ), a surrogate marker of cardiomyocyte diameter. 5Aortic stenosis (AS) is a condition that can lead to diffuse interstitial fibrosis.T 1 mapping can be used to measure fibrosis, using ECV. 6Regression of diffuse fibrosis is observed following aortic valve intervention 7,8 and outcomes following aortic valve intervention may be linked to the presence and extent of myocardial fibrosis. 7A preclinical study in a rodent model of trans-aortic constriction (TAC) 5 used a 2SXM to validate the intracellular lifetime of water as a surrogate marker of cell size.We chose patients with severe AS as a human model to investigate the influence of WX on ECV estimates.Since myocardial cell size is increased in severe AS, 9 WX is likely to be slower.The aim of this study was to investigate whether WX influences estimates of ECV in the myocardium of patients with severe AS.

Cardiovascular magnetic resonance imaging
CMR was performed at 3 T (Siemens Magnetom Prisma, Erlangen, Germany).T 1 measurements were undertaken (a) before contrast, (b) 4 min after a 0.05 mmol/kg bolus dose of gadobutrol (Gadovist; Bayer AG), and (c) 4 min, (d) 10 min, and (e) 30 min after an additional 0.1 mmol/kg gadobutrol which was delivered ∼8 min after the first injection (Figure 1).Resting myocardial blood flow (MBF) with automatic inline perfusion mapping was performed at the time of the 0.05 mmol/kg dose of gadobutrol using the same three short-axis myocardial slice locations used for T 1 measurement (see below). 10 Gadobutrol was used due to its higher relaxivity compared to other contrast agents. 11T 1 measurements used a multiparametric SAturation-recovery single-Shot Acquisition (mSASHA), 12 using six images of variable saturation recovery and two images with T 2 preparation to generate both T 1 and T 2 maps.mSASHA was used rather than a MOLLI acquisition because of its higher accuracy and precision and reduced sensitivity to T 2 , which is important as the T 2 of blood changes markedly following contrast agent administration.Three short-axis (basal, mid and apical) 8 mm slices were acquired with a free-breathing protocol 13 with FOV 360 mm × 270 mm, matrix 256 mm × 192 mm, voxel size, 1.4 mm × 1.4 mm, TR/TE: 904.0/1.26ms, saturation time (TS): 1 image with no preparation and 6 at 550 ms, maximum flip angle: 100 • , GRAPPA 2. In addition to T 1 measurements, a standardized imaging protocol was used consisting of short axis cine stack imaging of the left ventricle, short and long axis late gadolinium enhancement (LGE) imaging (performed 5-15 min after the cumulative dose of 0.15 mmol/kg), and standard long axis views of the left ventricle (two-chamber, three-chamber, and four-chamber).After an initial TI scout to derive the optimal inversion time for myocardial nulling, a short axis 15 slice stack of single slice phase sensitive inversion recovery motion corrected balanced steady state free-precession acquisition was acquired with FOV: 400 mm × 300 mm, matrix: base resolution

CMR analysis
Image analysis was undertaken using cvi42 software (Circle Cardiovascular Imaging Inc, Calgary, Canada, release number 5.9.4) by an experienced operator (EACVI level 3 CMR accreditation, NS).For T 1 mapping, short axis T 1 maps were manually contoured at the endocardial and epicardial borders.Partial volume effects of blood were minimized by setting an automatic offset of 10% from the endocardial and epicardial borders.Left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), myocardial mass, and maximal wall thickness (MWT) were calculated with LVEDV and myocardial mass measurements indexed to body surface area (BSA). 14To calculate BSA, the Mostellar formula was used. 15Short axis, mid ventricular T 1 maps together with the patient's hematocrit were used to calculate ECV.Areas of myocardial infarction or focal fibrosis (identified on LGE images) were excluded from analysis.In cases where myocardium and blood differentiation were difficult due to partial volume effects and a thin left ventricular (LV) wall, a region of interest in the basal septum was used.Three LM-based ECV estimates were calculated using pairs of T 1 measurements (a and b), (a and d) and (a and e) and these were compared to 2SXM ECV estimates made using all five T 1 measurements.Details of the fitting performed and an example spreadsheet to enable such a fit are provided in Supporting Information.Rest MBF values for the entire mid-ventricular slice were reported using the automatic inline perfusion maps generated as previously described. 10

Statistical analysis
Statistical analyses were carried out using SPSS software version 27 (IBM, Armonk, New York) and Excel version 16.66.1 (Microsoft).Normality of data was checked using the Shapiro-Wilk test.Continuous normally distributed data were expressed as mean ± SD, and continuous non-normally distributed data as median (range).
Comparison between quantitative variables was performed by independent-sample parametric (Student's t-test) or non-parametric (Mann-Whitney) statistical tests, as appropriate.p < 0.05 was considered significant.

Ethics approval and consent to participate
All the patients provided written informed consent for their inclusion.The study was approved by the local ethics committee, (Leeds East Research Ethics Committee), IRAS number 245109, REC reference 18/YH/0169.

Baseline characteristics
All 25 patients and 5 healthy controls completed CMR scanning.Patients and controls were well matched for age and sex (Table 1).Patients with severe AS had significantly higher indexed LV mass (81 ± 18 g/m 2 vs 55 ± 11 g/m 2 in controls), p = 0.004, and maximal LV wall thickness (14 ± 2 mm vs 10 ± 2 mm in controls), p = 0.002.

LGE and rest MBF
Analysis revealed two patients (8%) with evidence of ischemic LGE with a mean 1.5 segments affected.There were six patients (24%) who had evidence of non-ischemic LGE (mostly affecting the basal lateral segment) with a mean of 1.83 segments affected.Seventeen patients (68%) and all controls had no LGE abnormalities.Patients had resting MBF values of 0.73 ± 0.21 mL/g/min with no significant difference from controls who had values of 0.71 ± 0.20 mL/g/min (p = 0.79).

ECV analysis
Analysis of T 1 maps acquired in patients and controls are given in Table 1 and an example set of ECV fits are shown in Figure 2. Median ECV estimated using the LM 4 min following a 0.05 mmol/kg dose of gadobutrol was 25% in patients compared to 26% in controls.Median ECV estimated using the LM at 10 min after a cumulative dose of 0.15 mmol/kg gadobutrol was 21% for patients and 23% for controls, and at 30 min was 22% for patients and 22% for controls.LM ECV estimates for patients increased between 10 and 30 min following a cumulative contrast dose of 0.15 mmol/kg (p = 0.0001; Wilcoxon signed-rank test) but were highest 4 min after a low dose of contrast (0.05 mmol/kg).Figure 3 shows the increase in ECV estimates in patients using a LM as the concentration of contrast agent in the ECV decreases.These estimates of ECV are compared to 2SXM ECV estimates which were 25% for patients and 26% for controls.Cell residence time (τ ic ) was higher, though not significantly, for patients with AS (median 0.21 s) compared to controls (median 0.12 s) consistent with larger cardiomyocytes.

T A B L E 1
Baseline characteristics and CMR findings of patients and healthy controls.

F I G U R E 2
An example of the non-linear association between R 1 (1/T 1 ) of the myocardium and blood in a patient with severe aortic stenosis.In this example the residence time of water in the myocytes was estimated using the 2SXM at 215 ms and the ECV at 30%.LM ECV estimates at 10 and 30 min after 0.15 mmol/kg and at 4 min after 0.05 mmol/kg (represented by the slopes of the straight lines in the figure) were 24%, 25%, and 28%, respectively.

F I G U R E 3
A box-and-whisker plot showing LM ECV estimates in patients as a function of contrast agent concentration as a result of dose (and time) compared with ECV estimated by the 2SXM.

Water exchange and underestimation of ECV
WX between tissue compartments (intravascular, interstitial, and cellular) under physiological conditions is usually fast enough that any difference in the underlying relaxation rates of those compartments is averaged out and the myocardium can be considered to have a single T 1 relaxation time. 16However, contrast agents decrease the T 1 of water in the spaces in which they distribute and WX may no longer be fast enough to average out the subsequent relaxation rate differences.The measured tissue T 1 begins to depend on the rate of WX between compartments and transcytolemmal WX can play a pivotal role in determining myocardial T 1 post-contrast. 2 We used the 2SXM as the reference standard for measurement of ECV as it accounts for WX effects.In this study we obtained a range of T 1 values over time, resulting from different blood concentrations of contrast agent.We gave a small initial dose of gadobutrol to produce a small increase in myocardial R 1 , and then the remainder to produce a peak value of myocardial R 1 .We subsequently measured T 1 as the contrast agent was filtered from the blood and washed out of the myocardium over the next 30 min (Figure 1).We have shown that LM ECV estimates vary as a function of contrast agent concentration in the blood (Figure 2); estimates obtained between 10 and 30 min following a cumulative dose of 0.15 mmol/kg gadobutrol are underestimated by up to 12% in patients with severe AS compared with estimates of ECV obtained using a 2SXM which accounts for limited transcytolemmal WX (Figure 4).Similar results were presented by Dabir et al in a large study of healthy subjects (see Table 3 in 17 ).Their measures of ECV increased as the dose of gadobutrol decreased, though the authors did not recognize this as a consequence of limited WX.Coelho-Filho et al applied the 2SXM to data acquired in mouse models of TAC and hypertensive heart disease (L-NAME; a nitric oxide synthase inhibitor) 5 and showed that WX effects are more pronounced when the ECV is expanded and in the presence of ventricular The absolute difference between 2SXM and LM estimates of ECV (y-axis) increases as the residence time of water in the myocytes (x-axis) increases.Orange values represent data for patients and blue values represent data for healthy controls.
hypertrophy.The 2SXM predicts a sub-linear dependence of R 1 in tissue on R 1 in blood, the degree of which is determined by τ ic (as seen in our Figure 2).In patients with AS we observed hypertrophy (increased wall thickness and myocardial mass) but no evidence of interstitial fibrosis; ECV in the patients was no larger than ECV estimated in controls (Table 1).

Residence time of water in cardiomyocytes
It is well established that in severe AS cell size is increased. 9,18Using two mouse models to validate their findings, L-NAME and TAC, Coelho-Filho et al showed that the cell residence time of water (τ ic ) is a measure of cardiomyocyte volume to surface area ratio, an index of minor cell diameter in the case of cardiomyocytes. 5e obtained similar estimates of τ ic in our patients and showed that as τ ic increases (as the myocytes get bigger and WX slows down) the difference between LM and 2SXM estimates of ECV increases (Figure 4).

Contrast agent steady state
There are differing opinions on when equilibrium of contrast agent between the ECV and blood pool is established.In a study evaluating myocardial contrast uptake in patients with dilated cardiomyopathy, multiple T 1 measurements after injection of 0.1 mmol/kg Gd-DTPA and a plot of R 1 tissue vs R 1 blood did not show any significant deviation from a linear relationship. 19The authors suggested that a contrast agent steady state was achieved after 4 min.Another study showed that contrast agent in the ECV of normal myocardium, reaches equilibrium with the blood around 5 min after injection, while contrast agent in infarcted myocardium takes 15-20 min to reach a steady state (0.15 mmol/kg Gd-DTPA). 20Klein et al supported these findings using 0.2 mmol/kg Gd-DTPA, suggesting contrast agent steady states can be achieved almost immediately in normal tissue, compared to up to 20 min in scarred myocardium. 21Goldfarb and Zhao 22 similarly showed that contrast agent in infarcted myocardium took 15 min to reach a steady state when analyzed using a LM following a 0.

Limitations
Data from this preliminary study are limited to patients with AS and the results cannot be generalized or extended to other myocardial pathologies without further research.
Although there is no "gold standard" to estimate ECV, such as endomyocardial biopsy, we used the 2SXM as the reference standard because it models the effect of WX though we were not able to determine whether the 2SXM was preferred over the LM in any given heart.In this study, no histological samples were obtained and τ ic remains to be validated as a marker of cardiomyocyte size in humans, although our patient group with severe AS can be compared with the TAC model used for τ ic validation by Coelho-Filho et al. 5 There was considerable variability in our residence time estimates.The precision with which τ ic can be estimated depends on the dose of contrast agent injected.A dose of 0.5 mmol/kg was used in the murine TAC model study. 5We chose to use a clinically acceptable dose of 0.15 mmol/kg and this likely had an impact on the precision of our estimates of τ ic , though not on the precision of our estimates of ECV which were similar to the precision of those obtained using the LM.We did not investigate contrast agent doses between 0.05 and 0.15 mmol/kg, estimates of ECV using a low dose at times beyond 4 min, or contrast agents with lower relaxivity which will be less sensitive to limited WX.In this study we measured T 1 using mSASHA, however future studies looking into the effect of WX should compare mSASHA with other techniques of measuring T 1 such as MOLLI or saturation pulse prepared heart rate independent inversion recovery (SAPPHIRE).

CONCLUSIONS
Current guidelines for contrast agent dosing and timings for T 1 mapping leads to underestimation of ECV, by up to 12% compared to 2SXM estimates, in patients with severe AS, due to limited transcytolemmal WX.Our results suggest that, in patients with severe AS, waiting 10-30 min post contrast before T 1 mapping may be unnecessary since a contrast agent steady state is reached by 4 min.The LM ECV underestimates are a result of limited WX and not a delayed contrast agent steady state.A T 1 map following low contrast dose (0.05 mmol/kg) may provide more reliable estimates of ECV when using a LM.
The sequence of prototype multiparametric saturation-recovery single-shot acquisition (mSASHA) T 1 maps made after different times and doses of gadobutrol.(B) Time points (A-E) in Panel A used to measure blood R 1 over time in a healthy control.256 mm × 144, pixel size 1.56 mm × 2.08 mm, slice 8 mm, flip angle/TR/TE: 50 • /904 ms/1.14 ms, 8 averages.

Patients (n = 25) Healthy controls (n = 5) p-Value
Note: Continuous variables are presented as mean ± SD for normally distributed data or median (range) for non-normally distributed data, indicated by + .Dichotomous variables are presented as number (%).Appropriate comparison test was undertaken depending on normality of data distributions.p-Value (paired t test or Mann-Whitney test) is considered significant at <0.05 level and indicated by *.Abbreviations: 2SXM, two-site exchange model; BSA, body surface area; ECV, extracellular volume; LM, linear model; LVEDV, left ventricular end-diastolic volume; LVEDV(i), left ventricular end-diastolic volume (indexed); LVEF, left ventricular ejection fraction; MWT, maximal wall thickness; τ ic (cell residence time of water).