Dilated cardiomyopathy (DCM) is increasingly recognized as a heterogenous disease with distinct phenotypes on late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) imaging. While mid-wall striae (MWS) fibrosis is a widely recognized phenotypic risk marker, other fibrosis patterns are prevalent but poorly defined. Right ventricular (RV) insertion (RVI) site fibrosis is commonly seen, but without objective criteria has been considered a non-specific finding. In this study we developed objective criteria for RVI fibrosis and studied its clinical relevance in a large cohort of patients with DCM.We prospectively enrolled 645 DCM patients referred for LGE-CMR. All underwent standardized imaging protocols and baseline health evaluations. LGE images were blindly scored using objective criteria, inclusive of RVI site and MWS fibrosis. Associations between LGE patterns and CMR-based markers of adverse chamber remodeling were evaluated. Independent associations of LGE fibrosis patterns with the primary composite clinical outcome of heart failure admission or death were determined by multivariable analysis.± 14  with a mean left ventricular (LV) ejection fraction (LVEF) of 37%. At a median of 1061 days, 129 patients (20%) experienced the primary outcome. Any abnormal LGE was present in 306 patients (47%), inclusive of 274 (42%) meeting criteria for RVI site fibrosis and 167 (26%) for MWS fibrosis. All with MWS fibrosis showed RVI site fibrosis. Solitary RVI site fibrosis was associated with higher bi-ventricular volumes , lower bi-ventricular function , and higher extracellular volume (ECV). Patient with solitary RVI site fibrosis experienced a non-significant 1.4-fold risk of the primary outcome, increasing to a significant 2.6-fold risk when accompanied by MWS fibrosis.The mean age was 56 RVI site fibrosis in the absence of MWS fibrosis is associated with bi-ventricular remodelling and intermediate risk of heart failure admission or death. Our study findings suggest RVI site fibrosis to be pre-requisite for the incremental development of MWS fibrosis, a more advanced phenotype associated with greater LV remodeling and risk of clinical events.The online version contains supplementary material available at 10.1186/s12968-021-00761-0. Patients with dilated cardiomyopathy (DCM) are recognized to have a significant prevalence of replacement myocardial fibrosis as identified by late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) –4. StudiThe RV pedicles are recognized to experience elevated wall tension during states that contribute to RV volume and/or pressure overload . Cohort We hypothesized that RVI site fibrosis reflects an adaptive expansion of extracellular matrix at the RV pedicles in response to altered RV loading conditions that are experienced during the development of DCM. We further hypothesized that this marker may herald more advanced stages of remodelling associated with greater likelihood of heart failure-related events. In a large, prospectively recruited cohort of 645 patients referred to LGE-CMR for DCM we examined the prevalence and associated phenotypic features of RVI site fibrosis and MWS fibrosis, inclusive of their associations with bi-ventricular chamber volumetry, function and left ventricular (LV) extracellular volume (ECV) fraction. Their respective influence on the future occurrence of heart failure admission or death was also explored.This was a prospective observational cohort study of subjects recruited between January 2015 and May 2018 and followed for a minimum of 12-months. The study was a pre-defined sub-cohort analysis of the Cardiovascular Imaging Registry of Calgary (CIROC) at the Libin Cardiovascular Institute of Alberta (NCT04367220). All patients in Southern Alberta referred for CMR imaging are recruited by, and data collected using commercial software . This is inclusive of tablet-based patient engagement and consent, deployment of standardized patient-reported health questionnaires, indication-based CMR protocolling, standardized test reporting and phenotype coding, and automated linkage to electronic health record data inclusive of laboratory test results and administratively coded outcomes.Patients were considered eligible for this study based on the following criteria; (i) referral for CMR-based evaluation of DCM: (ii) confirmation of LV ejection fraction (LVEF) ≤ 50% by CMR-based quantification, (iii) no prior clinical history of, or ICD-10 coded prior occurrence of myocardial infarction, percutaneous revascularization or coronary artery bypass grafting (CABG), and (iv) no LGE-based evidence of prior myocardial infarction, as defined by sub-endocardial pattern LGE in a typical coronary artery distribution. Patients with any clinically documented history of pulmonary arterial hypertension, congenital heart disease, severe valvular insufficiency/stenosis, or other cardiomyopathy diagnosis were excluded. Specifically, other excluded cardiomyopathy diagnoses included: acute myocarditis, cardiac sarcoidosis, cardiac amyloidosis, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy (ARVC), restrictive cardiomyopathy, or constrictive pericardial disease. Only patients completing at least 12-months of clinical follow-up were considered eligible. A post-hoc sub-study was performed with stratification of the referral population to those meeting objective dilation criteria, according to the consensus guideline for a body surface area (BSA)-indexed LV end-diastolic volume (LVEDVI) .The study was approved the Conjoint Health Research Ethics Board at University of Calgary and all subjects provided written informed consent. All research activities were performed in accordance with the Declaration of Helsinki.CMR imaging was performed using 3T clinical CMR scanners . Standardized CMR imaging protocols were used routinely inclusive of cine imaging using a balanced steady-state free precession (bSSFP) pulse sequence and LGE imaging using a standard inversion recovery gradient echo pulse sequence both performed in standard 2, 3, and 4-chamber long axis views and sequential short-axis slices. LGE imaging was performed 10-min following intravenous administration of gadolinium contrast . Typical imaging parameters were: 6 mm slice thickness, 2 mm gap, TE 3.9 ms, flip angle 20°, matrix 256 × 205, segments 13–21, iPAT 2. T1 mapping was performed using a modified Look-Locker inversion recovery (MOLLI) pulse sequence in basal and mid-level short axis views prior to, and 15-min following intravenous contrast injection.Quantitative image analysis was performed using commercially available software  and with use of standardized operational procedures adherent to published Society for Cardiovascular Magnetic Resonance (SCMR) recommendations . Short aA standardized reporting interface  was used to code the presence, location and extent for all observed patterns of replacement myocardial fibrosis Fig. . PatternPre- and post-contrast T1 mapping was evaluated to estimate the ECV of mid ventricular tissue. Regions of interests were drawn in the myocardium and blood pool of mid-ventricular short-axis image, as shown in Fig. myo_pre and T1myo_post are native and post-contrast measures of myocardial T1, and T1blood_pre and T1blood_post are native and post-contrast measures of T1 in the blood pool.Clinical outcomes were collected by way of administrative data matching followed by adjudication of all identified events through medical chart review. Heart failure admission was defined according to occurrence of an ICD-10 coded heart failure admission (154.X) in conjunction with hospital admission ≥ 24 h duration. All-cause mortality was determined by combined consideration of data matching from Vital Statistics Alberta and In-hospital coded deaths. No patients were lost to follow-up.All descriptive statistics were expressed as mean ± standard deviation (SD) and categorical variables were expressed in counts with percentages. Comparison between two groups was performed with the independent Student t-test or Mann–Whitney U test, comparison between three groups was performed using ANOVA or Kruskal–Wallis test depending on the distribution. Pairwise comparison with Bonferroni procedure was performed as post-hoc analysis for three group comparison. Fisher exact test was used to compare categorical data. Missing variables, when identified, were specified in Table Univariable associations between demographic or CMR characteristics and the outcomes were performed using Cox proportional hazards regression. Multivariable Cox regression analysis was performed to assess associations between with and without the most prevalent patterns of LGE, these being RVI site fibrosis or MWS fibrosis, and the outcome. Covariates with a p-value < 0.1 were considered eligible for adjustment within each multivariable model. All models were assessed for collinearity and proportional hazards assumption.Intra- and inter-observer reliability was tested using 30 randomly selected studies stratified to achieve a balanced representation of normal, RVI site positive and MWS pattern positive cases. Two observers scored all studies, followed by repeat scoring of cases in random order by the second observer. Agreement was described by Cohen’s kappa coefficient.Statistical analyses were performed using RStudio 1.2.5019  and SPSS for Macintosh .Six hundred and forty-five patients were consecutively recruited and met study eligibility criteria. Baseline clinical characteristics for all subjects, as well as those with and without the primary outcome are provided in Table The mean LVEF was 37 ± 11%, and the mean RVEF was 46 ± 12%. All additional CMR-based chamber measurements are summarized in Table Replacement myocardial fibrosis was observed in 306 (47%) patients Table . This inAmong patients with RVI site but no MWS fibrosis  secondary patterns of fibrosis were observed in 9 (8%): 6 sub-epicardial and 3 mid-wall patchy fibrosis. Patients with combined RVI and MWS fibrosis  showed secondary patterns in 24 (14%): 23 sub-epicardial and 1 mid-wall patchy fibrosis.2 without RVI fibrosis; p < 0.01), higher RVESVI , and lower RVEF . Similarly, these subjects showed higher LVEDVI , higher LVESVI  and lower LVEF . LA volumes were significantly higher among patients with RVI site fibrosis .As shown in Additional file 2, p = 0.01); LVEF ; RVEDVI ; RVESVI ; RVEF .Sub-stratification of patients into groups with RVI fibrosis alone versus those with RVI plus MWS was subsequently performed, as shown in Fig. ± 4% . By comparison, patients with isolated RVI fibrosis (RVI+MWS−) or combined pattern fibrosis (RVI+MWS+) showed significant elevation in mid-ventricular ECV .A sub-group of 80 subjects underwent parametric T1 mapping to identify associations between the presence of RVI site fibrosis and LV myocardial ECV. Subject selection for this analysis was based upon availability of high-quality T1-mapping image datasets with prevalence of respective phenotypes as follows; RVI−MWS− (n = 34), RVI+MWS− (n = 19), and RVI+MWS+ (n = 27). ECV was lowest among RVI−MWS− subjects with a mean ECV of 31 During a median follow up of 1061 days (IQR = 674 days), 129 patients (20%) experienced the primary composite outcome of heart failure hospitalization or death . Univariable associations between baseline characteristics and the primary outcome are shown in Table RVI fibrosis (with or without MWS) remained an independent predictor of the primary outcome in three different multivariable models, these designed to provide robust adjustment for patient comorbidities (Model 1), medication use Model 2), and balanced adjustment of both (Model 3), as shown in Table , and balIdentical multivariable analyses were performed replacing any RVI fibrosis with the less frequently observed combined pattern of RVI plus MWS fibrosis. This provided corresponding adjusted hazards of 2.02  for Model 1, 1.93  for Model 2, and 1.78  for Model 3.Kaplan–Meier analysis curves for patients with and without any RVI are shown in Fig. Exploratory subgroup analyses were undertaken to compare associations between solitary RVI fibrosis  and the combined RVI and MWS fibrosis pattern  with the primary outcome. As shown in Fig. Post-hoc analyses were conducted to explore the influence of LV dilation on the prognostic utility of RVI fibrosis in the referral population. Patients were stratified by BSA-indexed LVEDV according to standard deviation (SD) from sex-matched healthy reference values . This reGood reproducibility of RVI site fibrosis was observed using the objective criteria. Respective kappa coefficients for intra- and inter-observer ratings were 0.93 and 0.88 respectively. By comparison, kappa coefficients for MWS fibrosis were lower at 0.91 and 0.82, respectively.This prospective study was designed to explore the prevalence, phenotypic associations and prognostic significance of RVI site fibrosis in the context of MWS fibrosis for a DCM referral population. Using objective criteria, any RVI site fibrosis was identified in 42% of DCM patients, and was associated with increased bi-ventricular chamber volumes, reduced ejection fractions, and a 2.1-fold unadjusted risk of heart failure admission or death. This fibrosis pattern was observed both in isolation (39% of cases) and accompanied by MWS fibrosis (61% of cases). Isolated RVI site fibrosis was associated with objective markers of bi-ventricular remodeling and a non-significant 1.4-fold increased risk of heart failure admission or death. Specifically, this subgroup showed an 18% elevation in LVESVI, 4% absolute reduction in LVEF, 24% increase in RVESVI, 5% absolute reduction in RVEF, and 3% absolute elevation in mid septal ECV. Patients with incremental evidence of MWS fibrosis showed worsening of LV remodeling and a significant 2.3-fold risk of heart failure admission or death.Our observations confirm that the RVI sites commonly experience a need to expand collagen content in the setting of DCM, and that this is associated with objective bi-ventricular remodeling and diffuse ECV expansion. While longitudinal studies are desirable, the ubiquitous presence of RVI site fibrosis in patients with MWS fibrosis strongly supports that its isolated presence represents an antecedent phenotype. As a requisite stage it is represented by objective bi-ventricular remodeling not yet sufficient to confer significantly elevated risk of heart failure admission or death. Therefore, while MWS fibrosis reflects an advanced clinical stage where major cardiovascular events are frequent , 2, 4–7,Our findings are in agreement with the study previously reported by Yi, et al. that, in a detailed cohort study of 360 DCM patients undergoing CMR, showed isolated RVI site fibrosis to convey a non-significant trend towards elevated event risk . Upon coThe factors that contribute to the development of RVI site fibrosis in patients with DCM have not been defined and therefore remain speculative. However, meaningful insights can be gathered from prior studies performed across other disease states. Most notably, chronic elevations in RV systolic pressures have been associated with a higher prevalence of RVI site fibrosis in patients with pulmonary hypertension –13. SimiExpanding evidence supports that patients with left-sided heart disease may similarly demonstrate irreversible remodeling of the RV myocardium , 25. NitCompared to patients without identifiable RVI fibrosis (RVI−MWS−), patients with the RVI+MWS− and RVI+MWS+ phenotypes demonstrated incremental elevation in chamber volumes, reduction in chamber function, and elevation in mid ventricular ECV. The latter finding has been associated with higher mortality in patients with DCM , and proLimitations of this study are recognized as patients were recruited from a single tertiary care institution. Accordingly, our results require confirmation using a multi-center study design. While our referral cohort was of substantial size, females were under-represented and sub-group analysis was limited by the number of events accrued for each sub-group. The latter is relevant to conclusions surrounding the prognostic significance of isolated RVI site fibrosis, where a non-significant 1.4-fold increased risk of the primary outcome (p = 0.16) was observed. Within a larger cohort study this may emerge to be significant. Further, through our detection of clinical events using administratively coded data, we may under-estimate event rates.Our study focused on evaluating patients referred to CMR with a clinical diagnosis of DCM, this defined by criteria that did not mandate performance of invasive coronary angiography or stress perfusion testing. Accordingly, we cannot exclude the potential for sub-clinical ischemia in our population. Also, we did not exclude patients based on dilation criteria at time of CMR. To address this, we performed post-hoc analyses stratifying our referral population by CMR-based measures of LVEDVI. This demonstrated any RVI site fibrosis remained prognostic among patients with a LVEDVI within 2SD and between 2 and 4SD above CMR-based reference values. However, significance was lost for patients with severe (> 4SD) dilation. Accordingly, the incremental value of LGE-based phenotyping among patients with severe structural remodeling may be limited. We did not assess the impact of device-related therapy and, while excluding patients with known pulmonary hypertension, did not have consistent access to echocardiographic-based measures to exclude sub-clinical elevations in pulmonary arterial pressures. Finally, we acknowledge that the clinical outcome of heart failure admission can be influenced by regional practice patterns.Our study design limits conclusions regarding sequential occurrence of, or mechanistic relationships between RVI site and MWS fibrosis patterns in patients with DCM. Longitudinal cohort studies inclusive of serial imaging are required to address such hypotheses. Histologic studies validating our objective criteria for RVI site fibrosis would also be of benefit, however, was beyond the scope of this study.Among patients referred for CMR-based evaluations of DCM, RVI site fibrosis (scored by objective criteria) was common and ubiquitously seen in patients with MWS fibrosis. Isolated RVI site fibrosis was associated with objective evidence of bi-ventricular remodeling. When employed as a solitary phenotypic risk marker, any RVI site fibrosis was associated with a 2.1-fold increased risk of heart failure or death. Incremental consideration of MWS fibrosis permits sub-classification into RVI+MWS− and RVI+MWS+ sub-groups, these respectively experiencing a non-significant 1.4-fold and significant 2.6-fold risk of the primary outcome versus RVI−MWS−.Overall, our results offer support for RVI site fibrosis being an antecedent phenotypic stage to MWS fibrosis that is objectively associated with bi-ventricular remodeling and intermediate risk. Future studies may therefore be justified evaluating targeted therapeutic strategies to mitigate phenotype progression in patients with isolated RVI site fibrosis.Additional file 1. Baseline clinical and CMR characteristics for patients with and without right ventricular insertion (RVI) site fibrosis.


No difference was seen between controls and HFpEF (p > 0.34). Non-normalized forces displayed no differences between controls and HFpEF  and did not correlate with EF (p = 0.36). Left ventricular hemodynamic force analysis, whether indexed to LV volumes or not, is not ready for clinical trials on HFpEF assessment.Hemodynamic force analysis has been proposed as a novel tool for early detection of subclinical systolic dysfunction in heart failure with preserved ejection fraction (HFpEF). Here we investigated the ability of hemodynamic forces to discriminate between healthy subjects and heart failure patients with varying degrees of systolic dysfunction. We studied 34 controls, 16 HFpEF patients, and 25 heart failure patients with mid-range (HFmrEF) or reduced ejection fraction (HFrEF) using cardiac magnetic resonance with acquisition of cine images and 4D flow at 1.5 T. The Navier–Stokes equation was used to compute global left ventricular hemodynamic forces over the entire cardiac cycle. Forces were analyzed for systole, diastole, and the entire heartbeat, with and without normalization to left ventricular volume. Volume-normalized hemodynamic forces demonstrated significant positive correlation with EF (r Hemodynamic forces were initially studied in heart failure patients with dyssynchronous left ventricles, where certain force patterns have been suggested to convey prognostic information regarding the response to cardiac resynchronization therapy (CRT)3. Early studies used echocardiographic particle imaging velocimetry (Echo-PIV) to compute hemodynamic forces in the apical three-chamber view, resulting in a limited approximation of the total force field. Later developments saw the application of cardiovascular magnetic resonance 4D flow to compute three-dimensional hemodynamic forces for the entire left ventricle (LV) and right ventricle (RV) over the cardiac cycle, a method now considered to be the reference standard5. Employing this reference method, several studies uncovered new mechanistic information regarding the rerouting of blood flow through both ventricles in both health and disease8.Hemodynamic forces, essentially the exchange of forces between intracardiac blood and surrounding myocardium, have garnered attention as potential quantitative markers of cardiac health. Hemodynamic forces indicate the functional performance of the ventricle, and have been proposed as a new, noninvasive measure of ventricular function as well as a possible driving mechanism for cardiac remodeling9 and validated10. While not as precise or accurate as the 4D flow-based approach, the numerical method could potentially be retrospectively applied to large cohorts of clinical data, to search for prognostic markers in various stages of structural heart disease even when 4D flow data has not been acquired. In a promising recent study, Lapinskas et al. used the numerical method to compute LV hemodynamic forces in healthy controls and heart failure with preserved ejection fraction (HFpEF) patients11. The authors found decreased longitudinal  hemodynamic forces in HFpEF patients despite having unaltered longitudinal and circumferential strain, suggesting a potential for early detection of subclinical myocardial systolic dysfunction, as well as enabling evaluation of therapeutic response in this patient group. To date, no studies have used reference-standard 4D flow measurements to evaluate hemodynamic forces in HFpEF.In parallel to the more direct flow-based methods, a numerical framework for approximation of hemodynamic forces from regular long-axis steady-state free precession (SSFP) images has been developedThe aim of the current study was therefore to quantify hemodynamic forces in patients with heart failure with varying degrees of systolic dysfunction using the best available measurement technique, testing the hypothesis that hemodynamic forces are quantitatively altered in HFpEF patients compared to both healthy controls and heart failure patients with reduced ejection fraction.This cross-sectional study was performed at Skåne University Hospital . All participants provided written consent after receiving written and oral information about the study. Study participants were divided into two groups: healthy controls and heart failure patients.12, which is a contemporary, prospective population-based cohort study including echocardiography and coronary computed tomography (CT) angiography image acquisition at the baseline examination12. Controls were defined as having no history of heart failure symptoms, normal echocardiogram and electrocardiogram (ECG), no signs of coronary vessel disease on a cardiac CT scan, blood pressure ≤ 140/90 mmHg, no systemic disease, no lung disease, and no medications.Healthy controls were recruited by invitation after participating in the Malmö cohort of the Swedish CArdioPulmonary bioImage Study (SCAPIS)Heart failure patients were recruited from the Cardiology Clinic, Skåne University Hospital, after being diagnosed by a cardiologist. The assessment included patient history of heart failure symptoms, clinical examination, and lab workup. Patients were subsequently classified according to LV EF as measured from CMR images. Heart failure with preserved ejection fraction was defined as EF ≥ 50%, HFmrEF as EF below 50 but ≥ 40%, and HFrEF as having EF < 40%.Exclusion criteria were atrial fibrillation, more than mild aortic regurgitation or stenosis, inability to undergo CMR exam , and known systemic disease apart from heart failure. Patients with mitral insufficiency were not excluded as this is a common secondary manifestation of heart failure with LV dilatation.All subjects underwent imaging at 1.5 T using a Magnetom Aera or Sola . The scan protocol included balanced steady-state free precession (bSSFP) cine images in the 2-, 3-, 4-chamber and short axis views, through-plane flow measurement in the proximal aorta, first-pass perfusion imaging during adenosine stress, postcontrast 4D flow sampled from a box covering the entire heart and proximal great vessels, and late gadolinium enhancement (LGE) imaging for assessment of myocardial viability. All images were acquired using retrospective ECG gating.14. Scanning was performed with a respiratory navigator prepulse centered on the right liver lobe in 9 subjects, and without navigator in 66 subjects to decrease scan time. A previous study found that quantification of hemodynamic forces was not sensitive to the use of respiratory gating14. Typical imaging parameters for the 4D flow: TE/TR 3.5/5.7 ms, flip angle 8°, VENC 100 cm/s, acquired temporal resolution 46 ms, 40 reconstructed time phases, spatial resolution 3 mm isotropic, no slice gap, partial Fourier factor 0.75 in phase and slice directions, no slice oversampling, GRAPPA factor 4 (factor 2 in anterior–posterior phase encode direction and 2 in superior-inferior slice encode direction) and temporal segmentation factor 2. 4D scan times were not systematically measured but were typically in the 6–10-min range, primarily dependent on cardiac dimensions, heart rate, and when used, respiratory gating efficiency.4D flow was acquired using a previously validated gradient recalled echo sequence (WIP 785 K) with Cartesian readout15. Residual background phase errors were corrected by subtracting a first- or second-order polynomial fit of static tissue velocities16, where correction order was selected to provide the best overall background nulling near the heart in the three phase encoding directions. Phase wraps were unwrapped automatically17. Spatial alignment between bSSFP and 4D flow images was evaluated visually and manually adjusted when necessary.Image analysis was performed in Segment 2.3 R8408  using freely available custom pluginsLate gadolinium enhancement images were used to stratify patients according to the presence of ischemic scar and/or non-ischemic fibrosis and to determine heart failure etiology (ischemic or nonischemic). Images were classified as ischemic LGE, non-ischemic LGE, or negative. Ischemic LGE was defined as subendocardial or transmural areas with increased intensity and within a vessel territory. Non-ischemic LGE was defined as subepicardial or mid-mural areas of increased intensity, or transmural areas in areas where bSSFP images demonstrated normal regional function, ruling out infarction. Slight fibrosis in the right ventricular septal insertion points was considered a nonspecific finding and was classified as LGE negative.14. Figure g within the LV for each timeframe:v is the velocity measured using 4D flow. Blood density was set to ρ = 1.05 g/cm2 and viscosity was set to µ = 4 × 10–3 Ns/m2. The instantaneous hemodynamic force was computed by integration of g over the LV.Global hemodynamic forces were computed as previously described8. The apical-basal direction was defined as being perpendicular to the AV plane. The anteroseptal-inferolateral direction was orthogonal to the apical-basal direction and oriented to intersect the mitral annulus and LV outflow tract; this plane aligns with the Society for Cardiovascular Magnetic Resonance (SCMR) recommendations for 3-chamber view orientation18.Forces were decomposed into three orthogonal components using eight atrioventricular valve (AV) plane reference landmarks, as previously described11. We also computed volume-normalized forces by dividing the force magnitude with LV volume for each timeframe, to facilitate comparison with previous results. Root mean square (RMS) force was computed for systole and diastole separately as well as for the entire cardiac cycle, in the transverse plane and the apical-to-basal direction. Average forces (FRMS) and peak forces (Fpeak) were quantified for the entire cardiac cycle and for systole/diastole separately, with and without volume normalization.Lapinskas et al. suggested normalizing forces to ventricular volume, resulting in a volume-related force index19.To facilitate comparison between subjects independent of heart rate, hemodynamic forces were resampled to a common time axis with end systole set to 0.42, the average in the study population. End systole was determined individually by extrapolation of the downslope of the aortic flow curveStatistical analysis was conducted using GraphPad Prism 9.1  and significance was assigned at p < 0.05 for all analyses. Continuous variables are expressed as average ± 1SD unless otherwise noted.RMS and Fpeak between groups, as well as for population characteristics, and post hoc analysis using Dunn’s multiple-comparisons test where appropriate. Sex differences between controls and patients was evaluated using Fisher’s exact test.We used the Kruskal–Wallis test to investigate differences in the main outcome measures FThis study was conducted in accordance with the Helsinki declaration and was approved by the Ethical Review Board, Lund, Sweden . All subjects provided written informed consent.Imaging was performed on 96 subjects, of which 21 were excluded from the study after initial analysis: 8 due to poor image quality or missing data, 6 due to aortic regurgitation, 3 due to other systemic diseases, 3 patients with impaired systolic function who did not fulfill criteria for heart failure diagnosis, and one due to severe aortic stenosis.We included 75 subjects in the final analysis. Subject characteristics are summarized in Table Examples of volume-normalized hemodynamic forces are shown in Fig. RMS for the entire heartbeat (panel B). When volume normalization was not performed, the systolic forces clustered closer together (panel C), and no correlation was seen between EF and non-normalized FRMS for the entire heartbeat (panel D).Average values for longitudinal  forces are shown in Fig. RMS and Fpeak values between groups in both systole and diastole, we found no differences between controls and HFpEF patients whether LV volume normalization was performed or not  inherently introduces a correlation to EF and will separate groups because of volumetric differences alone when forces are similar. In the present study we examined both normalized and non-normalized forces and found ventricular volume to be the driver of the differences seen. We also did not find any significant differences in forces among controls and patients with normal ejection fraction. Hemodynamic forces are therefore not likely to improve detection of early myocardial systolic dysfunction. Depending on the specific research question, indexing forces to stroke volume or cardiac output may be a better option, since these variables relate more directly to the effective mass transfer of cardiac pumping and hence force output.We found that volume-normalized force in the apical-basal direction correlated with ejection fraction, but importantly the same was not seen for non-normalized force. A trend was seen towards lower forces in the apical-basal direction in patients with impaired ejection fraction, explained by their larger ventricular volumes. Lapinskas et al. previously demonstrated a correlation between EF and volume-normalized force11 computes hemodynamic forces using numerical calculation of mass inflow and outflow from a three-dimensional mesh and known or assumed inflow conditions. In healthy hearts, the majority of ventricular pumping occurs as the result of a longitudinal shortening of the ventricles, known as atrioventricular plane displacement20. This mode of contraction is attenuated in acute myocardial injury as well as in heart failure, but remains an important driver of ventricular pumping24. While the 4D flow method for force quantification is unlikely to be affected by the underlying mechanism of contraction, it is possible that the endocardial dynamics-based model is sensitive to such alterations10.Considering the methodology, the approach used herein constitutes the reference standard for obtaining hemodynamic forces. In comparison, the method employed by Lapinskas et al.26. While statistically significant intergroup differences may be found by increasing the study size, it would be of greater clinical utility to use stress imaging to unmask latent differences in cardiac performance. More studies are required to investigate the normal biological inter- and intrasubject variability of hemodynamic forces in different diseases and during altered cardiac loading conditions.Despite studying a larger population than the previous publication, and using the best currently available method of measurement, we were unable to detect significant differences between HFpEF and controls. The still limited population size may be a contributing factor here, but it is also noteworthy that all examinations were carried out at rest. Furthermore, all HFpEF patients were in a state of compensation during the examination, as indicated by normal oxygen saturation levels, normal blood pressure, and no symptoms. There is gathering evidence for the application of stress testing in suspected heart failure29, and while controls were significantly younger than the aggregated HF group, the difference was driven by the HFpEF patients. This is in keeping with the commonly observed characteristics of HFpEF patients, who are typically older, more frequently diabetic, and less often suffering from ischemic heart disease compared to HFrEF patients31. Given that diastolic function is the key culprit in HFpEF33, any difference in hemodynamic force between controls and HFpEF patients would therefore be expected to be amplified rather than attenuated by the age difference. Despite this, we saw similar hemodynamic forces in HFpEF and controls, further supporting that force analysis is likely not a robust measure of ventricular function in the absence of decreased ejection fraction.Diastolic function is thought to deteriorate with age36, manifested as a transient atrioventricular pressure gradient which is affected by alterations in both systolic and diastolic performance39. Further studies into filling dynamics using hemodynamic force analysis would likely reveal additional information regarding atrioventricular interactions, as the pressure gradient which causes LV filling necessarily involves atrial blood19 and blood in the pulmonary veins41. This gradient has been studied both using invasive measurements37 and noninvasive 4D flow-derived approaches42, but was beyond the scope of the current study.It is well known that the early diastolic acceleration of blood (E-wave) is caused by ventricular suction43 as well as from regular cardiovascular magnetic resonance (cine SSFP) images10. The SSFP method was compared to reference-standard 4D flow and found to approximate forces in the longitudinal (apex-base) direction  and in the inferolateral-anteroseptal direction to a lesser degree . Of note, the SSFP method performed worse in patients than in healthy volunteers. In contrast, the 4D flow method has been validated against laser particle imaging velocimetry with good accuracy and precision for both peak and RMS forces (R2 = 0.96–1.0), high reproducibility and good agreement between scans with and without respiratory gating, for different field strengths, and for different LV segmentation methods14.For our measurements we used a prototype 4D flow sequence. Today, one major MR vendor offers 4D flow as part of the standard clinical package, and the other two are actively supporting research applications. If 4D flow is unavailable, forces can be computed from echo-PIV data46, it represents the current state of the art and was performed similarly for all groups, minimizing the risk of intergroup bias in the flow measurements.A remaining issue with 4D flow is the sensitivity to background phase offsets, which we countered by first- or second-order correction. While this is an imperfect solutionA descriptive, cross-sectional study should use the most precise and accurate method of measurement available, to minimize noise and focus on physiological rather than methodological interpretations of data. Therefore, we have used state-of-the-art imaging to demonstrate that a significant part of the previously described intergroup differences arise from the normalization of measured hemodynamic forces to ventricular volumes rather than from underlying differences in ventricular mechanics or other physiological phenomena.Left ventricular hemodynamic force analysis, whether indexed to LV volumes or not, fails to distinguish between healthy subjects and patients with heart failure and preserved ejection fraction. While force analysis remains a promising concept for specific applications, the current findings do not support a clear role for hemodynamic forces in HFpEF assessment.


Modifications of the myocardial architecture can cause abnormal electrical activity of the heart. Fibro-fatty infiltrations have been implicated in various cardiac pathologies associated with arrhythmias and sudden cardiac death, such as arrhythmogenic right ventricular cardiomyopathy (ARVC). Here, we report the development of an MRI protocol to observe these modifications at 9.4 T. Two fixed ex vivo human hearts, one healthy and one ARVC, were imaged with an Iterative decomposition with echo asymmetry and least-square estimations (IDEAL) and a magnetization transfer (MT) 3D sequences. The resulting fat fraction and MT ratio (MTR) were analyzed and compared to histological analysis of the three regions (“ARVC triangle”) primarily involved in ARVC structural remodeling. In the ARVC heart, high fat content was observed in the “ARVC triangle” and the superimposition of the MTR and fat fraction allowed the identification of fibrotic regions in areas without the presence of fat. The healthy heart exhibited twice less fat than the ARVC heart . Localization of fat and fibrosis were confirmed by means of histology. This non-destructive approach allows the investigation of structural remodeling in human pathologies where fibrosis and/or fatty tissue infiltrations are expected to occur. In the context of atrial fibrillation, structural remodelling initially develops as a consequence of the electrical, mechanical and hemodynamical disturbances, but becomes, as the pathology progresses, a key determinant in arrhythmia complexity and therapeutic success. Recent studies using high resolution MR imaging on samples of explanted human atria revealed significant fatty infiltrations and fibrotic tissue2. Excessive tissue remodelling, such as the accumulation of intramuscular adipose tissue in arrhythmogenic right ventricular cardiomyopathy (ARVC), can cause ventricular tachy-arrhythmias and sudden cardiac death3. More recently, subtle localized tissue alterations have been implicated in arrhythmogenesis and sudden cardiac death in patients suffering from primary electrical disorders or idiopathic ventricular fibrillation5. Although histology performed on a limited number of biopsies have shown fibro-fatty tissue infiltrations, these tissue alterations remain undetectable with current imaging approaches and risk prediction remains poor.Various cardiac arrhythmias, such as atrial fibrillation, ventricular tachycardia and ventricular fibrillation can be caused and maintained by alterations in the tissue architecture and intercellular coupling, through for example fibro-fatty infiltrations6. In this pathology, structural modifications mainly appear in the RV inflow tract, the outflow tract (RVOT) and apex of the RV; these are regarded as the “ARVC triangle” or “triangle of dysplasia”8. RV myocardial scarring initially produces regional wall motion abnormalities, but later may involve the free wall and become global; it may then produce RV dilatation and myocardial thinning. Tissue replacement can also occur in the left ventricle (LV) in the lateral and posterior walls; this is characterized by myocyte loss and fibrosis with relative sparing of the septum10. Fat infiltrates the free wall of the RV and the apex of the LV, such that both ventricles exhibit similar amounts of fat12. Clinical diagnosis of this pathology relies mainly on imaging assessment of cardiac wall kinetics, combined with histological analysis of local endocardial biopsy of the RV and the study of the genetic background of the pathology14; these together provide a limited description of the 3D extent of fibro-fatty infiltrations. However, for precise visualization of fat and fibrosis in the RV, current spatial resolution of clinical MR images remains limited15.In the case of ARVC, arrhythmia originate pre-dominantly from the right ventricle (RV)17, including the use of chemical shift saturation18, longitudinal relaxation time weighted images19, and multi-echo methods such as the Dixon technique20. Reeder et al.22 developed iterative decomposition with echo asymmetry and least-square estimations (IDEAL) imaging to separate water and fat with chemical shifts; this approach exhibits better robustness against field inhomogeneity, compared to the conventional Dixon technique20.Several MRI techniques have been developed to separate fat and water in cardiac tissue in vivo23, kidney25, and intestinal tissue26; this contrast mechanism is sensitive to collagen content, as confirmed by histological analysis.The magnetization transfer (MT) technique was proposed to visualize fibrosis in vivo and ex vivo in liver26 sequences, the IDEAL sequence22, and the hierarchical IDEAL27 processing at an isotropic resolution of 200 µm. MRI results were analyzed and compared with those of histological analysis in regions corresponding to the ARVC triangle.The objective of this study was to develop and evaluate a set of high-resolution 3D MRI acquisition sequences to localize and characterize fatty infiltrations and fibrosis in ex vivo formalin-fixed human hearts. Two hearts were included in the study to assess the specificity of the protocol. One from a patient with ARVC, where fibro-fatty infiltrations are expected, and a heart from a patient without known cardiac pathology. We present 3D images of the hearts obtained at 9.4 T from the MT3, was obtained from a 36-year-old man with known ARVC undergoing cardiac transplantation at Bordeaux University Hospital. This patient was included in a heart donor program before heart transplant. Diagnosis of ARVC resulted from echography examination . A second ex vivo human donor heart, with dimensions 13.9 × 8.1 × 7.9 cm3, was obtained from an 83 years-old patient through a human organ donor program at the Bordeaux University Hospital. No significant cardiac pathology was revealed during the clinical evaluation performed before explanting the heart: cardiac function was preserved without diastolic dysfunction, normal right and left pressures, a small mitral regurgitation without left atrium dilation was observed and no other valvulopathy was identified. This heart was thus labelled “healthy” in the rest of the manuscript. Both hearts were fixed with 1 L formalin 10% containing 2 ml gadolinium Dotarem, by means of retrograde perfusion from the aorta at 25 rpm for 48 h. They were continuously mechanically agitated in the fixative solution for 2 days at 4 °C, then stored in the same solution at 4 °C.Two human hearts were included in this study. An ex vivo human donor heart, dimensions 13 × 10 × 9.6 cm1H MR signal, which has magnetic susceptibility similar to that of the tissue; it is used to attenuate susceptibility artefact at the borders of cardiac chambers28.For MRI acquisition, both the hearts were removed from the solution a few minutes before scanning to allow formaldehyde to evaporate under a ventilation hood; the hearts were then immersed in Fomblin (Aldrich) inside a plastic container. Fomblin is a perfluoropolyether with no detectable −1 s−1 maximum linear slew rate and 300 mT m−1 maximum gradient strength) were used to image the cardiac samples.All experiments were performed using a 9.4 T in a horizontal open-bore access of 30 cm . A 7-channel transmission/reception array coil (165 mm inner diameter) and a B-GA20S gradient insert of 200 mm inner diameter . Acquisition parameters were: repetition time (TR) = 30 ms; echo time (TE) = 3.08/3.31/3.54 ms; number of averages (NA) = 4; flip angle = 17°; field of view (FoV) = 125 × 110 × 110 mm3; matrix = 625 × 550 × 550 resulting in an isotropic resolution of 200 µm; bandwidth = 250 kHz; and GRAPPA = 2 in the Y phase encoding direction, for a total acquisition time of 9 h.An IDEALon) was implemented using a modified fast low angle shot sequence. The MT preparation module consisted of 60 hard pulses of 5 ms each, with 10 µs interpulse delay , a B1 amplitude of 10 µT, and a frequency offset of 3 kHz. Acquisition parameters were TR/TE = 2000 ms/9 ms, NA = 1, flip angle = 90°, FoV = 125 × 110 × 110 mm3, matrix = 312 × 275 × 275 resulting in an isotropic resolution of 400 µm, bandwidth = 250 kHz, and GRAPPA = 2 in the Y phase encoding direction. A reference 3D volume was acquired with identical parameters, without the MT preparation module (MToff). Each acquisition required 22 h 45 min.The MT 3D data set  sequence was used to determine T1 relaxation time. For this, the saturation recovery technique was used with varying TR to collect a series of T1-weighted images. Acquisition parameters were 6 TR arrays = 200, 400, 800, 1500, 5000, and 10,000 ms; TE = 30.6 ms; FoV = 110 × 110 mm2; matrix = 275 × 275; slice thickness = 2 mm; RARE factor = 8; and bandwidth = 50 kHz for a total acquisition time of 10 min 15 s. A multiple gradient echo (MGE) sequence with 8 echoes was used to measure T2* in the same slice. Acquisition parameters were TR = 100 ms, NA = 1, FoV = 110 × 110 mm2, matrix = 275 × 275, slice thickness = 2 mm, and bandwidth = 100 kHz; the first TE began at 4 ms with an echo spacing of 5 ms, for a total acquisition time of 27 s.After each sample had been positioned in the magnet, scout view images were obtained to center the 3D volume, based on the ventricles. First, volumetric Boff and MTon images using the parameters described above.3D water-fat images were acquired, followed by MT1 and T2* maps were calculated based on the RARE and MGE datasets with the built-in Paravision 6.0 image data processing by doing pixel-wise exponential fittings:All the MR data were fully reconstructed using ParaVision 6.0 . T1 and T2* for fat and cardiac tissues were estimated from a selected region of interest (ROI) in the tissue and in the pericardial fat.The means of TFor the IDEAL sequence, a complex 3D image was reconstructed for each coil element and each echo. These images were post-processed in Matlab  as follows:27. Proton density images of water (Mw) and fat (Mf) were computed from water and fat using the following formulas, where α is the flip angle, T1w and T2*w were relaxation times of cardiac tissue, while T1f. and T2*f were relaxation times of adipose tissue.For each echo, the magnitude image was produced by a root-mean-squared over the different coils; the phase image was reconstructed by addition of the phase of each coil element for each pixel. The relative phase shift between each echo was then computed by subtraction of the phase of the shortest echo. Water and fat 3D images were then reconstructed from this dataset using the hierarchical IDEAL algorithm29 was calculated using the following formula:From these images, the Proton Density Fat Fraction 3D map (PDFF)30 in a manual ROI drawn in a central slice outside of the heart. This mask was then corrected to include pixels at the external border of pericardial fat, using a level tracing segmentation tool available in 3D Slicer31 (www.slicer.org). A final manual correction was performed on each slice to remove potential residual pixels.A 3D mask was generated to remove pixels with low intensity to analyze the fat content in each region of the heart. The square root of the sum of squares of the magnitude of the 3 IDEAL echoes was computed; a 3D mask was initially generated by using a threshold equal to three times the standard deviation of the noise20. 3D renderings were processed using VolView .This mask was applied to the 3D PDFF and a cumulative histogram with 1000 bins was computed. This histogram was fitted with a Gaussian mixing model, initialized with 4 peaks corresponding to water (peak with highest intensity on the histogram) and fat (peak with second highest intensity on the histogram) signals and residuals on the histogram. The upper bin value corresponding to 99.99% of the first Gaussian function was selected as the threshold to identify voxels containing water alone. A second threshold was defined using the classic Dixon water-fat separation technique, in which voxels > 50% were tagged as fat and voxels < 50% were tagged as wateron) and without transfer module (MToff) were zero-filled by a factor of 2 before fast Fourier transformation to match the IDEAL image resolution. Then, the magnetization transfer ratio (MTR) map was reconstructed as followsK-spaces of fast low angle shot acquisitions with . Note that RV apex septum sample contains both the RV apex and tissue from the LV. Following the same protocol, the healthy heart was also dissected with approximate dimensions of 1.4 × 1.1 × 1.2 cm3, 2 × 1.7 × 1.1 cm3 and 1.7 × 2.4 × 1.1 cm3 respectively.The pathological heart was dissected to assess the presence of fat and fibrosis. Three samples were extracted from regions of the ARVC triangle, with approximate dimensions of 1.4 × 1 × 0.6 cm32 for structural identification: collagen fibers in tissues were green; nuclei were black; and myocytes were red/pink. Slices were examined at 10x magnification on a scanner Axio Scan Z1 . Images were visualized using Zen lite software .Samples were first rinsed in phosphate-buffered saline, then agitated at 4 °C for 24 h. After samples had been dehydrated, each sample was embedded in paraffin and sectioned at 6 µm in the transmural direction. Tissue sections were stained with Masson’s trichrome33. The analysis was performed by the same person to avoid inter-observer variability. The resulting masks were applied to count the number of pixels attributed to fat and to the cardiac tissue. The fat ratio was then computed for each slice of a given region and averaged (mean ± SD). The number of slices in ARVC and healthy hearts were  and  for the RV free wall, RV apex and RVOT, respectively.The fat quantification was performed by thresholding the RGB color histogram of the histological slices using ImageJ software (NIH)3, 2.6 × 2 × 2.6 cm3, 2.2 × 1.4 × 2.3 cm3, respectively. Their dimensions were chosen to include the volume of the samples based on the histology of the ARVC heart and were applied identically on both hearts. Anatomical landmarks for positioning the boxes were defined as follow: for the RVOT, the upper bound of box was chosen under the pulmonary valve; for RV free wall the upper bound of box was placed under the lower bound of the RVOT box; for the RV apex septum, the lower bound of box was chosen at the junction between the RV wall and the apex region of the septum. Within these boxes, we computed the total number of pixels included into the 3D mask (volume of tissue) defined above and the relative content of water and fat.To study the ARVC triangle in both hearts, identical 3D boxes were drawn on the PDFF map, MT images, and MTR map. These boxes surrounding the RVOT, the RV free wall and the RV apex septum were 1.6 × 2.2 × 1 cmAll experiments were conducted in accordance with the declaration of Helsinki. The two hearts included in this study were obtained through two research programs: The heart from the patient suffering from ARVC was obtained in the context of heart transplantation by the University Hospital of Bordeaux. The heart was collected as a human biological sample resulting from standard care activities and dedicated to scientific research , after obtaining written informed consent from the patient. This program did not require additional approval by external review board. The heart from the 83 y.o. patient was obtained through the Human donor program "CADENCE"  approved by the French "Agence de la Biomédecine". After obtaining written informed consent from the patient's family, the heart was collected as a human biological sample for scientific research.None of the experiments involved hearts procured from prisoners.Figure C) and Dixon technique (red arrow TD). The number of voxels included between both thresholds represents 6.42% of the total number of voxels over the entire volume. Images displayed in Fig. C (40%) and TD (50%) thresholds defined in Fig. C determined from the Gaussian function is 41%.Figure Figure Figure 40%) shows that this hyperintense signal in the MTR does not correspond to the presence of fat. In the histology image  confirm that regions with PDFF < 40% (no fat) correspond to areas where MT is present.Figure In the histological results Fig. b, the le40%) emphasize that regions where PDFF is < 40% (no fat) show a MT ratio > 90% (green arrows); they also show regions with fat. Corresponding histological results are shown in Fig. A coronal view of the RV septum sample is shown in Fig. Table 3 vs. 306.6 cm3), but with different fat ratios (36.0% vs. 17.2%). Fat ratios according to the MRI data in the RVOT, RV free wall and RV apex septum for the ARVC and healthy hearts were 76.0% vs. 31.9%, 66.5% vs. 28.7% and 4.5% vs. 1.3%, respectively. The histological analysis of the fat ratios in the three regions led to 79.6 ± 9.6% vs. 39.2 ± 6.3%, 61 ± 5.6% vs. 28.8 ± 7.2% and 7.2 ± 3.8 vs. 5.7 ± 2.5% for the ARVC and healthy hearts, respectively.Both hearts had similar volumes  and sample imaging conditions (use of fomblin for MR-imaging at high field) is unlikely expected to modify the fat content or fibrosis.To the best of our knowledge, this is the first MR study to present high-resolution 3D images of fat and fibrosis obtained by analysis of intact ex vivo human hearts, at an isotropic resolution of 200 µm. Higher spatial resolutions (50 µm isotropic) were reported on hearts from small animals22 and associated hierarchical IDEAL27. This method is preferable to the conventional Dixon35 approach, because it is more resilient to the B0 inhomogeneity that is inherent to large sample imaging at high-field intensity. Our results showed good agreement between MRI results and histology data in each of the three ARVC triangle regions. As expected, large amounts of fat were observed in the RV free wall and RVOT. Despite the fat content appearing to be lower in the RV apex septum, this particular sample also contained a large part of the LV septum. In the pipeline of image processing, we chose a threshold (TC) to discriminate voxels with high fat content based on the PDFF histogram . We report a good correspondence between the fat ratios retrieved from the PDFF and the histological analysis in the three observed regions (Table 3D fat fraction maps were computed from water and fat images obtained with the IDEAL technique37. This approach to detect macromolecules (e.g. collagen) has been used to observe fibrosis in liver23, kidney38, and in intestinal tissues26. When combined with the PDFF, this MT acquisition technique allows the identification of areas where the MTR denotes the presence of macromolecules and the PDFF excludes the presence of any fat content. Indeed, comparison with histology findings confirmed the presence of collagen in areas where the MTR was > 70% and PDFF was < 40%. Both of these techniques, when used in combination, help to discriminate areas of fat from areas of fibrosis; they also help to identify areas where fat is already present, and fibrosis is emerging.We also developed an MT acquisition sequence to identify fibrotic areas based on a preparation module with off-resonance pulses, which could saturate a specific region of the proton spectrum containing macromolecules.39 presented analyses of left human atria; they compared T1 maps with 3-point Dixon-based fat fraction maps to differentiate fibrosis from adipose tissue. This method was proposed to discriminate fibrosis, fat, and fibro-fatty infiltrations in human left atria wall at the tissue level, based on the Dixon method (fat) and analysis of the T1 distribution (fibrosis). However, these analyses were not performed in 3D and included 5 slices of 1 mm thickness at 200 × 200 µm2 in-plane resolution. In the present study, we proposed 3D isotropic imaging of whole heart at 200 µm. We proposed a different approach for identification of fibrotic tissues using MT; acquisition of 3D T1 maps was reportedly challenging when using large samples, due to different sources of error42. Eliav et al.43 and Kim et al.44 demonstrated that the collagen matrix greatly contributes to the MT; moreover, there was a linear correlation between the MTR ratio and percentage of collagen in vitro. It was recently shown that MTR varies in the heart as a function of the collagen type (I vs. III)45. Indeed, the relative proportion of type I and type III collagen is expected to evolve with age and pathologies in the cardiac tissue. These factors contribute significantly to the observed cardiac fibrosis46. A more thorough analysis of the MTR contrast mechanism for quantifying the fibrosis using this technique is thus required and was considered out of the scope of the present work.Bouazizi et alIn our study, pathological regions of interest were extracted from whole heart 3D MR acquisition. Given the MRI FoV and resolution, we reported the distances and positions on the ex vivo sample to guide dissection for histology. Our results showed that MRI and histology were in good agreement in terms of both fat quantification and fibrosis localization for each sample within the “ARVC triangle”.47. Indeed, a loss of > 40% of myocytes with fibrous replacement of the RV in the free wall is a major criterion for tissue characterization, consistent with the proportion of 66.6% fat (Table 10. The RVOT exhibited the highest fat content (Table In this pathological heart with ARVC, we found abnormalities in the short axis view, such as RV wall thinning characterized by a loss of cardiac muscle in the whole RV free wall. These abnormalities are consistent with the Task Force Criteria for ARVCat Table  observedat Table , consistnt Table . These r51.An additional animated GIF file presents the PDFF and MT ratio volumes covering the whole ARVC heart (See Supplementary Fig. 53. This can open the possibilities to apply IDEAL and MT to such a resolution that fibro-fatty infiltrations could be observed in clinical situations.This study had several limitations. First, only two hearts were included, inherent to the difficulty to access such unique intact whole hearts. Although MT and fat fraction imaging are technically feasible in vivo, achievement of the necessary spatial resolution in vivo represents a significant technical challenge. The strong efforts of the MRI community toward 3D in vivo imaging of the heart is expected to result in more spatially resolved images. Some studies have reported the development of intra-cardiac MRI with catheter micro-coils to increase the image spatial resolution in restricted field of view and the cardiac gating to remove movement artefactIn this study, the fat fraction map combined with MT imaging provided 3D volumes at an isotropic resolution of 200 µm, allowing to differentiate healthy myocardium, fat, and fibrosis. Data from MRI showed good agreement with those from histological analysis. This non-destructive approach allows the investigation of potential alterations of the whole human heart in pathologies where fibrosis and/or fat infiltrations are expected to occur.Supplementary Figures.


Background: Ventricular arrhythmias are associated with sudden cardiac death (SCD) in hypertrophic cardiomyopathy (HCM). Previous studies have found the late gadolinium enhancement (LGE) on cardiac magnetic resonance (CMR) was independently associated with ventricular arrhythmia (VA) in HCM. The risk stratification of VA remains complex and LGE is present in the majority of HCM patients. This study was conducted to determine whether the scar heterogeneity from LGE-derived entropy is associated with the VAs in HCM patients.Materials and Methods: Sixty-eight HCM patients with scarring were retrospectively enrolled and divided into VA (31 patients) and non-VA (37 patients) groups. The left ventricular ejection fraction (LVEF) and percentage of the LGE (% LGE) were evaluated. The scar heterogeneity was quantified by the entropy within the scar and left ventricular (LV) myocardium.Results: Multivariate analyses showed that a higher scar  was independently associated with VA, after the adjustment for the LVEF, %LGE, LV maximal wall thickness (MWT), and left atrium (LA) diameter.Conclusion: Scar entropy and %LGE are both independent risk indicators of VA. A high scar entropy may indicate an arrhythmogenic scar, an identification of which may have value for the clinical status assessment of VAs in HCM patients. Cardiac magnetic resonance imaging is a valuable tool for the risk stratification of sudden cardiac death (SCD) in hypertrophic cardiomyopathy (HCM). The current gold standard for the visualization of a scarred myocardium is the late gadolinium enhancement (LGE) during cardiac magnetic resonance imaging (CMR)  and prevMyocardial fibrosis is a significant cause of arrhythmogenesis in HCM patients , 6. Howevia telephone. The study protocol was approved by the Sir Run Run Shaw Hospital ethics committee and complied with the Declaration of Helsinki.Sixty-eight patients with a diagnosis of HCM , in whomVentricular arrhythmias included previous aborted cardiac arrest, documented sustained ventricular tachycardia (VT) , and nonPatients were imaged using 1.5 T CMR scanners . All CMR images were acquired with electrocardiographic gating and breath-holding. Typical LGE CMR protocol parameters were applied: a repetition time of 4.8 ms, an echo time of 2.3 ms, and an inversion time of 200 adjusted to 300 ms. The LGE imaging was conducted 10 min after a cumulative intravenous injection of 0.2 mmol/kg of gadopentetate dimeglumine. A breath-hold segmented magnetization-prepared turbo gradient echo sequence was used with the inversion time chosen to null the normal myocardial signal. The images were visualized in 8–14 matchings short-axis (8 mm thick with 2 mm spacing) and three radial long-axis planes. The presence of LGE as a categorical value was first assessed by two radiologists who were blinded to the clinical data of the patients. The left ventricular ejection fraction (LVEF), left atrium (LA) diameter, maximal wall thickness (MWT), and %LGE were measured by standard volumetric techniques and analyzed with commercially available software . Late gadolinium enhancement was defined as the areas with adjusted gray-scale threshold ≥5 SD above the mean of the normal myocardium  are reported. The optimal cut-off values were determined by calculating the Youden index. Kaplan-Meier curves and log-rank tests were used to explore the association of the LGE and entropy with VAs. Receiver operator characteristic (ROC) curves were created and the value closest to the upper left corner determined the optimal sensitivity and specificity to discriminate between HCM patients with VAs. The inter-and intra-observer variabilities were expressed by the intra-class correlation coefficients. The inter-observer variability for quantitative scar and left ventricular entropy was assessed in a subset of 20 randomly selected CMR studies. Two readers (WLZ and CLP) independently quantitated the LGE without prior knowledge of the clinical data and were blinded to the previous results. For the intraobserver variability, two readers (CQ Z and ZP J) independently quantitated the entropy in a subset of 20 randomly selected CMR studies. The inter-rater agreement was evaluated using a Bland-Altman plot and linear regression analysis. All tests were two-tailed and p <0.05 was considered significant.The continuous variables are presented as mean ± SD, and the categorical data are summarized as frequencies and percentages. The data that did not follow a normal distribution are presented as median and quartile (interquartile range). The differences in baseline characteristics between the patients were analyzed using a Student's n = 13) had HCM with VA, and of the latter, 43.9 % (p = 0.81) (n = 18). In HCM with VAs, the 5-year SCD risk score was much higher among those with VA than those without VA {[5.2  vs. 1.80 ]; p <0.001; The baseline characteristics for all patients were presented in p = 0.63). The mean entropy within the scar was significantly higher in the VA group . There was no significant difference in the mean LV entropy between the two groups  but none between LGE and LV entropy .The data for the left cardiac function and myocardial entropy data are summarized in o groups . All theo groups . There wp = 0.005) and thicker MWT  compared with those without VA.Ventricular arrhythmias were documented in 31 patients (45.6%). Of these, 4 patients (12.9 %) had VA with aborted cardiac arrest, 7 (22.6 %) had sustained VT, and 20 (64.5%) had NS-VT. The HCM patients with VAs had poorer LVEF   . The mulp = 0.175). The efficiency of scar entropy in predicting the occurrence of VAs was evaluated by the ROC curve analysis (p = 0.175 vs. %LGE). The red curve represents the effect of adding entropy to the %LGE values and producing a better prediction of VAs in the HCM patients  , combined with %LGE > 6.14% had significant risk stratification for VAs compared with those with a 5-year risk score ≥ 4% (At a median follow-up time of 25 months (IQR 13–13), no patients died. Among these, 12 of the HCM patients with VA had received implantable cardiac defibrillation (ICD) devices. During the follow-up, the median time of the VAs after CMR was 1 month (IQR 0–4.75). Among the HCM patients without VA, only one received an ICD for primary prevention.We found a good reproducibility of scar and LV entropy measurements for the intra- and inter-observer variability and the intraclass correlation coefficient (ICC) values for entropy were over 0.9. The summaries of the ICC values in both the intra- and inter-observer reproducibility are shown in Our study demonstrated that the LGE-CMR-based calculations of entropy within the scar were associated with the occurrence of VA. The incorporation of the scar entropy values provides a significant refinement of the % LGE-modified 5-year SCD risk score. This modification has the potential to greatly assist the identification of high-risk HCM patients using LGE-CMR.Currently, available risk stratification indices only identify a limited proportion of HCM patients at risk of SCD. The current study used univariate analysis to show that the mean entropy within the scar, %LGE, MWT, LA diameter, and LVEF were all associated with the occurrence of VAs . The HCMThe current study showed that the HCM patients with VAs had worse LVEF by univariate analysis although no significant association could be shown by the multivariate analysis. The LVEF is not incorporated into the ESC risk model . Previoup = 0.005)  and %LGE higher than 6.14% (log-rank p < 0.001) had VAs more frequently. There was no significant difference between the %LGE and scar entropy by DeLong's test (p = 0.175). The efficiency of scar entropy in predicting the occurrence of VAs was further evaluated using ROC curve analysis (p = 0.175 vs. %LGE) and the red curve represents the scar entropy combined with %LGE with a C-statistics value of 0.900 . Using the optimal cut-off values from the ROC analyses, the patients with a scar entropy higher than 6.166 had VAs more frequently (sensitivity: 0.903% and specificity: 0.459%). A value for %LGE> 6.14% significantly refined the risk stratification for VAs when added to the conventional SCD Risk stratification score ≥4% .We demonstrated that a higher entropy within the scar was the CMR-derived parameter most closely associated with the occurrence of VAs  . Multivaanalysis . The greMany new techniques and modifications of CMR require further research. Myocardial texture analysis helps in the detection of myocardial disease  and thisLate gadolinium enhancement is an independent predictor of SCD in HCM patients  and the . The current study showed a very weak association between LGE and scar entropy and no association between LGE and LV entropy in HCM patients. Multivariate analysis identified the entropy within the scar but not that of the total LV myocardium as the LGE-derived parameter associated with the occurrence of VAs. As such, the entropy calculated two-dimensional LGE MR imaging seems to be a promising parameter to indicate the presence of an arrhythmogenic scar. The LGE extent is not a satisfactory predictor of risk due to the variable phenotype of HCM. The current retrospective study has shown that some patients with similar LGE extents experienced different outcomes. This may be partially explained by the heterogeneity of fibrosis which can be equated with scar entropy.The current study identified the novel marker, scar entropy, which refines the uncertainty in the LGE signal . Recent p < 0.001).Entropy independently predicts adverse cardiac events and all-cause mortality in HCM patients , 29. HigThe CMR assessments of LGE constitute a promising tool for SCD risk in HCM patients . HoweverWe found that scar entropy was higher in the HCM patients with VAs and constituted an independent predictor of arrhythmias, indicating a continuum of risk assessed by this parameter. We suggest that a noninvasive identification of inhomogeneous scars and their evaluation by scar entropy is a superior tool to the mere presence and extent of LGE for risk stratification in HCM patients.Ventricular tachycardia is an important risk factor of SCD in large HCM studies and is most frequently assessed by Holter monitoring. However, this approach carries a risk of missing and underestimating events within the timescale available for registration. The present study shows that scar entropy could help assess the arrhythmic risk in HCM patients, assisting with the identification of high-risk individuals.We acknowledge several limitations to our study. First, this study was retrospective, including relatively low numbers of HCM patients with both DCG and CMR results from a single medical center. Second, our 24 h monitoring period may be too short to accurately represent the overall picture of the arrhythmic burden of a patient making it quite limited in terms of follow-up. Though most patients had NS-VT and a high percentage had VAs in our HCM cohort, all had scarring identified by LGE-CMR. Third, risk stratification within these narrow parameters is quite difficult and challenging and further studies are needed to confirm the validity of the information contributed by our new technique. Future prospective investigations including larger cohorts with outcomes including VA and SCD are required to confirm our findings. Fourth, we did not get the information of the genetic testing results as this could also affect accurate risk stratification for a clinical diagnosis of HCM. Mapping techniques might add more values for risk stratification in HCM, which could be evaluated in future studies.The entropy used to quantify scar tissue in homogeneity within the scar was independently associated with VAs and therefore seems to be a promising marker of an inhomogeneous and arrhythmogenic scar in HCM patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Sir Run Run Shaw Hospital Ethics. The patients/participants provided their written informed consent to participate in this study.DWL, HJH, and GSF conceived and designed the experiments. YY, ZPJ, WLZ, CLP, CQZ, XHH, CC, YXS, QH, WJZ, YEQ, HR, FDY, CYJ, YKM, and BW analyzed and interpreted the data. YY, ZPJ, and JBA wrote or edited the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the National Natural Science Foundation of China (Grant Number 81873908), Key Research and Development Program of Zhejiang Province (Grant Number 2019C03022), Medical Science and Technology Project of Zhejiang Province (Grant Number 2020KY220 and 2022506537), Chinese Medicine Research Program of Zhejiang Province (Grant Number 2020ZA083), and the funding from the Clinical Research Project of Zhejiang Medical Association (Number 2016ZYC-A28).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Z-score ≥ 2.0]. Children with congenital heart disease or hypertension were excluded. Underlying diagnosis, echocardiographic and follow-up data were extracted from patient files. Seventy-one infants with CH were included. An underlying cause of CH was identified in two-thirds (n = 47). Most common etiologies of CH were malformation syndromes  and maternal diabetes mellitus (n = 13). Less common causes were congenital hyperinsulinism (n = 3), metabolic- (n = 5), sarcomeric- (n = 2) and neuromuscular disease (n = 1). In half of the identified causes (n = 22) an association with hyperinsulinism was described, including maternal diabetes mellitus (n = 13), malformation syndromes with insulin resistance (n = 6) and congenital hyperinsulinism (n = 3). CH associated with hyperinsulinism was echocardiographically characterized by lower LVPW thickness, higher IVS:LVPW ratio and more frequent sole involvement of the IVS . CH associated with hyperinsulinism normalized more often (41 vs. 0%) with lower mortality rates (14 vs. 44%) compared to CH not associated with hyperinsulinism . Nowadays, an etiology of CH can be identified in the majority of infants. The development of CH is often associated with hyperinsulinism which is mainly characterized by focal hypertrophy of the IVS on echocardiography. Prognosis depends on the underlying cause and is more favorable in CH associated with hyperinsulinism.This study aimed to describe the variety of etiologies currently identified in infants with cardiac hypertrophy (CH) and investigate whether there is a relation with hyperinsulinism, echocardiographic characteristics and prognosis. This retrospective cohort study included infants born between 2005 and 2018 with CH measured by echocardiography [interventricular septum (IVS) and/or left ventricular posterior wall (LVPW) thickness with  In previous literature CH is often incorrectly described as HCM which might be explained by the fact that CH cannot be distinghuished completely from HCM using echocardiographic examination. Therefore in the remainder of this article both CH and HCM will be reffered as CH3.In infants cardiac hypertrophy (CH) is a rare finding on echocardiographic examination. It can be either a sign of enlarged cardiomyocytes or of hypertrophic cardiomyopathy (HCM) in which there is histological and functional disruption of the myocardial structure/composition in the absence of abnormal loading conditions, as congenital heart disease or hypertension8. The degree and distribution of CH vary between the different etiologies and include diastolic interventricular septal (IVS) hypertrophy and/or left ventricular posterior wall (LVPW) hypertrophy with or without left ventricular outflow tract obstruction (LVOTO)9. The prognosis is variable and depends on the underlying disorder. Children with CH diagnosed before 1 year of age seem to have higher mortality rates than those diagnosed after 1 year of age12.CH in infants has an incidence of 30 per million and is diagnosed with echocardiography requiring a left ventricular wall thickness of more than 2 standard deviations greater than the predicted mean corrected for body surface area (Z-score ≥ 2.0)13. Known underlying causes of CH in infants include malformation syndromes (like Noonan), metabolic diseases , sarcomeric diseases  and neuromuscular disorders (such as Friedreich ataxia). In our recent literature review we already emphasized the importance to additionally consider hyperinsulinism in the differential diagnosis as hyperinsulinism is widely associated with CH. We report an occurrence of CH in 13–44% of infants of diabetic mothers, in approximately 40% with congenital hyperinsulinism and in insulin resistence syndromes14.The etiology of CH in young childhood is diverse and the mechanisms of development and the distribution of etiologies in young children are supposedly different from CH developing later in childhood16. Therefore, the main objective was to describe the current spectrum of identified etiologies of CH in our population of infants over the past years. In addition we aimed to investigate whether echocardiographic characteristics might help to distinghuish between underlying causes and relate to the prognosis of CH in infants.Unfortunately, literature reports that in a large group of young children the etiology of CH remains unknown, while determining the right diagnosis is important since this has direct implications for prognosis and possible medical treatmentThis is a tertiary single center retrospective study performed in Wilhelmina Children’s Hospital, Utrecht, The Netherlands. Echocardiographic reports of all consecutive infants (≤ 1 year) born between 2005 and 2018 and at least 1 year of follow-up were screened for the presence of CH. Their echocardiograms were reassessed to determine whether they met the criteria of CH. Children with CH as a result of hypertension or underlying congenital heart disease were excluded. Data of infants with CH were extracted from patient files. The study was performed in accordance with relevant guidelines and regulations. Study quality was checked before submission to the Medical Research Ethics Committee (MREC) Utrecht by the Division of Pediatrics, UMC Utrecht. The need for informed consent was waived by the Division of Pediatrics, UMC Utrecht. The institutional MREC approved the study as non Medical Research Involving Human Subjects Act (WMO) (No. 20-031/C).18. Ultimately, CH was diagnosed if IVS and/or LVPW had a Z-score ≥ 2.04. LV systolic function was assessed qualitatively on visual inspection “eyeballing” and/or quantitatively by fractional shortening  and/or ejection fraction  by an experienced pediatric cardiologist and was reported as normal or decreased. LV diastolic function was reevaluated in cases with thickening of both the IVS and LVPW using mitral inflow Doppler and Tissue Doppler Imaging (TDI) if this could be measured reliably. Diastolic dysfunction was characterized by an inversion of the mitral early and late diastolic E/A Doppler waves with a ratio < 0.8 or a mitral valve Doppler E/A ratio > 2. Tissue Doppler imaging signs of diastolic dysfunction were an IVS or LVPW E’ velocity < 8 cm/s19.EchoPAC was used to screen echocardiographic reports and for echocardiographic reassessment. All echocardiographic measurements were performed according to recommendations from the American Society of Echocardiography. Echocardiographic measurements were performed on 2-dimensional parasternal long-axis views at the end of the diastole with closed mitral and aortic valve. Two-dimensional guided linear internal measurements of the IVS and LVPW were obtained perpendicular to the left ventricular long axis and measured at the level of the mitral valve leaflet tips. Z-scores corrected for body surface area were calculated automatically with a cardiac Z-score calculator, which has been validated in a large cohort of healthy infants3. For each infant with idiopathic CH it was examined whether genetic screening/diagnostics was performed including the outcome. Echocardiographic data included age of first echocardiography with CH, the distribution of left ventricular hypertrophy (Z-score ≥ 2 IVS and/or LVPW), the ratio between IVS and LVPW, asymmetric septal hypertrophy (ratio IVS:LVPW ≥ 1.5), decreased systolic and diastolic left ventricular function (LVF) and LVOTO20. Prognostic variables included were normalization of CH and mortality within the first year of life.Demographic and anthropometric characteristics, clinical signs and echocardiographic and prognostic data were collected from the patient files for each infant with CH. Etiologies of CH were subdivided into: maternal diabetes mellitus, malformation syndromes, metabolic disease, sarcomeric disease, congenital hyperinsulinism, neuromuscular disease and idiopathic CH. Each underlying cause was assessed for an association with hyperinsulinism and a subdivision was made in etiologies associated and not associated with hyperinsulinismU test (continuous data) and Fisher’s Exact Test . One year survival of each etiology of CH was presented by a Kaplan–Meijer curve. Significance was determined as P value < 0.05.Data were analyzed with IBM SPSS Statistics . The Shapiro–Wilk test and Q–Q plots were used to test whether variables were normally distributed. Data were presented as median including percentiles 25–75, since almost all data were not normally distributed. Where appropriate, data were presented as number of patients with percentages. Differences between etiologies of CH associated and not associated with hyperinsulinism were analyzed by the Mann–Whitney Seventy-one infants with CH were included. Indications for echocardiography were prenatally diagnosed CH (n = 6), heart murmur (n = 9), enlarged heart on chest X-ray (n = 9), clinical suspicion of congenital heart disease or CH (n = 27) and screening for (suspected) syndromic disorder (n = 7) and others/unknown (n = 13).maternal diabetes mellitus (n = 13) was diagnosed early after birth and mainly characterized by septal hypertrophy. In almost all infants with CH by maternal diabetes mellitus normalization occurred within the first months. The most common cause of CH in infants were malformation syndromes (n = 23). Both IVS and LVPW were hypertrophic in most infants with malformation syndromes. One third had LVOTO. The infant with Beckwith–Wiedemann was the only one in which CH normalized within the first year. In 2 infants CH was caused by sarcomeric disease. The IVS was extremely thickened and CH normalized in neither of the infants with sarcomeric disease. Metabolic disease was the cause of CH in 5 infants. CH was diagnosed relatively late. Both IVS and LVPW were extremely hypertrophic. Most infants were symptomatic. In none of the infants with metabolic disease CH normalized. Three girls had CH due to congenital hyperinsulinism. One infant had a heterozygous KCNJ11 mutation and insulin levels ranging from 71 to 152 mIU/L . Another infant with hyperinsulinism (insulin level 59 mIU/L) and CHARGE association showed decreased LVF and LVOTO at presentation. CH normalized with a betablocker. A third infant with hyperinsulinism after extreme dysmaturity needed high glucose intake, diazoxide and hydrochloorthiazide. One infant showed CH of the LVPW as a result of a neuromuscular disorder. In 24 infants the cause of CH remained idiopathic. Median gestational age at birth for infants with idiopathic cardiac hypertrophy was 37.0 weeks (IQR 25–75: 31.4–39.2). Ten infants with idiopathic cardiac hypertrophy were born preterm , including moderate to late (n = 4), very (n = 4) or extremely preterm (n = 2), respectively. The diagnosis was made relatively early after birth. IVS Z-scores were relatively low. In the majority CH normalized. In 5 infants with idiopathic CH genetic testing was performed. In 2 siblings a variant of unknown significance (VUS) and a deletion in the RYR2-gene and a paternal variant in the MYH7-gene was found. In 1 infant a VUS in the MYH7-gene was found. DNA-testing was normal for the other 2 infants.An underlying cause was identified in two thirds of infants with CH. CH by 3. Infants with an underlying cause of CH associated with hyperinsulinism were diagnosed at a younger age, showed more often sole hypertrophy of the IVS, had a less hypertrophic LVPW, and a higher IVS:LVPW ratio. In infants with an etiology of CH associated with hyperinsulinism CH normalized more often and faster.In half of the identifiable causes of CH an association with hyperinsulinism was described including maternal diabetes mellitus (n = 13), malformation syndromes  and congenital hyperinsulinism (n = 3)Overall mortality rate in the first year was 24% (n = 17) and the median age of death was 3.2 months. Infants with malformation syndromes, sarcomeric and metabolic disease showed high all-cause mortality rates, whereas infants with CH by maternal diabetes mellitus, congenital hyperinsulinism, neuromuscular disorder and idiopathic CH had better survival chances. Nine infants (13%) developed severe progressive CH and died as a result of cardiac failure. CH as cause of death was based on clinical assumptions in 6 infants including Noonan (n = 2), Pompe (n = 2), VLCADD (n = 1) and Leprechaunism (n = 1), and on both clinical assumptions and the autopsy report in 3 infants including Noonan (n = 1), MYBPC3-gene sarcomeric disease (n = 1) and idiopathic CH (n = 1). Infants who died as a result of CH had a thicker IVS and LVPW and showed more often decreased left ventricular systolic function at initial presentation.3. Echocardiographic characteristics helped to differentiate between underlying causes of CH. Hyperinsulinism associated CH was diagnosed earlier and characterized by lower LVPW thickness, higher IVS:LVPW ratio and more often sole involvement of the IVS compared to CH not associated with hyperinsulinism. Normalization of CH and survival chances depended on the underlying cause and were more favorable in CH associated with hyperinsulinism compared to CH not related to hyperinsulinism. These findings confirm that establishing the correct diagnosis in infants with CH is extremely important since this has direct consequences for medical treatment and prognosis.This study highlights the etiologies of CH in infants and their associations with hyperinsulinism, echocardiographic characteristics and prognosis. In this study, in the majority of infants with CH an underlying cause could be identified, including maternal diabetes mellitus, congenital hyperinsulinism, malformation syndromes, metabolic-, sarcomeric or neuromuscular disease. In half of these cases there was an association with hyperinsulinism12. Compared to the study of Colan et al. we found a lower incidence of metabolic disease (7 vs. 15%), higher incidence of malformation syndromes (32 vs. 15%) and similar incidence of neuromuscular disease (1 vs. 1%). In both the study of Colan et al. and our study the causes that predominated in metabolic disease and malformation syndromes were Pompe disease and Noonan syndrome respectively. In our cohort only one infant with the neuromuscular disorder nemaline myopathy (mutation ACTA1-gene) was found, while Friedrich ataxia was the main cause of neuromuscular disease in the cohort with children of Colan et al.11 The relatively high incidences of metabolic disease and malformation syndromes and low incidence of neuromuscular disorders in our cohort of infants might be explained by the fact that metabolic or syndromic CH usually presents in infancy or early childhood, whereas those with neuromuscular disorders are more frequently diagnosed in adolescence13.The heterogeneous etiology of CH in infancy is illustrated by the observation that in our cohort a total of 18 specific causes were identified in 71 infants. Although this sample size of infants with CH in our study was smaller in comparison to the Pediatric Cardiomyopathy Registry study of Colan et al. (n = 328), we focussed specifically on children below the age of 1 year and reported detailed results in terms of etiology specific echocardiographic findings and prognosis. In addition, in our study we identified underlying causes of CH in a larger proportion of our population when compared to the studies of Colan et al. and the population based longitudinal cohort study of Nugent et al. 21. In contrast, in our cohort 2 infants (3%) were diagnosed with a MYBPC3-gene mutation. Variants and a deletion of unknown significance were found in 3 infants in the RYR2 and MYH7-gene. Importantly, only 5 infants with idiopathic CH (21%) underwent genetic testing. In the majority of idiopathic cases no indication for genetic testing existed, since CH normalized during follow-up. However, it may be possible that underestimated causes as sarcomeric and Danon disease (LAMP2 mutation) or currently undiscovered mutations could explain some of our idiopathic cases22. Analysis of a predefined panel of CH-related genes using next generation sequencing is generally recommended, including the most commonly involved genes in HCM 23. In severe phenotypes of CH, more than one mutation can be found25. Interestingly, in a recent study by Aye et al., preterms showed a disproportionate CH during the first 3 months after birth, and greater changes in ventricular mass were associated with lower gestational age. This might explain CH in 10 of the idiopathic cases born prematurely26. In addition, the association of transient CH and corticosteroid treatment was recently described in a case report, and might possibly explain one of our currently idiopathic cases27.In one-third of our infants with CH the specific cause remained unknown. Over 50% of cases of apparently idiopathic CH in childhood are caused by mutations in cardiac sarcomere protein genes of which 17% of sarcomeric mutations were diagnosed before 1 year of age3. Previous studies including our literature review did not yet report echocardiographic findings and their differences between underlying causes of CH. Distinghuishing between etiologies associated and not associated with hyperinsulinism is of importance because of differences in outcome and therapeutic consequences, as CH by hyperinsulinism can respond favorably to insulin lowering therapies3.Our study is the first to describe clear differences in echocardiographic findings of CH between infants with and without hyperinsulinemic disease. This attracted our interest, since we recently published a literature review in which we reported that CH is found in a broad spectrum of hyperinsulinemic diseases, including maternal diabetes mellitus, malformation syndromes with insulin resistence  and congenital hyperinsulinism11. This corresponds with the outcomes of our study were 24% of the infants died and all within the first year of life. The findings that infants with CH due to metabolic disease (death/heart transplantation 48% at 1 year) and malformation syndromes (19%) had worse survival rates than children with neuromuscular disorders or idiopathic CH were further substantiated in our study11. However, mortality rates in metabolic disease (80 vs. 48%) and malformation syndromes were higher (35 vs. 19%) in our study compared to death/heart transplantation rates in the study of Lipshultz et al., which might be explained by more serious cases in our cohort. In the longitudinal cohort study of Nugent et al. survival or no need for transplantation was 83% and 76% at 5 and 10 years after presentation with CH respectively12.Both Colan et al. and Lipshultz et al. studied the outcomes of CH in young childhood based on Pediatric Cardiomyopathy Registry data. Infants had significantly poorer survival (death/heart transplantation 19% at 1 year) than children presenting with CH after the age of 1 yearWe showed that underlying etiologies of CH can be identified in the majority of infants and include malformation syndromes, maternal diabetes mellitus, congenital hyperinsulinism, metabolic-, sarcomeric and neuromuscular disease. Noteworthy, underlying causes of CH were frequently associated with a broad range of hyperinsulinemic diseases and therefore hyperinsulinism should be included in the differential diagnosis of CH. Age at diagnosis and echocardiographic characteristics can help to distinghuish between the various underlying causes of CH associated and not associated with hyperinsulinism. Prognosis depends on the underlying cause of CH and is more favorable in infants with CH associated with hyperinsulinism. The results of this study are of added value in making the correct underlying diagnosis of CH in infants. This is of importance for prognosis and corresponding medical policy.Supplementary Information.