The upstroke of optical action potentials (APs) recorded from intact hearts are generally recognized to be slower than those recorded with microelectrodes. This is thought to reflect spatial signal averaging within the volume of tissue that makes up the optical signal. However, to date, there has been no direct experimental study on the relationship between conduction velocity (CV) and optical AP upstroke morphology in the intact heart. Notably, it is known that sodium channel block and gap junction inhibition, which both slow CV, exert differential effects on the upstroke velocity of microelectrode-recorded APs. Whether such differences are evident in optical APs is not known. The present study sought to determine the relationship between tissue CV and optical AP upstroke velocity in intact mouse hearts.max) and tissue CV were assessed in control conditions, during an acute period of low-flow ischemia, and following perfusion of flecainide (1–3 μmol/L), a sodium channel blocker, or carbenoxolone (10–50 μmol/L), a gap junction inhibitor.Isolated, perfused mouse hearts were stained with the potentiometric dye Rh-237. Fluorescent signals were recorded from across the anterior surface of the left and right ventricles during constant pacing. Maximum rate of change in fluorescence . Low-flow ischemia resulted in a time-dependent slowing of ventricular CV, which was accompanied by a concomitant reduction in optical AP upstroke velocity. All values returned to baseline on tissue reperfusion. Both flecainide and carbenoxolone were associated with a concentration-dependent reduction in CV and decrease in optical AP upstroke velocity, despite distinct mechanisms of action. Similar responses to carbenoxolone were observed for low- (156 μm pixel with) and high- (20 μm pixel width) magnification recordings. Comparison of data from all interventions revealed a linear relationship between CV and upstroke dF/dt.During epicardial pacing, an anisotropic pattern was observed in both activation and dF/dtIn intact mouse hearts, slowing of optical AP upstroke velocity is directly proportional to the change in CV associated with low-flow ischemia, sodium channel block, and gap junction inhibition. Cardiac optical mapping, using potentiometric dyes and fluorescent-light-sensitive digital cameras, allows researchers to study the electrophysiological properties of the heart at unparalleled spatial resolution. In intact heart tissue, the morphology of action potentials (APs) recorded with optical mapping typically exhibit reduced upstroke velocity and longer rise times compared to those recorded from the same tissues with microelectrode techniques  though . Meanwhimax) , reduced sodium channel availability, and reduced gap junction coupling. Sodium channel block reduces the number of available sodium channels and decreases the transmembrane sodium current, which slows tissue CV. It is well established that concurrently with slowing CV, sodium channel block reduces the rate of maximum AP depolarization (dV/dtmax) . Meanwhimax) . Model smax) , 1988. Tmax)  or dispumax)  by experIn principle, the rate of change of an optical AP upstroke is a function of (1) the intrinsic AP upstroke within each individual myocyte, (2) the activation delay across and through the tissue integrated by each camera pixel, and (3) the photon scattering effects of the tissue  . WhetherAll procedures were undertaken in accordance with ethical guidelines set out by the United Kingdom Animals (Scientific Procedures) Act 1986 and Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Studies conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health under assurance number A5634-01. Studies were approved by the University of Birmingham Welfare and Ethical Review Board.2) with concomitant intraperitoneal injection of heparin (100 units injected 5-min before heart isolation). Hearts were retrogradely perfused via the aorta at a perfusion pressure of 70–80 mmHg with an oxygenated crystalloid buffer, containing (in mM); NaCl 114, KCl 4, CaCl 1.4, NaHCO3 24, NaH2PO4 1.1, glucose 11.0 and sodium pyruvate 1.0 . Blebbistatin was added to the perfusate at a concentration of 15 μmol/L and solutions were continuously passed through a nitrocellulose filter (5 μm pore diameter). Once contraction had abated, the potentiometric dye Rh-237 was loaded by injection into the perfusion line. 100 mL of stock solution (1.25 mg/mL in DMSO) was injected over a period of 5-min. Final DMSO concentration was 0.001%. Hearts were illuminated at 530 ± 25 nm and emitted light >630 nm was collected via an Olympus MVX10 stereomicroscope and Evolve Delta 512 × 512 EMCCD camera. Images were taken from the anterior left and right ventricular surface. Unless stated, data was collected at an acquisition sampling rate of 1 kHz with a pixel width of 156 μm.Male mouse  hearts were isolated under isoflurane induced anesthesia . Recording time was 5-s. Hearts were subjected to interventions to alter ventricular CV. Namely, these were (i) 3-min of low-flow global ischemia  (ii) increasing concentrations of the sodium channel blocker flecainide  (iii) increasing concentrations of the gap junction inhibitor carbenoxolone . The pacing threshold was selected to ensure electrical capture during low-flow ischemia protocols.1 . As described in previous studies, the fractional level that dF/dtmax occurs at was defined as VF∗ (0.1 – dF/dt0.9). Unless stated otherwise, cubic spline interpolation was applied to increase the effective sampling rate from 1 to 16 kHz. Local tissue activation times were measured as the time of the maximum upstroke velocity  ] is affected by heterogeneities in dye loading and tissue illumination; which is a general limitation of single-wavelength fluorescent indicators. To quantify the AP upstroke morphology, we computed the maximum differential value of the optical AP upstroke (dF/dtd as VF∗ . In someand VF∗) . CV was and VF∗) .F∗ from the entire analyzed field of view was calculated for each individual heart. The mean data (n = 1 for each heart) was then used for statistical tests, i.e., n = 14 hearts for ischemia-reperfusion, n = 7 for flecainide/carbenoxolone treatment, respectively. Comparisons were paired (within experiment) and thus analysis was performed using paired t-tests, or repeated measures one- or two-way ANOVA with Bonferroni post hoc tests. Statistical significance was taken as p < 0.05. Unless otherwise stated, data is presented as Tukey’s boxplots.Hearts with signs of thrombi or with an initial heart rate less than 250 beats per minute were terminated and excluded. Hearts were randomized to different treatments prior to the experiments, but the nature of the paired (within heart) experimental design meant that blinding was unfeasible. However, excluding the initial determination of the region of interest, data were processed automatically with the same analytical steps and without user input, thus limiting sources of bias and error. Data analysis and statistical analysis was performed in GraphPad Prism . In all data presented, the mean value of dF/dt, CV or VF∗ is the fractional amplitude at which the optical AP upstroke has its fastest rate of rise and is shown in previous studies to reflect the subsurface orientation of depolarizing wavefronts as they spread through the myocardium  and the pattern of electrical propagation in the intact heart. F∗ map in the same heart (F∗ was low (0.5 or less) along the longitudinal axis, and high (greater than 0.5) in the transverse direction (Vocardium . Here weme heart . Consistirection .max) map. dF/dtmax was measured as the maximum of the first derivative of the normalized optical AP trace . Faster rates of changes are observed to align along the faster, longitudinal, conduction path. Differences in the morphology of APs in longitudinal and transverse conduction paths are shown in the example traces in F∗ is high, and steep along the longitudinal conduction path, where VF∗ is low (max before and after treatment with 2 μM flecainide in this representative mouse heart (see below for further results on the effect of flecainide). A positive linear correlation is observed, with faster local CV areas exhibiting larger dF/dtmax.∗ is low . The cormax) and at different fractional AP amplitudes (dF/dt0.1 – dF/dt0.9).The experiments described below were designed to quantify the effects of interventions that cause conduction slowing on the maximum rate of change of the optical AP upstroke , is dependent on the transmural wave orientation, with VF∗ ∼ 0.5 reflecting parallel conduction to the epicardial surface . F∗, with increased sampling rates resulting in increased measured VF∗. F∗, with lower magnification (larger pixel with of 156 μm) increasing VF∗. Pixel binning however, did not alter VF∗.max, we would therefore expect at higher magnification a carbenoxolone induced increase in dF/dtmax. However, data presented in We hypothesized that at smaller pixel sizes (higher-magnification), the intrinsic AP upstroke within a single myocyte would play a more prominent role in determining AP upstroke morphology. If gap junction uncoupling by carbenoxolone does indeed increase dV/dtmax. However, at kernel sizes of 5 × 5 pixels and larger , spatial filtering kernel size does not impact on measured dF/dtmax. Temporal filtering reduces dF/dtmax in a frame size dependent manner. max increases with interpolation, however, interpolation to extreme high effective sampling rates (to 256 kHz) does not substantially alter measured dF/dtmax values from “moderate” interpolation up to 16 kHz.It has been recognized for some time that the heterogeneity of activation of the myocardium is an important determinant of the rate of change and rise time of optically recorded APs  The presmax maps, with more rapid AP upstroke velocities aligned along the direction of the most rapid rates of conduction (and vice versa). This observation differs from previous reports utilizing microelectrode recording techniques, where it is established that the rate of change of the AP upstroke is slowest along the fastest conduction path  would become more dominant with smaller pixel widths, but we found this not to be the case. A likely explanation for this finding is the contribution of signals from within the ventricular wall, as well as the distortion of optical signals caused by fluorescent photon scattering within the tissue . IlluminF∗) is a function of the subsurface orientation of electrical wavefronts in the heart . 16 kHz spline interpolation was applied as it was observed that lower effective frame rate reduced measured dF/dtmax values, while higher order interpolation (up to 256 kHz) did not change dF/dtmax. However, the Nyquist theorem suggests that 1 kHz sampling rate is only sufficient to accurately resolve signal frequency content less that 500 Hz, meaning interpolation to 16 kHz may be inaccurate. Furthermore, only one interpolation method (cubic spline) was tested. Hence, further investigation is required, potentially utilizing “ground truth” computer models of optical APs in intact tissue, to optimize data processing for measurement of maximum and fractional dF/dt.We show that AP upstroke velocity is dependent on the temporal sampling rate and spatial resolution, and for the same reason will be affected by signal processing techniques like spatial and temporal filtering and ensemble averaging. For example, we found that temporal filtering with a third order Savitizy-Goaly filter reduced baseline dF/dtmax and tissue CV will be species- and setup- dependent.The present studies were performed in isolated mouse hearts, and additional studies are required to establish the properties of the optical AP upstroke in other species. It seems highly probable that our findings in the mouse ventricle will be more broadly applicable, though the absolute relationship between optical AP upstroke dF/dtIn intact mouse hearts, slowing of optical AP upstroke velocity is directly proportional to the change in CV associated with low-flow ischemia, sodium channel block, and gap junction inhibition.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the University of Birmingham Animal Welfare and Ethical Review Board.JW conducted the majority of studies, developed the analysis software, and conducted the analysis. CO’S integrated the software into ElectroMap and conducted the analysis. KR and DP supervised CO’S. All authors wrote and reviewed the manuscript.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.


In the clinical setting, current native T1 and T2 methods with the specific voxel sizes at 1.5 T could not differentiate between the infarcted and salvaged myocardium, whereas ECV could differentiate between the two. ECV was also higher in the salvaged myocardium when compared to the remote myocardium.We used multi-parametric cardiovascular magnetic resonance (CMR) mapping to interrogate the myocardium following ST-segment elevation myocardial infarction (STEMI). Forty-eight STEMI patients underwent CMR at 4 ± 2 days. One matching short-axis slice of native T1 map, T2 map, late gadolinium enhancement (LGE), and automated extracellular volume fraction (ECV) maps per patient were analyzed. Manual regions-of-interest were drawn within the infarcted, the salvaged and the remote myocardium. A subgroup analysis was performed in those without MVO and with ≤75% transmural extent of infarct. For the whole cohort, T1, T2 and ECV in both the infarcted and the salvaged myocardium were significantly higher than in the remote myocardium. T1 and T2 could not differentiate between the salvaged and the infarcted myocardium, but ECV was significantly higher in the latter. In the subgroup analysis of 15 patients, similar findings were observed for T1 and T2. However, there was only a trend towards ECV The AAR refers to the territory supplied by the infarct-related artery that would have been irreversibly injured without reperfusion. Once the AAR and MI size are known, MSI can be calculated using the formula: myocardial salvage index = (AAR − MI size)/AAR4.Myocardial salvage index (MSI) is considered a more sensitive measure for assessing the efficacy of a cardioprotective strategy than an absolute reduction in myocardial infarct (MI) size, as it normalizes the reduction in MI size to the area-at-risk (AAR)7. It can also provide information on the edema-based AAR using T2-weighted imaging9, T2 mapping11, T1 mapping12, early gadolinium enhancement13, or post-contrast steady-state-free-precession (SSFP) cine images14. However, none of these techniques have been established as the reference standard in the clinical setting for the AAR by CMR. Controversies exist on whether edema is confined to the infarcted myocardium only15, or extends to the salvaged myocardium16.Cardiovascular magnetic resonance imaging (CMR) is considered the reference standard technique for MI size quantification4. Native T1 and T2 mappings are acquired prior to contrast administration. A recent pre-clinical study16 using a canine model of reperfused MI has reported that native T2 values were higher in infarcted myocardium when compared to salvaged myocardium. However, the same group reported no difference in native T1 values between the infarcted and salvaged myocardium, but post-contrast T1 was able to differentiate between the infarcted and salvaged myocardium, 8 minutes following contrast injection17. So far, no clinical studies have interrogated the infarcted and salvaged myocardium in the same patients using the latest T1, T2 and ECV mapping technology. If we were able to differentiate between the infarcted and salvaged myocardium using native T1 and T2 mapping, we could potential quantify both the AAR and MI size accurately without contrast on these maps by applying different thresholds, which would significantly shorten scan time for these patients and expand the availability to those with contraindications to the gadolinium chelate.Conventionally, MI size is quantified on the late gadolinium enhancement (LGE) imaging following contrast administrationIn order to clarify whether the infarcted myocardium can be distinguished from the salvaged myocardium in the acute setting, we used multiparametric CMR with native T1 maps, T2 maps and automated extracellular volume fraction (ECV) mapping to interrogate the infarcted and salvaged myocardium in patients shortly after ST-segment elevation myocardial infarction (STEMI) reperfused by primary percutaneous coronary intervention (PPCI).25 but no prior publications have addressed multiparametric mapping in the salvaged and the infarcted myocardium. In brief, the London-Harrow Research Ethics Committee approved this study. Fifty STEMI patients were prospectively recruited from August 2013 to July 2014 following informed consent. This study complied with the declaration of Helsinki and was approved by the local ethics committee. Forty-eight patients completed the first CMR at 4 ± 2 days post-PPCI. A subgroup analysis was performed in those patients without late microvascular obstruction (MVO) (as this would interfere with the T1 and T2 of the infarcted myocardium) and with ≤75% transmural extent of infarct  in the salvaged myocardium).Patients included in this study have been previously reportedAll CMR scans were performed on a 1.5 Tesla scanner  using a 32-channel phased-array cardiac coil. The imaging protocol included short axis coverage of cines, native Modified Look-Locker Inversion Recovery (MOLLI) T1 mapping, T2 mapping, LGE and post-contrast T1 mapping. All the short axis images were aligned with the cine short axis slice position.26.An SSFP-based MOLLI sequence with a sampling protocol of 5 s(3 s)3 s was used for the T1 maps. The acquisition parameters were: flip angle = 35°; pixel bandwidth 977 Hz/pixel; matrix = 256 × 144; echo time = 1.1 ms; voxel size = 1.5 × 2.0 × 6.0 mm. Motion correction and a non-linear least-square curve fitting of the set of images acquired at different inversion times were performed inline by the scanner to generate a pixel-wise colored T1 map27 after acquiring 3 single-shot images at different T2 preparation times  and the acquisition parameters were: flip angle = 70°; pixel bandwidth 930 Hz/pixel; matrix = 116 × 192; echo time = 1.1 ms; repetition time = 3xR-R interval; voxel size = 2.0 × 2.7 × 6.0 mm.Pixel-wise colored T2 maps were generated inline following motion correction and fitting to estimate T2 relaxation times28.LGE images were acquired 10 to 15 minutes after the injection of 0.1 mmol/kg of Gadoterate meglumine , using either a standard segmented ‘fast low-angle shot’ two-dimensional inversion-recovery gradient echo sequence LGE phase-sensitive inversion recovery (PSIR) sequence or a respiratory motion-corrected, free-breathing single-shot SSFP averaged PSIR sequence29) 15 minutes after contrast injection (0.1 mmol/kg of Dotarem) using similar acquisition parameters (voxel size = 1.5 × 2.0 × 6.0 mm) as for native T1 maps.Post-contrast T1 maps were obtained using the 4 s(1 s)3 s(1 s)2 s sampling protocol 30 using the following formula31:The previously described and validated automated method for producing a pixel-wise ECV map was used25. In brief, MI size (from LGE images) and AAR (from T2 maps) were quantified from the whole LV coverage short axis acquisitions using signal intensity thresholds of 5 and 2 standard deviations (SD) above the normal remote myocardium32, respectively, and expressed both as a percentage of the left ventricle (%LV) and in grams. Transmural extent of infarct (TEI) was quantified by averaging the values from 100 chords from each short axis slice to obtain the mean transmural extent of LGE for each of the 16 American Heart Association (AHA) segments and expressed in as a continuous variable and then objectively classified into groups of “no LGE”, “1–25%TEI”, “26–50%TEI”, “51–75% TEI” and “76–100%TEI25”.All imaging analysis was performed using CVI42 software (V.5.1.2 (303), Calgary, Canada) as previously described16. After identifying the infarcted and salvaged myocardium on LGE, ROIs were placed in the corresponding regions on the native T1 maps and copied to the T2 and ECV maps, with minimal manual adjustments when required to avoid partial volume effects. The remote myocardium was defined as the region that did not display any LGE, native T1 or T2 abnormalities in a segment that was 180° from the infarct-related territory. In those with >75%TEI, no ROI were available for the salvaged myocardium. Patients without MVO  and with ≤75%TEI  were included in the subgroup analysis. In order to evaluate whether partial volume effect may impact on our results, we also excluded those patients with TEI 51–75% in a further subgroup analysis.For representative parametric data in the infarcted, salvaged and remote myocardium for each patient, one short-axis slice per patient of matching native T1 map, T2 map, ECV map through the region of wall motion abnormality was analyzed and the corresponding LGE image was used as the reference. ROIs were manually delineated in the infarcted, salvaged and the remote myocardium as shown in Fig. Statistical analysis was performed using SPSS version 22 . Normality was assessed using Shapiro-Wilk test. Continuous data were expressed as mean ± SD or median (interquartile range), and compared with the paired Student t-test/Wilcoxon signed rank test or unpaired Student t test/Mann–Whitney U test, where appropriate. Categorical data were reported as frequencies and percentages. To compare T1, T2 and ECV values in the remote, salvaged and infarcted myocardium, a linear mixed model was used with the T1, T2 or ECV being the dependent variable; the patients being a covariate; and the territory within patients  being a fixed factor, to take into account interaction among segments within the same patients. Pairwise comparison was performed using Bonferroni correction. All statistical tests were 2-tailed, and P < 0.05 was considered statistically significant.Baseline characteristics of the patients are shown in Table 403/768 (53%) myocardial segments had no LGE. Out of those segments with LGE, the TEI were as follows: 1–25%TEI: 89/365 (24%); 26–50%TEI: 100/365 (27%); 51–75% TEI: 90/365 (25%); and 76–100%TEI: 86/365 (24%). 15 patients without MVO and with ≤75%TEI were included in the subgroup analysis (Table infarct (1253 ± 73 ms) and T1salvage (1219 ± 66 ms) were both higher than T1remote  with no significant difference between T1infarct and T1salvage (P = 0.15)  and T2salvage (64 ± 6 ms) were significantly higher than T2remote  with no significant difference between T2infarct and T2salvage (P = 0.42)  was significantly higher than ECVsalvage (38 ± 4%) and ECVremote . Furthermore, ECVsalvage was also significantly higher than ECVremote (P < 0.001)  and T1salvage (1235 ± 61 ms) were significantly higher than T1remote  with no significant difference between T1infarct and T1salvage (P = 0.38).A similar pattern was observed for T1 and T2 in the subgroup of patients without MVO and with ≤75%TEI. T1infarct (64 ± 5 ms) and T2salvage (62 ± 7 ms) were significantly higher than T2remote  and there was no significant difference between T2infarct and T2salvage (P = 0.45).T2infarct (60 ± 10%) was significantly higher than ECVsalvage  and ECVremote . However, there was only a trend towards ECVsalvage being higher than ECVremote but this comparison did not reach statistical significance (P = 0.07).As for ECV, ECVinfarct; 1293 ± 86 ms; T1salvage: 1243 ± 63 ms; T1remote: 993 ± 54 ms), T2  and ECV . There was no significant difference between T1infarct and T1salvage (P = 0.27) and between T2infarct and T2salvage (P = 0.99) but there was a significant difference between ECVinfarct and ECVsalvage (P = 0.001).In order to evaluate whether partial volume effect may have contributed to some extent to the above results, we further excluded those patients with TEI 51–75% (n = 3). The same findings were seen for T1 , and for our understanding of salvage.19. Therefore, the larger variability in T1 in the remote myocardium in this cohort is unlikely due to the precision of the T1 mapping technique used, but rather changes in the remote myocardium that us19 and other groups34 have reported upon previously.Although the standard deviation of the T1 in the remote myocardium appeared relatively high (±53 ms), we have previously reported reference values by MOLLI in a healthy control group to be 1000 ± 25 ms35. The potential explanation for our observations could be related to the difference in severity of the intracellular and extracellular edema in the infarcted and salvaged myocardium. Native T1 and T2 measure edema both in the intracellular and extracellular compartments, whereas ECV measures edema in the extracellular compartment only. Native T1 and T2 may be similarly affected by intracellular edema, and may not be able to differentiate the severity of extracellular edema in some patients. Following contrast administration, the kinetics of the gadolinium chelate wash-in and wash-out at pseudo-equilibrium would be different in the infarcted (likely to predominantly consist of cardiomyocytes with ruptured cell membranes and extracellular edema) and salvaged myocardium (likely to predominantly consist of cardiomyocytes with intact cell membrane and with both intracellular and extracellular edema). A larger concentration of contrast would be present in the infarcted myocardium than in the salvaged myocardium (guided by the severity of extracellular edema) and this would explain the difference in the findings between pre-contrast and post-contrast mapping parameters. It is not surprising that ECV was significantly different between the infarcted and salvaged myocardium and this likely reflects the different severity of extracellular edema in those two territories. In those with significant myocardial salvage, the extracellular edema may be less severe as shown by the ECV values in our subgroup analysis where there was some overlap between the salvaged and remote myocardium.Our results are consistent with the findings of previous studies showing that the increase in native T1 and T2 values in the infarct-related artery corresponded to the edema-based AAR in the clinical settinget al.16 recently reported in a canine model of reperfused MI that T2 values in the infarcted myocardium were higher than in the salvaged myocardium, and both were significantly higher than the remote myocardium. They only included 11 animals, and they all underwent mid left anterior descending artery ligation for 2 hours, and CMR was performed at similar times for all animals. The difference between the pre-clinical and clinical findings may be related to the animal species used  or the heterogeneity of the patient selection . Furthermore, the occlusion of the infarct-related artery was abrupt in the animal model whether was this process is slower and stuttering in the clinical setting with downstream embolism of plaque debris and microemboli. Although we also performed a subgroup analysis by excluding patients with MVO  and >75% TEI , some patients may still have had minute areas of MVO and haemorhage within the infarcted myocardium that were not visible to the naked eye and may have affected the native T1 and T2 values within the infarcted myocardium.However, Hammer-Hansen et al.17 also showed that native T1 was similar in the infarcted and salvaged myocardium, and our findings confirm this. If T1 and T2 mapping could differentiate between infarcted and salvaged myocardium at 1.5 T in the acute setting, then by applying a particular threshold, it would possible to delineate the acute MI size without contrast. Interestingly, Liu et al.36 recently showed that native T1-mapping could identify acute MI size. However, their study was performed on a 3 T scanner and only 58 short-axis T1 maps without microvascular obstruction were analyzed in that study36. Further work is required with a larger number of patients to evaluate whether native T1 mapping can delineate the acute MI size.In the clinical setting, Hammer-Hansen et al.37 previously showed that a threshold of ECV >0.42 or 0.44 could be used to quantify the extent of scars in the setting of chronic MI and hypertrophic cardiomyopathy. Our findings are consistent with those from Hammer-Hansen et al.17, who showed that the ECV of the infarcted myocardium was higher than that of the salvaged myocardium. They also showed that the ECV of the salvaged myocardium was higher than that of the remote myocardium. Most recently, Garg et al.38 showed that ECV maps could be used for the AAR and chronic MI size by applying different thresholds. However, the limits of agreement with the T2-derived AAR from the T2-weighted imaging were quite wide at ±10.4%39. Whether the wide limits of agreement in edema measures seen in the study by Garg et al.39 could be due to ECV not identifying the edema-based AAR accurately in some patients with less severe extracellular edema, warrants further investigation.ECV mapping at pseudo-equilibrium provides pixelwise quantification of the ratio of the extracellular compartment to the total myocardial volume. Hwang Partial volume effects may have affected these results. Further analysis of those with TEI up to only 50% resulted in finding consistent with our prior findings. However, this does not necessarily suggest that partial volume effects along the slice direction are not important. Despite this, this highlights the fact that in the clinical setting, using current T1 and T2 mapping with the current voxel sizes at 1.5 T, salvaged myocardium cannot be differentiated from the infarcted myocardium.40. Our delineation of the infarcted and salvaged myocardium was based on the concept of ‘wave-front phenomenon’ where the ischemic injury is thought to progress radially from the subendocardium to the epicardium with increasing duration of coronary occlusion41, and therefore the epicardial side of the infarcted myocardium was considered as salvaged myocardium. This is the same definition used by Hammer-Hansen et al.16. We only analyzed one short axis slice of T1, T2 and ECV maps per patient although this is similar to previous studies17. The spatial resolution of T1, T2, and ECV maps were not the same.The number of patients included in our study was small and therefore any further in-depth analysis of any temporal trend, relationship with other predictors and indicators of myocardial injury  were not performed. However, a retrospective sample size calculation to detect a 25% difference between the infarcted and remote myocardium showed that the study was adequately powered. CMR was performed at different days within the first week post-STEMI and may be a source of bias, given that edema has been recently shown to be dynamicIn this cohort of STEMI patients, current native T1 and T2 methods with the specific voxel sizes at 1.5 T could not differentiate between the infarcted and salvaged myocardium, whereas ECV could differentiate between the two. ECV was also higher in the salvaged myocardium when compared to the remote myocardium. Whether this is due to a true pathophysiological observation or partial volume effect warrants further investigation.


Hypertensive heart disease (HHD) and hypertrophic cardiomyopathy (HCM) are both associated with an increased left ventricular (LV) wall thickness. Whilst LV ejection fraction is frequently normal in both, LV strain assessment could differentiate between the diseases. We sought to establish if cardiovascular magnetic resonance myocardial feature tracking (CMR-FT), an emerging method allowing accurate assessment of myocardial deformation, differentiates between both diseases. Additionally, CMR assessment of fibrosis and LV hypertrophy allowed association analyses and comparison of diagnostic capacities.1 mapping and late gadolinium enhancement (LGE). Global longitudinal strain (GLS) was assessed by CMR-FT .Two-hundred twenty-four consecutive subjects  underwent 1.5T CMR including native myocardial T1 , and LGE volume . Discrimination between HHD and HCM by GLS  was similar to LV mass index , global myocardial native T1 , and LGE volume .GLS was significantly higher in HCM patients . GLS was associated with LV mass index , and LV ejection fraction . In HCM patients, GLS was also associated with global native TCMR-FT GLS differentiates between HHD and HCM. In HCM patients GLS is associated with myocardial fibrosis. The discriminatory capacity of CMR-FT GLS is similar to LV hypertrophy and fibrosis imaging markers. Considering an aging population with an increased prevalence of hypertension, the differential diagnosis for patients with increased left ventricular (LV) wall thickness (LVWT) represents a diagnostic challenge often encountered in cardiovascular imaging laboratories. Next to hypertensive heart disease (HHD) , hypertrUntil late disease stages, HHD and HCM exhibit normal systolic function by conventional measurements, such as LV ejection fraction or fractional shortening –4. Yet aIn hypertensive rats, diffuse subendocardial fibrosis correlates with reduced longitudinal contractility, whilst midwall fibrosis occurs only in later disease stages and correlates with a decline in circumferential strain . Corresp1 mapping [On histology, HHD and HCM patients share myocyte hypertrophy and fibrosis ,19. The  mapping , respect mapping  and to b mapping .Recently, CMR myocardial feature tracking (CMR-FT), a software solution similar to STE , has beeConsidering the different pathophysiological processes in HHD and HCM and their related cardiac mechanics, we hypothesized that CMR-FT global longitudinal strain (GLS) would differentiate between diseases. We also postulated that CMR-FT strain would be associated with myocardial fibrosis and hypertrophy measurements.1 imaging to differentiate between HHD and HCM and was reported in [The study was approved by the Beth Israel Deaconess Medical Center’s Institutional Review Board (Protocol Number: 2001P-000793). Written consent was obtained. Two hundred and twenty-four consecutive subjects, referred for CMR at our center between July 2014 and March 2018 and meeting the criteria described below, were included in this retrospective study. The same dataset was previously used for radiomic analysis of myocardial native Torted in . In the orted in –30.Patients with HCM (n = 107) demonstrated either normal LV cavity size with maximum LVWT ≥15 mm, or LVWT above the normal range ≥12 mm)  in the c mm  in tCMR images were acquired with a 1.5T scanner  equipped with a 32-element cardiac coil. Breath-hold, retrospectively electrocardiogram (ECG)-gated cine, balanced steady state free-precision (bSSFP) images were recorded in the LV 2- and 4-chamber long-axis views, and a short-axis stack covering the entire LV (8-mm slices with 2-mm gaps).2 in men and ≥62 g/m2 in women [CMR images were interpreted using ViewForm software . LV and right ventricular volumes were quantified by manually tracing the end-diastolic and end-systolic endocardial contours and applying a summation of discs method. Slices at the base of LV were included if >50% of the blood pool was encircled by myocardium. LV dimensions or the maximum LVWT were quantified in the short-axis view at the level of the chordae or at the slice with largest endo- to epicardial border distance, respectively. LV mass was calculated as the total myocardial volume without papillary muscles multiplied by the specific gravity of myocardial tissue (1.05 g/mL). LV mass index (LVMI) ≥81 g/min women  were usein women  were usein women  with a sin women  or segmein women , respect1 values [CMR-FT analysis was carried out using CVi42 . LV endo- and epicardial borders were manually traced at end-diastole in ECG-gated bSSFP 4- and 2-chamber long-axis sequences using a point-and-click approach. The automatic border tracking algorithm of the software was applied to track image features throughout the cardiac cycle. Tracking was visually reviewed and manually corrected by border adjustment with consecutive reapplication of the algorithm if necessary. To adjust for morphological differences between the HHD and HCM cohorts, GLS was also investigated in the previously reported “equal LVWT subgroup” that was matched for LVWT, gender, LVH, and assembled with similar age, LVMI, and T1 values .LV LGE images were obtained using a three dimensional (3D) phase sensitive inversion-recovery (PSIR) sequence with spectral fat saturation pre-pulses during the end-diastolic phase approximately 15 minutes after administration of 0.1 mmol/kg body weight gadobenate dimeglumine . LGE presence and percentage (%LGE) were measured by an experienced (level 3 trained) reader (U.N.) blinded to clinical and laboratory data using an automated LV contour and LGE area quantification algorithm . Accurat1 mapping was conducted using the slice-interleaved T1 mapping (STONE) sequence, which allows acquisition of 5 slices in the short-axis plane during a 90 seconds free breathing scan, as previously describe [1 maps using custom software . An adaptive registration of varying contrast-weighted images enabled motion correction [1 maps were averaged to obtain the global native T1 value. Septal T1 and apical T1 were calculated as the average value of segments 2, 3, 8, 9 or 13-16/17 of the American Heart Association 16/17 segment model [1 quantification.Native myocardial Tdescribe . A 2-parrrection . The fivnt model , respect1 and LGE extent were tested based on 50 randomly selected cases and LGE quantification available in clinical reports (n = 26), respectively.CMR-FT was performed by a single investigator (U.N.). For reproducibility purposes, 20 randomly selected cases  were reanalyzed by the same reader ≥ 4 weeks later and by a second blinded investigator (L.M.). Both were blinded to clinical information. Reproducibility of global native TData were analyzed using SPSS . Normality of data distribution was determined using the Kolmogorov-Smirnov test and visual inspection of Q-Q plots. The Chi-squared test, two-sample t-test or the Mann-Whitney U-test was conducted as appropriate. Correlation between variables was tested by Pearson and Spearman ρ correlation coefficients as appropriate. Univariate and multivariate logistic regression was used to test the ability of CMR measurements to discriminate between HHD and HCM. To test for independent association of CMR measurements in the context of demographic characteristics and >1 maging marker multiple logistic regression analyses were conducted. Specificity, sensitivity, and discriminatory accuracy, cut-off values and area under the curve, were derived from receiver-operating characteristics (ROC) curve analyses using the Youden’s index. Areas under ROC curves were compared using the DeLong method. The intraclass correlation coefficient for a 2-way mixed- or random-effects model with absolute agreement was calculated to assess the intra- and interobserver reproducibility. Statistical significance was defined as a 2-sided P-value <0.05 and was Bonferroni corrected for multiple cohort comparisons.The clinical characteristics are summarized in 2, P = 0.002), maximum LVWT , LGE volume  and global native T1 . These differences remained significant after adjustment for age and body surface , whilst it was not in the subgroup with LVH :-13.8±3.8 vs. -13.2±3.8%, P = 0.653; LVH- or HHDLVH+ patients . Comparisons of LV mass index in each subgroup showed no significant difference between HHD and HCM . The GLS difference between LVH+ and LVH- patients was significant for both disease . GLS was significantly higher in the LGE positive HHD cohort , as well as the LGE positive HCM cohort . The maximum LVWT and prevalence of LVH were significantly greater in HHD and HCM patients in whom LGE was detected by CMR (P<0.05). GLS was different between concentric (-16.4±4.1%) and apical HCM , but not in the comparison of asymmetric (-14.4±3.4%) with concentric or apical HCM after adjustment for multiple cohort comparison . In the “equal LV wall thickness subgroup” [Global longitudinal strain (GLS) was significantly higher in HCM compared to HHD patients . The HHDubgroup”  GLS did 1, and LGE volume , revealed the independent role that extent of hypertrophy plays in the attenuation of GLS in HCM  and in HHD patients . Global native T1 did not contribute significantly (P>0.05) to these regression models. An exchange of global native T1 with LGE volume provided similar results in HCM patients , and showed a significant contribution of LGE to the regression models .Two multiple linear regression models, each including 1 variable representing extent of hypertrophy  and global native T1 (P = 0.131), LGE volume (P = 0.100), or LV mass index (P = 0.967) showed no significant differences. In a multivariate binary logistic regression analysis including the prevalence of LVH and GLS, a trend for GLS to independently discriminate between HCM and HHD was observed (P = 0.066). With a sensitivity of 68% and a specificity of 64%, the model had a better test accuracy than each of its parts. In a multivariate logistic regression with forward selection of all markers represented in 1 (P = 0.015) and LGE prevalence (P = 0.018) remained significantly associated with disease prediction.In univariate analysis, GLS had diagnostic accuracies similar to hypertrophy and fibrosis markers , Fig 4. 1  was excellent as well.The intra-  and interobserver agreement  for CMR-FT GLS measurements was excellent. Bland-Altman analyses showed narrow limits of agreement for GLS on intra- and interobserver level . The intOur work investigated the role of CMR-FT in the assessment of patients with increased LVWT and showed that GLS differentiates between two of the most prevalent disease with this phenotype, HHD and HCM. This finding is consistent with publications employing STE strain assessment –12. ConsLVH-, and HHDLVH+ patients, but similar to HHDLVH- patients.In subgroup analysis, the distinction between HHD and HCM was limited to patients with increased LVWT in the absence of LVH. Difficulties to differentiate diseases by CMR-FT GLS in the presence of LVH have been reported by Rodrigues et al., where HHD patients had a 17% higher LV mass index when compared to HCM patients . In hype1 and LGE, respectively. These findings were consistent with published STE and CMR-FT data [Our findings demonstrate that hypertrophy has a larger impact on CMR-FT GLS than diffuse and replacement fibrosis, as measured by native T-FT data ,23. Inte-FT data ,43. Rela-FT data –46, GLS -FT data .1 [, diastolic dysfunction [We provide evidence for CMR-FT GLS’s capacity to differentiate between HHD and HCM. Conclusions regarding the magnitude of its diagnostic capacity require consideration of other cardiac imaging markers. For instance, both diseases have focal LGE ,47–50, i1 ,47, diasfunction , as wellfunction ,49 in cofunction .Focal fibrosis has been detected in both disease ,47–50. M1 mapping is limited by the overlapping data distribution between patients with different cardiac pathologies [1 is associated with LV mass index and LVWT [2. Thus, relatively small phenotypic differences influenced probably our results. Furthermore, myocardial native T1 values and LVWT are correlated on segmental level [1 values despite regional hypertrophy. The diagnostic capacity of myocardial native T1 in our study differed from data reported by Hinojar et al. [1 mapping sequences differ in their bioequivalence for discrimination between different cohorts. Therefore the use of different T1 mapping sequences, such as the modified Look-Locker imaging (MOLLI) sequence [1 mapping (STONE) sequence [Comparable to GLS –12 and Lhologies ,51. In Hand LVWT ,52. In or et al. . Next tor et al. , T1 mappsequence  or the ssequence , could rSeveral studies reported differentiation between HHD and HCM by STE longitudinal strain –13. AlsoOur study has several limitations. To adjust for the diagnostic criteria of HCM, LVWT ≥15 mm rather than LV hypertrophy , we defiOur study demonstrated that CMR-FT GLS differentiates HHD from HCM. This distinction is mostly observed in patients with increased LVWT and absence of LVH. GLS’s diagnostic accuracy is similar to CMR markers of myocardial fibrosis and LVH. However GLS’s discriminatory ability limits its clinical application and emphasizes the difficulty to differentiate the two diseases based on cardiac imaging alone.S1 TableLV, left ventricular; LVWT, LV wall thickness. * P<0.001 compared with HHD; † P<0.01 compared with HHD; ‡ P<0.02 compared with HHD; § P<0.001 compared with healthy controls; ǁ P<0.01 compared with healthy controls; # P<0.02 compared with healthy controls. P values were Bonferroni corrected (0.05/3) to account for multiple cohort comparisons.(DOCX)Click here for additional data file.S2 TableGLS, global longitudinal strain; LGE, late gadolinium enhancement; LV, left ventricle; LVWT, LV wall thickness.(DOCX)Click here for additional data file.S3 TableLGE, late gadolinium enhancement; LV, left ventricular; RV, right ventricular. * P<0.001 compared with asymmetric HCM; † P<0.02 compared with asymmetric HCM; ‡ P<0.001 compared with concentric HCM; § P<0.02 compared with concentric HCM. P values were Bonferroni corrected (0.05/3) to account for multiple cohort comparisons.(DOCX)Click here for additional data file.S4 TableHCM, hypertrophic cardiomyopathy; HHD, hypertensive heart disease; LVWT, left ventricular wall thickness. Clinical characteristics of the equal LVWT subgroup have been reported elsewhere .(DOCX)Click here for additional data file.S1 Data(XLS)Click here for additional data file.


Disturbed fetal haemodynamics often affects cardiac development and leads to congenital cardiac defects. Reduced left ventricular (LV) preload in the fetus may result in hypoplastic LV, mitral and aortic valve, mimicking a moderate form of hypoplastic left heart complex. We aimed to induce LV hypoplasia by occluding the foramen ovale (FO) to reduce LV preload in the fetal sheep heart, using percutaneous trans-hepatic catheterisation. Under maternal anaesthesia and ultrasound guidance, hepatic venous puncture was performed in six fetal lambs (0.7–0.75 gestation). A coronary guidewire was advanced into the fetal inferior vena cava, right and left atrium. A self-expandable stent was positioned across the FO. An Amplatzer Duct Occluder was anchored within the stent for FO occlusion. Euthanasia and post-mortem examination was performed after 3 weeks. Nine fetuses were used as age-matched controls. Morphometric measurements and cardiac histopathology were performed. Compared with controls, fetal hearts with occluded FO had smaller LV chamber, smaller mitral and aortic valves, lower LV-to-RV ratio in ventricular weight and wall volume, and lower number of LV cardiomyocyte nuclei. We conclude that fetal FO occlusion leads to a phenotype simulating LV hypoplasia. This large animal model may be useful for understanding and devising therapies for LV hypoplasia. Consequently, almost all stages of heart development and growth take place in a working heart with active blood flow. Haemodynamic forces are critical for normal cardiac development, and interference in haemodynamics often leads to congenital heart defectset al. described the induction of hypoplastic left heart complex (HLHC) in chick embryos by obstruction of blood flow through the left atrioventricular canal2. A similar effect was observed by Fishman et al. 5 years later in fetal sheep when inserting a balloon in the LA obstructed LV filling and produced the phenotype of HLHC, however all of the fetal lambs died within a few days of the procedure3. Others described induction of a HLHC by reducing LV filling due to LA ligation in the chick embryo5. Interestingly, such induced LV hypoplasia can be rescued by an increased volume loading of the left heart at a later timepoint6.Experimental manipulation of flow through developing left-sided cardiac structures exerts profound effects on left atrium (LA), left ventricle (LV), and mitral and aortic valve development. Already in 1973, Harth 8, others described LV dilatation and fibro-elastosis9. While RV dilation, tricuspid regurgitation, pericardial effusion, pleural effusion, in-utero heart failure and hydrops fetalis as well as early and late pulmonary hypertension have also been reported14, there is substantial evidence that early (primary) closure of the FO results in hypoplasia of left heart structures and that normal fetal left heart development is dependent on a normal volume of flow though the FO15.In the fetus, LA and LV filling is almost completely dependent on right to left shunting of oxygenated blood from the placenta through the foramen ovale (FO) since the right ventricular (RV) output is mostly bypassing the lungs through the ductus arteriosus and LA filling by pulmonary venous return is only marginal. Premature closure or restriction of the FO, eliminating or restricting the fetal right to left atrial shunt, has been described to have variable consequences, probably depending on the timing and the presence or absence of accompanying cardiac malformations. Several authors have reported RV to LV growth discrepancy and mild to moderate hypoplasia of the mitral and aortic valves as well as the aortic arch17. Our technique of in-utero cardiac catheterisation allows occlusion of the FO without trauma or puncture into the fetal heart, and study of the effects of FO occlusion during fetal development. We hypothesized that by reducing volume loading of the left sided heart structures, a fetus with obliterated or obstructed blood flow through the FO would develop the phenotype of HLHC.Here, we report the effects of in-utero occlusion of the FO in the fetal lamb in mid-gestation, using a novel fetal cardiac catheterisation method with trans-hepatic access that we have described previouslyTotal of 26 fetuses had the transhepatic catheterisation. Access to the fetal venous system, followed by passage of catheter and guidewire though the RA and FO into the LA was successfully in all animals. Anchoring the occluder within the stent at FO was successful in 16 out of 26 fetuses. The 16 animals were then monitored with fetal ultrasounds regularly until the planned euthanasia at least 3 weeks later. Two fetuses died within 1–2 days after the procedure, with intra-abdominal haemorrhage found at post-mortem examination. One fetus died the day before the planned euthanasia, and post-mortem examination revealed the stent and occluder had dislodged into the mitral valves. On post-mortem examination after the planned euthanasia, the occluder was found to be in various oblique positions within the stent and even partly in the left atria in 7 animals, and therefore did not effectively obliterate FO blood flow. These 7 fetuses had variable structural cardiac changes from the partial FO occlusion  diameters of 5.4 (0.7) mm and 4.7 (0.8) mm respectively. The distances along the IVC from the RHV and LVH to the right atrium were 32.8 (3.9) mm and 32.4 (3.8) mm respectively. The IVC and FO had a diameter of 7.2 (0.8) mm and 6.5 (0.9) mm respectively. Other intra-cardiac measurements are shown in Table After the FO occlusion, ultrasonographic measurements showed significantly reduced diameters of the mitral valve and width of the left ventricle Table , Fig. 1cExternal examination showed that the fetal hearts with occluded FO had a smaller LV apex compared to the control Fig. . Body weFetuses with occluded FO had a lower LV chamber volume with significantly lower LV to RV chamber volume ratio compared to controls Table . While tCardiac morphometry data of the fetuses with partially occluded FO due to suboptimal and variable occluder positions are shown in Supplementary Table Cardiac histopathology was performed in 4 fetuses with occluded FO and 7 age-matched controls Table .Compared to the controls, animals with occluded FO had significantly lower LV + S cardiomyocyte nuclei count after adjusting for biventricular weight and, accordingly, the LV + S to RV ratio of cardiomyocyte nuclei count was significantly lower in animals with occluded FO compared to controls Table .Table 4CAfter adjusting for individual ventricular weight Table , fetusesThere was no significant difference between fetuses with occluded FO and the controls in the longitudinal cross-sectional area of cardiomyocytes in the LV + S and RV Table .There was no significant difference between fetuses with occluded FO and the controls in the level of cardiac interstitial fibrosis in the LV + S (1.8 ± 1.3% vs 3.0 ± 1.4%), RV (2.2 ± 1.1% vs 2.6 ± 1.2%) or LV + S to RV fibrosis ratio (1.1 ± 0.8 vs 1.2 ± 0.4) respectively.18, we present here a further novel interventional technique, which to our knowledge has not been performed before. We have previously described that stenting of a large FO in a sheep fetus seems to be possible only by a specially designed self-expandable stent which is deliverable through a 4 F sheath but expands to reach a stent lumen width of 8 mm17. The 8-mm stent lumen, together with its additional open-cell design, enables secure fixation in a widely opened FO with floppy atrial septum tissue around. Furthermore, the secured stent allows anchorage of a nitinol occluder to occlude the FO and create a model to induce the fetal HLHC. The interventional techniques and devices used in this study, all certified with the CE-mark for human use, could possibly be applied in treating other fetal cardiac malformations in future.Based on our previous experience of fetal cardiac interventions in a sheep model8. At the cellular level, the significantly lower LV-to-RV ratio of cardiomyocytes and reduced LV cardiomyocytes per biventricular weight in the fetuses with occluded FO also suggested LV hypoplasia with reduced cardiomyocyte proliferation. Interestingly, these animals showed reduced RV cardiomyocyte density when corrected for the slightly increased right ventricular weight, suggestive of either RV cardiomyocyte hypertrophy and/or increased fibrosis. However, we did not identify any difference in cardiomyocyte cross-sectional area or percentage of cardiac fibrosis in comparison to the controls. Notably, the cardiomyocyte area was a 2D measurement and may not represent the cell volume, and that acquired cardiac fibrosis is a rare phenomenon in fetal as well as neonatal heart19.Our study showed that FO occlusion in fetal sheep at 0.70–0.75 gestation led to reduced LV and increased RV strain on echocardiography, indicating altered ventricular mechanics in the context of acutely modified haemodynamics of both ventricles. Within 3–4 weeks the FO occlusion produced a phenotype of LV hypoplasia with smaller LV chamber volume, small but morphologically normal mitral and aortic valves, lower LV-to-RV ratio in ventricular weight and wall volume. The significant lower LV-to-RV ratios in weight and wall volume after FO occlusion suggest that the weight and wall volume were mildly reduced for the LV, but mildly increased for the RV, though the individual RV and LV measurements did not reach statistical significance when compared with the controls. The findings imply a combination of LV hypoplasia and RV hypertrophy. This phenotype bears a striking resemblance to a report in human babies born with closed FO, hypoplasia of LV, reduced mitral and aortic valve area as well as RV hypertrophy and dilatation22. Nowlen et al. proposes that early (primary) closure of the FO, due to abnormal development of the atrial septum, results in LV hypoplasia9 and that late FO closure is more of a secondary event to aortic stenosis and LV dilation. Considering the “chicken and egg” phenomenon, we agree that a secondary closure of the FO has to be differentiated from primary FO closure or obstruction of blood flow through the FO. However, our work shows that even late primary cessation of FO flow is disruptive to LV growth.One might speculate that an isolated occlusion of the FO without additional restrictive lesions would affect mostly the development of left heart structures as a result of poor LA and LV filling, whereas additional restriction on either the RV outflow or the ductal level might favor a phenotype of RV dilation, tricuspid regurgitation and hydrops fetaliset al. demonstrated that FO closure occurring late in pregnancy did not affect development of the left heart structures but rather resulted in changes of the right heart and pulmonary vasculature23. A case report of an infant with late FO closure due to a aneurysmatic changes of the intra-atrial septum and mild LV hypoplasia  seems to further support this notion24. Schall et al. speculated that his observed phenotype8, which is mimicked by our experiments here, represents the “missing link” between the majority of reported cases with early FO closure and significant deformities of the left heart, and the few cases with late antenatal FO closure as reported by Naeye et al.Notably, in a study on prenatal narrowing or closure of the FO in 12 deceased human infants of whom 10 were live-born, Naeye 26. In our study, there is supportive evidence for RV hypertrophy rather than hyperplasia, suggesting that the switch from the hyperplastic potential already occurred at 0.7 gestation. This is supported by the observation that second trimester human fetal intervention for aortic stenosis has not been shown to fully prevent LV hypoplasia27. Conceivably, the time-window for preferential hyperplastic remodeling in the human may be prior to the earliest currently feasible fetal intervention.During human fetal development, cellular proliferation (hyperplasia) is the main mechanism of cardiac growth, while the mature heart grows through hypertrophy but has very limited capacity for hyperplasia. Accordingly, there is a replacement of preferential hyperplasia by preferential hypertrophy at some point during development28.The group of fetuses with LV hypoplasia who are most likely to benefit from fetal interventions and undergo hyperplastic remodeling are those with a “borderline” (i.e. moderately hypoplastic) LV. Our study in fetal sheep has produced the phenotype of LV hypoplasia which matches the findings in the moderate clinical spectrum of human HLHC, and represents a significant proportion of patients. This large mammalian model of LV hypoplasia induced without prior direct surgery or scarring on the fetal heart, comparable to human in size and circulatory pattern, has the potential to be used in investigation of disease pathophysiology and devising treatment strategies, eg. maternal hyperoxygenation27, and our technique of in-utero cardiac catheterisation allows us to manipulate the FO and study the effects of such an intervention during fetal development. In this study, fetuses with occluded FO were compared to aged-matched control fetuses which did not undergo the experimentation. In our published experimental data in the fetal sheep on the procedures of percutaneous transhepatic access both with and without positioning of a stent at the FO18, we did not observe any compromised haemodynamics on the post-procedural echocardiography, or any altered LV development on examination of the hearts at term birth. An additional sham group of fetuses was therefore not performed in the interest of limiting animal sacrifice.Our study has limitations such as the small number of animals as well as a relatively large range of gestational age at the time of the intervention. Anchoring the occluder within the stent and positioning the occluder discs to obliterate FO blood flow was difficult. However, animal models to study the effect of FO occlusion or obstruction in the fetus are rareWe have established a large mammalian model of LV hypoplasia mimicking moderate HLHC, using percutaneous fetal cardiac catheterisation and novel interventional techniques to occlude the fetal foramen ovale, without any direct surgery or scarring on the fetal heart. The model can potentially be applied in future studies to understand the pathophysiology of and develop new therapies for LV hypoplasia.All procedures in this study were performed using sterile surgical techniques and in accordance with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, established by the National Health and Medical Research Council of Australia. The Monash University Animal Ethics Committee approved this study.2. The ewe received maintenance intravenous fluid (130 ml/h) of 0.18% saline + 4% glucose for the duration of anesthesia. Antibiotics (ampicillin 500 mg and gentamicin 80 mg) were administered during the procedure and repeated daily for 3 days.Fetal lambs at 104–117 days gestation  were used. All ewes were brought into the animal house 1 week before the procedure for acclimatisation. The ewes were initially anaesthetised with an intravenous injection of sodium thiopentone , and then placed supine, intubated and ventilated. Anaesthesia was subsequently maintained using 2% Isoflurane in OUltrasound  was used to determine the fetal number and position, and optimal access point. When required, suboptimal fetal position was managed by external manipulation of the fetus to improve access. No fetus was excluded or suspended from the procedure due to a suboptimal position. Ultrasound was used to measure the distance from the skin to the right hepatic vein (RHV) or left hepatic vein (LHV), the diameters of the RHV, LHV, inferior vena cava (IVC), FO, tricuspid valve, mitral valve, pulmonary valve and aortic valve, width of the RV and LV in end-systole, and the length of IVC between the insertion of the RHV or LHV and the right atrium (RA).16. Depending on the position of the fetus and the ease of access, the RHV or LHV was selected for puncture and sheath insertion. Hepatic access and sheath insertion was achieved using either a 13.3-cm, 14 G intravenous catheter  or a 4.5 F (ID) sheath  inserted into the fetal RHV or LHV. The catheter sheath was positioned close to the junction of the hepatic vein and IVC , whereby both discs of the occluder with an expanded width of almost 10 mm occluded the stent within the atrial septum . A video of the complete procedure is available as Supplementary Material.Access to the fetal venous system was obtained using a percutaneous puncture through the maternal abdominal and uterine wall and into the fetal abdomen, under continuous ultrasound guidance as we described previouslyIVC Fig. . Under uIVC Fig.  as previtum Fig. . The Amp31. We assessed global longitudinal peak systolic strain (LPSS) for both ventricles in the fetal lamb as previously described31. The frame rate for analysis was 60 fps. According to the consensus paper for the assessment of LPSS, endocardial strain was used to calibrate the LPSS32. In the offline analysis, one fetal heart cycle was selected by anatomical M-Mode. In a 3-point-analysis, an experienced operator set the endocardial borders of every cardiac cavity . This technique of speckle-tracking echocardiography offline analysis was recently described for the assessment of the atrial and biventricular strain of human fetal hearts33. Due to limitations of the echocardiographic console used, systolic strain rate and diastolic parameters were not analysed, as what were performed in human assessments34.In one fetus, data on myocardial tissue deformation (strain) were collected immediately before and after FO closure using 2D speckle tracking for both ventricles , equipped with a 1–5 MHz curved array probe (PVT 375 BT) as we previously describedAfter recovery from the general anaesthesia, the fetuses were monitored with daily ultrasound for the first 3 days and then weekly ultrasound to check fetal well-being.The ewes and fetuses were euthanised at 3–5 weeks after the FO occlusion, by pentobarbital sodium overdose. For twin pregnancies, only 1 fetus had the FO occlusion performed, and the twin was used as control for comparison. For singleton pregnancies, gestational age-matched fetuses were euthanised as controls for comparisons in cardiac morphology and histopathology.35. The saline infusion was followed by freshly prepared 4% formaldehyde. The fixed hearts were stored in 10% buffered formalin prior to tissue sampling.At post-mortem, the fetuses were weighed. The hearts were excised with the ascending aorta and weighed. Freshly excised hearts were retrogradely perfusion-fixed via the aorta. Prior to flushing the hearts with saline, we infused heparin to prevent clotting, papaverine to dilate the cardiac vasculature, and KCl to arrest the hearts in diastoleThe fixed hearts were weighed after removal of connective tissue and fat. The atria were carefully dissected and the FO was examined for positions of the stent and occluder. The atria were then removed.Each heart was assigned an arbitrary number to permit blinding to the experimental group in subsequent analyses. The volume of each ventricular chamber was measured by amount of fluid required to fill it up to the level of the atrioventricular valves. The fixed hearts were then transversely sliced at 5 mm thickness, with the first slide at the level of the aortic valves and the second slide at the level of the atrioventricular valves.35. The wall volumes of the ventricles and septum were determined using the Cavalieri principle36.The annuli of the aortic, mitral and tricuspid valves were measured in the heart slices. Morphometric measurements were made on digital images of transverse sections of the heart (as described above) using image analyser . Images were used to determine septal and ventricle wall thickness and area, and atrioventricular valvular areasDue to the haemodynamic differences between the ventricles before and after birth, the LV and RV were analysed separately. The LV was sampled together with the adjacent septum (S) as the septum is structurally similar to the left ventricular wall.37. The selected samples of RV and LV + S were embedded in either glycolmethacrylate or paraffin.Sampling of heart tissue for stereologic analyses was performed using a smooth fractionator approachth section stained with hematoxylin in a 1000 W microwave oven at 50% power for 2–4 min. This ensured adequate nuclear staining throughout the sections. Cardiomyocyte nuclei number was estimated using an optical disector–fractionator approach; we used a light microscope  coupled with a motorised stage and a z-axis sensor. Every second section was systematically sampled and projected onto a computer screen. An unbiased counting frame (329.6 mm2) was superimposed on the image using C.A.S.T  software . Nuclei were counted when they came into clear focus in the disector area (so long as no part intersected the forbidden lines) within a 10 mm depth in the middle of the section. The total numbers of cardiomyocyte nuclei in the RV and LV + S were estimated by multiplying the number of nuclei counted stereologically by the inverse of all sampling fractions35.Glycolmethacrylate blocks were sectioned at 20 µm and every 1036.Paraffin-embedded blocks of the RV and LV + S were cut at 40 µm. A 5 µm section from each block was cut initially for haematoxyline stain and examination to ensure longitudinal orientation of the cardiomyocytes in the paraffin block, and re-embedding was performed if required. Three paraffin sections from each of RV and LV + S of each animal were stained with 10 mg/L of wheat germ agglutinin-Alexa Fluor 488 conjugate  and 1:5000 YOYO-3 (Molecular Probes Invitrogen). Wheat germ agglutinin-Alexa Fluor 488 conjugate stains the cell membranes and YOYO-3 stains the nucleus. We used a Leica SP5 broadband multi-photon confocal microscope  with a Spectra-Physics MaiTai Ti:Sapphire multi-photon source  to examine cardiomyocyte nuclearity. Volocity Version 5 software  was used to visualise and measure the cardiomyocyte longitudinal cross-sectional area, by tracing the boundaries of cardiomyocytes in which the nuclei could be seen in the center. One hundred mononucleated and 100 binucleated cardiomyocytes were measured in each RV and LV + S for each animal35.Paraffin-embedded blocks of RV and LV + S were sectioned at 5 µm and stained with picrosirius red after pre-treatment with phosphomolybdic acid. Three sections from each of RV and LV + S were used and the percentage of collagen within the interstitium was quantified using image analysis Data is expressed as mean ± SD. All data were compared between the fetuses with FO occlusion and the controls using an independent sample two-tailed t-test for parametric data, or Mann-Whitney U test for non-parametric data. Ultrasonographic measurements in the fetal hearts before the FO occlusion were compared with those taken 30 mins after the FO occlusion using the paired t-test. Statistical significance was defined as a p-value of <0.05.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Video.Supplementary Table and legend for the Supplementary video.


Left ventricular (LV) ejection fraction (LVEF) assessed by two-dimensional echocardiography (2DE) is the most widely used parameter for clinical decision-making, but reproducibility and accuracy problems remain. We evaluated the usefulness of a novel training program based on cardiac magnetic resonance (CMR) imaging to obtain more reliable values of 2DE-derived LVEF and LV volumes.Fifty-four sonographers from five hospitals independently measured LV volumes and LVEF using the same 2DE images from 15 patients who underwent CMR and 2DE. After receiving a lecture from an expert on how to properly trace the LV endocardium, each sonographer voluntary performed the measurements using the same datasets, and was invited to perform the same analysis for additional patients. The effect of the training intervention was evaluated using the coefficient of variation (CV) and coverage probability (CP).p < 0.001) for the LVEF showed significant improvement. However, the degree of improvement differed among institutions, and the CV actually became worse in two hospitals after the intervention. Level of experience and self-practice was associated with the reproducibility after the intervention.Before the intervention, the LV volumes were significantly underestimated and the LVEF was significantly overestimated compared to the CMR results; however, these differences were reduced after the intervention. In particular, the CP (0.52 vs. 0.76, A training program using CMR as a reference improved the accuracy of 2DE-determined LV measurements. Since the degree of improvements differed among hospitals, individualization of training programs and periodical objective evaluation may be required to reduce inter-institutional variability. Echocardiography plays a pivotal role in the diagnosis and management of cardiovascular diseases and other pathologies , 2. In pCardiac magnetic resonance (CMR) imaging is a reference standard to measure both LV volumes and LVEF . If bothAccordingly, we aimed to (1) clarify the inter-observer and inter-institutional variability of 2DE-determined LV volumes and LVEF in a large number of examiners and to assess their accuracy against CMR measurements; (2) evaluate whether intervention via a training program including expert guidance would improve reproducibility and accuracy; and (3) determine sonographer’s characteristics on reproducibility after the intervention.This study was approved by the Institutional Review Board of the University of Occupational and Environmental Health, School of Medicine. All examiners provided informed consent for participation in this study.We selected a consecutive series of 15 patients who underwent clinically indicated CMR examinations and also agreed to undergo 2DE examinations on the same day among the CMR database of our laboratory. We did not exclude any patients who had poor 2DE image quality. We then aimed to select an additional 15 patients with propensity score matching of LV end-diastolic volume (LVEDV) and LVEF on the CMR and 2DE image quality from the same CMR database. However, one of the patients among the first 15 cases did not have an appropriate propensity score match in the database. Thus, we did not use this patient for the comparative analysis before and after the intervention.All 2DE images were acquired by one expert sonographer using a commercially available ultrasound machine and equipment . Echocardiographic image acquisition was systematically performed according to the American Society of Echocardiography guidelines  with breCMR imaging was performed using a 3-T scanner  with a phased-array cardiovascular coil. Retrospective electrocardiography-gated localizing spin-echo sequences were used to identify the long axis of the heart. Steady-state free precision (SSFP) dynamic gradient-echo cine loops were acquired by retrospective electrocardiographic gating and parallel imaging techniques during 10- to 15-s breath-holds with the following general parameters: 8-mm slice thickness of the imaging planes, 40 × 40-cm field of view, 200 × 160-scan matrix, 50° flip angle, 3.8/1.7-ms repetition/echo times, and 20 reconstructed cardiac phases. Eight to 16 short-axis slices from the base of the heart to the apex, and three standard long-axis views were recorded in each patient.CMR LV volumes and LVEF were measured by the same operator  via feature tracking analysis . In three apical long-axis SSFP images, the LV endocardial border was manually traced at the end-diastolic frame. Then, the feature tracking software propagates the endocardial contour and follows its tissue features throughout a cardiac cycle to generate LV volume curves from which LVEDV, LVESV, and LVEF were determined automatically.We invited cardiac sonographers working in five different hospitals to participate in the study. We defined an expert sonographer as one with > 10 years of experience in echocardiography and a novice sonographer one having ≤10 years’ experience [Anonymized 2DE apical 4- and 2-chamber DICOM images for 15 patients were sent to each participating hospital. There were only two DICOM images for each patient, with three consecutive cardiac cycles for each image. Each sonographer selected one cardiac cycle from the three beats and determined both the end-diastolic and end-systolic frames. Subsequently, the sonographer traced the LV endocardial border, which was used for calculation of LVEDV, LVESV, and LVEF. After finishing the measurements, all measurement data were sent to the core laboratory. Differences in LV volumes and LVEF between 2DE and CMR examinations were determined for each participant. We also analyzed the inter-reader and inter-institutional variability.The LVEDV, LVESV, and LVEF values for all 15 patients determined by CMR were given as feedback to each sonographer. An expert (MT) then visited each hospital and gave a 4-h hands-on lecture on how to best trace the LV endocardium border to obtain similar values to the CMR measurements. The lecture was also video recorded and uploaded to a webpage where each participant was able to access. We allocated 3 months for the sonographers to individually or jointly practice LV border tracing using the same datasets of protocol 1. This practice period was completely arbitrary and individually based. When the 3-month practice period was finished, we sent the propensity score matched 2DE apical 4- and 2-chamber DICOM images from the additional 14 patients to the same sonographers who were invited to repeat the analysis. The same statistical analysis was performed in the core laboratory on the data as conducted for protocol 1 to assess the impact of the intervention. The study protocol is schematically summarized in Fig. p value < 0.05 was defined as statistically significant. All statistical analyses were performed using commercial software  and R version 3.1.0.  with the party package.Continuous data are expressed as the mean ± standard deviation (SD). Categorical data are presented as a number or percentage. The t-test or Wilcoxon rank sum test was used to evaluate the differences in continuous variables between two groups. Friedman’s analysis with post-hoc comparison was used to determine differences in the 2DE assessment between each hospital and from the CMR results. The coefficient of variation (CV) was used to evaluate reproducibility , definedn = 1) and moving to a different hospital (n = 2). Thus, we used the results from 51 sonographers.A total of 54 sonographers agreed to participate the study. Table LV volume and LVEF measurements of each hospital and the CMR examination are summarized in Table Figure Table Figure CP analysis revealed that the effect of the intervention was different among the hospitals. Although the tracing habit of the sonographers working at sites C, D, and E obviously changed following the intervention, the effect was much smaller at sites A and B. Figure p = 0.801). The prevalence of the expert sonographer was not different (p = 0.409) (Table n = 414) and followed by Site E (n = 650).Years of experience for echocardiography was not different among five hospitals p = 0.80. The preAll but four sonographers attended the hands-on lecture or watched the web video lecture. However, less than half of sonographers (47%) actually performed the self-practice for LV endocardial border tracing before the protocol 2.Table The main strengths of our study include the relatively large number of participants, new insights concerning CP usage, and provision of a novel training program incorporating CMR as a reference.Inter-observer variability is a major limitation of echocardiography, especially for assessing LV volumes and LVEF , 14, 15.When the LV endocardial border is traced using the biplane Simpson method, the eyeball EF  must also be taken into consideration . LVEF deWe also analyzed the relationship between sonographers’ characteristics and reproducibility. Expert sonographers (> 10 years’ experience) showed better CVs of LVEDV and LVESV after the intervention than novice sonographers. CVs of LVEDV and LVESV in sonographers who perform more than 1000 examinations per year markedly improved after the training. Importantly, sonographers who performed the self-practice showed significantly lower CV values before and after the intervention compared with the sonographers who did not perform the self-training. Our results suggest that not only clinical experience but also voluntary self-practice are important to improve tracing habitus with keeping the range of CV values among the groups. Medvedofsky et al.  has alreThis study further demonstrates the potential utility of CP as a quality control parameter. CP has been used as an indicator of inter-observer variability of echocardiographic measurements –13. We uThis study has several limitations that should be acknowledged. First, we had no data concerning the longitudinal effect of the training program. We do not know how long the effect of training remains, and thus, it is difficult to determine when retraining is required to maintain the participants’ measurement ability at a constant level. The longitudinal effect may be influenced by the experience of each sonographer; thus, further study is required to address this point. Second, our intervention only focused on manual tracing of the LV endocardial border, which is only one aspect of quality control. Patient selection and image quality could also affect the outcome, although we used propensity score matching for the selection of the patients. Third, we used one ultrasound vendor’s machine to acquire all of the 2DE datasets. Therefore, our results cannot be generalized to other ultrasound vendors . Fourth,A training program for 2DE determined LV volumes and LVEF incorporating CMR as a reference improved the accuracy of measuring LV volumes and LVEF in the majority of participants. However, the degree of the learning effect differed among the hospitals, and reproducibility even became worse in two of five hospitals after the intervention. To improve accuracy and reproducibility, individualization of the training program and a periodical objective evaluation in each subject for each hospital with a standardization of the practice period is required.Additional file 1:Table S1. Difference of echocardiographic measurements between hospitals


Marfan syndrome is a rare cause of heart failure due to primary or secondary cardiomyopathy. Recently, sacubitril/valsartan—an angiotensin receptor blocker-neprilysin inhibitor—has been added in clinical practice as a standard therapy for heart failure. To our knowledge, there are no data on sacubitril/valsartan's effects on cardiovascular outcomes in patients with Marfan syndrome.A 24-year-old man was admitted to our Internal Medicine Department due to dyspnea, ascites, and leg swelling. Arterial blood gas analysis revealed severe hypoxemia with respiratory and metabolic alkalosis. Hilar congestion was highlighted on chest x-ray.Recurrent acute decompensated heart failure with reduced ejection fraction despite optimal medical therapy in Marfan-related cardiomyopathy.Sacubitril/valsartan was added to optimal medical therapy after hemodynamic stabilization allowing progressive clinical, laboratoristic, and echocardiographic improvement. Patient maintained a free survival from heart failure and a good quality of life until 9-month follow-up.Sacubitril/valsartan should be effective on pathophysiologic mechanisms and cardiovascular outcomes of Marfan syndrome–related cardiovascular complications. Marfan syndrome (MFS) can be listed as a rare cause of HF.,2 MFS diagnosis is made by the use of 2010 revised Ghent-2 nosology criteria based on the presence of family history along with other specific diagnostic criteria.,3 Single- and multi-organ involvement  and absence of a curative therapy are responsible for the chronic, severe, and life-threatening course of the disease. Nevertheless, amelioration in medical and surgical management produced an increase in life expectancy from 32 to >50 years.MFS is a connective-tissue autosomal dominant inherited disorder characterized by 400 individual mutations in the gene of fibrillin-1 with a prevalence of 1.5 to 17.2 per 100,000 person in the general population. Furthermore, clinically relevant cardiovascular manifestations consisting in root and descending-aorta dissection and rupture due to progressive aortic dilatation represent the main cause of mortality.Patients with MFS present primary cardiomyopathy for heart muscle fibrillin-1 deficiency in 3.08% of cases  and/or cardiac involvement secondary to ischemic complication of cardiovascular surgery in 8.07% of cases.–9 Indeed, sacubitril/valsartan influences the 2 HF pathophysiological mechanisms. While valsartan inhibits renin-angiotensin-aldosterone system (RAAS) through angiotensin II type-1 (AT1) receptor blockage, sacubitril inhibits neprilysin and increase natriuretic peptides-atrial natriuretic peptide, brain natriuretic peptide (BNP), C-type natriuretic peptide, bradykinin, and adrenomedullin values.Recently, the angiotensin receptor blocker-neprilysin inhibitor sacubitril/valsartan has been introduced in clinical practice as treatment for HF showing its superiority to ACE inhibition alone in reducing the risks of death and hospitalization.We report a case of a III–IV New York Heart Association (NYHA) class, stage C reduced ejection fraction HF in young patient with MFS occurred despite optimal medical therapy and successfully treated with sacubitril/valsartan once reached persistent hemodynamic stabilization.2A 24-year-old man with MFS-related cardiovascular complications was admitted to our Internal Medicine Department because of 3 consecutive episodes of acute decompensated HF with reduced ejection fraction (ADHFrEF) from 2014 through 2017. His remaining medical history includes arterial hypertension, dyslipidemia, hyperthyroidism, anaphylactic shock due to flecainide, and previous tabagic habit.MFS diagnosis was made in 2010 when ascending aorta and aortic valve substitution with mechanical prosthesis were performed due to type A acute aortic dissection in presence of family history—mother, father, and 3 sons with MFS. During intraoperative period, inferior acute coronary syndrome occurred and was treated with coronary artery bypass graft surgery.On May 2014, mechanical descending aorta and aortic arch prosthesis implantation were performed due to chronic dissecting aneurysm. Peri-aortic hematoma and medullary ischemia complicated surgery and caused lower limb paraplegia, dolorific and thermal hypoesthesia, and rectal incontinence. After 2 months, during his first admission for ADHFrEF, laboratory tests were notable for N-terminal pro-BNP (NT-proBNP) of 12,000 pg/mL and transthoracic echocardiogram showed normal prosthesis function, dilatation of all cardiac chambers (mainly the left ones) with moderate mitral and tricuspid insufficiency, left ventricular hypertrophy, inferior wall akinesis, septal dyskinesis, and hypokinesis of the remaining walls with severe left ventricular ejection fraction (LVEF) reduction −20%. He was treated with intravenous diuretics and internal cardiac defibrillator  without cardiac resynchronization therapy (electrocardiogram criteria not met) and discharged on maximal medical therapy .New episode of ADHFrEF occurred on august 2017. Notable admission laboratoristic values included NT-proBNP of 2719 pg/mL and midregional-proadrenomedullin (MR-proADM) of 1.61 nmol/L, whereas transthoracic and transesophageal echocardiogram showed an LVEF of 35%, normal prosthesis function, severe tricuspid regurgitation, and rupture of the anterior leaflet of the chordae tendineae with severe mitral regurgitation. Patients were treated yet with intravenous diuretics and digoxin and discharged on optimal medical therapy .Lastly, he was readmitted to our Internal Medicine Department on November 2017 due to third episode of ADHFrEF and infected mediastinal fluid collection secondary to 1 month before severe valvular insufficiency surgical correction with mitral mechanical prosthesis implantation (31-mm ST Jude) and De Vega tricuspid annuloplasty.2 saturation 87% on room air with orthopneic obligatory position, and Marfanoid habitus . Cardiopulmonary evaluation revealed metallic second heart sound, stony dull percussion with reduced tactile vocal fremitus and crackles at the basis of the lungs, widely diminished vesicular breath sounds, presence of abundant ascites, and leg swelling.Physical examination was notable for blood pressure 120/80 mm Hg, pulse 82 bpm, respiratory rate 26 apm, ONotable laboratoristic values included NT-proBNP of 10,132 pg/mL and MR-proADM of 2.36 nmoL/mL.2 46 mm Hg, pCO2 24.8 mm Hg, HCO3− 21.9 mmol/L, alveolar-arterial gradient 31 mm Hg). Although electrocardiogram highlighted atrial fibrillation with ventricular rate of 80 bpm, left ventricular hypertrophy, and inferolateral subepicardial ischemia, transthoracic echocardiogram showed the same features as the previous except for LVEF of 30%, presence of mitral mechanical prosthesis, with paravalvular leak and mean gradient of 9 mm Hg, and mild tricuspid insufficiency , echocardiographic-LVEF increase (42% vs 30%), left ventricle end-systolic diameter, left ventricle end-diastolic diameter, left ventricular mass, and left ventricular mass index reduction, and quality of life improvement without new episodes of ADHFrEF until 9-month follow-up  compared with enalapril.,9Sacubitril/valsartan effect are confirmed by improvement of echocardiographic features, LVEF increase of 5%, left ventricle end-systolic diameter, left ventricle end-diastolic diameter, left ventricular mass index reduction-free survival from HF (about 1–2 years), and quality of life.FBN1 mutation leads to fibrillin-1 deficiency causing activation of transforming growth factor-β (TGF-β) signaling pathways with improvement of collagen synthesis and elastic fiber disruption in cardiac and vessel wall connective tissue. This remodeling increases aortic stiffness and decreases vasoreactivity resulting in aortic dilatation, impaired diastolic ventricular relaxation, and hypertrophic remodeling.In patients with MFS,  and that neprilysin inhibition by sacubitril causes an increase in BNP values , echocardiographic LVEF increase (42% vs 30%), left ventricle end-systolic diameter, left ventricle end-diastolic diameter, left ventricular mass and left ventricular mass index reduction, and quality of life improvement without new episodes of ADHFrEF until 9-month follow-up Fig. .To our knowledge, this is the first reported case of sacubitril/valsartan administration in patients with MFS-related cardiomyopathy. This case report highlights the beneficial effect of sacubitril/valsartan on pathophysiologic mechanisms of MFS-related cardiovascular complications and its possible administration in III–IV NYHA class, stage C reduced ejection fraction HF, once the hemodynamic stabilization was reached.Authors thank Valeriani Stefano for English language revision.Conceptualization: Silvia Spoto, Sebastiano Costantino, Massimo Ciccozzi, Silvia Angeletti.Data curation: Silvia Spoto, Emanuele Valeriani, Luciana Locorriere, Giuseppina Beretta Anguissola, Angelo Lauria Pantano, Maria Caterina Bono, Sebastiano Costantino, Massimo Ciccozzi, Silvia Angeletti.Formal analysis: Silvia Spoto, Luciana Locorriere, Giuseppina Beretta Anguissola, Angelo Lauria Pantano, Francesca Terracciani, Maria Caterina Bono, Sebastiano Costantino, Silvia Angeletti.Investigation: Luciana Locorriere, Giuseppina Beretta Anguissola, Angelo Lauria Pantano, Francesca Terracciani, Sebastiano Costantino.Methodology: Silvia Spoto, Emanuele Valeriani, Sebastiano Costantino, Silvia Angeletti.Supervision: Sebastiano Costantino, Silvia Angeletti.Writing – original draft: Silvia Spoto, Massimo Ciccozzi.Writing – review and editing: Emanuele Valeriani, Sebastiano Costantino, Massimo Ciccozzi, Silvia Angeletti.