Clinically serious congenital heart valve defects arise from improper growth and remodeling of endocardial cushions into leaflets. Genetic mutations have been extensively studied but explain less than 20% of cases. Mechanical forces generated by beating hearts drive valve development, but how these forces collectively determine valve growth and remodeling remains incompletely understood. Here, we decouple the influence of those forces on valve size and shape, and study the role of YAP pathway in determining the size and shape. The low oscillatory shear stress promotes YAP nuclear translocation in valvular endothelial cells (VEC), while the high unidirectional shear stress restricts YAP in cytoplasm. The hydrostatic compressive stress activated YAP in valvular interstitial cells (VIC), whereas the tensile stress deactivated YAP. YAP activation by small molecules promoted VIC proliferation and increased valve size. Whereas YAP inhibition enhanced the expression of cell-cell adhesions in VEC and affected valve shape. Finally, left atrial ligation was performed in chick embryonic hearts to manipulate the shear and hydrostatic stress in vivo. The restricted flow in the left ventricle induced a globular and hypoplastic left atrioventricular (AV) valves with an inhibited YAP expression. By contrast, the right AV valves with sustained YAP expression grew and elongated normally. This study establishes a simple yet elegant mechanobiological system by which transduction of local stresses regulates valve growth and remodeling. This system guides leaflets to grow into proper sizes and shapes with the ventricular development, without the need of a genetically prescribed timing mechanism. Congenital heart disease is the most common birth defects and affects about 0.5–2.0% of the general population . CongeniThe flowing blood generates shear and hydrostatic stress on valves. The shear stress is in direct contact with VECs, while the hydrostatic stress causes compression and tension in valves and can be transmitted to VICs. Multiple mechanosensitive signaling pathways have been identified in adult tissues,  but theiHere, we explored the mechanism by which the shear and hydrostatic stress regulate valve growth and remodeling. We used in-vitro and ex-vivo models to decouple the effects of shear and hydrostatic stress on the size and shape of valves. We also studied the role of YAP mediated mechanotransduction in those effects by gain- and loss-of-function tests. To verify our findings in vivo, we performed left atrial ligation (LAL) in chick embryonic hearts. The four-chambered chick hearts develop in a manner that mirrors human heart development. The LAL manipulates mechanical forces in vivo and has been shown to replicate some important features of congenital heart defects . By combWe collected embryonic hearts at different developmental stages from wild type mice and examined the YAP activation in heart valves. We found that YAP was expressed in both mesenchyme and endothelium of outflow tract (OFT) and atrioventricular (AV) cushions at E11.5 . YAP actWe also examined the YAP activity in chick embryonic hearts at Hamburger–Hamilton stage (HH) 25, HH30 and HH36 . It follWe further examined YAP transcriptional activity to identify the upstream of YAP activation. YAP target genes THBS1, ANKRD1 and PTX3 were elevated by almost 10-fold at E14.5 and HH31 when compared to E11.5 or HH25 cushions, respectively . The expIn addition to the co-effector of the Hippo pathway, YAP is also a key mediator in mechanotransduction. Indeed, the spatiotemporal activation of YAP correlated with the changes in the mechanical environment. During valve remodeling, unidirectional shear stress (USS) developed on the inflow surface of valves, where YAP was rarely expressed in the nuclei of VECs . On the 2) promoted YAP nuclear translocation in VEC (2) restrained YAP in cytoplasm.To study the effect of shear stress on the YAP activity in VECs, we applied USS and OSS directly onto a monolayer of freshly isolated VECs. The VEC was obtained from AV cushions of chick embryonic hearts at HH25. The cushions were placed on collagen gels with endocardium adherent to the collagen and incubated to enable the VECs to migrate onto the gel. We then removed the cushions and immediately applied the shear flow to the monolayer for 24 hr. The low stress OSS  condition refers to the osmotically balanced media. Notably, in vivo hydrostatic pressure is generated by flowing blood, while in vivo osmotic pressure is generated by cardiac contractility and plays a critical role in the mechanotransduction during valve development . DespiteTo study the function of YAP in valve growth and remodeling, we added a pharmacological inhibitor of YAP, verteporfin (VP), into the CS (pro-growth) and U conditions. The VP inhibits the interaction between YAP and TEAD, which in turn, blocks transcriptional activation of targets downstream of YAP . We cultFor YAP gain-of-function study, a small molecule PY-60 was added into TS (pro-compaction) and U conditions. The PY-60 treatment promoted the association of YAP and TEAD . Cell viTo assess the role of YAP in valve stiffness, we employed micropipette aspiration to measure the strain energy density of the cushion explants cultured with VP or PY-60 treatment. Micropipette aspiration applies a local vacuum stress and monitors resultant tissue displacement within the tip . We haveTo manipulate mechanical forces in vivo, we performed LAL at early stages (HH24) when AV cushions were growing. We collected the hearts during cushion remodeling (HH31) when the valves begin to take shape. The LAL restricted the blood flow in the left ventricle, resulting in a reduced hemodynamic stress and shear stress on left AV valves but augmented forces on the right AV valves. As a result, the LAL valves and hearts  had smalCardiac valves form in response to mechanical forces generated by the flowing blood. These forces include shear and hydrostatic stress. Our results reveal that they can regulate YAP activity in valvular cells, and the mechanically regulated YAP activity can affect the size, shape and stiffness of valves. First, the shear stress regulates YAP activity in the VEC: OSS (oscillatory shear stress) promotes YAP nuclear translocation while USS  restricts YAP in cytoplasm. The hydrostatic stress regulates YAP activation in the VIC: CS (compressive stress) activated YAP while TS (tensile stress) deactivated YAP. Secondly, these mechanoresponsive YAP activities have morphological functions. YAP activation in VICs promotes their proliferation and increases valve size, YAP deactivation in VICs leads to valve compaction. In terms of shape, cushion explants tend to form a sphere in a way similar to the clustering of cells to minimize the surface tension . By contIn short, shear stress can control valve shape: USS promotes an elongated shape while OSS promotes a globular shape, and hydrostatic stress modulates valve size: CS increases the size while TS leads to compaction. In fact, the simple interactions between mesenchymal and endothelial provide basic elements for tissue morphogenesis . Here weOn the other hand, disrupted flow with abnormally developed forces may cause errors in valve growth and remodeling. For example, a combination of delayed OSS and insufficient CS would result in a globular shape and small size, which are the features of underdeveloped valves. Whereas the delayed OSS and overloaded CS could cause a hypertrophic phenotype with globular shape and abnormally big size. In addition, early occurrence of high USS and TS would promote a compacted and elongated shape but with an insufficient length. Such malformations have been verified by in vivo mechanical manipulation. LAL diverted blood flow from the constricted left ventricle toward the untreated right ventricle, decreasing the hemodynamic stress on the left ventricle wall and cushions. This decreased CS in left AV cushions led to insufficient growth. Furthermore, according to the Law of Laplace, the decreased stress also results in a diminished ventricle wall tension that delays the transition from CS to TS, thus hindering cushion compaction. The LAL also altered shear stress environments with a decrease of 40% and over 50% in mean WSS applied on superior cushions and inferior cushions . The levIn general, YAP works like a mechanobiological switch, converting mechanical signaling into the decision between growth and maturation. When YAP is activated the growth programs are turned on and the maturation programs are suppressed. When YAP is inhibited the growth programs are paused and the maturation programs are released. Unlike the pro-growth function, the pro-maturation side of YAP has been less studied. During valve remodeling, cushions elongate into mature leaflets with increased stiffness. Although both YAP activation and inhibition increased valve stiffness, the stiffness of YAP activated valves was only about half of that of YAP inhibited valves, and only YAP inhibited valves exhibited an in vivo-like stiffness increase. We have previously shown a linear relationship between valve stiffness and valve maturation . This suThis mechanism explains a major difference between contributions of cell lineage to the EMT and post-EMT growth and remodeling. During the EMT, cell lineage is a determinant factor. Cells derived from the second heart field make the major contribution to the OFT walls, and neural crest cells make a major contribution to the OFT cushions but a minor contribution to AV and intercalated cushions . HoweverIn conclusion, our study shows that the spatiotemporally coordinated mechanotransduction of shear and hydrostatic stress is required for proper valve growth and remodeling. The shear and hydrostatic stress can regulate VEC tensions and VIC proliferation by YAP pathways and thus determine the valve size and shape. Malfunctional YAP signaling could cause valve malformation, but improper local mechanical signaling imposes a more important malformation risk, even if the YAP signaling is fully functional and no genes are mutated. The presented mechanobiological system could also open an opportunity to control valve growth and change valve shape by influencing forces or targeting YAP pathway at a specific stage.2, respectively. The components were clamped together with binder clips. The female Luers of the channel slide were connected to the silicone tubing with a 3.2 mm inner diameter .The bioreactor included a histology microscope slide, biocompatible double-sided tape (W.W. Grainger), 5-mm-thick silicone sheet (McMaster-Carr) and a sticky-Slide I Luer (Ibidi). Wells with 4 mm diameters were created in the silicone sheet with a 4 mm disposable biopsy punch (Miltex). The magnitude of shear stress is determined by the height of the sticky-Slide I Luer and have been calculated and validated by Ibidi. The sticky-slide I Luer creates a 5 mm x 50 mm channel with various heights. 0.8 mm and 0.4 mm high sticky-slide I Luer were used to create shear stresses of 2 and 20 dyne/cm2 within an incubator.For unidirectional shear stress (USS) experiments, flow was generated using Masterflex L/S Brushless variable-speed digital drive; Masterflex L/S 8-channel, 4-roller cartridge pump head; and Masterflex L/S large cartridges (Cole-Parmer). The peristaltic pump was connected to a pulse dampener (Cole-Parmer) to maintain non-pulsatile unidirectional flow over the samples. The pulse dampener was then connected to the bioreactors. The media was stored in a polycarbonate bottle with a filling/venting cap  with 80 mL of M199 culture medium, with 1% insulin-selenium-transferrin, Pen/Strep, and 3% chick serum. The cells were exposed to a flow rate of 21.1 mL/min for 24 hr at 37 °C and 5% CO2 within an incubator.For oscillatory shear stress (OSS) experiments, flow was generated using a NE-1000 syringe pump . The bioreactors were connected to a 20 mL syringe (BD Biosciences) that was controlled by the syringe pump. Cells were exposed to shear stress in the forward direction for one-half of a one-second cycle and in the reverse direction for the other half of the cycle at a flow rate of 21.1 mL/min for 24 hr at 37 °C and 5% CO2. The dissected outflow tracts were then placed on top of the collagen gel, and excess media was pipetted off to allow for the valve primordia to come in contact with the collagen gel. After 6 hr of incubation at 37 °C and 5% CO2. the valve endocardial cells are repolarized, delaminated, and attached to the surface of the collagen constructs. These endocardial cells were then exposed to USS or OSS at 2 or 20 dyne/cm2 for 24 hr.Collagen gels at a concentration of 2 mg/mL collagen were made using 3 x Dulbecco’s Modified Eagle’s Medium (Life Technologies), 10% chick serum (Life Technologies), sterile 18 MΩ water, 0.1 M NaOH, and rat tail collagen I (BD Biosciences). An aliquot of the collagen gel solution was pipetted into the wells in the silicone sheet and allowed to solidify for 1 hr at 37 °C and 5% COAtrioventricular cushions (HH25) were dissected from the myocardium of embryonic chick hearts. The explants were cultured in M199 culture medium, 3% chick serum, and 1% insulin-transferrin-selenium. YAP inhibitor verteporfin (10 µg/ml) were purchased from Sigma Aldrich. The explants were placed in 20 µl hanging drops, settled at the apex of the droplets and cultured upside down for 24 hr.Fertilized White Leghorn chicken eggs were incubated in a 38 °C forced-draft incubator to Hamburger- Hamilton (HH) stage 21 . The embryo was cultured in an ex-vivo platform previously described [RG 49]. Briefly, an overhand knot of 10–0 nylon suture loop was placed across a portion of either the right atrium and tightened, partially constricting the left AV orifice. This diverted flow from the constricted inlet toward the untreated inlet, decreasing hemodynamic load on the one side and increasing it on the other . At D7 and D10 , hearts and/or AVs were fixed, and paraffin sectioned for immunohistochemistry.Chick and mouse hearts and mouse embryos were fixed in 4% paraformaldehyde for overnight, washed with TBS, embedded with paraffin, and sectioned. Sections were deparaffinized and rehydrated. Antigen retrieval was completed using citrate buffer at pH 6.0. Samples were then washed with TBS, permeabilized with 0.3% Triton-X 100 in TBS, and blocked with 3% BSA, 20 mM MgCl, 0.3% Tween 20, 0.3 M Glycine, and 5% Donkey serum in 1xTBS. Samples were then incubated with the primary antibodies at 1:100 dilution with the blocking solution overnight. Primary antibodies used include YAP , Lef1 , pHH3 , VE-cad (abcam ab33168), MF-20 (mouse DSHB MF 20), IB4 (Vector B-1205-.5), Phalloidin (Invitrogen A12379). Samples were washed with 0.3% Triton-X 100 in TBS and incubated with secondary antibodies at 1:100 dilution with 5% BSA in TBS at room temperature for 1 hr. Secondary antibodies used include species-specific Alexa Fluor 568 or 647. Samples were then washed again for 3x10 min with TBS and stained with DAPI. Images were taken with Zeiss LSM 710 confocal microscope.p)/rp was defined by normalizing the aspirated length to the pipette radius. The experiment stretch ratio is a measure of geometry change during aspiration, which is related, but not identical to the local stretch of the tissue. The ΔP versus λ curves were presented.Mechanical properties of cushion explants were measured by micropipette aspiration. A glass micropipette (rp ≥35 μm) was placed adjacent to the cushion surface collinear with the AV canal axis. Vacuum stress was incrementally applied via a 200 μL pipette calibrated with a custom manometer. Previous strain history was mitigated by preconditioning with 20 cycles of low pressurization (<1 Pa). The preconditioning step ensured the tissue and pipette tip were in full contact. Incremental stress loads were then applied, at which images were captured for each static load at ×150 magnification using a Zeiss Discovery v20 stereo microscope. The aspirated length was measured using calibrated images in NIH ImageJ. An experimental ‘stretch ratio’, λ= and RNA was reverse transcribed to cDNA using the SuperScript III RT-PCR kit with oligo(dT) primer (Invitrogen). Sufficient quality RNA was determined by an absorbance ratio A260/A280 of 1.8–2.1, while the quantity of RNA was determined by measuring the absorbance at 260 nm (A260). Real-time PCR experiments were conducted using the SYBR Green PCR system  on a Biorad CFX96 cycler, with 40 cycles per sample. Cycling temperatures were as follows: denaturing, 95 C; annealing, 60 C; and extension, 70 C. Expression of mouse genes are normalized to GAPDH, and chick genes are normalized to 18 S. Sequences of primers are included below.Images were analyzed using ImageJ. Results are presented as mean ± SD and compared using either an ANOVA tests with Tukey post hoc paired or two-tailed student t-tests. Differences were considered significant at p≤0.05. For immunofluorescence staining, heart sections from n≥5 embryonic hearts were used for quantification. For cushion explants culture, n≥5 independent cultures per treatment condition and four to six dozen chick embryos pooled for each experiment. For shear flow experiments, n≥5 endocardial patches per shear flow condition. For LAL surgery, n≥3 survival embryos for LAL control and sham control. This study examines Yap signaling in mouse and chicken embryonic valve development with supporting studies of Yap pathway manipulation and mechanotransduction in valve primordial explants. Calculations of endogenous Yap nuclear/cytoplasmic ratios support conclusions regarding Yap activation status during valve development and under different biomechanical conditions. These studies are novel and provide a clear picture of Yap signaling in embryonic heart valve morphogenesis relative to fluid forces. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Julien Vermot (Reviewer #3).Thank you for submitting your article "Shear and hydrostatic stress regulate fetal heart valve remodeling through YAP-mediated mechanotransduction" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Improved analysis of Yap activation status relative to valve areas under different mechanical stresses is needed. Immunostaining for total Yap is inadequate to support the major findings in the study.2) Additional manipulation of Yap activation is needed to support the major conclusion that Yap-medicated mechanotransduction has a role in fetal heart valve remodeling response to shear and hydrostatic stress.3) Additional major conclusions in the study are not supported by rigorous data. Please see the reviewers' comments.Reviewer #1 (Recommendations for the authors):1. Additional data are needed to determine Yap activation status as might be indicated by nuclear/cytoplasmic ratios and Yap phosphorylation. The Yap immunostaining is widespread and does not seem to relate to localized mechanical forces. Other indicators of Hippo signaling status might be informative.2. There are no clear conclusions regarding Yap signaling status, cell proliferation, or valve morphogenesis under different biomechanical conditions. This is described in Figures 3A, B, but localized Yap activation in specific regions or cell types is not clear in these panels. The data are confusing largely due to the widespread Yap expression shown in VIC, VEC, and other cell types throughout the valves.3. Conclusions regarding endothelial folding and altered junctions between endothelial cells are not supported by data.4. It is not clear what tissue rounding or increased "stiffness" of valve explants means in the context of leaflet elongation, ECM remodeling, cell density, or growth.5. In the chicken embryo heart unloading study , Yap is widely expressed in the controls, but regions of Yap activation are not apparent. It is not clear how the ratios of positive Yap were calculated or if there were specific regions of activation in response to specific blood flow dynamics.6. Figure 7 is described as a "pseudo model" but it should probably be just a "model". The dynamics of Yap activation shown in the model were not clearly demonstrated in the figures.Reviewer #2 (Recommendations for the authors):Addressing the weaknesses outlined below would greatly strengthen the significance and impact of the work.Identified weaknesses of the manuscript include:– Figure 1 demonstrates dynamic YAP expression in the developing mouse; however, all experimental studies are performed in chick. Comparative temporal and spatial expression studies in the avian model should be performed– The relevance of YAP1 and LEF1 co-staining (or lack of) in Figure 2 adds little value to the study. At E11.5, yellow cells are identified suggesting that there is an overlap. The authors comment that the data supports "that YAP has different functions at early EndMT than later remodeling stages". However, based on this expression data, this conclusion is difficult to interpret and support.– In Figure 3, data presented in panel D should be quantitated. In addition, what are the axis referring to on the graphs of Figures 3E, F?– In the text referring to Figure 3, the authors discuss examining the "YAP signaling pathway" but this is not being examined here. In addition, the authors claim that USS restrains YAP in the cytoplasm, but this is not detectable.Throughout the study, the authors solely rely on the quantitation of nuclear YAP expression based on IHC. An addition supporting experimental readout is highly recommended, including nuclear/cytoplasmic extraction and western blot to detect YAP in cellular fractions for Figures 3C, and 3D.– For Figures 4E, 4F, 4G, 5E-H, the US control is needed.– In Figure 4B, there is a large decrease in circularity with YAP inhibition, yet proliferation changes appear minimal. What other YAP-dependent mechanisms might contribute to this drastic change in valve morphology? Likewise, Figure 4F indicates 10% pHH3 reactivity, yet this is not represented in Figure 4C. Which cells are proliferating?– The phenotype in Figure 4D is interesting and unusual. The authors conclude that loss of YAP leads to endothelium folding, but again, there is little evidence to support this. If this is true, then additional supporting readouts are needed including another endothelial cell marker. How do the authors explain an increase in VE-cadherin with so little pHH3 reactivity in the VECs?– On Page 4, the authors comment that VP treatment led to "successful YAP inhibition" but this data is not included.– When referring to Figure 5, the authors comment that PY-60 treatment dose-dependently promoted the association of YAP and TEAD. This data is not included.– Figure 5E is confusing and suggests that YAP gain of function has no effect on stress-induced increases in circularity, yet the authors conclude that YAP activation reverses the compaction of valves under TS and U conditions.– The conclusion of findings from Figure 5D is that "VE-cadherin in the YAP activated endothelium was significantly weaker…showing that YAP activation led to a relaxed endothelium." Again, this conclusion needs to be supported by additional data.– Figure 5H is mislabeled in the text.– The authors should comment further on how and why YAP inhibition decreases the strain energy density. How can YAP influence the material properties of avian valves? Reviewer #1 (Recommendations for the authors):1. Additional data are needed to determine Yap activation status as might be indicated by nuclear/cytoplasmic ratios and Yap phosphorylation. The Yap immunostaining is widespread and does not seem to relate to localized mechanical forces. Other indicators of Hippo signaling status might be informative.We thank the reviewer for the constructive suggestion. We have now analyzed the intensity ratios of YAP expressions in nuclei versus cytoplasm as suggested to quantify YAP activation status and revised Figure 1, Figure 2 , and Figure 5 .We have also added qPCR data of YAP target genes THBS1, ANKRD1 and PTX3, as well as YAP upstream in Hippo cascade LATS1 and LATS2, into a new supplementary Figure 1—figure supplementary 2. Expressions of THBS1, ANKRD1 and PTX3 in mouse cushions markedly upregulated from E11.5 to E14.5 but fell sharply from E14.5 to E17.5. By contrast, LATS1 and LATS2 did not change statistically. Fold changes of those genes in chick cushions followed the same trend. These data were also consistent with YAP activation measured from immunofluorescence. The results showed that YAP was independent of the Hippo pathway. It is worth noting that the PCR data mainly reflect gene transcriptions of the VIC, as it was the dominant cell population in valves.We have also revised the first two sections of Results:“YAP expression is spatiotemporally regulatedWe collected embryonic hearts at different developmental stages from wild type mice and examined the YAP activation in heart valves. We found that YAP was expressed in both mesenchyme and endothelium of outflow tract (OFT) and atrioventricular (AV) cushions at E11.5 . YAP activation in VIC increased significantly at E14.5  then dropped at E17.5 . This decrease in YAP activation during later remodeling stages was significant in AV valves but insignificant in SL valves, as the development was not uniform across all valves. In VECs on the outflow side, nuclear YAP expression (triangles) increased significantly during later remodeling stages . Whereas in VECs on the inflow side, cytoplasmic YAP expression (arrows) was stronger throughout all stages.We also examined the YAP activity in chick embryonic hearts at Hamburger–Hamilton stage (HH) 25, HH30 and HH36 . It followed the same spatiotemporal pattern. YAP activation in VICs at HH30 then dropped at HH36. VECs on the outflow side had an upregulated nuclear YAP expression with development while YAP expression in VECs on the inflow side was mainly in cytoplasm.We further examined YAP transcriptional activity to identify the upstream of YAP activation. YAP target genes THBS1, ANKRD1 and PTX3 were elevated by almost 10-fold at E14.5 and HH31 when compared to E11.5 or HH25 cushions, respectively . The expressions of those genes then reduced significantly during later stages of remodeling. In comparison, gene expressions of LATS1/2, the upstream of YAP in the Hippo pathway, had little change during the valve growth and remodeling. This result supports that YAP activation is operated independently of the Hippo pathway.”2. There are no clear conclusions regarding Yap signaling status, cell proliferation, or valve morphogenesis under different biomechanical conditions. This is described in Figures 3A, B, but localized Yap activation in specific regions or cell types is not clear in these panels. The data are confusing largely due to the widespread Yap expression shown in VIC, VEC, and other cell types throughout the valves.We used the dash lines for clarity of surface flow domains, but these may have covered YAP expression in the VEC. We have now improved the annotations in Figure 2A and 2B. In Figure 2A, solid lines are used for the USS domain and dash lines for the OSS domain. Solid arrows highlight nuclear YAP, and dash arrows highlight nuclei without YAP. In Figure 2B, the CS domain is highlighted by a solid circle with force direction and the TS domain by a dash circle with force direction. We have also changed the color scheme in Figure 2 for a better visualization.3. Conclusions regarding endothelial folding and altered junctions between endothelial cells are not supported by data.We appreciate the reviewer for pointing out our lack of clarity. The term “endothelial folding” was poorly chosen. In fact, by “folding”, we meant the shrinkage of surface areas. Due to the compaction of cushion explants, their surface areas became muchd/D)2. Even a small compaction will cause a large reduction in surface areas. As a result, the endothelium at 0-hour was geometrically too large for the cushions at 24-hour. Therefore, the endothelium could be forced to cluster into multiple layers. Indeed, we observed extra thick and densely packed VE-cadherin positive layers in YAP inhibited valves. Another possibility is that YAP activity can affect cell-cell junctions between VECs. For example, some studies reported that YAP can break cell-cell junctions between VECs via SMADs mediated TGF-β signaling during the EMT process. The detailed mechanisms by which YAP affects the cell-cell junctions and endothelium behaviors is important but requires further studies to elaborate.smaller, and the shrinkage is approximately proportional to , into the CS (pro-growth) and U conditions. The VP inhibits the interaction between YAP and TEAD, which in turn, blocks transcriptional activation of targets downstream of YAP. (31) We cultured cushion explants under CS, CS+VP and U+VP. We confirmed that the concentration of VP we used (5mM) does not influence cell viability . Successful YAP inhibition  reduced the cushion size , regardless of the media condition. In contrast to the spherical shape of cushions cultured under CS, cushions cultured in media with VP maintained their trapezoidal shape, which was characterized by circularity . Further investigation demonstrated that loss of YAP significantly reduces the expression of the proliferation maker, pHH3 in the VIC . In addition, the YAP inhibition significantly strengthened the expression of VE-cadherin between VECs . Cushions normally just had a single layer of endothelium, but the YAP inhibited valves showed five or more layers of endothelium."The revised title of fourth section of the Results and Figure 4 is:“Activation of YAP promoted cell proliferation and inhibited valve elongation”The new first paragraph of the Discussion is as follows:“Cardiac valves form in response to mechanical forces generated by the flowing blood. These forces include shear and hydrostatic stress. Our results reveal that they can regulate YAP activity in valvular cells, and the mechanically regulated YAP activity can affect the size, shape and stiffness of valves. First, the shear stress regulates YAP activity in the VEC: OSS (oscillatory shear stress) promotes YAP nuclear translocation while USS  restricts YAP in cytoplasm. The hydrostatic stress regulates YAP activation in the VIC: CS (compressive stress) activated YAP while TS (tensile stress) deactivated YAP. Secondly, these mechanoresponsive YAP activities have morphological functions. YAP activation in VICs promotes their proliferation and increases valve size, YAP deactivation in VICs leads to valve compaction. In terms of shape, cushion explants tend to form a sphere in a way similar to the clustering of cells to minimize the surface tension (32). By contrast, YAP inhibition promotes valve shaping and leaves a densely packed endothelium. The densely packed endothelium could be a result of the large shrinkage in surface area, during which the endothelium at 0-hour could be geometrically too large for the valves at 24-hour. Consequently, the endothelium could be forced to cluster into multiple layers. Another possibility is that YAP activity can affect cell-cell junctions between VECs. For example, studies reported that YAP can break cell-cell junctions via SMADs mediated TGF-β signaling during the EMT process (33).”4. It is not clear what tissue rounding or increased "stiffness" of valve explants means in the context of leaflet elongation, ECM remodeling, cell density, or growth.During valve remodeling, cushions elongate into mature leaflets with increased stiffness. We have previously shown a nearly linear relationship between strain energy and leaflet elongation in-vivo.The new fourth paragraph of Discussion is as follows:“In general, YAP works like a mechanobiological switch, converting mechanical signaling into the decision between growth and maturation. When YAP is activated the growth programs are turned on and the maturation programs are suppressed. When YAP is inhibited the growth programs are paused and the maturation programs are released. Unlike the pro-growth function, the pro-maturation side of YAP has been less studied. During valve remodeling, cushions elongate into mature leaflets with increased stiffness. Although both YAP activation and inhibition increased valve stiffness, the stiffness of YAP activated valves was only about half of that of YAP inhibited valves, and only YAP inhibited valves exhibited an in-vivo-like stiffness increase. We have previously shown a linear relationship between valve stiffness and valve maturation (32). This suggests that YAP inhibition promotes a more mature phenotype.”5. In the chicken embryo heart unloading study , Yap is widely expressed in the controls, but regions of Yap activation are not apparent. It is not clear how the ratios of positive Yap were calculated or if there were specific regions of activation in response to specific blood flow dynamics.The control was at HH31, when the YAP activation surged to the highest level. This was consistent with the qPCR data that the fold change of YAP target gene expressions was highest at E14.5 or HH31. We have now used the Intensity ratios of nuclear to cytoplasmic YAP to clarify this confusion.6. Figure 7 is described as a "pseudo model" but it should probably be just a "model". The dynamics of Yap activation shown in the model were not clearly demonstrated in the figures.We thank the reviewer for this reminder. We have now removed the word “pseudo”. we have also revised Figure 6B-6E  to reflect the dynamics of YAP activity during remodeling.Reviewer #2 (Recommendations for the authors):Addressing the weaknesses outlined below would greatly strengthen the significance and impact of the work.Identified weaknesses of the manuscript include:– Figure 1 demonstrates dynamic YAP expression in the developing mouse; however, all experimental studies are performed in chick. Comparative temporal and spatial expression studies in the avian model should be performedWe apologize if these requested experiments and results weren’t suitably called out in the manuscript. Immunostainings of chick embryonic heart valves at different developmental stages were given in Figure S4, which has now been moved to Figure 1—figure supplementary 1. We have also revised the first section of Results to present the data in Figure 1—figure supplementary 1 right after Figure 1:“YAP expression is spatiotemporally regulatedWe collected embryonic hearts at different developmental stages from wild type mice and examined the YAP activation in heart valves. We found that YAP was expressed in both mesenchyme and endothelium of outflow tract (OFT) and atrioventricular (AV) cushions at E11.5 . YAP activation in VIC increased significantly at E14.5  then dropped at E17.5 . This decrease in YAP activation during later remodeling stages was significant in AV valves but insignificant in SL valves, as the development was not uniform across all valves. In VECs on the outflow side, nuclear YAP expression (triangles) increased significantly during later remodeling stages . Whereas in VECs on the inflow side, cytoplasmic YAP expression (arrows) was stronger throughout all stages.We also examined the YAP activity in chick embryonic hearts at Hamburger–Hamilton stage (HH) 25, HH30 and HH36 . It followed the same spatiotemporal pattern. YAP activation in VICs at HH30 then dropped at HH36. VECs on the outflow side had an upregulated nuclear YAP expression with development while YAP expression in VECs on the inflow side was mainly in cytoplasm.We further examined YAP transcriptional activity to identify the upstream of YAP activation. YAP target genes THBS1, ANKRD1 and PTX3 were elevated by almost 10-fold at E14.5 and HH31 when compared to E11.5 or HH25 cushions, respectively . The expressions of those genes then reduced significantly during later stages of remodeling. In comparison, gene expressions of LATS1/2, the upstream of YAP in the Hippo pathway, had little change during the valve growth and remodeling. This result supports that YAP activation is operated independently of the Hippo pathway.”– The relevance of YAP1 and LEF1 co-staining (or lack of) in Figure 2 adds little value to the study. At E11.5, yellow cells are identified suggesting that there is an overlap. The authors comment that the data supports "that YAP has different functions at early EndMT than later remodeling stages". However, based on this expression data, this conclusion is difficult to interpret and support.We understand the reviewer’s comments regarding LEF1, and we have removed the related sections in Results as requested.– In Figure 3, data presented in panel D should be quantitated. In addition, what are the axis referring to on the graphs of Figures 3E, F?we apologize if this information wasn’t suitably called out in the manuscript. We have revised the Yap analysis as requested by Reviewer 1. Figure 3E is the quantification of YAP activation in the monolayer VEC experiments . Figure 3F is the quantification of YAP activation in the ex-vivo valve explant experiments . The area ratios that describe the valve compaction is not included here, as we have published detailed compaction data in previous studies:Bassen D, Wang M, Pham D, Sun S, Rao R, Singh R, et al. Hydrostatic mechanical stress regulates growth and maturation of the atrioventricular valve. Development. 2021;148(13).Therefore, in this study, compaction levels under different stress conditions were given directly. We have also revised the Figure 3 , the axis of E and F refers to the intensity ratio of nuclear vs. cytoplasmic YAP expressions.– In the text referring to Figure 3, the authors discuss examining the "YAP signaling pathway" but this is not being examined here. In addition, the authors claim that USS restrains YAP in the cytoplasm, but this is not detectable.As requested by Reviewer 1, we have further conducted qPCR for YAP target genes, the results showed that the YAP staining well represents the “YAP signaling pathway”. We have revised also the “YAP signaling pathway” to “YAP activation”. In terms of detecting whether YAP is in the nucleus or cytoplasm, we have changed the color scheme in Figure 2C. There are clearly green nuclei (solid arrows) and plenty of green in the cytoplasm (dash arrows) outside blue nuclei.Throughout the study, the authors solely rely on the quantitation of nuclear YAP expression based on IHC. An addition supporting experimental readout is highly recommended, including nuclear/cytoplasmic extraction and western blot to detect YAP in cellular fractions for Figures 3C, and 3D.We thank the reviewer for the constructive suggestion. As also requested by reviewer 1, we have used the intensity ratios of YAP nuclear/cytoplasmic expressions to quantify YAP activation status for Figure 1, 2, 5. We have also added new Figure 1—figure supplementary 2, which includes qPCR data of YAP target genes THBS1, ANKRD1 and PTX3, as well as YAP upstream in Hippo cascade LATS1 and LATS2.– For Figures 4E, 4F, 4G, 5E-H, the US control is needed.We have added US control in Figure 3 (previously 4) and Figure 4 (previously 5).– In Figure 4B, there is a large decrease in circularity with YAP inhibition, yet proliferation changes appear minimal. What other YAP-dependent mechanisms might contribute to this drastic change in valve morphology? Likewise, Figure 4F indicates 10% pHH3 reactivity, yet this is not represented in Figure 4C. Which cells are proliferating?Circularity describes the shape, while proliferation changes the size. These are independent parameters that don’t necessarily correlate with each other. We have revised Figure 3 and Figure 4 with higher magnifications to highlight the areas of interest.– The phenotype in Figure 4D is interesting and unusual. The authors conclude that loss of YAP leads to endothelium folding, but again, there is little evidence to support this. If this is true, then additional supporting readouts are needed including another endothelial cell marker. How do the authors explain an increase in VE-cadherin with so little pHH3 reactivity in the VECs?d/D)2. Even a small compaction will cause a large reduction in surface areas. As a result, the endothelium at 0-hour was geometrically too large for the cushions at 24-hour. Therefore, the endothelium could cluster into multiple layers Therefore, the increased VE-cadherin expression does not require VEC proliferation.We appreciate the reviewer for pointing out this issue. The term “endothelial folding” was poorly chosen. In fact, by “folding”, we meant the shrinkage of surface areas. In fact, by “folding”, we mean the shrinkage of surface areas. Due to the compaction of cushion explants, their surface areas became much smaller, and the shrinkage is approximately proportional to :767-75.– Figure 5E is confusing and suggests that YAP gain of function has no effect on stress-induced increases in circularity, yet the authors conclude that YAP activation reverses the compaction of valves under TS and U conditions.We understand the reviewer’s concern, as circularity and compaction may sound correlated. In fact, however, circularity evaluates whether the valves are spherical, while compaction evaluates how small valves became. YAP gain of function maintained a large size, probably due to enhanced cell proliferation, but its shape became round.– The conclusion of findings from Figure 5D is that "VE-cadherin in the YAP activated endothelium was significantly weaker…showing that YAP activation led to a relaxed endothelium." Again, this conclusion needs to be supported by additional data.We again appreciate the reviewer for raising this issue. As mentioned in previous answers, the terms “endothelium folding” and “relaxed endothelium” were chosen. Here, the “relaxed endothelium” meant the opposite to the “clustered endothelial geometry”. We have also moved this discussion from the first section of the Results to the Discussion, we have also revised titles of the third and fourth sections of the Results and titles of Figure 3 and Figure 4.The revised third section of the Results, as well as the title of Figure 3 are as follows:“Loss of YAP limited cell proliferation and promoted valve shapingTo study the function of YAP in valve growth and remodeling, we added a pharmacological inhibitor of YAP, verteporfin (VP), into the CS (pro-growth) and U conditions. The VP inhibits the interaction between YAP and TEAD, which in turn, blocks transcriptional activation of targets downstream of YAP. (31) We isolated HH34 OFT SL cushion explants and cultured them under CS, CS+VP and U+VP. We confirmed that the concentration of VP we used (5mM) does not influence cell viability . Successful YAP inhibition  reduced the valve size , regardless of the media condition. In contrast to the spherical shape of valves cultured under CS, valves cultured in media with VP maintained their trapezoidal shape, which was characterized by circularity . Further investigation demonstrated that loss of YAP significantly reduces the expression of the proliferation maker, pHH3 in the VIC . In addition, the YAP inhibition significantly strengthened the expression of VE-cadherin between VECs . Valves normally just had a single layer of endothelium, but the YAP inhibited valves showed five or more layers of endothelium.”The revised title of fourth section of the Results and Figure 4 is:“Activation of YAP promoted cell proliferation and inhibited valve elongation”The new first paragraph of the Discussion is as follows:“Cardiac valves form in response to mechanical forces generated by the flowing blood. These forces include shear and hydrostatic stress. Our results reveal that they can regulate YAP activity in valvular cells, and the mechanically regulated YAP activity can affect the size, shape and stiffness of valves. First, the shear stress regulates YAP activity in the VEC: OSS (oscillatory shear stress) promotes YAP nuclear translocation while USS  restricts YAP in cytoplasm. The hydrostatic stress regulates YAP activation in the VIC: CS (compressive stress) activated YAP while TS (tensile stress) deactivated YAP. Secondly, these mechanoresponsive YAP activities have morphological functions. YAP activation in VICs promotes their proliferation and increases valve size, YAP deactivation in VICs leads to valve compaction. In terms of shape, cushion explants tend to form a sphere in a way similar to the clustering of cells to minimize the surface tension (32). By contrast, YAP inhibition promotes valve shaping and leaves a densely packed endothelium. The densely packed endothelium could be a result of the large shrinkage in surface area, during which the endothelium at 0-hour could be geometrically too large for the valves at 24-hour. Consequently, the endothelium could be forced to cluster into multiple layers. Another possibility is that YAP activity can affect cell-cell junctions between VECs. For example, studies reported that YAP can break cell-cell junctions via SMADs mediated TGF-β signaling during the EMT process (33).”– Figure 5H is mislabeled in the text.We thank the reviewer for pointing out this mistake, we have corrected the text.– The authors should comment further on how and why YAP inhibition decreases the strain energy density. How can YAP influence the material properties of avian valves?We hypothesize that YAP inhibition promotes pro-maturation programs, which increase valve stiffness. We have now added a discussion in the new fourth paragraph of the Discussion:“In general, YAP works like a mechanobiological switch, converting mechanical signaling into the decision between growth and maturation. When YAP is activated the growth programs are turned on and the maturation programs are suppressed. When YAP is inhibited the growth programs are paused and the maturation programs are released. Unlike the pro-growth function, the pro-maturation side of YAP has been less studied. During valve remodeling, cushions elongate into mature leaflets with increased stiffness. Although both YAP activation and inhibition increased valve stiffness, the stiffness of YAP activated valves was only about half of that of YAP inhibited valves, and only YAP inhibited valves exhibited an in-vivo-like stiffness increase. We have previously shown a linear relationship between valve stiffness and valve maturation (32). This suggests that YAP inhibition promotes a more mature phenotype.”


The manifestations of the athlete’s heart can create diagnostic challenges during an echocardiographic assessment. The classifications of the morphological and functional changes induced by sport participation are often beyond ‘normal limits’ making it imperative to identify any overlap between pathology and normal physiology. The phenotype of the athlete’s heart is not exclusive to one chamber or function. Therefore, in this narrative review, we consider the effects of sporting discipline and training volume on the holistic athlete’s heart, as well as demographic factors including ethnicity, body size, sex, and age. Exercise training is associated with an array of morphologic and functional cardiac adaptations and termed the ‘athlete’s heart’ (AH) . These mThe multifactorial nature of AH morphology Fig.  and funcNumerous studies have attempted to quantify the extent of left ventricular (LV) adaptation/remodelling and its association with the type of exercise stimulus. The first recognised proposal from this research was the development of the Morangroth hypothesis over 45 years ago. This stated that endurance trained (ET) athletes exhibit eccentric LV hypertrophy whereas resistance trained (RT) athletes exhibit concentric LV hypertrophy . EccentrThere is a widespread agreement that ET elicits LV dilation with increased LV mass, LVEDV and LV end-diastolic diameter (LVEDD), whereas neither concentric hypertrophy nor remodelling are now thought to be expressed in RT athletes –16. UtomA more recent cross-sectional study on 139 elite Rugby Football League (RFL) athletes found that values for LV mass and mean WT were greater in RFL athletes compared to sedentary controls  . However2 vs. 90 ± 14 g/m2; 160 ± 24 ml vs. 129 ± 32 ml respectively) [Recent studies have focussed on examining the effects of varying training volume and intensity on the AH. It has been found that athletes with high levels of training volume showed a more pronounced structural remodelling with significantly higher LV mass and LVEDV than those with a lower training volume per week (99 ± 19 g/mctively) . Additioctively) . This deLower resting LV ejection fraction (EF) in endurance athletes has been demonstrated in previous research (12% found to have an LVEF of < 52%) . This caThe application of strain imaging in the assessment of the AH has increased significantly in tandem with the evolution of this technology. It has been reported that longitudinal strain (GLS) and basal circumferential strain are lower in RT athletes compared to ET athletes . HoweverAthletes demonstrate a superior ability to augment systolic function during exercise . This thDiastolic function assessment is multiparametric as Doppler parameters are reliant on both atrial and LV pressures . UnsurprAlthough longitudinal data is scarce, some studies have demonstrated that neither ET nor RT elicit changes in global diastolic function in a period of 6-months training , 18 wherNearly a quarter of SCDs in athletes have been attributable to AVC , a condiGlobal resting RV function is maintained and almost always normal when assessed by RV fractional area change, Tricuspid Annular Plane Systolic Excursion (TAPSE), RV myocardial tissue velocities S’ and peak RV free wall strain , 39, 40.Patients with cardiomyopathy often present with left atrial (LA) dilation due to a combination of increased LV pressures and atrial myopathy , yet bi-Although two studies suggest that there are no differences for any atrial strain parameter between athletes of varying dynamic disciplines and sedentary groups several other studies have suggested that LA reservoir strain and bi-atrial strain rate during atrial contraction are lower in athletes compared to sedentary controls , 51, 53.Larger aortic root dimensions tend to be present in some athletes compared with sedentary controls, albeit values usually fall within normal clinical cut offs . It is uOur knowledge pertaining to the AH and the LV has been predominantly based on Caucasian athletes (WA) with these data being the driving force used to distinguish normal limits for LVWT in both males and females . In a co2 vs. 101 ± 21 g/m2 respectively) [A higher prevalence of LVH and increased LVWT in adult male, female and adolescent BAs when compared with their white counterparts has also been demonstrated , 66–71. ctively) . This stctively) . The stuctively) . HoweverSystolic blood pressure at peak exercise has been found to be a predictor of LV mass and a study found adolescent BAs exhibit a higher systolic blood pressure during exercise compared to WAs and, hypothesised this could be a mechanism underpinning a more pronounced LVH response , 67.In a recent review, athletic training has been found to be associated with increased LV trabeculation and can therefore overlap with criteria for LVNC . It has Compared with sedentary controls, Male Arab athletes demonstrate increased LV mass and LVWT . HoweverCompared to other ethnicities, research governing LV adaptation in Asian athletes is scarce. However, both male and female Chinese athletes have been found to display a similar prevalence and magnitude of LV cavity dilatation and hypertrophy compared with their white counterparts . HoweverData on other ethnic groups are scarce. Pacific Islanders are indigenous people from Melanesia, Micronesia, and Polynesia. In a study examining Pacific Islander rugby league players, they were found to have an increased LV mass and RWT when compared to white players .Adolescent mixed ethnicity footballers, specifically African and Caucasian descent, present phenotypical similarities with BAs with significant increases in LVWT compared to WAs but a smaller magnitude when compared to BAs , 79.Studies have demonstrated that RV structural adaptation in BAs and mixed ethnicity are similar to WAs , 81, witIt is of interest that if AVC criteria are considered, 9.9% of adolescent black footballers fulfil the structural criteria for definite or borderline AVC diagnosis whereas this is less likely in mixed ethnicity and WAs (3.9% and 0.6% respectively) . Right hTo the best of our knowledge there is only one study that has assessed LA dimensions in African/Afro-Caribbean athletes. Compared to WAs, BAs have larger LA dimensions although the clinical significance of this finding remains unclear . FurtherWhilst there are no studies conducted on the aorta between athletes of different ethnicities, there is evidence to suggest that the size of the aorta differs between ethnicities in non-athlete populations with black ethnicities having smaller BSA-indexed ascending aortic dimensions compared to white ethnicities  Table 22.Table 2b), hence refuting the first assumption. Many AH studies have sought to formulate a suitable b exponent to facilitate the comparison of LV mass between different studies and to accurately remove the impact of body size [b exponent values generated differ between 1.97 and 3, with variances in cohort sex, age and physical fitness [one-size-fits-all value has been highlighted via a similar range of b exponents being described for indexing to body mass [It is well established that heart size is dependent on body size, and it is convention in echocardiography to scale structural indices to a relevant body size scalar . Due to ody size , 84. Usiody mass .2.7 [In addition, it has been proposed that BSA is an appropriate scalar to represent muscle mass i.e., metabolically active tissue. Generally, in an athletic population, fat mass is low and hence body mass and derived BSA may act as a reasonable surrogate for fat free mass (FFM). However, in obese populations, fat mass significantly confounds BSA. Studies have therefore produced data pertaining the efficacy of FFM as an indexing variable , 85–88. 2.7 . As the 2.7 , 82. Thi2.7 . Interpr2.7 .In a study assessing asymptomatic athletes without a family history of SCD it has been found that BSA has a strong linear relationship with LV dimensions . They dib exponents [Literature examining the scaling of RV structural parameters are scarce, which could be representative of the challenging geometry of the RV , 84. Geoxponents , 37. Frob exponent provided more acceptable body size-independent values [To the authors knowledge, only 2 studies have examined scaling of the athlete’s LA, with no data governing indexing of the RA. The LA appears to conform to conventional geometrical scaling similar to the LV . Also obt values . Furthert values .To the best of our knowledge there are three studies pertaining to the effects of body size on aortic dimensions in athletes. Indexed aortic root size have been shown to have correlations with ratiometric scaling by BSA and LV mass, with greater values in men compared with women , 91. It 0.5 and FFM−1 [Female athletes consistently display smaller LV cavity dimensions and WT compared with their male counterparts , 92, 93.Global systolic function is analogous between female and male athletes, with disparities in absolute LV stroke volume being removed when scaling to FFM . AdditioStudies have also attempted to highlight differences in diastolic function but these are unremarkable with exception of a single study that highlighted a lower early diastolic longitudinal strain rate in males compared with their female counterparts (1.56%/s vs. 1.815/s) . FurtherMuch like the LV, smaller RV structural dimensions are observed in female athletes when compared with males , 90. TheLong-term ET promotes an increase in all RV indexed dimensions and induces a more spherical RV shape in both male and female athletes . This stTraining-induced biatrial dilatation has been observed in male and female athletes, with larger absolute dimensions being displayed in male athletes . In addiIn highly trained competitive athletes, males have been found to have larger aortic root dimensions than females , 99, 100In adolescent athletes of variable ethnicity, LVWT, LVEDD, left ventricular end diastolic volume index and LV cavity enlargement are greater when compared to age-matched controls , 102–108Exercise training has a more profound effect on the adolescent BAs heart compared to WAs or mixed ethnicity , 98. 7% Senior athletes demonstrate a more pronounced enlargement of the LV cavity and increases in WT due to a greater physical maturity and increased cumulative training hours compared to the adolescent athletes . A lifelThere are no differences in EF between masters athletes, sedentary older individuals, junior athletes, sedentary young individuals and pre-adolescent athletes , 113–116Global diastolic function is similar between age-matched controls and junior athletes , 107. A Similar to that of the LV, RV cavity size is observed to increase throughout adolescence in young athletes due to both increased training volume and maturation , 121. FuRV systolic strain rate in the mid and apical wall has been shown to be reduced in adolescent athletes when compared to older athletes and sedentary controls, suggestive of RV systolic reserve, whilst RV S’ and RV peak strain have been found to decrease with age –123. FurTo the authors’ knowledge, only two papers describe the effects of age on RV diastolic function. It has been found that with increasing age of the athlete, e` and the trans-tricuspid E/A ratio decline with the ratio being coupled with a decreased ability to augment diastolic function during exercise stress , 123.LA cavity size, bi-atrial volumes and RA area have been found to be greater in adolescent athletes compared to sedentary controls , 106. AdAortic diameter is greater in adolescent athletes compared with age-matched sedentary controls , 126. InIt has been reported that in elite and Olympic athletes who withdraw from training a reduction in RV and LV cavity dimensions, mass and WT occur, which can therefore be suggestive of a reversibility of any hypertrophy that is induced by athletic training –129. FurThe assessment of LV mass is fundamental to defining geometry in the AH and yet the validity and reproducibility of echocardiographic is debatable with the use of different methods and modalities i.e. m-mode versus 2D. It is therefore important to acknowledge the variability applied to the empirical studies seen presented in this literature review  to assess athletes who have tested positive for COVID-19. Echocardiography is a vital diagnostic tool in initial return to play assessment post-infection. This is due to its efficacy in evaluating global LV function, localized wall motion abnormalities, valvar dysfunction, and pericardial effusions . If any There is a consensus that athletes appear to have an overall low risk of Covid-19 induced cardiac abnormalities. These cardiac abnormalities—myocarditis, pericarditis and pericardial effusion—have been found to affect between 0.2% and 4% of athletes with a single study having a prevalence of 40% of athletes –141. ThaIn a study conducted on 455 football players, 3% demonstrated de-novo ECG changes post COVID-19 infection, with 88% of these athletes being diagnosed with cardiac inflammation . FurtherThe AH is a complex phenotype with unique structural and functional characteristics as determined by sporting discipline, training volume, ethnicity, body size, sex, and age. Identifying normal physiology for each individual athlete is imperative when undertaking preparticipation echocardiographic screening. Our understanding of normality helps to improve sensitivity and specificity for the detection of cardiac disease and hence reduces anxiety or the unnecessary disqualification of an athlete, whilst ensuring that pathology is accurately detected. Furthermore, with the new complex challenge of COVID-19, understanding the AH is even more vital in protecting the health of our athletes.


To explore the additional value of cardiac magnetic resonance (CMR) post-contrast T1 mapping in the detection of myocardial infarction, compared with late gadolinium enhancement (LGE).A CMR database of consecutive patients with myocardial infarction was retrospectively analyzed. All patients were scanned at 3 T magnetic resonance; they underwent conventional CMR (including LGE) and post-contrast T1 mapping imaging. Two radiologists interpreted the CMR images using a 16-segment model. The first interpretation included only LGE images. After 30 days, the same radiologists performed a second analysis of random LGE images, with the addition of post-contrast T1 mapping images. Images were analyzed to diagnose myocardial scars, and the transmural extent of each scar was visually evaluated. Diagnoses retained after LGE were compared with diagnoses retained after the addition of post-contrast T1 mapping.P = 0.008), the percentage of uncertain segments was lower after the addition of post-contrast T1 mapping , and the percentage of uncertain transmural extent of scarring was lower after the addition of post-contrast T1 mapping .In total, 80 patients  were included in the final analysis. After the addition of post-contrast T1 mapping, eight previously unidentified subendocardial scars were detected. Compared with LGE images, the percentage of infarcted segments was higher after the addition of post-contrast T1 mapping images (21.7% vs. 22.3%, The addition of post-contrast T1 mapping after LGE helps to improve the detection of myocardial infarction, as well as the assessment of the transmural extent of scarring. Cardiovascular disease is the greatest threat to human health worldwide, and myocardial infarction is the leading cause of death , 2. The Over the past two decades, rapid developments in magnetic resonance technology have helped cardiac magnetic resonance (CMR) to become an essential method for evaluation of cardiovascular diseases. CMR is a non-invasive imaging method with that exhibits high resolution, good contrast, and no ionizing radiation; it can be used for multi-sequence imaging .CMR can also characterize histopathological changes in the myocardium, such as edema, necrosis, and fibrosis. Late gadolinium enhancement (LGE) is an essential method for non-invasive examinations of myocardial pathology; it has been widely used to assess myocardial infarction. LGE uses an inversion recovery (IR) sequence to attenuate the signal of normal myocardium; it can distinguish a bright myocardial scar from normal black myocardium. Ischemia-induced myocardial infarction initially affects subendocardial fibers of the myocardium, then gradually extends to the epicardium through the ventricular wall  .In LGE images, the high signal of left ventricular blood may lead to poor contrast between a subendocardial scar and adjacent blood. This may hinder identification of the subendocardial scar or reduce diagnostic confidence . BecauseIn recent years, quantitative mapping technology has played an increasingly important role in the evaluation of cardiovascular diseases. Myocardial mapping technology provides specific parameters of the myocardium , which can reflect changes in myocardial tissue composition . Post-coPost-contrast T1 mapping has shown promise in detecting myocardial scars and quantifying the extent of scarring in previous studies , 13. HowThe institutional ethics review committee at our institution approved this study and waived the requirement for informed consent. In total, 128 consecutive patients with myocardial infarction who underwent CMR imaging in our department from June 2019 to February 2022 were included in this study. The clinical diagnostic criteria for myocardial infarction were based on the "Fourth Universal Definition of Myocardial Infarction (2018)" ; specifiAll patients underwent CMR on a 3-T magnetic resonance scanner  with an 18-channel body coil and a 32-channel spine coil. LGE was scanned 10 min after the injection of 0.15 mmol/kg gadolinium contrast agent, post-contrast T1 mapping was scanned immediately after the LGE scan, and all LGE and post-contrast T1 mapping scans were performed 10–20 min after contrast agent injection. The stack of 8–12 short-axis slices covered the left ventricle from base to apex. Subsequently, images were scanned with a breath-hold of 7–14 s (depending on the patient's heart rate) during the diastolic period.2, slice thickness = 8 mm, and bandwidth = 770 Hz. The selection of the inversion time (TI) value was based on a TI-scout sequence using multiple inversion times. The optimal TI value was adapted to null the signal of normal myocardium, typically in the range of 280 to 330 ms.LGE was performed using a segmented phase-sensitive inversion recovery (PSIR) sequence. The number of k segments per one cardiac cycle was 28, and 10 cardiac cycles were necessary for one slice. The main scanning parameters were as follows: repetition time = 3.2 ms, echo time = 1.2 ms, matrix = 224 × 150, flip angle = 55°, field of view = 360 × 320 mm2, slice thickness = 8 mm, and bandwidth = 1,085 Hz; three inversion pulses were used . The acquired original images were fitted inline by motion correction and a non-linear least-squares curve to generate a pixel-wise colored T1 map.Post-contrast T1 mapping was performed immediately after LGE using the MOLLI sequence with the acquisition protocol 4b(1b)3b(1b)2b, where b represents heartbeat. The main scanning parameters were as follows: repetition time = 2.84 ms, echo time = 1.2 ms, matrix = 256 × 172, flip angle = 35°, field of view = 360 × 320 mmLGE and post-contrast T1 mapping images were divided into 16 myocardial segments (excluding the apex), in accordance with the American Heart Association segmentation model . Two radt-tests  or the Mann-Whitney U test . Categorical variables were analyzed using the McNemar chi-squared test when for frequencies ≥ 5 and Fisher's exact test for frequencies < 5. Interobserver and intraobserver agreements were analyzed using Cohen's kappa coefficient . SPSS 21.0 software  was used for statistical analysis. p-values < 0.05 were considered statistically significant.Continuous quantitative data were expressed as means ± standard deviations or medians (interquartile ranges); categorical data were expressed as frequencies (percentages). The Shapiro-Wilk test was used to assess whether continuous quantitative data conformed to a normal distribution. Continuous variables were analyzed using independent samples For patients with acute or subacute myocardial infarction, CMR was performed 3 days after coronary angiography (CAG) or PCI; for patients with chronic myocardial infarction, CMR was performed during the 3-month follow-up period after CAG or PCI. Overall, 80 patients  were included in this study; their clinical characteristics are shown in P = 0.008); the percentages of uncertain segments and uncertain transmural extent of scarring were both lower after the addition of post-contrast T1 mapping . A comparison of LGE alone and the addition of post-contrast T1 mapping is shown in Figures 2–After the addition of post-contrast T1 mapping, eight previously unidentified scars were detected. Among these eight segments, four 50%) were located in the basal segment, one (12.5%) was located in the middle segment, and three (37.5%) were located in the apical segment. The percentage of infarcted segments was higher after post-contrast T1 mapping than after LGE alone (22.3% vs. 21.7%, 0% were lThis study retrospectively evaluated the additional value of CMR post-contrast T1 mapping in the detection of myocardial infarction. The results showed that the addition of post-contrast T1 mapping to LGE can improve the detection of infarcted segments, reduce the percentage of uncertain myocardial segments, and reduce the percentage of uncertain extent of transmural scarring.When LGE nulls normal myocardium through the IR sequence, subendocardial scars and blood both exhibit high signals, which may cause scars to be missed because of the lack of contrast. Additionally, the selection of an appropriate TI value and presence of cardiac motion artifacts during LGE imaging may affect image quality and scar detection. The addition of post-contrast T1 mapping can resolve some limitations of LGE. The principle of T1 mapping is that myocardial tissue characteristics are represented by specific T1 values. T1 maps constitute pixel-wise, color-coded quantitative maps that can directly reflect subtle changes in T1 values and provide additional diagnostic information for myocardial assessment , 17.Because of the shortening effect of the gadolinium contrast agent, the T1 value of a scar is lowest 10–20 min after contrast agent injection, and the scar is visible on post-contrast T1 mapping. Importantly, post-contrast T1 mapping avoids dependence on the selection of an appropriate TI value. Compared with LGE using segmented PSIR, T1 mapping with motion correction can reduce motion artifacts in some images with poor breath holding, thereby facilitating evaluation of scarring.The results of the present study will allow clinicians to acquire additional information regarding patient prognosis, which could influence patient management and treatment strategies. Because scar size is closely associated with many adverse outcomes , 19 and To improve contrast between scars and blood, researchers have used various preparation pulses that produce dark or gray blood LGE by inhibiting the signal of left ventricular blood –27; thesHoltackers et al.  found thSome challenges remain in the imaging-based detection of myocardial infarction. This type of imaging constitutes an area of research in which a new method can improve decision making, risk stratification, and management. Ischemia-induced myocardial necrosis initially affects subendocardial fibers, then gradually progresses from the subendocardium to the epicardium. Therefore, accurate assessment of subendocardial scars requires attention, and the use of multiple sequences is essential. In our institution, T1 mapping has become a routine CMR scan. Considering the additional diagnostic value of post-contrast T1 mapping, we believe that the addition of 3–4 min of scanning time for patients with myocardial infarction is acceptable. We also recommend careful consideration of the diagnostic value of post-contrast T1 mapping among institutions that have implemented post-contrast T1 mapping scans.This study had several limitations. First, the scars identified by post-contrast T1 mapping and LGE were not pathologically confirmed. To reduce false positives, this study only included patients with clinically diagnosed myocardial infarction; ischemia-induced myocardial infarction scars initially affect subendocardial tissue. In this study, all additional myocardial infarction scars identified after the addition of post-contrast T1 mapping originated from subendocardial tissue. Second, post-contrast T1 mapping may produce less common artifacts, such as errors related to blood-derived partial volume contamination, which must be carefully assessed. Third, extracellular volume maps were not included in this study, and their value should be explored in future research. Finally, this study only considered scars in patients diagnosed with myocardial infarction. Post-contrast T1 mapping may be useful in the diagnosis of scars caused by other diseases, such as hypertrophic cardiomyopathy, sarcoidosis, or myocarditis. Therefore, further analyses are needed concerning the clinical role of post-contrast T1 mapping in different populations and diseases.In patients with myocardial infarction, the addition of post-contrast T1 mapping after LGE helps to improve the detection of myocardial infarction, as well as the assessment of the transmural extent of scarring.


Rheumatic fever is still a major cause of mitral valve (MV) stenosis in the developing world. Few patients with critical rheumatic MV stenosis can present with acute cardiogenic shock (CS) that requires urgent treatment with circulatory support and definitive valvular repair or replacement.2, mean diastolic gradient of 17 mmHg, Wilkins score 7). His Society of Thoracic Surgery score and EuroScore were 50.1% and 12.1%, respectively. Thus, a percutaneous transcatheter mitral commissurotomy (PTMC) was decided as the definitive treatment in a multidisciplinary team meeting. Following the procedure, the patient’s circulatory support was gradually weaned off, and he was successfully extubated with a marked improvement in his renal functions. The patient achieved a complete recovery without any long-term sequelae.A 37-year-old gentleman was admitted with heart failure, CS Society for Cardiovascular Angiography and Interventions D, and atrial fibrillation with a rapid ventricular response. He had no prior medical history. He had multiple organ failures and required intubation, two DC shocks of 200 joules without haemodynamic improvement, continuous renal replacement therapy, and medical and mechanical circulatory support using extracorporeal membrane oxygenation (ECMO). His echocardiography showed severe rheumatic mitral stenosis , and relief of the MV obstruction. Percutaneous transcatheter mitral commissurotomy can be the preferred option in this setting if the valve is pliable. This case illustrates the importance of rapid diagnosis of valvular heart disease in the setting of cardiogenic shock (CS).This case demonstrates the best available choices of mechanical circulatory support devices for CS caused by severe mitral valve stenosis.Multidisciplinary team decisions are of extreme paramount to determine the best management pathway of critically ill patients with valvular heart disease.1 The mainstay of the management is to support the circulation medically and mechanically until the underlying cause has been identified and treated.3 Although ischaemic heart disease is considered the most common cause of CS,4 structural heart disease and valvular dysfunction are considered important causes of CS that necessitate prompt management.5 This article highlights a case of severe rheumatic mitral valve (MV) stenosis presenting with tachyarrhythmia and refractory CS successfully supported with extracorporeal membrane oxygenation (ECMO) and treated with percutaneous transvenous mitral commissurotomy (PTMC).Cardiogenic shock (CS) is a life-threatening condition where the cardiac output is significantly reduced with haemodynamic instability that compromises the tissue perfusion. Cardiogenic shock carries a high mortality risk of more than 40–50%.The emergency medical services brought in a 37-year-old Asian man with an unremarkable past medical history or cardiovascular risk factors because of shortness of breath and palpitations. His symptoms started a few months ago and progressed to the point that he became dyspnoeic at rest. He also had occasional haemoptysis and chest discomfort—no history of chest pain or loss of consciousness.2O, positive end-expiratory pressure 5 cm H2O, and 100% FiO2. He had diffuse crackles bilaterally, raised jugular venous pressure, and cold extremities with peripheral oedema. His electrocardiogram showed atrial fibrillation (AF) with a rapid ventricular response . Chest X-ray showed cardiomegaly and pulmonary oedema .On examination, he looked sick, cyanosed, and in respiratory distress. He was afebrile; his respiratory rate was 42 breaths/min, pulse rate was 140 b.p.m. and irregular, blood pressure was 94/77 mmHg, and oxygen saturation was 98% on BiPAP pressure support 6 cm HTable 1). He also developed anuric AKI, for which continuous renal replacement therapy (CRRT) was started.As he was in severe respiratory distress, rapid sequence intubation was done with ketamine 100 mg and rocuronium 100 mg. Then, he received 40 mg IV furosemide, 2.5 mcg/kg/min norepinephrine, and 5 mcg/kg/min dopamine for haemodynamic support. He also received two transoesophageal echocardiogram (TEE)-guided direct current shocks of 200 joules without a response and 150 mg amiodarone and 0.5 mg digoxin for the AF, after which sinus rhythm was restored with tachycardia without much improvement in the haemodynamics. The initial laboratory findings showed multiple organ failures with acute kidney injury (AKI), acute liver injury with transaminitis, and a deranged coagulation profile  dilatation , and moderately reduced right ventricular (RV) function EF 37%  (see Videos S1 and S2).Transthoracic echocardiogram (TTE) revealed moderately reduced systolic left ventricular (LV) function, moderate global hypokinesis of LV, an ejection fraction (EF) of 38%, severe rheumatic mitral stenosis (MS) [mitral valve area (MVA) 2D of 0.7 cmThe CS persisted and became refractory, Society for Cardiovascular Angiography and Interventions (SCAI) D, he was placed on ECMO and intra-aortic balloon pump (IABP) with prompt improvement in his haemodynamics and a decrease in the requirements of dopamine from 10 to 2 mcg/kg/min and norepinephrine from 3 to 0.1 mcg/kg/min. His Society of Thoracic Surgery (STS) score and EuroScore were 50.1% and 12.1%, respectively, for MV replacement. The invasive haemodynamics showed a pulmonary capillary wedge pressure (PCWP) of 21 mmHg and a mean pulmonary artery pressure of 22 mmHg.Figure 3), without significant mitral regurgitation (MR). A follow-up TTE on the same day demonstrated MVA 2D improvement to 1.9 cm2 and tricuspid annular plane systolic excursion (TAPSE) improvement to 1.2 cm, indicating residual mild RV function impairment and LV EF improvement to 48%. His haemodynamics improved afterward, and he was weaned off ECMO, decannulated on Day 6, and extubated on Day 14. His CRRT was switched to intermittent haemodialysis, and eventually, his renal function fully recovered on Day 23, and he did not require haemodialysis or diuresis afterward. He was then transferred to the rehabilitation centre for physical therapy as he developed critical care myopathy during his hospital stay. After 2 months of his stay in the rehabilitation centre, the patient was discharged home with normal cognitive function and independent mobility (ambulation and stair climbing). He was discharged on bisoprolol (dose) for rate control, warfarin for valvular AF to target international normalized ratio (INR) level of 2–3, and amlodipine (dose) for hypertension and planned to further optimize his heart failure medications during his follow-up clinical visits. The patient will be followed up in the structural cardiology and heart failure clinics with repeated transthoracic echocardiography and further workup for a reduced EF if it persisted.On Day 5, the patient underwent PTMC after the multidisciplinary team discussion determined that he had extreme surgical risk and his valve was pliable for percutaneous intervention. The procedure was performed with transoesophageal guidance. Due to the shortage of dedicated PTMC balloons, the procedure was performed with a 26 mm × 40 mm Numed balloon over Safari wire after obtaining transseptal access using a steerable guide sheath. After one balloon inflation, his mean LA pressure (LAP) and mean diastolic MV improved from 17 to 9 mmHg and from 9 to 2 mmHg, respectively  is veno-arterial (VA) ECMO or LA–VA ECMO.10 This would be a bridgeport for the definitive management, which is a valvular repair, and as the patients are critically ill, minimally invasive procedures are considered first. In severe MS, PTMC has been reported to alleviate patients with severe pulmonary oedema, intractable heart failure, and CS. It is regarded as a feasible and lifesaving procedure.13The typical management of CS caused by valvopathies requires inotropic and vasopressors and, in advanced stages, adjunctive mechanical circulatory support as a bridge until further intervention is sought. As there are different types of mechanical circulatory supporting devices, the selection of an appropriate device hinges upon various factors, including the availability of options and the expertise of the clinical team.In this case, ECMO was chosen to provide both respiratory and circulatory support. Intra-aortic balloon pump (IABP) was added to help reduce the afterload and reduce the risk of LV overdistention. Alternatively, LA–VA ECMO or tandem heart with external oxygenation membrane could have been considered in this case. Meanwhile, definitive management was sought in a multidisciplinary team fashion involving cardiac surgeons, cardiac anaesthesia, and interventional cardiologists. As the patient was fragile with unstable haemodynamics and the valve was pliable for percutaneous intervention, catheter-based management was determined for him with excellent outcomes.Cardiogenic shock related to severe rheumatic MV stenosis requires multidisciplinary team management with prompt diagnosis, initiation of the most appropriate mechanical support device (e.g. ECMO or tandem heart), and relief of the MV obstruction. Percutaneous transcatheter mitral commissurotomy can be the preferred option in this setting if the valve is pliable.As a future cardiologist, Dr Ashraf Ahmed has a burning desire to learn as much as possible about the field of cardiology through clinical research. He hopes to do this through connecting with like-minded professionals and mentors. Dr Ahmed is a POCUS coach at Hamad Medical Corporation, Affiliated with Weil Cornell Medicine in Qatar. He also achieved the Paul Dudley White International Award at the AHA Conference 2021. In 2022, Harvard T.H. Chan School of Public Health recognized him as an outstanding Teaching Assistant-I for the Principle and Practice of Clinical Research Course.European Heart Journal – Case Reports online.Supplementary material is available at ytad553_Supplementary_DataClick here for additional data file.


In a subset of patients who underwent baseline speckle tracking echocardiography, pre-procedural inward displacement was compared with left ventricular regional echocardiographic strain (n = 15). Results: Inward displacement of basal and mid-cavity left ventricular segments increased by 27% (p < 0.001) and 37% (p < 0.001), respectively, following left ventricular reconstruction. A significant overall decrease in both the left ventricular end systolic volume index and end diastolic volume index of 31% (p < 0.001) and 26% (p < 0.001), respectively, was detected, along with a 20% increase in left ventricular ejection fraction (p = 0.005). A significant correlation between inward displacement and speckle tracking echocardiographic strain was noted within the basal  and mid-cavity left ventricular segments , respectively. Inward displacement resulted in relatively larger measurement values compared to speckle tracking echocardiography, with a mean difference of absolute values of −3.33 and −7.41 for the left ventricular base and mid-cavity, respectively. Conclusions: Obviating the limitations of echocardiography, inward displacement was found to highly correlate with speckle tracking echocardiographic strain to evaluate regional segmental left ventricular function. Significant improvements in basal and mid-cavity left ventricular contractility were demonstrated in ischemic HFrEF patients following left ventricular reconstruction of large antero-apical scars, consistent with the concept of reverse left ventricular remodeling at a distance. Inward displacement holds significant promise in the HFrEF population being evaluated pre- and post-left ventriculoplasty procedures.Background: Hybrid minimally invasive left ventricular reconstruction is used to treat patients with ischemic heart failure with reduced ejection fraction (HFrEF) and antero-apical scar. Pre- and post-procedural regional functional left ventricular assessment with current imaging techniques remains limited. We evaluated ‘inward displacement’ as a novel technique of assessing regional left ventricular function in an ischemic HFrEF population who underwent left ventricular reconstruction with the Revivent System. Methods: Inward displacement adopts three standard long-axis views obtained during cardiac MRI or CT and assesses the degree of inward endocardial wall motion towards the true left ventricular center of contraction. For each of the standard 17 left ventricular segments, regional inward displacement is measured in mm and expressed as a percentage of the maximal theoretical distance each segment can contract towards the centerline. The left ventricle was divided into three regions, obtaining the arithmetic average of inward displacement or speckle tracking echocardiographic strain at the left ventricular base (segments 1–6), mid-cavity (segments 7–12) and apex (segments 13–17). Inward displacement was measured using computed tomography or cardiac magnetic resonance imaging and compared pre- and post-procedurally in ischemic HFrEF patients who underwent left ventricular reconstruction with the Revivent System ( TM therapy  has evolved to effectively target ischemic HFrEF patients with antero-apical scar folding and volume reduction via pledgeted anchors, while the AccuCinch® Ventricular Restoration System  typically targets the LV base in non-ischemic HFrEF via endocardial anchoring of a non-circumferential ring that is cinched to reduce the septo-lateral LV diameter ). Categorical outcomes were expressed as frequencies and percentages. Preoperative and postoperative continuous, normally distributed data of the same patients were compared using the paired Student’s Out of a total group of 86 patients who underwent LVR with the Revivent System in the CE-mark cohort, 21 patients were included for InD analysis pre- and post-LVR. Out of 30 patients who were operated on at the St. Antonius Hospital with the Revivent System, 15 patients were included for InD analysis pre- and post-LVR. InD was thus derived from CT- and/or CMR-images and analyzed both pre- and postoperatively at the LV base (segments 1–6) and mid-region of the LV (segments 7–12) in 36 patients . The incp < 0.001) (p < 0.001). LVESVi decreased from 68.2 ± 27.6 to 46.4 ± 24.3 ; left ventricular end diastolic volume index (LVEDVi) decreased from 96.8 ± 30.7 to 71.5 ± 26.4  (p = 0.005).Preoperatively, InD was derived from CT in 9 cases (25%) and from MRI in 27 cases (75%). Postoperatively, InD was derived from CT in 18 cases (50%) and from MRI in 18 cases (50%). Follow-up CT or MRI were performed after a median period of 11 months (IQR 7–27). Comparing InD pre-LV reconstruction with corresponding post-LV reconstruction imaging in 36 patients, mean InD increased significantly in the basal LV region from 15.2 ± 5.8 to 19.3 ± 6.3  a. InD in< 0.001) b. In thip < 0.001 and R = −0.65, p = 0.004. Bland Altman analysis showed that InD measurements result in larger absolute values compared to STE measurements, with a mean difference of absolute values of InD and STE of −3.33 for the LV base, with 95% limits of agreement of −10.93 to 4.27  was found in basal LV contractility in addition to a 37% increase in contractility of the mid-cavity (p < 0.001), with an overall 31% and 26% reduction in LVESVi and LVEDVi, respectively, that coincided with a 20% LVEF improvement. Furthermore, InD correlated significantly with STE measures of longitudinal shortening on TTE.Thus far, only a single case report has been published describing CT-derived InD as a tool for quantification of segmental LV wall motion abnormalities . The curEventually, InD might supersede STE as the gold standard for assessing regional/segmental LV function, since crisp endocardial border detection and precise means of pre- and post-procedural image comparisons using an automated image processing system minimize the shortcomings of TTE at two (or more) time points. The present Bland Altman analyses suggest InD values tend to be greater than corresponding values measured with STE, which is not surprising given the lack of foreshortening with CT and CMR. Further validation in identifying the InD reference ranges for each of the 17 LV segments across larger numbers of control subjects is currently underway to better place the current observations into clinical context. Overall, these nascent observations demonstrate InD to hold significant promise in the evaluation and planning of left ventriculoplasty therapies for HFrEF patients, as well as for post-procedural assessment of loco-regional treatment efficacy, overcoming the shortfalls and limitations of surface echocardiography.The present analysis is an observational retrospective study. As the availability of CT and CMR images differed per patient, two different imaging modalities were used for InD analyses. Moreover, as pre- and postoperative CT or CMR images were not made in all patients, InD data were not available for all patients, finally resulting in the inclusion of 36 patients in this study. Furthermore, STE images were not available for the CE-mark cohort, preventing a larger analysis of correlations between baseline STE and InD in this study. Although the number of patients in both cohorts remains relatively small, the present analysis represents the single and largest patient population to date in which InD has been measured, comprising the only currently available data of its application in a HFrEF population that underwent a left ventriculoplasty intervention, enabling the unique opportunity to assess regional LV segmental remodeling at a distance. As functional symptomatic status  was not available, it was not possible to translate improvement in InD to an improvement in clinical functional outcome. However, the InD findings post-left ventriculoplasty do correlate well with the significant LV volume reductions observed in the patient population, which serves as an important surrogate endpoint of clinical efficacy in the HFrEF population independent of LVEF . As InD InD, measured using CT or CMR, successfully demonstrated the concept of reverse LV remodeling at a distance when an ischemic HFrEF population with large anteroseptal and/or apical scars underwent a hybrid LVR procedure. The ability of InD to reliably and reproducibly measure specific LV segmental function at multiple time points will allow us to better understand the mechanism and efficacy of a range of left ventriculoplasty procedures that are currently being investigated in clinical trials with other similar device trials on the horizon. The strong correlations of InD improvement with LV volume reductions, along with STE, provide early mechanistic reassurance of the ability for InD to ultimately emerge as the gold-standard for regional LV functional assessment, overcoming many of the shortfalls of TTE. Further clinical validation in both control subjects as well as HFrEF patients pre- and post-left ventriculoplasty along with correlations with clinical and surrogate endpoints of efficacy will be required before InD emerges as the preferred means of screening and monitoring HFrEF patients undergoing assessment for device-based LV interventions.