There were no differences in other CMR parameters, hemodynamic parameters, or cardiovascular risk factors, except for more frequent use of statins in the MINOCA patient group compared to volunteers. In conclusion, global stress perfusion is lower in MINOCA patients during follow-up, compared to age- and sex-matched volunteers, suggesting that CMD may be a possible pathophysiological mechanism in MINOCA.Myocardial infarction with nonobstructive coronary arteries (MINOCA) has several possible underlying causes, including coronary microvascular dysfunction (CMD). Early cardiovascular magnetic resonance imaging (CMR) is recommended, however cannot provide a diagnosis in 25% of cases. Quantitative stress CMR perfusion mapping can identify CMD, however it is unknown if CMD is present during long-term follow-up of MINOCA patients. Therefore, this study aimed to evaluate presence of CMD during long-term follow-up in MINOCA patients with an initial normal CMR scan. MINOCA patients from the second Stockholm myocardial infarction with normal coronaries study (SMINC-2), with a normal CMR scan at median 3 days after hospitalization were investigated with comprehensive CMR including stress perfusion mapping a median of 5 years after the index event, together with age- and sex-matched volunteers without symptomatic ischemic heart disease. Cardiovascular risk factors, medication and symptoms of myocardial ischemia measured by the Seattle Angina Questionnaire 7 (SAQ-7), were registered. In total, 15 patients with MINOCA and an initial normal CMR scan , and 15 age- and sex-matched volunteers, underwent CMR. Patients with MINOCA and an initial normal CMR scan had lower global stress perfusion compared to volunteers (2.83 ± 1.8 vs 3.53 ± 0.7 ml/min/g, Clinical Trial Registration: Clinicaltrials.gov identifier NCT02318498. Registered 2014-12-17. MINOCA has several possible underlying causes, including coronary microvascular dysfunction (CMD)5. CMD encompasses functional and structural abnormalities in the coronary microvasculature, leading to insufficient increase in myocardial perfusion in response to increasing oxygen demands, causing myocardial ischemia6. Early cardiovascular magnetic resonance imaging (CMR) is recommended for differential diagnosis following MINOCA presentation10, and provides prognostic information11. However, standard clinical CMR is normal in 25% of MINOCA cases and thus fails to provide a diagnosis enabling specific treatment13. New fully quantitative stress CMR perfusion mapping adds the possibility to assess regional and global myocardial perfusion (ml/min/g)15. Quantitative stress perfusion mapping can detect CMD as reduced global stress perfusion and/or myocardial perfusion reserve (MPR)16 and can differentiate CMD from obstructive coronary artery disease (CAD)17. CMD has been shown in acute MINOCA and is associated with poor outcome both in the acute stage and long-term18. However, long-term presence of CMD following acute MINOCA presentation has to the best of our knowledge not been examined previously. Moreover, multiparametric CMR including quantitative stress perfusion has not been utilized in the evaluation of MINOCA patients, despite its ability to non-invasively and without radiation assess cardiac function, structure and tissue properties. Therefore, we aimed to evaluate presence of CMD in MINOCA patients with an initial normal CMR scan in long-term follow-up, using comprehensive multiparametric CMR, including quantitative stress perfusion mapping. The hypothesis was that MINOCA patients would have lower global stress perfusion compared to sex- and age-matched volunteers with similar comorbidities but without symptomatic ischemic heart disease (IHD), indicating CMD.Myocardial infarction with nonobstructive coronary arteries (MINOCA) is a myocardial infarction (MI) without coronary artery stenosis ≥ 50% on coronary angiography (CAG)13. Inclusion criteria included fulfillment of the diagnostic criteria of MI1 and nonobstructive coronary arteries (stenosis < 50%) on CAG, determined visually. Exclusion criteria included lack of sinus rhythm on admission electrocardiogram (ECG), pulmonary embolism, previous MI, known cardiomyopathy, severe asthma or chronic obstructive pulmonary disease, decreased kidney function (serum creatinine of > 150 μmol/l), pacemaker, or claustrophobia. All patients were invited to follow-up CMR 6 months after index event19, the last follow-up scan was performed in September 2019.MINOCA patients were identified from the second Stockholm Myocardial Infarction with Normal Coronaries (SMINC-2) study, which was a prospective, non-randomized study of MINOCA patients performed during 2014–2018 in five hospitals in Stockholm, Sweden. SMINC-2 utilized early CMR including cine imaging, native T1 and extracellular volume (ECV) mapping and late gadolinium enhancement (LGE) imaging for differential diagnosis of MINOCA20. Ethical approval has been granted for all procedures in the study by the Swedish Ethical Review Authority , all study procedures were carried out in accordance with relevant guidelines and regulations as per the Declaration of Helsinki for involving human participants, and all participants provided written informed consent.Patients were invited to participate in the present study if their initial CMR scan and 6 months follow-up scan were read as normal by two independent level 3 CMR physicians. Furthermore, sex- and age-matched volunteers without IHD history were recruited by advertising at Karolinska Institutet Fig. . Stress 2, slice thickness 8.0 mm, echo time (TE)/repetition time (TR) 1.19/37.05 ms, matrix size 256 × 144 and field of view (FOV) 360 × 270 mm2.CMR was performed supine with a Magnetom Aera® 1.5 Tesla (T) scanner , with a phased-array 18-channel body matrix coil and a spine matrix coil. A venous blood sample was drawn to determine hematocrit and blood creatinine prior to imaging. Full coverage retrospective electrocardiographic (ECG)-gated balanced steady state free precession (bSSFP) cine imaging was acquired in standard three long-axis and short-axis slices. Typical imaging parameters were flip angle (FA) 68°, pixel size 1.4 × 1.9 mm21 and the Gadgetron inline perfusion mapping software was used to generate the perfusion maps14.Typical imaging parameters were bSSFP single shot readout, FA 50°, slice thickness 8.0 mm, TE/TR 1.04/2.5 ms, bandwidth 1085 Hz/pixel, FOV 360 × 270 mm2 and saturation delay/trigger delay (TD) 95/40 ms.Using first pass perfusion imaging, quantitative perfusion maps (ml/min/g) were acquired in three short-axis slices  during administration of an intravenous contrast agent bolus , during adenosine infusion ) and in subsequent rest. Adenosine response was assessed clinically based on symptoms and heart rate response and was evaluated by the rate pressure product (RPP) in rest and stress. Adenosine and contrast agent were administered in two different cannulas. Subjects abstained from caffeine for 24 h and nicotine for 12 h prior to CMR examination. A distributed tissue exchange model was used to compute the perfusion maps2, slice thickness 8.0 mm, imaging duration 167 ms, TE/TR 1.12/2.7 ms, matrix size 256 × 144 and FOV 360 × 270 mm2. Following a bolus of contrast , post-contrast T1 maps were acquired with the same slice position as the native T1 maps. Extracellular volume (ECV) maps in three short-axis slices were generated from native T1 and post-contrast T1 maps, calibrated by the hematocrit23. Moreover, LGE images were acquired in three long-axis and short-axis slices post-contrast using a free breathing phase-sensitive inversion recovery (PSIR) sequence with segmented fast low angle shot (FLASH) read out. Imaging parameters were image matrix 256 × 156, voxel size 1.3 × 1.3 × 7 mm3, slice thickness 8 mm, FOV 340 × 276 mm, TR 8.25 ms, TE 3.4 ms and FA 50°.Using an ECG-gated Modified Look-Locker inversion recovery (MOLLI) 5s(3s)3s prototype sequence, native T1 maps were acquired in three short-axis slices. Typical imaging parameters were single shot SSFP in end-diastole, FA 35°, pixel size 1.4 × 1.9 mm2, slice thickness 8.0 mm, acquisition window 700 ms, TE/TR 1.06/2.49 ms, matrix size 144 × 256 mm2.Using a T2-prepared sequence (Siemens MyoMaps product sequence), native T2 maps were acquired in three short-axis slices. Typical imaging parameters were FA 70°, pixel size 1.4 × 1.4 mm25. Left ventricle (LV) mass and volumes were quantified by delineating the endo- and epicardial borders in end-diastole and end-systole in the cine short-axis stack. Native T1, native T2, ECV and perfusion maps were analyzed by careful manual delineation of the endo- and epicardial borders of the LV in the respective short-axis stack and values were acquired in a 16-segment model of the LV26. To avoid blood pool and adjacent tissues contaminating the analysis, a 10% erosion margin was set for both endo- and epicardial borders. Inter-observer variability was assessed in all 30 study-participants by two independent observers and intra-observer variability was assessed by one observer re-analyzing 10 subjects, with regards to native T1, native T2, ECV and stress and rest myocardial perfusion maps.All images were anonymized and analyzed offline with the freely available software Segment 27. Global native T1, native T2, ECV, and rest and stress perfusion were acquired by averaging segmental values per subject. The RPP was calculated as heart rate multiplied by systolic blood pressure. The MPR was calculated as stress perfusion divided by rest perfusion. CMR findings between baseline, 6 months follow-up and long-term follow-up were compared using the paired t-test. Continuous variables were compared between the MINOCA patients and the volunteers with the independent t-test in normally distributed data and the Mann–Whitney U test in non-normally distributed data. Categorical data were compared between the MINOCA patients and the volunteers with Fisher’s exact test. Subgroup analysis was performed using the independent t-test or the Mann–Whitney U test. Perfusion and MPR were compared in MINOCA patients and volunteers without hypertension, and pooled MINOCA patients and volunteers were compared based on sex, presence of hypertension or statin treatment. Inter- and intra-observer agreement was calculated for global native T1, native T2, ECV and perfusion in rest and stress as well as MPR, and presented as intra-class correlation coefficient (ICC). ICC was 0.92–1.00 . Based on previously published data on healthy subjects, an a priori power calculation showed that 16 subjects were needed to detect a 0.78 ml/min/g difference in stress perfusion28 with 80% power and alpha of 0.05. Statistical analysis was performed with Microsoft Excel  and IBM SPSS Statistics . In all statistical analyses, the significance level was defined as p < 0.05.Continuous data were presented as median [interquartile range] or mean ± standard deviation (SD) and categorical data were presented as numbers (percentages). The Shapiro–Wilk test was used to assess normality. LV mass and volumes were indexed to body surface area (BSA), which was calculated using the Mosteller formulaThe study has been approved by the Swedish Ethical Review Authority, DNR 2014/131-31/1, 2017/2415-32/1, 2021-06837-02, and all patients provided written informed consent.19, and the infarcted segments were excluded from analysis at long-term follow-up. The other MINOCA patients suffered no major adverse cardiac event (MACE) during follow-up.Clinical characteristics and CMR findings of the MINOCA patients at baseline, 6 months follow-up and long-term follow-up are presented in Table Clinical characteristics including comorbidities and medications of the MINOCA patients at long-term follow-up and the age- and sex-matched volunteers without IHD are presented in Table p = 0.02), however there were no differences in rest perfusion or MPR, as shown in Table p = 0.04), however there were no differences in rest perfusion or MPR . There were no differences in rest or stress perfusion, or MPR, between pooled MINOCA patients and volunteers compared with regards to sex or presence of hypertension or statin treatment .The MINOCA patients had lower global stress perfusion compared to volunteers  has been shown in acute MINOCA, which was associated with increased risk of MACE during 2 years follow-up5. Interestingly, increased CAG-derived IMR has recently been shown across different subtypes of MINOCA, indicating CMD to be a unifying pathophysiological feature of several different clinical entities35. The only prior CMR study evaluating CMD in acute MINOCA, using qualitative perfusion analysis, showed focal rest and stress perfusion abnormalities, which were often co-localized with T2 + and/or ischemic or non-ischemic LGE4. MPR index (MPRI) was low in areas both with and without LGE and T2 + 4. Thus, it is unclear if ischemia caused LGE and T2+, or if acute edema caused LGE and T2 + as well as hypoperfusion by compressing micro-vessels4. This contrasts with the present follow-up study, where the only difference was reduced stress perfusion. Since native T1, native T2 and ECV did not differ between MINOCA patients and volunteers, the reduced stress perfusion cannot be attributed to edema or fibrosis from previous myocarditis or MI. One MINOCA patient in our study had LGE from a previous MI, occurring during initial 6 months follow-up19 and affected segments were excluded from analysis. The MINOCA patients had a nonobstructive CAG at presentation, and no patient had focal perfusion defects on stress perfusion CMR. Therefore, MI or obstructive CAD cannot explain reduced stress perfusion in the MINOCA patients. As the MINOCA patients had no other pathophysiological explanation detectable at index and were compared to age- and sex-matched volunteers with similar cardiovascular risk factors but without a history of IHD, we theorize that their initial presentation may be attributable to CMD which remained at follow-up.Early CMR is recommended for differential diagnosis in MINOCA and provides a diagnosis in most cases, which carry prognostic importance with regards to future risk of MACE and potential specific treatment36, heart failure with preserved ejection fraction (HFpEF)37, dilated cardiomyopathy (DCM)38, and microvascular angina pectoris (without obstructive CAD)41. Moreover, quantitative CMR perfusion mapping can detect obstructive CAD42 and differentiate CMD from three-vessel CAD which causes more pronounced reduction in myocardial perfusion without focal perfusion defects17.Novel fully quantitative adenosine stress CMR perfusion mapping adds the possibility to identify CMD, presenting as reduced global stress perfusion and/or MPR, which has been shown in patients with post-covid37. Stress perfusion and MPR is reduced in DCM while rest perfusion is augmented, with lower rest and stress perfusion in segments with LGE38. The increased rest perfusion in both studies may indicate functional CMD, as reduced resting tone and higher rest perfusion has been shown invasively in functional CMD, causing reduced stress perfusion due to impaired vasodilatory reserve43. However, presence of fibrosis indicates structural CMD, possibly coexisting and contribution to each other. In microvascular angina, stress perfusion and MPR is reduced while rest perfusion is unaffected39. Moreover, reduced stress perfusion and MPR, without differences in T1, ECV or rest perfusion, has been shown in microvascular angina40. These studies report lower stress perfusion and MPR than the current study, possibly reflecting more pronounced CMD also contributing to clinical ischemic symptoms. Moreover, women have higher perfusion values, which decreases with age44. Two studies included more men of higher age39 and one included more females of similar age compared to the current study40. Furthermore, the results of these studies may reflect structural CMD, as normal rest perfusion and impaired stress perfusion has been shown invasively in structural CMD, due to impaired vasodilatation and/or increased microvascular resistance43. Overall, the compared studies show the complexity of CMD as common pathophysiological feature in many different clinical entities, with many different mechanisms and presentations ultimately impairing myocardial perfusion.CMD is common in HFpEF, as shown by reduced MPR despite unaffected stress perfusion due to increased rest perfusion, and ECV is increased39. The MINOCA patient group was well-characterized, presenting with an MI with nonobstructive coronary arteries as determined visually on CAG and a normal CMR scan at index hospitalization and 6 months follow-up. One individual had an MI during initial 6 months-follow-up19, and the infarcted segments were excluded from analysis at long-term follow-up. Optical coherence tomography or intravascular ultrasound was not performed at index hospitalization and therefore, plaque erosion or rupture could have been overlooked. Moreover, it is unknown if CMD was present before and at index hospitalization or developed during follow-up. However, the MINOCA patients were compared to volunteers with no history of IHD despite matching age and sex, and similar cardiovascular risk factors. The MINOCA patients were more frequently treated with statins and had a numerically higher prevalence of hypertension. Hypertension and hyperlipidemia are known risk factors for CMD, decreasing stress perfusion and/or MPRI49. The small sample size in the current study does indeed limit the utility of subgroup analysis. However, also normotensive MINOCA patients had lower stress perfusion than normotensive volunteers and there were no differences comparing pooled MINOCA patients and volunteers based on hypertension or statin use, with regards to stress and rest perfusion as well as MPR. Intra- and interobserver agreement was excellent, indicating that quantitative perfusion mapping is a robust method for evaluating myocardial perfusion. The loss to follow-up during this long-term study with the resultant small sample size constitutes major limitations. Sample sizes were larger in the compared studies40, however the sample size calculation prior to study start established 16 patients as sufficient based on previous stress perfusion values. Unfortunately, T2 maps were missing in one MINOCA patient and rest perfusion maps of one healthy volunteer were excluded due to residual hyperemia. Neither T2 values nor rest perfusion were the primary scope of this study, thus the effect of these missing values is small, as there is no missing data for stress perfusion which was the primary aim of this study. Furthermore, perfusion values of the volunteers were similar to previously reported normal values50, thus possibly reflecting healthy perfusion values. Further multi-center studies in larger groups both at admission and during long-term follow-up are needed to further elucidate the role of CMD in MINOCA, and the results of this study may be considered as hypothesis generating for future studies to come.Fully quantitative stress CMR perfusion mapping avoids risks and limitations related to CAG and positron emission tomography (PET), the current gold-standard modalities for assessing CMDGlobal stress perfusion is lower in MINOCA patients with a normal initial CMR scan, during long-term follow-up, compared to age- and sex-matched volunteers with similar cardiovascular risk factors but without a history of IHD. Therefore, it is possible that CMD may be a pathophysiological mechanism in MINOCA.


To correlate 3-D Echo and CMR RV parameters and to verify whether they are similarly related to the clinical conditions of patients with pulmonary arterial hypertension (PAH), a disease in which the RV plays a crucial prognostic role.We enrolled 34 consecutive PAH patients followed by our PAH clinics. All patients underwent a 3-D Echo and CMR assessment of RV volumes and functions in the same day. The presence or absence of correlation between major findings was investigated; functional RV parameters were also analyzed in relation to 6-min walking test (6MWT) results and BNP/Nt-proBNP plasma levels. Twenty-four subjects served as controls.r = 0.72, P < 0.0001), RV-end-systolic volume (ESV) ] and function  for all the subjects of the study. These correlations were stronger in PAH patients than in control subjects. Importantly, 3-D Echo and CMR RV-EF and RV to pulmonary arterial coupling (RV-ESV/SV) similarly correlated with BNP/Nt-proBNP levels and with functional capacity measured at 6MWT in the PAH patients group.Good agreement was found between 3-D Echo and CMR measures of RV volumes [RV-end-diastolic volume (3-D Echo demonstrated a significant agreement with CMR in the assessment of RV volume and function in PAH patients. Both techniques showed a similar correlation with clinical and prognostic parameters. The use of 3-D Echo should be amply boosted in the real-world clinical evaluation of PAH patients. Demographic characteristics (age and sex), clinical data , classification of PAH and laboratory findings (BNP and NT-proBNP) were recorded, when available. Both walking endurance and aerobic capacity data were acquired from a 6-min walking test (6MWT), whereas hemodynamic data  were derived from RHC; lastly, information about PAH-specific therapy and the number of drugs were also obtained , a wide waist circumference (>150 cm) or a weight greater than 130 kg, arrhythmias such as atrial fibrillation, frequent ventricular extrasystoles or ventricular tachycardia, claustrophobia, stage 4 and 5 CKD (eGFR < 30 mL/min).The investigation conforms to the principles outlined in the Declaration of Helsinki. The Ethical Committees gave the approval for the analysis of the RELY registry . All patients signed an informed consent agreement approved by the Institutional Review Board of both centers for observational, nonpharmacological, nonsponsored studies, which complies with Italian legislation on privacy .A complete transthoracic echocardiogram was performed with an ultrasound system Vivid E95  Healthcare, Little Chalfont, United Kingdom), whereas the postprocessing analysis of data was conducted using the pc-software GE EchoPAC. Both echocardiographic acquisition and offline analysis were performed by two experienced cardiologists and echocardiographers; for each patient, the acquisition of the images was repeated several times  to minimize acquisition errors and to enhance image quality. The analysis of 28 3-D Echo was performed by three different experienced cardiologists in order to calculate the inter-observer variability. Intra-observer variability was performed by the same three experienced cardiologists on offline data at different times.From a practical point of view, after performing a full two-dimensional echocardiographic examination, the 3-D ultrasound examination of the RV was conducted through the transducer GE Healthcare 4V-D Probe Collector and two main phases. The first phase consisted of two different steps:(1)Image acquisition was performed with the probe placed on the left hemithorax at the level of the fifth or sixth intercostal space, with the ultrasound beam directed toward the right shoulder and the transducer marker pointing toward the left side; in this way, an apical four-chamber view was obtained.(2)With the focus shifted to the right ventricle (RV) and a widening of the ultrasound beam sector, a ‘multislice’ and ‘multibeat’ 3-D acquisition was performed with a frame rate of four frames per second.Then there was the second phase performed offline in the absence of the patient, which began with the transfer of the images from the ultrasound system to a separate pc-workstation. All the images were processed using the GE EchoPAC software.(1)In the apical four-chamber, three-chamber, two-chamber and short-axis view some landmarks necessary for the correct interpretation of the images by the software have been identified  Fig. a.(2)Then there was the ‘Beutel revision phase’ consisting of endocardial edge detection of the RV in the end-diastolic phase by the software; since that process has not always been particularly accurate, a manual review of each measurement was performed Fig. b.(3)In the so-called ‘tracking revision phase’, the intracavitary perimeter was identified by the software during the entire cardiac cycle Fig. c.(4)Finally, based on the measurements made, the software quantified the volumes of the RV and produced a 3-D reconstruction of the chamber Fig. d.For each patient, the RV measurements provided by 3-D Echo were: end-diastolic volume , end-systolic volume , stroke volume  and EF (%).2), ESV indexed , indexed SV ] and to calculate cardiac output  and cardiac index .Knowing the patient's weight, height and heart rate, it was also possible to index all the parameters for the BSA [EDV indexed  with a phased array surface coil and ECG-gated sequences.In all patients a noncontrast evaluation was performed; after the T2-based sequences, a steady-state free precession (SSFP) cine (end-diastolic) analysis was achieved of both ventricles.All the images obtained were postprocessed and analyzed by an expert CMR radiologist on a pc-workstation Siemens Leonardo . The global biventricular function and volumes were determined following the manual identification of the left ventricle endocardial and epicardial edges and RV endocardial edge, both in the end-diastolic and end-systolic phase, on contiguous 8-mm-thick sections in short-axis view Fig. .2), ESVi (mL/m2), SVi (mL/m2), CI (L/min/m2).The parameters provided by the software were: EDV (mL), ESV (mL), SV (mL), EF (%), CO (L/min), EDVi (mL/mP was <0.05. A Bland–Altman plot (difference plot) was used to analyze the agreement between 3-D Echo and CMR measurements in PAH patients and control subjects. Correlation analysis between imaging parameters and functional capacity was performed by Pearson's test (r) and coefficient of determination (r squared), considering significant values if >0.8.Continuous variables were expressed as mean and standard deviations while discrete variables were expressed as numbers and proportions. Regression analysis using the Least Squares Method was performed, considering a significant regression line slope if the r .All analyses were performed with the ‘STATA12’ software from Stata Corp LLC and MedCalc (version 12.5.0.0). Intra- and inter-observer variabilities were calculated by intra-class correlation coefficient P < 0.001), without evidence of structural heart disease.Overall, 34 PAH patients and 27 controls were included in our study. No subject was excluded. Main baseline characteristics of the patients and control subjects (when available) are noted in Table 3-D Echo and CMR parameters are listed in Table 3-D Echo systematically underestimated RV volumes; the average difference for RV-EDV and RV-ESV was 69.6 and 32 mL, respectively. At regression analysis the data were not well distributed around the identity line ; it was slightly lower for RV-SV  and for RV-EF   and for RV-ESV , but not so for RV-SV  (Table All parameters exhibited good intra- and inter-observed correlation. Intra-observer variability was excellent for the three 3-D Echo measurements of RV-EDV and RV-ESV (respectively: 1) Table . Inter-oThe main result of the present study is the finding of a good agreement between indices of RV function obtained at CMR and 3-D Echo, including not only RV-EF, but also the RV to pulmonary arterial coupling. Importantly, CMR and 3-D Echo parameters of RV function similarly correlated with BNP/NT-proBNP and with functional capacity of PAH patients, thus meaning that the two techniques may portend a similar clinical value in this disease.4 Deriving and validating imaging biomarkers for an accurate evaluation of RV function is considered a major research priority to be addressed in the next 5 years.19 Although 3-D Echo is emerging as an interesting source of prognostically relevant data in PH, the few studies in the literature which have attempted to validate 3-D Echo in adult populations of PAH patients have reported contrasting results.18The background to our study is that, despite extensive literature reporting that several echocardiographic variables may provide significant prognostic information in PAH patients, imaging of the right heart with 2-D Echo (due to the inherent limits) has not yet been considered as providing sufficient prognostic information to serve as an integral part of the treatment goal.In the present study, 3-D Echo substantially underestimated the ESV and EDVs of the RV. This result is consistent with data in the literature obtained in normal subjects and patients with congenital heart disease. Interestingly, the correlation between volume measurement with both techniques was weaker in the control group rather than in PAH patients. This can be explained if we hypothesize a greater precision in recognizing the endocardial edges of the RV at echocardiography when the RV chamber is dilated and thus the RV trabeculae are better identifiable.As expected, the measurements performed by the same operator showed a linear correlation, very close to perfection. On the other hand, a certain discrepancy of measurements obtained by different operators (inter-observer variability) emerged, especially as regards SV, EDV and ESV but the inter-observer correlation of the RV-EF proved to be good.20The discrepancy in volume measurements can be explained by the poor resolution of the echocardiographic images of some patients, which makes difficult the tracing of endocardial edges, especially in the end-diastolic phase of the cardiac cycle. On the contrary, the accuracy between the two techniques was substantially greater in the assessment of RV functional measurements, RV-EF and RV to arterial coupling. This can be explained considering that discrepancies occurring both in end-diastole and in end-systole are eliminated when both are included in a ratio, as in the calculation of EF and of SV/ESV. Importantly, with the limit that the present population includes a relatively small number of subjects, the analysis shows that there is no systematic bias in the measurement of EF and ESV/SV with 3-D Echo as compared to CMR. To substantiate the clinical validity of 3-D Echo measurements is the observation that 3-D and CMR parameters similarly correlated with the distance walked at 6MWT and BNP/NT-proBNP plasma levels. The correlations were better with BNP/NT-proBNP plasma levels than with the distance walked at the 6MWT, most likely because exercise capacity does not depend only on RV function, but also on several other factors such as age, sex, physical deconditioning and comorbidities. Overall, these correlations were quite poor; however, the relation between 6MWT and clinical, hemodynamic, and neurohumoral parameters in PAH patients has never been reported to be very robust, even though the distance walked is a well known predictor of survival in PAH patients.The take-home message of the study may be clinically relevant because there is a widespread agreement in the literature that prognosis in PAH patients is strictly dependent on the function of the RV, but despite the convincing and long-standing evidence that CMR is the gold standard for morphological and functional assessment of the RV, CMR is not part of the routine clinical practice to date in patients with PAH. The lower costs and the ease of access to the examination make echocardiography the most widely used imaging technique in PAH patients in the real world.The small number of patients enrolled in this study is a limitation; however, it must be considered that PAH is a rare disease. Although there was a significant difference in age between PAH patients and controls, this difference should not impact the correlation between imaging of the RV with the two techniques. Finally, the study did not aim at identifying which index of RV function (RV-EF or RV-SV/ESV) is a better predictor of the clinical conditions or of the exercise capacity in PAH patients; the clinical value of each of these indices of RV function in PAH was out of the scope of the present study.3-D Echo demonstrated a good agreement with CMR in the assessment of RV volumes and in particular of RV function in PAH patients. Both techniques showed a similar correlation with other clinical prognostic parameters. The use of 3-D Echo should be amply boosted in the real-world clinical evaluation of PAH patients.R.D.P. has received lecture fees from Biosense Webster and Biotronik and his Institution has received an educational grant from Medtronic, Biotronik, Boston Scientific, Biosense Webster, Abbott.None for the other authors.


Most studies on single ventricle (SV) circulation take a physiological or anatomical approach. Although there is a tight coupling between cardiac contractility and metabolism, the metabolic perspective on this patient population is very recent. Early findings point to major metabolic disturbances, with both impaired glucose and fatty acid oxidation in the cardiomyocytes. Additionally, Fontan patients have systemic metabolic derangements such as abnormal glucose metabolism and hypocholesterolemia. Our literature review compares the metabolism of patients with a SV circulation after Fontan palliation with that of patients with a healthy biventricular (BV) heart, or different subtypes of a failing BV heart, by Pubmed review of the literature on cardiac metabolism, Fontan failure, heart failure (HF), ketosis, metabolism published in English from 1939 to 2023. Early evidence demonstrates that SV circulation is not only a hemodynamic burden requiring staged palliation, but also a metabolic issue with alterations similar to what is known for HF in a BV circulation. Alterations of fatty acid and glucose oxidation were found, resulting in metabolic instability and impaired energy production. As reported for patients with BV HF, stimulating ketone oxidation may be an effective treatment strategy for HF in these patients. Few but promising clinical trials have been conducted thus far to evaluate therapeutic ketosis with HF using a variety of instruments, including ketogenic diet, ketone esters, and sodium-glucose co-transporter-2 (SGLT2) inhibitors. An initial trial on a small cohort demonstrated favorable outcomes for Fontan patients treated with SGLT2 inhibitors. Therapeutic ketosis is worth considering in the treatment of Fontan patients, as ketones positively affect not only the myocardial energy metabolism, but also the global Fontan physiopathology. Induced ketosis seems promising as a concerted therapeutic strategy. Children with complex congenital heart disease (CHD) and single ventricle (SV) physiology typically undergo several-step surgical palliation with the aim of a total cavopulmonary connection. In this so-called Fontan circulation, the subpulmonary pump is missing. Instead, the vena cavae are anastomosed directly to the pulmonary arteries, causing elevated systemic venous pressure and chronically decreased cardiac output ,2,3,4.Even if the outcome of SV patients is steadily improving, particularly their largest subgroup—i.e., SV patients with aortic atresia and a hypoplastic left heart  and a mPharmacological interventions for both patients with a failing biventricular (BV) and patients with a failing SV heart currently target either hemodynamics or the neuro-hormonal axis, with conflicting evidence regarding the clinical benefit ,22,23,24Reviewing the literature, we compare the energy metabolism in patients with healthy, or with a failing BV heart with that reported in SV patients after Fontan palliation. Focusing on lipid and ketone metabolism, and focusing on the role of therapeutic ketosis in BV HF, a potential role of metabolism-targeted therapy in Fontan patients will be discussed.The heart is a biomechanical pump with complex hemodynamics. Ninety percent of the cellular adenosine triphosphate (ATP) is used to sustain the contraction-relaxation of the cardiac muscle . MitochoThe “omnivore heart” and its diverse substrate consumption. Cardiomyocytes have the capacity to oxidize fatty acids, carbohydrates, amino acids, ketones, and lactate [ lactate . The sub lactate , and dep lactate ,54. Chan lactate , reachin lactate . Due to  lactate ,58  through pyruvate or beta-oxidation. Acetyl-CoA is fed into the Krebs cycle. Under normal conditions, mitochondrial oxidative phosphorylation matches 90–95% of the myocardial ATP demand with glycolysis filling the gap [ the gap .From ATP to contractile work: creatine phosphate reserve of ATP. The creatine kinase (CK) energy shuttle plays a crucial role in maintaining energy balance in cells with high and fluctuating energy demands, such as cardiomyocytes: It comprises the conversion of creatine to phosphocreatine (and back), which serves as a rapid and reversible energy storage system. This pathway enables the efficient transfer of energy from the mitochondria, where ATP is produced by oxidative phosphorylation, to the cytosol, where it is consumed during contraction.Regulation of metabolism through gene expression. Control and regulation of cardiac metabolism are complex and include overexpression or deletion of metabolically relevant proteins, such as receptors, regulators, transporters, or enzymes.Peroxisome proliferator activated receptors. PPAR are a family of nuclear receptor proteins that play a crucial role in regulating the expression of genes involved in lipid and glucose metabolism, inflammation, and cellular differentiation. PPAR is an abundantly expressed key regulator of cardiac substrate switching [witching ,106,107,witching . PPAR wawitching .Adenosine monophosphate-activated protein kinase pathway, GLUT and CPT. The adenosine monophosphate-activated protein kinase (AMPK) pathway is a critical cellular signaling pathway that plays a crucial role in regulating energy homeostasis in cells. The activation of the AMPK pathway leads to the phosphorylation of numerous downstream targets involved in metabolism, transcription, and protein synthesis. Through this pathway, CPT1, medium-chain acyl-CoA dehydrogenase, cluster of differentiation 36, and fatty acid transport protein 1 are decreased in HF [ed in HF . Inhibited in HF .Histone acetylation/deacetylation. Acetylation is a protein post-translational modification controlling expression and transcription of genes, regulating embryonic development, postnatal fatty acid oxidation maturation, and heart hypertrophy. It enables the cell to quickly and effectively react to cellular stress [r stress . Histoner stress . Histoner stress . HDAC car stress . All tisr stress ,114. Actr stress .Disturbed substrate consumption. Once the heart has reached its metabolic adulthood, the main substrates used for ATP production are fatty acids (60–70%), followed by pyruvate (glucose/lactate), ketone bodies, and amino acids. In early stages of HF, myocardial fatty acid utilization may be unchanged or augmented. In advanced stages, the myocytes switch from fatty acid to glucose oxidation, returning to a fetal pattern of energy substrate metabolism [tabolism ,59,60  ,73,74,75Reduced energy production. In the failing BV heart, electron transport chain activity is altered  and tring in HF .Insufficient energy transfer to ATP-consuming reactions. CK energy transfer shuttle works by transferring high-energy phosphate groups from creatine phosphate to ADP to produce ATP. It was the initial mechanism of energy starvation discovered in HF, with creatine deficiency [ficiency : In HF, ficiency ,77,78,79ficiency . Reducedficiency . Acting ficiency ,123.Epigenetic and transcriptional changes: reactivation of fetal gene expression in the failing BV heart. The activation of fetal cardiac genes which encode proteins involved in contraction, calcium management, and metabolism, is associated with pathological heart hypertrophy. A deterioration in heart function is associated with such reprogramming. Conversely, improvement in cardiac function is associated with normalization of cardiac gene expression in the failing heart [ng heart . The matng heart . Lysine ng heart . In presng heart . Histoneng heart . The HDAng heart . Anotherng heart . Both clMetabolomics as a bridge between gene and metabolism. Metabolomics is more and more used to analyze metabolites and intermediates in cardiology [rdiology , as wellrdiology ,133. IntKetolytic metabolism was described as being increased in HF . In atri2+ [In fetal circulation, the right ventricle (RV) is responsible for up to 60% of the cardiac output . Thickne2+ . The mol2+ ,141. Met2+ . Dysfunc2+ . Additio2+ .RV failure shares with LV failure the perturbation in substrate consumption. Glucose homeostasis as well as fatty acid metabolism are impaired ,147,148.Untypical substrate consumption. PAH is a disease resulting in RV failure. Initially regarded an isolated disease of the pulmonary circulation, evidence is accumulating that PAH is to be considered from the metabolic perspective [spective ,154.Abnormal glucose oxidation. Abnormal glucose metabolism is observed in patients with PAH, even without manifest diabetes mellitus [mellitus ,83,84  was found to correlate with better clinical health in PAH [h in PAH . This ish in PAH . Aarhus h in PAH . There ah in PAH .While our article was submitted, a non-reviewed article from the Lillehei heart institute (Minnesota university) describes that compensatory ketosis is absent in RV failure, in contrast to LV failure. The therapeutic stimulation of ketolytic activity is improving RV function, suppresses NLRP3 inflammasome activation and blunts myocardial fibrosis . Those aOxidative stress. The electron transport chain in PAH shows pronounced alterations [erations ,90. Proterations ,92.Decreased creatine kinase shuttle. Alterations in CK were found in diastolic dysfunction in an animal model with RV HF [th RV HF ,157.Alterations in signaling pathways. Histone deacetylase inhibitors are acting differently in LV, or RV hypertrophy [ertrophy ,159. Dueertrophy .Metabolomics findings. In right-sided HF, metabolomics analysis found specifically elevated blood levels of L-carnitine, acetyl-L-carnitine and long-chain acylcarnitine. Alteration of beta-oxidation of fatty acids increases the concentration of acyl-CoA, thus increasing acylcarnitine levels. The conclusion is that an increased level of acylcarnitine may reflect significant inhibition of the mitochondrial fatty acid beta-oxidation in PAH [n in PAH .Altered substrate consumption—glucose oxidation. Pyruvate metabolism might be altered in SV patients, especially in those with HF. One metabolomics study found elevated levels of circulating pyruvate in this group of patients, which might indicate an alteration of pyruvate metabolism and glucose oxidation [xidation   through an increase in AMPK and PPAR-gamma coactivator 1 alpha, and that control of fatty acid oxidation is not impaired in the hearts of HLHS children [children . This suchildren .Surprisingly, in a very recent metabolomics study functional analysis of the mitochondrial CPT system demonstrated significantly decreased activity of the mitochondrial CPT transporters, suggesting that the diminished myocardial acylcarnitine is related to an overall decreased capacity of the failing SV to oxidize long-chain fatty acids, resulting in a diminished rate of cardiac ATP production . Those fn = 14), and the study includes patients with severe atrioventricular valve regurgitation or a history of protein-losing enteropathy.Another recent metabolomics study suggests differences in 2-oxoglutarate, isocitric acid, malic acid, and cis-aconitic acid that could reflect alterations of the Krebs cycle . The autInduced ketolysis in single ventricle physiology—early reports on the use of Sodium-glucose co-transporter-2 inhibitors. The decreased activity of CPT2 in SV patients without HF, and CPT1/CPT2 in SV patients with HF [ with HF , as well with HF  might in with HF ,164. Thi with HF . An alte with HF ,170,171, with HF . Althoug with HF , and is  with HF .While SGLT2 inhibitors’ mechanism still is not completely understood, one of the main hypotheses is that SGLT2 inhibitors are increasing circulating ketone levels . The impInflammation and oxidative stress. In HLHS patients, disorders of cellular respiration are suspected to be present [ present . In an a present ,177. The present . Other i present . We hypo present ,97,98. A present . InteresSystemic metabolic changes. Abnormalities in Fontan patients’ glucose and lipid metabolism as well as in the neurohumoral axis have been reported, and this even in ‘stable’ Fontan patients with good exercise capacity and without signs of imminent Fontan failure [ failure ,184,185, failure . Moreove failure , similarInitially being an intuition in biochemistry , evidencKetones as a substrate for the failing heart. Early experimental studies showed that BHB is not only a fuel, but a super fuel for the heart [he heart ,193,194.he heart . It was he heart . Clinicahe heart ,196,197.Ketones and oxidative stress. Oxidative stress is involved in the progression of congestive HF, showing a positive correlation between elevated oxidative stress and myocardial dysfunction [function ,198,199.function . Oxidatifunction . Reducedfunction .Ketones and inflammation. Inflammation is associated with cardiac remodeling and HF [g and HF ,203,204.g and HF . BHB is g and HF . In ketog and HF . In neurg and HF . Consistg and HF . High-faKetones and mitochondrial respiratory complex activity. In an experimental model, ketogenic diet normalized complex I and improved complex II-III activities in rats [ in rats . This waKetones, myocardial contractility, and ventricular ejection fraction. Infusion of 3-hydroxybutyrate to patients with HF with reduced EF increased the EF by 8% [EF by 8% . At the Ketones, myocardial remodeling, and prevention of cardiac hypertrophy. Inefficient myocardial fuel consumption can cause pathological hypertrophy [ertrophy ,212,213.ertrophy ,214. HDAertrophy . Overexpertrophy . In an eertrophy . In precertrophy . Chronicertrophy . In a siertrophy ,218. Thiertrophy .Ketones, endothelial function, and vascular resistance. Keeping pulmonary vascular resistance low is necessary in Fontan circulation [culation ,220. In culation ,222. Mecculation . Under Bculation ,224. BHBculation . Ketone culation .Antiarrhythmic potential of ketones. Ketone oxidation, membrane excitability, and arrhythmogenesis are interrelated. BHB contributed to a 24% improvement in cardiac efficiency, mitochondrial function and the stabilization of cellular membrane potential, enhancing the antiarrhythmic potential of the myocardial cell [ial cell .Ketones and oxygen consumption. Ketone oxidation spares oxygen consumption and is neuroprotective through two mechanisms, oxygenation improvement and decreased blood carbon dioxide [ dioxide ,227. Com dioxide ,229.Ketones in RV failure. Nutritional ketosis improved PAH through reversal of the metabolic syndrome [syndrome . In a sisyndrome . A clinisyndrome  , or braerations  and odd-erations ,267. Octerations . It is cerations . It is uerations ,271.SV malformation is a rare disease. Most studies on cardiac and systemic metabolism of Fontan patients were conducted on a limited number of patients. The etiology of all SV malformations might not be identical, and metabolism as well as gene transcription might vary. Furthermore, metabolomics results should be treated with caution as interpretation can be complex. Large-scale (multicenter) studies are necessary to further explore the metabolic impairment of the SV heart and the ketones‘ therapeutic potential in the respective patients.Our literature review compares the metabolism of patients with an SV circulation after Fontan palliation with that of patients with a healthy BV heart, or different subtypes of a failing BV heart, by Pubmed review of the literature on cardiac metabolism, Fontan failure, heart failure, ketosis, metabolism, published in English from 1939 to 2023.per se may become a cause of circulatory failure. Evidence accumulates that stimulating ketone oxidation as a targeted metabolic therapy might be a therapeutic strategy to address HF in BV patients. Therapeutic ketosis may be worth considering also in the treatment of Fontan patients, as ketones positively affect not only the myocardial energy metabolism, but also the global Fontan pathophysiology. Induced ketosis seems promising as a therapeutic strategy for chronic ventricular failure and low-grade inflammation, as well as diseased liver, kidney, and intestines. Ketone esters, 1,3 butadeniol, or octanoate might provide a third fuel to the SV heart, and a well-formulated ketogenic diet taking into account the micronutritional status inherent to Fontan patients may have an integrative, concerted effect on the several complications of Fontan circulation.Evidence is growing that SV circulation after Fontan palliation not only is a hemodynamically challenging state, but also raises a metabolic issue with its alterations of fatty acid oxidation as well as glucose oxidation, similar to those reported in the failing BV heart, generating metabolic instability and disturbed energy production that


Bronchial asthma is a global health problem with rising prevalence in developing countries. Children with severe asthma can experience cor pulmonale later in life, but little is known about the cardiac changes that might be present earlier in mild or moderate severity of disease. This study aimed to evaluate biventricular function among children suffering from persistent asthma by Tissue Doppler Echocardiography (TDE).S’ velocity and peak E’) were significantly reduced (14.55 ± 2.30 and 14.69 ± 2.30) versus controls  but with preserved LV function. The lateral tricuspid annulus (S’ velocity and peak E’) was significantly reduced (11.53 ± 3.24 and 11.56 ± 3.18) versus controls , while E/A and IVRT were significantly increased  with impaired RV function. Peak expiratory flow rate (PEFR) was negatively correlated with the IVRT of the tricuspid annulus  and to the E'/A' . All TDE variables of lateral tricuspid annulus of severe subgroups were significantly  changed versus moderate or mild subgroups.Thirty-five asthmatic children enrolled from Alexandria Children’s Hospital between September 2021 to May 2022; they were compared to 35 healthy matched children. Chronic respiratory disease, cardiac disease, or other comorbidities were excluded. The mean age of cases was 8.87 ± 2.03 years with a male/female ratio of 54.3%:45.7%. There were 28.3% mild cases, 45.7% moderate, and 25.7% severe. Conventional echocardiographic variables of cardiac function were normal for both ventricles. The TDE indices of medial mitral annulus (Tissue Doppler echocardiography is the recommended modality for early detection of biventricular cardiac dysfunction among children with different levels of asthma severity. Periodic screening is advised through the use of IVRT especially for RV. Bronchial asthma is a common respiratory disorder among children, worldwide. Asthma is characterized by chronic inflammation and remodeling of the airways induced by recurrent exposure to hypoxemia that leads to repeated tissue injury and repair. The interaction between respiratory diseases and cardiovascular function is complex , 2.Cardiac dysfunction can be attributed to pulmonary hypertension (PH) secondary to recurrent hypoxia in patients with bronchial asthma. PH affects the pulmonary vasculature by releasing various cytokines leading to pulmonary vasoconstriction and enhancing the remodeling process with muscularization and proliferation of the vascular media and intima –5. OtherChildren with severe bronchial asthma can experience cor pulmonale later in life, but little is known about the early cardiac changes that might be present during childhood especially for mild or moderate persistent asthma. Some studies have reported right ventricular dysfunction as the earliest hemodynamic change among those cases , 8. OtheThis was a case–control study conducted at the Main Children’s Hospital from September 2021 to May 2022. Ethical approval was received from the ethical and research committee of Faculty of Medicine No: 0305278. Informed consent was taken from the caregivers of all participants.We calculated the sample size using G Power 3.1.9.7, 2020. Based on a mean TDE mitral E′ velocity among asthmatic patients of 0.17 ± 0.03 and among controls of 0.19 ± 0.03, and a mean TDE tricuspid S′ velocity among asthmatic patients of 0.08 ± 0.022 and among controls of 0.10 ± 0.028 (1), alpha error of 0.05, power of 80%, a ratio of cases to controls of 1:1, the minimum required sample size was calculated to be 68 (34 asthmatic patients and 34 controls). The sample size was calculated based on a previous study [Group I (patients): This group included 35 asthmatic children diagnosed according to updated GINA guidelines 2019 with different levels of asthma severity whether controlled or not controlled on medications [ications . The casInclusion criteria: The age of the enrolled cases ranged between 5 and 15 years (to avoid transient wheezes). The duration of the disease was at least one year. The cases had been on inhalation steroids for at least six months, and the last exacerbation was at least one month. The minimum oxygen saturation was 91%, and the hemoglobin level was 11 g/dl.Exclusion criteria: We excluded patients with acute asthma exacerbation, patients with chronic respiratory comorbidities, e.g., interstitial lung disease, congenital lung anomalies, patients with clinical evidence of right ventricular failure, patients with other comorbid diseases, positive cardiac history of congenital heart disease or acquired heart disease, gastroesophageal reflux, obesity, sleep apnea, and/or anemia or polycythemia.Group II (control): Thirty-five healthy children of matched age and gender were included as controls.The study was conducted over two groups of children.Pulmonary function testsPeak expiratory flow rate was evaluated using a mini-Wright peak flow meter. The best reading from three forced expirations was recorded.Spirometry was measured via forced expiratory volume in 1 s (FEV1).All participants had a history and physical examination. Group I was further subdivided according to degree of asthma severity: mild, moderate, and severe. Transthoracic echocardiography and pulmonary function tests were also done , 12.Pulm2.Echocardiography study Pulmonary function testing (PFT) was performed using CareFusion Germany 234 GmbH pulmonary function apparatus according to new American Thoracic Society (ATS) and European Respiratory Society (ERS) standardization guidelines for performance of spirometry .2.EchocaConventional echocardiographyTransthoracic echocardiography was performed by a single experienced pediatric cardiologist blinded from the respiratory status of the case at the cardiology clinic of the Main Children University Hospital. The studies were performed for cases and controls using a Philips machine HD 11, 2–5 MHz two-phase array imaging transducer. These studies aimed to assess parameters of systolic and diastolic function of both ventricles based on the recommendations of the American Society of Echocardiography. The left and right ventricular functions were assessed via two-dimensional echocardiography: M-mode, color-flow imaging, standard pulsed-wave Doppler and TDE according to Guidelines of the American Society of Echocardiography .ConventiVmax2 × 4) + RAP. Normal systolic arterial pressure is up to 30 mmHg at rest [Tissue Doppler Echocardiography (TDE)Conventional echocardiography  measured the following parameters: ejection fraction , fractional shortening , tricuspid annular plane systolic excursion (TAPSE), E velocity , A velocity , and the E/A ratio of mitral and tricuspid valves. Pulmonary artery pressure was estimated (if possible) through calculations of the Bernoulli equation after measuring tricuspid and pulmonary regurgitation and estimating the pressure gradient. Normal RAP was considered 5 mmHg. PSAP = tricuspid regurgitation gradient + RAP. PSAP = , E' velocity , A' velocity , the E’/A’ ratio, and isovolumetric relaxation time IVRT, from the end of the S-wave to the beginning of E-wave. These were measured at lateral leaflets of mitral and tricuspid annuli and at the medial leaflets of the mitral annulus. At least three clearest successive cycles were used for calculations. These steps were done in addition to estimating medial and lateral mitral annuli pressure .Data were fed to the computer and analyzed using IBM SPSS software package version 20.0 . Qualitative data were described using number and percent. The Kolmogorov–Smirnov test was used to verify the normality of distribution quantitative data described using range (minimum and maximum), mean, standard deviation, and median. The significance of the obtained results was judged at the 5% level. Student’s t test was used for normally distributed quantitative variables and to compare between two studied groups. An F-test (ANOVA) was used for normally distributed quantitative variables and to compare between more than two groups. A post hoc test (Tukey’s test) was used for pairwise comparisons. Pearson’s coefficient was used to correlate between two normally distributed quantitative variables.P < 0.001*). There was no statistically significant difference between mild asthmatics and the control group (224.7 ± 42.7).We enrolled 35 asthmatic children: 19 males (54.3%) and 16 females (45.7%) with a mean age of 8.87 ± 2.03 years. The characteristics of the asthmatic groups and controls were similar in terms of age, gender, weight, height, and body mass index (BMI) as shown in Table P > 0.05) as shown in Tables Conventional echocardiographic variables of LV and RV  such as EF, FS, TAPSE, E velocity, A velocity, and E/A did not differ significantly between patients and controls . Similarly, the E' velocity of the medial mitral annulus (14.69 ± 2.30) was significantly lower for asthmatics than the control group . The E’/A’ ratios at the medial mitral annulus were significantly greater in patients than the control group. The IVRT was significantly (P < 0.001) greater in the asthmatic group at both medial and lateral leaflets of mitral annulus  in relation to control group  as summarized in Table A' velocity and E'/A' ratio did not differ significantly among the studied groups at lateral mitral annulus. There was a positive correlation between S' and E' velocities of medial mitral annulus and PFT (PEFR and FEV1). Negative correlation was found between E/E' and PFT  (P < 0.001) as shown in Fig. Concerning the TDE study of LV at mitral annulus, the mean of the S' and E' velocities at medial mitral annulus and higher E/E' ratios versus controls as well as between mild and moderate disease (Table P < 0.001) for all subgroups even in the mild group versus controls. IVRT was the only parameter of lateral mitral annulus significantly greater among the moderate and severe subgroups when compared to the control group  versus controls , respectively (P = 0.002 and r = −0.503*) (P = 0.036 and r =−0.355*). It also clarified the negative correlation between RV function (S' and E') and LV filling pressure (E/E').Figure E', and/or A' velocity at the tricuspid annulus [S' values (systolic), and 56.2% had abnormal E’ and A’ values (diastolic). Although the IVRT of the mild group was significantly greater than the controls, none of the mild group showed abnormal LV or RV function. In contrast, the LV function indices were still normal for all asthmatic groups.Here, about 90% of severe asthmatics had impaired RV function . They had lower values than the recommended cutoff values for children at either S' wave velocity,  annulus . MeanwhiBronchial asthma is a significant global health problem with rising prevalence especially in developing countries. It increases the cost of care and raises the burden on patients and society , 14. AstE wave velocity, peak A velocity, and E/A ratio) were insignificantly different among asthmatic patients and control cases (P > 0.05) [Cases and controls were properly matched in terms of age, height, and weight with no statistically significant differences in respiratory rate and/or heart rate. This is consistent to the study done by Shedeed et al. and Mahmoud et al. , 15. Pul > 0.05) . On the  > 0.05) . The conS' velocity and peak E)' were significantly reduced, while the IVRT and E/E' were significantly increased in asthmatics versus controls. The only significant change of mitral lateral annulus indices was in prolongation of IVRT among asthmatic. This was consistent with many studies such as Mohammed et al. at 2021 and Zeybek et al. in 2007 [A’ velocity) of both septal and lateral mitral leaflets in the asthmatic group [The LV function was preserved for all cases. Studies of TDE revealed that the medial mitral annulus indices of asthmatics  and the PEFR and FEV1 (P > 0.5). This supports the indirect effect of poor pulmonary function tests in asthmatic children linked to the degree of severity of asthma on impairing the preload of the heart and hence the diastolic and/or systolic dysfunction.We found a positive correlation between A' velocity of tricuspid lateral annulus of asthmatic group versus controls. There was a significant increase in IVRT and E'/A' versus the control group . These findings result in subclinical RV dysfunction among 74% of the asthmatic group. These data highlight the role of TDE in early detection of ventricular impairment. Our results are consistent with many studies [The TDE study of RV revealed significant reductions in the S' velocity peak E' and the peak  studies , 18, 19.El Masry et al. suggested that asthmatic children may experience bouts of transient pulmonary hypertension during exacerbations of respiratory symptoms, which may exert a cumulative effect leading to RV hypertrophy and may explain subclinical RV dysfunction . Thus, pP > 0.5) and the E'/A' ratio and IVRT, respectively, for the tricuspid lateral annulus, which reflects the impact of poor pulmonary function tests on diastolic function of RV. These data agree with by Ozdemir et al. who reported the same correlation in asthmatic children [Our study showed a significant negative correlation between PEFR (children .E/E1) of LV and the  of RV. This explains the RV to LV link, referring to the effect of impaired systolic and/or diastolic function of RV on impairing the left ventricular filling pressure, and thus the LV preload [Interestingly, we found a significant negative correlation between the . These results are similar to other studies reporting early RV dysfunction in severe and or moderate asthmatics [The moderate asthmatic patients represented 45.5% of cases. About 56% of them had impaired diastolic function, and 33% had impaired systolic function of RV. The moderate subgroup showed significantly different TDE variables for the lateral tricuspid (thmatics , 9, 18. Here, the IVRT measured by TDE played an essential role for evaluation in many aspects. It is correlated with pulmonary function and is a tool for comparison between asthma subgroups. The IVRT is also an early marker of impaired cardiac performance. Our data for the mild asthmatic subgroup were surprising: A significant prolongation of IVRT was seen for both RV and LV versus controls with preserved biventricular function. This reflects the multiplicity of contributing factors underlying impaired cardiac diastolic or systolic function among asthmatics. The severity of chronic asthma is not the only risk for cardiac dysfunction—even mild asthmatics are not safe. We noted impairments in mild patients might be attributed to previously poor controlled severe exacerbations. These impairments might affect the cardiac function during severe exacerbation and remain hidden until our study. Our results were similar to De-Paula et al. and Ozde , 24. In Cardiac dysfunction was detected by Tissue Doppler Echocardiography rather than conventional echocardiography in chronic asthmatic children. Right ventricular dysfunction was earlier and more aggressive than LV dysfunction and directly related to poor pulmonary function tests. Therefore, TDE is the recommended imaging modality in asthmatic patients regardless of the degree of asthma severity. These cases should be screened by IVRT measured by TDE together with regular PEFR for early detection of cardiac and pulmonary dysfunction because these metrics are significantly and negatively correlated with each other and also easy to perform.


Following bipolar voltage mapping, AMA was utilized to reveal zones of decelerated conduction velocity vectors (CVV) and this information was superimposed onto the 3D reconstructions and compared with the presence of scar. Mapping time was 28.1 ± 10 min for the endocardial reconstruction of the left ventricle (LV) and 17 ± 5.4 min for the epicardium (n = 6 patients). The mean area of LV low voltage was 13.9 ± 15%  and 11.9 ± 5.7% . Decelerating CVV zones were revealed in all patients (mean conduction velocity threshold of 39.3 ± 13%). Sustained VA have been terminated through ablation and substrate modification was performed in all patients. Correlation between the presence of CVV deceleration zones and areas of abnormal low voltage from bipolar mapping was revealed in only 37.5% of patients, but there was good correlation between scar from unipolar voltage mapping and the presence of CCV deceleration zones . The novel AMA may improve the understanding of individual VA substrates due to the visualization of decelerated CVV zones and their correlation with abnormal low voltage predominantly from unipolar mapping.A new automated vector-based mapping algorithm (AMA) for 3-dimensional (3D) mapping has been introduced. The aim of this study was to present our experience using AMA to recognize additional catheter ablation targets in patients with ventricular arrhythmias (VA). A total of 16 patients (ICM; ischemic cardiomyopathy,  A new automated mapping algorithm (AMA) for 3-dimensional electroanatomical mapping  has recently been introduced . This novel feature aims to visualize the electric-wave propagation over the myocardium by means of color-coding and direction vectors , 2, to iVentricular tachycardia (VT) is a major health issue in patients with structural heart disease and myocardial scar as well as fibrosis play a crucial role in the genesis and maintenance of VAs. The coexistence of surviving myocardial fibers within scarred fibrotic tissue leads to the formation of slow conduction zones and to a dispersion of activation and refractoriness which contributes to a milieu for tachycardia circuits. AMA might have the potential to discriminate corresponding regions of interest within the scar tissue or at the border zone between healthy and scarred tissue to assist VT ablation and to define potential additional ablation targets.The aim of this proof of concept manuscript was to present our initial experience using AMA to guide the mapping process in patients with VAs. AMA was utilized to visualize potential ablation targets beyond the arrhythmia substrate from ultra-high-density mapping in multiple VT etiologies. The novel mapping approach aimed to improve the understanding of the slowest conduction zones during sinus rhythm or right-ventricular pacing according to an adjusted conduction velocity vector analysis.This prospective observational study included consecutive patients undergoing VA ablation guided by CARTO PRIME and the novel AMA at our center between February 2020 and June 2021. All patients signed a written informed consent. Prior to ablation, all patients received transthoracic echocardiography to rule out LV-thrombus formation. Transesophageal echocardiography (TEE) was performed in patients with elevated risk for left atrial (LA) or LA appendage (LAA) thrombus. The ICD/CRT devices were interrogated prior to ablation and VA therapies have been deactivation during the ablation procedure.Catheter ablation was performed under general anesthesia. Two diagnostic catheters were introduced via the femoral veins and positioned in the coronary sinus  and the right ventricle (RV) . For an antegrade approach, venous access was obtained via the right femoral vein. A single transseptal puncture was performed under fluoroscopic guidance using a modified Brockenbrough technique and an 8.5-Fr transseptal sheath  and an additional retrograde access to the LV was obtained via the right femoral artery.Endo- and epicardial mapping was performed using a 3D-mapping system  and a multipolar mapping catheter . Ultra-high density electroanatomical reconstruction of the RV and LV was conducted aiming for > 1000 mapping points. A low-voltage area suggestive for endocardial myocardial scar was defined as endocardial bipolar voltage of 0.1–1.5 mV. A low-voltage area suggestive for epicardial myocardial scar was defined as epicardial bipolar voltage of 0.1–1.5 mV and endocardial unipolar voltage of 5–8 mV. Tissue Proximity Indication applied in all cases of LV and RV mapping.The amount of LA low voltage (from uni- and bipolar mapping) was measured using the area measurement tool. For the epicardial approach, access was gained via a subxiphoid anterior puncture using a micropuncture needle followed by the introduction of a guidewire. Afterwards a steerable sheath  was inserted into the epicardial space. Prior to ablation, the course of the left-sided phrenic nerve was constituted using pacemapping.Following bipolar voltage mapping, the AMA algorithm was utilized to reveal zones of decelerated conduction velocity vectors (CVV) based on manual thresholding enabling the dynamic view function. The novel AMA has been previously described in detail –4. The tRadiofrequency current (RFC) was delivered in the power-controlled mode with a maximum power of 40 W, a maximum temperature of 43 °C, and a flow rate of 30 mL/min using an open-irrigated tip-ablation catheter . LV and RV ultra-high-density mapping was conducted as described above followed by programmed ventricular stimulation when the patient was not in sustained VT at the beginning of the procedure. When VA was sustained and hemodynamically tolerated, activation map was conducted, and critical isthmus sites were identified using entrainment mapping. If no sustained VA was inducible, comprehensive substrate ablation was performed targeting all low-voltage areas, as well as abnormal electrograms such as local abnormal ventricular activity (LAVA) and late potentials (LP). After ablation, pericardial effusion was ruled out and patients were monitored at our intensive care unit (ICU) for more than 24 h. The ICD/CRT device was reactivated before hospital discharge.Patients were routinely followed up in our outpatient clinic at 3, 6 and 12 months. In addition, continuous device (ICD/CRT) interrogations were performed via telemedicine. At each follow-up visit, symptom-specific interviews, physical examination, echocardiography and on-site ICD/CRT interrogations were performed. In case of recurrent VA following catheter ablation, all episodes were obtained and analyzed by the treating physicians and redo-ablation was scheduled if applicable.P value smaller than 0.05 were consider significant.Statistical analysis was performed using SPSS for Windows . We performed descriptive statistics using Mann–Whitney U-test and chi square test. The values are given as mean and Standard deviation. A n = 7 transvenous ICD; n = 1 subcutaneous ICD; n = 8 CRT-D). The patients’ baseline characteristics and are summarized in Table A total number of 16 male patients with a median age of 66.1 ± 12.4 years suffering from VA underwent catheter ablation in our hospital. The cohort consisted of six patients with history of ischemic cardiomyopathy (ICM) and ten patients with non-ischemic cardiomyopathy (NICM). All patients were scheduled for VT ablation due to drug-refractory ventricular arrhythmias and/or electrical storm and all patients have had a history of repetitive ICD therapies. No previous VA ablation was performed. At the time of ablation, all patients were on ongoing optimal antiarrhythmic drug therapy. The mean LVEF at the time of ablation was 34.3 ± 13.9%. All patients have had an ICD . The mean area of low-voltage was 13.9 ± 15% from endocardial mapping and 11.9 ± 5.7% from epicardial mapping. Decelerating CVV zones were revealed in all patients using a mean conduction velocity threshold of 39.3 ± 13%. The individual distribution of low voltage suggestive for scarred tissue and their relationship with areas of slow conduction from AMA is demonstrated in Table Mean procedure duration (skin-to-skin) was 160 ± 26.9 min, mean fluoroscopy time was 11.8 ± 6.2 min. and mean fluoroscopy dose was 2.078 ± 2.749 Substrate modification targeting all late potentials and local abnormal ventricular activation (LAVAs) in scar border zones were performed in all cases. The findings from AMA were also integrated into the diagnostic process when there was its match with low-voltage area from uni- or bipolar high density mapping. Mean radiofrequency (RF) ablation time was 39.8 ± 16 min. We observed no procedure related complications requiring intervention. After follow-up (16 ± 5.1 months) three patients from NICM group (30%) and two patients from ICM (33%) group had VT recurrence.The correlation between CVV deceleration zones from AMA and areas of abnormal low-voltage from endocardial uni- and bipolar mapping was analyzed as follows:p = 0.0008), , Table .. In addDespite these findings inside the scarred arrhythmia substrate, additional zones of decelerated CVV haven been observed also in close relationship to the border area between normal and scarred myocardium especially in endocardial bipolar maps of ICM patients Fig. . In DCM This study has following major findings: first, AMA mapping has been easily integrated into a routine workflow and appears to be useful. Second, areas of decelerated CCV have been revealed in all patients. Third, distribution and location of decelerated CCV areas differ in patients with ICM and DCM. Fourth, correlation between the findings from AMA and the individual arrhythmia substrate was superior when utilizing the information from unipolar mapping.Recently, Anter et al. demonstrated, that AMA resulted in improved identification of atrial arrhythmia substrates and origin in terms of focal and localized reentry. AMA also identified a higher number of localized reentrant circuits that were incorrectly identified as focal tachycardias by the standard activation mapping algorithms . This isIn this context, Hoshiyama et al. demonstrated that slow CVV zones matched arrhythmia substrates in a patient with multiple VTs. The authors concluded that AMA might help to understand the relationship between electrograms and critical slow conduction areas, even in multiple and complex VTs .Based on the reported findings mentioned above –3, 7, weA potential explanation for this finding can be the fact that unipolar voltage mapping is suggested to improve the visualization of scar tissue especially in the midmyocardial or epicardal layer. To create a global pattern of activation in the chamber of interest, coherent mapping uses anatomical data and LAT data using both unipolar and bipolar electrograms based on the Wavefront algorithm and thus can show areas of slow conduction in all layers of the myocardium.Moreover, information from unipolar voltage mapping can visualize areas representing the extension and border of dense endocardial scar tissue resulting in an improved understanding of intramural or even epicardial exits of individual VT circuits , 6. An aThe present study is of observational design and therefore has typical limitations. The patient cohort is relatively small and not all patients received epicardial mapping. Nevertheless, this study is the first to provide information on CVV zones based on AMA in patients with VAs. This study was provided for assessment of the new AMA as an additional tool for better understanding of the VT substrate. Therefore, ablation strategy was not changed according to the AMA findings in this study cohort and will be investigated in further studies.The novel AMA improves the understanding of individual VA substrates due to the visualization of decelerated CVV zones and their correlation with abnormal low voltage predominantly from unipolar mapping. The utilization of AMA in conjunction with evidence for scar tissue might result in additional intramural or epicardial targets for VA ablation and improved freedom from arrhythmia recurrence.


Left atrial ligation (LAL) of the chick embryonic heart is a model of the hypoplastic left heart syndrome (HLHS) where a purely mechanical intervention without genetic or pharmacological manipulation is employed to initiate cardiac malformation. It is thus a key model for understanding the biomechanical origins of HLHS. However, its myocardial mechanics and subsequent gene expressions are not well-understood. We performed finite element (FE) modeling and single-cell RNA sequencing to address this. 4D high-frequency ultrasound imaging of chick embryonic hearts at HH25 (ED 4.5) were obtained for both LAL and control. Motion tracking was performed to quantify strains. Image-based FE modeling was conducted, using the direction of the smallest strain eigenvector as the orientations of contractions, the Guccione active tension model and a Fung-type transversely isotropic passive stiffness model that was determined via micro-pipette aspiration. Single-cell RNA sequencing of left ventricle (LV) heart tissues was performed for normal and LAL embryos at HH30 (ED 6.5) and differentially expressed genes (DEG) were identified.After LAL, LV thickness increased by 33%, strains in the myofiber direction increased by 42%, while stresses in the myofiber direction decreased by 50%. These were likely related to the reduction in ventricular preload and underloading of the LV due to LAL. RNA-seq data revealed potentially related DEG in myocytes, including mechano-sensing genes , myosin contractility genes , calcium signaling genes , and genes related to fibrosis and fibroelastosis . We elucidated the changes to the myocardial biomechanics brought by LAL and the corresponding changes to myocyte gene expressions. These data may be useful in identifying the mechanobiological pathways of HLHS.The online version contains supplementary material available at 10.1007/s10439-023-03187-0. Hypoplastic left heart syndrome (HLHS) is a congenital heart malformation characterized by an underdevelopment of the left ventricle (LV) and ascending aorta. Thus, no blood is supplied to the systemic circulation. This is a clinically significant disease as it accounts for 2–3% of congenital heart malformations , 32 and The etiology of HLHS is currently unclear. It is known that genetic faults cause a minority of congenital heart malformations , and offLeft atrial ligation (LAL) of the chick embryonic heart at early development is a model of HLHS . The ligHemodynamics of the chick embryonic heart following LAL has previous been investigated by Salman et al. , who fouHowever, on top of flow biomechanics, the resulting malformation is also likely to be associated with myocardial biomechanics due to LAL, which needs further studies. Tobita et al.  measuredA previous study have also attempted to characterize the gene expressions in the LV after LAL through microarray analysis, to assist in pinpointing the mechanobiological pathways that may be causing LV hypoplasia . A totalIn the present study, we analyzed the biomechanics of the normal and LAL myocardium via analysis of myocardial strain function derived from the 2D stacks of high-frequency ultrasound images of embryonic hearts and cardiac finite element (FE) modeling. Further, we performed single-cell RNA sequencing to obtain gene expressions that are potentially important to the mechanobiology leading to the malformation. Our study aims to improve the understanding of biomechanical origins of HLHS, to identify potentially new pharmacological gene targets to treat HLHS, and to inform fetal heart interventions on how to rescue abnormal biomechanics.Fertilized White Leghorn chicken eggs were used to obtain embryonic heart images at HH25 (embryonic day 4.5). Animal data were obtained from our previous studies , 19, whiThe experimental procedures for performing LAL and imaging chick embryonic hearts were provided in our previous publications , 19. Dat® 100 ultrasound transducer gel  was placed over the polyurethane membrane without contacting the embryo. The egg was held by a holder secured onto a micro-adjustable stage, which was used to manually shift the position of the embryo at accurate translational distances. A 75 W infrared heat lamp was placed close to the egg to maintain body temperature during scanning. B-mode images were acquired in a plane-by-plane manner with a regular spacing of 50 microns to cover the 3D space at and around the heart. At each plane, more than 20 cardiac cycles were imaged. This thus formed a stack of 2D cine-images covering a 3D space.Imaging was performed via a high-frequency ultrasound  with the MS700 transducer (30–70 MHz), with the experimental setup shown in Fig. ®. Resulting segmentations are in Supplementary Fig. 1. From the segmentation, an estimate of the average wall thickness of the left and right side of the common ventricle was obtained via manual measurements at 30 random uniformly distributed locations per chamber.From the images, whole image auto-correlation was used to synchronize timing of different frames to obtain the cardiac phase, while whole image cross-correlation was used to synchronize the timing of neighboring image planes as previously described , 19. No https://github.com/WeiXuanChan/motionSegmentation. Validation was performed on human adult echocardiography images with MRI truths, chick embryonic heart ultrasound images and zebrafish embryonic heart microscopy images. Figure A validated cardiac motion estimation algorithm was used to extract cardiac motions from the arithmetic ensemble averaged echo images . which iGallus gallus domesticus) were incubated at 37.5 °C and 65% relative humidity until HH 25 (4.5 days), when the embryonic heart was extracted and washed several times with PBS. The heart tissue was immobilized into the petri dish using syringe needle and it was placed under an optical coherence tomography (OCT)  set-up. A glass capillary pipette (inner radius of 60 μm) controlled by a micromanipulator was brought to the epicardial surface of the ventricle, and vacuum pressure was incrementally applied via a calibrated pipettor (pressure range of 0 to − 60 mmHg). Vacuum pressures were recorded with a servo-null pressure system  in synchrony with OCT images acquisition, and OCT images were analyzed with ImageJ . Seurat plots for each cluster (N = 4) is provided in Supplementary Document 1 at 10.6084/m9.figshare.22227772.v2. Cell type annotation was based on detected differentially expressed markers and informative terms from the GO enrichment analysis (FDR < 5%). Out of 391 cells , 130 were endothelial cells, 171 were myocardial cells, 59 were fibroblasts, and the rest were “others”.Single-cell RNA sequencing of chick embryonic heart ventricles for normal and LAL embryos at HH30 (6.5 days) was performed at the Genome Institute of Singapore. This embryonic stage is chosen as it is the known timing of the manifestation of the hypoplastic LV morphology. Tissue dissociation and sorting into single cells were done within 1 h of harvest, and cells were then frozen until all samples were collected, before sequencing is performed. Left ventricle tissue were extracted from embryonic heart and dissociated with Papain Dissociation System Kit . The left ventricle tissue was immersed in 500 μl of Papain-DNAse I solution (from the Papain kit) and incubated on a thermomixer at 30 °C and 600 rpm for 45 min and every 15 min to promote dissociation. The Fluidigm  automated microfluidic C1 system was used to isolate single dissociated cells. DAPI were added to the dissociated cardiac cells and were sorted with Fluidigm microfluidic chip. Wells in the Fluidigm chip were inspected via fluorescence microscopy and visual QC was performed to exclude potential doublets or debris captured in the chip capture sites. Single-cell whole-transcriptomic sequencing was performed with the Illumina Hiseq 2500 system (USA). Barcoding of cDNA of each cell was performed such that we could link sequencing results of each cell to the source of the sample. At the read level, our QC pipeline excluded cells with low sequence quality, < 50,000 reads or < 1000 detected genes. 391 cells out of 460 cells (85%) were kept. The data were normalized by library size using Seurat’s log-normalization model “others”  and the p = 0.0286), but mean RV wall thickness did not show statistically significant difference between LAL and controls .Figure p = 0.0286) and the RV , suggesting that contractile deformations were greater in LAL LV than normal LV.A representative reconstruction of the ventricular myocardium is shown in Fig. From the MPA measurements, the best matching C parameter in the Fung strain energy function Eq.  was founTmax) required for this was 6833 ± 378 Pa for LAL hearts, which was 10% less than that for healthy hearts, 7287 ± 725 Pa, even though this was not statistically significant   and RV , suggesting that LAL reduced the stresses experienced by the myocardium.Figure https://doi.org/10.6084/m9.figshare.22227772.v2). As our current study focus on tissue biomechanics, we analyze only differentially expressed genes for myocytes. However, gene expressions for all cell types are given in Supplementary Document 2. Among the list, we found several differentially expressed genes that were relevant to stress and stress sensing in the myocardium, including focal adhesion genes ITGα and ITGβ, PTK2, SRC and α-actinin, Glypicans, Cadherin and Notch1. Interestingly, NRG1 expression was also altered, collaborating with observed trabeculation compaction changes during LAL [A list of all differentially expressed genes between normal and LAL LV myocardial cells at HH30 is given in the Table ring LAL . There wring LAL . We alsoIn the first part of the current study, we performed biomechanical analysis of the chick embryonic ventricle during LAL at HH25, to understand alterations to the myocardial biomechanical loading and deformational characteristics that might be contributing to the hypoplastic LV in the animal model. There has been much interest in inferring myocardial biomechanics of this animal model via imaging, histology, and in vivo manipulations in the past , 43, 46.Our results indicated that with LAL, LV wall thickness increased while RV wall thickness slightly remain unchanged, myocardial strains were higher, and myocardial stresses and active tension were lower. In terms of thickness, a previous study by Tobita et al. showed that after LAL and at HH27, the compact layer thickness decreased in the RV but remain unchanged in the LV , but meaWe propose that our observations can be explained as follows. The reduction of flow from the atria after LAL led to reduced preload and ventricular end-diastolic volume. This led to myofibers being less stretched and thus less engaged in the subsequent contraction phase, in alignment with the Frank-Starling mechanism, thus leading to decreased active tension generation and lower systolic blood pressure. Lower pressures then translated to reduce myocardial stresses. Available data could support this proposal. Our measurements showed that the LAL ventricle was smaller than control ventricle at end-diastole (width of 1.37 ± 0.09 mm versus 1.44 ±0.07 mm) although the difference was not significant, suggesting a mild reduction in ventricular pre-load. Observations in previous studies supported our proposal, Tobita et al. found LAL LV to be significantly smaller in its major axis length with no change to the minor axis length , while SInterestingly, in a previous study investigating the effects of the unloading of mouse hearts via the transplantation of a mouse heart to another’s abdominal aorta, it was observed that consequent to the pressure unloading of the transplanted heart, there were increased fractional shortening, reduced cell sizes, and reduced contractility . In our The above-described biomechanical differences caused by LAL were likely to contribute to altered biological expressions and altered growth and remodeling, leading to the HLHS morphology at HH30. Although one would typically expect that increased LV strain would lead to an enlarged ventricle, however in this case, we speculate that effects of reduced pressure and myocardial stresses dominated, and were the main cause of the LV hypoplasia.Our studies also revealed that the embryonic heart had a statistical uniform distribution in the directions of contraction across the whole ventricle, and that there was not a distinction between LAL and normal ventricle at HH25 in this statistical sense. This corroborated findings of the previous study that utilized confocal imaging to quantify myofiber orientations , which sIn terms of gene expressions, we observed significant upregulation several fibrosis genes, including many types of Collagen, in LAL myocardium. These were in line with past reports of myocardial fibrosis  and incrAn earlier study by Sedmera et al.  on chickRitter et al. showed that increased expression levels of calcium ion signaling genes could be due to the under-loading of cardiac myocytes . This coThe results of the present study are certainly subject to limitations. Firstly, limited experimental sample size used in this study because of substantial time needed for segmentations, motion tracking and FEM simulation for each sample. Second, ventricle trabeculation was not modeled during the tissue segmentation because high-frequency ultrasound did not have sufficient resolution to properly visualize trabeculation details. Thirdly, an assumption was made that the LV and RV had the same passive material properties in the FEM simulations. Fourthly, the biomechanics analysis was done at embryonic day 4.5, while gene analysis was done at embryonic day 6.5. Biomechanics analysis was done earlier because our hypothesis was that biomechanics stimuli preceded and caused disease gene expressions. However, due to the time gap, the biomechanics conditions in the heart might have changed nearer to 6.5 days. We were unable to image the embryonic heart close to 6.5 days as the embryo sank deeper into the egg, out of the field of view of our ultrasound probe. Further, in our cardiac active tension model in FE modeling, some parameters were kept constant between normal and LAL hearts due to a lack of data on them, such as initial sarcomere length, and time to peak tension, potentially leading to errors. Future work on how LAL might have changed these parameters can be conducted.Supplementary file1 (MP4 887 KB)Supplementary file2 (MP4 2841 KB)Supplementary file3 (MP4 3947 KB)Supplementary file4 (DOCX 453 kb)Below is the link to the electronic supplementary material.