To evaluate ventricular synchronization and function in patients with right bundle-branch block after left bundle-branch-area pacing (LBBAP) by echocardiography.Forty patients who successfully received LBBAP were selected and divided into the right bundle-branch block group (RBBB group) and the non-RBBB group by pre-operation ECG. Echocardiography and follow-up were performed 1 month after operation. Interventricular synchronization was evaluated by tissue Doppler (TDI), tissue mitral annular displacement (TMAD), and interventricular mechanical delay. The tricuspid annular plane systolic excursion (TAPSE), right ventricular fractional area change (RVFAC), tricuspid annulus sidewall systolic velocity (TV-s’), left ventricular global ventricular longitudinal strain (GLS), right ventricular free wall longitudinal strain (LS-RV), standard deviation of left ventricular 18 segments peak time difference (SDt-L) and standard deviation of right ventricular free wall 3 segments peak time difference (SDt-R) were applied to evaluate intraventricular synchronization and ventricular function.TV-MV measured by TMAD, the difference of systolic time to peak of the tricuspid and mitral valves, namely ΔTsTV-MV measured by TDI, were statistically different between the two groups (P < 0.05). Compared with the non-RBBB group, there were no statistically significant differences in the GLS, RVFAC, LS-RV, TAPSE, TV-s’, SDt-L, SDt-R (P > 0.05).The difference of displacement peak time of the tricuspid and mitral valves, namely ΔPTEchocardiography technology including two-dimensional speckle tracking imaging (2D-STI), TDI, and TMAD can effectively analyze interventricular synchronization, intraventricular synchronization, and ventricular function. Although the movement of the right ventricular myocardium in the RBBB group was slightly later than that of the left ventricular myocardium after LBBAP, LBBAP could still be applied in RBBB patients with pacing indication.The online version contains supplementary material available at 10.1186/s12872-022-02818-z. Left bundle-branch-area pacing (LBBAP) is a kind of physiological pacing with a low and stable pacing threshold . After LThe ethics committee of The Third People's Hospital of Chengdu approved to carry out the study within its facilities, and all methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all subjects. This study was carried out in Chengdu Third People’s Hospital in patients who were indicated for pacing therapy according to 2013 ESC/EHRA Guideline. The criteria for exclusion were as follows: (1) Patients with left ventricular ejection fraction of less than 50%; (2) Patients with arrhythmia, such as atrial fibrillation; (3) Patients with congenital heart disease, valvular heart disease, and myocardial disease; (4) Unclear acoustic window. Patients completed an electrocardiogram to confirm whether they had RBBB and then were divided into the RBBB group or the non-RBBB group.All 40 patients were implanted with dual-chamber pacemakers using Medtronic RELIA REDR 01 pulse generator. The atrial electrode was fixed in the right auricle, and the ventricular electrode (3830 electrode) was placed transseptally from the right ventricular septum to LV septal subendocardium in the LBB region with the assistance of C315 sheath. The potential of the left bundle branch could be recorded. ‘W’ pattern with the notch closer to nadir in lead V1 may indicate ideal location of left ventricular electrode. After testing the conventional parameters of the pacemaker , we connected the pulse generator.The pre-optimized AVD was determined empirically by the cardiologist. The post-optimized AVD was determined under the assistance of echocardiography. Firstly, the cardiologist set the pacemaker to DDD mode, and increase the AVD from 80 ms at the increment of 20 ms until 240 ms or intrinsic cardiac rhythm. Secondly, velocity time integral of aortic valve (VTI), LVEF, mitral anterior blood flow spectrum, mitral and tricuspid regurgitation are recorded at each AVD. Finally, the optimal atrioventricular phase (AVDopt) is determined mainly based on VTI, supplemented by the rest of the parameters. The partial parameters between AVDopt and different AVDs in patients with LBBAP were listed in the Additional file Conventional echocardiography was performed by the Philips IE Elite color Doppler ultrasound diagnostic apparatus, equipped with an S5-1 probe, a frequency of 1–5 MHz, and a Qlab13 workstation. The conventional pre-operation echocardiography was performed in 3 days before the pacemaker implantation. One month after the implantation, the programmer set the pacing mode to the DDD unipolar pacing mode, and we optimized the atrioventricular interval according to LVEF, aortic velocity time integral, mitral regurgitation and tricuspid regurgitation. Under the optimal atrioventricular interval, the patient took the left side decubitus, synchronously connecting it to the electrocardiogram, and then performed image acquisition. To avoid beat-to-beat variance, the heartbeat fluctuation does not exceed 5 beats per min during the image acquisition. The specific operations were as follows: (1) conventional echocardiographic parameters measured according to the guideline of ASE: left atTV-MV  We measured the time from the beginning of QRS to the beginning of the blood flow spectrum on the pulmonary valve namely pulmonary pre-ejection interval (PPEI), and the time from QRS to the beginning of the blood flow spectrum on the aortic valve, namely artery pre-ejection interval (APEI). The difference of PPEI and APEI is IVMD; (2) We selected the apical four-chamber TDI dynamic image and entered the SQ plug-in, outlined the tricuspid valve (TV) sidewall and mitral valve (MV) sidewall, and obtained the myocardial motion curve of the right ventricular basal segment and the left ventricular basal segment. The difference of systolic time to peak of TV and MV were recorded as ΔTsThe evaluation of intraventricular systolic synchronization and systolic function was as follows: Two-dimensional speckle tracking imaging (2D-STI) of the Qlab 13 workstation was used to analyze the left ventricular global longitudinal strain (GLS) and longitudinal strain of right ventricular free wall (LS-RV) . Categorical variables are expressed as percentages. The differences between two groups were assessed with the chi-square analysis for categorical variables and the t-test or non-parametric tests for continuous data at baseline. P < 0.05 was considered statistically significant.P < 0.05). There was no statistical difference between the two groups in terms of age, gender, comorbidities, etiology, pacemaker parameters, or preoperative cardiac color Doppler ultrasound parameters (P > 0.05). See Table A total of 40 patients who successfully underwent left bundle-branch pacing were included in this study, including 22 males and 18 females. The average age of the enrolled patients was (70.88 ± 12.95) years; there were 26 patients with hypertension, 11 patients with diabetes, and 6 with coronary heart disease. Moreover, for the indication of pacemaker implantation, 10 patients are owing to high-grade atrioventricular block, 19 patients owing to third-degree atrioventricular block, 11 patients owing to a second-degree type II atrioventricular block. Among the non-RBBB group, there are 7 patients with LBBB, 14 patients with normal QRS. After LBBAP treatment, the pacing parameters were satisfied: The ventricular capture is at low output (< 1.5 V/0.5 ms), and the impedance is > 500 Ω by unipolar pacing. According to the preoperative electrocardiogram, the patients were divided into two groups, including 19 patients in the RBBB group and 21 patients in the non-RBBB group. The optimal AVD in RBBB group was shorter than those without right bundle branch block (P > 0.05). ΔTsTV-MV between two groups has statistical difference (P < 0.05). The ΔTsTV-MV of RBBB group and non-RBBB group were (47.29 ± 58.45) ms, (− 12.00 ± 49.91) ms respectively. ΔPTTV-MV measured was statistically different between two groups (P < 0.05). Moreover, the RBBB group was (28.14 ± 39.04) ms, and the non-RBBB Group was (− 28 ± 48.26) ms. Both the difference of ΔTsTV-MV and ΔPTTV-MV showed that in RBBB group the tricuspid side segment activated slower than the mitral side. See Table There was no significant difference in IVMD between the two groups ( were 47.9 ± 58.45P > 0.05)  Fig. . See TabThe measurement of IVMD, TMAD, TDI and 2D-STI showed good reproducibility. The complete data of inter-observer variability and intra-observer variability were shown in Table The right bundle branch originates from the His bundle and divides into three branches at the base of the tricuspid anterior papillary muscle. It runs along the low part of the ventricular septum, the anterior wall of the right ventricle, the free wall of the right ventricle, the posterior papillary muscle, and the lower right posterior part of the ventricular septum. The electrical excitement is quickly transmitted to each segment of the right ventricular wall through the three branches, ensuring the interventricular and intraventricular synchronous contraction. However, because the right bundle branch is slender, superficial, mostly supplied by a single branch of the left anterior descending branch, the right bundle-branch conduction system is prone to conduction disorders. Therefore, RBBB is common in clinical practice, with an incidence of 8%, which increases with age , 8. HereIn this study, we performed LBBAP on patients with and without RBBB who had no statistical difference in general conditions. In the beginning, we found a difference in the optimal AVD between the RBBB and non-RBBB group, which is in accord with our former study. After tAccording to the study by Junyu et al. , SDt-R oThis study also had certain limitations. Firstly, the small sample size may have caused some errors in statistics. Secondly, because the right ventricle has an irregular crescent shape, we only studied the strain of the right ventricle in the apical four chambers; Thirdly, the detailed preoperative ultrasonic data of patients was not collected, so the changes of preoperative and postoperative ultrasound parameters of patients could not be analyzed; Finally, the follow-up time was short, so this study could only reflect the short-term impact of LBBAP on right ventricular function and synchronization. With a prolonged implantation time, whether the mechanical and electrical remodeling of the heart will have a long-term improvement effect on RBBB remains unknown. Whether it can reduce atrial fibrillation and cardiovascular adverse events caused by RBBB , 23 alsoIn summary, after LBBAP, RBBB patients had a certain degree of asynchrony of left and right ventricular movements, but postoperative RBBB patients’ right ventricular synchronization and ventricular longitudinal strain were not statistically significant compared with the non-RBBB group. It indicates that LBBAP is an optional pacing strategy for RBBB patients with pacing indications.Additional file 1: Table S1. Comparison of parameters between AVDopt and different AVDs in patients with LBBAP


For LVEF-1 < 30%, the odds ratio for the presence of CAD was 22.67  in the logistic regression adjusted for age, sex, and body mass index. Finding LVEF-1 < 30% in an individual with normal LV myocardial contraction and preserved LVEF strongly suggests the presence of CAD.Left ventricular (LV) systolic function is often measured with echocardiography using LV ejection fraction (LVEF) or global longitudinal peak systolic strain (GLPSS). Global wasted work (GWW), global work efficiency (GWE), and first-phase ejection fraction (LVEF-1) are newer LV systolic function indices. We examined these parameters in 45 healthy individuals and 50 patients with stable coronary artery disease (CAD), normal LV contractility, and LVEF > 50%. Compared to healthy individuals, CAD patients had similar LVEF but increased GLPSS and GWW and reduced GWE and LVEF-1. The highest area under the receiver operating characteristic for detecting CAD was found for LVEF-1 (0.84; 95% CI 0.75–0.91;  Left ventricular (LV) dysfunction is a common consequence of coronary artery disease (CAD), particularly in individuals who have experienced a myocardial infarction or have chronic critical coronary artery narrowing. Assessing LV systolic function is crucial for patients’ diagnosis, treatment, and prognosis.The left ventricular ejection fraction (LVEF) is the most commonly used single parameter for evaluating LV function, but it has several limitations, such as reproducibility, image quality, and endocardial border definition. LVEF is a measure of global LV systolic function that can be assessed using various noninvasive methods, including two- (2D) and three-dimensional echocardiography, cardiac magnetic resonance imaging, computed tomography, and nuclear medicine imaging, such as single-photon emission computer tomography . LVEF caDifferent methods may produce slightly different LVEF values, and the choice of method depends on the individual patient and the specific clinical context . While tA diminished LVEF < 40% defines heart failure with reduced LVEF (HFrEF) . Based oGlobal longitudinal peak systolic strain (GLPSS) measured using speckle tracking echocardiography can detect subtle impairment of LV systolic function . Like LVGu et al.  introducRussel et al.  exploredTo date, no study has directly compared LVEF and GLPSS with GWW, GWE, and LVEF-1 in CAD patients with no LV contractile abnormalities and LVEF > 50%. As such, the present investigation aims to explore this issue. For this reason, the diagnostic features of standard and new echocardiographic measures of the LV systolic function in healthy control subjects were compared to patients with established CAD and preserved LVEF. Nonparametric comparison of unpaired data with the Mann–Whitney test.Statistical power set at 0.8 and two-sided alpha at 0.05.As there are limited data on LVEF-1 and MW in CAD patients with normal contractility, we have used GLPSS instead as the main parameter, which might differentiate CAD patients from healthy people. Additionally, GLPSS varies between vendors of echocardiographic systems, e.g., GE, Philips, and Tomtec. In our department, we work with GE echocardiographs. Therefore, we searched for papers using the same system for GLPSS studies. Based on our own experience and the study by Tsai et al.  . We used data from 45 healthy people and 50 CAD patients with no LV contractile abnormalities to limit the risk of insufficient statistical power of our analyses.The estimated minimum sample size was 38 people in each group (online tool at In the past (November 2018 and May 2019), we gathered healthy volunteers for the Department’s Echo Lab internal reference database for a newer echo machine  purchased in October 2018. Routinely, all people enrolled in our reference database undergo a clinical evaluation, which includes brachial blood pressure measurements and standard 12-lead ECG recording before transthoracic 2D echocardiography. We used the following criteria to define participants as healthy: no known acute or chronic illness, no signs and symptoms of any disease, and not taking any chronic medication except for oral hormonal contraception in women of reproductive age. Smokers were allowed to participate. Occasional use of non-steroid anti-inflammatory drugs for occasional pain  was allowed but not in the 48 h preceding ECG and echo studies. Sinus rhythm and normal findings on ECG, brachial blood pressure (<130/80 mmHg) at supine rest, and echocardiographic measurements were obligatory for all healthy people. For this study, we used anonymized data from our reference database from all consecutive volunteers who had available cine loops to measure LVEF-1 and MW indices. No other data selection criteria were applied, and the data were used retrospectively.−Good quality images and cine loops allowing the measurement of LVEF-1 and MW in the post-processing (directly on Vivid E95 or EchoPAC platform by GE)−No contractile abnormalities of the LV and right ventricle−Presence of a significant (over 50%) narrowing of the lumen of at least one coronary artery. All coronary lesions were assessed by invasive angiography according to the current guidelines of the European Society of Cardiology ,24. VisuFrom our clinical database of patients who have undergone clinically indicated coronary angiography (between 1800 and 2000 procedures per year), we selected data from 50 consecutive CAD individuals who also had performed routine 2D echocardiography on the Vivid E95 and fulfilled the following criteria:Individuals with any clinically significant valvular disease, defined as more than mild regurgitation or stenosis, were excluded from the study.Because the study had no experimental features and was retrospective, the local Ethics Committee waived the requirement to obtain patients’ agreement and permitted the use of data from our department’s databases. This approach is consistent with the Declaration of Helsinki for retrospective studies . Peripheral arterial blood pressure was measured using an Omron 705 IT device . Echocardiographic analyses were performed using a Vivid E95 equipped with an M5S 3.5 MHz transducer. Standard techniques outlined by the American Society of Echocardiography were used to obtain 2D, M-mode, and Doppler measures . Digital images were captured and stored for further offline analysis. LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), and left ventricular ejection fraction (LVEF) were calculated in four- and two-chamber views using the biplane modified Simpson’s method. A pulsed-wave Doppler (PW) was used to measure the peak velocity of the E wave, and Doppler tissue imaging for estimation of the mitral annular early diastolic velocity (E′) in both the septal and lateral annulus, and these were averaged to calculate the E/E′ ratio. LVEF-1 is calculated as the relative LV volume change from end-diastole to the time of maximal velocity of aortic flow (T1V): T1V is measured using a biplane-modified Simpson’s method. The time difference between the peak of the ECG R-wave and a maximal aortic flow velocity recorded by PW gate in the LV outflow tract, close to the aortic valve leaflets, from an apical five-chamber view, were used to estimate T1V. Echocardiographic continuous dynamic images of the LV apical four-chamber, three-chamber, and two-chamber views at a frame rate of at least 60 frames per second were obtained. Depth, sector width, and gain were optimized for adequate LV myocardium visualization. Automated Function Imaging software of the Vivid E95 ultrasound system  was used to perform speckle-tracking analysis. GLPSS and brachial blood pressure recordings measured before the echocardiography were used to quantify global MW indices through the noninvasive LV pressure-strain analysis . In thisQualitative data were coded as either 0  or 1 (for men or CAD patients) and then represented as numbers and percentages. The gender distribution between the control group and CAD patients was compared using the Fisher exact test. As most continuous data did not have a normal distribution , results are summarized as medians and the 25th and 75th percentiles (IQR). Comparisons between healthy individuals and CAD patients were made using the unpaired Mann–Whitney test. For exploratory purposes, only LV systolic function parameters that differed significantly were compared using analysis of covariance (ANCOVA), a priori adjusted for subjects’ age, gender, and body mass index (BMI). Only ANCOVA models with a normal distribution of residuals  were accepted and presented, even if the original covariates did not have a normal distribution. Nonparametric Spearman tests were performed to establish correlations between LVEF or GLPSS and newer LV systolic function indices. Multivariate linear regression was analyzed to study the association of LV systolic function indices with sex, gender, blood pressure, heart rate, and LV mass index. Receiver operating characteristic (ROC) characteristics with the area under the curve (AUC) were computed for various measures of LV systolic function to determine their potential for differentiating control subjects from CAD patients.Additionally, the obtained ROC curves were compared using the method of DeLong et al.  to test As some critical parameters, such as gender distribution and BMI, were not comparable between the control and CAD groups, we used sex for the case-control matching. The results of comparisons of continuous data for the 31 case-control matched pairs by the paired Wilcoxon test are shown in the p-value < 0.05 was considered statistically significant. MedCalc® Statistical Software version 20.110  and PQStat Software version 1.8.4.136  were used for statistical analyses. A p = 0.0008). Fifteen (30%) healthy individuals and 19 (38%) CAD patients were active smokers. Following the inclusion criteria, no healthy subjects had any chronic disease or took pharmaceutical agents regularly . The proportion of men in the control group was significantly lower than in the CAD group  had a previous myocardial infarction, 14 (28%) had undergone percutaneous coronary intervention (PCI) in the past, one (2%) had undergone coronary artery bypass grafting (CABG), 42 (84%) had hypertension and all were well controlled, 11 (22%) had type 2 diabetes, and three (6%) had suffered either a transient ischemic attack (TIA) or ischemic stroke in the past. Thirty-two (64%) patients had single-vessel CAD, 16 (32%) had two-vessel CAD, and 2 (4%) had three-vessel CAD. Among them were three patients with a significant narrowing of the left main coronary artery . All CAD patients were taking at least one antiplatelet drug and lipid-lowering therapy (a statin or ezetimibe), 36 (72%) were taking either an angiotensin-converting enzyme inhibitor or an angiotensin-2 receptor blocker, 34 (68%) were on a beta-blocker, 16 (32%) on a calcium antagonist, 3 (6%) on a mineralocorticoid antagonist, and 11 (22%) were taking a diuretic. Only LV systolic function indices that were significantly different in the ANCOVA were subjected to ROC analysis. The estimated cut-off values for univariate logistic regression analysis were either unadjusted or adjusted for participants’ age, gender, and body mass index. In both analyses, abnormal values of all LV systolic function indices were significantly associated with an increased odds ratio for CAD. LVEF-1 outperformed all remaining parameters, particularly in the adjusted regression logistic models, with participants exhibiting LVEF-1 < 30% being 22 times more likely to have CAD than individuals with higher LVEF-1 .The newer measures of LV systolic function significantly differ between patients with stable CAD and normal LVEF and healthy individuals. Regardless of the comparable LVEF in both groups, GLPSS, GWW, GWE, and LVEF-1 indicate poorer LV systolic function in CAD patients. Larger LV mass correlates with worse systolic function, which also declines (except for LVEF-1) if heart rate and blood pressure increase. LVEF-1 outperforms the diagnostic properties of other LV systolic function measures—people with reduced LVEF-1 < 30% are at the highest risk for CAD regardless of age, gender, and BMI. An impaired LV systolic function with LVEF > 50% was demonstrated with GLPSS in studies on patients with aortic stenosis, hypertension complicated by HF with diastolic dysfunction, or those treated with certain chemotherapeutics ,31,32,33GLPSS is a direct measure of the LV walls’ deformation during systole. On the other hand, LVEF quantifies the relative contribution of the blood pumped from the LV  to the overall LV end-diastolic volume. Although LVEF does not reflect myocardial contractions and deformation, it is an indirect measure of LV systolic function. A normal LVEF provides approximate information that the LV global pumping function is preserved. Nevertheless, an increased GLPSS detects some early abnormalities in myocardial deformation. In CAD patients with no visible contractile impairment and normal LVEF, deformation abnormalities could be caused by active myocardial ischemia. However, our patients had stable CAD with no angina during the examination. Long-term consequences of chronic CAD  might contribute to this finding. We also show that GLPSS increases with higher DBP, HR, and LVMI. Our CAD patients had significantly larger LVMI, whereas their HR and blood pressure were comparable to healthy people.LVEF-1 measures the early systolic shortening of cardiomyocytes during the most dynamic phase of LV contraction, which is associated with blood ejection. At this time, the highest myocardial wall stress is generated. As Gu et al.  suggestep = 0.0001—data not presented in Results). As shown by Gu et al. [Our study found that LVMI negatively impacts all newer LV systolic function indices. Wasted MW increased, while GWE and LVEF-1 decreased with a larger LV mass, which alters LV geometry, raises myocardial wall stress, and changes LV systolic performance. Higher GWW appears to be a consequence of LVEF-1 reduction. At normal LVEF, the LV pumping function is preserved at the expense of diastolic function when some myocardial segments continue their contraction. In our study, LVEF-1 and GWW were negatively correlated . The CAD group consisted of consecutive patients who met certain criteria, including having no contractile abnormalities, LVEF > 50%, and available 2D echocardiography recordings on the same echo machine. Although the minimum sample size was estimated to be 38 people per group, data from 45 healthy individuals and 50 patients with CAD were used. Despite the expected clinical differences between healthy individuals and those with CAD, the sex distribution and BMI significantly varied between the two groups. This resulted from using consecutive data from healthy people and CAD patients. Therefore, an additional case-control matching based on sex was performed  for GLPSS. Additionally, for LVEF-1, the statistical power was even stronger (0.9998). Our CAD patients share several common features, such as having stable CAD, no LV contractility impairment, and normal LVEF > 50%. However, this group consists of quite heterogeneous patients. Some of them have survived MI, TIA, or stroke, undergone PCI (one person had CABG) in the past, and have diabetes. Among them are single-, two-, and three-vessel CAD patients. All of these factors might be considered bias factors. Unfortunately, it is impossible to control and adjust for all of these factors in the multivariate analyses with a group of 50 people and obtain reliable results. However, we acknowledge the presence of potential biases and that their effects should be explored in more detail in a larger CAD group.It is important to note that our findings may not be applicable to other cardiac patients, as the study specifically focused on the diagnostic properties of newer LV systolic function indices for a specific subpopulation of CAD patients . Additionally, as the study population was predominantly Polish and of Caucasian race, the results may not be generalizable to other racial groups.Finally, this study utilized 2D echocardiography to measure cardiac structure and function. While other imaging modalities such as 3D echocardiography, cardiac magnetic resonance, or cardiac computed tomography may be more accurate, they also have limitations . Additionally, LVEF-1 and MW indices analyzed in this study were initially developed for use with 2D echocardiography. Overall, we demonstrate that GLPSS, GWE, GWW, and particularly LVEF-1 help distinguish subjects with CAD and normal LVEF from healthy people. LVEF-1 focuses mainly on the early part of the LV’s blood ejection, while GWW partially covers the early diastole. The newer echocardiographic indices emphasize the importance of going beyond global LV systolic function analysis and using other parameters that better explore other phases of LV contraction and possibly the early diastole. It is unclear which pharmaceutical agents may target the early ejection or early diastole, but those interacting with ATP metabolism and mitochondria are the most promising. Drugs acting at this time might and targeting cellular and mitochondrial energy production might significantly improve the treatment of CAD and possibly prevent its progression to HF. In summary, contemporary markers of LV systolic function aid in assessing subjects with CAD and normal LVEF; indeed, GWW, GWE, and LVEF-1 demonstrate better functional phenotyping of CAD patients with normal LVEF but early signs of systolic dysfunction.


Rapid screening and accurate diagnosis of acute myocardial infarction are critical to reduce the progression of myocardial necrosis, in which proteolytic degradation of myocardial extracellular matrix plays a major role. In previous studies, we found that targeting the extracellular matrix metalloprotease inducer (EMMPRIN) by injecting nanoparticles conjugated with the specific EMMPRIN-binding peptide AP9 significantly improved cardiac function in mice subjected to ischemia/reperfusion.In a porcine model of coronary ischemia/reperfusion, we tested the theragnostic effects of administering 0.1 mg/kg gadolinium-containing nanoparticles conjugated with AP9 (NAP9), a synthetic peptide that targets EMMPRIN or a control nanoparticle (NAPSC). Cardiac magnetic resonance assessment of the infarct progression, ventricular function, and nanoparticle distribution was performed the next 7 days. We also measured the infarcted area of the heart and cardiac remodeling at 7 or 21 days after ischemia/reperfusion.After 21 days of ischemia/reperfusion, NAP9 reduced the extension of cardiac necrosis (14.1±9.7 versus 35.5±1.8) and the levels of collagenolytic activity of MMPs , along with a significant reduction in collagen deposition (7.5±4.5 versus 41.3±20); including the ratio of type I versus III collagen fibers in the necrotic myocardium. In terms of cardiac function, the response to NAP9 administration resulted in a significant improvement of cardiac performance overtime, as evidenced by the left ventricle ejection fraction (64.0±7.8), when compared with those present in the NAPSC group (47.3±4.7). As shown by magnetic resonance imaging, noninvasive molecular imaging of NAP9 enabled us to find a significant reduction in cardiac necrosis, myocardial edema, hemorrhage, and microvascular obstruction, suggesting that NAP9 may reduce myocardial injury and preserve left ventricular function, at least, by preventing the effect of EMMPRIN on extracellular matrix degradation.Our data point towards NAP9 as a promising theragnostic tool in managing acute myocardial infarction, by inhibiting EMMPRIN-induced extracellular matrix degradation and allowing noninvasive visualization of cardiac necrosis progression over time. The use of nanotechnology applied to the generation of new molecular targets represents one of the greatest technological challenges in cardiovascular imaging, providing significant advantages that include a lack of undesired immune responses. Nanoparticles show theragnostic features combining both diagnostics and therapeutics properties in one single compound, and noninvasive molecular imaging may represent a significant breakthrough in the study of myocardial infarction enabling high-resolution tissue visualization but also featuring imaging detection of a target molecule over time. Such is the case of extracellular matrix metalloproteinase inducer, a potent activator of proteolytic degradation of the extracellular matrix which expression increases at the onset of myocardial ischemia, as one of the most powerful effectors of cardiac necrosis. By using gadolinium-containing nanoparticles conjugated with specific extracellular matrix metalloproteinase inducer-binding peptides, we delimited the myocardial ischemic area and inhibited myocardial fibrosis, myocardial extracellular volume, microvascular obstruction, intramyocardial hemorrhage, and edema, and at the same time, the nanoparticles inhibited extracellular matrix degradation, and hence myocardial necrosis, improving cardiac contractility. Our preclinical research in large animal models of myocardial infarction takes a further step towards technological development for a new recent upcoming diagnosis and treatment methods of acute myocardial infarction.Thackeray & HessSee Editorial by 1 In recent years, multiple therapeutic approaches have emerged, but currently, only a few reduce myocardial injury. Cardiac magnetic resonance imaging (MRI) is the noninvasive and most reliable procedure to study myocardial injury, and it provides valuable information in the search for new therapies.2Early diagnosis and treatment are critical to reduce harmful outcomes in patients after acute myocardial infarction (AMI).3 In this context, the ECM metalloproteinase inducer , a glycoprotein that promotes MMP -induced ECM degradation when is highly glycosylated EMMPRIN, is a potential target that plays a key role in the inflammatory response to cardiac ischemia, by regulating the expression of MMP-9 and MMP-13.7Targeting extracellular matrix (ECM) degradation is a promising therapeutic approach, given that ischemia-induced ECM proteolytic activation often leads to extensive cardiac cell necrosis, adverse ventricular remodeling, and heart failure.9 mice deficient for genes involved in cardioprotection10 or, more recently, specific nanoparticles conjugated with the EMMPRIN-binding peptide AP9 in mice,11. However, no large animal studies have been reported so far of the potential benefit of using EMMPRIN as a therapeutic target in cardioprotection.To study whether EMMPRIN may serve as a therapeutic tool, many laboratories have targeted EMMPRIN through different approaches. We and others have used antibodies against EMMPRIN,13 infectious diseases,14 central nervous system diseases,15 or even cancer.16 The rationale of using micelle or iron oxide-based nanoparticles lies on the significant advantages over other strategies. The use of nanoparticles tends to reduce in many cases the adverse effects associated with undesired immune responses, as when using antibodies, and in general, given their small size and chemical composition, they exhibit very low or no toxicity, as we previously reported in mice.11 Once more, testing in large animal models of disease is, in most cases, pending of investigation.The use of nanoparticles has emerged for their theragnostic capacity against pathologies that include myocardial infarction,11 effectively improved the cardiac function of hearts subjected to ischemia/reperfusion (IR). In this work, we extend our study to a porcine model of coronary IR to evaluate its potential use in clinical practice.In a mouse model of AMI, we previously reported that nanoparticles conjugated with AP9 (NAP9), a nanoparticle conjugated with the specific EMMPRIN-binding peptide AP9,We hypothesized that targeting EMMPRIN with NAP9 is a promising theragnostic tool by allowing noninvasive visualization of myocardial infarction while inhibiting ECM degradation and improving left ventricular (LV) function. We found that NAP9 reduces infarction size and locates preferentially in the infarcted tissue compared with pigs injected with NAPSC.The data that support the findings of this study are available from the corresponding author upon reasonable request.All the surgical procedures were performed in the Experimental Surgery Department of the Hospital Universitario La Paz in Madrid and The Medical Research Institute Hospital La Fe (IIS La Fe) in Valencia, in accordance with the following institutional guidelines: Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , and the Animal Welfare Ethics Committee and complied with the EU Directive on experimental animals (63/2010 EU) and related Spanish legislation (RD 53/2013). IRB approval was obtained (PROEX 138/17) to perform the animal procedures (no informed consent was required by the participants).Evans Blue, triphenyl tetrazolium chloride, hematoxylin-eosin, sirius red, Triton X-100, calcium chloride, zinc chloride, and acetic acid were from Merck . The Masson trichrome staining kit, anti-EMMPRIN, anti-CD68, HRP/DAB IHC kit, and HRP-conjugated secondary antibodies were from Abcam . Anti-MMP-9 and anti-MMP-13 were from Santa Cruz . Ketamine was from Pfizer ; the isoflurane was from Abbvie ; the propofol was from Fresenius ; the fentanyl was from Kern Pharma ; the diazepam was from Roche ; and the amiodarone was from Sanofi Aventis .All the surgical procedures were performed in the Experimental Surgery Department of the Hospital Universitario La Paz in Madrid and The Medical Research Institute Hospital La Fe (IIS La Fe) in Valencia, in conforming to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , and the Animal Welfare Ethics Committee and complied with the EU Directive on experimental animals (63/2010 EU) and related Spanish legislation (RD 53/2013). PROEX 138/17.Supplemental Methods for nanoparticle cytotoxicity assay), prepared as previously described.11 After 7 days of reperfusion, animals were euthanized to extract the heart. Immunoblot samples were immediately frozen at −80 °C, and histology samples were included in 4% Formalin.Twenty-eight Yorkshire pigs (36±5 kg) were housed 1 week before surgery to avoid unease or stress associated with the new environment. Before the surgical intervention, animals were anesthetized with intramuscular ketamine 10 mg/kg and midazolam 0.5 mg/kg. Anesthesia was induced by inhaled isoflurane and maintained with continuous infusion of propofol 2 mL/kg per hour, fentanyl 50 µg/kg per hour, and diazepam 10 µg/kg per hour. After intubation and mechanical ventilation with 100% oxygen saturation, 5000 IU of heparin and amiodarone 2 mg/kg per hour were administered to avoid blood clotting of catheters and malignant cardiac arrhythmias, respectively. Before complete occlusion, the hearts were subjected to ischemic preconditioning by blocking the left anterior descending artery for short periods . IR was produced by occluding left anterior descending artery for 45 minutes using a JL3 6F catheter and an angioplasty balloon. The complete myocardial ischemia was confirmed by ST-segment elevation. The balloon was then removed to reopen the artery, and animals received Placebo , NAP9, or NAPSC  with multi-transmission technology, using a 32-channel XL Torso coil specifically for human torso acquisitions. Acquisitions were made before ischemia, basal cardiac magnetic resonance (CMR), and immediately after reperfusion. Nanoparticles were injected, and postinjection acquisition was performed at times 0-hour, 24-hour, 72-hour, and 7-day postinjection. The number of animals subjected to MR were 8 (4 per group).To estimate LV function, contiguous images were acquired covering the LV from the base to the apex with a parallel imaging Balanced Turbo Fast Field Echo Multi-Slice sequence, with a 45° flip angle, 2.9 ms repetition time and 1.4 ms echo time. The field of view was 280×280 mm with a resolution of 1.8 mm in plane and 6 mm in slice thickness.The quantification of edema as the area at risk (AAR) was performed on a T2-weighted short-TI inversion recovery turbo fast spin-echo sequence (repetition time=1429 ms and echo time=80 ms).Myocardium characterization was extended with the acquisition of a Multi-Eco sequence Multi-slice mFFE sequence for the study evaluation of the transverse relaxation time (T2*) and ratio (R2*). The precontrast and postcontrast Modified Look-locker Imaging sequences allowed us to obtain the longitudinal relaxation time (T1) and calculate the extracellular volume (ECV).The contrast delayed enhancement was evaluated after the administration of gadolinium (0.2 mmol/kg), applying an inversion recovery/fast echo gradient sequence .The LV ejection fraction (LVEF) was obtained by delimiting the perimeter of the endocardium and epicardium at the moments of systole and diastole on the balanced turbo fast field echo sequence.The volume of regional edema and myocardium at risk was quantified after its detection by semi-automatic region recognition algorithms, based on thresholds and contour detection on the T2-weighted short-TI inversion recovery turbo fast spin-echo sequence.The transverse relaxation time (T2*) was calculated voxel-by-voxel from the different described regions to obtain the T2* and R2* maps over the areas of interest. The calculation of the transverse relaxation times was made as follows:We obtained the T1 relaxation time calculation from the Modified Look-locker Imaging sequence as a T1 map for each voxel of the myocardium, which will serve as potential additional information for detecting the myocardial AAR and fibrosis.The edema volume and myocardial AAR were quantified through semi-automatic algorithms for region recognition, based on thresholds and contour detection on the delayed enhancement images. The ECV map was obtained to get quantitative information about the state of the myocardium to estimate viability on each segment.Echocardiography was performed to determine LV function using a Vivid Q ultrasound system from GE Healthcare , equipped with a 1.9-4 MHz scan head. Parasternal long and short-axis-view images of the heart were taken before the surgery, at the end of ischemia, and at the end point to determine LV function worsening and recovery. The parameters studied using the on-site software cardiac package were: Diastolic interventricular septum thickness, systolic and diastolic left ventricle internal diameter, systolic and diastolic left ventricle posterior wall thickness, LVEF, LV shortening fraction, heart rate, cardiac output, and LVEF. The same operator performed data acquisition and analysis to avoid the interobserver error.The necrotic area of the hearts was measured by Evans Blue/triphenyl tetrazolium chloride staining. The left anterior descending artery was reoccluded in the same location as day 0. Then, 200 mL of 5% Evans Blue solution was injected using a 5F fenestrated Pigtail catheter within the LV to distribute the compound across the entire cardiovascular system, excepting the blood-deprived area of the heart. After 2 minutes, the animal was sacrificed, and the heart extracted and frozen at −20 °C for 24 hours. Eight pigs (4 per group) were euthanized 7 days after the surgery and 8 pigs (4 per group) were euthanized 21 days following the same method described before. The next day, hearts were cut into 0.8 cm slices and incubated in 1% triphenyl tetrazolium chloride for 20 minutes at 37 °C and next in 10% paraformaldehyde solution. The necrotic area was relativized to the AAR to avoid interexperiment variability concerning the ischemic area.Heart morphology  was visualized by hematoxylin/eosin staining, and Masson trichrome staining detected collagen deposition. Collagen fibers were stained with sirius red for 1 hour and washed using acetic acid. Total collagen was assessed under bright-field microscopy and type I and III collagen fibers were visualized and differentiated under normal and polarized light.CD68 expression was detected using the Mouse and Rabbit HRP/DAB Detection IHC kit (ab64264) from abcam following the manufacturer’s instructions.Proteins from 10 hearts (5 per group) were extracted to measure EMMPRIN, MMP-9, and MMP-13 expression. Twenty micrograms of total protein were loaded into 10% polyacrylamide gels. After the electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% BSA in T-TBS. Membranes were incubated for 1 hour with anti-EMMPRIN 1:500 and anti-Rabbit HRP-conjugated secondary antibody. Then, protein bands were visualized by chemiluminescence and studied using image analysis software.Protein lysates prepared from 10 hearts (5 per group) were resolved on 7.5 % SDS-polyacrylamide gels containing 1 mg/mL gelatin. Gels were incubated for 30 minutes with 2.5% Triton X-100 buffer, and then incubated over 16 hours with developing buffer . Gels were stained with Coomassie blue for 30 minutes, and gelatinolytic activity was visualized after incubation with unstaining solution . Optical densities were analyzed using image analysis software and collagenolytic activity was measured by analyzing the active/zymogen ratio.To estimate the sample size for each group we performed a comparative study of 2 independent means from similar studies, assuming a pooled SD of 5 units. Therefore, we projected a sample of 4 pigs per group (N=8) to achieve a power of 80% and a level of significance of 5% (2 sided), for detecting a true difference in means between the test and the reference group of 10  units.P<0.05. Error bars represent±SD. The number of animals used in each experiment is specified in the text.Unless otherwise specified, data are expressed as mean±SD. The samples were normally distributed, and homogeneity of variances between groups was verified by performing a Levene test. After confirming equal variance across samples, mean values between study groups were compared by using a Wilcoxon rank-sum test. Differences were considered significant at To further map the distribution of nanoparticles within the LV, we used MRI to study NAP9 and NAPSC relaxation rates (ΔR1) in each segment of the LV, following the American Heart Association 17-segment model. We used the Spearman rank correlation to calculate the correlation between native T1 relaxation times and NAP9 ΔR1 for each segment at days 1, 3, and 7 after IR, analyzing the presence of 5 different points/pig, located in the following segments:Point 1: anterior/septal location (segments 7 and 8). Point 2: inferior/septal location (segments 9 and 10). Point 3: lateral location (segments 11 and 12). Point 4: anterior/septal location (segments 13 and 14). Point 5: inferior/lateral location (segments 15 and 16). These points were defined to discriminate the location of the ischemic tissue in the 4 walls of the heart .Animals were subjected to coronary IR and treated with 0.1 mg/kg NAP9 or NAPSC as depicted Figure A and 2B Figure S2A), as well as inflammatory macrophages present in the group of NAPSC . NAP9 also decreased collagen deposition as detected by Masson trichrome staining of the same sections  24-hour post-IR Figure A. SubseqTo test whether in pigs NAP9 may also prevent proteolysis, we injected 0.1 mg/kg NAP9 or NAPSC into pigs subjected to IR, finding that in the necrotic area, a significant reduction in high (highly glycosylated: active EMMPRIN) and low glycosylated EMMPRIN, MMP-9, and MMP-13, were detected by immunoblot with protein extracts isolated after 7 days of IR Figure A. IntereWe also analyzed myocardial ECV by MRI, as a matter to quantification of cardiac remodeling, finding that administration of 0.1 mg/kg NAP9 effectively prevented the increase of myocardial ECV post-IR. Simultaneously, hearts from the NAPSC group also showed a significant increase at 1- and 3-day post-IR Figure C.Following coronary IR, the risk of experiencing major adverse cardiovascular events is increased, and the presence of microvascular obstruction (MVO), intramyocardial hemorrhage (IMH), and edema are strong predictors of major adverse cardiovascular event. To determine whether NAP9 might reduce reperfusion-mediated major adverse cardiovascular event, we analyzed native T1, T2, and T2* values to address the presence of MVO (native T1 1150–1250 ms and T2* <20 ms), IMH (T2* <20 ms), and edema (T2 >70 ms). Cardiac IR induced a 20% increase of MVO , a more significant acquisition of NAP9 was found in hyperintense T1 sections.Figure S3).To determine the distribution of the nanoparticles within the LV, we studied NAP9 and NAPSC relaxation rates (ΔR1) in each section of the LV, following the American Heart Association 17-segment model. We represented native T1 values and nanoparticles ΔR1 following a color code of 7 to 16 to compare native T1 hyperintense and the nanoparticles enriched areas. Gray-colored graphics represent native T1 values of the LV corresponding lighter tones to hypointense sections (<1200 ms) becoming darker, while the signal increase . Concerning nanoparticles ΔR1, NAP9 graphics were depicted in blue, and NAPSC was colored in red. Lighter tones correspond to low ΔR1 (<4 1/s) and darker tones to higher ΔR1 . Graphics show results from days 1, 3, and 7 since data from day 0 showed no significant effects , suggesting that accumulation of NAPSC could be at least partly explained by IMH or edema. To verify these results, we studied the correlation between native T1 relaxation times (x axis) and NAP9 ΔR1 (y axis) for each segment  and NAPSC ΔR1, indicating that NAPSC aggregation was not specific in T1 hyperintense segments  was defined for each animal by days 1, 3, and 7 by the Spearman rank correlation (ρ) between NAP9 relaxivity versus native T1 relaxation times. LVEF (y axis) of the same hearts was measured by day 1, 3, and 7 ,34 suggesting that NAP9 could prevent the early induction of MMPs during the inflammatory response.Recently, Sado et al36 together with MVO as a recurrent sequel after AMI, a strong predictor of adverse cardiac function and remodeling.36 Administration of NAP9 successfully prevented IMH, edema, and MVO, although we are not certain about the exact mechanism underlying this phenomenon. However, the inflammatory process is strongly related to these events, and EMMPRIN is the natural ligand of cyclophilin A, a key inflammatory element in the heart that partakes in microvascular injury as Jin et al26 demonstrated in a mouse model of ischemic stroke. In addition, previously we also found that nitric oxide prevents overexpression of EMMPRIN in a murine model of AMI9 and during carotid atherosclerosis, and therefore MVO after endothelial dysfunction, which is related to a depletion of NO availability, may be prevented after inhibition of EMMPRIN.28 However, further studies must be performed to explore role of NAP9 in these contexts.IMH and edema associated to AMI can be detected in T2 sequences thanks to deoxyhemoglobin paramagnetic properties produced after blood degradation and the increase in water content at the edematous location,17 there was no significant CMR signal detected in the hearts of NAP9 treated animals, which could be explained by a low concentration of gadolinium-containing NAP9. Thus, we explored alternative approaches, starting with CMR sequences to detect myocardial relaxation rates (ΔR1), which are relative to the nanoparticle concentration.37 We detected that NAP9, but not NAPSC, specifically aggregates in the necrotic tissue, providing valuable evidence about the diagnostic reliability of using NAP9, and allowing in vivo visualization and measurement of the myocardial infarction. This is of significant interest, since as some authors point, heterogeneity between patients is a main problem in the development of new therapies.27 Hence, precise measurement tools may help to avoid these biasing, thus improving precise diagnosis and forecast progression of disease.We studied whether NAP9 could be detected in CMR postcontrast T1 maps, as it contains gadolinium as a principal component, but unexpectedly, and although we previously found the rhodamine containing NAP9 by confocal microscopy in the heart,Limitations of the study may include the reduced sample size affecting at the clinical translation of results, which is related to the budget required to perform these studies. Another limitation is the animal model itself, since a porcine procedure o IR is, for several reasons, the best experimental approach.Noninvasive targeting of ECM remodeling is a reliable strategy and, to our knowledge, this is the first time in which a theragnostic tool is used in this context. Our results open a new range of possibilities for the treatment of AMI, as aiming ECM degradation and cardiac remodeling is not only a reliable approach to measure and visualize infarction but also this approach is designed to induce cardiac protection as well. Although further studies must be done to deeply understand NAP9 contribution, we show strong evidence of functionality to consider using NAP9 in future clinical studies as a new treatment against AMI.This work was supported by C. Zaragoza: Universidad Francisco de Vitoria (grant numbers 2017/18), Fundación BBVA Ayudas a Equipos de Investigación científica (2017), and Proyectos de i+D+I, from the program Investigación orientada a los retos de la sociedad, cofounded by Fondo Europeo de Desarrollo Regional (FEDER) A way to achieve Europe (MINECO/AEI/FEDER/EU SAF2017-87342-R), Dr Saura: Ayudas para realización de proyectos de investigación Junta de Comunidades de Castilla La Mancha SBPLY/19/180501/000055, cofounded by Fondo Europeo de Desarrollo Regional (FEDER). A way to achieve Europe. M. Filice: Comunidad Autonoma de Madrid and Universidad Complutense de Madrid for research project No.2017-T1/BIO-4992 Accion de Atracción de Talento.None.Supplemental MethodsFigures S1–S3


Spasticity is defined as a velocity-dependent increase in tonic stretch reflexes and is manually assessed in clinical practice. However, the best method for the clinical assessment of spasticity has not been objectively described. This study analyzed the clinical procedure to assess spasticity of the elbow joint using an electrogoniometer and investigated the appropriate velocity required to elicit a spastic response and the influence of velocity on the kinematic response pattern.This study included eight healthy individuals and 15 patients with spasticity who scored 1 or 1+ on the modified Ashworth Scale (MAS). Examiners were instructed to manually assess spasticity twice at two different velocities (slow and fast velocity conditions). During the assessment, velocity, deceleration value, and angle [described as the % range of motion (%ROM)] at the moment of resistance were measured using an electrogoniometer. Differences between the slow and fast conditions were evaluated. In addition, variations among the fast condition such as the responses against passive elbow extension at <200, 200–300, 300–400, 400°/s velocities were compared between the MAS 1+, MAS 1, and control groups.Significant differences were observed in the angular deceleration value and %ROM in the fast velocity condition (417 ± 80°/s) between patients and healthy individuals, but there was no difference in the slow velocity condition (103 ± 29°/s). In addition, the deceleration values were significantly different between the MAS 1 and MAS 1+ groups in velocity conditions faster than 300°/s. In contrast, the value of %ROM plateaued when the velocity was faster than 200°/s.The velocity of the passive motion had a significant effect on the response pattern of the elbow joint. The velocity-response pattern differed between deceleration and the angle at which the catch occurred; the value of deceleration value for passive motion was highly dependent on the velocity, while the %ROM was relatively stable above a certain velocity threshold. These results provide clues for accurate assessment of spasticity in clinical practice. Spasticity is a positive sign of upper motor neuron syndrome caused by cerebrovascular or spinal cord injury and is defined as “a motor disorder characterized by a velocity-dependent increase in tonic stretch reflexes (“muscle tone”) with exaggerated tendon jerks, resulting from hyperexcitability of the stretch reflex, as one component of the upper motor neuron syndrome” . In the Manually evoked spastic muscle responses are evaluated using clinical scales. The Ashworth Scale (AS) , modifieOne possible reason for the recurrent debate on the reliability of these clinical scales may be related to ambiguity in how the spasticity-testing technique is defined , 15. ForThere is a more concrete description of the practical technique used in the MAS, described by Bohannon et al.  as to “eThis study aimed to clarify the influence of velocity on the kinematic response patterns in poststroke spasticity, especially in patients with mild spasticity. We sought to do this by using an electronic goniometer to measure the influence of velocity on the resistance-response pattern against passive movement.Participants were recruited from among those patients who underwent rehabilitation at Fujita Health University in Toyoake, Japan, between February and April 2020 and from the local community. The study population comprised a total of 23 participants, of whom 15 were patients with stroke with hemiplegia and spasticity (spasticity group) and eight were healthy individuals without any history of central nervous system or musculoskeletal diseases (control group).Inclusion criteria for the spasticity group were as follows: (1) existence of spasticity in the elbow flexor muscle group and (2) ischemic or hemorrhagic stroke. Exclusion criteria included: (1) presence of a manually detected increase in muscle tone during slow stretching; (2) motor, cognitive, visual, or hearing dysfunction that hindered spasticity measurements; and (3) limitation in the passive range of motion (ROM) in the paretic upper limb. Participant details are presented in For all assessments, the participant was placed in a sitting position with a backrest, and an electronic goniometer  was attached to the elbow joint. Two of six randomly selected physiotherapists and occupational therapists  performed all examinations. The examiner held the participant's elbow joint in maximum flexion for 2 s and then moved it to maximum extension and measured the elbow joint angle during this period. Examiners were instructed to test joint mobility under two different velocities: (1) a slow-speed condition to examine the ROM and (2) a fast-speed condition to evaluate spasticity. Two examiners performed the test twice under each speed condition for each participant. Thus, we obtained four sets of time-angle data at low-speed conditions and four datasets at high-speed conditions for each study participant. The minimum speed among the four datasets of the low-velocity condition was set as the representative value for the slow condition (henceforth referred to as Slow), and the maximum speed among the four datasets for the high-speed condition was set as the representative data for the fast condition (henceforth referred to as Fast) for each participant.We calculated the velocity, deceleration value, and %ROM during passive movement using the gathered time-angle data. Each index value was defined as follows:c): the elbow joint angle that indicates the occurrence of the “catch.” Although the “catch” is typically considered the stop of motion where the velocity goes to zero, such a complete cessation of movement may not always occur, depending on the velocity and the degree of spasticity. To detect a wider range of dynamic spasticity responses, the angle of “catch” in this study was defined as the angle from the start of joint motion to the point where deceleration by braking begins. To eliminate the influence of small fluctuations in acceleration value, we defined the zero value of acceleration by a range of ±3 standard deviations (SD); the start of the movement and the start of the deceleration were defined as the points at which deceleration value exceeded the mean ± 3 SD of the acceleration at rest and the point when it returned to within the mean ± 3 SD, respectively.“Catch” angle (θMaximum velocity (°/sec): maximum value of velocity2): maximum absolute deceleration valueMaximum deceleration value at the “catch” (°/secc) / maximum flexion angle (θmax) × 100 (%ROM (%): “catch” angle (θx) × 100 .Examples of the index values are shown in post-hoc multiple comparisons were performed using Tukey's HSD test, when significant. The significance level was set at 5%. The average data was shown with the SD.The deceleration value and %ROM values at the Slow or Fast conditions were compared between the spasticity and control groups. Then, the 15 patients in the spasticity group were classified into two subgroups according to the severity of their measured spasticity (MAS1 and 1+), and the deceleration value and % ROM during passive movement of the MAS 1, MAS 1+, and control groups were compared. To investigate the relationships between spastic response and velocity, the measurement data of the deceleration value and % ROM  were classified into four velocity conditions  according to the velocity actually applied and compared within each condition. If there were two or more datapoints for the same participant within the same velocity condition, we used the average of these datapoints for our calculations. We used two-way repeated measures analysis of variance (ANOVA) for statistical analysis, and P < 0.001). Repeated-measures ANOVA of %ROM with group factor (patient or control) and velocity factor (Slow or Fast) showed significant effects for group factor , velocity factor , and group × velocity interaction . The post-hoc multiple comparison test indicated that the %ROM was significantly greater in the Fast condition compared to the Slow condition in the spasticity group . In the Slow condition, there was no significant difference in %ROM between the spasticity group and control group. However, in the Fast condition, %ROM was significantly higher in the spasticity group compared to the control group  and eight healthy individuals participated in this study. All the participants underwent complete measurements. The average maximum velocities in the Slow and Fast conditions were 103 ± 29 and 417 ± 80°/s, respectively (mean ± SD). No significant differences were observed between the spasticity and control groups . The maxF = 23.1, df = 1, P < 0.001), velocity factor , and group × velocity interaction . Repeated-measures ANOVA of %ROM with MAS factor  and velocity factor  showed significant effects for MAS factor , velocity factor , and group × velocity interaction . The post-hoc tests indicated a significant difference in the deceleration value between all velocity conditions in the spasticity group with MAS 1+ . In the spasticity group with MAS 1, there were significant differences between all the velocity conditions except for velocity conditions <200 vs. 200–300°/s . We observed significant differences between the spasticity group with MAS 1+ vs. the one with MAS 1, MAS 1 vs. the control group in 300–400 and 400 < °/s conditions (MAS 1+ vs. MAS 1: both P < 0.001).As there was marked variation in the measured velocity applied by the examiners, we analyzed the data using four different velocity groups . Unlike deceleration value, the value of %ROM did not increase with the velocity increase, and there was no significant difference in the %ROM between the 200–300, 300–400, and 400 < °/s conditions. The control group showed no difference between all velocity conditions. Comparison of %ROM between the groups in each velocity condition showed significant differences between the spasticity subgroups with MAS 1+ and MAS 1 vs. the control group at 200–300, 300–400, and 400 < °/s . Moreover, there were significant differences between the spasticity group with MAS 1+ vs. MAS 1 in only the 200–300°/s conditions (P = 0.002). There were no differences between the three groups at <200°/s conditions.In %ROM, there were significant differences between <200°/s and the other three conditions in the MAS 1+ and 1 spasticity groups  than that in the healthy participants (control group), and there were significant differences between the patients and healthy participants in the Fast condition, while there were no differences in the Slow condition. Moreover, significant differences in deceleration value between the MAS 1 and MAS 1+ groups were observed in the conditions where the speed was faster than 300°/s. Movement velocity also affected the %ROM at which the catch occurs. However, the response patterns differed from that of the deceleration value; the %ROM was higher in the Fast condition than that in the Slow condition, but no significant difference was observed between the conditions with velocity faster than 200°/s. A significant difference between MAS 1 and MAS1+ in %ROM was observed at the velocity of 200–300°/s.The deceleration value as resistance to the movement represents braking against the movement due to the spastic response, reflecting the magnitude of resistance force. Previous studies have shown that electromyographic responses and resistance increase with increasing velocity in a passive extension of spastic joints, which is consistent with the results of the present –22. In tThe difference in %ROM between patients and healthy subjects was evident for passive exercise in the Fast condition; however, not apparent in the Slow condition. In addition, no significant differences in %ROM between the velocity conditions of 200–300, 300–400, and 400 and above were observed in either of the subgroups . These results show that there is a ceiling effect for the velocity that induces a catch. Calota et al. reported that there is a correlation between velocity and the angle at which the spastic response appears, and that the tonic stretch reflex threshold (TSRT), the intercept calculated from the linear regression, is a reliable indicator of spasticity in patients with moderate to severe spasticity . SimilarOn the other hand, the velocity-angle relationships shown in this study may be more complex in more severe cases that experience spastic dystonia, which is the most frequently observed symptom in patients with poststroke hypertonia . SpasticFirst, the movement velocities in each test varied because the participants of this study underwent manual testing rather than automated testing that is inherently objective. We did not define the condition in advance since we endeavored to obtain clues from the clinical testing procedure. Although this method replicated the actual evaluation of spasticity in clinical practice, it might be insufficient to examine the effect of velocity in detail. Further studies that examine resistance response at a uniform velocity will promote our understanding of the relationship between movement velocity and spastic response.Second, the study population was limited to patients with mild spasticity, rated as MAS 1 or MAS 1+. We opted to enroll in our study patients with mild spasticity because patients with severe spasticity usually present with increased stiffness in soft tissue and enhanced neural response, either of which would require a more complex analytical procedure than that examined here, as mentioned above. Future studies with patients with more severe spasticity are needed to evaluate this point.Lastly, electromyography was not conducted in this study. We focused on the kinematic response that was directly related to the clinician's clinical maneuver. However, to investigate the mechanism behind this velocity-response relationship further, especially on the contribution of spastic dystonia, an electromyography study is necessary.While spastic dystonia in patients with MAS2 and above is a relatively clear-cut symptom that can be observed at rest, the detection of a velocity-dependent response such as catch in mild spasticity including MAS1 and MAS1+ may vary greatly depending on the examiner's technique. The present results showed a possible great variation in catch detection in clinical maneuvers; the strength of response and the catch angle varied extensively with velocity. Given the large variation in velocity in measurement procedures seen in the present study, it is possible that reliability problems with clinical scales such as MAS and MTS may be linked to the variations in procedures and therefore a strict definition of measurement conditions may improve the reliability of clinical scales.In particular, the strong dependence of the extent of braking response on the movement velocity seen in this study may indicate that the clinicians should not be influenced by the strength of braking response in manually evaluating the severity of spasticity where the speed of movement varies. For example, the strength of braking response in MAS1 patients may be greater than that in MAS1+ patients if the velocity was faster by 100–200°/s. However, the apparent differences in deceleration values between the groups in high-velocity conditions (faster than 300°/s) imply that the measurement of deceleration value can be useful when the measurement is performed in strictly fixed velocity conditions, which may be achieved by mechanical measurement.In %ROM, the differences between MAS 1+, MAS 1, and control could be detected in the velocity of 200–300°/s. Interestingly, there was no significant difference in %ROM between the conditions with a velocity of 200–300, 300–400, and >400°/s. Therefore, when the velocity is sufficiently high, the %ROM should be considered a stable indicator of spasticity in manual testing, where the velocity can vary among different testers. This may also support the reliability of existing clinical scales that depends on the angle of the catch in their assessment. However, the definition of the velocity used in the testing procedure may have to be updated. Previously, the MAS testing procedure was defined as being done by “passively stretching the muscles through their available range of motion over a period of about 1 s” . HoweverIn summary, kinematic and dynamic analysis of passive elbow movements at varying speeds in patients diagnosed with mild spasticity confirmed the velocity dependence of hypertonia, which include spasticity and possible spastic dystonia, and provided more details of symptoms than clinical scales at the individual patient level. A novel result of the present study is that kinematic measures, especially angles, are good markers of hypertonia at velocity of at least 200°/s. This finding suggests that the velocity of passive movement, when subjecting patients to MAS, should be updated; in addition, this velocity requirement may also be better considered in using MTS to improve its reliability.Here we examined the resistance response of spastic muscles to manual passive movement in clinical practice based on indices such as deceleration value and %ROM. Our results suggest that a catch against the passive movement is steadily observed in velocities faster than 200°/s and that the values of maximum deceleration in resisting the passive extension of the elbow joint are highly velocity-dependent. Conversely, the angle at which the catch against the passive joint movement occurred did not change significantly with the velocity of the movement in velocities faster than 200°/s. Therefore, the joint angle at which the resistance reaction occurs can be a stable indicator of the severity of the spasticity. Future studies that include patients with increased static muscle stiffness are warranted to further understand the velocity-dependent response to passive movements in patients with spasticity.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Fujita Health University Ethics Committee. The patients/participants provided their written informed consent to participate in this study.KF, MM, SI, HM, RI, DN, and HT designed and conducted the study, participated in data collection, and conducted data analysis. KF and MM prepared the manuscript draft with important intellectual input from YK, ES, and YO. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients with a functionally univentricular heart represent one of the most common severe cardiac lesions with a prevalence of 3 per 10,000 live births. Hemodynamics of the singular ventricle is a major research topic in cardiology and there exists a relationship between fluid dynamical features and cardiac behavior in health and disease. The aim of the present work was to compare intraventricular flow in single right ventricle (SRV) patients and subjects with healthy left hearts (LV) through patient-specific CFD simulations.Three-dimensional real-time echocardiographic images were obtained for five SRV patients and two healthy subjects and CFD simulations with a moving mesh methodology were performed. Intraventricular vortex formation and vortex formation time (VFT) as well as the turbulent kinetic energy (TKE) and ventricular washout were evaluated.The results show significantly lower values for the VFT and the TKE in SRV patients compared with healthy LV subjects. Furthermore, vortex formation does not progress to the apex in SRV patients. These findings were confirmed by a significantly lower washout in SRV patients.The study pinpoints the intriguing role of intraventricular flows to characterize performance of SRVs that goes beyond standard clinical metrics such as ejection fraction.The online version contains supplementary material available at 10.1007/s13239-021-00598-9. In Germany, the prevalence of CHD is 1.1% of live births. 12% of these patients have severe lesions that require treatment immediately after birth.29Univentricular heart (UVH) is the most common severe CHD in Germany with a prevalence of 0.03% and takes a major role in recent studies. The term functional UVH is used for heart defects in which only one ventricle is functioning and the other is absent or hypoplastic. Biventricular repair of this pathology is not possible.15If a severe hypoplasia of the left ventricle is present in functional UVH, it is referred to as hypoplastic left heart syndrome. Consequently, only one functional right ventricle is present and is described as singular right ventricle (SRV). This anomaly must be treated within the first days after birth with palliative measures by adjusting the blood supply to the lungs. Afterwards, during the first four years of the patient's life, the so-called Fontan circulation is achieved by means of several surgical interventions. The purpose of this procedure is to divide the single cardiac circulation into two. The Fontan procedure allows the systemic circulation to be supplied by only one ventricle. In this procedure, deoxygenated blood is diverted from the superior and inferior vena cava to the lungs by means of the total cavopulmonary connection (TCPC). Thus, only oxygenated blood is pumped into the body through the one functional ventricle.6 Patients with Fontan failure can develop progressive functional impairment, such as gradual systolic and diastolic ventricular dysfunction.10The Fontan procedure is a palliative strategy for patients with single-ventricle circulation. However, its inherent limitations and deficits are becoming apparent as more and more patients are present with failing Fontan physiology.36There is a need to increase the understanding of ventricular function and physiology of Fontan patients to improve therapeutic approaches in the future. It is well known that intraventricular hemodynamics play a central role for the description of ventricular function.in vivo studies with imaging techniques (ultrasound and MRI), in vitro experiments with particle image velocimety (PIV), as well as numerical simulations have been performed.27Many studies have been performed over the years to investigate fluid dynamics in the heart and especially in the left ventricle. Among these studies, 33 A central aspect of such studies is the investigation and evaluation of vortex formation within the ventricles.11 Furthermore, flow analyses focus on quantitative flow energetics and blood transit properties.33 For instance, the study by Loke et al.21 has shown that vorticity and energy loss are correlated to right ventricular dysfunction in Tetralogy of Fallot patients using 4D flow MRI. The same group also used CFD simulations based on cardiac MRI data to further investigate flow parameters in this patient cohort.20Recent studies aimed to perform flow simulations using a moving mesh method or fluid-structure interaction (FSI). As flow is strongly influenced by the motion and shape of the heart, these methods are expected to provide a more realistic representation of blood flow within ventricles. In the moving mesh method, ventricular wall motion is captured patient-specifically by imaging techniques and integrated as a boundary condition in CFD flow simulations.et al.26 observed differences in kinetic energy between ventricles of Fontan patients and healthy volunteers through 4D flow MRI. Wong et al. analysed through 4D MRI the flows and kinetic energies in moving singular ventricles.36 Finally, the study by Sjöberg et al.31 determined lower diastolic ventricular kinetic energies in patients with Fontan circulation with 4D flow MRI as well. A combination of echocardiography and computational fluid dynamics (CFD) simulations was used by Chen et al.5. Real-time 3D echocardiography is a commonly used method for recording ventricular motion because it allows examination of patients with less discomfort and in less time compared to other imaging methods.3A few recent studies have focused on the UVH and flow imaging. Rutkowski et al.5 that the cardiac performance for both ventricles should be in the same range. The findings of this study underline that 3D modeling based on echocardiography is suitable to represent the morphological and functional abnormalities in the SRV during the cardiac cycle. Furthermore, CFD can detect abnormal flow patterns and vortices in the SRV compared with healthy LV. A limitation of Chen et al. was the consideration of three characteristic time steps throughout the diastole and steady-state simulations at these time points. In our study we expand the work by integrating ventricular motion into CFD analyses of healthy subjects (LV) and SRV from Fontan patients. We hypothesize that there are differences in fluid dynamic markers between these two groups.The pathological SRV and the healthy LV both have to transport blood through the systemic circulation. Therefore, it was assumed in Chen Time-discrete ventricular volumes of five patients with single right ventricle (SRV) circulation and two subjects with healthy left ventricle (LV) were acquired from transthoraxic real-time 3D-echocardiography measurements performed at the University Hospital Bonn (UKB). The ultra-sound device iE33  was used to record the volumetric shape of the ventricle over one cardiac cycle. Between 12 and 28 frames per heartbeat were recorded. The study was approved by the local ethics institutional review committee (Registration No. 226/06 and No. 007/15) and conformed to the principles of the Declaration of Helsinki as well as German law.et al. and represent different ages, end-diastolic volumes (EDV), and ejection fractions (EF).19 The LV patients were selected from Krell et al. and represent small (pediatric) ventricles with EDV 40–60 mL.14 Table The SRV group was selected from data of Linden The geometric data from the echocardiography was translated into a unicode character database (UDC) format using the program ImageArena . For each time step a 3D model with a constant number of cells and associated cell nodes was created. Afterwards, the UCD data was translated using Matlab into an STL file format for the application of the moving mesh model.All STL files had the same number of nodes to facilitate interpolation between the individual time steps and the integration of continuous ventricle surface movement during CFD simulations. Additional mesh smoothing was performed with Blender 2.82.y + value of < 5 was taken as a measure for appropriate resolution of the boundary layer flow. Depending on the EDV, the element number ranged from 485,000 up to 3,476,000.Meshing of the geometry was performed with the ANSYS Meshing tool from ANSYS 2019 R1  with unstructured tetrahedral elements supported by a "Patch Conforming Method”. The independence of the near-wall flow mesh was determined based on the area average velocities at the inlets and outlets and the maximal wall shear stress during peak diastolic flow (E-wave) and changes in the variables of < 5% between two mesh refinement steps were considered independent. A et al.1 with a density of ρ = 1056.4 kg/m3 (hematocrit level of 44%) was applied. In addition, the pressure-based solver was selected and set in coupled mode and solved with ANSYS Fluent. The flow was assumed to be turbulent and the k–ω–SST model was applied. For the convergence of the velocity, k- and ω-residuals the criteria were set to 0.001 and for the continuity to 0.0003. The maximum number of iterations was 30. Resulting time steps ranged between 0.0018 and 0.004 s. The simulations started in the end systolic state of the cardiac cycle and ran for five cycles. The cycle independence was obtained by looking at the averaged and maximum velocity at the inlet and outlet and independence was identified after the second cycle.For the simulations the non-Newtonian blood model by Ballyk et al.19). The pressure curves for the healthy LV have been calculated via a lumped parameter model (LPM) of the UKB.35 Valves were not modeled and a wall or a fully open inlet/outlet was set as the zone function.As inlet and outlet conditions, patient specific pressure curves were set as boundary conditions for the SRV patients. These curves were taken from clinical data of UKB  and, after temporal and spatial interpolation of these geometries, ventricular wall movement was implemented through the mesh motion capabilities of ANSYS Fluent by using user defined functions. A detailed explanation of the workflow with the according scripts is included in the Supplementary Material.For the evaluation of the blood flow characteristics, vortex structure formation, vortex formation time, turbulent kinetic energies and ventricular washout were investigated.Vortex structure formation was visualized by the Q-criterion within the ventricle.13The vortex formation time (VFT) was calculated as the time integral of the velocity from the start of the diastole to the peak of the E-wave. VFT is a dimensionless parameter describing the process of vortex formation during the beginning of diastole. It is a characteristic value for ventricular filling and it has been shown that reduced VFT correlates with deteriorated diastolic ventricular function.13 The TKE was calculated in ANSYS Fluent based on the transport equation for the k–ω-SST turbulence model.34The turbulent kinetic energy [TKE (J/kg)] is a direction-independent measure of the intensity of turbulence and represents the kinetic energy of the fluctuating velocity field.The value of TKE within the ventricle was calculated for each subject as an integral value after median averaging of the last four cycles. In addition, the ratio of systolic/ diastolic TKE to total TKE was examined.32 These simulations were performed with two fluids  with identical rheological properties (see above).Finally, the efficiency of blood exchange within the ventricle was evaluated using washout simulations, as described in.old_blood and new_blood with a volume fraction of 1 at the inlet was set throughout the simulations. The flow simulation with new_blood was performed for three cycles gradually replacing the old_blood. The Volume of Fluid methodology was used to describe the interaction between the two fluids, see32 for further details. Results were investigated after cycle independency has been reached.The entire domain was initialized with t-test and a significance threshold of p = 0.01. The simulations were performed on a 64GB RAM system using 10 physical cores. Depending on the volume of the ventricles, the simulation duration was between 40 and 360 core h.The results of the fluid analysis for the SRV group are shown as mean values and standard deviation. Furthermore, statistical comparison between the SRV group and the individual LV was performed with a one-sample Figure The presented SRV of subject #3 has an EDV of 55 mL and an EF of 51%. The beginning of the E- and A-wave can be recognized by a developing vortex at the tricuspid valve (inlet). During diastasis, the annular vortex formed at the E-wave remains only in the basal part of the ventricle. During the A-wave, another circular vortex is formed, which spreads further down than that of the E-wave, but also remains only in the basal part. Throughout systole, the vortex ring of the A-wave develops into an elongated vortex that is ejected at the end. The presented SRV-case is exemplary for the examined SRV-patients. In all cases, the vortex formation during the diastasis does not extend to the apex of the ventricle.Exemplary blood flow in a healthy ventricle is also shown in Fig. Of note, TKE is almost an order of magnitude higher in the healthy LV subject #7 in comparison to SRV subject #3.Figure The results for the flow analysis for five cardiac cycles of each patient are summarized in Table new_blood (red) into the SRV during the first cycle reaches only the basal part of the ventricle. The repeated injection of new_blood during the second cycle leads to progression of the fluid towards the apex. However, at the end of the third cycle, residual old_blood (blue) is still visible in the apical part of the ventricle. The SRV of patient #3 contains 74% of new_blood after three cycles. A lack of fluid exchange in the apical part is observed in all simulated SRV patients. In contrast, the injection of new_blood during diastole of the healthy LV (#7) in the first cycle already reaches the apical part. After the third cycle the ventricle is 95% filled with new_blood.The washout characteristics of SRV subject #3) and healthy LV (subject #7) are shown in Fig.  and healp < 0.01 for washout after three cycles.In the graph on the right side of Fig. In this study, the blood flow inside pathological SRVs and healthy LVs was analyzed using real-time 3D echocadiographic data. For this purpose, data from five Fontan patients and two healthy subjects were implemented in a moving mesh simulation model. The results obtained from numerical analysis of the intraventricular flow showed significant differences in VFT, TKE_tot, and washout between SRV and LV.The investigation of blood flow within the ventricle was based on the moving mesh model, which includes the movement of the ventricular inner wall as a boundary condition. The model was used to perform patient-specific analyses of cardiac flows and to investigate the differences between SRV and LV for seven subjects.22 With the SRV, one vortex forms at the beginning of each of the E and A waves at the inlet, splits into several elongated vertebrae, and remains in the middle of the ventricle until the end of systole. Observation of vortex formation using the Q-criterion shows that blood is not completely drawn to the apical region during diastole, which was also observed in Lampropoulos et al.17 The amount of vortex formation was qualitatively lower in the SRV compared to the LV.The vortex formation in healthy LV compared to pathological SRV, showed a more distinct development in healthy subjects. According to the literature, a main vortex forms in the middle of the ventricle up to the apex.24 In the normal left ventricle, a value above 50% is taken as an indication of a patient considered healthy.18 In Fontan patients where the SRV takes over the function of the systemic ventricle, the mean EF was 46.4%.EF is one of the basic evaluation factors in echocardiography for cardiac function.22 The examined SRV subjects had VFT values of 1.16 ± 0.53, indicating a weak diastolic vortex formation and filling phase. The diastolic vortex stores kinetic energy, which initiates the ejection of blood during late diastasis. The formation of a weak diastolic vortex was also evident from the results of TKE. SRV subjects have significantly lower total TKE than the compared subjects with healthy LV. Furthermore, the ratio of diastolic TKE to total TKE is 45.7% in SRV subjects and 56–61% in LV subjects. Optimal filling and formation of TKE during diastole is necessary for sufficient energy for ventricular ejection during systole.17VFT is a dimensionless factor that has been used in several studies as an indication of an optimally forming vortex during diastole. Healthy subjects have a VFT around 4.22 The remaining blood in the apex could indicate that although the required stroke volume is provided by the ventricle, the overall fluid exchange and therefore the transport of oxygenated blood is reduced. The washout simulations in this study deliver an additional method for representing the performance of a ventricle.The washout simulations illustrate the weak forming vortex during diastole and the associated inadequate filling of the entire ventricle. It was observed that residual blood remains longer in the apex of the SRV compared to the LV. After three cycles, SRV subjects have an average residual volume of 24% in the ventricle. Healthy subjects with LV have a residual volume between 5 and 7% after three cycles, similar to.During the study, a relationship between TKE and VFT that deviated from the mean was observed in two SRV subjects . VFT, which serves as a parameter for vortex formation during diastole, was below the mean of 1.16  for the two subjects . This indicates extremely weak diastolic filling of the ventricle. In contrast, these two subjects showed an increased total TKE  compared with the mean of 0.881 J/kg of all SRV, indicating comparatively improved overall ventricular performance. Additionally, an increased ratio of systolic TKE to total TKE  was observed compared with the mean ratio of 54.3%. Overall, the results in these two patients show impaired diastolic filling and increased systolic ejection, which indicates a diastolic dysfunction of the ventricle and suggest a compensation of this dysfunction during the systole. These findings should be verified in the future by a larger subject number.The advantage of the acquisition technique with the iE33 Philips ultrasound scanner is the possibility of patient-friendly and time-discrete imaging of a cardiac cycle. Real-time 3D echocardiography is a non-invasive and reliable technique that can be used at the bedside. Disadvantages of this imaging technique (related to its application in CFD simulations with a moving mesh methodology) are on the one hand the quality of the outer mesh for the geometric form. During ventricular contraction, the triangular mesh, which is constant for all time steps, causes strong deformations of the individual cells. This can lead to distorted cell nodes or illogical shapes. Manual editing of the geometry outer surface allows to obtain feasible mesh models for numerical analysis, but is time-consuming. On the other hand, the lack of proper resolution of the atrioventricular and aortic valve shows up a second disadvantage. Here additional data with e.g. transesophageal echocardiography might be an imaging modality for good resolution of the valve.30 To reduce inaccuracies in the spatial description of the motion due to the low time resolution of the data, the position parameters were primarily integrated over the total ventricular volume and cardiac cycles.Regarding this study, one of the most critical limitations was the lack of geometric information about the valvular morphology. For the data used in this study, the field of view was limited for the transthoracic ultrasound images, and the focus of the currently used software was on volumetry. The dynamics of the valve and the geometry of the atrium affect the specific properties of the inflow jet.Another challenge in the evaluation of univenticular hearts is the inhomogeneous Fontan patient population. The complexity of the pathology and the surgical history of these patients is often highly individualized. Because of this limitation, we have focused only on the anatomy of singular right ventricles in this study for the time being.Despite the small number of analyzed patients, the results already show significant differences for washout, TKE and VFT.in vivo flow imaging such as 4D MRI.Further work should focus on evaluating and comparing a higher number of subjects with univentricular hearts and healthy hearts including additional information about valvular structure. The complex patient history of Fontan patients and the related anatomical conditions must always be kept in mind. To improve the usability of the acquired real-time 3D echocardiography images, methods for better mesh processing should be elaborated. Furthermore, it is important to integrate a directed flow into the simulation model, preferably under integration of heart valves. Subsequently, the simulation models require validation. This could be done in the form of a suitable PIV study or This study has shown significant differences in intraventricular flow between healthy subjects and Fontan subjects using real-time 3D echocardiography based CFD simulations. Weak vortex formation during diastole was characteristic for the examined SRV subjects. This observation was also evident in the significantly lower TKE and in the weaker washout. Numerical flow analysis presented additional parameters for the interpretation of cardiac performance besides the clinically obtained value of EF.The studied SRV subjects had an average EF of 46%. Considering the SRV as a systemic ventricle analogous to the healthy LV, this EF suggests a mildly abnormal performance. However, the numerical flow analyses of VFT, TKE, and washout demonstrated strongly deteriorated cardiac performance. While EF is a pure volume measure, numerical analysis provided additional information on blood flow within the ventricle. By expanding the number of analyzed ventricles, these relations should be further investigated.Supplementary file1 (DOCX 149 KB)Below is the link to the electronic supplementary material.