Current non-invasive electrocardiographic imaging (ECGi) methods are often based on complex body surface potential mapping, limiting the clinical applicability. The aim of this pilot study was to evaluate the ability of a novel non-invasive ECGi method, based on the standard 12-lead ECG, to localize initial site of ventricular activation in right ventricular (RV) paced patients. Validation of the method was performed by comparing the ECGi reconstructed earliest site of activation against the true RV pacing site determined from cardiac computed tomography (CT).This was a retrospective study using data from 34 patients, previously implanted with a dual chamber pacemaker due to advanced atrioventricular block. True RV lead position was determined from analysis of a post-implant cardiac CT scan. The ECGi method was based on an inverse-ECG algorithm applying electrophysiological rules. The algorithm integrated information from an RV paced 12-lead ECG together with a CT-derived patient-specific heart-thorax geometric model to reconstruct a 3D electrical ventricular activation map.2 and the mean circular LE area was 7.0 ± 5.2 cm2 resulting in a relative LE of 5.0 ± 4.0%.The mean geodesic localization error (LE) between the ECGi reconstructed initial site of activation and the RV lead insertion site determined from CT was 13.9 ± 5.6 mm. The mean RV endocardial surface area was 146.0 ± 30.0 cmWe demonstrated a novel non-invasive ECGi method, based on the 12-lead ECG, that accurately localized the RV pacing site in relation to the ventricular anatomy. Along with QRS morphology from the 12-lead electrocardiogram (ECG), fluoroscopy is the main guiding tool for right ventricular (RV) pacemaker lead implantation. However, several studies have demonstrated the inaccuracy of both fluoroscopy and QRS morphology to determine RV lead implantation site . InterinNon-invasive electrocardiographic imaging (ECGi) provides 3D reconstruction of the cardiac electrical activity overcoming some of the limitations from the standard 12-lead ECG, potentially increasing the diagnostic value . HoweverThe aim of this pilot study was to evaluate the ability of a novel non-invasive ECGi method, based on the standard 12-lead ECG, to localize initial site of ventricular activation in RV paced patients. Validation of the method was performed by comparing the earliest site of activation estimated from the reconstructed ventricular activation model against the true RV pacing site determined from cardiac computed tomography (CT).de novo implantation of a dual chamber pacemaker due to advanced atrioventricular block at Aalborg University Hospital between December 2014 and December 2017 were retrospectively included. Patients with suspected fusion pacing or patients who had been upgraded to cardiac resynchronization therapy after primary pacemaker implantation were not considered eligible. Following data were acquired at time of study inclusion: clinical characteristics from electronic medical records, a contrast-enhanced cardiac CT scan, an RV paced 12-lead ECG including a 3D photography showing the ECG electrode positions and a transthoracic echocardiography.Thirty-four patients who underwent n = 30), a study specific cardiac CT was acquired using a second-generation dual source scanner . During breath hold, the contrast-enhanced retrospective ECG gated scan was timed with contrast filling of both ventricular cavities. Reconstruction was done in diastole with a mean slice thickness of 0.95 ± 0.1 mm for the narrow field of view focused on the heart. The mean total dose length product (DLP) for the study-acquired CT scans was 114.9 ± 65.3 mGy cm and mean contrast dose (Iopromide 370 mg/ml) was 67.4 ± 9.6 ml.If no contrast-enhanced cardiac CT, showing the RV lead position, was available . The echocardiograms were analyzed using EchoPAC software . Analyses included estimation of left ventricular ejection fraction (LVEF) and left ventricular (LV) volumes .The ECGi method was based on an inverse-ECG algorithm that reconstructs the electrical activity of the ventricles by integrating information from an RV-paced 12-lead ECG, location of surface ECG electrode positions together with a CT-derived patient-specific heart-thorax geometric model .For each patient, a standard 12-lead ECG during RV pacing was digitally recorded at time of study inclusion using the Cardiovit AT-102 plus resting ECG machine . All ECG recordings were imported to the MUSE Cardiology Information System . In MUSE, QRS onset and thus duration was manually adjusted on median beats formed by version 23 of the Marquette 12SL algorithm to exclude the pacing spike. A single QRS onset was defined across the 12 leads as the earliest positive or negative deflection after a pacing spike. Furthermore, a 3D photography of the patient thorax was recorded documenting the position of the ECG electrodes at time of the ECG recording.The patient specific heart-thorax geometric models were created from the cardiac CT scan using specialized research software  . The modApplication of the algorithm was automated using specialized software . The alga)The first step was to determine the QRS axis. The heart-thorax model was used to derive the vectorcardiogram (VCG) from the recorded 12-lead ECG, using the method described by Boonstra et al. . The meab)The Fastest Route Algorithm, together with a realistic myocardial propagation velocity (0.7–0.85 m/s), was used to compute depolarization times between the discrete nodes on the closed triangulated modeled myocardial surface mesh . In thisc)The cardiac depolarization sequence was used to compute ECGs applying the equivalent dipole layer model as a source model, where local transmembrane potentials simulated the local currents generated by the heart , 18. Thed)Subsequently, the improvement procedure was started in which additional extra late break through points (foci) were added in the left cavity, mimicking (late) breakthroughs from the His-Purkinje system. The location and timing of the initial RV stimulation site and the added His-Purkinje nodes was optimized in an iterative procedure, matching the recorded ECG and simulated ECGs. The simulated ECGs were the result of the depolarization sequence generated from the location and timing of the combination of different foci.e)Finally, the depolarization sequence resulting in a simulated ECG best matching the recorded ECG was chosen for final analysis.Performance of the reconstructed electrical activation was assessed by calculating the localization error (LE) defined by the geodesic distance (shortest distances between two points on a curved surface) between the RV CT-marker and the reconstructed initial site of activation (rISA) . FurtherThe rISA was constrained to the discrete nodes of the ventricular geometric model whereas the marker for RV lead position was localized on the CT scan and imported into the heart model without this constraint. This created a potential default distance between the RV marker and the rISA. This was adjusted for by calculating a corrected localization error (cLE) defined as the distance from the rISA to the discrete node closest to the RV marker on the path between the rISA and the RV marker. A cLE of 0 mm meant that rISA was located in the triangle to where the RV lead marker was projected on to the endocardial model surface.Performance of the reconstructed activation sequence was assessed comparing it with an activation sequence forced to initiate from the known RV lead insertion site. This was done to assess how sensitive the overall activation sequence was to change in initial site of activation. Lastly, the correlation between the recorded ECG and the ECG fitting the reconstructed model was assessed.t-test was used for binary independent variables and fractional polynomial regression was used for continuous independent variables. To compare different activation maps within one patient the Pearson correlation coefficient was used. p < 0.05 was considered statistically significant. STATA version 17 was used to perform the statistical analyses.Continuous variables are reported as mean ± standard deviation (range) unless severely skewed. Categorical values are reported as absolute numbers and percentages. For analyzing associations, unpaired Patient characteristics  showed t2 and the mean circular LE area was 7.0 ± 5.2 cm2 resulting in a LE error of 5.0 ± 4.0% relative to the RV endocardial area.The mean LE was 13.9 ± 5.6 mm (range 4.3–28.6 mm) , 5. For p = 0.02) among those with a septal lead position. This association remained significant for the cLE and in a sensitivity-analysis excluding the outlier.Several variables possibly affecting local cardiac conduction properties, myocardial propagation velocity and quality of the input data were investigated for their potential to affect the accuracy of the localization algorithm. Following variables were tested for an association with LE: body mass index, gender, age, use of beta-blocker, ischemic heart disease, LVEF, LV end-diastolic volume, RV lead segmental position, noise in the recorded ECG, heart rate and QRS duration. Only the segmental RV lead position was significantly associated with the LE. The mean uncorrected LE among those with a non-septal lead position was 15.2 mm (95% CI 13.1–17.4) compared to 10.3 mm  . The cor > 18 mm . Mean cor = 0.88 (range 0.62–0.99)]. Correlation was > 0.8 in 28 (82%) patients. The correlation between the fitted ECG and the recorded ECG was not associated with the LE (p = 0.22).Overall, the fitted ECG was highly correlated with the recorded ECG [median Having loaded the patient-specific model and 12-lead ECG into the specialized software system, the mean algorithm computation time used for reconstructing the ventricular activation was 1.1 ± 0.4 s per ECG using a standard laptop (Intel CORE i7 CPU).In this pilot study, we developed and validated a novel non-invasive ECGi method based on the 12-lead ECG for reconstructing ventricular activation during RV pacing and showed: (1) The rISA was accurately and effectively localized in relation to ventricular anatomy. (2) The reconstructed ventricular activation sequence was robust to small changes in initial site of activation.Current ECGi methods are often based on more complex body surface potential mapping (BSM) derived from up to 256 leads . Only fer = 0.92, range 0.73–0.99) indicating that despite varying degrees of LE, the reconstructed activation pattern is robust to smaller changes in initial site of activation. The used regularization method in the final optimization, minimizing the L2 error norm of the ECG signals, maximizes the correlation between measured and simulated ECG signals. Due to the smoothness of the initial estimate based on a physiological based activation pattern, no large variations are expected in the error function. Consequently, the found initial correlations are equivalent to the resulting fitting errors in the modeled activation pattern. However, it was not possible to compare the reconstructed activation sequence with invasive electrophysiological mapping and the accuracy of the reconstructed activation sequence needs to be confirmed in future studies using invasive electrocardiographic mapping for comparison.It was not possible to evaluate the accuracy of the ECGi method using invasive electrocardiographic mapping as ground truth data, as it was not available for comparison . HoweverECGi is not a direct measurement of electrical activity of the heart, it is a reconstruction estimated by an algorithm using different input data. Regardless of the ECGi method used, there is some inherent sources of error depending on the algorithm used and the quality of the input data , 25, 26.Our ECGi method assumed a uniform conduction velocity throughout the myocardium. However, we do not know how well the method works when the assumptions in the algorithm are violated. Elderly pacemaker patients often suffer from ischemia, diabetes or hypertension with hypertrophic myocardium and fibrosis resulting in areas of slower conduction velocities , 28. ThiBased on these promising results, further work to improve the accuracy of the method seems encouraging. The reconstructions are calculated almost real-time. Thus, potentially enabling intraprocedural model-assisted evaluation of ventricular activation and lead position in relation to ventricular anatomy ensuring optimal pacemaker lead implantation for the individual patient. Furthermore, providing a detailed electrical activation pattern, ECGi is highly relevant as a tool to guide and optimize cardiac resynchronyzation therapy (CRT) implantation , 29–31.As described in detail above, comparison with ground truth data from invasive electrocardiographic mapping was not available and the quality of the input data was varying possibly limiting the accuracy. The current method includes creating a patient-specific heart-thorax model, which is a cumbersome process requiring a cardiac CT scan. Future studies should investigate the feasibility of using a generic model to reduce the workload and avoid the need for a CT scan, making the method more manageable in a clinical setting.In this pilot study, we demonstrated a novel non-invasive ECGi method, based on the readily available 12-lead ECG, which accurately and effectively localized initial site of activation in relation to ventricular anatomy and provided an estimate for the ventricular activation sequence during RV pacing.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.PF: conceptualization, investigation, methodology, formal analysis, data curation, writing—original draft, and visualization. PV: conceptualization, methodology, software, visualization, and writing—review and editing. AS, JM, and TZ: conceptualization and writing—review and editing. SL-C: conceptualization, formal analysis, and writing—review and editing. PS: investigation and writing—review and editing. CG: conceptualization, methodology, data curation, and writing—review and editing. SR: conceptualization, methodology, writing—review and editing, and supervision. All authors contributed to the article and approved the submitted version.


A 38-year-old man was admitted to our hospital after ventricular tachycardia. Endocardial bipolar and unipolar voltage mapping were performed and findings were integrated with data from intracardiac echocardiography (ICE) right ventricular (RV) speckle-tracking analysis. A reduction in the strain analysis was stored in correspondence of the fragmented electrogram area. The definitive diagnosis was arrhythmogenic RV cardiomyopathy (ARVC). The integration of ICE-derived RV strain and voltage mapping could represent a successful strategy to improve the results of ablation in ARVC. Three hundred milligrams of amiodarone was then administered intravenously, resulting in the restoration of sinus rhythm. Twelve-lead resting electrocardiography (ECG) was performed, which revealed a suspected notch as an epsilon wave at V1 , with negative T-waves observed in leads V1–V3.A 38-year-old man was admitted to our hospital after a sustained ventricular tachycardia (VT) with a left bundle branch block (LBBB) morphology with an inferior axis, QRS transition in V5. The cycle length of the VT was 250 ms Transthoracic echocardiography (TTE) revealed a right ventricle (RV) with bulging of the free-wall mid-segment from the 4-chamber apical view. Speckle-tracking echocardiography (STE) analysis performed on TTE clips revealed a reduction in RV longitudinal strain (LS) in all 3 myocardial layers in the sub-tricuspid region..During the observation, the patient underwent cardiac magnetic resonance imaging, which showed an RV with slightly reduced ejection fraction (EF) of 45%, dyskinesia of the mid-segment of the free wall, and a non-ischemic subepicardial pattern of late-gadolinium enhancement at the mid-basal anterior septum and inferior biventricular junction In-hospital ECG monitoring documented sporadic ventricular premature beats (VPBs), couples, and triplets with 2 different morphologies, the first of which had an LBBB pattern with an inferior axis, comparable to the morphology of the VT previously observed, and the second of which had an LBBB pattern with a superior axis.2 ARVC was diagnosed by the combination of an epsilon wave, negative T-wave from V1–V3 and non-sustained VT with LBBB and a superior axis (major criteria), mildly increased RV dimension, and reduced EF using cardiac magnetic resonance imaging (minor criteria). Genetic testing confirmed the presence of a known pathogenic mutation in the DSP gene, a gene in which truncating alleles have been reported when ARVC is present.3According to the 2010 Task Force Criteria,, and a 3D fast anatomical map was then created and merged with the 3D echo map. Bipolar voltage definitions of abnormal and normal myocardium were based on previously validated data in the RV . Detailed maps of the endocardial RV surface during sinus rhythm were obtained with a dedicated electroanatomic mapping (EAM) system and a 4-mm standard tip catheter or 3.5-mm open irrigated-tip catheter and multipolar catheter using bipolar (bandpass-filtered at 10–400 Hz) and unipolar (bandpass-filtered at 1–240 Hz) voltage mapping. The endocardial regions with a bipolar electrogram amplitude > 1.5 mV were defined as “normal,” “scar” was defined as an area with an amplitude < 0.5 mV, and “abnormal” myocardium was defined as a region with a bipolar electrogram amplitude of 0.5–1.5 mV or unipolar amplitude between 3.5–5.5 mV . The voltage map was obtained using a total of 6,000 map points, and unipolar and bipolar voltage mapping of the RV showed low-voltage areas and corresponding fragmented potentials from the tricuspid annulus to the inferior apex  and 3H. In the RVOT, the bipolar voltage mapping was normal, while the unipolar mapping showed low-voltage areas in the anteroseptal outflow tract. An offline map was also used to perform STE on intracardiac RV and standard echocardiography (TTE).The patient underwent an invasive electrophysiologic (EP) study by a dedicated mapping software and intracardiac echocardiography (ICE), obtaining a 3-dimensional (3D) map. Two morphologies of VPBs were identified at baseline during sinus rhythm, originating from the RV inferior wall and the RV outflow tract (RVOT), respectively. In the ICE 3D-RV reconstruction, the multipolar catheter performed a voltage map reconstruction without fluoroscopy Pace-mapping found a 97% match for the morphology from the inferior wall and a 95% match for the morphology from the RVOT. Isoprenaline infusion was then started, inducting sinus tachycardia and aberrant atrioventricular conduction with an RBBB and superior axis morphology. After a programmed ventricular stimulation , non-sustained VT was induced with both an LBBB pattern and inferior axis and an LBBB pattern and superior axis. With TTE, a reduction of the RV LS was shown from the tricuspid annulus  to the inferior free wall.. Interestingly, the endocardial LS was reduced from sub-tricuspid segments to the RV apex in accordance with the fragmented potentials stored during voltage mapping. On the contrary, at the anterior RVOT wall, the unipolar voltage mapping showed fragmented potentials, and the STE analysis revealed a reduced epicardial LS.STE analysis on ICE views showed a reduction of the RV LS in the segments below the tricuspid valve in the 3 different myocardial layers . During catheter ablation, an irritable VT occurred, and the corresponding ECG morphology was the same as that observed in the first clinical VT. Three days after catheter ablation, the patient underwent implantation of a subcutaneous implantable cardioverter-defibrillator for secondary prevention of sudden cardiac death. He was discharged 12 days after admission on a low dose of β-blocker therapy (2.5 mg of bisoprolol daily). Six months of subcutaneous implantable cardioverter-defibrillator follow-up confirmed no shock delivery.Considering the arrhythmic substrate in an ARVC patient, we decided to proceed with a 30-W catheter ablation using power control for a total of 2 min at the level of each fragmented potential of the inferior wall, where low voltage had been documented by unipolar and bipolar mapping 4 In ARVC, a fibrofatty replacement involves the basal and mid-apical segments of the RV, and the so-called “triangle of dysplasia” refers to a small region involving the RV inflow tract, RVOT, and RV apex, where structural abnormalities are typically found in the early stages of the disease.2ARVC still remains one of the main causes of sudden cardiac death, particularly in the younger population.5 Moreover, TTE indices tend to be progressively more abnormal with a likelihood of ARVC as demonstrated in adolescents.6TTE has been the first-line tool used in these situations, although it has low specificity. In addition to RV dimensions, indices commonly impaired are the M-mode of the tricuspid annulus in the lateral, septal, and posterior positions; tissue Doppler velocity during early and late diastole in the same positions; systolic annular velocity in the lateral annulus; and a decreased E:A ratio.The arrhythmic substrate of ARVC can be recorded during voltage mapping as a low-amplitude, fractionated signal reflecting a pathologic conduction such that electroanatomic voltage mapping can identify the scars that correlate with pathognomonic histopathological features of the disease. The RV regional and transmural EP heterogeneities constitute the arrhythmogenic substrates, targets of catheter ablation in ARVC patients. Several studies have addressed the ability of unipolar and bipolar EAM to identify dysplastic areas identified by echocardiography in ARVC populations.9 RV mechanical dispersion in ARVC can detect segments with subtle dyskinesia also in earlier stages of the disease and might reflect an arrhythmic risk.11 In particular, Kirkels et al. showed that abnormal deformation patterns and a higher mechanical dispersion, both assessed through STE parameters, are independently associated with ventricular arrhythmias in ARVC.12 However, compared to TTE, ICE has a better imaging resolution; moreover, it allows real-time integration with EAM and can provide an optimal RV inflow/outflow view for RV anatomy and function evaluation and strain deformation assessment.13Previous studies applying speckle-tracking TTE to ARVC patients have provided promising results, with RV myocardial-deformation indices demonstrating significantly reduced strain with relevant predictive value.As highlighted in this clinical case, the use of ICE-derived RV strain could guide the use of EAM in order to detect and delimit with more sensitivity the areas of RV whose ablation leads to more successful results. To our knowledge, our report is the first case to describe the relationship between LS and cardiac electrograms during voltage mapping. Moreover, the use of ICE facilitates a precise definition of the RV boundaries of the tricuspid and pulmonary fibrous annuli and a reduction in fluoroscopy use for diagnostic or therapeutic EP studies and catheter ablation. However, other studies are currently needed to better characterize the combined approach of STE using ICE and voltage mapping guided by a dedicated software of reconstruction.


Although Chagas cardiomyopathy is related to thromboembolic stroke, data on risk factors for cerebrovascular events in Chagas disease is limited. Thus, we assessed the relationship between left ventricular (LV) impairment and cerebrovascular events and sources of thromboembolism in patients with Chagas cardiomyopathy.This retrospective cohort included patients with chronic Chagas cardiomyopathy who underwent cardiovascular magnetic resonance (CMR). CMR was performed with a 1.5 T scanner to provide LV volumes, mass, ejection fraction (LVEF), and myocardial fibrosis. The primary outcome was a composite of incident ischemic cerebrovascular events (stroke or transient ischemic attack—TIA) and potential thromboembolic sources , atrial flutter, or intracavitary thrombus) during the follow-up.A total of 113 patients were included. Median age was 56 years (IQR: 45–67), and 58 (51%) were women. The median LVEF was 53% (IQR: 41–62). LV aneurysms and LV fibrosis were present in 38 (34%) and 76 (67%) individuals, respectively. The median follow-up time was 6.9 years, with 29 events: 11 cerebrovascular events, 16 had AF or atrial flutter, and two had LV apical thrombosis. In the multivariable model, only lower LVEF remained significantly associated with the outcomes . Patients with reduced LVEF lower than 40% had a much higher risk of cerebrovascular events and thromboembolic sources (HR: 3.16 95% CI: 1.38–7.25) than those with normal LVEF. The combined incidence rate of the combined events in chronic Chagas cardiomyopathy patients with reduced LVEF was 13.9 new cases per 100 persons-year.LV systolic dysfunction is an independent predictor of adverse cerebrovascular events and potential sources of thromboembolism in patients with chronic Chagas cardiomyopathy.The online version contains supplementary material available at 10.1186/s12968-022-00885-x. Trypanosoma cruzi (T. cruzi), transmitted to human beings primarily by contact with blood-sucking triatomine bugs. However, other transmission routes are still possible, such as blood transfusion, solid organ transplantation, congenitally from mother to fetus, and oral infection through ingestion of contaminated food or drink [T. cruzi worldwide, while 75 million people are at risk of infection [Chagas disease is caused by infection with the protozoan parasite or drink . Despitenfection . Furthernfection , 4.Chronic Chagas cardiomyopathy, the most severe and frequent manifestation of Chagas disease chronic phase, may lead to heart failure, arrhythmias, and systemic and pulmonary thromboembolic events . Even thHowever, data about the association of LV impairment with cerebrovascular events in patients with Chagas disease is limited, so a debate continues about the best strategy for preventing and managing stroke and transient ischemic attack (TIA) in this clinical setting . DespiteThus, the purpose of this study was to investigate the relationship between LV morphological and functional parameters, assessed by cardiovascular magnetic resonance (CMR), with the incidence of ischemic cerebrovascular events and potential sources of thromboembolism in patients with chronic Chagas cardiomyopathy.. Exclusion criteria were evidence of other types of cardiomyopathies, known obstructive epicardial coronary artery disease, primary valvular heart disease, or contraindication for CMR, including cardiac implantable electronic devices and claustrophobia, as previously described [In this time-to-event analysis, we retrospectively collected data from 140 adult patients aged ≥ 18 years and with chronic Chagas cardiomyopathy who underwent CMR from October 2009 to December 2013. All patients were followed in the outpatient clinics at the Hospital das Clínicas de Ribeirão Preto, University of São Paulo, Brazil. Chagas disease was defined by the positivity of at least two different serological tests: enzyme-linked immunosorbent assay, indirect immunofluorescence, or hemagglutination. CMR was requested according to a prior study protocol from our group, published elsewhere [Images were acquired using a 1.5 T scanner , with a 5-element coil SENSE. Cine imaging was obtained by balanced steady-state–free precession pulse sequence electrocardiographic (ECG) gated, with a ventricular short-axis slice thickness of 8 mm and a gap of 2 mm between slices. Myocardial late gadolinium enhancement (LGE) evaluation was assessed using a T1-weighted inversion recovery fast gradient echo pulse sequence, applied 10 min after intravenous infusion of 0.2 mmol of gadolinium-based contrast , with ventricular slices thickness of 10 mm and no gap between them. Detailed CMR protocol parameters are reported in Additional file The study follow-up time was defined from the CMR examination to the last clinical visit until December 31st, 2020. Demographics and clinical data were obtained when the participants underwent the CMR. All participants were followed in a specialized clinic for Chagas disease, with visits performed at least once a year but more frequently if clinically necessary. Clinical management was performed according to the Latin American Guidelines for the Diagnosis and Treatment of Chagas Heart Disease . As a roThe study endpoint was a composite of incident ischemic cerebrovascular events (stroke or TIA) and potential thromboembolic sources . According to a hierarchical order, only one outcome was computed for two or more overlapping incident events, beginning with stroke or TIA, followed by intracavitary thrombus and AF or flutter. Cerebrovascular events were identified from medical records reviewed by two physician members of the study group, with a third senior physician to resolve disagreements, if necessary. Diagnosis of stroke was determined as a focal neurologic deficit of sudden onset, persisting for more than 24 h, without a known alternative to a vascular cause, and confirmed by clinical examination or radiological findings according to Stroke Data Bank and World Health Organization criteria , 16. TIAContinuous variables are expressed as mean ± standard deviation if normally distributed and as median and interquartile range (IQR) if not normally distributed. Normality of the data was examined by histograms and the Shapiro–Wilk test. The CMR-derived independent variables (exposures) were LV end-diastolic and end-systolic volumes, LV mass, LVEF, detection and quantitation of LV myocardial fibrosis, and LV aneurysm. LV volumes were indexed to body surface area. LVEF was assessed continuously and based on a sensitivity analysis to determine the best cutoff point related to the highest risk prediction model performance. LV fibrosis was expressed as the total amount of myocardial fibrotic mass, percentage of LV mass, and categorically based on its presence or absence . LV fibrBaseline characteristics of the 113 patients included in the study are presented in Table Hypertension was the most frequent comorbidity (38%), while dyslipidemia, diabetes, and current smoking were present in 24%, 9%, and 9% of the patients, respectively. Most participants (57%) were in use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, while beta-blockers and amiodarone were in use by 36% and 19%, respectively. Right bundle branch block was the most common ECG abnormality (50%).CMR-derived LV parameters are summarized in Table During a median follow-up time of 6.9 years (IQR: 6.1–11.0), 29 (26%) patients presented with the primary outcome: 11 had cerebrovascular events , 16 had AF or atrial flutter , and two had LV apical thrombosis identified by echocardiography (n = 1) and invasive ventriculography (n = 1). Another patient had an LV thrombus at the apex identified by the baseline CMR and, hence, was not accounted for in the time-to-event analysis.A detailed description of the cerebrovascular events is provided in Table In the univariate analyses, age and all CMR-derived parameters were significantly associated with the primary outcome Table . Sex, hyThe multivariable models Table  includedSince the multivariable model included five parameters, we considered possible overfitting. Given that the primary question is to determine which of the CMR parameters is the most important predictor, we performed a sensitivity analysis including only LVEF, LV scar (both continuous variables), and LV aneurysms in a multivariable model. LV mass was not included in the model due to the correlation between LV mass and LVEF and because LV mass seemed relatively weaker on univariate analysis. In this additional model, LVEF was significantly related to the outcomes , but not either LV scar  or LV aneurysms .The overall incidence rate of the combined event in our cohort was 4.2 new cases per 100 persons-years. In the group with reduced LVEF, the incidence rate for those events was 13.9 new cases per 100 persons-year, significantly different from those with preserved LVEF . The incidence rate of cerebrovascular events in patients with reduced LVEF was 7.5 new cases per 100 persons-year.This study demonstrates that LV systolic dysfunction is a significant predictor of cerebrovascular events and potential sources of thromboembolism in patients with Chagas cardiomyopathy. Although LV fibrosis and LV aneurysms have been associated with the combined outcome in the univariate analysis, this relationship was not significant in the adjusted models. Notably, our cohort’s overall incidence rate of stroke or TIA was relatively low. In addition, during the regular clinical follow-up, a higher proportion of potential sources of thromboembolic events, such as AF or flutter and LV mural thrombus, were identified, warranting preventive oral anticoagulation. Although thromboembolism is a well-recognized phenomenon in individuals with Chagas disease, data about risk factors for cerebrovascular events in this clinical condition are scarce, mostly from autopsy and clinical case series or case–control investigations , 21. HenFirst, using CMR, a reference method to assess LV structure and function, we found that reduced LVEF was significantly related to incident cerebrovascular events and potential sources of thromboembolism in patients with Chagas disease. This result is consistent with prior investigations using echocardiography to assess LV function. Sousa et al. found that LV systolic dysfunction was associated with cardioembolic ischemic stroke in a large cohort of patients with Chagas disease . Nunes eSecond, we tested the role of LV myocardial fibrosis in predicting cerebrovascular events and thromboembolic sources in Chagas disease Fig. . AlthougThird, the presence of LV aneurysm was not related to the primary combined outcome in our study. In a sensitivity analysis, including only LV apical aneurysms, after excluding aneurysms of the inferolateral wall, the results did not significantly change. Although LV apical aneurysm is a well-known site of intramural thrombi formation, its role as an embolic event predictor in the absence of LV systolic dysfunction is less established. In a case–control analysis, Carod-Artal et al. showed that apical aneurysms were independently related to stroke in Chagas disease patients . SimilarIn our view, these conflicting findings may result, at least in part, from the different methods and definitions used to describe LV aneurysms in those studies. Apical aneurysms in Chagas disease can range from small “hollow punch” lesions to large areas of akinetic myocardium thinning Figs.  and 6. , 26 In oFinally, our study has described the incidence rate of cerebrovascular events and potential sources of thromboembolism in patients with Chagas disease and provided these data stratified by LV systolic dysfunction. The incidence of cardioembolic ischemic stroke in Chagas disease has been poorly described . In anotGiven the retrospective nature of our study design, we acknowledge that causality cannot be inferred from our data. The diagnosis of stroke and TIA was primarily based on the medical reports. In one of the cases that reached the primary study outcome, we did not have access to the computed tomography images and results because the exam was performed in an external medical service. Nevertheless, we still could apply the World Health Organization criteria for stroke since this patient had persistent hemiparesis and dysarthria. One patient with a cerebrovascular event had normal LVEF and neither AF nor intracardiac thrombus at the time of the outcome, while images of cranial arteries were not available. Although this patient had a low 10-year atherosclerotic cardiovascular disease risk at the event time, we recognize that a non-cardioembolic cause for the cerebrovascular event could not be excluded. The other patients who had neither AF nor intracardiac thrombus at the time of the cerebrovascular event showed global LV dysfunction and no significant stenosis on cranial arteries imaging. For those, a cardioembolic cause is highly probable, although not definitive. A detached thrombus or paroxysmal AF may be a possible explanation for these events. Given the low-intensity, slowly progressive but incessant myocarditis in chronic Chagas cardiomyopathy, a serial evaluation with CMR would provide important insights into Chagas disease’s natural history . In our LV systolic dysfunction is a significant and independent predictor of cerebrovascular events and potential sources of thromboembolism in patients with chronic Chagas cardiomyopathy. The predictive value of LV fibrosis and aneurysm for these events needs further clarification, particularly in those with normal LVEF. A regular follow-up of patients with Chagas disease, with attention to LV systolic dysfunction and other risk factors for cardioembolic events, such as AF and intracavitary thrombus, might effectively reduce cerebrovascular events in this clinical condition. Nevertheless, further studies are needed to ascertain the potential benefit of this more frequent surveillance for preventing thromboembolic events in patients with chronic Chagas cardiomyopathy.Additional file 1. CMR pulse sequence parameters.Additional file 2. Harrell's C-statistic for the primary combined outcome according to different LVEF cutoff points.Additional file 3. Pearson's correlation coefficient between CMR-derived parameters.


Hypertrophic obstructive cardiomyopathy (HOCM) can bring a high risk of sudden cardiac death in young people. It is particularly urgent to understand the development and mechanism of HOCM to prevent unsafe incidents. Here, the comparison between pediatric and adult patients with HOCM has been performed to uncover the signaling mechanism regulating pathological process through histopathological analysis and immunohistochemical analysis. We found SMAD proteins played an important role during myocardial fibrosis for HOCM patients. In patients with HOCM, Masson and HE staining showed that myocardial cells were diffusely hypertrophied with obvious disorganized myocardial fiber alignment, and myocardial tissue was more damaged and collagen fibers increased significantly, which come early in childhood. Increased expressions of SMAD2 and SMAD3 contributed to myocardial fibrosis in patients with HOCM, which happened early in childhood and continued through adulthood. In addition, decreased expression of SMAD7 was closely related to collagen deposition, which negatively expedited fibrotic responses in patients with HOCM. Our study indicated that the abnormal regulation of SMAD signaling pathway can lead to severe myocardial fibrosis in childhood and its fibrogenic effects persist into adulthood, which is a crucial factor in causing sudden cardiac death and heart failure in HOCM patients. Some studies confirmed the high burden of myocardial fibrosis in both young patients who suffered sudden cardiac death2 or older patients with HOCM suffering from advanced heart failure3. Our previous histopathological study demonstrated that pediatric patients with HOCM may present with severe myocardial fibrosis and reduced microvascular density4.Sudden cardiac death and progressive heart failure are considered to be the most significant complications of hypertrophic obstructive cardiomyopathy (HOCM), while myocardial fibrosis is the main cause of lethal arrhythmias and heart failure in patients with HOCM6. The members of the Smad family are considered the best characterized intracellular effectors of TGF-β, which can be divided into three functional classes: (a). Receptor-activated Smads (R-Smads): including Smad1, Smad2, Smad3, Smad5 and Smad8, responsible for the TGF-β superfamily signaling pathway; (b). the co-mediator Smads (Co-Smads), the Smad4 forms a signaling complex with R-Smads; (c). the inhibitory Smads (I-Smads): Smad6, Smad7, involved in the negative regulation of the R-Smad-mediated cascades7. Extensive experimental evidences suggest fibroblast-specific deletion of Smad2/3 from cardiac fibroblasts prevented the gene program for fibrosis and extracellular matrix (ECM) remodeling8. Activation of Smad2/3 cascade is critical in ECM gene expression and regulation of fibrous tissue deposition9. Smad3 is a key activating signal for cardiac fibroblast to function at inducing myofibroblast conversion, stimulating the transcription of extracellular matrix proteins, and promoting a matrix-preserving phenotype10. In vitro studies also suggest that Smad2 plays a significant role in activation of a fibrogenic transcriptional program7. Smad2 knockdown was proved to inhibit incorporation of α-SMA into myofibroblast stress fibers9. In contrast, Smad3 appears more important than Smad2 in mediating fibroblast activation and fibrosis in vivo8, Smad7 can negatively feedback block the activation of Smad2/Smad3, so the Smad7 has a protective role in MF and ventricular remodeling11. Aberrant Smad family expression can occur in a variety of cardiac pathophysiological states. Fibroblasts, vascular cells and cardiomyocytes, the major cellular effector cells of cardiac fibrosis, are highly responsive to TGF-β and play a critical role in regulating the fibrotic process by activating Smad-dependent signaling pathways.The transforming growth factor-β (TGF-β) superfamily proteins play a critical role in regulation of cardiac fibrotic responses, which is mediated via intracellular effectors (the Smads), or via activation of Smad-independent cascadesIn this study, we analyzed the expression levels of SMAD proteins in septal myocardial tissue samples from pediatric and adult patients with HOCM after Morrow procedure using immunohistopathological methods, focusing on the effect of SMAD signaling on myocardial fibrosis to further elucidate the evolution of myocardial fibrosis in patients HOCM.Pediatric and adult patients with hypertrophic obstructive cardiomyopathy who underwent transaortic septal myocardial resection (Morrow procedure) were selected from March 2004 to December 2018 at the heart center of the First Hospital of Tsinghua University. The control group was obtained from ventricular septal myocardial tissue of pediatric and adult autopsy individuals who died of non-cardiac disease. Diagnostic criteria for hypertrophic obstructive cardiomyopathy: left ventricular wall thickness, or septal thickness ≥ 15 mm, left ventricular outflow tract pressure difference at rest > 30 mmHg, or > 50 mmHg at provocation, and can exclude other causes of ventricular hypertrophy. In pediatric patients, the increase in LV wall thickness exceeded the mean LV wall thickness plus 2 standard deviations  for children of the same age, sex, and body surface area, and other conditions causing increased cardiac load were excluded. Exclusion criteria: coronary artery disease (> 50% stenosis on coronary angiography), uncontrollable hypertension (defined as blood pressure above 140/90 mmHg), valvular heart disease, infection, or renal insufficiency. Twelve adult and five pediatric patients with hypertrophic obstructive cardiomyopathy were included in this study. Control myocardium from the LV septal wall was collected at autopsy of five adults and four pediatric individuals, who died of non-cardiac causes. Clinical information was obtained from the hospital electronic medical record system . Parameter acquisition was performed according to the American Society of Echocardiography guidelines14.The septal myocardium samples were fixed in 10% formalin, embedded in paraffin. The samples were cut into 5 μm sections and stained with hematoxylin and eosin (H&E) to analyze the morphology. The samples were also stained with Masson trichrome to analyze the extent of myocardial fibrosis2O2. Then, the sections were incubated with 10% goat serum for 1 h at room temperature, followed by different primary antibodies at 4 °C overnight: anti-Smad2 , or anti-Smad3 , or anti-MADH7/SMAD7 , or anti-Collagen I . The sections were subjected to horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG  for 1 h at room temperature and then visualized with 3,3-diaminobenzidine (DAB) solution (ZSGB-BIO). Finally, the sections were counterstained using hematoxylin.Septal myocardial tissue specimens were fixed in 10% buffered formalin, embedded in paraffin, and cut into 5um thick tissue sections for immunohistochemical staining. After deparaffinization and rehydration with graded concentrations of ethanol to deionized water, the sections were heated in EDTA antigen retrieval buffer  in a pressure cooker for 2.5 min for antigen retrieval. Endogenous peroxidase activity was bloked with 3% (vol/vol) HImages were captured using Motic EasyScan 60 and analyzed with Image -pro plus software . Ten areas per section were randomly selected, and the images were magnified × 40 magnification, and the positive expression areas were brown in color. Image-Pro Plus 6.0 image analysis software was used to calculate the integrated optical density (IOD) value, IOD indicates the protein expression of each index, the higher the IOD value, the higher the protein expression.t-test. p values less than 0.05 were used as a test for significant differences.Statistical data were analyzed using SPSS 25.0 . Data were expressed as mean ± standard deviation. Continuous variables were compared between the two groups using an independent The study was supported by the Ethics Committee of the First Hospital of Tsinghua University, and informed consent was obtained from patients or legal guardians. All experiments were performed in accordance with relevant guidelines and regulations.Demographic and clinical baseline characteristics of the study population are presented in Table HOCM is a genetic disorder characterized by thickened septum between the ventricles, which is usually accompanied by dynamic left ventricular outflow tract obstruction. First, we depicted the changes of cell gross morphology, muscle fiber arrangement, and organization of the architecture in the hearts with HOCM using histological analysis. HE staining showed that in the adult control and pediatric control groups, myocardial cells were structurally intact and well-arranged; however, in the adult HOCM and pediatric HOCM groups, myocardial cells were diffusely hypertrophied with obvious disorganized myocardial fiber alignment Fig. a. MassonTherefore, in patients with HOCM, myocardial cells were diffusely hypertrophied with obvious disorganized myocardial fiber alignment, and myocardial tissue was more damaged and collagen fibers increased significantly, which come early in childhood.15, we hypothesized that expressions of SMAD2 and SMAD3 might be upregulated in patients with HOCM. Using immunohistochemical staining, we found expressions of SMAD2 and SMAD3 in myocardial tissue of adult HOCM patients were significantly higher than those of their controls  or TGF-β receptor II (TβRII). Activated TβRI recruits and phosphorylates downstream receptor-regulated Smad proteins (Smad2 and Smad3), and activated Smad2 and Smad3 bind to Smad4 to form Smad complexes and enter the nucleus, initiating the expression of fibrosis-related transcription factors and proteins23. Smad7 activation acts as a TGF-β-induced endogenous inhibitory signal that prevent myocardial fibrosis. Decreased Smad7 expression results in cardiac fibrosis in the infarcted rat heart, whereas Smad7 overexpression in cardiac fibroblasts inhibits collagen synthesis24. In our study, we found that SMAD7 expression significantly reduced in myocardial tissue of pediatric and adults with HOCM while SMAD2/3 expression increased, which is consistent with a previous report that Smad7 loss accentuated Smad2/3 activation25. Thus, with respect to patients with HOCM, the steady state controlled by the antifibrotic effects of SMAD7 and the fibrotic responses of SMAD2/3 might be disturbed.Smad7 serves as a negative regulator of TGF-β1/Smads signaling pathway, which binds to activated TβRI to inhibit the phosphorylation and activation of R-Smads and antagonize TGF-β1 signaling26. In hypertrophic cardiomyopathy, the most typical ECM components are collagens I and III, and the collagen content is heavily aggregated, affecting cardiac diastolic function27. In this study, we found that myocardial tissue collagen I expression was significantly elevated in pediatric and adults with HOCM, while SMAD2 and SMAD3 increased and inhibitory SMAD7 decreased in myocardial tissue, which was closely related to myocardial fibrosis described above. Therefore, activated SMAD signaling may be the direct cause of the fibrotic outcome of HOCM. And in our previous study, we found that patients with HOCM had reduced microvascular density and impaired myocardial perfusion4. The main role of MMP is to degrade type I and III collagen, and its elevated activity can cause increased fibrillar collagen degradation, extracellular matrix remodeling, and ventricular dilation28. Bi et al. found in patients with HOCM that myocardial MMP-2/TIMP-1 ratio was elevated by reduced microvasculature and that high plasma PICP/ICTP and MMP-2/TIMP-1 ratio were independent predictors of adverse outcomes in patients with HOCM29. So, our results have extended the understanding of the mechanism of HOCM, which helps to explore the new therapeutic treatments for HOCM patients as early as possible.The characteristic pathophysiological changes in hypertrophic cardiomyopathy are myocardial cell abnormalities, disorganized arrangement and interstitial fibrosis. The main components of the extracellular matrix (ECM) are collagen and glycosaminoglycans (GAGs)30. Transcription factors (TFs) can control the molecular network that regulates cardiomyocyte hypertrophy. Among them, SP1 is able to affect cardiomyocyte hypertrophy by SP1/GATA4 signaling pathways30, MRTF-A-Sp1-PDE5 axis31, and ROCK1-Sp1-PKCγ axis32. The study earlier reported that SP1 plays a crucial role in the pathogenesis and the progression of human glomerulonephritis probably via cooperation with pSmad2/3 and p30033. More importantly, many of the functions of TGF-β are mediated through Sp1 in cooperation with Smad signaling34. p300 is a transcriptional co-activator that may be involved in the activation of Smad complexes35. So, in our further research, Sp1 may be an important candidate that help uncover the more complicated pathogenic mechanism of HOCM.To date, more and more important molecules and crosstalk involved in the development of HOCM were revealed by bioinformatics technology, such as lncRNAs , TFs (SPI1 and SP1), and miRNAs (has-miR-29b-39 and has-miR-29a-3p)This study also has some limitations. As a retrospective study, the research methods restricted to the sample size and sample types (only apply to the relevant examinations). First, the number of cases in this manuscript is slightly less, particularly the samples from pediatric patients with HOCM are very rare, but they are crucial to understand the development and mechanism of HOCM. Second, formalin/PFA- fixed paraffin-embedded tissues are long-lasting and easy to archive, but these are only fit for immunohistochemical investigations, which limits its applications. For these reasons, we will try to use some advanced technologies to the small but rare sample, such as using single cell RNA sequencing (scRNA-seq) to display comprehensive gene expression profiling of different cell types from the HOCM myocardial tissue, and to reveal the gene regulatory network or the crosstalk among different cell types by combining with bioinformatics technology. Also, we will collect RNA samples and protein samples from fresh tissues for data validations. Further studies with multi-level confirmations using various sample types, correlations between gene or transcript levels, and clinical prognosis will be carried out in the next part of our plans.In myocardial tissue from patients with HOCM, SMAD2/3 and type I collagen expression levels are elevated, whereas inhibitory SMAD7 protein expression levels are reduced, and their expression levels do not differ significantly between pediatric and adult patients with HOCM. These indicate that myocardial fibrosis due to SMAD signaling pathway activation occurs in childhood and that its fibrogenic effects persist and are a crucial factor in causing sudden cardiac death and heart failure in HOCM patients.


Primary mitral valve regurgitation (MR) results from degeneration of mitral valve apparatus. Mechanisms leading to incomplete postoperative left ventricular (LV) reverse remodeling (Rev–Rem) despite timely and successful surgical mitral valve repair (MVR) remain unknown. Plasma exosomes (pEXOs) are smallest nanovesicles exerting early postoperative cardioprotection. We hypothesized that late plasma exosomal microRNAs (miRs) contribute to Rev–Rem during the late postoperative period.n = 19; age, 45–71 years) underwent cardiac magnetic resonance imaging and blood sampling before (T0) and 6 months after (T1) MVR. The postoperative LV Rev–Rem was assessed in terms of a decrease in LV end-diastolic volume and patients were stratified into high (HiR-REM) and low (LoR-REM) LV Rev–Rem subgroups. Isolated pEXOs were quantified by nanoparticle tracking analysis. Exosomal microRNA (miR)-1, –21–5p, –133a, and –208a levels were measured by RT-qPCR. Anti-hypertrophic effects of pEXOs were tested in HL-1 cardiomyocytes cultured with angiotensin II .Primary MR patients (p = 0.016), who showed lower cardiac mass index . Postoperative exosomal miR-21-5p values of HiR-REM patients were higher than other groups (p < 0.05). In vitro, T1-pEXOs isolated from LoR-REM patients boosted the AngII-induced cardiomyocyte hypertrophy, but not postoperative exosomes of HiR-REM. This adaptive effect was counteracted by miR-21-5p inhibition.Surgery zeroed out volume regurgitation in all patients. Although preoperative pEXOs were similar in both groups, pEXO levels increased after MVR in HiR-REM patients (+0.75-fold, High levels of miR-21-5p-enriched pEXOs during the late postoperative period depict higher LV Rev–Rem after MVR. miR-21-5p-enriched pEXOs may be helpful to predict and to treat incomplete LV Rev–Rem after successful early surgical MVR. Mitral valve regurgitation (MR) is one of the most common cardiac valve diseases leading to blood regurgitation into the left atrium (LA) and to left ventricular (LV) dilatation, also defined as “LV adverse remodeling,” following increased preload up to the onset of heart failure (HF) . PrimaryMost patients with severe primary MR and LV dysfunction undergoing timely MVR –9 are atPreoperative echocardiography is now commonly used as a method for screening postoperative outcome in terms of long-term survival –23, MR rPlasma exosomes (pEXOs) are smallest membrane bound extracellular vesicles (40–150 nm in diameter) released by different cells, which may play a key role in intercellular communication by regulating the magnitude of postoperative LV Rev–Rem. It is conceivable that combining analysis of pEXOs with CMRI parameters may enable more precise and informative assessment of late LV Rev–Rem. Indeed, the previous studies have demonstrated the role of pEXOs in mediating cardioprotection through anti-inflammatory and anti-apoptotic pathways , 38 regaExosomal microRNA (miR), small non-coding RNA molecule (∼20 nucleotides) regulating gene expression, is effective mediator of the adaptive paracrine responses in cardiomyocytes exposed to different stressors , 44. TheThe evidences above well support the use of pEXOs and corresponding microRNAs to depict the extent of LV Rev–Rem in our MR patients during the late postoperative period. Since some reports have focused on their predictive value in dogs with MR  and in pin vitro model of cardiac hypertrophy  method, and (3) regurgitant volume more than or 60 ml. The patients with echocardiography evidence of leaflet tethering  or calcific degenerative restricted motion of leaflets were excluded. In the case that, during surgery, the surgeon decided to convert intervention to valve replacement, the patients were then excluded from the study. Other exclusion criteria comprehended the following: chronic kidney disease (CKD) defined as glomerular filtration rate less than 50 ml/m2, the previous cardiac surgery or history of congenital heart disease, the current or past myocardial ischemia/severe coronary artery disease, LV ejection fraction (LVEF) less than 40%, other-than-mitral valve diseases more than mild, and any contraindications to CMRI. The current cardiovascular risk factors, functional capacity according to the New York Heart Association (NYHA) classification   in accord with the principles outlined in the Declaration of Helsinki. We obtained signed informed consent from each patient. Aiming to select patients with higher mitral valve repair probability, the inclusion criteria were as follows: Age (35–75 years); presence of severe primary MR due to prolapse or flail leaflet as assessed by transthoracic echocardiography, sinus rhythm, and clinical indication to surgery. According to clinical guidelines, severe primary MR was diagnosed when at least one of the following parameters was detected by transthoracic echocardiography –58: (1) fication , presencThe complete clinical profile of patients is described in We performed combined CMRI and blood sampling for pEXO isolation before surgery (baseline) and at 6 months after surgery. The timing of the experimental protocol was in accord with the previous studies , 61. Onlvia a small right incision in the third or fourth intercostal space—a periareolar  access or axillary  minithoracotomy was performed. The median sternotomy was the alternative surgical approach when the minimally invasive approach was not possible (All patients underwent same anesthetic protocol and standard hypothermic cardiopulmonary bypass (CPB) procedure. Successful surgical repair of the mitral valve was performed mainly through a minimally invasive endoscopic approach to the mitral valve , 62. Bripossible . An extrpossible . The sucTransthoracic echocardiography was performed with a commercial machine equipped with a 5–1 mHz phased array probe . Left ventricular ejection fraction was evaluated with the modified Simpson Biplane method. The postoperative residual mitral regurgitation was evaluated using a scale from 0 (nil) to 4 (severe), in all patients. All the measurements were performed according to the current standards , 64.i) were calculated for all parameters  was used to assess cardiac remodeling and valve function. Briefly, endocardial and epicardial borders of both ventricles and left atrium (LA) were manually delineated at end-diastole (ED) and end-systole (ES) by an experienced operator. For both ventricles, ED and ES volumes  were calculated from a stack of cine balanced steady-state free precession (bSSFP) short-axis images acquired orthogonally to the LV long axis, from mitral valve to apex, without gap between each slice. Left ventricular myocardial mass was calculated by subtracting the total endocardial volume from the total epicardial volume then multiplying for myocardial density (1.06 g/ml). Left ventricular and right ventricular (RV) stroke volumes  were calculated as EDV– ESV. The body–surface area indexed values  while patients with values above the median value were included in the Low LV reverse remodeling group (LoR-REM).The patients with a percentage of change lower than the median value were included in the Phase contrast sequences at the level of aorta and pulmonary artery roots were used to obtain a direct quantification of the blood volume pumped out of LV and RV at each systole, respectively (measured stroke volume). For each patient, the velocity of encoding was adapted to be the lower possible level that avoided aliasing noise. Regurgitant volume was obtained by subtracting the anterograde LVSV measured by the analysis of velocity encoded phase contrast images at the aortic root level . BrieflyLVSV was also measured in short axis cine bSSFP asand the mitral valve regurgitant volume was measured asThe myocardial fibrosis was assessed by Late Gadolinium Enhancement (LGE) technique . Brieflyg centrifugation for 15 min at 10°C and was stored at –80°C until analysis. pEXOs were isolated and purified from 1-ml plasma using an optimized 2-d ultracentrifugation protocol [adapted from the study discussed in Ref. 39]. Briefly, plasma was diluted with 15 volumes of filtered (0.2-μm pore) phosphate buffer solution (PBS) and centrifuged at 3,000g for 15 min at 10°C. Supernatant was filtered (0.2-μm pore) and subjected to the following centrifugation series: 10,000g for 15 min at 10°C (discarding pellet), 20,000g for 30 min at 10°C (discarding pellet), then 100,000g for 5 h at 4°C. The pellet from the ultracentrifugation was re-suspended in 1 ml of PBS and stored at 4°C overnight. The day after, it was vortexed thoroughly and centrifuged at 10,000g for 15 min at 10°C. The supernatant obtained was diluted with 15 volumes of filtered PBS, and centrifuged at 20,000g for 30 min at 10°C. Eventually, the supernatant was ultracentrifuged at 100,000g for 5 h at 4°C. The final exosomal pellet was re-suspended in 100 μl of cold filtered PBS and was stored at –80°C until analysis.A 12-ml volume of peripheral venous EDTA-treated blood was collected from MR patients and controls. Plasma was isolated with a 10,0007)–(1 × 109)] particles/ml}. Three 60-s videos were acquired for each sample maintaining a camera level, 10; and the videos were analyzed at detection threshold, 3. The modal particle size and particles concentration/ml was measured by the software. The pEXOs concentration was inferred from the particle size distribution up to 150-nm size, it was normalized by resuspension volume and expressed relative to the starting plasma volume.The particle size and concentration were measured by nanoparticle tracking analysis (NTA) using a NanoSight LM10 NTA instrument with NTA 3.2 software for data acquisition and analysis . The pEXOs samples were diluted with filtered PBS to match the recommended particle concentration range {20–120 particles/field; [ ethanol. Each sample was transferred into RNeasy® Mini columns (Qiagen). The RNA binding, washed and eluted according to the manufacturer’s instructions. Moreover, RNA was eluted in 30 μl of RNAse-free water. Template RNA (7 μl) was reverse transcribed using the miScript II Reverse Transcription (RT) kit (Qiagen) on a 20 μl final volume in line with the manufacturer’s instructions. Also, miScript HiSpec Buffer was used in each reaction to selectively target the mature miRNA forms. Before proceeding to quantitative PCR, the cDNA was diluted 12.5 times in nuclease free water. The quantitative PCR was carried out on 5-μl diluted cDNA using the QuantiTect SYBR Green PCR reaction mix (Qiagen) on a 20-μl final reaction volume according to the manufacturer’s instructions. Forward primers for each selected miR were inferred from mature miRNA sequences and purchased from Sigma  following the manufacturer’s guidelines with few adaptations. Briefly, the isolated pEXOs were lysed by incubation with 5 volumes QIAzol reagent for 5 min, then 5 μl of 0.33-nM CelMir39 spike-in control (1.65 fmol) and 1 μL of 20 μg/μL molecular grade mussels’ glycogen (Sigma) were added to each sample, followed by one volume of chloroform. The samples were vortexed briefly, incubated for 3 min at room temperature and centrifuged at 12,000om Sigma . The miSe method . For eac® Premix Ex Taq™ reagent (Takara). The following primers, obtained from Sigma, were employed in the analysis at 500-μM concentration: Atrial natriuretic peptide (ANP) 5′-TCGTCTTGGCCTTTTGGCTT-3′ (forward) and 5′-AGGTGGTCTAGCAGGTTCTTGAAA-3′ (reverse); Brain natriuretic peptide (BNP): 5′-CGTTTGGGCTGTAACGCACT-3′ (forward) and 5′-TCACTTCAAAGGTGGTCCCAG-3′ (reverse); Cardiac β-myosin, β-MHC (MYH7) 5′-TCCTGCTGTTTCCTTACTTGCT-3′ (forward) and 5′-GCTGAGCCTTGGATTCTCAAAC-3′ (reverse); Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) 5′-CCTTTGCCGCTCATTTTCCAG-3′ (forward) and 5′-GGCTGCACACACTCTTTACC-3′ (reverse); β-Actin 5′-GGCACCACACCTTCTACAATG-3′ (forward) and 5′-GGGGTGTTGAAGGTCTCAAAC-3′ (reverse). All reactions were performed on a Rotor-Gene Q real-time PCR cycler (Qiagen) at the following thermocycling conditions: 30 s at 95°C followed by 40 cycles of 15 s at 95°C and 30 s at 60°C as suggested by the manufacturer’s guidelines. The relative quantification of mRNA expression was performed by applying the 2–ΔΔCt comparative method , following the manufacturer’s instructions. Furthermore, RNA was re-suspended into 40 μl of RNAse-free water and quantified by NanoDrop spectrophotometry (Thermo Fisher Scientific); 1 μg of the total RNA was reverse transcribed with the PrimeScript RT kit (Takara) on a 20-μl final volume, following manufacturer’s guidelines. The cDNA obtained was diluted 1:10 and analyzed by quantitative PCR using the TB Greene method , with β-2. For the experimental purposes, HL-1 cells were seeded at a concentration of 5,000 cells/well on fibronectin pre-coated  8-well chamber slides  and let adhere for 24 h before the treatment. Cardiomyocytes were then exposed to 1-μM angiotensin II  in complete Claycomb medium for 48 h (9 particles/ml) isolated from each group of patients or miRNA inhibitors (30 nM) were added 24 h after the beginning of AngII treatment, and the treatment was maintained for the remaining 24 h. Sterile filtered (0.2-μm pore) PBS vehicle was employed as a negative control.Murine cardiomyocyte cell line HL-1  was used to evaluate anti-hypertrophic role of human pEXOs. The HL-1 cells were cultured in Claycomb Medium  (Sigma) for 48 h   for 24 h, following manufacturer’s instruction. All procedures were performed in antibody-free Claycomb medium.Furthermore, MiRVANA miRNA miR-21-5p inhibitor and miRVANA miRNA inhibitor negative control #1 were purchased from Thermo Fisher Scientific. To perform direct miR-21-5p inhibition in HL-1 cardiomyocytes, cells were transfected with 30 nM miRNA-21 inhibitor/negative control using Lipofectamine8 particles in PBS), then 30-μl of 1-M CaCl2 were added, and the final volume was adjusted to 300 μl with PBS. The mixture was incubated on ice for 30 min, then heat shocked for 60 s at 42°C and placed again on ice for 5 min. Treated pEXOs were then collected again by differential centrifugation as follows: After a 15-min clear-up step at 10,000g at 4°C, exosomes were isolated with a 5-h ultracentrifugation at 100,000g, performed at 4°C. The pEXOs collected (approximately 5 × 108 particles) were then re-suspended in 100 uL PBS. The amount of pEXOs obtained with this procedure is sufficient to treat HL-1 cardiomyocytes in one 8-well chamber slide well, in a final volume of 500 μl (1 × 109 particles/ml). All procedures were again performed in antibody-free Claycomb medium.As a different approach, isolated pEXOs were pre-loaded with miR-21 inhibitor then added to HL-1 cells as described above. The protocol for oligonucleotides loading was adapted from Zhang et al. . Briefly1 and expressed as pixels.Actin filaments were stained with phalloidin to define HL-1 cell area . BrieflyF-test of equality of variances (2 groups) or the Brown–Forsythe test (more than 2 groups). Robust Regression and OUTlier removal (ROUT) method was employed to identify outliers , which are reported in the graphs as “×” symbols. Paired Student’s t-test or the Wilcoxon test were applied to identify significant differences between repeated measurements according to their distribution. Independent samples Student’s t-test  or the Mann–Whitney test were employed between two independent groups according to their distribution. One-way ANOVA  or the Kruskal–Wallis test, depending on sample distribution, were applied when more than two groups were compared. All statistical tests were performed as two-sided, statistical significance was considered for p < 0.05.Continuous clinical variables and CMRI measurements are presented as mean ± SD. Categorical clinical variables are presented as percentages of the total. The pEXOs concentration and miRNA expression are presented as median with interquartile range (IQR). Fisher’s exact test was used to reveal differences between the frequencies of categorical variables. Regarding continuous variables, normal distribution was evaluated by the Kolmogorov-Smirnov test. Homoscedasticity was evaluated by the Receivers operating characteristic (ROC) curves were constructed with easyROC open source online software  using ea2DS2-VASc Score = 0, 52.6%; n = 5; HiR-REM, n = 2). No other postoperative complications were observed in the study population. All patients underwent to a transthoracic echocardiography within 1 month after surgery, showing a good postsurgical outcome with nil  or mild  residual mitral regurgitation and preserved LV EF .During a 1-year timeframe (from January 2018 to February 2019), 23 surgical MR patients met inclusion criteria and were enrolled in the study. All the patients underwent to successful MVR. The time of extracorporeal circulation was 134 (114–166) min and the cross-clamp time was 84 (75–101) min; all surgeries were carried out through the initial incision with no need for sternotomy conversion. We had no cases of conversion to replacement during intervention. Four patients were excluded after surgery because one patient died early after the intervention and three patients voluntarily withdrew from the study. Therefore, the final population consisted of 19 patients. As shown in p = 1.90 × 10–6; p = 0.0055), RV end-diastolic volume index  and RV end-systolic volume index RVESVi (p = 0.035) were lower at T1 compared to T0 (p = 7.60 × 10–6) and volume (p = 1.30 × 10–5), as well as RA area (p = 0.001), were reduced in accord with the previous findings , even if its preoperative values were higher than 50% for most patients . Postoperative cardiac index values were also lower than ones at T0 (p = 7.40 × 10–5), which remained within physiological range; while higher resting heart rate was observed after surgery  and 6-months (T1) after surgery to assess cardiac phenotype in all patients . Hallmared to T0 . The LA findings  ; otherwise, patients with low degree of LV Rev–Rem showed %Δ LVEDVi values significantly reduced by 20.23 ± 9.87%  of LVEDVi at T1 compared to T0 using a cut-off value of –30% . PatientTo evaluate the role of preoperative cardiac parameters to predict postoperative LV reverse remodeling, we first compared the clinical and CMRI-derived features at T0 in HiR-REM and LoR-REM groups. We found that both groups have similar clinical profile . Moreovep = 0.011), LVEDVi (p = 0.021), indexed LA volume (p = 0.037), LVSVi (p = 0.0013) and cardiac index (p = 0.007) showed high level of postoperative Rev–Rem  and even more in patients with mitral leaflets prolapse. Myocardial fibrosis is a hallmark known to be associated to adverse postsurgical outcomes and arrhythmic events , 77. In N = 8, control). Plasma exosomes (pEXOs) were positive for canonical markers CD63, CD81, and TSG101  than LoR-REM patients  and healthy subjects , while in seven patients the exosomal levels decreased significantly from T0 to T1  of pEXOs at T1 compared to T0. In 12 patients, pEXOs levels were significantly higher after surgery  and for T1-pEXOs was 0.765 , indicating reliable predictive power of the postoperative pEXO levels , and tended to be higher than preoperative values  levels of exosomal miR-21-5p were higher in the entire MR population than in healthy controls  and healthy subjects  and 3.0-fold (p = 0.046), respectively  and patients with minimal reverse remodeling (LoR-REM patients). At T1, exosomal miR-21-5p levels in HiR-REM group were higher than those in the LoR-REM patients , for T1-miR-21-5p was 0.763  and for T1-miR133a was 0.792  indicating fair predictive power at 6 months after MVR, Therefore, postoperative exosomal miR levels may be reliable enough to depict HiR- and LoR-REM patients.As showed in In vitro, we have tested the anti-remodeling effects on murine cardiomyocyte of a single dose of pEXOs isolated from HiR-REM and LoR-REM patients. As showed in Because miR-21-5p levels are 10-fold higher than those of other exosomal miRNAs and peak only in pEXOs isolated from HiR-REM patients, we assessed miR-21-5p role during cardiomyocyte response to chronic AngII stimulation. For this purpose, we have performed additional experiments by co-treating cardiomyocytes with miR-21-5p inhibitor. As showed in In the last decade, plasma exosomes have gained increasing interest as perioperative diagnostic, therapeutic and prognostic factors related to different types of cardiac surgery , 39. YetImportantly, we observed an elevated pEXOs values only in patients with a higher grade of reverse remodeling of the left ventricle characterized by lower values of LVEDVi, LVESVi, and ventricular mass index. Since the release of exosomes can be induced by external factors, we can assume that pre-existing diseases and perioperative factors did not affect late exosomal levels after heart valve surgery. All surgical patients with severe primary MR showed similar preoperative LVEF values and were treated with the same drugs and anesthetics. Indeed, preoperative pEXOs level did not change among patients and were independent of the magnitude of regurgitant volume. Of note, preoperative regurgitation volume was higher in patients who exhibited the greatest postoperative LV Rev–Rem. It is conceivable that with the severity of mitral valve disease, the extent of preoperative regurgitant volume associated with adverse remodeling, co-morbidities, as well as co-medications, did not influence the pEXOs level before surgery. We cannot exclude that late release of exosomes was part of a stress response following exposure to additional, as yet undefined, intra- and postoperative factors. In our previous study, indeed, we ruled out that anesthesia and minimally invasive on-pump mitral valve surgery lead to an early increase in postoperative pEXOs level in patients without ischemic heart disease . Yet, thOur study suggests that rising postoperative pEXOs level is a reliable indicator of the long-term adaptive response of the heart after mitral valve repair regardless of LV ejection fraction. Indeed, at 6 months after early rescue surgery, among patients with null mitral regurgitation volumes and preserved systolic function, the HiR-REM group showed smaller ventricular volumes and cardiac mass index than the LoR-REM patients. In light of the finding of partial restoration of the left ventricular structure despite full recovery of mitral valve function, we suggest that low postoperative pEXOs level may hinder the maintenance of normal ventricular chamber geometry and mass over time in LoR-REM patients. This finding is clinically relevant because our study population with severe primary chronic MR had adverse tissue remodeling before surgery, as evidenced by the presence of myocardial fibrosis assessed using CMRI. In fact, adverse remodeling is ongoing even if the ventricle is normocontractile and patients are asymptomatic as demonstrated by the presence of myocardial fibrosis .in vivo kinetics of heterogeneous circulating exosomes is still difficult to investigate, the previous experimental evidence has also ascertained that not all cells release highly cardioprotective exosomes. In this regard, we have demonstrated that higher dose of exosomes released from cardiac progenitor cells provides sustained anti-remodeling effects in vivo  established indicators of cardiac hypertrophy . Third, our follow-up was limited to 6 months after surgery. Further investigations are required to assess the relationship between miR-21-5p-rich plasma exosome levels and magnitude of reverse ventricular remodeling during longer follow-up after early rescue MVR. Finally, the definition of the cellular source of miR-21-5p-rich plasma exosomes and better understanding of molecular pathways regulated by exosomal miR-21-5p in cardiomyocytes deserve further ad hoc investigation in vivo.Some limitations of this study should be considered to promote next investigations. First, the study population consists of a low number of patients. This was in part due to the relatively low number of patients with isolated primary MR who underwent surgery within the timeframe of the study and partly due to the low patient adherence to the experimental protocol, mainly driven by the refusal of the CMRI examination due to claustrophobia. However, the high accuracy and reproducibility of CMRI can partly compensate for the small sample size. In fact, each patient studied with CMRI is equivalent to 10 patients studied with echocardiography, whose measurements are less reliable. Second, our population consists mainly of surgical patients in the early stage of adverse cardiac remodeling induced by primary MR, with mild-to-moderate degrees of LV dilatation. In accord with latest recommendations for acting in elective surgical MVR , we cannThe original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of “G. Monasterio” Foundation . The patients/participants provided their written informed consent to participate in this study.FP, GF, and VL contributed to the conception and design of the study; GF and VC advised in physiological mechanisms. SS, DC, MS, GB, and SM advised in physiological topics and surgical approach. FP and GF performed the statistical analysis. VL analyzed the results and wrote the first draft of the manuscript. GF, GA, FP, MM, and VL wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.


Pressure-strain loop (PSL) is a novel method to quantify myocardial work in many cardiovascular diseases. To investigate the value of myocardial work parameters derived from PSL for evaluating cardiac function and clinical prognosis in patients with pulmonary hypertension (PH).A total of 52 patients with PH and 27 healthy controls were enrolled in this prospective study. PSLs determined by echocardiography were used to calculate global work index (GWI) of left ventricle (LV) and right ventricle (RV). Global constructive work (GCW) comprised the sum of myocardial work performed during shortening in systole and during lengthening in isovolumic relaxation. Global wasted work (GWW) comprised the sum of myocardial work performed during lengthening in systole and during shortening in isovolumic relaxation. Global work efficiency (GWE) was defined as GCW/(GCW + GWW).P < 0.001). LVGWE, LVGWI, LVGCW, and RVGWE were lower in patients than controls . Myocardial work parameters correlated well with clinical and other conventional echocardiographic assessments . In binary logistic regression analysis, the combination of RVGWE and estimation of pulmonary arterial systolic pressure (ePASP) was the best model to predict clinical outcomes . Receiver operating characteristic curv demonstrated the combination of RVGWE and ePASP was the best predictor of adverse events with 100% sensitivity and 76.3% specificity .LVGWW, RVGWI, RVGCW and RVGWW were significantly higher in patients than controls (all Myocardial work parameters derived from PSL are emerging markers of cardiac function. And the combination of RVGWE and ePASP is a useful predictor of clinical outcome in PH patients. Pulmonary hypertension (PH) is a pathological state or disease caused by abnormal increase of pulmonary circulation pressure due to various factors or diseases . AccordiIt is well acknowledged that cardiac function is directly related to the prognosis of patients. A great number of studies have shown that PH patients in World Health Organization functional class (WHO-FC) IIIWH have worse outcomes in the long-term prognosis , 4. PrevEchocardiography is widely used to evaluate cardiac function in PH patients at the advantage of non-invasion . Global No previous study has assessed myocardial work in PH patients. Thus, in this study, we aimed to quantify LV and RV myocardial work in PH patients by non-invasive PSL, and explore the value of myocardial work parameters of evaluating cardiac function and clinical prognosis.Fifty-two patients diagnosed with PH between January 2019 and June 2020 in Shanghai Renji Hospital were consecutively enrolled in this prospective study. Given PH prevalence and incidence were mostly in middle-aged women, we recruited twenty-seven age- and gender- matched healthy controls from medical center for further study. According to 2015 ESC/ERS Guidelines of PH , RHC data including pulmonary arterial systolic pressure (PASP), pulmonary arterial diastolic pressure (PADP), mPAP, PAWP, cardiac output (CO), cardiac index (CI) and PVR, as well as the specific drug therapy were recorded from all patients. Patients underwent RHC and echocardiography within 3 days of each other. Before echocardiography, all patients’ blood pressure (BP) by a brachial artery cuff were measured immediately. In order to evaluate the cardiac function, all patients underwent 6-minute walking distance (6MWD) to identify function capacity and functional class was determined by clinical investigator based on the WHO-FC .Standard transthoracic echocardiography connecting with electrocardiogram were performed in all of the 52 PH patients and 27 controls using a Vivid E95 ultrasound machine  equipped with an M5S probe by an experienced doctor. As recommended by the American Society of Echocardiography , images Images from apical four-chamber, apical two-chamber and apical long axis views were put into offline workstation  to yield LV global longitudinal strain (LVGLS). RV global longitudinal strain (RVGLS) was assessed by using an apical four-chamber view focusing on the RV in offline workstation.Ventricular myocardial work was quantified by a novel non-invasive PSL method which used GLS combined with estimated non-invasive pressure . The PW LV pressure was replaced by the cuff systolic pressure. Because of the particularity of RV, in order to improve the accuracy of pulmonary arterial systolic pressure obtained by echocardiography, other right cardiac function parameters obtained from echocardiography, including ePASP, TAPSE, FAC, RAP, RAA, RV/LV, LVGLS and RVGLS, were used for adjusting, then the calculated pulmonary arterial systolic pressure  were obtained. In the absence of left/right bundle branch block, the left and right heart could be considered as synchronous contraction. Similarly, the estimation of real time RV pressure was acquired by using an empiric reference curve which was adjusted by stretching it in time according to valvular event times  by echocardiography and in amplitude according to PASPcal. LVGWE, LVGWI, LVGCW, LVGWW, RVGWE, RVGWI, RVGCW, and RVGWW were eventually obtained by the algorithm above-mentioned.The interobserver reliability of GLS and myocardial work parameters was assessed using the measurements of 10 randomly chosen PH patients and 5 randomly chosen controls by two experienced cardiologists.All patients with PH were followed up regularly at 3-month intervals by telephone calls to ascertain present symptom, therapy, and cardiac function. The deadline of the last visit of the last patient was May 2021. Major adverse events defined as all-cause of mortality, hospitalization and need of new specific drug therapy or enhancement on the original therapy basis were recorded.t-test or Mann-Whitney U test was used to compare variables between PH patients with controls, while Pearson’s Chi-Squared test or Fisher exact test to compare the categorical variables . Categorical variables were expressed as numbers (percentage). Independent sample ariables . PASPcalP = 0.947; age 44.1 ± 13.2 vs 43.0 ± 13.7 years, P = 0.748) were included in this clinical study. Main clinical characteristics of patients were shown in A total of 52 PH patients and 27 controls . The formula was as follows: PASPcal = 0.609*ePASP + 0.187*TAPSE − 0.182*FAC + 0.153* RAP + 0.12*RAA − 0.128*RV/LV − 0.153*LVGLS + 0.135* RVGLS. Bland-Altman plot also showed great agreement between PASPcal and PASP , RAA , RV/LV , LVGLS , RVGLS  and lower TAPSE , FAC  than controls. However, there was no significant difference in LVEF . Compared to the controls, LVGWW , RVGWI , RVGCW  and RVGWW  were significantly increased in PH patients. LVGWE , LVGWI , LVGCW , and RVGWE  were significantly lower in PH patients than controls.The representative PSL and global work curves of participants were presented in r = 0.406, P = 0.003), and FAC , but a negative correlation with WHO-FC , and RV/LV . LVGWI significantly correlated with 6MWD , WHO-FC , FAC , RV/LV . The correlations between LVGCW and 6MWD , WHO-FC , and FAC  were also significant. LVGWW was only correlated with 6MWD , WHO-FC  and FAC . As for RV function, RVGWE showed a negative correlation with BNP , WHO-FC , and RV/LV  and a positive correlation with 6MWD , and FAC . In addition, RVGWI correlated well with BNP , TAPSE , and FAC . The correlations of RVGWW with 6WMD , WHO-FC , FAC  and RV/LV  were significant as well.We used linear correlation analysis to assess relationship between myocardial work parameters and other clinical data in PH patients, which were described in 2 = 4.84, P = 0.775). The formula was as follows: logit (P) = 12.586 − 0.22*RVGWE + 0.051*ePASP. In the model, RVGWE had an OR of 0.803  and ePASP had an OR of 1.052 . Furthermore, based on the model, ROC curves showed the combination of RVGWE and ePASP had the biggest area under curve (AUC) with 0.910 (P < 0.0001), which were described in PH patients were followed for a median of 515.0 days  at termination. During the follow-up period, adverse events occurred in 14 patients (26.9%): 12 patients (23.1%) had hospitalization and 2 patients (3.8%) needed enhancement on the basis of original therapy. Best binary logistic model for evaluating adverse events was shown in PSL was firstly proposed by Urheim et al.  to quantNon-invasive PSL refers to estimated ventricular pressure and strain derived from echocardiography. According to Russell et al. , LV peakIn our study, RVGWI, RVGCW and RVGWW substantially increase in PH patients compared to healthy controls while RVGWE decrease to a certain extent. RV is not composed of a single layer of myocardium, but mainly composed of superficial myocardium from the basal part of interventricular septum (IVS) and deep longitudinal myocardium. Under normal conditions, the contraction patterns of RV are mainly as follows: contraction of LV and IVS pull the free wall of RV to move inward, resulting in passive contraction of RV myocardium; the deep longitudinal myocardium of RV free wall contract, causing the tricuspid annular plane to approach to the apex ; IVS rotLV and RV are closely related in structure and function, and there is an interaction between two ventricles . In HardIn this study, as an emerging quantitative marker of cardiac function, myocardial work parameter has potential value to definite present cardiac function in PH patients. In clinical practice, WHO-FC remains a determinant part for assessing cardiac function, as it provides cardiologists with valuable information for determining disease severity, improvement, deterioration or stability. A follow-up cohort study containing 982 PH patients with WHO-FC III at baseline in the REVEAL Registry have shown that patients who improve from WHO-FC III to I/II have better prognosis than those who remain III or worsen to IV by Kaplan-Meier estimates of 3-year survival . WHO-FC Management of cardiac function is critical to the prognosis of patients with PH. Guidelines put emphasis on the importance of RV function in PH patients , as RV fThere are some limitations in the present study. First, this is a single-center and small population cohort study, which may produce a selection bias. Because of a relatively short follow-up period, the prognostic use of myocardial work by PSL method needs to be further demonstrated. Second, in the absence of myocardial work analysis before specific drug therapy, we have no chance to explore the short- and long-term effect of specific drug therapy on myocardial work parameters.Non-invasive PSL is feasible for quantifying myocardial work in patients with PH. Myocardial work parameters derived from PSL method are the emerging markers of cardiac function and the combination RVGWE and ePASP is a useful predictor of the clinical outcome in patients with PH.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Clinical Ethics Committee of Shanghai Renji Hospital. The patients/participants provided their written informed consent to participate in this study.JW: original manuscript drafting and writing—review and editing. CN: statistical analysis and figures graphing. MY and XZ: data collection and literature research. BR and LS: clinical studies. XS and JS: conceptualization, supervision, project administration, and funding acquisition. All authors have studied concepts/study design or data acquisition or data analysis/interpretation, manuscript drafting or manuscript revision for important intellectual content, approval of final version of submitted manuscript, and agreed to ensure any questions related to the work are appropriately resolved.