This study aimed to evaluate the twist/untwist parameters of the left ventricle (LV) in patients with heart failure with preserved ejection fraction (HFpEF) measured by ultrasonic two‐dimensional speckle tracking echocardiography (STE) and to examine the correlations between twist parameters and serum N‐terminal pro b‐type natriuretic peptide (NT‐proBNP) as well as conventional two‐dimensional echocardiography (2DE) indexes.Changes in twist/untwist parameters can be used to evaluate LV function in HFpEF patients.In 63 HFpEF patients and 40 healthy controls, we analyzed LV twist/untwist parameters by STE, cardiac function by 2DE, and serum NT‐proBNP by enzyme‐linked immunosorbent assay (ELISA). The correlations between twist/untwist parameters and 2DE parameters and serum NT‐proBNP were examined by Pearson correlation analysis.Left ventricular end diastolic inner diameter and ejection fraction in HFpEF patients were within the normal range, whereas other 2DE parameters including left ventricular posterior wall end diastolic thickness, interventricular septal thickness, left atrial volume index, E, E/A, and E/e' differed significantly between HFpEF patients and control subjects. The twist/untwist parameters such as peak apical rotation (Par), peak untwisting velocity (PUWV), and isovolumic diastole untwisting percentage (Iutw%) were significantly decreased in HFpEF patients compared with control participants. Positive correlations between PUWV/Iutw% and E/A/E/e' and a significant negative correlation between PUWV/Iutw% and left atrial volume index (LAVI) were observed. The plasma NT‐proBNP concentration was positively correlated with LAVI, but negatively correlated with PUWV and Iutw%.Changes in twist/untwist parameters correlate well with conventional 2DE parameters and plasma levels of NT‐proBNP, and can be used to evaluate LV function in HFpEF patients. Par is sensitive to the LV myocardial function damage. Diagnosis of HFpEF was based on the presence of symptoms and signs of heart failure and diastolic dysfunction without LV systolic dysfunction or dilatation. A “preserved” EF was defined as Left ventricular ejection fraction (LVEF) ≥50%. The definition of diastolic dysfunction included NT‐proBNP >220 pg/mL, the ratio of mitral valve early diastolic peak flow rate (E) to the tissue Doppler mean between the septal and lateral wall' (E/e′) >15, and/or other objective evidence of cardiac functional and structural alterations underlying Heart failure (HF), as defined by the 2015 consensus statement of the International Journal of Cardiology.The demographic and basal clinical characteristics of HFpEF patients and controls were obtained through retrieval from the hospital database or inquires. This study was approved by the ethics committee of our hospital, and all participants provided written informed consent.2.22DE was performed using a GE Vivid E9 ultrasound system with a M5S transducer , with a frame rate ≥50 frame/sec. The participants were in a calm state and in the left lateral position while 2DE was performed. The conventional 2DE parameters recorded included the left ventricular end diastolic inner diameter (LVDD), left ventricular posterior wall end diastolic thickness (LVPW), interventricular septal thickness (IVS), EF, left atrial volume index (LAVI), E, late diastolic peak flow rate (A), E/A, and E/e'.2.3All participants were instructed to hold their breath at the end of exhalation, and short‐axis dynamic images of the LV apical and the basal plane of the heart were recorded for three consecutive cardiac cycles with a stable heart rhythm. Image acquisition was guided by the following internal landmarks: the apical plane was proximal to the underside of the papillary muscle at a level where it was not yet visible, and the basal plane was identified by the tip of mitral valve leaflets. The ventricular cavity was kept round as much as possible. The angular displacement of the LV in each short‐axis plane was defined as “LV rotation.” Apical rotation was defined as a positive value, and basal rotation was defined as a negative value. The net difference in LV rotation between the apical and basal plane was defined as “LV twist.” Thus, peak LV twist (Ptw) = peak apical rotation (Par) − peak basal rotation (Pbr). Untwisting, the directional reversal of systolic twist motion during diastole, and its angular displacement, are the same as twist, but the peak untwisting velocity (PUWV) is different from twist in systole. Most untwisting was completed during the isovolumic diastole period, so the percentage of untwisting in isovolumic relaxation (Iutw%) may reflect whether untwisting is delayed.Twist parameters were analyzed offline by an experienced echocardiographer who blinded to clinical diagnosis and NT‐BNP, using the STE software . The parameters included Par, basal rotation angle peak value (Pbr), twist angle peak value (Ptw), twist speed peak value (PtwV), and twist angle peak time as a percentage of systolic duration (TPK%). The measurement also included two diastole untwist parameters: PUWV and Iutw% Figure .2.4The plasma NT‐proBNP levels were measured by enzyme‐linked immunosorbent assay (ELISA) in all participants on the same day that 2DE was performed.2.5P value less than .05 was considered statistically significant.All statistical analyses were performed by the SPSS 13.0 software system . The data were expressed as mean ± SD, and the variance test was used to compare differences in data between the two groups. Pearson correlation analysis was performed to determine the relationships between twist/untwist parameters and conventional ultrasonic indexes as well as between twist/untwist parameters and the plasma NT‐proBNP concentration. A 33.1A total of 63 HFpEF patients  with an average age of 60.44 ± 8.43 years (range: 49‐74 years), and 40 controls  with an average age of 58.05 ± 7.19 years (range: 45‐70 years), were enrolled in this study. Among the 63 patients, 37 had hypertension, 59 had coronary heart disease, and 17 had diabetes. No significant differences in the demographic and baseline clinical characteristics were observed between the two groups (Table 3.2P > .05), but the HFpEF group had significantly higher values of LVPW, IVS, LAVI, and E/e' as well as lower E and E/A values compared with the control group (P < .01); , Par was significantly decreased during the contraction period in the HFpEF group compared with that in the control group (P < .01). Also, the diastolic untwist parameters PUWV and Iutw% in HFpEF patients were decreased significantly compared with those in control subjects (P < .01) , Ptw, PtwV, and TPK% were comparable between the HFpEF and control groups  but not with Pbr, PtwV, or TPK. The diastolic untwist parameters PUWV and Iutw% were positively correlated with E/A  Figure .3.5P < .05). Correlation analysis showed that the plasma NT‐proBNP concentration was positively correlated with the LAVI , but negatively correlated with PUWV and Iutw%  (Figure As expected, the plasma NT‐proBNP concentration in the HFpEF group was significantly greater than that in the control group (1247.24 ± 530.38 pg/mL vs 50.24 ± 15.86 pg/mL, 4Although the pathophysiologic mechanisms underlying HFpEF remain unclear, HFpEF patients are more likely to have LV hypertrophy or concentric remodeling, LV diastolic dysfunction, and enlargement of the LA.Conventional 2DE has become a routine technique for assessing LV diastolic dysfunction and holds advantages such as noninvasiveness, simple operation, and accuracy. The conventional 2DE parameters most commonly assessed include E/A, E/e', and LAVI. The change in E/A reflects the degree of impairment of LV diastolic function, but the value of this index is easily affected by other factors such as heart rate, myocardial systolic and diastolic state, and valve regurgitation. E/e' is a valuable evaluation index for LV diastolic dysfunction and is not affected by changes in front and post load, heart rate, and other hemodynamic factors.The main pathological change in HFpEF is cardiac diastolic dysfunction, that is, LV stiffness increases, while the LA expands simultaneously, thus impairing the cardiac diastolic function. The main manifestation of LA dysfunction is the increase in its volume, which is an indicator of the severity of HFpEF and plays an important role in disease progression in HFpEF patients.LV twist is defined as the wringing motion whereby the LV apex rotates with respect to the LV base around the LV long axis.The LV stores force in systole and releases it during the isovolumic relaxation by recoiling and taking blood rapidly into the LV during mitral valve opening. Untwist movement in the vast majority of healthy people is completed during the isovolumic diastole period and is related to the rapid release of untwist force into the extracellular matrix and the restoration of the original length of the shortened sarcomere.NT‐proBNP is the amino acid residue of brain sodium urea precursor and is mainly secreted by the heart; especially the LV plasma NT‐proBNP levels may be used to help with diagnosing and evaluating the severity of cardiac diseases and holds a prognostic value for the outcomes of HF patients. An elevated plasma NT‐proBNP concentration suggests a cardiac hemodynamic disorder.4.1In the present study, we did not take into account the patients' medication history, and this might may affect the results to some extent, because some medications might have influenced the LV systolic and diastolic functions. Also, our study had a limited sample size, which might have contributed to the finding of nonsignificant differences in the rotation angle, twist angle, and velocity and peak time percentage of the bottom of the heart between the groups.5Twist/untwist parameters may be used to assess the severity of LV diastolic dysfunction in HFpEF patients in a new viewpoint, although conventional 2DE shows that their EF is in the normal range. The systolic apical rotation angle is very sensitive to changes in LV myocardial mechanics and can be used as an early diagnostic indicator of myocardial dysfunction in HFpEF patients.The data that support the findings of this study are available from the corresponding author upon reasonable request.The authors declare no potential conflict of interest.Supplementary Figure 1The diastolic untwist parameter PUWV in the HFpEF group is significantly lower than that in the control group.Click here for additional data file.Supplementary Figure 2The diastolic untwist parameter Iutw% in the HFpEF group is significantly lower than that in the control group.Click here for additional data file.Supplementary Table 1Comparison of demographic and basal clinical characteristics as well as conventional ECG parameters between the control and HFpEF groupsClick here for additional data file.Supplementary Table 2Comparison of twist and untwist parameters between the control and HFpEF groupClick here for additional data file.


Background: Survivors of childhood cancer are at risk for anthracycline- and/or radiotherapy-induced cardiotoxicity.Aims: The aim of this study was to assess clinical, laboratory, and imaging parameters of subclinical cardiovascular disease in childhood cancer survivors.Methods: Patients underwent cardiopulmonary exercise test (CPET), laboratory testing, transthoracic echocardiography (TTE) with tissue doppler imaging (TDI) and speckle tracking. A subset of patients also underwent cardiovascular magnetic resonance imaging (CMR). Findings were correlated to cumulative anthracycline and exposure to mediastinal irradiation during cancer treatment. In a subgroup analysis, TTE and CMR findings were compared to data from 40 gender- and age-matched patients with childhood onset hypertrophic cardiomyopathy (HCM).Results: Cardiac evaluation was performed in 79 patients  at 11.2 ± 4.5 years after cancer treatment. Oncologic diagnosis at a median age of 12.0 years was Hodgkin lymphoma in 20, sarcoma in 17, acute leukemia in 24, relapse leukemia in 10, and others in 8 patients. Cumulative anthracycline dose exceeded 300 mg/m2 in 28 patients. Twenty six patients also received mediastinal irradiation. Decreased peak respiratory oxygen uptake in % predicted on CPET, increased levels of N-terminal pro-brain natriuretic peptide (NTproBNP), increased global longitudinal strain on TTE speckle tracking, and diastolic dysfunction on TDI were the most prominent findings on detailed cardiology follow-up. In contrast to HCM patients, childhood cancer survivors did not show left ventricular hypertrophy , hyperdynamic systolic function on TTE , or fibrotic myocardial changes on CMR  at time of follow-up. There was no correlation between chest radiation exposure and abnormal cardiac findings. Cumulative anthracycline dose was the only significant independent predictor on multivariate analysis for any cardiovascular abnormality on follow-up (p = 0.036).Conclusion: Increasing cumulative anthracycline dose during cancer treatment correlates with subclinical cardiac dysfunction in childhood cancer survivors best detected by elevated cardiac serum biomarkers, decreased exercise capacity on CPET, and abnormalities on echocardiographic speckle tracking and TDI. Due to advancements in treatment, survival rates for childhood cancer have substantially improved during the past decades with an overall 5-year survival rate of over 80% today –3. More The primary prevention of long-term adverse effects of childhood cancer treatment includes modification of therapeutic exposures –33, the Secondary prevention after completion of anticancer treatment, such as improved surveillance, appropriate screening strategies, and preventative therapies for cardiovascular complications, require an interdisciplinary approach between oncologists and cardiologist.Standardized clinical evaluation with regular follow-up and assessment of the entire cardiovascular system is necessary in childhood cancer survivors . HoweverData are scarce with regards to the optimal use of serum biomarkers, the mode and sensitivity of distinct imaging modalities, and optimal management strategies once subclinical cardiotoxicity has been detected, specifically in children.The aim of the study was to assess laboratory, cardiopulmonary exercise testing, echocardiographic, and cardiac magnetic resonance imaging findings in clinically asymptomatic childhood cancer survivors on long-term follow-up. The identification of monitoring parameters shall optimize risk stratification, identify subclinical cardiovascular disease, and enable preventative treatment measures in time before overt clinical disease in childhood cancer survivors becomes evident.In this retrospective single tertiary care center study, medical charts were reviewed from all childhood cancer survivors followed at the pediatric cardiology outpatient unit at the German Heart Center Munich between March 2015 and October 2018. Demographic, clinical, laboratory, transthoracic echocardiographic (TTE), cardiopulmonary exercise testing (CPET), cardiovascular magnetic resonance (CMR), and other patient-related data were collected retrospectively by review of patients' charts at last follow-up.Data from a total of 79 patients with a primary diagnosis of childhood cancer and initial cancer treatment between 0 and 18 years of age were analyzed. For subgroup analysis, TTE and CMR findings were compared with 40 gender- and age-matched patients with childhood onset hypertrophic cardiomyopathy (HCM). The diagnosis of HCM was based on clinical evaluation and cardiac catheterization or TTE detecting myocardial hypertrophy (defined as a z-score of >2) in the absence of another cardiac or systemic disease causing the degree of left ventricular hypertrophy identified , 43. Thi2 max), evidence of myocardial ischemia or arrhythmia and escalation of heart rate or blood pressure during or after exertion were recorded (2 max (% norm)], a cut-off level of <80 was determined as “abnormal.”CPET was performed in sitting position on a bike-ergometer. A standardized testing protocol was performed and physical working capacity, peak oxygen uptake (VOrecorded . For theThe serum biomarkers cardiac troponin T (cTNT) and N-terminal pro-brain natriuretic peptide (NTproBNP) levels were determined as marker for early systolic dysfunction or myocardial stress. Values >0.01 ng/dL and >100 ng/dL, respectively, were considered as “elevated.”http://parameterz.blogspot.com/2008/09/z-scores-of-cardiac-structures.html, (2 was considered as enlarged (Echocardiograms were obtained at the time of outpatient visits and were remeasured by blinded study staff (AK). TTE was performed using standard equipment in routine clinical practice and using standard views according to the American Society of Echocardiography guidelines –48. Echoes.html, ]. Z-scores.html, . Global es.html, . Referenes.html, , 51–53. es.html,  were conenlarged , 55.CMR was performed on a 1.5 Tesla MR scanner . Cine images  were acquired in short axis and four chamber orientations in breath hold to evaluate the ventricular volume, ventricular mass, ejection fraction, and regional wall anomalies. Late Gadolinium Enhancement (LGE) was acquired using a T1-weighted phase-sensitive inversion recovery sequence 10 min after intravenous administration of an extracellular MR contrast agent (Gadopentetat) to detect focal fibrosis .Native and post contrast T1 mapping, using a modified look-locker inversion recovery sequence (MOLLI) , with noThe study was conducted in accordance with the Declaration of Helsinki (revision 2008) and the Good Clinical Practice guidelines. The study protocol was approved by the local ethical board of the Technical University Munich .t-Test and Mann-Whitney-Test were used according to data distribution to compare parameters between the two groups. ANOVA  or Kruskall-Wallis-test were used to compare differences between multiple groups (oncologic diagnosis) according to data distribution. Fisher's Test or Pearson chi-square was used for comparison of categorical variables. Pearson correlation was used to assess the correlation between anthracycline dose, radiation exposure, age at cancer treatment, age at follow-up, follow-up time, and cardiovascular findings. Binary outcome variables  were created using cut-off for the respective variables. Backward multivariate binary logistic regression analysis was done for the categorical variable “Any cardiovascular abnormality” defined as decreased exercise capacity, NTproBNP level, or increased septal E/E' ratio on TDI as dependent variable and cumulative anthracycline dose, radiation exposure, gender, age at cancer treatment, age at follow-up, and follow-up time as covariates. Cut-off levels of cumulative anthracycline dose predictive for any cardiovascular abnormality on follow-up were derived from the Receiver operating characteristic (ROC) curve. The significance level of p-value was set at <0.05. Data were analyzed on IBM SPSS Version 25.Data are presented as mean and standard deviation or as median and minimum / maximum according to distribution. Kolmogorov-Smirnoff Test was used to assess distribution of variances. Independent N = 24) or relapse leukemia (N = 10), Hodgkin lymphoma (N = 20), sarcoma (N = 17), nephroblastoma (N = 4), neuroblastoma (N = 3), and hepatoblastoma (N = 1). Median age at primary oncologic diagnosis was 12.0 years. Mean cumulative anthracycline dose was 261±104 mg/m2 and exceeded 300 mg/m2 in 36% of patients. Patients with sarcomas received significantly higher doses of anthracycline compared to the remaining patients. Twenty-six patients, most of them with Hodgkin lymphomas and relapse ALL, also received mediastinal irradiation with heart exposure. All patients with relapse ALL and one patient with ALL received mediastinal irradiation in the setting of total body irradiation for preparation to bone marrow transplant  of <80 on CPET was evident in a third of patients. Cardiac troponin T levels were normal in all patients. NTproBNP levels >100 ng/dL were measured in 29% of childhood cancer survivors. Whereas, left ventricular systolic function (EF) and left ventricular dimensions were normal on TTE in most patients, there were abnormal GLS measurements in off-line echocardiographic speckle tracking analysis in about 28% of patients. No left atrial enlargement was documented on TTE, but pulse wave and tissue Doppler examination showed signs of diastolic dysfunction in 21, 24, and 11%, respectively  or interstitial (increased ECV on T1 map) myocardial fibrosis on CMR imaging  or the MYBPC3  gene. Other genes affected included TNNT2, TNNI3, ML2, TPM1 in 7 of 40 HCM patients (17.5%) and multiple mutations were detected in 4 of 40 HCM patients (10 %). Molecular diagnostic testing was negative in 3 of 40 HCM patients (7.5 %). Left ventricular outflow tract obstruction was present in 14 of 40 HCM patients (35 %). In contrast to HCM patients, childhood cancer survivors did not show left ventricular hypertrophy or hyperdynamic systolic function on TTE (TTE and CMR data of childhood cancer survivors were compared to age- and gender-matched patients with an underlying diagnosis of childhood hypertrophic cardiomyopathy (HCM) secondary to mutations in sarcomere genes. The majority of HCM patients carried a pathogenic variant in either the n on TTE . Fibrotin on TTE . Evidencn on TTE . EnlargePatients were grouped into five categories according to oncologic diagnosis and variance analysis was carried out to assess if there were differences of abnormalities in cardiovascular findings between groups. There were significant differences between oncologic groups concerning decreased exercise capacity, NTproBNP levels and evidence of diastolic dysfunction on echocardiographic tissue Doppler imaging . Elevated NTproBNP levels and evidence for diastolic dysfunction were mostly observed in patients treated for sarcoma .There was no influence of age of diagnosis, age at follow-up and follow-up time on cardiac features on Pearson's correlation in univariate analysis.2 vs. 231 ± 95 mg/m2, p = 0.007, independent t-Test).There was a positive correlation between increasing cumulative anthracycline doses and decreased exercise capacity on CPET . Septal There was no significant correlation between cumulative anthracycline dose and decreased systolic echocardiographic function as shown by TTE ejection fraction (EF) on follow-up.There was no correlation between exposure to mediastinal irradiation or exposure to total body irradiation and abnormal cardiac findings in univariate analysis. Patients with relapse ALL who were all exposed to mediastinal radiation in the setting of TBI for bone marrow transplantation conditioning had also received higher cumulative anthracycline doses, so that mediastinal radiation exposure was eliminated as independent predictor on multivariate analysis (see below).p = 0.036). Cut-off levels of the cumulative anthracycline dose associated with any cardiac abnormality on follow-up examination on the ROC-curve were 261, 269, and 305 mg/dL for a true positive prediction rate (sensitivity) of 62.2, 59.5, and 54.1%, and a false-positive prediction rate (1- specificity) of 30, 20, and 17.5%, respectively  after elimination of influencing factors, such as oncologic diagnosis, gender, age at diagnosis, age at follow-up, follow-up time or exposure to irradiation, on multivariate logistic regression analysis  (E/E') was increased in patients with subclinical cardiovascular impairment in the current study, correlating with the amount of cumulative anthracycline exposure as a sign of subclinical diastolic dysfunction. This was also shown by others , 82–84 aIn summary the presented data provide evidence of subclinical cardiovascular disease in childhood cancer survivors exposed to high doses of anthracyclines. Subtle changes may precede clinical manifestation and can be evaluated by laboratory testing (biomarkers) and focused echocardiographic imaging techniques including speckle tracking and tissue Doppler.Of course, focus should always be on the primary prevention of cardiotoxicity  by limitLong-term childhood cancer survivors exposed to cardiotoxic therapy show subtle impairment of cardiovascular function best detected by decreased exercise capacity, elevated NTproBNP levels, increased global longitudinal strain on speckle tracking, and abnormal mitral valve annular septal tissue doppler velocities in the absence of gross morphological changes, such as myocardial fibrosis on CMR, or echocardiographic decreased ejection fraction, enlarged left ventricular chamber sizes or left atria. Increased cumulative anthracycline dose for cancer treatment is the only independent predictor for the presence of those abnormal cardiovascular findings. Regular application of blood biomarkers and new imaging technologies can optimize risk stratification of childhood cancer survivors and can facilitate identification of subclinical cardiovascular disease and enable preventative treatment measures in time before overt clinical disease in high risk childhood cancer patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the Technical University of Munich Institutional Review Board . Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.Data were in part presented at the Annual Meeting of the Association for European Pediatric and Congenital Cardiology in Athens, 9 May – 12 May 2018.CW, RO, PE, IS, and JW designed the study and data collection instruments, coordinated and supervised data collection, statistical analysis and interpretation of data, drafted, reviewed and critically revised the manuscript. BR, AK, AH, JM, and CM collected cardiovascular data. BR and AH analyzed cardiopulmonary exercise testing data. AK and AH performed measurements on transthoracic echocardiography, specifically of diastolic dysfunction on tissue Doppler and speckle tracking for global longitudinal strain analysis. CM performed and analyzed cardiovascular magnetic resonance imaging and contributed to interstitial fibrosis quantification by determination of extracellular volume fraction on T1 map as well as quantification of late gadolinium enhancement and critically revised the article for important intellectual content regarding cardiovascular magnetic resonance imaging. IS collected oncologic data. CW carried out statistical analyses and drafted the manuscript. All authors listed have made substantive intellectual contributions to this study. Each author has provided final approval of the version to be published and has given agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Indications of cardiac resynchronization therapy (CRT) do not include exercise‐induced left bundle branch block, but functional impairment could be improved with CRT in such cases. A 57‐year‐old woman with idiopathic dilated cardiomyopathy (ejection fraction 23%) presented with New York Heart Association Class IV and recurrent hospitalizations. During heart transplant evaluation, a new onset of intermittent left bundle branch block was observed on the cardiopulmonary exercise test. CRT was implanted, and 97% resynchronization rate was achieved. In 12 month follow‐up, both clinical and prognostic exercise parameters improved. In patients with heart failure with reduced ejection fraction and no left bundle branch block at rest, exercise test can uncover electromechanical dyssynchrony that may benefit from CRT. She was mostly limited by exertional fatigue and dyspnoea, despite being mostly euvolemic not dependent on high doses of diuretics. Rest electrocardiogram (ECG) showed sinus rhythm at a rate of 70 bpm and a QRS duration of 80 ms. Left ventricular ejection fraction was 23%, with a dilated left ventricle (end‐diastolic diameter 6.7 cm) and diffuse hypokinesis. A cardiopulmonary exercise testing (CPET) demonstrated severely decreased exercise capacity, with a peak oxygen uptake (VO2 peak) of 12 mL/kg/min (or 16.46 mL/kg/min when corrected for lean body mass), along with other variables indicating adverse heart failure prognosis, including minute ventilation/carbon dioxide production (VE/VCO2) slope of 42 and periodic breathing. At the second minute of exercise, at a heart rate of 131 bpm, the patient developed LBBB with widening of the QRS to 200 ms. This finding persisted into the recovery phase  to approach the EI‐LBBB. Cardiac magnetic resonance imaging performed prior to CRT insertion showed only a small area of late gadolinium enhancement at the mesocardium basal septum, but no patterns suggestive of specific aetiologies other than non‐ischaemic dilated cardiomyopathy were noted. Initially, biventricular pacing was set at a minimal heart rate of 75 bpm with atrioventricular delay of 105–80 ms, and the ECG showed QRS duration of 145 ms.FigureTableSubsequent to CRT implant, the patient improved to New York Heart Association II, and no recurrent heart failure hospitalizations occurred. Medical therapy was relatively unchanged after CRT, except for the introduction of amiodarone due to ventricular arrhythmias and a change from metoprolol to carvedilol and introduction of hydralazine, both to enhance hypertension management, as blood pressure levels increased and tolerance to medications improved after CRT. Pacemaker interrogation showed 94% atrial pacing and 100% biventricular pacing. A few months later, repeat CPET at 6 and 12 months demonstrated significant and sustained amelioration in exercise capacity, as depicted in 3To the best of our knowledge, this is the first report of CRT implantation in a patient with advanced heart failure and severe exercise limitation attributed to EI‐LBBB. In the present case, CRT was associated with sustained functional capacity and prognostic markers improvement. The patient received at least 5‐year guideline‐based treatment, and no major changes in medications were performed after CRT. This observation highlights the relevance of exercise evaluation in patients with advanced heart failure to better understand their symptomatology, which may be related to the broadening of the QRS complex during exertion; in fact, this patient was severely limited under minimal activities, the exercise test uncovered a EI‐LBBB. Both exercise stress ECG and echocardiography can unravel dynamic electromechanical dyssynchrony that may eventually benefit from CRT.Exercise‐induced LBBB is a rare condition reported only in 0.5% in patients who underwent exercise testing and is associated with twofold increase in all‐cause mortality CRT is known to promote left ventricular reverse remodelling. In the Resynchronization Reverses Remodelling in Systolic Left Ventricular Dysfunction trial, which evaluated left ventricular remodelling after CRT, the average absolute improvement in ejection fraction was 3.6% in a follow‐up of 1 year.Left ventricular dyssynchrony during exercise occurs in a third of patients with heart failure with reduced ejection fraction being associated with poor outcomes.There are few published reports of CRT for the management of cardiac symptoms related to EI‐LBBB. Prystowsky et al. reported a case in which severe shortness of breath was coincident with LBBB development at a heart rate of 140 bpm, in the absence of evidence of ischemia or systolic dysfunction. She was initially treated with beta blockers, but there was significant improvement in symptoms only after CRT.In conclusion, although EI‐LBBB is a relatively rare finding, it should be actively sought in patients with heart failure, even in those with more advanced structural and functional abnormalities. While there is no guideline‐oriented indication for CRT in EI‐LBBB, it could be considered in selected individuals with advanced heart failure with the aim to improve symptoms, particularly in those with left ventricular dyssynchrony.None declared.


We hypothesized that DD patients would have an increased risk for common heart disease. We performed a cross-sectional case-control clinical study on 25 DD patients and 25 matched controls; and a population-based cohort study on 935 subjects with DD and matched comparison subjects. Main outcomes and measures were N-terminal pro-brain natriuretic peptide, ECG and heart diagnosis . DD subjects showed normal clinical heart phenotype including heart failure markers and ECG. The risk for heart failure was 1.59  times elevated in DD subjects, while no major differences were found in myocardial infarcation or arrhythmias. Risk for heart failure when corrected for cardivascular risk factors or alcohol misuse was 1.53 (1.11–2.11) and 1.58  respectively. Notably, heart failure occurred several years earlier in DD patients as compared to controls. We conclude that DD patients show a disease specific increased risk of heart failure which should be taken into account in patient management. The observation also strenghtens the clinical evidence on the important role of SERCA2 in heart failure pathophysiology.Human data supporting a role for endoplasmic reticulum (ER) stress and calcium dyshomeostasis in heart disease is scarce. Darier disease (DD) is a hereditary skin disease caused by mutations in the ATP2A2 gene encoding the sarcoendoplasmic-reticulum Ca2 Herein, we examined increased risk of heart disease as a comorbidity of the rare skin disorder Darier disease (DD).Rare monogenic diseases provide opportunities to study human physiology and pathology from unique perspectives. Besides the importance of diagnosing and successfully treating patients with a rare disorder, major scientific breakthroughs resulting from investigation of rare diseases have often provided insight into more common disorders6. It is an autosomal-dominant skin disorder caused by mutations in the ATP2A2 gene, which encodes for sarcoendoplasmic-reticulum Ca2+-ATPase isoform 2 (SERCA2)7. DD is characterized by a peculiar keratinization of the epidermis, nails, and mucus membranes resulting in a persistent hyperkeratotic papules8. The prevalence of the disease has been estimated to be 1 in 30,000–55,000 and the onset is usually before the third decade of life with complete penetrance in adults9. DD never remits, but oral retinoids may reduce hyperkeratosis. Interestingly, DD has been reported to be associated with epilepsy and neuropsychiatric problems including mild intellectual disability, bipolar disease and schizofrenia11.Darier (Darier-White) disease (DD) was first described independently by Darier and White in 18892+ is released from the sarcoplasmic reticulum (SR) via the ryanodine receptor 2 (RYR2), thus raising the cytosolic Ca2+ concentration and activating cardiac muscle contraction. The Ca2+ is then pumped back into the SR by SERCA2a lowering the cytosolic Ca2+ concentration to baseline levels and causing relaxation12. SERCA2 protein has three isoforms; SERCA2a, 2b and 2c. While SERCA2b is expressed ubiquitously in most tissues, SERCA2a is predominantly expressed in cardiomyocytes12. Heart failure (HF) is a highly morbid condition with increased incidence after the age of 65, affecting 1 in 100 individuals13. While efforts have been devoted to investigate the therapeutic value of various Ca2+ channels and proteins that regulate cytosolic Ca2+ concentrations, SERCA2a has received the most interest in recent years. Indeed, it was shown that the expression of SERCA2a is downregulated in HF, which subsequently contributes to severe systolic and diastolic dysfunction16. Consequently, restoring SERCA2a expression by gene transfer, both in HF patients18, as well as in animal models19, is elegantly described; however, with contradicting results. Thus, the role of SERCA2a is still unclear and further investigation is required.In the healthy heart, Ca2+ homeostasis can trigger endoplasmic reticulum (ER)/SR stress20. The latter is defined as an imbalance between the protein load and the folding capacity of the ER, which results in accumulation of misfolded proteins further activating an adaptive response termed the unfolded protein response (UPR). UPR is executed by a signaling network comprised of three ER-localized transmembrane proteins , which aim to restore the protein-folding homeostasis. Principally, for efficient cardiomyocyte contractile function, protein quality control is essential, and any perturbations in protein homeostasis can affect functionality and contractility of the myocytes, resulting in cell death23. Indeed, reduced SERCA2 expression or function causes the phenotype of HF also through induction of ER stress24.In heart disease, pathological events such as overproduction of reactive oxygen species (ROS), ischemia/reperfusion, release of inflammatory cytokines and toxins, as well as perturbations in Ca2+ dyshomeostasis and ER stress induced by endogenously reduced SERCA2 function28, are prone to heart failure.With this background, we hypothesized that DD patients, who are characterized by ER Ca29 in addition to its use in detecting myocardial infarction.A cross-sectional case-control study design was implemented in which we at one time-point measured cardiac function by heart failure markers N-terminal pro-brain natriuretic peptide (NT-proBNP), Galectin-3, suppression of tumorigenicity 2 (ST2), and troponin T as well as ECG and blood lipids . The rational for including troponin T was that it can be elevated in heart failureATP2A2 mutations by whole exome and Sanger sequencing as previously described30.The study included 25 patients with DD and 25 healthy volunteers matched by age, sex and body mass index (BMI). Age matching was made in ±5-year intervals and BMI was matched according to four categories: <18.5, 18.5–24.99, 25–29.99 and >30. During 2017–2018 DD subjects were recruited mainly from the Dermato-Venereology clinic at Karolinska University Hospital in Stockholm, Sweden, and through study participants since it is most often a familial condition. Control subjects were recruited through advertisements by e-mail and online as well as from a primary healthcare clinic in Stockholm. Inclusion criteria for the study were typical skin symptoms combined with histopathology-verified DD or family history of DD. Exclusion criteria were pregnancy, oral corticosteroids, recent acute illness (past 4 weeks), active substance abuse and severe kidney or liver disease. All patients but one was previously tested for Patients underwent a history taking focused on DD debut and family history. Family history was defined as having a first or second degree relative with DD. DD is most often treated with acitretin, an oral retinoid. Subjects who had been on oral acitretin treatment at some point during the 6 months prior to participation were classified into the acitretin category. Weight (kg) and height (cm) was measured to calculate BMI. The skin was inspected to verify the DD clinical diagnosis.33, subjects on oral acitretin discontinued the medication 7 days before the visit, longer wash-outs were not considered ethically.Blood samples were collected after an overnight (10 h) fast. Since acitretin has a half-life of approximately 50 hours and is known to influence triglycerides and cholesterolPlasma NT-proBNP and troponin T was measured by immunochemistry and ElectroChemiLuminescence ImmunoAssay, and plasma triglycerides, cholesterol and HDL measured by enzymatic reaction and photometry . Serum Galectin-3 and ST2 were determined using ELISA and photometry . LDL and HDL/LDL ratio were estimated from total cholesterol and HDL.34 contains discharge diagnoses for all inpatient care episodes since 1973, and for outpatient treatment since 2001. Diagnoses are assigned by physicians according to the WHO’s classification systems ICD-9 and ICD-1037. DD was defined by a diagnosis of the disorder according to the ICD-9 (757D) or ICD-10 (Q82.8E) in the National Patient Register. A total of 935 individuals with DD were identified. and matched with up to 100 comparison individuals selected randomly from the general population. Matching variables were birth year, sex and county of residence at the time of the first diagnosis in the DD index subject. Comparison individuals did not have a diagnosis of DD at the date of the first diagnosis of their respective index subjects with DD. Heart diseases were defined as a diagnosis of myocardial infarction (I21), heart failure (I50), or arrhythmia (I49) in ICD-10. While we are not aware of any validation of these specific diagnoses, most diagnoses of chronic diseases in the National Patient Register typically have positive predictive values between 85–95%34.We conducted a cohort study based on linkage between several Swedish registers. The Total Population Register contains demographic information on all individuals registered as Swedish inhabitants since 1968. The National Patient RegisterP-values < 0.05 were considered statistically significant. Demographic data (genetic background and presence of acitretin) were analyzed using two-way ANOVA and Bonferroni to correct for multiple comparisons. Data was analyzed by GraphPad Prism version 7.03 for Windows , and SPSS version 24.0 for Mac . The matched cohort design was used to examine associations between DD and heart disease. Odds ratios were estimated with conditional logistic regressions in SAS version 9.3 . In this design, odds ratios can be regarded as risk ratios, due to the incidence density sampling procedure. In additional analyses, we adjusted the results for the potential effects of cardiovascular risk factors of heart diseases, and alcohol misuse.In the clinical study descriptive analyses were performed to show frequencies, means, upper and lower limit and standard deviations. Since there were a small number of observations, Wilcoxon signed-rank test was used to compare continuous variables between groups. Differences between categorical variables were tested for significance using Fisher’s exact test. The study was approved by the Regional Ethics Committee in Stockholm and carried out in accordance with relevant guidelines and regulations such as the Helsinki declaration. All patients provided written confirmed consent for the clinical study.Comparisons of baseline clinical parameters between subjects with DD and healthy control subjects are given in Table 39 (Table 29. All these markers were within the normal range and with no significant differences between DD patients with or without mutation and control subjects (Table We analysed several biomarkers for heart failure including the gold standard NT-proBNP as well as the novel heart failure biomarkers ST2 and Galectin-3ts Table .Table 2HA resting ECG was recorded to detect alterations in cardiac electrophysiology and analysed as shown in Table 33 (Table Blood lipids were measured due to their importance in heart disease and possible increase due to retinoid treatment33 Table . AlthougDD patients in general had a 59% higher risk of heart failure diagnosis, compared to undiagnosed matched individuals; risk ratio 1.59, 95% confidence interval 1.16–2.19 (Table While our clinical study found no heart abnormalites in DD patients our population study indicates that DD patients are more prone to develop HF and at an earlier age. Possible reasons behind the observed excess risk of HF in DD are outlined below.ATP2A2 gene7, which encodes for the SR/ER Ca2+ pump- SERCA2. To better understand and further characterize the consequenses of SERCA2 dysfunction in DD patients, we searched for more common conditions in which SERCA2 plays a central role and chose to focus on heart disease, as the importance of SERCA2 in heart function is well established. In both animal and human heart failure, SERCA2a expression is significantly decreased, which leads to abnormal Ca2+ handling and a deficient contractile state and HF40. Interestingly, SERCA2 haplosufficent mice were reported to develop HF when crossed with a with a transgenic model of increased myofibrillar Ca2+-sensitivity43, thus suggesting that DD patients may be more susceptible to HF. Further, in attempts to rescue SERCA2a deficiency phenotype, a gene-targeted mouse with full substitution of the cardiac isoform SERCA2a by SERCA2b was described44. The 2b isoform has a nearly 2-fold higher affinity for Ca2+ but a lower maximal catalytic turnover rate than 2a, which resulted in a mild concentric heart hypertrophy and impaired contraction-relaxation. This suggests that also dysfunctional and not only defiency in SERCA2 may cause HF. Importantly, Atp2a2−/+ mice develop squamous cell carcinomas but no DD-like lesions, thus mouse SERCA2 pathophysiology may therefore differ substantially from humans45. Our study presents the first evidence that HF is accociated with DD which adds to the growing body of evidence that DD is a systemic condition not only confined to the skin. This finding also adds a novel angle to understanding of SERCA2 pathophysiolog in human HF.DD is mainly caused by a mutation in the 47. While we do not know what subtype of HF is overrepresentend in DD female patients, these gender differences in HF may be explained by the presence of dysfunctional SERCA2 as it has been shown in multiple separate studies that increased oxidative stress observed in damaged myocardium lead to impairment of the SERCA pump followed by Ca2+ dyshomeostasis and prolonged diastolic relaxation, which contributed to the females’ predominent heart failure type43.Our findings indicates that DD female patients have higher risk for heart disease in general and probably specifically for heart failure. In fact, over the past decade, one of the most robust findings across numerous HF studies was a distinct difference in gender distribution. Namely, women significantly outnumber men, leading to a gender ratio of approximately 2:1 in HF, with preserved ejection fraction48. Among the side effects associated with this drug, it has been reported that treatment with acitretin increases serum lipids and triglycerides in 25–50% of patients50, however the DD-HF association did not change significantly after correction for cardiovascular risk factors including hyperlipidemia and hypertriglyceridemia (Table 51. Because the national registers in the cohort study lacked potentially important individual lifestyle characteristics, the results from these analyses warrant replication in a more informative data set where statistical adjustment for such factors are possible.An alternative explanation to the DD-HF correlation might be the long term usage of acitretin to treat DD, that makes these patients more susceptible to HF. Acitretin is a vitamin A derivative and a second generation retinoid which has anti-inflammatory and immunomodulatory effectsDD is likely a syndrome with multi-organ involvement. The association between DD and HF we demonstrated here can be further understood in several ways. Most important will be to characterize what kind of heart failure DD patients have, in order to tailor treatment. Careful assessment of heart function in healthy individuals would further increase our understanding of SERCA2 in normal heart physiology. Importantly, it may be that DD would require systemic treatment from early age to combat multi-organ involvement however before that can be realized we must further map the its’ organ involvement. Moreover, there is currently no therapy available for such treatment.The increased risk of heart failure should be considered in patients with DD that although being a rare disease affects around 200,000 patients worldwide. While speculative, it may be that heart failure in DD show special characteristics.This study adds significant human evidence for a role of SERCA2 in heart failure and adds to the growing body of literature suggesting SERCA2 as a potential terapeutic target.Supplementary Information.


Volume indices and left ventricular ejection fraction (LVEF) are routinely used to assess cardiac function. Ventricular strain values may provide additional diagnostic information, but their reproducibility is unclear. This study therefore compares the repeatability and reproducibility of volumes, volume fraction, and regional ventricular strains, derived from cardiovascular magnetic resonance (CMR) imaging, across three software packages and between readers.Seven readers analysed 16 short-axis CMR stacks of a porcine heart. Endocardial contours were manually drawn using OsiriX and Simpleware ScanIP and repeated in both softwares. The images were also contoured automatically in Circle CVI42. Endocardial global, apical, mid-ventricular, and basal circumferential strains, as well as end-diastolic and end-systolic volume and LVEF were compared.Bland-Altman analysis found systematic biases in contour length between software packages. Compared to OsiriX, contour lengths were shorter in both ScanIP (-1.9 cm) and CVI42 (-0.6 cm), causing statistically significant differences in end-diastolic and end-systolic volumes, and apical circumferential strain . No differences were found for mid-ventricular, basal or global strains, or left ventricular ejection fraction . All CVI42 results lay within the ranges of the OsiriX results. Intra-software differences were found to be lower than inter-software differences.OsiriX and CVI42 gave consistent results for all strain and volume metrics, with no statistical differences found between OsiriX and ScanIP for mid-ventricular, global or basal strains, or left ventricular ejection fraction. However, volumes were influenced by the choice of contouring software, suggesting care should be taken when comparing volumes across different software. Accurate and reproducible quantification of cardiovascular properties is imperative for correct identification and management of many cardiovascular diseases. Prognosis post myocardial infarction is partially based on measurements of end-diastolic and end-systolic volumes, or left ventricular ejection fraction (LVEF), as these reflect adverse remodelling of the left ventricle –4. Non-iCMR images can be used as non-invasive diagnostic tools to assess LVEF and, more recently, ventricular strains. Ventricular strains quantify ventricular deformation and are dependent on the change in endocardial or epicardial lengths throughout the cardiac cycle. They have been shown to be sensitive markers for myocardial dysfunction , 7, 8. SA large range of commercial software packages have become available over the last decade, many with in-built image analysis tools, which can operate on an automatic, semi-automatic, or manual basis. Through machine learning and greyscale thresholding techniques, vendors have been able to incorporate automatic and semi-automatic contouring of the left ventricle into software packages, with accurate results which need minimal manual alterations . CalculaA cross-centre study for CMR centres showed that there can be large variations in both endocardial and epicardial contouring even amongst expert readers , confirmThis study aimed to investigate the variability introduced to clinical metrics by different readers and different pieces of software when using CMR images. The clinical metrics under investigation were end-diastolic volume (EDV), end-systolic volume (ESV), LVEF, global circumferential strain (GCS), and three regional circumferential strains; apical, mid-ventricular, and basal.Cine CMR images from one porcine specimen (age = 7 months) suffering from induced heart failure with dilated cardiomyopathy were selected. This data collection, and analysis presented here, was part of a study demonstrating a closed-chest porcine model of myocardial infarction and reperfusion, assessed by serial imaging and biomarker measurements, and proteomics tissue analysis . Those aCMR images were acquired in both short- and long-axes slices, using a CMR image scanner  with a spatial resolution of pixels of 1.6 mm x 1.6 mm. Temporal resolution was 30 frames per cycle. FISP functional cinematic images were acquired in the three long axis planes  of the left ventricle and in a contiguous stack of short axis slices from base  to the apex. The acquisition parameters are as follows: slice thickness 8mm, base resolution 208, phase resolution 80%, TE 1.39 ms, TR 46.5 ms, flip angle 45o, bandwidth 1335 Hz/Pixel, calculated phases  30, data segments per R-R 15. For short-axis images the slice thickness was 8 mm with a 2 mm gap, which is similar to that used in other studies –19. The Seven readers from one institution  independently analysed the data set on four separate occasions, twice on each of two software packages (OsiriX and ScanIP) with at least one interval passing between each occasion. The whole workflow is shown as a schematic in 2 respectively. In OsiriX, typically one cine CMR image slice is viewed through all frames, showing the user the same location at different points in the cardiac cycle as references to guide their contouring.OsiriX  is a DICOM viewer commonly used in clinical settings. The user places a number of markers along the border of interest with the software then interpolating a spline curve between these points to generate the contour . The lenSimpleware ScanIP  is a 3D image segmentation and processing software commonly used in biomedical engineering, and which has a comprehensive environment capable of handling and complying with DICOM standards. Segmentation/contouring is based on thresholding the greyscale values of the DICOM images, and individual pixels can be inserted or removed using a ‘paint’ tool . There iMost readers had prior knowledge of at least one of the pieces of software or were otherwise trained on the software prior to the study taking place. Two users had prior knowledge of delineating LV contours and a training session on this process was provided to the other users prior to testing. However, four of the users did have prior knowledge of the ScanIP software (one for LV contouring) and one user prior knowledge with OsiriX again for LV contouring. The experienced reader was equally experienced in both software packages. All contours were placed manually in both OsiriX and ScanIP, following the SCMR guidelines of standardised image interpretations and post-processing of DICOM CMR images , with boSixteen consecutive short axis CMR frames from the original 30 were chosen for endocardial contouring, with the first frame occurring just before end-diastole (ED) and the last frame just after end-systole (ES). Readers contoured all 9 slices from the 16 timeframes provided to them, covering the majority of the LV, from base to apex. All contours were assessed by an experienced reader to ensure they followed the SCMR guidelines of the experiment and were otherwise clinically acceptable.Once all contours were drawn, the data were extracted from OsiriX and ScanIP and processed using an automated MATLAB script. Volumes for each frame were calculated for both OsiriX and ScanIP. OsiriX automatically calculates the area enclosed by a contour for each slice and frame. These areas were multiplied by the sum of the image slice thickness (8 mm) and gap (|2 mm), i.e. 10 mm, and summed across the LV to give the total LV volume. This is the composite midpoint integration method described by O’Dell et al.  and was convhull returned the area within the convex hull derived for each contour. These areas were similarly summed for each slice and frame, and then multiplied by 10 mm to find the volumes at each time-point. For the calculation of clinical metrics, ED was defined as the time with maximum LV volume, and ES was defined as the time with minimum LV volume. From these volumes, LVEF was calculated.For ScanIP the MATLAB function nϵ, was defined using the clinical standard, in L0 is the endocardial perimeter reference length at ED, and nL is the endocardial perimeter length at time n for a given cine slice. These short axis image slices were then grouped into three vertical regions in the LV, apex, mid-ventricle, and base, with each region containing three slices. The means of the circumferential strains in each region were then calculated to give: apical circumferential strain (ACS), mid-ventricular circumferential strain (MCS), basal circumferential strain (BCS) [Endocardial circumferential strain was also calculated using the perimeter length data for every slice and frame for both OsiriX and ScanIP. Strain, in (BCS) . GCS wasCVI42 , which has an in-built automatic contouring function, was also tested on the same CMR images. Contouring was performed once using the software’s automated algorithm and these contours were then used to calculate endocardial strain as before. CVI42 has the functionality to calculate clinical indices such as LVEF from these contours, and although it can also calculate myocardial strain, the strain definition it uses is quite different to that defined here and thus would not form a suitable comparison. Using CVI42’s automatic contouring of the endocardium in this way does however still provide an ‘automated method’ for computing the strain metrics of ACS, MCS, BCS, and GCS for comparison with that derived from OsiriX and ScanIP. Perimeter lengths, areas, volumes, and strains were determined as before, with all measurements and post-processing undertaken by one experienced reader. The results from this CVI42-based method were then directly compared to those from OsiriX and ScanIP.Data are presented as means, standard deviations, and 95% confidence intervals for each software package and repetition. Mean differences and standard deviations between the four groups are also presented.Statistical analyses were run on all variables of interest: ACS, MCS, BCS, GCS, EDV, ESV, and LVEF. Paired t-tests were used to compare means, either between ScanIP and OsiriX or between reader contouring repetitions. T-tests pairing OsiriX repetition one with OsiriX repetition two, and pairing ScanIP repetition one with ScanIP repetition two were performed to assess within-software variability. T-tests pairing OsiriX repetition one with ScanIP repetition one, and OsiriX repetition two with ScanIP repetition two were performed to assess inter-software package differences. A p-value of 0.05 was the desired level of statistical significance, however, as several parameters were being assessed, Bonferroni corrections were performed to decrease the likelihood of type I errors , yieldinBland-Altman analysis was performed to evaluate the variation and bias in the endocardial perimeter length measurements across all the readers, with limits of agreement defined at 95%. The Bland-Altman analysis data points were split into test repetitions when comparing ScanIP to OsiriX (test 1 and test 2). As CVI42 had only one test repetition, the assumption was made that CVI42 could be considered 100% repeatable in the delineation of the endocardial contours, as they were generated automatically. As such, the CVI42 data was treated as both the first and second test repetitions for comparison with the first and second test repetitions in ScanIP and OsiriX. All statistical analyses were performed using Stata  and MATLAB.After visual assessment of all contours from all readers by one expert reader, no unacceptable contours were found in ScanIP or OsiriX. It was also noted qualitatively that the locations of large disagreement between readers were most noticeable at the apex. CVI42 performed well for the porcine data, although it is not currently validated for use in animal models. Manual alteration of these automatically derived contours was performed when required, mainly in the apex. It was also found that CVI42 had not accurately identified the time points corresponding to ES and ED from the images, so these were judged by one expert reader.Means of each variable, for each group,  and their standard deviations are presented in The magnitude of the standard deviation of a variable relative to the corresponding mean for a particular software and attempt gives an indication of the reproducibility of that variable between users. For instance, the reproducibility between users for the strain variables MCS, BCS, and GCS was good, with the standard deviations (SD) for these three variables being less than 10.4% of the mean value for all groups . The repPaired means, and mean differences between the four groups and their standard deviations, are presented in The results for all variables from the CVI42-based method lay within the same ranges for the equivalent variables when calculated using OsiriX Figs  & 4A–4C.Investigation of the paired t-tests showed that EDV had highly statistically significant differences between the two software packages for both sets of repetitions . Systematic errors present in the absolute metrics were ‘cancelled out’ in the relative metrics making them repeatable across software packages. This occurred as, on average, if a user had higher EDV values they also had larger ESV values, and the same was true for smaller ESV and EDV values for a given user. When calculating LVEF ((EDV-ESV)/EDV), the stroke volume (EDV-ESV) remained approximately consistent, whether ‘over’ or ‘underestimating’ the volumes. In this manner, the relative metrics LVEF  were more repeatable as the systematic ‘over’ or ‘underestimation’ was cancelled out.This effect was particularly strong for the global metrics GCS and LVEF, in which differences for individual contours cancelled out over the whole ventricle. For MCS and BCS no statistically significant differences were found at either the software or attempt level, suggesting there was no bias in the mean values. For ACS, a difference between OsiriX test two and ScanIP test two was found , together with the relatively small standard deviation of the mean differences in comparison to the paired groups means showed that intra-software repeatability was higher than inter-software repeatability  which isClearly certain results were affected by the software package, most noticeably with Bland-Altman analysis showing there were systematic differences between contour lengths measured in ScanIP and OsiriX, with a mean bias of -1.9 cm to ScanIP , and conEDV and ESV are used as absolute measures, as well as measures of temporal change, to guide pharmacotherapy, device implantation, and surgical intervention . Large dIt is also possible that the context in which the images were viewed, contributed to differences in manual contour placement. In OsiriX, the same cine-slice was seen through 16 points in the cardiac cycle, whereas in ScanIP all slices from one time-point were seen in sequence. It has been noted elsewhere that the context of an image, either viewing one slice through time (2D), or considering 3D methods segmenting multiple slices a volume, changes the resultant perception of the contour whether contouring is done manually or automatically .There are limitations to this study. Only one porcine left ventricle was investigated, however the total number of image stacks (16) and individual images (144) that were analysed is comparable to other studies , 11. As The sample of readers (n = 7) was small, however, this is comparable to the number of readers in similar studies , 11 and This study investigated the repeatability of ventricular circumferential contours, and their derived volume and strain metrics, for a porcine heart failure model. Two software packages were used by seven readers and on two separate occasions, and a further automated software was also tested on one occasion. No statistically significant difference in circumferential strain measurements , or relative volume (LVEF), were found , despite systematic biases in contour length between the software packages OsiriX and ScanIP. However, these biases did result in differences in EDV and ESV values between software packages. Differences between software packages were also found for ACS due to the difficulties in capturing the shape of the apex. Intra-software differences for variables were found to be lower than inter-software differences. These results will benefit future studies that use contouring methods and volume & strain metrics derived from these contours, by providing associated error estimates, guidance to improve repeatability, and aiding comparison of results with other studies.


Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of  The primary function of the heart is to pump blood throughout the body via the circulatory system, delivering oxygen and nutrients to the tissues, and removing carbon dioxide and waste simultaneously. The heart is composed of four chambers, each separated by uni-directional valves, that synchronously work to cycle blood through the systemic and pulmonary circulation. The function of the heart relies on the action of contractile cells, known as cardiomyocytes, specialized conducting cells that facilitate coordinating rhythmic contraction, extracellular matrix that provide mechanical support, as well as veins, arteries, and microvasculature to supply blood to the working muscle. Importantly, the heart vascular network, known as the coronary circulation, maintains perfusion of myocardial tissue with hemodynamics that are out-of-phase to the systemic circulation .Almost all diseases of the heart involve an immune response with a high degree of spatial and temporal regulation that is orchestrated by functionally varied leukocyte populations. This dynamic nature of leukocytes, coupled with the fact that contractile motion at the cell and tissue level is an essential function of the heart, present a unique and challenging environment to study the immune system. Given that the heart is highly specialized and metabolically active, containing the highest oxygen consumption rate per unit of tissue in the human body , it is hex vivo perfused heart, cell dissociation or isolation and analysis such as flow cytometry). These approaches have led to enormous progress in our fundamental understanding of leukocyte biology in the heart over the past century. However, these approaches fail to capture simultaneous and interacting processes at the cell-scale. The study of leukocyte dynamics in the majority of organ systems, including brain  or use reduced preparations  occurs when there is a deficiency in the relaxation and filling capacity of the heart chambers (diastolic dysfunction) while maintaining a normal ejection fraction , 56. HFpA major advantage of using intravital microscopy to study cardiac disease, is the ability to visualize fast and dynamic behaviors of leukocyte sub-types that can influence tissue repair. Such interactions have been described in other tissues, including neutrophil mediated dismantling of damaged vessels and the creation of channels for regrowth in liver , and patApproaches to imaging the rodent heart at cell resolution need to consider three separate aspects—surgical access, image acquisition, and pre- or post-processing of images. Previous approaches to imaging leukocytes in the heart at cell resolution used varied strategies to address these three aspects, and are summarized in Figure 3A). In combination with transgenic cell ablation studies (diphtheria toxin receptor targeted expression), this technique has been used to demonstrate that tissue-resident, CCR2-expressing cardiac macrophages promote monocyte recruitment after transplantation  and is not well-suited for studying leukocyte dynamics that involve fine cellular features. Image resolution and achievable depth rely on many factors including acquisition speed and fluorescence excitation source (multiphoton or single photon excitation), however the approximate lateral and axial resolution of GRIN lenses are 1 and 12 μm, respectively, with ≤ 0.6 NA objectives, compared to sub-micron resolution with approximately 1.0 NA objectives  interaction of two photons with a fluorescent molecule to excite fluorescence  microscopy, because it provides fluorescence imaging with microscopic resolution at depth in intact tissues . In contrescence . The emi5 μm/min), and the leukocytes remain in the field of view over multiple heartbeats, imaging can be gated to limit acquisition during stable portions of the cardiac cycle , enables structural imaging and some measurements , 78, 88.in vivo imaging using laser scanning microscopy through a single cardiac cycle using this method (The key challenge in active motion compensation is generating a feedback signal that indicates the motion of the tissue. With an appropriate feedback signal, the motion of the tissue can be matched by moving either the focal plane of the microscope, such as the microscope objective using actuators for fast motion, or moving the mouse using the microscope stage for slower motion. The feasibility of these methods was shown in early approaches using stroboscopic illumination of epicardial microvessels in combination to image, and a computer-controlled electromechanical micromanipulator that moved a micropipette in synchrony with the heart to capture phasic changes in microvascular pressure in the left ventricle of cats . To genes method . Early ss method . A thirds method  and corrs method . ReflectThe current maximum 2PEF imaging depth within the mouse heart is ~200 μm, limiting visualization to functions within the epicardial layer. Although this provides a good first step to studying immune cell dynamics in the heart, there are both structural and functional differences within deeper layers of the heart. Structurally, specialized conduction fibers, known as Purkinje fibers, are located in the subendocardial space, and larger coronary arteries lie deeper within the mouse myocardium. Given the recent discovery that resident macrophages facilitate electrical conduction in the heart (Recent advancements in mid-infrared laser sources now make deeper imaging feasible using three-photon microscopy . By utilDeeper imaging is also impaired by sample-dependent optical aberrations. The use of an adaptive optical element such a deformable mirror can greatly improve multiphoton microscope performance –98. By pOther organ systems have benefitted from chronic implantable windows to enable time-lapse imaging, making it possible to follow the same regions at the micro-scale during disease progression. This includes the cranial window , 99, 100in vivo environment which can be improved with intravital, multiphoton microscopy.With continued advancements in technology to access and image the murine heart, we are sure to make strides toward increasing our understanding of leukocyte dynamics during diseases of the heart. Targeting the immune system with therapies has proven to be successful in other diseases, especially immunotherapies for cancer . HoweverDS conceived the idea for the review, performed literature search, and wrote the manuscript. NA-R wrote the active motion compensation section. ML wrote the deeper imaging section. NN and WC provided direction for the manuscript. All authors contributed to reviewing and editing 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.


Right ventricular (RV) function is impaired in patients with Tetralogy of Fallot due to right bundle branch block and volume overload by pulmonary regurgitation. As a consequence, paradoxical septal motion develops. Daily practice suggests left ventricular (LV) function is also reduced. Paradoxical septal motion could be one of the mechanisms causing diminished LV function.The purpose of this CMR study was to measure paradoxical septal motion and its effect on LV function in patients with Tetralogy of Fallot.Twenty nine patients  with Tetralogy of Fallot with a mean age of 29 ± 10 years were included. MR imaging was performed on Siemens Avanto 1.5 Tesla scanner. Data-analysis was performed with the MR Analytical Software System  to measure end-diastolic (EDV) and end-systolic (ESV) volumes and calculate ejection fraction (EF) for both ventricles. Paradoxical septal motion was quantified in end-diastole by the curvature of the septum, defined as 1 divided by the radius of the curvature and a negative value corresponds to septal motion towards the LV cavity. MR phase-contrast flow quantification was performed to assess pulmonary regurgitation. ECG analysis for QRS duration was performed to diagnose right bundle branch block. Pearson correlation tests were done to assess correlations between variables.RV EDV and ESV were 227.0 ± 53.3 and 148.4 ± 47.7 ml, respectively. LV EDV and ESV were 119.4 ± 24.5 and 55.9 ± 17.9 ml, respectively. RV function was reduced (EF = 35.3 ± 10.2%) and LV function was also mildly reduced (EF = 53.5 ± 9.5%). All patients had reduced curvature (0.14 ± 0.08), but in only 1 case the septum moved towards the LV cavity. Mean pulmonary regurgitation fraction was 43.6 ± 17.6%. Mean QRS duration was 144 ± 30 msec. Significant correlations were found between curvature and pulmonary regurgitation fraction , pulmonary regurgitation fraction and RV EDV , RV ESV volumes and QRS duration  and between QRS duration and RVEF . No significant correlations could be found concerning LV function.Patients with Tetralogy of Fallot have dilated right ventricles with impaired function due to pulmonary regurgitation and right bundle branch block leading to paradoxical septal motion. Paradoxical septal motion was seen in all patients with Tetralogy of Fallot. Although in this population with only mildly reduced LV function and mild paradoxical septal motion a correlation between both could not be demonstrated, further research in a more heterogeneous population is warranted.


Ejection fraction (EF) is a measurement of heart function that reflects the portion of pumped out blood from the filled left ventricle per each heartbeat. The current study aimed to investigate the prevalence of left ventricular thrombus in patients with EF lower than 35% by using Transthoracic Echocardiography (TTE).st to December 31st 2017 at the Military Cardiac Centre in Sana’a, Yemen.In this prospective study, 82 cardiac patients underwent TTE procedure in order to assess the presence of left ventricular thrombus (LVT) from January 1Out of 82 patients enrolled in this study, the mean age was 49.13 ± 14.8 years and 87.8% were male. The mean of EF was 31.16% and LVT was found in 6.1%. The spontaneous contrast was seen in 25.6% of patients indicating strong relationship with low EF (p < 0.001). Among patients with low EF, ischemic heart disease (IHD) was identified in 50%, hypertension in 30.5%, diabetes mellitus (DM) type 2 in 23.2%, and hyperlipidemia 12.2%. Exactly 80% of LVT were detected in IHD patients with dilated cardiomyopathy (DCMP) and 80% of detected LVT were apical in site.Cardiac patients with low ejection fraction developed left ventricular thrombosis, and most of the affected patients were ischemic heart disease with dilated cardiomyopathy. Interestingly, spontaneous contrast was found high significantly in these patients, which may reflect the continuous process of thrombus formation. Ejection fraction (EF) is the portion of pumped out oxygen-rich blood from a filled left ventricle to the aorta per each heartbeat. It measures the pumping ability and function of the heart. The cutoff of normal EF value is poorly-defined, however 50% of volume of blood in the left ventricle (LV) is considered as the lower boundary of normal.11This study is intended firstly to assess the incidence of LV thrombus in patients with low EF, and secondary to assess the relationship between common habits in Yemeni population such as khat chewing, smoking, shamma using with reduced EF of the heart. Khat is a plant that is chewed for its stimulant action which is popular in Yemen.12Shammah is a snuff of smokeless tobacco that is used in the middle east especially Saudi arabia, Sudan and Yemen.st to December 31st 2017 and met the inclusion criteria of this study were collected. The inclusion criteria were:This cross-sectional, prospective study was conducted at Military Cardiac Center in Sana’a, Yemen. Data of 82 patients who underwent echocardiography in the outpatient clinic from January 1Patients with EF less than 35% and more than 12 years old age.The Exclusion criteria were:Patients with rheumatic heart disease (RHD) who were under anticoagulant drugs.Patients with atrial fibrillation (AF).This study was approved by the Cardiac Center Research ethics committee. Full institutional ethical approval was obtained before beginning the study. Verbal consent from each patient was also obtained before examination. Confidentiality of all patient information was assured.A pre-tested questionnaire was prepared to include all relevant information. Researcher through direct interview filled the questionnaire with the patients. Explanation of the study purpose was explained to each patient, and consent was obtained. The following variables are included: sociodemographic, clinical history for cardiac disorders causes, risk factors such as hypertension (HTN), diabetes mellitus (DM), smoking, hyperlipidemia, drug in-use and disease duration, severity and complications.Physical examination was performed for each patient looking for signs of congestive heart failure (CHF) as increase jugular venous pressure (JVP), hepatomegaly, lower limb edema. Investigations were done included complete blood count (CBC), cardiac enzymes, liver function tests (LFT), renal function test (RFT), electrocardiogram (ECG), and chest x-ray (CXR). Patients with EF less than 35% underwent another TTE to assess the presence of left ventricular thrombus (LVT).The ECHO procedure was performed with the same ultrasound imaging machine  using a standard protocol. All selected patients were examined by the same cardiologist with 25 years’ experience in echocardiography.LVT was defined as a hyperechoic mass with definite margins adjacent to the myocardium on multiple plane views through the cardiac cycle. Regional wall motion abnormalities were considered present whether hypokinesia, akinesia or dyskinesia were observed in at least two segments of the left ventricular wall. Left ventricular function was assessed by echocardiographic examination. LV end diastolic and end-systolic volumes and ejection fraction were determined from apical 2 and 4 chamber views using Simpson`s biplane formula according to recommendation of the American Society of echocardiography: EF=(EDV-ESV)/EDV.2EF: Ejection fractionEDV: End Diastolic Volume (ml)ESV: End Systolic Volume (ml)Data analysis was performed using SPSS version 21 (IBM). Data are presented as frequency and percentage for continuous variables and mean ± Standard Deviation (SD) for descriptive variables. Quantitative data were analysed using One sample student t-test, and qualitative data were compared using Chi square test. P-value was assumed to be significant when P < 0.05.In total, 82 patients who had EF <35% included in this study. The mean age at diagnosis was 49.13 ± 14.8 years; 87.8% were male and 12.2% were female. Echocardiographic findings showed the mean of EF was 31.16% and LVT was present in 6.1%, which is statistically not significant (p = 0.61). The spontaneous contrast picture was seen in 25.6% of patients (p < 0.001) -. Among pKhat chewer and smokers were the most common popular habits in the patients of low EF then Khat chewing only but no significant relationship (p=0.96) . ExactlyLV thrombus is a serious complication of heart failure. In this study, we assessed the prevalence of LV thrombus in patients with heart failure with low EF. Disertori et al., reported that patients with LVEF less than 35% have a high risk of death due to evolution of HF.22It was a single-center study and the sample size was limited by 82 patients.Cardiac patients with low ejection fraction developed left ventricular thrombosis and most of the affected patients were of dilated cardiomyopathy. Interestingly, spontaneous contrast was significantly high in patients with low ejection fraction which is a continuous process of thrombus formation. This make starting anticoagulant therapy in this group of cardiac patients is an individual decision based on patient and physician discussion.NMA: Conceptualization, and data collection.ZAM: data analysis.SAA: writing the original draft and is responsible for integrity of research.FHA: manuscript review & editing.EF:ejection fraction,ASE:American society of echocardiography,EACVI:European association of cardiovascular imaging,LV:left ventricle,LVEF:left ventricular ejection fraction,HFpEF:heart failure with preserved ejection fraction,HFrEF:heart failure with reduced ejection fraction,MI:myocardial infarction,DCMP:dilated cardiomyopathy,AF:atrial fibrillation,TTE:Transthoracic echocardiography,TEE:trans-esophageal echocardiography,RHD:rheumatic heart disease,HTN:hypertension,DM:diabetes mellitus,CHF:congestive heart failure,JVP:jugular venous pressure,CBC:complete blood count,LFT:liver function tests,RFT:renal function test,LVT:left ventricular thrombus,ECG:electrocardiography,LVT:left ventricular thrombus,EDV:end diastolic volume,ESV:end systolic volume,SPSS:statistical package for the social sciences,IBM:international business machines,SD:standard deviation.