Echocardiographic evaluation of right ventricular (RV) function is a challenge due to the complex anatomy of the RV. Several transthoracic echocardiographic methods have been suggested for the quantification of RV function. However, many of the parameters are time consuming and need dedicated hardware and software. We suspected that the majority of the established markers are not used on a wide basis. In a multinational online survey, we evaluated the use of current clinical standards for the quantification of RV function in clinical practice. Through the network of an Ultrasound Online Teaching Platform, echocardiographers were invited to participate in an open online survey. The participants were asked about the parameters  they used in clinical practice. A total of 1150 participants from 109 countries completed the survey. Only eyeballing (72%), TAPSE (69%), and S′ (31%) were commonly used in clinical routine. These methods were applied significantly less common in low-income economies when compared to high-income economies. Twenty-three percent of all participants stated to rely on eyeballing only, when evaluating RV function in clinical routine. New technologies, such as global longitudinal strain (3%) and 3D echocardiography (1%) were rarely applied independent of region and economic strength. Eyeballing and TAPSE are the most widely used methods in echocardiography for the assessment of RV function. Although advanced parameters such as longitudinal strain and 3D echocardiography were shown to be highly accurate, they are rarely used in clinical routine. Correct assessment of right ventricular (RV) function is an essential part of every transthoracic echocardiographic (TTE) examination. It plays an important role in the diagnosis and management of many diseases and conditions.TTE evaluation of RV function (RVF) remains challenging due to the complex anatomy of the RV. In contrast to the left ventricle, there is no exact geometric model for the RV that would permit volumetric measurements based on 2D echocardiographic images. Right ventricular ejection fraction (RVEF) derived from cardiac magnetic resonance imaging (MRI) remains the gold standard for functional RV assessment.Several parameters have been suggested for echocardiographic evaluation of the RV. The simplest and most commonly used parameter is visual assessment. The reliability of visual quantification has been evaluated in previous studies , 2; the Guidelines for TTE assessment of the right heart demand the use of at least one quantitative parameter in addition to visual gradation . SeveralHowever, we lack knowledge about the gap between guideline recommendations and the actual assessment of RV function in clinical practice. For the present study, we performed a multinational online survey to determine those methods most frequently used by echocardiographers for the quantification of RV function.Through the network of an Ultrasound Teaching Platform (123sonography.com), echocardiographers were invited to participate in an open online survey. All answers received between April 1 and July 31 2017 were analyzed. The study was approved by the ethics committee of the Medical University of Vienna (EK #1288/2016). The study protocol is in conformity with the ethical guidelines of the Declaration of Helsinki.The participant’s name and the name of the institution were not included in the questionnaire. Baseline demographic data were obtained, such as age, the country in which participants practiced, profession, work setting , and experience in TTE.Participants were asked about the parameters they used for the assessment of right ventricular function. They answered two questions, (1) which methods of RV quantification they apply occasionally as part of their individual diagnostic repertoire, and (2) which methods they apply in daily clinical routine on most of their examinations.Common methods  were given as possible answers in a multiple-choice format. When applicable, the respondents could mention other techniques in a free-response box.The main objective of this study was to determine which methods for the evaluation of right ventricular function are applied in daily clinical practice in a real-life sample of echocardiographers from all over the world.The World Bank divides the countries of the world into four income classes depending on the gross national income per capita ranking (GNI). Low income economies were defined as those with a GNI of $1005 or less in 2016. Lower middle-income economies were defined as those with a GNI between $1006 and $3955. Upper middle-income economies were defined as those with a GNI between $3956 and $12,235. High-income economies were defined as those with a GNI of above $12,235 .According to the World Bank, we divided the world into the following seven regions to analyze regional differences: East Asia and Pacific, Europe and Central Asia, Latin America and the Caribbean, Middle East and North Africa, North America, South Asia, and Sub-Saharan Africa.2 test and Fisher’s exact test as appropriate. A p value of < 0.05 was considered statistically significant. SPSS Version 24  was used for all analyses.Categorical data are presented as absolute numbers and percentages. Comparison of descriptive data between groups was performed using A total of 1150 participants from 109 countries completed the survey. Sixty-three percent of the participants were from high-income economies, 37% were from lower income economies . Thirty-nine percent were cardiologists, 23% were sonographers, and 19% internal medicine specialists. Twenty-eight percent worked in university hospitals and 48% in non-tertiary hospital settings. Nineteen percent of the participants described themselves as beginners in echocardiography, 45% as moderately advanced, 29% as advanced, and 7% as experts. Eleven percent of the participants worked in the United States of America, 7% in Germany, 5% in Austria, 5% in India, and 5% in the United Kingdom. Full descriptive statistics are shown in Table Seventy-nine percent of the participants (n = 906) stated that they used eyeballing to assess right ventricular function. TAPSE was used by 82% of the participants, S′ by 48%, FAC by 26%, and RV-GLS by 11%. RIMP, 3D echocardiography, and dp/dt was used by 7, 4, and 6% respectively. Twenty percent of the participants used one method  to assess right ventricular function, 29% used two methods, and 51% three or more methods.Only eyeballing 72%), TAPSE (69%), and S′ 31%) were mentioned as frequently used methods. Thirty-nine percent of the participants applied only one method  to assess right ventricular function in daily clinical practice, 35% used two methods, and 26% more than two methods. Eyeballing was applied as a single parameter by 23% of the participants , S′ (p = 0.01), FAC (p = 0.004), 3D-EF (p < 0.001), and RV-GLS (p < 0.001) were significantly more often mentioned as part of the individual diagnostic repertoire in the university setting. Those practicing at universities also used significantly (p < 0.001) more methods to assess RVF. However, the analysis of the respondents’ answers concerning daily clinical routine revealed no significant differences between the two settings (see Table Sixty percent of the respondents stated that they regularly examined patients with severe pulmonary hypertension (PH). A significant (p < 0.001) difference was registered between the PH-center and the non-PH-center group with regard to the number of methods used in clinical routine. At PH centers, 31% of the respondents reported the use of three or more parameters  to assess right ventricular function in clinical routine, as opposed to 16% at non-PH-centers. All imaging parameters were used more frequently at PH centers; the difference was statistically significant for S′ (p < 0.001), FAC (p = 0.001), and RIMP (p = 0.006). Full data about imaging at PH centers are summarized in Table Eyeballing was used significantly more frequently by non-cardiologists, while TAPSE was used significantly more often by cardiologists. The differences in all other parameters did not reach statistical significance (data not shown).Results were compared between high income economies on the one hand and low income, lower middle-income, and upper middle-income on the other hand. There were significant differences between the two groups. While eyeballing, TAPSE, S′, and FAC are the four most commonly applied parameters in both groups, all of the methods were applied significantly more often in the high-income economies as opposed to the lower income economies (data not shown).Eyeballing 72%) and TAPSE (69%) are the most commonly applied parameters in all regions of the world. TAPSE is used less frequently in the Middle East and North Africa (49%) and in Sub-Saharan Africa (44%), S′ is used less frequently in South Asia (17%) and in Sub-Saharan Africa (13%). Furthermore, there were differences in the number of parameters applied for RV function gradation. Eyeballing as a single parameter was used in less than 20% of the participants from East Asia and Pacific, Europe and Central Asia, and Latin America and the Caribbean, and by 30% of those from North America, by 37% by those from the Middle East and North Africa, by 28% of the participants from South Asia, and by 48% of those from Sub-Saharan Africa  on a global scale. Eyeballing (79%) and TAPSE 82%) were the most commonly used parameters, followed by the tissue Doppler parameter S′, which was applied by 31% of respondents. All other parameters were used infrequently. Notably, the novel parameters such as RV-GLS and 3D echocardiography have not yet been established in clinical routine—neither in the academic setting nor in non-tertiary hospitals. Guidelines recommend that, in addition to eyeballing, at least one other parameter should be used to assess right ventricular function. Eyeballing alone is not sufficient for the quantification of right ventricular function [% were thThis gap between current scientific knowledge and actual real-world echocardiography deserves attention and should be addressed by the echocardiographic community.TAPSE and S′, as measures of longitudinal motion of the basal lateral free wall of the RV, have several limitations. They only reflect the motion of the basal segments of the RV and may lead to a significant over- or underestimation of right ventricular function in the presence of regional differences in contraction. Displacement of the apex, resulting in passive motion of basal right ventricular segments, may result in the overestimation of RVF using TAPSE. TAPSE does not reflect true right ventricular function in many clinical conditions, including patients who have undergone cardiac surgery, those with Tetralogy of Fallot, and those with tricuspid regurgitation –25. AparFAC is a quantitative parameter that reflects volume change during contraction. Nine percent of our respondents used this parameter in clinical routine. The meager use of this procedure is surprising in view of the fact that FAC can be measured with every echo machine; no Doppler recordings or additional software is needed. Moreover, the clinical value of the parameter has been confirmed in several studies –14.Eleven percent of those who worked at PH centers used FAC in clinical routine, as opposed to 5% at non-PH centers (p = 0.001). The significantly more frequent use of FAC at PH centers indicates the valuable additional information provided by this method, especially in patients with severely dilated right ventricles and reduced FAC, but preserved longitudinal contraction. The rare use of the method at non-PH centers may be explained by the additional time taken to perform the procedure, and uncertainty about correct tracing of the right ventricular endocardial border.RIMP and dP/dt reliably predict reduced right ventricular function. Among our echocardiographers, both methods were used by a mere 2% in clinical practice. Dp/dt and RIMP require the recording of special Doppler signals which might not be a part of the standard imaging protocol at many echocardiography laboratories. Acquisition is not as straightforward as the simple measurement of distance (TAPSE), area (FAC), or peak velocity (S′). The infrequent use may be related to difficulties in understanding physiologic and pathologic principles as well as the additional time taken for the assessment.As expected, new and advanced parameters of RV quantification are used more frequently in the university setting than in the non-university setting. However, the difference did not persist in daily clinical routine.Longitudinal strain of the free lateral wall of the RV is a promising new technology to asses RVF. It measures the deformation of the myocardium and permits regional as well as global assessment of the free lateral wall of the right ventricle. RV longitudinal strain is occasionally used by 11%, and regularly used by 3% in clinical routine. Strain analysis requires dedicated software, good image quality, ECG tracing, and additional time for analysis. The method is easy to use but expensive. Our data showed that although strain analysis has been available for several years, it is rarely used in daily clinical practice.3D volume assessment yields end-diastolic and end-systolic volumes as well as ejection fraction and stroke volume. It is not affected by regional variations in RV contractility or passive right heart motion. 3D echocardiography was occasionally used by 4% of the respondents and regularly used by 1% in clinical routine. 3D echocardiography requires dedicated software and hardware, which may not be available at many centers. Advanced and expensive imaging systems are available at university hospitals and usually do not exist in smaller hospitals or medical offices. Thus, it was no surprise to note that 3D echocardiography is available more frequently in university hospitals than in other settings . Time and experience also play a role. A multi-beat 3D dataset of the right ventricle including offline analysis requires significantly more time than TAPSE and S′ and—in addition—the skill to acquire loops with very good image quality. It is used significantly less often by beginners than by advanced echocardiographers.RV-GLS and volume assessment of the RV by 3D echocardiography are promising new tools for the assessment of right ventricular function. The present investigation showed that significant effort will be needed in the future to implement these methods in daily routine. Echocardiographers will have to be trained in, and convinced of, the use of these parameters. Recent promising developments in 3D technology have accelerated and improved the acquisition and interpretation of RV-3D datasets by introducing a single-beat method for RV volume analysis . The indEyeballing and TAPSE are the most commonly applied parameters in all regions of the world. TAPSE is used less frequently in the Middle East and North Africa and in Sub-Saharan Africa, S′ is used less frequently in South Asia and in Sub-Saharan Africa.There were significant differences between high-income and lower income economies. While eyeballing, TAPSE, S′, and FAC are the four most commonly applied parameters in both groups, all of the methods were applied significantly more often in the high-income economies as opposed to the low-income economies. As TAPSE and FAC are parameters solely based on 2D measurements which can be performed by every echo machine, higher costs for more sophisticated imaging systems do not explain the observed differences. The differences can be explained through less reachable training in echocardiography, different health care systems with less capacities for the diagnosis and treatment of complex diseases and therefore more focused examinations.Data were collected anonymously through an online survey. Therefore, accuracy of the individual responses could not be verified; we had to rely on the honesty of the participants. However, it may be assumed that the results overestimate true clinical practice and not vice versa. The anonymous nature of the survey permitted a large number of persons to participate in the study. The study participants were recruited from the users of an online teaching platform, which may have signified a selection bias in favor of echocardiographers interested in continuing medical education and new technologies. Again, it may be assumed that the results overestimate the application of parameters of right ventricular function.Due to the mode of recruitment this study analyzes a heterogenic sample. Participants were contacted through the network of an online teaching website, including social media platforms. Therefore, the rate of participation in relation to those contacted cannot be determined. Thus, the analyzed group is not a representative sample but rather a large real-life group of echocardiographers. Considering the homogenous results showing a similar distribution of applied methods in all countries, professional groups, and levels of expertise, it can be assumed that the presented data nevertheless represents the real world daily clinical practice.More information on the reasons for the meager use of modern parameters would have been of great interest. This survey focused on the assessment of the current state of clinical practice, further questions regarding reasons for certain answers were not asked. The nature of the short questionnaire allowed for a large number of participants.Eyeballing and TAPSE may lead to misinterpretations of right ventricular function. However, the two parameters are applied most frequently for the quantification of right ventricular function. New methods such as 3D echocardiography and RV-GLS are employed by some physicians, mainly in the university hospital setting. Although these advanced parameters were shown to be highly accurate, they are rarely used in daily clinical routine.Our investigation revealed that the assessment of right ventricular function by health care professionals does not conform to current guidelines. This is especially true of the non-academic setting. It therefore remains the task of the echocardiographic community to continue teaching about the importance of thorough assessment of right ventricular function.


Functionally, PAH ECFC had higher proliferative rates compared to control in vitro, although inter-patient variability was high. ECFC proliferation was inversely related to RV end diastolic volume , but not pulmonary vascular resistance. Tube formation-ability was similar among donors. Normal and highly proliferative PAH ECFC were transplanted in pulmonary trunk banded rats. While no effect on hemodynamic measurements was observed, RV vascular density was restored. In conclusion, we found that ECFC outgrowth associates with high clinical severity in PAH, suggesting recruitment. Transplantation of highly proliferative ECFC restored myocardial vascular density in pulmonary trunk banded rats, while RV functional improvements were not observed.In pulmonary arterial hypertension (PAH), lung-angioproliferation leads to increased pulmonary vascular resistance, while simultaneous myocardial microvessel loss contributes to right ventricular (RV) failure. Endothelial colony forming cells (ECFC) are highly proliferative, angiogenic cells that may contribute to either pulmonary vascular obstruction or to RV microvascular adaptation. We hypothesize ECFC phenotypes  are related to markers of disease severity in a prospective cohort-study of 33 PAH and 30 healthy subjects. ECFC were transplanted in pulmonary trunk banded rats with RV failure. The presence of ECFC outgrowth in PAH patients was associated with low RV ejection fraction, low central venous saturation and a shorter time to clinical worsening (5.4 months (0.6–29.2) vs. 36.5 months (7.4–63.4),  In pulmonary arterial hypertension (PAH) angio-proliferation, inflammation and vasoconstriction of the small arteries of the lungs progressively increase pulmonary vascular resistance (PVR) ,2. The rIn response to tissue damage, inflammation or hypoxia, endothelial progenitor cells (EPC) are recruited to the peri-vasculature following a gradient of cytokines and chemokines. EPC arise from the mononuclear cell fraction (MNC) of blood, and as a class EPC contain several cell populations, including late outgrowth endothelial colony forming cells (ECFC) . As oppoPrevious studies have shown altered numbers of circulating EPC in PAH and have also suggested abnormal ECFC behavior ,13,14,15Previous research has suggested that transplantation of autologous EPC has a beneficial effect on hemodynamic and functional parameters of PAH patients ,17, and In this prospective cohort study, we aimed to investigate the clinical significance of ECFC (dys-) function in PAH. ECFC were isolated from 33 patients with PAH and from 30 healthy control subjects. Blood outgrowth, proliferation and tube formation were quantified. Our hypothesis was that because ECFC participate in pulmonary vascular and/or right heart remodeling, ECFC functional read-outs will correlate with clinical characteristics and markers of disease severity. PAH ECFC functional read-outs were compared to clinical data, including right heart catheterization, cardiac magnetic resonance (CMR) imaging and time to clinical worsening (TTCW) over a period of 3 years. Because ECFC proliferative capacity correlated inversely with RV function but not PVR, we subsequently transplanted PAH ECFC in rats with chronic RV failure induced by pulmonary trunk banding (PTB) to determine effects on RV function and vascular density.p = 0.032, see ECFC were isolated from 33 patients with PAH and 30 healthy control subjects. Out of 33 PAH patients, 21 had outgrowth of at least one ECFC colony (64%). Likewise, 20 control subjects gave outgrowth to ECFC (67%). All ECFC-lineages expressed mature endothelial cell markers . The quap-interaction = 0.018, t = 8 days , indicating patients with highly proliferative ECFC were more likely to have preserved RVEDV C. No relECFC were seeded on fibrin matrices and stimulated with tumor necrosis factor (TNF-α), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) to induce tube formation A. AveragTwo PAH ECFC donors were selected for ECFC transplantation to rats: one donor with a highly proliferative and tube formation ability, and one donor with a normal proliferative and low sprouting ability. Highly and normally proliferative PAH ECFC were transplanted to rats with chronic RV failure induced by PTB. Two weeks after PTB, all animals showed signs of RV failure on echocardiography. Tricuspid regurgitation was present in all PTB animals, but not in sham animals. Tricuspid annular plane systolic excursion (TAPSE) was significantly lowered in PTB animals, as were stroke volume and cardiac output . At eval2, and a shorter TTCW during 3 years of follow up. Somewhat paradoxically, in patients whose blood yielded ECFC, a high proliferative ability of these cells was related to preserved RV function, as indicated by a low RVEDV. Transplantation of highly proliferative ECFC to rats with severe RV failure induced reversal of vascular rarefaction but did not result in an improved RV function.To improve the understanding of the clinical significance of ECFC (dys-)function in PAH, we related ECFC function  to markers of lung vascular remodeling and RV function. Our finding that blood-outgrowth of ECFC is more prominent in PAH patients with a severe clinical phenotype suggests that clinical severity is related to an increased recruitment of ECFC from the bone marrow. Accordingly, the presence of outgrowth was associated with lower RVEF, lower SvOIn our study, the blood of two thirds of PAH patients gave outgrowth to ECFC colonies. While selective outgrowth of ECFC in PAH was reported previously ,14, our How disease severity influences the actual presence of ECFC in the bloodstream remains elusive. Recruitment of ECFC (and other EPC) from the bone marrow to the blood stream occurs upon release in tissues of bone-marrow targeting molecules, such as GM-CSF and SDF-1, in response to damage, inflammation or hypoxia ,32. It iWe observed substantial inter-donor variation in proliferative rates of ECFC from patients with PAH. This finding confirms previous studies from Toshner et al.  and AsosWhile the proliferative rate of ECFC had no relationship with pulmonary vascular remodeling or PVR in our cohort of PAH patients, we found a moderate inverse relationship between proliferation rate of PAH ECFC and RV dilatation. One possible explanation for this finding is a direct hypertrophy and growth suppressing effect of inflammatory cytokines on both the RV and ECFC. For example, serum levels of TNF-α and IL-6, which are increased in PAH, have been attributed with cardio-depressive effects ,44,45 asAlthough we found no relationship between ECFC proliferative capacity and PVR, it remains possible that highly proliferative angio-proliferative ECFC contribute to pulmonary vascular remodeling and plexiform lesion formation. Accumulation of c-kit positive cells  and SDF-An imbalance in oxygen supply by cardiac vessels and oxygen demand by cardiomyocytes may lead to myocardial hypoxia, apoptosis, and contractile dysfunction in the development of hypertrophic cardiomyopathy . EvidencIndeed, we observed that in a rat model of RV failure, transplantation of highly proliferative ECFC enhanced the capillary network in the RV. The fact that this increase in capillary density was not accompanied by an improvement in RV function could indicate that group sizes were too small, or that capillary rarefaction is not critical to the function of the pressure overloaded RV. Alternatively, the enhancement in capillarization could have been insufficient to result in functional improvement at the time of assessment. Whether repetitive transplantations and a longer follow-up would have resulted in more positive effects remains to be determined. In a follow-up study, stereology would be the best option to solidly prove increased myocardial revascularization.In conclusion, we found donor dependent variation in outgrowth, proliferative, and tube formation ability of ECFC derived from patients with PAH. Outgrowth and proliferative capacity were related to markers of disease progression and RV-function. Transplantation of highly proliferative ECFC with tube formation ability reduced capillary rarefaction of the RV myocardium in rats after banding of the pulmonary trunk, although no improvement of RV function was observed. Restoring ECFC angio-proliferative ability could be a target for therapy.WHO group 1.1, 1.2, and 1.4 PAH patients were recruited from the Pulmonary Hypertension clinic at the VU Medical Center, Amsterdam, The Netherlands. Control subjects were healthy volunteers as well as patients undergoing a right heart catheterization for diagnostic reasons but who were found to have normal hemodynamic values. The Institutional Ethical Review Board of the VU Medical Center  reviewed and approved the study . Since the intervention consisted of a single blood draw, no signed informed consent was obligate before 7th of July 2017 by our Institutional Ethical Review Board. After this date, all patients gave informed consent. Comparison of baseline characteristics of PAH patients and control subjects are shown in 2) were obtained from patient files. Magnetic resonance imaging was performed as described previously [Right heart catheterization and pressure measurements were performed as described previously , and meaeviously . RV end Time to clinical worsening (TTCW) was obtained from patient records and defined by the time from ECFC isolation until death, emergency admittance to a hospital for re-evaluation or other cause attributable to PH, a decrease of ≥10% of 6 min walking distance .2, 5% CO2. Culture medium was refreshed every other day. After 3 days, a washing step was performed to remove non-adherent cells. ECFC colonies appeared in culture after 7–28 days and were subsequently re-plated and expanded in a ratio of 1:5 until passage 4–7 was reached in order to perform experiments. Isolated cells were characterized as ECFC by presence of endothelial cell markers CD31  VE-cadherin  VEGFR2  and lack of CD45  using flow cytometry as previously described [ECFC were isolated and cultured as described previously . In shorescribed   was defined by the slope of the proliferation curve, obtained by linear regression of the linear part of the sigmoidal-shaped growth curve covering a minimum of 3 subsequent data points and R2 ≥ 0.97. PAH ECFC that exceeded the average proliferative speed of control ECFC by >2 SD were classified as being “high proliferative” PAH ECFC, while PAH ECFC donors that follow the growth curve of control ECFC were classified as “normal proliferative” PAH ECFC.Proliferation was quantified over a period of 8 days. Primarily, ECFC were seeded in a density of 1000 cells/cm2 on 3D human fibrin matrices and subsequent growth factor stimulation as described previously [Sprouting ability was quantified from ECFC seeded in triplicate in a seeding density of 20,000 cells/cmeviously ,58. Folln = 12) or sham-surgery (n = 2). All animals were anesthetized after which a lateral thoracotomy was performed and the pulmonary trunk carefully separated from the ascending aorta. In PTB animals, the banding was made with a horizon ligating clip applier modified to compress a titanium clip to a pre-set inner diameter of 0.5 mm around the pulmonary trunk.Male Wistar rats  were treated according to the Danish National Guideline, and all experiments were approved by the Institutional Ethics Review Board and conducted in accordance with the Danish Law for animal research . Rats were housed two per cage with free access to water and standard rat chow  in a room with a 12-h light-dark cycle and a temperature of 23 °C. At time of surgery, rats weighed 186 ± 28 g. PTB was performed as described previously by Andersen et al. . Brieflyn = 4), to transplantation with normal proliferative PAH ECFC with little tube formation (n = 4), or to saline injections (n = 4). Animals received transplantations via the tail vein at baseline and after 1.5 weeks after the first transplantation. Prior to transplantation, animals received an heparine bolus i.v. via a venflon in the tail vein, after which 1.5–2.0 × 106 ECFC were administrated suspended in 0.4 mL warm 1% BSA/saline. Lines were flushed with heparinized saline. 2 sham and 2 PTB/saline animals were euthanized 1.5 weeks post sham/PTB procedures. All other animals were euthanized 3 weeks post-sham/PTB procedure. As no difference in hemodynamic, cardiac or anatomic data was observed, the 1.5 and 3 week time point post sham/PTB procedure were combined in order to increase power. All animals (sham and PTB) received daily subcutaneous injections with cyclosporine (15 mg/kg) to avoid ECFC rejection.Two weeks after PTB, baseline echocardiography was performed ) to assess tricuspid annular plane systolic excursion (TAPSE), and tricuspid regurgitation. After the echocardiography, PTB rats were randomized to transplantation with PAH ECFC of a highly proliferative donor with in vitro tube formation . Measures of RV volumes were obtained from a series of cine short axis images, and cardiac output assessed using flow measurements from the pulmonary trunk obtained from a phase contrast sequence. Invasive pressure-volume measurement was performed using a small catheter  installed in the left carotid artery and the RV. Slow occlusion of the inferior vena cava allowed simultaneous recordings of RV pressures and volumes with decreasing pre-load. Load independent measures of RV contractility, including end systolic elastance (Ees) and ventricular arterial coupling (Ees/Ea) were calculated from pressure volume-loops using LabChart Software . Subsequently euthanasia was performed by extraction of the circulating blood volume, and organs were harvested.RV tissue sections were deparaffinized and stained for CD31  and subsequent DAB  and H&E staining. Small, non-muscularized vessels were manually counted by two independent, blinded observers using BF microscopy-images from 6 randomly selected, transversal sections. RV tissue area was calculated with ImageJ software , and counted vessels were corrected for area.t-test or ANOVA was reported. Correlations were tested using univariate linear regression. For non-normally distributed data, median with interquartile range (IQR) and Mann-Whitney non-parametric test was reported. For dichotomous variables chi-quadrate was performed, or, in case of low numbers Fisher’s exact test. Cox-regression and −2log likelihood test were used for TTCW. A p value of ≤0.05 was considered significant. All tests were performed with SPSS®. IBM software, Armonk, NY, USA or GraphPad Prism 6®, GraphPad Software, La Jolla, CA, USA.Continuous variables were visually inspected for normal distribution. If normally distributed mean with standard deviation (SD), and two-tailed independent student We choose a range of 1.5 year from time of ECFC isolation as a threshold for inclusion of hemodynamic and RV data. Because this is an observational study not all patients met this criterion. If so, the subjects were excluded from specific analysis.


To assess the inter-technique agreement of relative cerebral blood volume (rCBV) measurements obtained using T1- and T2*-perfusion MRI on 3T scanner in glioma patients.A total of 49 adult patients with gliomas underwent both on T1- and T2*-perfusion in the same scanning session, and rCBV maps were estimated using both methods. For the quantitative analysis; Two independent observers recorded the rCBV values from the tumor as well as contralateral brain tissue from both T1- and T2*-perfusion. Inter-observer and inter-technique rCBV measurement agreement were determined by using 95% Bland-Altman limits of agreement and intra-class correlation coefficient (ICC) statistics.Qualitative analysis of the conventional and perfusion images showed that 16/49 (32.65%) tumors showed high susceptibility, and in these patients T2*-perfusion maps were suboptimal. Bland-Altman plots revealed an agreement between two independent observers recorded rCBV values for both T1- and T2*-perfusion. The ICC demonstrated strong agreement between rCBV values recorded by two observers for both T2*  and T1  perfusion and similarly, good agreement was noted between rCBV estimated using two methods . ROC analysis showed that rCBV estimated using T1- and T2*-perfusion methods were able to discriminate between grade-III and grade-IV tumors with AUC of 0.723 and 0.767 respectively. Comparison of AUC values of two ROC curves did not show any significant difference.In the current study, T1- and T2*-perfusion showed similar diagnostic performance for discrimination of grade III and grade IV gliomas; however, T1-perfusion was found to be better for the evaluation of tumors with intratumoral hemorrhage, postoperative recurrent tumors, and lesions near skull base. We conclude that T1-perfusion MRI with a single dose of contrast could be used as an alternative to T2*-perfusion to overcome the issues associated with this technique in brain tumors for reliable perfusion quantification. Perfusion-MRI is used in the daily clinical practice for the evaluation of hemodynamic changes in brain tumors for more than a decade and serves as a surrogate marker for tumor angiogenesis ,2. Two mT2*-perfusion commonly uses single-shot gradient echo based echo planar imaging (EPI) sequence which is highly sensitive to susceptibility as it aims to detect small signal loss due to a contrast bolus . HoweverOn the other hand, T1-perfusion is less susceptible to the presence of blood products including hemorrhage, calcification, and artifacts associated with postsurgical changes and expected to give a more accurate assessment of the lesions associated with these issues. ,15. In TDespite, the availability of these advanced perfusion-MRI methods in a clinical setting for brain tumor diagnosis and treatment assessment for more than decades, the systematic comparison of these two approaches for evaluation of brain tumors is relatively scarce. Only a few studies have used both the techniques for assessment of gliomas, and very rarely hemodynamic metrics derived from both T1- and T2*-perfusion have been compared ,22–25.This study aimed to compare the performance of T1- and T2*-perfusion MRI for the evaluation of glial neoplasm and also to evaluate whether T1-perfusion could be a valid alternative to the T2*-perfusion for the quantitative evaluation of brain tumor in a routine clinical setting.The National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, India, Institutional Ethics Committee approved this study protocol. A retrospective review of hospital records and PACS revealed a total of 58 adult patients with histopathologically proven gliomas who had undergone both T1- and T2*-perfusion MRI as a part of brain tumor evaluation. The informed consent was waived off for by ethics committee for this retrospective study. Nine patients were excluded from the final analysis due to incomplete or poor perfusion data quality. Hence, the final data analysis was performed in 49 cases, these patients presented with symptoms related to a space-occupying intracranial mass lesion.2, matrix = 400x272]; T2-weighted images . The acquisition parameters for the 3D FLAIR imaging were the following ; T2-weighted GRE ; Susceptibility weighted imaging (SWI) ; respectively.All patients underwent conventional and perfusion-MRI (T1- and T2*-) in the same scanning session on 3.0T MRI  using 32-channel head-coil. MR imaging protocol included the following sequences: T1-weighted images [TR/TE = 2,000/20ms, inversion time (TI) = 800ms, number of slices = 28, slice-thickness = 5mm, FOV = 230×184mm0, slice-thickness = 6.0mm, FOV = 240×240mm2, matrix = 128×128, dynamics = 32, slices = 12, with temporal resolution of 3.9s), followed by T2*-perfusion using a gradient echo T2*-weighted EPI sequence . For both perfusion-MRI examinations, gadolinium-diethylenetriamine Penta-acetic acid , was administered intravenously (0.1mmol/kg) as a bolus through an autoinjector  after 3rd dynamic in T1-based (11.7s), and 10th dynamic in T2*- (16s) at the rate of 3.0ml/sec, and 5.0ml/sec respectively, followed by a bolus injection of 30ml of the physiological saline solution at each time. Contrast agent leakage through BBB breakdown can reduce T2* signal decay because of T1 shine effect related signal increase in extravascular space. Therefore, the rationale for the performing T1-perfusion before T2*-perfusion was to preload contrast agent during the T1-perfusion which can reduce the T1 shine effect and T2* contrast agent leakage effects on the measurements of rCBV from the T2*-perfusion MRI [Initially, T1-perfusion data was acquired using a 3D turbo field echo (TFE) sequence;  blinded to clinical and histopathology results independently evaluated the conventional MRI data on a PACS workstation.Conventional MRI and rCBV maps were visually inspected to look for the presence of susceptibility within the lesions or arising due to the proximity of the lesion to the skull base or in the vicinity of the paranasal sinuses. Both reviewers individually analyzed the images and saw if the perfusion evaluation of the whole tumor could be accomplished or only partial evaluation was possible.T2*-perfusion data was analyzed, and rCBV maps were computed using vendor supplied Intellispace Portal 9.0 , which included motion correction, and registration followed by a manual AIF selection, and deconvolution. rCBV maps generated from T2*-perfusion were further assessed in the Intellispace portal, and multiple ROIs are ranging from 3–6 with each measuring 5–10 mm in diameter was drawn on the rCBV map and selecting the regions showing the highest value of the CBV within the tumor from each patient. While recording the rCBV values care was taken to avoid placement of ROI on large vessels and areas with frank susceptibility. For this purpose, the rCBV maps were coregistered with FLAIR/T2-weighted, post-contrast T1-weighted or SWI/GRE images and thus avoiding inappropriate placement of the ROI. Contra-lateral ROIs were placed on the healthy brain white matter avoiding vessels to obtain the normalized rCBV values. The parts of the tumor showing susceptibility artifacts on gradient/SWI images were not used for placement of ROIs.1 map was computed using a previously described method based on T1, T2, and PD-weighted TSE images [All T1-perfusion data were transferred to an offline workstation dedicated to post-processing and quantification. T1-perfusion data were registered concerning precontrast T1-weighted images. T1-perfusion data were analyzed using an in-house developed software ,27. The E images . After pE images ,17. ThesE images . CBV mapE images . The samThe variables were tested for normal distribution. The maximal rCBV values for both T2*- and T1-perfusion images recorded by two observers were compared using a paired sample test. For T2*- and T1-perfusion derived rCBV values recorded by the two observers, inter-observer agreement was assessed using Intraclass correlation coefficient (ICC), which is categorized as the ICC<0.40 = poor, 0.40–0.59 = fair, 0.60–0.74 = good, and ICC>0.74 or above = excellent .Variance in the values recorded by two observers was also assessed by Pitman’s test of difference in variance. Also, Bland-Altman plots were constructed to look for “agreement limits” between the rCBV values dervied from the two modality (T2* and T1- perfusion). We also looked for agreement limits between the rCBV values recorded by two independent observers. If this interval presents a wide range of variation, it is supposed that there is no concordance between the two measurements and the reproducibility hypothesis is rejected. A visual inspection of the graph shows the data dispersion. If there is a concordance between two measures, the different values scatter along the zero line. The relationship between the T2*- and T1- derived rCBV was computed using Spearman non-parametric correlation.Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of rCBV individually in discriminating between grade-III and grade-IV gliomas by comparing the area under the curve (AUC). The sensitivity and specificity were established to discriminate between grade-III and grade-IV gliomas.All the statistical computations were carried out using Statistical Package for Social Sciences . A p-value of ≤0.05 was considered statistically significant.A total of 49 patients with supratentorial gliomas were included in this study. All patients were histopathologically confirmed and classified according to the latest WHO classification . Six patOn T2*-perfusion, 16/49 (32.65%) patient’s evaluation was suboptimal due to the presence of the high susceptibility artifacts arising due to various reasons and entire tumor could not be evaluated on T2*-perfusion data from these patients; however, all these cases could be adequately evaluated by using T1-perfusion. Geometrical distortions and signal loss causing marked image degradation on the T2*-perfusion corresponding to FLAIR signal abnormality were considered as suboptimal T2*-perfusion. Such lesions influenced by susceptibility image degradation and had a poor diagnostic value of the rCBV maps derived from the T2*-perfusion, and tumor evaluation was reported suboptimal or difficult by both reviewers. In these patients, the highest rCBV values were recorded from the parts of the tumor which were not affected by the presence of susceptibility. On both T1- and T2*- perfusion, there was no significant difference in the maximum rCBV values recorded by two observers as well as between two methods. The ICC with a 95% confidence interval for interobserver agreement on T2*-perfusion was excellent measuring 0.96 for maximum rCBV values (p = 0.040) . The mea2 = 0.4733). The Bland-Altman plots also showed good agreement between the rCBV values recorded by two independent observers for measuring rCBV derived from T2*- and T1-perfusion methods; respectively  . The meaectively . A signiThe inter-technique agreement for maximum rCBV measurements on T2*- and T1-perfusion maps were good and showed an ICC value of 0.74 (p<0.001). Bland-Altman plots showed a good agreement between the rCBV values obtained using these two methods i.e. T2*- and T1- perfusion .Comparison of grade-III and grade-IV tumors showed significantly higher rCBV values in grade-IV gliomas. The rCBV values derived from T2*- and T1-perfusion showed an AUC of 0.767; p = 0.040 and 0.723; p = 0.026; respectively for discrimination of grade-III and grade-IV gliomas. T2*-perfusion measured the rCBV cutoff value of 4.73 could discriminate grade-III from grade-IV gliomas with sensitivity and specificity of 81% and 72%; respectively. While T1-perfusion derived, rCBV showed a cutoff value of 5.47 discriminating grade-III and IV gliomas. It could discriminate the two tumor grades with a sensitivity and specificity of 78% and 63%; respectively . CompariIn this study, we compared the rCBV values derived independently using T1- and T2*-perfusion MRI in the patients with gliomas, and both techniques showed a good agreement with the comparable diagnostic performance for the discrimination of grade III and grade IV glioma. T2*-perfusion data was suboptimal in 32.63% of the patients due to the presence of significant intratumoral susceptibility arising from blood products, adjacent bone or sinuses in case of tumors located near to the skull base or post-surgical changes; on the other hand, all these tumors could be evaluated entirely by using T1-perfusion MRI.As previously reported by other investigators that the T2*-perfusion data has some inherent limitations and it is more susceptible to the magnetic field inhomogeneity. On the other hand, T1-perfusion is less sensitive to the magnetic field inhomogeneity compared to T2*-perfusion and could be advantageous for brain tumor assessment especially in cases where T2*-perfusion images are not interpretable. However, limitations of T1-perfusion include a low temporal resolution, limited coverage, and lack of commercially available post-processing tools. Estimation of hemodynamic parameters using T1-perfusion method is considered problematic because the signal changes induced by the intravascular component of the tracer have T1-relaxivity in order of the magnitude smaller than for T2* images, and the small blood volumes in brain tissue do not allow for significant tissue concentrations .In our study, we noted the higher number of suboptimal T2*-perfusion scans as compared to some of the previous studies ,30,31. OFew studies in the past have compared the T1- and T2*-perfusion-based methods for glioma evaluation have used the hemodynamic parameters derived from T2*-perfusion and tracer kinetic parameters (Volume transfer coefficient (Ktrans), fraction of EES (Ve), fraction of plasma volume (Vp) from T1- perfusion data. CBV estimation using T1-perfusion method is less common because of low temporal resolution and the signal changes induced by the intravascular component of the tracer have T1-relaxivity in order of magnitude smaller than for T2* image, and the small blood volumes in brain tissue do not allow for significant tissue concentrations . In prevrd of the T2*-perfusion scans being suboptimal, possible because most of the lesions were large and had enough non-hemorrhagic areas where no susceptibility changes were noted, and perfusion could be assessed with confidence using T2*-perfusion which in turn led to correct grade information. However, it becomes essential when assessing small lesions; planning image-guided biopsy of lesions and preoperative assessment as the whole tumor needs to be assessed to identify areas of increased perfusion correctly.Similarly, Santarosa et al. reported improved diagnostic performance of T1-perfusion method for brain tumor evaluation especially in the presence of susceptibility within the tumor . RevieweA significant limitation of the current study is the inclusion of a relatively small number of patients. In the current perfusion study, the T1-perfusion technique has limited coverage that is likely to be addressed in the future by using techniques like compressed sensing which can improve both temporal and spatial resolution . AnotherWe conclude that T1-perfusion with a single dose of contrast could be used as an alternative to T2*-perfusion to overcome the issues associated with this technique in brain tumors for reliable perfusion quantification. This is especially true for hemorrhagic, and post-treatment follow-up cases of gliomas.


The left (LA) and right (RA) atria undergo adaptive remodeling in response to hemodynamic stress not only induced by endurance exercise but also as part of several cardiovascular diseases thereby confounding differential diagnosis. Echocardiographic assessment of the atria with novel speckle tracking (STE)-derived variables broadens the diagnostic spectrum compared to conventional analyses and has the potential to differentiate physiologic from pathologic changes. The purpose of this study was to assess and categorize baseline values of bi-atrial structure and function in elite rowers according to recommended cutoffs, and to assess the cardiac changes occurring with endurance training. Therefore, fifteen elite rowers underwent 2D-echocardiographic analysis of established variables of cardiac structure and function as well as STE-derived variables of bi-atrial function. Measurements were performed at baseline and after eleven weeks of extensive training. 40% of athletes displayed mildly enlarged LA and 47% mildly enlarged RA at baseline, whereas no athlete fell below the lower reference values of LA and RA reservoir strain. Average power during a 2000 m ergometer rowing test (P2000 m) improved from 426 ± 39 W to 442 ± 34 W (p = 0.010) but there were no changes of echocardiographic variables following training. In elite rowers, longitudinal bi-atrial strain assessment indicates normal resting function of structurally enlarged atria and thereby may assist to differentiate between exercise-induced versus disease-associated structural cardiac changes in which function is commonly impaired. A vast amount of data in athletes reveals a balanced structural enlargement of all cardiac chambers3 in connection with endurance exercise. Exercise-induced left (LA) and right (RA) atrial remodeling has recently received more scientific attention, because atrial fibrillation (AF) is more common in middle-aged athletes participating regularly in endurance events compared to non-athletes5. A complex interplay culminates in the increased prevalence of AF in individuals performing chronic intensified endurance exercise. Thereby, autonomic disturbances, atrial dilatation and possibly atrial fibrosis seem to play a decisive role6. In addition, current literature indicates that LA and RA are not only passive transport chambers but actively modulate ventricular filling and contribute to global cardiac performance7 via three repetitive functional phases, i.e. reservoir, conduit and contraction phase8. However, data on atrial function in athletes are scarce partly due to limitations of transthoracic echocardiography (TTE), the primary diagnostic tool for assessment of the atria9. In this regard echocardiographic B-Mode- and Doppler-derived analyses insufficiently extract intrinsic atrial function from ventricular interdependence and especially the assessment of distinct atrial phases. Recent advances in echocardiographic imaging, i.e. two-dimensional speckle tracking echocardiography (2D-STE)10, enables a more independent insight into atrial function by visualising and calculating each of the three phases separately11. Feasibility and reliability of speckle-tracking-derived atrial strain assessment depends on echocardiographic image quality improving automatic tracking of acoustic markers12 and cardiac sonographer/lab experience. In addition to the common superiority of echocardiographic image quality in athletes, our own data demonstrates good reliability of 2D-STE-derived bi-atrial strain assessment in different hemodynamic conditions13. LA reservoir strain cutoffs have even been suggested to catagorize the severity of ventricular diastolic dysfunction14 and risk evaluation of AF15. At the same time, RA reservoir strain has the potential to non-invasively evaluate the severity of right ventricular (RV) dysfunction16. Thus, 2D-STE-derived echocardiographic bi-atrial strain assessment seems promising as a non-invasive and reliable diagnostic tool for assessing atrial function and may assist to differentiate between exercise-induced versus disease-associated structural cardiac changes in which function is commonly impaired. Notably, existing echocardiographic studies on atrial structure and function in athletes i) are typically cross-sectional, ii) are restricted to the LA only, iii) do not assess all phases of atrial function independently and iv) have been examined only in a heterogeneous cohort of athletes in which physiologic variables as well as training volume and intensity differ17. This study aimed to investigate baseline values and the adaptive changes of established structural and functional echocardiographic variables as well as comprehensive bi-atrial STE-derived reservoir, -conduit and -contraction strain following an eleven weeks preparation period training in elite endurance athletes.Endurance training is associated with hemodynamic alterations which, depending on type and intensity of exercise, lead to adaptive changes in cardiac structure and function. The cardiac adaptation in connection with endurance training are summarized in the term “athlete’s heart”18). Table Fifteen rowers competing at national or international level participated in this prospective study which was originally designed to compare the effects of different training intensity distributions  Endurance: other kinds of endurance training like running, cycling, swimming, etc., (iii) Strength: strength and resistance training, machine-based or weight lifting, and (iv) Other: stretching, stability training, etc. To quantify training volume and intensity, all rowers updated the mandatory online training diary of the German Rowing Federation daily. The diary included information about training mode, duration, distance, and intensity as well as information on days off and illness or injury. After completion of the study, all data were exported (.csv files) and subsequently analyzed.Training was divided into four modes, namely (i) 19. 21.A licensed sports physician examined all rowers medically during the first visit and diagnosed all free from cardiovascular disease and eligible to take part in the study. The medical examination included electrocardiographic assessment at rest  and a comprehensive echocardiogram described in detail below. Afterwards, all conducted a series of rowing ergometer  tests on two consecutive days. On the first day, maximal oxygen uptake (22.Hydration status was assessed from hematocrit (Htc) and hemoglobin concentration [Hb] from venous blood samples taken in the morning of the examinations to account for the impact of intravascular volume status of echocardiographic variables. Changes in blood and plasma volume (%ΔPV) were estimated employing the equation by Dill and Costill23. An electrocardiogram connected to the ultrasound system recorded heart rate during the examination. All images were saved on a mass storage device in raw Digital Imaging and Communications in Medicine format and analyzed offline using commercially available software .All rowers underwent transthoracic echocardiography using a commercially available ultrasound system  with a 1.0–5.0 MHz sector array transducer  at baseline and after the eleven weeks of preparation. The same experienced cardiac sonographer performed all acquisitions the day prior to 23), severity partition cutoff values only exists for left ventricular (LV) ejection fraction (EF), -size and -mass as well as LA maximum volume. For other parameters, the mean value plus 1.96-fold standard deviation (SD) of gender-, age-, and body surface area (BSA)–normalized parameters classify the upper limit of normal and were applied as cutoff values to analyze the echocardiographic results of the present study. We used the upper cutoff value of normal LV EDV indexed to BSA (i.e. >74 ml/m²) to identify participants with classic feature of exercise-induced cardiac remodeling in order to perform group comparison of our strain results. Reference values for LA and RA reservoir strain were employed according to current recommendations25.According to the latest recommendations about normal values of cardiac chambers in adults (as proposed by the the American Society of Echocardiography and the European Association of Cardiovascular Imaging (ASE/EACVI)Image quality optimization, linear, and volumetric measurements of cardiac chambers were performed according to the current recommendations. Briefly, LV and LA volumes were assessed using biplane disk summation algorithm by manually tracing endocardial borders in the apical 4- and 2-chamber views. When tracing the LA borders, the confluences of the pulmonary veins were excluded, and the atrioventricular interface was represented by the mitral annulus plane. LA maximal volume was measured just before the opening of the mitral valve. RV areas were measured by manual tracing of RV endocardial border. RA volumes were measured using the single-plane  method of disks.26.The echocardiographic report contains all linear and volumetric measurements in absolute numbers as well as indexed to BSA calculated using the formula by Mosteller27. Tricuspid annular plane systolic excursion (TAPSE) was assessed by placing an M-mode beam through the lateral tricuspid annulus27. The myocardial performance index (MPI) was calculated as the ratio of isovolumetric time divided by ejection time and derived from TDI analyses27.Conventional Doppler and Tissue-Doppler Images (TDI) were obtained from the apical 4-chamber view. For assessment of transmitral flow, the pulsed-wave Doppler sample volume was placed between the mitral leaflet tips in order to obtain peak early (E) and late diastolic (A) flow velocities. For TDI assessment of peak early diastolic (e’), late diastolic (a’) and systolic (s’) LV excursion velocities ultrasound beam was aligned to the longitudinal motion of the LV and the pulsed-wave sample volume placed at the basal septum and basal lateral wall. Peak velocities from the septal and lateral side of mitral annulus were averaged. e’, a’, s’ of RV excursion velocity were assessed by placing the pulsed-wave TDI sample volume in the tricuspid annulus of the RV free wall from a standard apical 4-chamber window28 and depending on the software package used in this study, LA reservoir, -conduit and -contraction strains were calculated with the first reference frame set at the onset of the QRS-wave of the surface ECG as illustrated in Fig. Longitudinal STE-derived LV and LA strain measurements were obtained from the apical 4- and 2-chamber views whereas RA strain was obtained from apical 4-chamber views. The researcher optimized image quality by positioning the focus at the region of interest and adjusting sector depth and width to include as little as possible outside the region of interest in order to maintain a frame rate between 50–80/sec. The same researcher performed offline analyses using a commercially available acoustic tracking software package (QLAB 9 (cardiac motion quantification (CMQ)); Phillips Medical Systems, Andover, MA, USA). Thereby, the region of interest was set at the myocardium using a point-and-click technique and the software divided the atrial wall into six equidistant segments. Orifices of the pulmonary venous component and segments in which inadequate tracing was observed were excluded from further analyses and the remaining segments were averaged. According to current practice recommendationst test compared pre- and post-protocol measurements after confirmation of normal distribution using the Shapiro-Wilk-test. A p-value ≤ 0.05 was considered to be statistically significant. Cohens d (dCohen) was calculated to estimate effect sizes with the thresholds for small, moderate, and large effects set at 0.20, 0.50, and 0.80, respectively29. Pearson’s r coefficient of correlation assessed correlations between variables.All statistical analyses were performed using SPSS 21 for Windows  with continuous variables presented as arithmetic mean ± SD and categorical variables as percentages. Student paired The datasets analyzed during the study are available from the corresponding author on reasonable request.The average training volume was 961 ± 82 min/week with 539 ± 77 min per week rowing, 163 ± 55 min per week strength training, 173 ± 59 min per week with endurance training and 84 ± 32 min other training activities. The power during the 2000 m all-out ergometer test improved significantly  after eleven weeks of preparation, whereas Structural and functional LV parameters at baseline and after eleven weeks of training are summarized in Table Structural and functional RV parameters are summarized in Table Using the current ASA/EACVI recommendations of normal values for cardiac chamber quantification in adults, absolute as well as indexed mean RV areas exceeded upper reference limits whereas mean linear RV dimensions remained within normal range. 86.7% of athletes presented with BSA-indexed end-diastolic areas above the upper reference limit  during baseline echocardiographic examination. Reduction in RV function was not detected using the reference values from the ASE/EACVI . No significant change of structural or functional parameters occurred between baseline and after eleven weeks of training  at baseline whereas no athlete fell below the lower reference value for LA reservoir strain. There was no significant change of structural atrial parameters between baseline and after eleven weeks of training Table .Figure 2Structural RA parameters are listed in Table LA reservoir strain did not correlate significantly with maximal LA volume  or LV end-diastolic volume . Likewise, RA reservoir strain did not correlate with maximal RA volume  and RV end-diastolic area .Change in average 2000 m ergometer test power output did not correlate significantly with change in LA reservoir , -conduit  and -contraction strain  nor with change in RA reservoir , -conduit  and -contraction strain . Change in The present study shows that (i) approximately half of our elite rowers exhibited bi-atrial enlargement while STE-derived bi-atrial strain parameters were within normal limits, (ii) no structural or functional adaptation occurred after eleven weeks of preparation training, (iii) the vast majority of athletes exceeded internationally accepted upper reference limits of RV enlargement.30. The average training volume of our athletes was 961 ± 82 min/week with 539 ± 77 min per week rowing and 163 ± 55 min per week strength training, pointing out the enormous exposure of our elite rowers to cardiac stress. Thus, elite rowers offer a unique opportunity to study new and evolving variables of cardiac structure and function assessment which might assist improving the differentiation between exercise-induced adaptive remodeling and cardiac pathology. Noteworthy, 40% of the athletes in our study exceeded the upper ASE/EACVI reference limit of normal LA volume (i.e. 34 mL/m²). In case of concurrent clinical symptoms as dyspnea, which is a common indication for which athletes are referred to echocardiography, their presumably adaptive atrial enlargement overlaps with atrial enlargement seen in different pathologic heart conditions.Rowing is unique within the broad spectrum of endurance disciplines, as it opposes the athlete not only to high isotonic but also exceptional high isometric cardiac stress, which are both important mediators for exercise-induced cardiac remodelling. Furthermore, endurance disciplines characterized by high concomitant isotonic and isometric stress appear to result in different and more pronounced structural and functional LV remodeling patterns than disciplines with more isolated isotonic stimuli, as distance running or soccer31 showed significantly reduced STE-derived LA reservoir (39.7 ± 8.4%) and -contraction (10.6 ± 3.8%) strains in healthy competitive adolescent male soccer players31 but not in female volleyball players 17. Our study extends these findings towards a cohort of healthy elite male rowers. Although we did not directly compare our athletes with a control group, our results (LA reservoir strain: 43.1 ± 5.3%) are in line with the mean LA reservoir strain from a large cohort of healthy non-athletic individuals (45.1 ± 11.4%)24 despite the pronounced adaptive hemodynamic stimulus during rowing. Noteworthy, the recent recommended lower reference value for LA reservoir strain is 22.7%24 and none of our athletes fell below this value. Though slightly reduced LA reservoir strain at rest may be caused by exercise-induced adaptive changes of heart function17, its assessment has the potential to support the diagnostic work-up in athletes with structural LA changes. Reference values for LA conduit and -contraction strain are not published yet and thus no comparison can be provided.Assessment of functional atrial parameters has become feasible since the advent of STE and holds promise to guide the diagnostics within this uncertain atrial gray zone. D’Ascenci32. D’Ascenzi showed a significant reduction of LA reservoir and -contraction strain after eight months training in male soccer players as well as after 16 weeks of training in female volleyball players. In contrast, none of the phasic LA strain parameters changed during eleven weeks of intensive preparation period training in our male rowers. These findings have important clinical implications, considering that middle-aged males who have been engaged in strenuous endurance training for more than 10 years and who are otherwise healthy, are at risk of developing AF5. Taking into account the overall health benefits and even reduced cardiovascular disease prevalence in former elite rowers33 continuing athletic lifestyle while reducing the risk of AF has the potential to further improve cardiovascular health of endurance athletes. In light of the reduced STE-derived LA strain in individuals with paroxysmal AF34, and considering the preserved resting strain results despite large volume of training in our study, monitoring LA function in endurance athletes has the potential to detect a drift in strain to evaluate the risk and induce early preventive strategies of AF in endurance athletes.In addition, this study extends two longitudinal-designed studies assessing STE-derived LA strain in team-sports athletes35, which is a common complication of arrhythmogenic right ventricular cardiomyopathy (ARVC)36. Thus STE-derived RA strain might serve as a diagnostic aid to differentiate between a potentially life-threatening condition and physiologic, exercise-induced RV adaptation. The recently recommended lowest expected value for RA reservoir strain is 24%25 and no athlete in our study was below this value. In addition, our STE-derived RA strain results (Reservoir strain: 42.9 ± 5.7%) are in agreement with data by Pagourelias et al.3 who did not observe any difference of RA reservoir and –contraction strain between three groups consisting of endurance athletes (Reservoir strain: 43.9 ± 15%), athletes from game sports, and a control group. Phasic RA strain parameters did not change during eleven weeks of intensive preparation period training in our study what is in line with the only longitudinal study investigating the impact of training on RA strain in female volleyball players17. Our study extends previous results17 with male endurance athletes and some trials concluded an even more modest atrial adaptation to training in women37. In parallel to our LA results, our data suggest that STE-derived resting RA strain assessment is no appropriate surrogate for exercise-induced atrial adaptation but has the potential to support diagnostic work-up in athletes with symptoms and suspected right heart disease.In parallel to the LA, the RA faces similar diagnostic uncertainties when assessing only structural parameters in athletes with clinical symptoms. In our study, 47% of athletes showed enlarged RA using the current reference values from the ASE/EACVI. RA reservoir strain at rest is reduced by 27.1 ± 11.6% in patients with pulmonary hypertension and the reduction is thought to reflect RV dysfunction38. Notably, 67% of athletes in our study fulfilled the minor ARVC criterion for BSA-indexed RV dilatation whereas none fulfilled the major ARVC criterion39. Recently new cutoffs derived from a large cohort of top-level athletes from different sports were published40 and 20% of our athletes even exceeded the suggested upper reference value . In line with the atrial results, reduction in RV function was not found in our cohort of elite endurance athletes. 53.5% even exhibited supranormal systolic RV function at baseline as indicated by an s’ > 13.4 cm/sec. Concerning the diagnostic gray zone between exercise-induced RV remodeling and ARVC as well as concerns about potential myocardial RV damage due to intense endurance exercise, the major ARVC Task Force criteria or reference values derived from an athletic population in combination with functional assessment should be employed in the interpretation of echocardiographic-derived structural RV parameters in endurance-trained athletes.In addition to the abovementioned structural atrial changes, 60% of our athletes yielded enlarged LV and 94% enlarged RV according to the current reference values. RV dilatation is a common phenotypic expression in ARVC, a disease in which competitive sports participation is a modifiable risk factor for sudden cardiac death41. Thus, the results of this study cannot be transferred to female endurance athletes.Our study has limitations worth indicating. From a methodological standpoint a larger sample size may be favorable. However, we aimed to investigate a selected cohort of elite endurance athletes competing at national and international level. Therefore, the number of subjects is limited by the nature of the highly selective study cohort. Adding more individuals into the study would have meant to integrate weaker athletes which were not the target of the study and with more athletes the individual monitoring of training load becomes more challenging. No follow up examinations were performed because athletes were competing in international races making group testing of all athletes logistically impossible. This study was conducted with male athletes only. Sex significantly affects the normal heart size in athletesIn summary, this study has shown that mildly enlarged LA and RA maximal volumes are common in elite rowers whereas STE-derived resting bi-atrial strain variables are within normal limits and do not change after eleven weeks of training. Hence, variables of atrial strain at rest are unsuitable to evaluate exercise-induced atrial remodelling. but have the potential to guide the diagnostic work-up in symptomatic athletes with structural atrial changes. Furthermore, considering the stable echocardiographic variables despite large volume of endurance training, our study further supports the cardiac safety of competitive rowing.


The three-dimensional rearrangement of the right ventricular (RV) myocardium during cardiac deformation is unknown. Previous in-vivo studies have shown that myocardial left ventricular (LV) deformation is driven by rearrangement of aggregations of cardiomyocytes that can be characterised by changes in the so-called E3-angle. Ex-vivo imaging offers superior spatial resolution compared with in-vivo measurements, and can thus provide novel insight into the deformation of the myocardial microstructure in both ventricles. This study sought to describe the dynamic changes of the orientations of the cardiomyocytes in both ventricles brought upon by cardiac contraction, with particular interest in the thin-walled RV, which has not previously been described in terms of its micro-architecture.The hearts of 14 healthy 20 kg swine were excised and preserved in either a relaxed state or a contracted state. Myocardial architecture was assessed and compared between the two contractional states by quantification of the helical, transmural and E3-angles of the cardiomyocytes using high-resolution diffusion tensor imaging.The differences between the two states of contraction were most pronounced in the endocardium where the E3-angle decreased from 78.6° to 24.8° in the LV and from 82.6° to 68.6° in the RV. No significant change in neither the helical nor the transmural angle was found in the cardiomyocytes of the RV. In the endocardium of the LV, however, the helical angle increased from 35.4° to 47.8° and the transmural angle increased from 3.1° to 10.4°.The entire myocardium rearranges through the cardiac cycle with the change in the orientation of the aggregations of cardiomyocytes being the predominant mediator of myocardial wall thickening. Interestingly, differences also exist between the RV and LV, which helps in the explanation of the different physiological capabilities of the ventricles.The online version of this article (10.1186/s12968-019-0547-2) contains supplementary material, which is available to authorized users. Structural remodelling of the myocardium has been attributed to the functional impairment observed in heart disease  and cardOverall, the right ventricle (RV) and left ventricle (LV) serve the same purpose of pumping blood against an afterload, but apart from this they differ in most other aspects. Their anatomy and mural thickness is far from identical, and the same is true of their physiology , 4, biocAlthough there are considerable data on the mechanisms of LV dysfunction and failure, the pathophysiological mechanisms underlying RV dysfunction and failure are only beginning to be understood . Heart fWe know ventricular wall deformation coincides with contraction of the cardiomyocytes, however, cell shortening alone is not sufficient to produce normal ejection fractions . Cardiac3, means the intricacies of wall deformation cannot be resolved and neither can the morphological details of the relatively thin walled RV. Questions remain regarding regional and transmural differences in deformation and the functional significance of the aggregations of cardiomyocytes. Ex-vivo imaging offers superior spatial resolution, and as a result, can provide novel insight into the rearrangement of all components of the cardiac mesh relative to cardiac phase, region, and wall depth in both the LV and RV.Rearrangement of the myocardial architecture through the cardiac cycle has been demonstrated previously using in-vivo diffusion tensor magnetic resonance imaging in the LV of both healthy , 18 and In the present study, using high resolution ex-vivo diffusion tensor CMR, we describe the changes in orientation of the cardiac mesh components in the LV and RV, and muscular ventricular septum, brought upon by cardiac contraction.We included 14 Danish landrace female swine in the study each weighing 20 kg. All experiments were approved by the Danish Animal Inspectorate license no. 2013-15-2934- 00869.Prior to experiments, the swine were kept in their usual farm environment, with unrestricted access to food and water. Artificial daylight was maintained for 12 h, from 8 am to 8 pm. Before transportation to the experimental laboratory, the swine were pre-anesthetised with intramuscular administration of 0.5 mg/kg of midazolam and 2.5 mg/kg of ketaminol. If needed this was repeated upon arrival to the laboratory to allow establishment of an intravenous access. Anaesthesia was supplemented by intravenous administration of 3 mg/kg of propofol to allow endotracheal intubation and coupling to a ventilator. Continuous anaesthesia was maintained using 2–3% inhalational sevoflurane. While anaesthetised a venous 6 French sheath was placed in the femoral vein using Seldinger technique guided by ultrasound.The animals were then transferred to our CMR facilities to perform CMR for determination of overall cardiac anatomy. To achieve maximal myocardial contraction within the normal physiological range, intravenous administration of dobutamine 40 μg/kg/min was introduced in order to simulate an exercise situation. The animals were then returned to our experimental operating theatre where their hearts were exposed through a median sternotomy while still fully anaesthetised. After intravenous administration of 10.000 units of heparin, the animals were euthanised by means of excision of the heart and great vessels. The hearts were subsequently randomised into fixation in either a relaxed state or a contracted state. In the group randomized to the relaxed state, 7 hearts were each perfused with one litre of potassium rich cold cardioplegic solution  immediately after excision directly through the coronary arterial orifices at a pressure of approximately 100 mmHg at the point of the catheter tip. Subsequently, a water-based CMR compatible polymer (Histomer) was injected into the ventricles via the atrioventricular valvar orifices to maintain the relaxed state. Residual polymer could escape via the pulmonary and aortic valvar orifices to avoid excess dilation. The polymer was allowed to harden for approximately 15 min, after which the hearts were perfused with formalin 10%  using the same method as with the cardioplegic solution. In the group randomized to contraction, 7 hearts were perfused with formalin immediately after excision of the hearts. No polymer was injected in this group. All hearts were kept in formalin for at least 48 h to ensure complete transmural fixation.CMR was performed in a 1.5 T CMR scanner . The animals were placed on the scanner bed in a supine position and anaesthesia was maintained using 2–3% inhalational sevoflurane. Scout images determined the orientation of the LV long-axis. LV function was assessed using a retrospective, electrocardiogram-triggered balanced steady state free precession (bSSFP) breath-hold cine sequence in the cardiac short-axis, vertical long-axis and horizontal long-axis plans. In the cardiac short-axis, LV volume was completely encompassed by contiguous 8 mm slices with a spatial resolution of 1.22 mm × 1.22 mm and a field of view of 288 mm × 288 mm. The following imaging parameters were used: repetition time 3.0 ms; echo time 1.5 ms; flip angle 60°; 30 heart phases. Heart rate and blood pressure was continuously recorded.2 and one additional with b = 0 s/mm2, 125 slices with 800 μm slice thickness and no gap and an in-plane-resolution of 400 × 400 μm2.Twenty-four hours prior to diffusion tensor CMR scanning, the hearts were perfused with phosphate buffered solution and stored at 4 °C. On the day of diffusion tensor imaging the hearts were allowed to adapt to room temperature before initiating the scan. Imaging was performed using an Agilent 9.4 T MR-system , equipped with 400 mT gradients and vnmrJ 4.0 software. Each heart was placed with LV long-axis aligned parallel to the axis of the main magnetic field. Scout imaging enabled precise adjustment of axis and rotation of the images. Measurements were performed using a standard multi-slice 2D spin echo sequence with diffusion gradients. The repetition time was 7000 ms and echo time was 30 ms. The scan time was approximately 16 h for each heart. We used 30 isotopically distributed diffusion directions with a b-value of 1000 s/mmTo evaluate the state of contraction of the excised hearts, ventricular wall thickness from the ex-vivo diffusion tensor imaging data, was compared with that of the in-vivo CMR cine data. In order to do so the ventricular myocardium was virtually subdivided into 23 zones as previously described in our group and illustrated in Fig. http://medviso.com/segment/) while determination of the ex-vivo wall thickness was done on the diffusion-weighted images using an investigator independent mathematica algorithm measuring the distance between the most epicardial voxel and the most endocardial voxel in the centre of the zones of interest. Ventricular mural thickness was used rather than ventricular volumes in order to reduce the risk of type two error when comparing the low-resolution in-vivo CMR data with the high-resolution ex-vivo data.Septal measures were taken in zones 8 and 9 whereas zone r4, r5 and r6 quantified the RV wall thickness. Mean wall thickness was measured in-vivo in end-diastole and end-systole both before and after dobutamine administration. All image analyses of in-vivo data were done manually using Segment version 2.0 R4942 , the septum (p <  0.001) and the RV (p <  0.001), confirming fixation in two significantly different contractional states  during exercise and 7.6 mm  in fixation, p = 0.4. In the RV, a significant difference was found between the diastolic exercise wall thickness of 1.9  and the mean wall thickness in the ex-vivo relaxed state of 3.4 , p <  0.0001.In the LV, no significant change was found between the mean diastolic exercise wall thickness of 6.24 mm  and the mean wall thickness in the ex-vivo relaxed state of 7.7 mm , p = 0.14. In the LV, a significant difference was found between the mean systolic wall thickness during exercise of 13.4  and the fixed contracted wall thickness of 17.9 , p <  0.001. In the RV, a significant difference was found between the systolic exercise wall thickness of 3.9  and the fixed contracted wall thickness of 5.9 , p <  0.001.No significant difference was found between the mean systolic septal wall thickness during exercise of 11.6  and that in the ex-vivo contracted state of 12.7 , Figure Furthermore, eigenvalue comparison of the LV and the RV can be appreciated in Fig. p = 0.02) and the RV wall (p = 0.004). In the septum, however, an even more obvious change in the distribution of helical angles was seen from relaxation to contraction (p < 0.001). Relative to myocardial wall depth helical angles changed significantly from relaxation to contraction in the LV and in the septum, but not in the RV  in the endocardium (Table p < 0.001). In the epicardium the mean helical angle did not change significantly from relaxation to contraction. No significant changes of the helical angles were found from relaxation to contraction in the RV myocardium as a whole. If, however, each zone of the RV myocardium was considered individually, significant changes in helical angles were found in several zones of the RV  and in the midwall an increase was found from 2.0° in the relaxed state to 19.1° in in the contracted state (p < 0.001), see Table In Fig.  RV Fig. . In the  RV Fig. . In the p = 0.007) and in the midwall this angle increased from 1.2° in the relaxed state to 5.6° in the contracted state (p = 0.013). No significant change was found in the epicardium of the LV . In the midwall the E3-angle decreased from 76.2° in the relaxed state to 25.3° in the contracted state (p < 0.001) and in the epicardium a decrease was found from 80.8° in the relaxed state to 9.6° in the contracted state (p < 0.001). In the RV, the E3-angle decreased from 82.6° in the relaxed state to 68.6° in the contracted state in the endocardium (p < 0.001). In the midwall a decrease from 78.0° in the relaxed state to 58.4° in the contracted state was seen (p < 0.001) and in the epicardium a decrease was found from 80.0° in the relaxed state to 60.4° in the contracted state (p < 0.001). In the septum the E3-angle decreased from 79.7° in the relaxed state to 6.3° in the contracted state in the endocardium and in the midwall a decrease from 76.8° in the relaxed state to 54.2° in the contracted state was found (p < 0.001). In the epicardium a decrease was found from 82.0° in the relaxed state to 66.0° in the contracted state (p < 0.001).In Table This is the first study describing the dynamic changes in the orientation of the cardiomyocytes within the RV myocardium between two contractional states. We, moreover, confirm existing knowledge on the dynamic changes of the myocardial architecture, that accompany LV deformation , 18, 20.In the RV, the proportion of E3-angles approaching 0° increased from relaxation to contraction as the aggregates assume a more radial or horizontal orientation in the contracted state. Similarly, in the LV, a shift towards smaller E3-angles was observed in the contracted state, which is in line with previous in-vivo studies , 18, 20.Few have investigated RV micro-architecture using diffusion tensor imaging; this may be due to the fact that the importance of the RV has been underestimated for many years , but it The present study shows no change in transmural angulation and only discrete changes in helical angle in the RV. But what mechanisms are then facilitating wall deformation from diastole to systole in the RV? Ejection of blood from the ventricles is provided by a combination of circumferential constriction and longitudinal shortening of the ventricle resulting in myocardial wall thickening . To achiOur observations provide new insights into the dynamic differences between the various parts of the heart. As mentioned in the introduction, the two ventricles differ in many aspects although serving the same overall purpose. Most notably, the gross anatomy differs greatly. The RV has a very complex shape often referred to as a flattened tube wrapped around the LV , which lIn addition to the anatomical differences described above, RV physiology differs markedly from that of the LV , 33. In The most relevant limitation to this present study is the lack of dynamic in-vivo diffusion tensor imaging. This has previously been performed on the human LV , 20. In According to our wall thickness comparison Fig. , some paThe remodelling of the RV myocardium is a novel field of research, and it can be argued that diffusion tensor imaging is only histopathologically validated in the LV. The technique has, however, been validated several times over the years using both two-dimensional and three-dimensional techniques, thus we consider the accumulated evidence of the validity of the technique very convincing and see no reason why it should not likewise apply to the RV –39.In addition, it can be argued that ventricular volumes should be used when comparing the contractional states. We were, however, facing the challenge of comparing low-resolution in-vivo data with high-resolution ex-vivo data. This introduced a considerable inaccuracy in the comparison, which increases for each dimension assessed. We are, therefore, reducing the risk of a type two error by using ventricular wall thickness as a measure of state of contraction.Further, we used the E3-angle as a pseudo-marker of the orientation of the aggregates of cardiomyocytes. This angle is assessed using the tertiary eigenvector, which is a mathematical derivative of the primary and secondary eigenvectors and is hence not a direct depiction of the actual structure. From Fig. The architecture of the entire myocardium changes through the cardiac cycle. This rearrangement is provided by continuous changes in the orientation of cardiomyocytes. The movement of cardiomyocytes is, however, different in the RV and LV and in the septum, potentially explaining the different physiological capabilities of the various parts of the heart. The current study provides new insight into the micro-structural mechanisms underlying RV contraction, suggesting that the change in the orientation of the aggregates of cardiomyocytes, quantified by the E3-angle, is the predominant mediator of myocardial wall thickening not only in the LV, but in the entire myocardium. This study also provides high-resolution validation of recent LV in-vivo measurements, paving the way for potential diagnosis of heart disease based on the function of ventricular micro-structure.Additional file 1:Rotatable 3D PDF showing the angle definitions. The local set of orthogonal planes for the assessment of cardiomyocyte orientation. The plane A is the local epicardial tangential plane and plane C is defined as the plane spanned by normal of plane A and the local epicardial horizontal (short axis) tangent. Plane B is orthogonal to planes A and C. The helical angle is defined as the angle between the cardiomyocytes (yellow rod) and plane C. The transmural angle is defined as the angle between the cardiomyocytes and the epicardial tangential plane A. The E3-angle is defined as the angulation between the aggregate plane (red box) and the epicardial tangential plane A. (PDF 100 kb)