Assessment of intraventricular pressure gradients (IVPG) using color Doppler M-mode echocardiography has gained increasing interest in the evaluation of cardiac function. However, standardized analysis tools for IVPG quantification are missing. We aimed to evaluate the feasibility, the test-retest observer reproducibility, and the inter-system variability of a semi-automated IVPG quantification algorithm. The study included forty healthy volunteers (50% were men). All volunteers were examined using two ultrasound systems, the Philips Epiq 7 and the General Electric Vivid 6. Left ventricular diastolic (DIVPG) and systolic (SIVPG) intraventricular pressure gradients were measured from the spatiotemporal distribution of intraventricular propagation flow velocities using color Doppler M-mode in standard apical views. There was good feasibility for both systolic and diastolic IVPG measurements . Intra and inter-observer test-retest variability measured with the intraclass correlation coefficient were 0.98 and 0.93 for DIVPG respectively, and 0.95 and 0.89 for SIVPG respectively. The inter-system concordance was weak to moderate with Lin’s concordance correlation coefficient of 0.59 for DIVPG and 0.25 for SIVPG. In conclusion, it is feasible and reproducible to assess systolic and diastolic IVPG using color Doppler M-mode in healthy volunteers. However, the inter-system variability in IVPG analysis needs to be taken into account, especially when using displayed data. LV ejection is facilitated by a positive systolic intraventricular pressure gradient (SIVPG) between the apex and the outflow tract. The SIVPG increases during physical exercise and inotropic stimulation, whereas it is attenuated by infusion of betablockers2. During the early diastole, LV active relaxation generates suction by developing a local negative diastolic pressure gradient between the mitral valve and the LV apex (DIVPG). This mitral to apical driving pressure was closely related to the rate of LV relaxation5, and it was shown to be sensitive to inotropic stimuli, loading variations, and acute myocardial ischemia6. For the past two decades, experimental and human studies have demonstrated the validity of color Doppler M-mode echocardiography to estimate non-invasively the instantaneous local pressure differences into LV during ejection and diastolic filling13. These authors have confirmed the close relationship between color Doppler-derived systolic and diastolic IVPGs and invasively measured indices of LV contractility and relaxation16. Several studies have evaluated the clinical applications of IVPGs assessment using color Doppler M-Mode in healthy subjects and patients with cardiomyopathies. Heart failure with preserved and reduced ejection fraction was associated with a loss of adrenergic augmentation of DIVPGs under pharmacological and physical stress19. These observations suggested that heart failure is associated with a reduced ability to generate an effective ventricular suction in such stress conditions, essential to ensure rapid LV filling without increase in atrial pressure. Similar observations of reduced LV apical suction in heart failure patients were reported in resting-state20. Inversely, reduction of left ventricular obstruction after ethanol septal reduction in a small group of patients with hypertrophic cardiomyopathy was associated with increased DIVPGs, suggesting an improvement in diastolic suction force12. The same authors have also demonstrated that the inability to augment DIVPGs in heart failure was strongly correlated with peak VO218.Left ventricular (LV) systolic contraction and early diastolic active relaxation generate forces that transmit regional pressure differences to the LV cavity. Hemodynamic investigations have demonstrated that the assessment of intraventricular pressure gradients (IVPGs) provides determinant insights in cardiac function21. This point is particularly relevant for clinical quantitative processing of color Doppler-derived IVPGs to characterize cardiac function in patient cohorts. The temporal, spatial and velocity resolutions differ between ultrasound vendors, some of whom restrict the access of raw proprietary velocity data. The lack of studies evaluating the inter-system reliability of a standardized method for IVPG quantification might limit the clinical interest of physicians. The present study aimed to assess the feasibility, the test-retest observer reproducibility, and the inter ultrasound system variability of systolic and diastolic IVPG measurements, using a semi-automated algorithm based on velocities extracted from color Doppler M-mode image file.These echocardiographic studies have used the same approach based on the Bernoulli equation to estimate non-invasively the instantaneous local pressure gradient from the spatiotemporal distribution of blood velocities along the LV long axis. However, these studies have used different ultrasound scanners for data acquisitions and different processing chains of the blood flow velocity data. To date, only one study addressed the impact of color-Doppler scanner resolutions when calculating IVPGs from color Doppler M-modeForty healthy volunteers (50% were men), aged between 18 and 35 years old, were prospectively enrolled among medical students at the university hospital center of Caen (France). The upper age limit was set at 35 years-old to limit the prevalence of cardiovascular diseases that could impact myocardial function. Participants were not taking any medication, and cardiovascular disease was ruled out by history, clinical examination, electrocardiogram, and echocardiography. Subjects with metabolic and chronic extracardiac diseases were excluded. All volunteers referred for a standard echocardiographic examination were recruited on the condition of an acceptable apical ultrasonic view. The study protocol was approved by the institutional ethical committee (Comité de Protection des Personnes Nord Ouest III – ID: A16-D11-VOL.27), and written informed consent was obtained from all participants. The medical procedures of this trial are in line with the most recent recommendations of the Helsinki Declaration.To assess the inter-system agreement of IVPG measurements, each participant was sequentially examined by the same sonographer on two different ultrasound systems: the Philips Epiq 7 equipped with an X5-1 transducer, and the General Electric (GE) Vivid 6 equipped with an M4S transducer. The ultrasound systems were identified randomly as system A and system B in this publication. Ultrasound machines were arranged on the same side of the examination bed. The sonographer position, scanning right-handed, did not change between examinations. For each volunteer, color Doppler M-mode acquisitions for systolic and diastolic IVPG assessment were performed consecutively in the same session using both ultrasound systems (time required to switch between probes from system A to system B). All examinations were made in the left lateral decubitus position. Left ventricular inflow and outflow recordings using color Doppler M-mode were obtained from all subjects on three consecutive cardiac cycles in apical four and five-chamber views during a brief apnea. Arterial blood pressure was measured during the echocardiographic study. Heart rate was monitored during the examinations. Two sonographers were able to perform echocardiographic examinations. The first sonographer (AH) was well trained in IVPG acquisitions. The second sonographer (LC) was less experienced. All participants were provided the option to choose the gender of the sonographer who would perform their examinations. Intra and inter-observer test-retest variability for IVPG measurements were determined on ten randomly selected subjects scanned by the two sonographers, who acquired each two independent SIVPG and DIVPG data sets, using the same machine. The sonographers analyzed their own data sets in a blinded fashion. For each data set, peaks IVPG (systolic and diastolic) were obtained as the average value of three consecutive cardiac cycles.All color Doppler M-mode acquisitions were stored as Dicom files, which contain raw and processed data displayed in Joint Photographic Experts Group (JPEG) format. IVPGs were measured using our independent software algorithm coded in Matlab (version R2017b) from displayed data.Color Doppler M-mode provided both a spatiotemporal map of greyscale anatomical information and a spatiotemporal map of color-encoded blood velocity distribution 11. The position of the mitral and aortic annulus had to be defined to design the integration limits. It was automatically positioned from the echo M-mode and could be manually corrected by the reader if the result was not judged as correct. Examinations that did not meet the quality criteria were not processed. The internal validation of image quality before processing was: 1/optimal alignment of the scan line with the direction of blood flow as described by Greenberg et al.8; 2/complete color Doppler flow map through the streamline; 3/presence of anatomical landmarks of LV in the color Doppler sector; 4/absence of flow obstruction.The velocity data were extracted from the displayed image by using the color encoding given by the Look-Up Table (LUT). To enable further quantification, the velocity data were processed with dealiasing and smoothing Fig. . We usedThe temporal resolution was set by default to acquire three successive complete cardiac cycles to guarantee the stability of the averaged IVPG measure. Consequently, the displayed image resolutions for system A were 285 Hz for the temporal resolution and 0.24 mm for the spatial resolution, and for system B 180 Hz for the temporal resolution and 0.42 mm for the spatial resolution. To study the potential impact of temporal sampling on the measure of pressure gradient differences, we assessed SIVPG and DIVPG at different temporal displayed resolution settings  on ten randomly selected subjects scanned with system A. For each subject, peaks IVPG were averaged during the same period of three cardiac cycles in systole and diastole.The greyscale gain was adjusted to optimize the color flow mapping on the displayed image. For that, we tested a range of the grayscale gain on 15 color Doppler M-mode acquisitions for both systolic and diastolic gradients. For each acquisition, the gain was increased by steps of 10% from 40% up to 90%. Figure 22), as well as Lin’s concordance correlation coefficient (CCC)23. Intra and inter-observer test-retest variability for SIVPG and DIVPG measurements were determined using the intraclass correlation coefficient (for average fixed observers) with a 95% confidence interval, and the coefficient of variation ± SD. A paired t-test was used to analyze the significance of the biases in IVPG measurements between systems. Mean values of IVPGs between men and women were compared using unpaired t-test. The level of significance was defined as P < 0.05.Statistical analysis was conducted using Medcalc version 13 . All variables were normally distributed (Shapiro-Wilk test) and expressed as mean ± SD. IVPG measurements were expressed as absolute values. The agreement between the ultrasound system acquisitions was estimated by calculating the mean and SD of the difference between system A and system B . The overall body mass index was 22.2 ± 2.3 kg/m². The mean arterial blood pressure was 86 ± 8.8 mmHg. The heart rate remained stable during system A and system B examinations . No gender differences were observed in arterial blood pressure nor heart rate. All subjects were in sinus rhythm. Cardiac function and structure were normal, without any valvular diseases.The mean age of the enrolled volunteers was 25.5 ± 3.7 years old  and 85% for diastolic acquisitions (34/40 subjects). The feasibility was different according to the experience of the sonographer who performed the examination. For the well-trained operator in IVPG acquisitions (AH), who scanned 22 subjects during the study, the feasibility was up to 91% (20/22 subjects) for both systolic and diastolic color Doppler acquisitions. Whereas, for the less-trained operator (LC), who scanned 18 subjects during the study, only 72% (13/18 subjects) of SIVPG and 78% (14/18 subjects) of DIVPG acquisitions met the validation criteria. Examples of IVPG acquisitions that did not meet the validation criteria are illustrated in Supplementary Fig. Regarding intra-observer test-retest variability, for DIVPG the intraclass correlation coefficient was 0.98 [IC 95%: 0.94–1], and the coefficient of variation was 5.2 ± 0.1%. For SIVPG the intraclass correlation coefficient was 0.95 [IC 95%: 0.83–0.99], and the coefficient of variation was 6.3 ± 0.2%. Regarding inter-observer test-retest variability, for DIVPG the intraclass correlation coefficient was 0.93 [IC 95%: 0.83–0.97], and the coefficient of variation was 10.5 ± 0.3%. For SIVPG the intraclass correlation coefficient was 0.89 [IC 95%: 0.74–0.95], and the coefficient of variation was 9.3 ± 0.3%.vs. 2.24 ± 0.83 mmHg for women, P = 0.17). Overall SIVPG measurements were significantly higher among men compared to women .Table To analyze if the displayed blood flow velocities were not more degraded by one of the two systems, we were able to test the concordance between the raw proprietary velocity data and the displayed velocity data estimated from the LUT on both systems for one set of color Doppler M-mode diastolic acquisition. The results are reported in Fig. The results are shown in Fig. 13, is feasible and highly reproducible. However, we observed an inter-system variability, which might be mostly explained by differences in the temporal displayed resolutions of processed velocity data between the two ultrasound systems.This study confirmed that IVPG quantification using color-Doppler M-mode echocardiography, previously validated in experimental and human studies18. Ohara et al.19 have shown the clinical utility and feasibility of DIVPG assessment in a large cohort of 166 patients with diastolic dysfunction undergoing stress echocardiography. Yet, the authors did not report whether there have been patients excluded from the study due to insufficient image quality to assess color-Doppler derived DIVPGs. Based on these observations, and in contrast with the alias-based slope method for measuring the color M-mode flow propagation velocity (Vp) which is no more recommended in the routine assessment of LV diastolic function due to low feasibility and reproducibility24, DIVPGs seem to provide a clinically applicable and reliable evaluation of LV suction.In the present study, we used a semi-automated software for processing IVPGs that resulted in high intra- and inter-observer test-retest reproducibility. Although IVPG assessment requires an experienced operator to correctly acquire color Doppler M-mode velocity profiles during systole and diastole, as suggested by a higher rate of feasibility for the well-trained operator in IVPG acquisitions, the overall feasibility for both systolic and diastolic IVPG measurements was acceptable. The choice of the operator did not significantly influence the concordance between systems A and B measurements for both SIVPG and DIVPG (data not shown). If the color Doppler M-mode acquisitions fulfilled the required quality criteria for IVPG assessment, the post-processing did not appear to be influenced by the operator’s experience, which was confirmed by our test-retest analysis. These results confirmed the good inter-reader reproducibility of echocardiographic diastolic and systolic IVPG measurements previously reported21. Rojo-Alvarez JL. et al.21 have demonstrated the importance of the temporal resolution in the calculation of IVPGs derived from flow color Doppler. The authors have estimated by an analytical approach that IVPG calculation requires an ideal image temporal resolution of at least 3000 Hz to ensure a risk of error of less than 10% considered clinically acceptable. However, this is significantly higher than the scanner temporal resolution used (200 Hz) in the first studies that have validated the technique by comparison to direct measurements with micromanometers10. In accordance, our results showed the impact of the temporal displayed resolution on color Doppler-derived IVPG quantification.The present study analyzed for the first time the inter-system variability of color Doppler M-mode derived IVPG assessment using two of the most common echocardiographic systems in echo labs (Philips and GE). The concordance between IVPG measurements acquired with the two ultrasound systems was weak to moderate. Sources of inter-system variability for color Doppler-derived IVPGs analysis can be multiple from the color Doppler velocity data acquisition to the image post-processing. The risk of errors caused by color-Doppler scanner resolutions has been investigatedBecause raw proprietary velocity data of color flow mapping are not routinely provided by the ultrasound systems that we tested, IVPG quantification was made on the same displayed format image. In the absence of recommendations for systolic and diastolic IVPG quantifications based on displayed JPEG data obtained from color Doppler velocity profiles, we showed that to prevent a significant underestimation of IVPGs Figs.  and 6 thAnother source of inter-system variability could also be related to the fact that IVPGs were calculated from the displayed blood flow velocity data extracted from the LUT. We ignored whether the velocity data encoded in JPEG were consistent with the raw velocity data. To test the accuracy of using displayed velocity data given by the LUT to calculate IVPGs, we analyzed the concordance between the raw proprietary velocity data and the displayed velocity data on both ultrasound systems. Our findings showed similar concordance between raw and displayed velocity data for both systems, suggesting that the use of displayed velocity data might not be responsible for major inaccuracies in IVPG measurements. However, IVPG quantification based on processed data is challenged by multivendor differences in the displayed resolution of the image.25 encouraging the learned society of echocardiography to focus on homogenizing the type of stored data used to quantify left ventricular strain. We believe that a similar reflection should be carried out on measurements based on flow color Doppler data.The present study has confirmed good overall feasibility and observer reproducibility of systolic and diastolic IVPGs assessed by an automated quantification tool using displayed data in a healthy population. However, even using a standardized method for IVPG calculation we observed low inter-system concordance, which could be related to inter-system specificities in color-Doppler signal acquisition and image resolutions. These observations raise the question of more uniformity in the color Doppler acquisition process between ultrasound vendors, and larger accessibility to raw proprietary velocity data. Our results are evocative of some concerns recently highlighted about speckle tracking-derived strain. This semi-recent technique has been widely demonstrated as a sensitive marker of LV dysfunction and patient outcomes. However recent reports have pointed out its high inter-vendor variability11. Another limation is related to our population. The small number of subjects included in our study may limit the statistical analysis. Further, our results of IVPG reliability cannot be generalized to patients with systolic dysfunction and pathological ventricular remodeling which could be associated with a modulation of the spatiotemporal velocity distribution of intraventricular flows26. However, previous studies have shown good feasibility of IVPG assessment using color Doppler M-mode in patients with heart failure and reduced ejection fraction20. In addition, we cannot exclude that the manual correction of the mitral and aortic annulus level left to the observer’s appreciation could participate in the inter-system variability. Although we have been careful to accurately assess LV landmarks during post-treatment, a misalignment near the valvular annulus plan where the velocities are the highest could induce variability in IVPG analysis. However, our quantification tool showed excellent test-retest reproducibility. To end, among other parameters that could have been a source of inter-system variability, we were not able to evaluate the equivalence of the value of priority given to the color flow between systems. We used the threshold value that was optimized by the ultrasound vendors on their commercially available machines.The first limitation of our study is related to the technique used for the non-invasive assessment of IVPGs. The validity of systolic and diastolic IVPG assessment based on the Bernoulli equation requires assuming a laminar intracardiac flow where the considered streamline coincides with the scanline. This assumption of a laminar flow has been previously validated in experimental and numerical studiesNon-invasive estimation of the instantaneous intraventricular pressure differences, closely related to the LV systolic contractile forces and early diastolic relaxation, has demonstrated its clinical potential. However, IVPG assessment using color-Doppler M-mode remains confined to research. Our automated quantification tool, which could be implemented on ultrasound machines, was able to assess systolic and diastolic IVPGs with good feasibility and high observer test-retest reproducibility in healthy volunteers. The inter-system variability, related to inter-vendor differences in image resolutions, needs to be brought to the attention of clinicians who are interested in quantitative assessment of IVPGs on displayed velocity data.Supplementary Figure 1.


To investigate the clinical value of heart failure echocardiography index (HFEI) in evaluating the cardiac function and predicting the prognosis of patients with different types of heart failure (HF).Four hundred eighty-nine consecutively admitted HF patients were divided into three groups: HF with reduced ejection (HFrEF), HF with mid-range ejection fraction (HFmrEF), and HF with preserved ejection fraction (HFpEF). The baseline characteristics and ultrasound indexes were compared between the three groups. The correlation between HFEI and one-year risk of adverse events was compared by multivariate logistic regression. The clinical value of HFEI and plasma level of NT-proBNP in assessing the prognosis of patients with chronic heart failure (CHF) was analyzed by the receiver operating characteristic (ROC) curve.HFEI in HFrEF was significantly higher than that in HFmrEF and HFpEF. Multivariate regression analysis indicated that HFEI and plasma level of NT-proBNP were independent risk factors for predicting the short-time prognosis of HF patients. The ROC curve indicated that the HFEI cutoff level of 3.5 and the plasma NT-proBNP level of 3000 pg/ml predicted a poor prognosis of CHF patients with a sensitivity of 64% and a specificity of 75% vs. 68 and 65%.HFEI can comprehensively evaluate the overall cardiac function of patients with various types of HF, and may prove to be an important index of assessing the prognosis of HF patients. Chronic heart failure (CHF) represents the final common pathway for the development and progression of most cardiovascular diseases and the leading cause of death , 2. CurrLeft ventricular ejection fraction (LVEF) and the left ventricular diastolic filling (LVDF) have long been used clinically to evaluate the LV systolic and diastolic functions respectively , 6. HoweThe 2016 European Society of Cardiology (ESC) has updated its guidelines for HF. In addition to retaining HF with reduced ejection (HFrEF) and HF with preserved ejection fraction (HFpEF), a new type named HF with mid-range ejection fraction (HFmrEF) has been added . Our stuThis study included 489 HF patients who were admitted to the Department of Cardiovascular Medicine of the Affiliated Hospital of Lanzhou University  between January 2016 and December 2018. They included 268 males and 221 females, with a mean age of 64 ± 14 years. Inclusion criteria were as follows: (1) the diagnosis of heart failure is confirmed by two or more experienced cardiologists according to the 2016 ESC guidelines;(2) we can collect comprehensive clinical data; and (3) the one-year follow-up data of the patients were complete. The exclusion criteria were as follows: (1) patients with kidney disease, liver disease or severe liver and kidney dysfunction;(2) tumor, nervous system disease; All patients signed the informed consent forms before enrollment.The demographic characteristics and comorbidities of the patients were obtained on admission. All hematological specimens including blood routine, blood biochemistry or NT-proBNP were collected within 24 h after admission and tested in the central laboratory of the hospital. All clinical data were recorded by one physician and reviewed by another.The GE Vivid 7 ultrasound system was used, with a probe frequency of 3.4–5.0 mHz. The measurement was performed by the same professional physician using the means of the results obtained from three consecutive cardiac cycles. Measurement indicators included: (1) the cardiac structure: LVEF, LV end diastolic diameter (LVEDD), interventricular septal thickness (IVST), LV posterior wall thickness (LVPWT), left atrial volume index (LAVi), diameter of left atrium (LAD) calculate relative ventricular wall thickness (RVWT) = 2 × LVPWTd/LVEDD, and LV mass index (LVMi) = LVM/body surface area; (2) cardiac function: peak velocity during early filing(E), late filling from atrial contraction (A), E/A ratio, left ventricular early diastolic flow propagation velocity (FPV), E/FPV, peak systolic mitral annular velocity(S′), E/E’S ratio, pulmonary arterial systolic pressure (PASP), early diastolic mitral flow deceleration time (DT), pulmonary venous flow (S peak systolic wave velocity and D peak diastolic wave velocity), and D/S ratio.According to the related indexes of echocardiography, the total score was HFEI. The HFEI scoring criteria are as follows: 1) 35 ≤ PASP< 50 mmHg, 1 point, PASP≥50 mmHg, 2 point; (2) LV systolic function: 30% ≤ LVEF< 45% or regional ventricular wall motion abnormality, 1 point, LVEF< 30%, 2 point; (3) LV diastolic function: ①E/A < 0.5, DT > 220 ms, D/S < 1 or E/A = 1–2, DT = 150-220 ms, D/S ≥ 1, 1 point; ②E/A > 2, DT < 150 ms, D/S > 1 or restrictive change, 2 point; (4) atrioventricular remodeling: ①56 mm < LVEDD< 66 mm, IVST or LVPWT≥13 mm, LAD≥45 mm, 1 point; ②LVEDD≥66 mm or right ventricular dysfunction; (5) valvular regurgitation or valvular stenosis: moderate, 1 point, serious, 2 point  was used for continuous variables which accord with the normal distribution and the homogeneity test of variance. The three groups of data were compared by the ANOVA test. Categorical variables were expressed as percentage and analyzed by the chi-square test. Univariate and multivariate logistic regression models were used to analyze the correlation between HFEI and the risk of one-year adverse events. The ROC curve was performed to evaluate the diagnostic performance of HFEI and NT-proBNP in patients with poor prognosis. A Four hundred and eighty-nine patients with HF were recruited and divided into three groups, including 170 in HFrEF, 171 in HFmrEF and 148 in HFpEF. There were no significant differences in age, diastolic blood pressure (DBP), blood urea nitrogen (BUN) and α-hydroxybutyrate dehydrogenase (α-HBDH) between the three groups of HF patients, while there were significant differences in HFEI and NT-proBNP between the three groups. The characteristics of the included patients are summarized in Table Analysis of the echocardiographic parameters in the three groups of patients showed that there were significant differences in the indicators that reflect the ventricular structure including LVEDD, LAD, LVMI and RWT, and in the indicators that reflect the ventricular function including E/FPV and E/E’S. No variances were found in the remaining indicators Table .Table 3Multiple logistic regression analysis was performed on variables that may affect the adverse prognosis of HF patients. It was found that NT-proBNP and HFEI were related to the risk of one-year adverse events. However, there was no correlation between LAVi, LVMi, α-HBDH and endpoint events Table .Table 4The sensitivity and specificity of NT-proBNP and HFEI to predict the risk of one-year adverse events in patients with HF were analyzed by plotting the ROC curve, and the Jordon index was calculated. When NT-proBNP was 3000 pg/ml, the area under the ROC curve was 0.698. The greatest area under the ROC curve of HFEI was 0.712, and the optimal cut-off value was 3.5  there were significant differences in HFEI between HFrEF, HFmrEF and HFpEF patients; (2) multivariate regression analysis indicated that HFEI and NT-proBNP were independent risk factors for the prognosis of one-year adverse events in HF patients; and (3) HFEI and NT-proBNP had a good value in predicting the short-term prognosis of HF patients.Clinically, LVEF and LV filling indicators are generally used to evaluate the cardiac function in HF patients, but single indexes cannot reflect the overall cardiac function, especially in patients with HFpEF and HFrEF, in whom LVEF does not always reflect the severity of HF. HFEI is a comprehensive ultrasound index for evaluating the cardiac function by taking into full account the changes in LV systolic and diastolic function, valve regurgitation and atrioventricular remodeling , 13. In NT-proBNP is a cardiac neurohormone secreted by the ventricles, playing an important role in the diagnosis and prognosis of HF , 15. HowThere are some shortcomings in this study. On the one hand, the study population was from a single center with a smaller sample size. On the other hand, the follow-up period was relatively short. Therefore, more studies with larger sample sizes and longer follow-up time are required to fully demonstrate the value of HFEI in the diagnosis and prognostic prediction of HF patients.In summary, the preliminary data obtained in this study suggest that HFEI could objectively predict the prognosis of patients with HF. HFEI and plasma NT-proBNP level are well correlated with the risk of adverse events, and combination of the two can improve the accuracy of prediction.


Echocardiography is commonly used for assessing cardiac structure and function in various species including non-human primates. A few previous studies reported normal echocardiographic reference intervals of clinically healthy rhesus macaques under sedation. However, these studies were under-powered, and the techniques were not standardized. In addition, body weight, age, and sex matched reference intervals should be established as echocardiographic measurements are commonly influenced by these variables. The purpose of this study was to establish reference intervals for complete echocardiographic parameters based on a large cohort of clinically healthy rhesus macaques with wide ranges of weight and age distributions using allometric scaling.b. On correlation matrix, body weight, age, sex, and heart rate were significantly correlated with various echocardiographic parameters and some of the parameters were strongly correlated with body weight and age. Multiple regression analysis revealed that heart rate and body weight statistically significantly predicted several echocardiographic parameters. Valve regurgitation including tricuspid, aortic, pulmonic, and mitral regurgitations without other cardiac structural and functional abnormalities are common in clinically healthy rhesus macaques under ketamine sedation.A total of 823 rhesus macaques  were enrolled. Of these rhesus macaques, 421 were males and 402 were females. They were assessed with a complete echocardiographic examination including structural and functional evaluation under sedation with ketamine hydrochloride. The reference intervals of the key echocardiographic parameters were indexed to weight, age, and sex by calculating the coefficients of the allometric eq. Y = aMIn this study, the reference intervals of echocardiographic parameters were established by performing complete echocardiographic examinations on a large number of clinical healthy rhesus macaques. In addition, allometric scaling was performed based on their weight, and further indexed to age and sex. These allometrically scaled reference intervals can be used to accurately evaluate echocardiographic data in rhesus macaques and diagnose structural and functional evidence of cardiac disease. Echocardiography is a noninvasive imaging technique commonly used for assessing cardiac structure and function. It includes the measurement of cardiac chamber size and ventricular wall thickness, and an assessment of valve structure and function, systolic and diastolic function, and blood flow patterns in the heart. In clinical settings, findings from complete echocardiographic assessment are used as important information to determine medical and surgical treatment of cardiac diseases. In research settings, echocardiographic evaluation of subjects is inevitable to fully assess the cardiac condition for cardiovascular and non-cardiovascular research.A few previous studies reported normal echocardiographic reference ranges of clinically healthy rhesus macaques under sedation , 2. In aThe purpose of the present study was to establish reference intervals of key echocardiographic parameters based on a large cohort of clinically healthy rhesus macaques with wide ranges of weight and age distributions and establish these reference intervals with allometric scaling to BW. They are also indexed to other patient characteristics including the sex and age. This cohort of rhesus macaques represents the largest sample size for establishing echocardiographic parameters in the literature in any other veterinary patients.A total of 823 rhesus macaques were enrolled in the present study. The average age (+/− standard deviation [SD]) and BW (+/−SD) were 7.8 (+/− 5.4) years and 8.49 (+/− 3.52) kg, respectively. The mean heart rate (HR) during the echocardiographic studies was 134 (+/− 24.6) bpm. Of 823 rhesus macaques, 421 rhesus macaques were male and 402 were female. The mean age (+/− SD) in males and females were 7.9 (+/− 5.4) years and 7.8 (+/− 5.4) years, respectively. The mean BW in males and females were 8.46 (+/− 3.54) kg and 8.46 (+/− 3.53) kg, respectively. The mean HR during the studies was 133 (+/− 24.6) bpm in males and 134 (+/− 24.6 bpm) in females.None of the rhesus macaques had evidence of significant cardiac structural abnormalities or moderate to severe valve regurgitation. None of the rhesus macaques had systolic dysfunction determined based on left ventricular fractional shortening (LV FS) and left ventricular ejection fraction (LV EF). However, some of the older animals in the study group had evidence of diastolic dysfunction without other structural and/or functional abnormalities, which could be attributed to the age-related findings based on previous studies in rhesus macaques and other species . Of the Intra-observer measurement variability for all echocardiographic variables measured in 10 rhesus macaques was between 0 and 6.5%, and interobserver measurement variability was between 0 and 10.7%.r = − 0.011, P = 0.8).Mean, SD, range, and reference interval with 90% confidence intervals of upper and lower limits for each two-dimensional 2D) and M-mode echocardiographic measurement are listed in Table D and M-mMultiple regression analysis was done to predict various echocardiographic parameters from weight, age, sex and HR. Among these animal characteristics, weight and HR statistically significantly predicted various echocardiographic parameters  of animals and lack of quantitative or qualitative structural heart changes expected with these conditions  determined prediction intervals, where a, t, and Sx,y represent the proportionality constant, desired Student’s t-statistic for n-2 degrees of freedom, and the standard error of the Y estimate, respectively.Linear echocardiographic variables were normalized to BW (kg) based on the constants from power equation, Y = aXP-value of < 0.05 was considered as significant for all analyses.Statistical analysis was performed using commercially available softwares . A Additional file 1: Supplement Table 1. Echocardiographic parameters showing significant correlations with age, body weight, heart rate, or sex listed as correlation coefficient (r) with the P-value in parenthesis. Supplement Table 2. Results of multiple linear regression analysis of echocardiographic parameters with age, body weight, heart rate, or sex. Supplement Table 3. Body weight-based means and 95% prediction intervals of 2D and M-mode echocardiographic parameters derived from allometric scaling parameters in 421 male rhesus macaques. Supplement Table 4. Body weight-based means and 95% prediction intervals of 2D and M-mode echocardiographic parameters derived from allometric scaling parameters in 402 female rhesus macaques. Supplement Table 5. Body weight-based means and 95% prediction intervals of 2D and M-mode echocardiographic parameters derived from allometric scaling parameters in 328 rhesus macaques in the range of 5 months to 4 years old. Supplement Table 6. Body weight-based means and 95% prediction intervals of 2D and M-mode echocardiographic parameters derived from allometric scaling parameters in 264 rhesus macaques in the range of 5 years to 9 years old. Supplement Table 7. Body weight-based means and 95% prediction intervals of 2D and M-mode echocardiographic parameters derived from allometric scaling parameters in 137 rhesus macaques in the range of 10 months to 14 years old. Supplement Table 8. Body weight-based means and 95% prediction intervals of 2D and M-mode echocardiographic parameters derived from allometric scaling parameters in 94 rhesus macaques over 15 years old.


Cardiac MRI determined blood oxygen level dependence oxygenation (n = 8) after 4 min of apnea was unaltered compared to rest, whereas cine-MRI demonstrated increased left ventricular wall thickness (LVWT). Arterial blood gases were collected after warm-up and maximum apnea in a pool. At the end of the maximum pool apnea (5 min), arterial saturation decreased to 52%, and lactate decreased 20%. Our findings contrast with previous MR studies of BHD, that reported elevated cardiac troponins and decreased myocardial perfusion after 4 min of apnea. In conclusion, we demonstrated for the first time with 15O-H2O-PET/CT and MRI in elite BHD during maximum apnea, that MBF and LVWT increases while lactate decreases, indicating anaerobic/fat-based cardiac-metabolism similar to diving mammals.Breath-hold divers (BHD) enduring apnea for more than 4 min are characterized by resistance to release of reactive oxygen species, reduced sensitivity to hypoxia, and low mitochondrial oxygen consumption in their skeletal muscles similar to northern elephant seals. The muscles and myocardium of harbor seals also exhibit metabolic adaptations including increased cardiac lactate-dehydrogenase-activity, exceeding their hypoxic limit. We hypothesized that the myocardium of BHD possesses similar adaptive mechanisms. During maximum apnea  It has also been demonstrated that similar to adaptions in the northern elephant seal, which can endure apnea for more than 120 min, BHD have a non-coupled maximum respiratory state in their skeletal muscles ~ 22–23% lower than matched aerobic athletes7. Hence, the skeletal muscles of BHD possess an oxygen conserving adaptation, and this might—at least partly—explain, the extreme performances of BHD holding their breath for more than 11 min and swimming as far as 300 m, or diving beyond 200 m in depth, all on a single breath of air (http://www.freedive-earth.com/aida-freediving-world-records). Similarly, samples of the skeletal muscles and the heart from adult harbor seals (Phoca Vitulina) shows up to sixfold higher β-hydroxyacyl-CoA dehydrogenase activities normalized to tissue-specific resting metabolic rate, and the heart of the seals possess the highest lactate dehydrogenase activity, compared to dog and rat, suggesting both a reliance on anaerobic metabolism during dives exceeding the animal's aerobic limit, but also a high aerobic capacity for lipid metabolism within the heart8. It could be hypothesized, that the hearts of BHD would possess similar adaptions. However, two cardiac magnetic resonance imaging (CMR) studies of BHD reported that after only 4 min of dry apnea concomitant with a reduction in arterial oxygen saturation ~ 23%, cardiac troponins increase, and CMR epicardial perfusion decreases about 60%10. We have reported of elite BHD enduring a reduced arterial oxygen saturation of more than ~ 33%4, and after competitions with pool static apneas of ~ 5 min (and subsequent 88 ± 21 m underwater swim), where cardiac troponins remains unchanged normal11. The explanation might be, that BHD who can endure apnea for more than 4 min have a lesser increase in ischemia-modified albumin (IMA), a marker of the release of reactive oxygen species (ROS) and a measure of resistance to hypoxemia, than compared to BHD who can endure less than 4 min of apnea12. This is in accordance with studies of adult seals are demonstrated to have a higher relative capacity for mitochondrial respiration than pups and juvenile seals13, and adult diving mammals also have resistance to release of ROS during dives to tolerate ischemia15, indicating that the hypoxic capacity is an adaption.The physiological adaptions in breath hold divers (BHD) to apnea, includes significant diving responses, attenuated post-apnea acidosis, reduced sensitivity to oxidative stress and, probably critical to their performances, reduced sensitivity to hypoxia15O-H2O-PET/CT and the resulting myocardial oxygenation as a sign of ischemia measured using BOLD CMR at rest and during a maximal voluntary apnea in elite BHD with a self-reported maximum static apnea of more than 5 min.Hence, it is hypothesized that similar adaptive mechanisms to hypoxia as observed in the hearts of adult diving mammals could be observed in the hearts of human elite BHD, who can endure apnea for more than 4 min. This study aimed to demonstrate this hypothesized adaptation in BHD. As myocardial ischemia is dependent on both blood oxygen levels, perfusion and the need of oxygen based on cardiac contraction, these parameters were quantified using arterial samples, cine CMR and This study included eight Danish healthy male non-smoking subjects and was approved by the Regional Ethics Committee of Copenhagen (H-1-2013-060). All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Informed consent, written and oral, were obtained from the participants.All subjects were elite BHD (age 42 ± 8 years) able to hold their breath for more than 5 min and ranked among the national top 10. Three ranked among the World top 10, one was a 2016 outdoor free-diving World champion, one reached third place at the same championship (no limit depth competition), and one was a World record holder.2 and CO2 concentrations in the expired gas were continuously measured breath-by-breath  during the test. The highest recorded 30 s average oxygen uptake (VO2) during the test was defined as VO2max. For recognition of true VO2max three of five criteria had to be met: individual perception of exhaustion, respiratory exchange ratio > 1.15, plateau of VO2 curve, heart rate approaching age-predicted maximum and inability to maintain a pedaling frequency above 70 rpm. A dual-energy X-ray absorptiometry scan  was performed to assess body composition.Subjects completed a standardized warm-up followed by an incremental cycling test starting at a workload of 150 W and increasing 25 W every minute until voluntary exhaustion. Pulmonary O4. Cine images were acquired at (1) rest during a short (< 20 s) apnea at end-expiration with open pharynx, (2) end of a apnea with max expiration and (3) the end of dry static apnea after glossopharyngeal insufflation (GPI)16. Images were collected shortly before end of apnea before breathing, and subjects were instructed to stay as calm as possible during imaging to avoid imaging artefacts. Cardiac chamber volumes and function were collected in the transversal and double-oblique short axis stacks with 8 mm thick slices and 25% gap. Cine imaging were performed with retrospectively ECG-gated steady-state free precession sequences (SSFP) reconstructed to 25 phases covering the entire cardiac cycle using the following settings: TR/TE 3.3/1.6 ms, flip angle 60°; and spatial resolution 1.3 × 1.3 × 8 mm3. For blood oxygen level dependence (BOLD) imaging a fast gradient-echo multi-echo black blood T2* sequence was used in the short axis plane18. The BOLD sequences were acquired at rest and after 240 s of apnea, to ensure image collection on all subjects, as the duration of this could be up to 20 s. BOLD had the following settings: TR 25.8 ms with effective repetition time 1600 ms and collecting 14 images with TE from 3.2 to 24.1 ms, flip angle 20°; and spatial resolution 1 × 2 × 1.2 × 8 mm3.For one day prior to the study, the subjects refrained from physical exercise and consumption of alcohol or caffeine. Imaging was performed in a 1.5 T MR imaging system  after a warm-up of three consecutive apnea to maximize the diving response19. LV wall thickness (LVWT) was determined from a midventricular short-axis slice from the epicardial and endocardial borders delineations of the left ventricle at end-diastole. Stroke volume (SV) was computed as the difference between end diastolic volume (EDV) and end systolic volume (ESV). Ejection fractions were computed as SV divided by EDV. T2* was determined from the BOLD images using a LV septal midwall region of interest carefully avoiding image artefacts and using a fitting algorithm in the Segment software as previously described20.Left and right ventricular (LV and RV) and atrial (LA and RA) volumes were determined in end-diastole (ED) and end-systole (ES) and used to determine the influence of apnea on cardiac volumes, function, filling and individual chamber outflow. Data were analyzed by one experienced level three CMR reader and one level one CMR cardiologist using dedicated software (Segment version 2.1) carefully avoiding image artefacts and imaging especially during involuntary breathing movements4, all subjects performed a maximum static pool apnea to simulate apnea during natural circumstances; (1) before, (2) after 4 min of apnea, and (3) just before end of maximum apnea, arterial blood samples were obtained through a catheter  in the radial artery of the non-dominant arm with connection to a transducer for continuous flow of saline . Blood gas analyses were performed immediately after sampling, using an automated self-calibrating blood gas machine  and evaluated for pH, bicarbonate, lactate, PaCO2, and the arterial oxygen tension (PaO2).On a separate day, after a warm-up of three consecutive apneas to maximize the diving responseArterial blood pressures and heart rates were recorded concomitantly using a Lifepack ® 20 monitor.Blood samples for lipid analysis were collected following 8 h of fasting from the radial artery as described above for arterial blood gas collection, and before the pool dive. Total Cholesterol, High-density lipoprotein, Low-density lipoprotein and Triglycerides were measured with an automated analyzer  using commercially available kits.15O-H2O-PET/CT study. The participants in the 15O-H2O-PET/CT sub-study were required to be able to hold their breath for 5 min while lying in the PET/CT scanner in the supine position with arms raised above the head. They were instructed to refrain from intake of chocolate, to refrain from strenuous physical exercise for one day and to be fasting for at least 6 h before the study.The six subjects with the longest pool maximum apneas, were recruited for an additional 15O-H2O-PET/CT data were obtained in list mode on a GE Discovery MI Digital Ready PET/CT system . The participants underwent three image acquisitions: (1) at rest, (2) during hyperemia induced by a dry static apnea after GPI16 and after a warm-up of three consecutive apneas to maximize the diving response4, and (3) third in the recovery phase 4 min after the apnea . For the stress acquisition, 15H2O was infused after 2 min (n = 3) or 4 min (n = 3) after initiation of the apnea with acquisition of data for at least an additional 2 min  at time points as specified above. The 4-min dynamic images were reconstructed in a 3.27 × 3.27 × 3.27 mm matrix utilizing all normal corrections  and the VPFX-S reconstruction algorithm. For subsequent analysis, the dynamic scan was divided into 21 frames . Kinetic analyses were performed using aQuant software  and a validated 1-tissue compartment model with image derived input from cluster analysis, corrections for spillover and automatic estimation of MBF and perfusable tissue fraction (PTF)21. In healthy subjects, PTF primarily reflects the incomplete estimation of true tissue radiotracer uptake , which is a particular challenge in subjects with diaphragmatic movements and therefore reported. This parameter is derived from the tracer inflow, whereas MBF is derived from the outflow curve  was quantified by 15O-H2O-PET/CT sub study resulted in a power of 80% to detect an expected increase in MBF of at least 1 ml/g/min with a standard deviation of 0.422.To test for imbalances in the variables in our study with a limited number of subjects, we performed a power calculation: A P-value < 0.05 was considered statistically significant. Given this level of significance, our sample size of 6 subjects in the Variables are presented as mean ± standard error of the mean (SEM). Data were analyzed by Sigma-Plot ® using one-way repeated measures ANOVA. Kruskal–Wallis with Tukey’s methods posthoc respectively were used to evaluate differences between the collected data during rest, apnea, and recovery. Time-related changes in MBF, PTF and MBF × PTF interaction in the 2- and 4-min groups were analyzed using a mixed model repeated measures ANOVA.2max) and lipids, are displayed in Table The morphometric variables age, height, body mass, whole-body aerobic capacity (VO2 rose from 5.2 ± 0.2 kPa to 7.0 ± 0.2 kPa (P < 0.001) concomitant with a decrease in PaO2 from 12.2 ± 0.5 kPa to 4.3 ± 0.4 kPa (P < 0.001), and a decrease in pH from 7.42 ± 0.029 to 7.35 ± 0.01 (P < 0.001) and a decrease in lactate from 1.70 ± 0.13 to 1.36 ± 0.18 (P = 0.013).After GPI and 326 ± 19 s of static apnea in a pool, heart rate (HR) decreased from 86 ± 5 bpm to 50 ± 13 beats/min  induced stress  at end of max pool apnea, systemic oxygenation decreased to 4.3 kPa ± 0.4 and pH decreases concomitant with a decrease in lactate in BHD. This has to our knowledge not previously been described. Hence, we demonstrated for the first time that BHD, similar to adult diving mammals, possess cardiac hypoxic resistance and increased myocardial blood flow with CMR and 15O-H2O-PET/CT during apnea ~ 6 min, and also lactate metabolization during max pool apnea. Our study also demonstrated that at end of max apnea with GPI, mean arterial blood pressure increased, and HR, biventricular EDV, CO and EF decreased, concomitant with an increase in LV wall diameter and an increase in PaCO2.The main and novel findings of our study are: After a warm-up of three consecutive apneas and 1) during 4 min of apnea, the hearts of BHD in this study were without signs of concomitant T2* BOLD assessed myocardial ischemia on CMR, 2) after 4 min of apnea 2 is increased, myocardial perfusion and oxygenation remains preserved or even increased during apnea episodes of 60 s in anaesthetised animals and during voluntary apnea of up to 117 s in healthy humans28: Longer apneas causes higher increases in BOLD-signal intensity and a decline in capillary PO2 is compensated through a CO2-mediated increase in MBF as a countermeasure for desaturation and prevention of compromised myocardial oxygenation during long effective apneas28. During repetitive apnea in BHD combined with 100 W exercise, it has been demonstrated that PaO2 decreases to 7.3 kPa and PaCO2 increases to 8.2 kPa4 concomitant with a decrease in pH to 7.35. In this study, PaO2 decreased to 4.3 ± 0.4 kPa while PaCO2 increased to 7.0 ± 0.2 kPa and pH decreased to 7.35 during max apnea (326 ± 19 s). In part, this adaption to hypoxia and hypercapnia may explain the findings of sustained myocardial oxygenation and the increased MBF during apnea in our study, and the latter is comparable to increases in MBF during max exercise in endurance trained athletes29.Guensch et al. demonstrated, that when PaCO10, and Eichhorn et al. reported that 4–5 h after a similar period of apnea and a decrease in PaO2, ~ 23% in BHD, cardiac troponins elevate9. This study could not confirm these observations in elite BHD with personal records of static apnea of more than 5 min, and after a similar decrease in PaO2, after 4 min of apnea and at end of max apnea. Our observations are also consistent with animal models demonstrating that after the onset of myocardial ischemia, cell death is not immediate but takes a finite period to develop: it has been reported that the level of cardiac troponins I (cTnI) starts to increase by 30 min with a peak value at 3 h after cardiac arrest in adult pigs30 and 4 h of hypoxia in rats has no effect on coronary flow rate or cardiac troponin T31. cTNI  also remain unaffected in competitive BHD 3 h after two consecutive maximum apneas (static apnea ~ 5 min and subsequent ~ 88 m underwater swim) . The question is, why the otherwise highly adapted BHD, possessing extreme tolerance for hypoxia7 including a mitochondrial adaption to hypoxia in their skeletal muscles similar to the northern elephant seal7, should display or suffer from cardiac injury after only 4 min of apnea10? However, high altitude permanent residents, chronically exposed to hypoxia, suffer more often from acute coronary syndromes than comparable low altitude populations, probably due to hyperlipidemia32 and hence arteriosclerotic coronary disease, and it may be, that the BHD studied by Kyhl et al. and by Eichhorn et al., were not as cardiac healthy as the subjects with normal lipids33 and low resting MBF34 in our study , a marker of the release of ROS during hypoxemia, increases less in the BHD who endured apnea for more than 4 min, than compared to those who endure less than 4 min of apnea12. Our study included only BHD who endured apnea for more than 5 min, and our results indicated that they have a similar resistance to cardiac hypoxia as observed in adult diving mammals8. Our results included increased myocardial perfusion and are in contrast to the studies by Eichhorn et al. and Kyhl et al. of BHD enduring apneas of 4 min or less and reporting signs of myocardial injury and reduced myocardial perfusion10. However, studies of restrained and sedated seals demonstrate severely reduced myocardial perfusion during static apnea35, but sedation depresses the cardiovascular system including the myocardial regional oxygen supply36, and cannot be compared to the foraging animal with an increased metabolic demand during their dives. Compared to the lung tissue and skeletal muscles, seal hearts have the lowest levels of Hypoxia-Inducible Factor-1alpha, an adaptive protein to protect against hypoxia38. This indicates, that the hearts of seals, also sensitive to localized ischemia39, do not have decreased myocardial perfusion during their dives, as it would cause myocardial damage. Elite BHD performing maximum apnea, also go through a struggling phase, with muscular tensions, hence also an increased metabolic demand, which in part may explain the increased myocardial perfusion at end of apnea in our study.Diving seals are exposed to repetitive cycles of ischemia and following reperfusion. Similarly sleeping seals experience sleep apnea, but do not increase production of ROS nor suffer systemic or local oxidative damage40. Gormsen et al. demonstrated that ketone bodies increased myocardial blood flow 75% without affecting myocardial fatty acid transport or oxidative capacity in the myocardium of healthy subjects41. A novel study demonstrated that ketone bodies can even increase CO and LVEF in patients with heart failure without impairing myocardial external energy efficiency42. These observations are in line with our results of elite BHD, demonstrating that after a warm-up and during a consecutive maximum apnea, myocardial perfusion increases, concomitant with a decrease in pH and also a decrease in lactate. A decrease in lactate may be the consequence of decreased production, a higher metabolic rate or both, whereas hypercapnia-induced respiratory acidosis during exercise promote a decrease in blood pH43, but a respiratory acidosis have negligible effects on the lactate response44. We interpretate the concomitant decrease in pH as a consequence of the increase in PaCO245 during decreasing lactate in this study, the latter may indicate lactate metabolization, as discussed below. Similarly, the hearts of harbor seals have a heightened lactate dehydrogenase activity indicating an adaptation for the anaerobic production of adenosine triphosphate (ATP) on dives beyond the animal's aerobic dive limit8. Lactate is cumulated during aerobic exercise, and can be metabolized after exercise by skeletal muscle, heart, liver, kidneys, brain, adipose tissue and lungs46. However, Kaijser et al. demonstrated that lactate is the primary substrate for oxidation of the heart muscle cells during short-term normoxic heavy exercise in normal young subjects at sea level47, and similarly the subjects in our study performed maximum apnea with increases in MBF comparable to the increases in MBF in endurance trained athletes during maximum exercise29. Sherpas living in high altitude with chronic hypoxic exposure have also been demonstrated to decrease lactate levels during maximum exercise suggestive of lactate metabolization49. Hence, we suggest, that the elite BHD in our study have an adaption to metabolize lactate during hypoxia similar to these high-altitude Sherpas and the harbour seal cardiac mitochondria8.Fatty acids are the major energy source for healthy myocardium, but lactate and ketone bodies, can be the energy substrate under certain circumstances51. Exposing the myocardium to acute ischemia and reperfusion causes initially mitochondrial dysfunction and subsequent excessive production of ROS by the electron transport chain50. The deleterious effects of ROS include damage to the mitochondria52, resulting in depolarization and apoptosis53. These processes may ultimately lead to a compromised cardiac function: Chronic ischemic hearts in animal models also possess a decreased capacity for oxidative phosphorylation57, whereas hypoxia in a hypobaric environment may trigger increased cardiac carbohydrate metabolism and enhanced mitochondrial respiratory capacity, even with preserved contractile function58. Stride et al. demonstrated that chronic ischemic human hearts possess a decreased intrinsic mitochondrial respiration59, which ultimately may lead to failure of the left ventricle60. In this study of elite BHD during hypoxia, no signs of myocardial ischemia could be demonstrated by CMR or myocardial perfusion deficits by 15O-H2O-PET/CT, despite a decline in ejection fraction, and hence declined myocardial contractile function concomitant with bradycardia at end of apnea. We suggest that the resistance to even more extreme cardiac hypoxia in our study, than found in previous similar studies10, at least in part confirms our hypothesis, of elite BHD possessing a cardiac adaptive resistance to hypoxia.Mitochondria are central to synthesis of ATP and ROS, and hence also important to myocyte death61. At the end of apnea in our study, LVEDV decreased by ~ 49%, concomitantly with an ~ 31% increase in LV wall thickness and a ~ 49% decrease in LVCO which is consistent with the findings in similar studies of BHD10. Our findings are also comparable to observations in diving adult harbor seals in which thermodilution measured stroke volumes at surface are almost twice the volumes compared to when submerged62.Swine LV volume in a CMR closed-chest model have been demonstrated to decrease ~ 53% during 25 min of untreated ventricular fibrillation, while the mean interventricular width or LV wall thickness markedly increasesIn conclusion, our study demonstrated for the first time cardiac hypoxic resistance and increased MBF in the hearts of elite BHD, during apnea of more than 5 min concomitant with systemic hypoxia below 4.3 kPa ± 0.4 and decreasing lactate. Our results indicate an increased ability for anaerobic and fat-based metabolism in the hearts of elite BHD during hypoxia similar to the harbor seal during routine diving. In elite BHD, biventricular EF and SV decreases at end of maximum apnea concomitant with bradycardia, similar to CMR of hypoxic diving mammals during apnea. Hence, the results of our study confirm, that the hearts of elite BHD have similar adaptions to hypoxia as air breathing diving mammals.Future studies may reveal myocardial oxygen extraction at end of apnea in elite BHD, and may confirm presumed hyperdynamic LVEF due to lactate metabolization after apnea in elite BHD.Furthermore, it is speculated that understanding of these mechanisms may help in the development of new treatments of severe clinical conditions caused by extreme hypoxia, e.g. cardiac surgery, organ transplantation and in the post-resuscitation care setting.15O-H2O-PET/CT and blood gases in the pool: BOLD-MR and blood gases were both collected at 4 min of breath-hold to be able to compare the results. 15O-H2O/PET-CT for absolute perfusion cannot be done at one time point but is collected over several minutes. Therefore, we collected data from 2 to more than 8 min of breath-hold. Subjects insisted on drinking coffee on the day for apneas before 15O-H2O-PET/CT, and therefore MBF may be impaired compared to fasting from caffein64. In one subject, resting and recovery 15O-H2O-PET/CT scans were lost due to technical difficulties.The number of subjects was low because the inclusion criteria required the BHD to have very long apnea, and this may give problems with statistical power. A correlation analysis would be of interest to find a possible graded difference in diving response between subjects, but this would need to have a larger number of subjects with larger differences in levels of apnea. However, the findings were for most analyses statistically significant because of the pronounced physiological effects and each subject being its own control. BOLD on the other hand was not different during apnea compared to baseline and thus this finding should be viewed with some caution due to few subjects. The length of the apneas were different for MR, 65. However, data collected in our study were both dry and wet static apnea, and a difference may be observed in the cardiovascular responses, if all data could be collected during wet apnea65.Apnea during exercise with facial immersion have been demonstrated to increase the cardiovascular responses compared to apnea and exercise without facial immersion


Since 2003 our unit has adopted an imaging protocol for all infants with hypoplastic left heart syndrome (HLHS), which includes CMR imaging for inter-stage assessment prior to the formation of a bidirectional cavo-pulmonary shunt. The aim of this study was to assess whether the CMR data acquired during this protocolised follow-up could help to stratify the risk for these patients.We assessed all locally followed patients, who had undergone the Norwood procedure for HLHS between January 2003 and May 2008, and who had undergone CMR imaging according to unit protocol.Imaging was performed under general anaesthetic, using a 1.5 T MR scanner, and a combination of cine sequences, phase contrast flow sequences and gadolinium-enhanced MR angiography. From short axis cine images, manual segmentation of the ventricles was completed, giving ventricular volumes, ejection fraction and cardiac output (CO). Arterial measurements were made from the isotropic angiographic data using 3D analysis software. The pulmonary artery (PA) measurements were made at the proximal native vessel and distally, just prior to the 1st lobar branch. Aortic measurements were made at the narrowest point of the proximal descending aorta (CoA) and at the diaphragmatic-level descending aorta. At each site the shortest and orthogonal cross-sectional diameters were averaged, to correspond with conventional 2D measurement methods. Additionally, the exact cross-sectional area of the vessel at each point was measured using manual planimetry. The coarctation (CoA) index was defined as the (CoA measurement/diaphragmatic aorta measurement), for vessel diameter and planimetered area respectively.st October 2008). Secondary, functional outcome measures were RV ejection fraction (RVEF), and CO.The primary outcome measure was survival to analysis date  days, and 5 (3.2 – 11) kg respectively. The planimetered area index of vessels was smaller than averaged orthogonal diameter index. The mean ratio of the CoA diameter index to the CoA area index was 1.4 (95% CI 1.25 – 1.55), indicating that diameter measurements may underestimate true narrowing. The PA size and indices of proximal stenosis did not correlate with outcome. The median CoA area index for all patients was 0.52 (0.22 – 1.0). Twenty-one (70%) patients had a CoA area index < 0.7, with no difference between the Sano-type and conventional Norwood surgery. There was a significant correlation between the CoA diameter index and cardiac output -1.09 (95%CI -2.17 – -0.02) (p = 0.04), but the CoA size indices were not correlated with RVEF. The median RVEF for this cohort was 53% (30 – 81%). Eight patients had RVEF < 50%. There were 7 deaths in this cohort, during a total follow-up time of 67 person years. The RVEF was strongly predictive of death, with a hazard ratio 0.92 (95% CI 0.86 – 0.99) (p = 0.02). Figure This study shows that death is a more likely outcome in HLHS patients with a lower RV ejection fraction at inter-stage CMR. Other CMR-measured factors such as CoA and PA size indices did not predict outcome. Measures to preserve RV systolic function, and CMR assessment of this, should be paramount in the complex management of these patients.


Cor pulmonale is the condition in which the right ventricle undergoes morphological and/or functional changes due to diseases that affect the lungs, the pulmonary circulation, or the breathing process. Depending on the speed of onset of the pathological condition and subsequent effects on the right ventricle, it is possible to distinguish the acute cor pulmonale from the chronic type of disease. Echocardiography plays a central role in the diagnostic and therapeutic work-up of these patients, because of its non-invasive nature and wide accessibility, providing its greatest usefulness in the acute setting. It also represents a valuable tool for tracking right ventricular function in patients with cor pulmonale, assessing its stability, deterioration, or improvement during follow-up. In fact, not only it provides parameters with prognostic value, but also it can be used to assess the efficacy of treatment. This review attempts to provide the current standards of an echocardiographic evaluation in both acute and chronic cor pulmonale, focusing also on the findings present in the most common pathologies causing this condition. Cor pulmonale can be defined as the clinical setting in which the right side of the heart, in particular the right ventricle (RV), is affected by a pressure overload that induces changes of RV function and morphology. Depending on the duration of time in which the increased RV afterload is established, it is possible to distinguish an acute cor pulmonale from a chronic form of the disease. The most common condition responsible for the acute type is acute pulmonary embolism (APE), whereas the chronic cor pulmonale is usually caused by chronic obstructive pulmonary disease (COPD) , followeAcute cor pulmonale might be described as the clinical setting in which the RV afterload rapidly increases, causing dilatation and/or impairment of RV function. In clinical practice, the most common condition responsible for this scenario is APE where an acute obstruction of 30–50% of the cross-sectional area of the pulmonary arterial bed, together with hypoxia-induced vasoconstriction, is responsible for the abrupt increase of pulmonary vascular resistance (PVR), which greatly affects RV function. Table According to the latest European Society of Cardiology Guidelines for the diagnosis and management of APE , echocarBesides the direct assessment of specific signs of embolism, such as mobile thrombi in the RV or right atrium (RA), which occurs in approximately 4% of patients , there aRV dilatation is one of the main indirect findings in APE  with the severity of the pulmonary vascular bed occlusion [cclusion .TAPSE and tricuspid s’ velocity have shown an inferior sensitivity and specificity and are sometimes normal in acute cor pulmonale. In fact, their reduction might be present only in cases with significant hemodynamic impairment [However, some standard echocardiographic parameters of RV longitudinal function, like pairment , and, aspairment , while spairment . This fipairment  and RV fpairment . The acupairment .Trans-tricuspid E/A ratio is significantly reduced in the presence of increased RV pressure, and it generally presents a gradual return to normal values once the obstruction is resolved, likewise RVFAC and TAPSE, even though the latter has been shown not to completely recover during follow-up [ollow-up .tricuspid e′ velocity is impaired even in the presence of normal RV systolic pressures, tricuspid s′ velocities and RV filling pressures, which might highlight its role as an early marker of RV dysfunction [Additionally, function .Besides its diagnostic role, RV dilatation and dysfunction, identified by echo in hemodynamically stable APE patients, play a key role in disease severity evaluation and risk stratification, in addition to laboratory markers of myocardial damage . Also, fRV Tei index and free wall RV strain by STE, have been independently correlated with mortality, whereas such correlations have not been shown for any RV diameter, TAPSE or RVFAC [Moreover, recent technological advances in echocardiography (such as 3DE and STE) have allowed, in patients with adequate acoustic windows, a more accurate evaluation of RV geometry, dimensions, and systolic function, both regional and global, thus overcoming many of the limitations and assumptions necessary for the standard 2D evaluation  is a manifestation of regional wall motion abnormalities and is more likely to occur in acute cor pulmonale due to a sudden pressure overload. Its assessment may play a key role in the differential diagnosis of acute from chronic cases, and the standardization of RV-MD quantitative measurement performed by STE could be superior to visual inspection or other qualitative assessments [essments , 23.McConnell’s sign, which consists of hypo-akinesia of the mid RV free wall with a normal function of the apex, show a lower absolute value of RV free wall apical segmental strain compared with controls [To this point, Vitarelli et al. demonstrated that in submassive APE, the impairment of RV segmental synchrony is associated with alterations of both right ventricular ejection fraction (RVEF), calculated by 3DE, and mid-free wall RV strain . This recontrols . This micontrols . Interescontrols .Regarding the RA, evidence of its role in acute cor pulmonale is less consistent compared with the RV. In intermediate-risk APE patients with different morphological and functional alterations have been identified, such as an increased RA area, reduced RA strain by STE, and impaired RA/RV mechanical coupling relationship . HoweverChronic cor pulmonale refers to the clinical setting in which the RV has to face a progressively increasing afterload, which then determines its extensive dilatation and remodelling. Although the underlying causes may be different, PH is the common denominator of all the diseases that leads to chronic cor pulmonale. PH is defined as an increased mean pulmonary arterial pressure (PAP) ≥ 25 mmHg at rest, assessed by right heart catheterization . CurrentRV end-systolic volume as a stronger predictor of outcomes compared with RV longitudinal strain [RV end-systolic remodelling index, defined as the ratio between end-systolic RV free wall and interventricular septum length, which was proven to have a strong prognostic value and could possibly help in the risk stratification of these patients [Several studies have demonstrated the prognostic role of echocardiography in the setting of PH, through parameters, such as TAPSE, RA size, the presence of pericardial effusion or RV longitudinal strain by STE , 33. Spel strain . Similarpatients . Furtherpatients . In thispatients . In addipatients . The detpatients .Fig. 4.MThe role of cardiac fibrosis in developing overt RV failure in PH is not unequivocal, with both positive and negative features associated, as thoroughly analyzed by Andersen et al. . In thisAnother aspect to consider is that the RV dysfunction might be concealed at rest and become evident during exercise, highlighting the role of stress testing in the assessment of the RV. In fact, exercise echocardiography provides useful information on how hemodynamic changes during exercise in patients affected by or at risk of PH . Indeed,The RA plays a key role in chronic cor pulmonale, since the augmented RV afterload induces a progressive RA dilatation, probably caused by impaired filling of the RV. Indeed, RA size has found to be a predictor of poor outcome in PH . In addiCOPD is a chronic and progressively disabling disease, characterized by persistent respiratory symptoms and an irreversible clinical course, usually caused by significant exposure of the airways and/or alveolar department to noxious agents . PH is aIPF is the most common among the idiopathic interstitial pneumonias , 65 and CTEPH is characterized by a progressive occlusion of branches of pulmonary arteries by organized thrombi, which consequently leads to the onset of PH and is classified in group 4 according to current guidelines . RegardiThe distinction between acute and chronic cor pulmonale is mainly based on patient history and clinical examination, although echocardiography always provides additional information and can sometimes play an important part. However, two conditions that might be hard to differentiate are APE and CTEPH. Whether echocardiography alone is able to distinguish one condition from the other is a difficult answer. In fact, current evidence is scarce and sometimes contradictory. Some studies that have tried to shed some light on this matter revealed that, both in the acute and chronic settings, the RV is affected by mechanical dyssynchrony , 23 thatIn the context of both acute and chronic cor pulmonale, it is essential to stress that echocardiography fulfils a role that goes beyond a diagnostic and prognostic purpose. Indeed, the echocardiographic exam could help the clinician to identify the patients that could benefit from a particular treatment and most importantly could attest the success of the adopted therapeutic strategy. In the acute setting, the reduction of RV size and its recovery, as well as the reduction of systolic PAP, are parameters that could indicate that the anticoagulant regimen has been effective. Moreover, after the acute episode, as the current guidelines recommend , it is iThe MESA COPD study defined a new RV phenotype in COPD patients, termed “cor pulmonale parvus,” defined by a lower RV volume, normal RV mass or RVEF, but with increasing functional impairment, as assessed by cardiac magnetic resonance imaging . AnotherEchocardiography plays a central role in the primary assessment of RV morphology and function in all forms of PH, providing an accurate insight into pathophysiological changes and carrying an important diagnostic and prognostic value.This review pointed out how several functional and morphological echocardiographic RV parameters, especially those derived from new techniques, represent valuable and easily accessible tools for the clinician in order to assess RV function in patients with cor pulmonale and periodically reassess functional stability, deterioration or improvement over time.Regarding future perspectives, further studies are required to investigate how new echocardiographic parameters could improve discrimination between acute and chronic cor pulmonale, which would be particularly useful in defining the optimal therapeutic strategy. Furthermore, it might be intriguing to establish the role of echocardiography in COPD patients, whether it is in aid of identifying patients with a proportionally greater lung destruction or an inflammation of the airways, or investigating the new phenotype “cor pulmonale parvus.”