Objective: During cardioplegia, which is often used in cardiac surgery, the heart is subjected to global ischemia/reperfusion injury, which can result in a post-operative impairment of cardiac function. Mitochondria permeability transition pores (MPTP) play a key role in cardiomyocyte survival after ischemia/reperfusion injury. It was shown in clinical settings that blockers of MPTP like cyclosporine might have a positive influence on cardiac function after cardioplegic arrest. Olesoxime, which is a new drug with MPTP blocking activity, has been introduced as a neuroprotective therapeutic agent. This drug has not been investigated on a possible positive effect in ischemia/reperfusion injury in hearts. Therefore, the aim of our study was to investigate possible effects of olesoxime on cardiac recovery after cardioplegic arrest.Methods: We evaluated 14 mature Chinchilla bastard rabbits of 1,500–2,000 g. Rabbit hearts were isolated and perfused with constant pressure according to Langendorff. After induction of cardioplegic arrest  the hearts maintained arrested for 90-min. Thereafter, the hearts were re-perfused for 60 min. At the end of each experiment left ventricular samples were frozen in liquid nitrogen for ATP measurements. Furthermore, heart slices were embedded in paraffin for histological analysis. During the entire experiment hemodynamic and functional data such as left ventricular pressure (LVP), dp/dt(max) and (min), pressure rate product (PRP), coronary flow, pO2, and pCO2 were also assessed.Results: Histological analysis revealed that despite the same ischemic burden for both groups markers of nitrosative and oxidative stress were significantly lower in the olesoxime group. Moreover, hearts of the olesoxime-group showed a significantly faster and better hemodynamic recovery during reperfusion. In addition, tissue ATP-levels were significantly higher in the olesoxime treated hearts.Conclusions: Olesoxime significantly protected the cardiac muscle from ischemia/reperfusion injury. During conventional cardiac surgery using heart-lung machine the heart is arrested by cardioplegic solutions. Depending on the type of operation cardiac arrest is maintained for up to 90 min or even longer. Although, cardioplegia and hypothermia provide certain protective effects regarding cardiac metabolic processes the heart is subjected to global ischemia/reperfusion injury. Therefore, heart protection during cardio-pulmonary bypass (CPB) is still a matter of debate. Experimental approaches range from improved management of cardiac cardioplegia, new cardioplegic solutions to improved flow profiles of the CPB  cannot be stored intracellularly the breakdown of ATP has serious consequences for function and survival of cardiomyocytes. Due to hypoxic conditions cardiac metabolic processes have to switch from aerobic to anaerobic glycolysis with the result of insufficient ATP-production and intracellular acidosis, which then in turn will lead to an activation of the NaHence, prolonged opening of MPTPs has negative effects on heart function . The investigation conforms to the Directive 2010/63/EU of the European Parliament as well as to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .For our experiments we used mature female and male Chinchilla rabbits weighting 1,500–2,000 g. Preparation of the hearts and perfusion according to the Langendorff-technique was performed as described earlier  Na+ 161, K+ 5.36; Ca2+ 1.8, Mg2+ 1.05; Cl− 148, 2 and 95% O2) and coronary flow (CF ml/min) was measured via the pulmonary artery. Left atrium was opened and the pressure balloon was introduced into the left ventricle and filled with water to achieve an end-diastolic pressure of 8.3 ± 0.18 mmHg. The pressure catheter was connected to a transducer and a two-channel bridge amplifier . Pressure traces were continuously recorded  to determine left ventricular systolic pressure (LVP), left ventricular end-diastolic pressure (EDP), dp/dt (max), and (min), basic cycle length (BCL) and heart rate (HR).In brief, animals were anesthetized with medetomidine (0.2 mg/kg body weight) and ketamine (20 mg/kg body weight). Heparine (500 IU/kg body weight) was administered intravenously. After induction of narcosis the rabbits were exsanguinated, the thorax was opened and the ascending aorta as well as the pulmonary artery were cannulated (1 min until aortic cannulation). Vena cava superior and inferior as well as the pulmonary veins were ligated separately. The hearts were quickly removed and connected to the Langendorff apparatus. Coronary arteries were retrogradly perfused at constant pressure of 75 cmHFour polyester plates with 64 electrodes each  were attached onto the surface of the heart in an elastic manner to allow a resonation of the electrode blocks with the spontaneously beating hearts. The electrodes were connected to a 256 channel mapping system HAL4  to measure surface ECGs from the right and left ventricular free wall and the anterior and posterior wall. Activation recovery interval (ARI), total activation time (TAT), PQ-interval, QRS width, corrected QT interval (cQT), and vector field similarity were assessed according to Dhein et al. .2 , pCO2  and coronary flow  (ml/min) were assessed during the entire experiment. Oxygen partial pressure was measured in Tyrode's solution  and in the outflow of pulmonary cannula (“venous”) and the “arterio-venous” difference was calculated which is a measure for the oxygen extraction of the beating heart.Additionally, via the pulmonary cannula pOFourteen rabbits were investigated and divided into two groups : cardioplegia group without olesoxime (CP-) and CP-group with 5 μmol/L olesoxime (CP+). The concentration of 5 μmol/L olesoxime was chosen according to Bordet et al. , 2010.2 and pCO2. Afterwards, cardioplegia (without olesoxime or with 5 μmol/L olesoxime) was initiated by coronary injection of 30 ml 4°C cold Custodiol®  containing (in mmol/L): NaCl 15, KCl 9, MgCl2 4, CaCl2 0.015, histidinehydrochloride 18, L-histidine 180, tryptophan 2, mannitol 30, and ketoglutarate/glutamic acid 1. A stock solution of 5,000 μmol/L olesoxime was prepared in DMSO (dimethyl sulfoxide), diluted in Custodiol® and added to the cardioplegia solution to achieve a final concentration of 5 μmol/L. The final concentration of DMSO within the cardioplegia solution was 1:1000. The surface temperature of the heart was measured by a thermometer placed on the cardiac surface. The temperature was maintained at 18°C by over-spilling the heart's surface with Tyrode's solution at 0.5 ml/h.After an equilibration period of 45 min baseline hemodynamic parameters and epicardial electrograms were recorded as well as arterial and venous pOAfter 90 min of cardioplegia, reperfusion with Tyrode's solution was initiated and the above described parameters were acquired at 5, 10, 20 30, and 60 min.Subsequently, the hearts were quickly removed from the Langendorff-apparatus and samples from the left ventricular apex were shock frozen in liquid nitrogen for ATP analysis. For histological analysis transverse slices of the heart were fixed in 4% neutral buffered formalin and embedded in paraffin.pH = 6) . Thereafter, histological specimen were blocked with BSA  and treated with either mouse monoclonal anti-nitrosine primary antibody , mouse monoclonal anti-PAR antibody , mouse anti-AIF primary antibody , with mouse monoclonal anti-HIF1α primary antibody , with mouse monoclonal HSP60 primary antibody  or with rabbit anti-cC3 primary antibody  over-night at 4°C.Histological evaluation of the ventricular probes was carried out according to Salameh et al. , in ordeAfter several washing steps, HRP-labeled secondary goat anti-mouse or goat anti-rabbit antibodies  were applied for 2 h. Subsequently, specimens were washed again and incubated with the red chromogen AEC  according to the manufacturer's instruction. Nuclei of cardiomyocytes were counter-stained with haemalum.The slides were investigated at 400 × magnification using a Zeiss Axiolab microscope  and photographs were randomly taken by a blinded investigator. The following heart regions were analyzed: for the right ventricle the mid-myocardial region of the right ventricular free wall (RV) was studied and for the left ventricle (LV) three regions of the free LV wall were assessed separately: epicardium, mid-myocardium and endocardium. These regions can be discriminated microscopically by the direction of the fibers. Endocardium makes about the inner 1/4 of the wall, epicardium the outer 1/4, and mid-myocardium the remaining 2/4 in the middle of the wall  was evaluated in relation to the total number of counted cells. Accordingly, 200 cells per heart and 1,400 cells per experimental group were investigated.Left ventricular specimen were homogenized at 4°C with 0.4 mmol/L perchloric acid and precipitated with KOH. Thereafter, the samples were centrifuged for 10 min and 20 μl of the supernatant was injected twice onto a pre-equilibrated RP18 column  as previously published  of t-test with Bonferroni correction for multiple measurements to detect statistical significance at a level of p < 0.05. ATP measurements were tested for statistical significance using ANOVA followed by Students t-test, and the non-parametric Kruskal-Wallis test was performed to analyse the histological data (p < 0.05).Hemodynamic data were evaluated with repeated measurements ANOVA followed by Students Prevalence of ventricular fibrillation in both experimental groups was tested using Fischer's exact test.For the statistical analyses Systat for Windows, version 13  was used.n = 7) and CP+ with 5 μmol/L olesoxime administered during cardioplegia . Both groups did not significantly differ in body and heart weight.Two groups of rabbits were evaluated: CP- .During the reperfusion period pCOP- Table  i.e., caP- Table . MoreoveP- Table . Heart rP- Table . At the Analysis of hemodynamic parameters during reperfusion revealed that cardioplegia itself induces significant decreases in LVP, PRP (pressure-rate product) and dp/dt (max) and (min) compared to baseline values. EDP was significantly increased in the CP- group and only slightly elevated in CP+. Administration of olesoxime during cardioplegia resulted in a significantly improved hemodynamic outcome of the hearts: i.e., inotropy and lusitropy was significantly better in the CP+ group compared to CP- Figures , 2. IndeAnalysis of electrophysiological data revealed a slight prolongation of ARI and cQT intervals during early reperfusion in both groups Table . Interesp = 0.192). Ventricular fibrillation could be mechanically terminated. Other ECG parameters like PQ-interval, QRS width, BCL (which reflects heart rate) and beat-to-beat vector similarity were not different between both experimental groups  after reperfusion indicates O2-deficit and consecutive metabolic autoregulation induced by the degradation of ATP.Our experimental data revealed that olesoxime significantly improved cardiac outcome after cardioplegia: left ventricular pressure, dp/dt (max) and (min), PRP -a measure for cardiac workload-, coronary flow, and ATP-levels were significantly higher in the olesoxime treated hearts. In the reperfusion phase after cardioplegia a slight prolongation of TAT occurred in the CP- group, which was accompanied by an enhanced incidence of ventricular fibrillation. Prolongation of TAT means a longer timespan for ventricular activation and thus slower ventricular conduction velocity. Slowing of conduction however is a well-known pro-arrhythmic factor. In contrast, olesoxime treated hearts did not exhibit prolonged TAT and did not develop ventricular fibrillation after cardioplegia. Moreover, cessation of ATP-synthesis due to cardioplegic arrest is known to result in failure of NaAssuming that olesoxime inhibits MPTPs would mean that ATP-synthesis is maintained  and RNS (reactive nitrogen species) production was diminished as was apoptosis after pretreatment with olesoxime (Martin et al., Mitochondria play a key role in ischemia/reperfusion injury of the heart and are also one of the main producers of ROS and RNS during ischemia and reperfusion injury (Akopova et al., Although it is known that cardioplegia induces AIF and cC3 translocation (Ramlawi et al., As mentioned above, ATP levels were higher in the olesoxime treated hearts, thus, if olesoxime has the capacity to maintain ATP-synthesis via blockade of prolonged MPTP-opening it should improve the metabolic state of cells and also should ameliorate cardiac function after cardioplegia. Indeed, we could detect an enhanced contractility and improved relaxation behavior of the left ventricle in the CP+ group. In conclusion, it can be said that olesoxime application during cardioplegia improved cardiac outcome and thus might be a therapeutic tool during this condition.Taken together, our data show that in isolated rabbit hearts cardioplegia results in delayed recovery of contractility and lusitropy together with reduced ATP content and slowed ventricular conduction, so that the hearts became more prone to ventricular fibrillation. These effects of cardioplegic arrest and the resulting functional defects could be significantly alleviated by olesoxime. Our findings that olesoxime treatment in addition to these effects also decreased PAR-synthesis, HSP60 expression and nitrotyrosine formation are in good accordance with the hypothesis, that the blockade of MPTP might be the underlying effect for the beneficial actions of olesoxime.The results of the present study -referring to ischemia-related changes- may encourage further studies targeted at local infarction and infarct size reduction.2 delivery and aspects regarding inflammatory responses can differ from the in vivo situation. Another aspect concerns the EDP. In cardiac surgery typically the heart is emptied during arrest and refilled at reperfusion. Due to the fact that the 256 electrodes had to stay in place we could not unload the balloon, repress and refill the balloon for technical reasons, since otherwise the geometry of the ventricle would be changed and the electrodes would alter their location by these manual manipulations.As all methods the isolated Langendorff heart has also limitations. Among these we should be aware, that the heart is perfused with saline solution and not with blood. Thus, aspects of OAlthough desirable we had no possibility to perform electron microscopy or to measure mitochondrial function and ROS production directly. However, we measured NT production as a surrogate parameter for nitrosative stress. Thus, the conclusions on an involvement of mitochondria are indirect at present and need further validation in subsequent studies targeted directly at mitochondrial function.AS, ID, and SD conceived and designed the experiments for this manuscript. AS, MK, and SD performed the experiments, analyzed and interpreted the data of this work. AS and SD wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anthracyclines are agents with a well known documented anti-tumoral activity. Cardiac side effects are the principal toxicity. Here we evaluate and monitor the onset of late anthracycline-induced cardiotoxicity with real-time CW-Doppler ultrasound cardiac output monitoring (USCOM®) and echocardiography in combination with serum biomarkers.Fifty-two patients without cardiac disease who had received an anthracycline-based regimen for various cancer types were included in this study. Patients’ hemodynamic parameters as stroke volume (SV USCOM (mL)) and ejection fraction (EF ECHOCARDIOGRAPHY (%)) were measured with USCOM and echocardiography and correlated to serum biomarkers (NT-pro-BNP and cTnT).Eighteen patients (34.6%) developed cardiac disease (NYHA I-III). An increasing cumulative anthracycline dose was associated with a decrease of the EF determined by echocardiography as well the SV by USCOM and with a higher NYHA class. Those patients who experienced cardiac disease showed a reduction of the EF and SV and increased serum biomarkers.Real-time CW-Doppler USCOM, is a fast and reliable method to monitor late hemodynamic changes as a symptom of anthracycline-induced cardiotoxicity comparable to the findings by echocardiography and serum biomarkers. Anthracyclines are high effective cytostatic with a broad spectrum of activity in various cancer types. The anti-tumor effect of anthracyclines based on DNA binding, production of free radicals and of causing irreversible DNA damage by inhibition of topoisomerase II , 2. CardPublished data on the incidence of anthracycline-induced cardiotoxicity are heterogeneous and lie between 2.2% and 26% . InternaThe most specific method to diagnose myocardial changes during treatment with anthracyclines is endomyocardial biopsy . But nonIn general, the prognosis of anthracycline-induced cardiomyopathy is described as relatively poor . In contANP and BNP are important serological parameters for diagnosis, prognosis and response to treatment in patients with acute or chronic heart failure -19. ANP Cardiac troponins are proteins with three subunits known as troponin T (cTnT), troponin I (cTnI) and troponin C. Cardinale et al showed that elevated levels of troponin during treatment with chemotherapy are associated with a decrease of left ventricular ejection fraction (EF). Contrary, patients with no elevation of troponin levels during chemotherapy may show a transient reduction of left ventricular EF which returns to baseline in the long-term follow up -24, cTnIUSCOM  is a non-invasive hemodynamic monitoring system based on continuous-wave (CW) Doppler-principle with a transportable touch-screen monitor and 2.2 MHz ultrasound probe. Knobloch and Co-workers have shown that combining real-time CW-Doppler ultrasound and serum biomarkers is feasible to monitor the hemodynamic changes to cardiotoxic agents like the anthracyclines or trastuzumab , 28.Aim of the study was to evaluate late cardiotoxicity with real-time CW-Doppler ultrasound cardiac output monitoring (USCOM), and echocardiography in combination with serum biomarkers in patients undergoing an anthracyclines based chemotherapy for at least 24 months.The present study was carried out in accordance with the Declaration of Helsinki of the World Medical Association and was approved by the Ethics Committee of the Medical Faculty of the Ludwig-Maximilians University in Munich. Informed consent was given by the patient prior to enter the study. The primary objective was to quantify late cardiotoxicity after anthracycline-based chemotherapy as a reduction of the stroke volume (SV) by USCOM, as well as the EF by echocardiography. Secondary objectives were the determination of myocardial markers (NT-pro-BNP and cTnT), taking into account the time interval between treatment and the cumulative dose and time of investigation.Patients who had received for at least 24 months an anthracycline-based regimen for hematologic malignancies or a solid tumor, a Karnofsky Perfomance Status of ≥ 70% and an age between 18 and 65 years were eligible. Cardiac EF determined by echocardiography had to be normal (EF ≥ 50%) prior to enter the study. Patients who had a positive history of coronary heart disease with cardiac dysfunction (> NYHA I), or an impaired left ventricular EF (LVEF) were not eligible.The New York Heart Association (NYHA) Functional Classification provides a simple way of classifying the extent of heart failure. Patients are categorized accordig to limitation of activities of daily living. The limitations/symptoms are in regards to normal breathing and varying degrees in shortness of breath and or angina pain. Class I: Cardiac disease, but no symptoms and no limitation in ordinary physical activity, for example, shortness of breath when walking, climbing stairs etc.; Class II: Mild symptoms (mild shortness of breath and/or angina) and slight limitation during ordinary activity; Class III: Marked limitation in activity due to symptoms, even during less-than-ordinary activity, for example, walking short distances (20 - 100 m). Comfortable only at rest; Class IV: Severe limitations. Experiences symptoms even while at rest. Mostly bed bound patients.USCOM is a noninvasive, continuous wave (CW) Doppler technology based device for the direct real-time measurement of cardiac output. The device calculates the velocity time integral (VTI), stroke volume (SV) and heart rate (HR), cardiac output (CO). Measurement was performed with the patient in the horizontal or sitting position. All measurements were performed by the same investigator. Extensive validation studies against invasive thermodilution  were performed in intensive ward settings, demonstrating high correlation and limits of agreement according to Bland-Altman analysis -31.As relevant cardiac markers hs-TnT  and NT-pro-BNP levels were determined on the same day of cardiac monitoring.The anthracycline equivalent dose was obtained multiplying the specific cumulative dose for a specific index value. Index for Doxorubicin, Epirubicin, Daunorubicin, Mitoxantrone was 1, 0.67, 0.83 and 4, respectively , 32, 33.The analysis was performed using descriptive statistical methods. Results are plot using Microsoft® Excel®.Between April 2008 and May 2009, fifty-two patients were enrolled. Median patients’ age was 60 years. Patients suffered from various cancer-types including 24 non-Hodgkin lymphomas (46%), 15 breast cancers (28.8%), 6 Hodgkin lymphomas (11.5%), 4 soft-tissue sarcomas (7.7%), 2 acute myeloid leukaemia’s (3.9%), and 1 chronic lymphatic leukaemia (2%). Two patients died a few weeks after the examination due to tumor progression. The median elapsed time after receiving an anthracycline-based regimen was 59 months (range: 25 - 123 months). Baseline characteristics are given in detail in A total of 18 of the 52 patients developed clinical signs of heart disease after receiving an anthracycline-based regimen, with 16 patient who were classified to NYHA class I-II (30.8%) and 2 patients to NYHA class II-III (3.8%). The inverse correlation of a higher NYHA class and a decrease in EF ECHO and SV USCOM is demonstrated in As the cumulative anthracycline dose is considered the most important risk factor for the development of an acute and chronic cardiotoxicity we tried to quantify late cardiotoxicity as a reduction of the stroke volume (SV) by USCOM, as well as the EF measured by echocardiography. The correlation between cumulative anthracycline dose and EF ECHO and SV USCOM is given in To investigate the relationship between anthracycline therapy and time, patients were divided into five groups depending on the elapsed time after receiving an anthracycline-based regimen and the hemodynamic investigations . The relationship between the time interval after treatment and EF ECHO and SV USCOM is illustrated in Secondary objective of this study was the determination of myocardial markers and their correlation with the cardiac function measured as EF ECHO and SV USCOM. For all patients hs-cTnT and NT-pro-BNP was measured after anthracycline therapy at the same time of the hemodynamic investigations. A higher NYHA class was associated with an increased median level of myocardial serum biomarkers. The objectives of the present study were to assess and to quantify late cardiotoxicity after anthracycline-based chemotherapy as a reduction of EF ECHO and SV USCOM determined by echocardiography and USCOM. The intention of routine cardiac monitoring during anti-cancer treatment is the early detection of restricted cardiac function with the consequence of dose reduction or interruption (and/or application of cardioprotectants). Due to the potential risk of a late onset cardiotoxicity, the cardiac function has to be monitored years after anti-cancer treatment , 35.USCOM is a noninvasive cardiac output monitor based on the transthoracic measurement of Doppler flow velocity over the aortic and pulmonary outflow tract. It is easy to operate, and hemodynamic parameters are displayed ‘beat by beat’. The technique is reported to be easily learned after a short learning period , 37. ValKnobloch and co-workers first have used CW-Doppler USCOM and NT-pro BNP measurements for monitoring the hemodynamic response in patients who received an anthracycline- or trastuzumab-based regimen for breast cancer , 28. MorThe value of serum biomarkers in patients with heart failure is well documented. Gustafsson and co-workers investigated the diagnostic and prognostic performance of NT-pro-BNP in primary care patients with suspected congestive heart failure. In summary, the mortality rate was higher in patients with NT-pro-BNP levels > 125 pg/mL than in patients with normal values (P < 0.002). This difference persisted after controlling for age, gender, and EF ECHO . KnoblocIn summary, the major findings of this study were: 1. Eighteen patients (34.6%) developed cardiac disease according to the NYHA classification. 2. We could show a trend towards a decreased EF ECHO and SV USCOM with increasing anthracycline doses in both, echocardiography and by USCOM, indicating a negative impact on myocardial function of the applied chemotherapy. 3. A higher NYHA class was associated with a markedly elevation of myocardial serum biomarkers. 4. Finally, we could demonstrate a trend towards an EF ECHO and SV USCOM decrease in relation to the elapsed time after the anthracycline-based treatment. 5. The correlation of USCOM to the ‘gold-standard’ echocardiography was evident in our trial. A decreased EF measured by echocardiography was paralleled by a corresponding reduced SV measured by USCOM.2 [Despite some striking observations, the results and conclusions of our study are hampered by several factors: 1). The present study was initiated as a pilot study with the aim to evaluate late cardiotoxicity with real-time CW-Doppler ultrasound cardiac output monitoring (USCOM), echocardiography and serum biomarkers after a median interval of 59 months after an anthracycline-based therapy. One can not exclude other factors that influence EF ECHO and SV USCOM as for example ischemic coronary heart disease. 2). The risk of developing cardiotoxicity increases considerably beyond a cumulative doxorubicin dose of 550 mg/m2 . Critica2 . 4). BioIn summary, the addition of laboratory parameters to hemodynamic-based measurement gives an important contribution in terms of reliable cardiac monitoring. All findings by echocardiography were paralleled by a corresponding finding by USCOM. The increase of serum biomarkers was correlated to a higher NYHA stage, indicating a negative impact on myocardial function of the applied chemotherapy. USCOM does not replace standard methods as echocardiography in the cardiac surveillance of patients undergoing an anthracycline-based chemotherapy. But USCOM is attractive in many ways. It is easy to use, and as an ultrasound technique safe so it can be used repeatedly to measure the trend over time. Moreover, by using the USCOM device the physician will obtain a result in an unbeatable period of time. USCOM deserves closer attention in the cardiac monitoring of a cancer patients population even in those who survived their cancers. The present study justifies further clinical studies evaluating USCOM in haematology and oncology in larger, well-defined patient cohorts.


Right ventricular dysfunction is a known predictor of mortality in patients with nonischemic cardiomyopathy (NICM), however predictors of right ventricular ejection fraction (RVEF) in this patient population are poorly understood. We sought to identify independent predictors of RVEF, measured using cardiac MRI, in patients with NICM.We retrospectively evaluated 140 consecutive patients with NICM who underwent delayed hyperenhancement cardiac MRI between 2002 and 2009. A Cox proportional hazards model was used to evaluate the association between RVEF and the composite outcome of death, left ventricular assist device implantation, or heart transplantation. Predictors of RVEF were evaluated using a multivariable linear regression model.The study population included 140 patients  = 33 ± 12%). There were 39 events  over a median follow-up time of 6.5 years. After adjusting for age, gender, and LVEF, RVEF was a significant predictor of the composite outcome . On univariate analysis, male gender, lower LVEF, higher LV myocardial fibrosis, mitral regurgitation (MR) severity ≥ 2 , and presence of right bundle branch block (RBBB) were significantly associated with lower RVEF. After adjusting for age and the aforementioned variables, male gender, LVEF, LV myocardial fibrosis, and presence of RBBB remained significant independent predictors of RVEF, however MR severity was no longer a significant predictor of RVEF . See Figure Right ventricular dysfunction has been shown to predict poor outcomes in patients with NICM, but predictors of RVEF in this cohort are not well defined. We found male gender, lower LVEF, higher LV myocardial fibrosis, and the presence of RBBB were independent predictors of lower RVEF. While MR severity was associated with RVEF on univariate analysis, this relationship was not significant independent of LVEF.


The hypertensive deoxy-corticosterone acetate (DOCA)-salt-treated pig  was recently introduced as large animal model for early-stage heart failure with preserved ejection fraction (HFpEF). The aim of the present study was to evaluate cardiovascular magnetic resonance (CMR) of DOCA pigs and weight-matched control pigs to characterize ventricular, atrial and myocardial structure and function of this phenotype model.V(int/LV)) and collagen (VV(coll/LV)). Rest, stress, and stress-to-rest differences of cardiac and myocardial parameters in DOCA and control animals were compared by t-test.Five anesthetized DOCA and seven control pigs underwent 3 T CMR at rest and during dobutamine stress. Left ventricular/atrial (LV/LA) function and myocardial mass (LVMM), strains and torsion were evaluated from (tagged) cine imaging. 4D phase-contrast measurements were used to assess blood flow and peak velocities, including transmitral early-diastolic (E) and myocardial tissue (E’) velocities and coronary sinus blood flow. Myocardial perfusion reserve was estimated from stress-to-rest time-averaged coronary sinus flow. Global native myocardial T1 times were derived from prototype modified Look-Locker inversion-recovery (MOLLI) short-axis T1 maps. After in-vivo measurements, transmural biopsies were collected for stereological evaluation including the volume fractions of interstitium (Vp < 0.001) and LV wall-thickness  were elevated. During stress, increase of LV ejection-fraction and decrease of end-systolic volume accounted for normal contractility reserves in DOCA and control pigs. Rest-to-stress differences of cardiac index (p = 0.040) and end-diastolic volume (p = 0.042) were documented. Maximal (p = 0.042) and minimal (p = 0.012) LA volumes in DOCA pigs were elevated at rest; total LA ejection-fraction decreased during stress (p = 0.006). E’ was lower in DOCA pigs, corresponding to higher E/E’ at rest (p = 0.013) and stress (p = 0.026). Myocardial perfusion reserve was reduced in DOCA pigs (p = 0.031). T1-times and VV(int/LV) did not differ between groups, whereas VV(coll/LV) levels were higher in DOCA pigs (p = 0.044).In DOCA pigs LVMM . Heart failure with preserved ejection fraction (HFpEF) is a multifactorial heterogeneous clinical syndrome that is recognized as an independent risk factor for mortality and cardiovascular morbidity –3. MechaCardiovascular magnetic resonance (CMR) is the standard of reference for non-invasive assessment of cardiac and myocardial function and morphology –22. EmplThe aim of our explorative study was to evaluate comprehensive CMR imaging of DOCA pigs and weight-matched control pigs at rest and during dobutamine stress to characterize ventricular, atrial and myocardial structure and function of this phenotype model of early-stage HFpEF, and to identify potential non-invasive imaging markers of the disease.The explorative study was approved by the local Bioethics Committee of Vienna, Austria (BMWF-66.010/0091-II/3b/2013) and conformed to the guide for the care and use of laboratory animals, US National Institute of Health . Thirteen female landrace pigs were enrolled. Arterial hypertension was induced in six animals by subcutaneous implantation of DOCA pellets  in combination with a high-salt, high-sugar, high- potassium diet. After 12 weeks of treatment, animals were examined by CMR imaging at rest and during dobutamine stress. One DOCA pig was excluded from analysis because of heart rate and blood pressure instability during the measurements, which could be attributed to florid pericarditis when attempting to acquire histologic samples. Seven weight-matched healthy animals served as controls. The characteristics of the DOCA and the control animals are summarized in Table −1), midazolam (0.25 mg · kg−1) and azaperone (5 mg · kg−1). Anesthesia was induced by 30–60 mg propofol  to allow endotracheal intubation. Pigs were mechanically ventilated  and anesthesia was maintained with sevoflurane (1.5–2.5 %), fentanyl (35 μg · kg−1 · h−1), midazolam (1.2 mg · kg−1 · h−1), ketamine (2–8 mg · kg−1 · h−1) and pancuronium (0.2 mg · kg−1 · h−1). Respiratory gases , heart rate and arterial blood pressure  were continuously monitored. Sheath accesses of the left internal carotid artery and jugular vein were surgically prepared. Blood samples collected from the arterial line were used to control oximetric and metabolic parameters . A balanced crystalloid infusion  was administered at a fixed rate of 10 ml · kg−1 · h−1 throughout the protocol. Oral temperature of animals was assessed by a sublingual thermometer and was maintained at 38 °C during CMR imaging via air ventilation and/or infusion of cold saline solution.Animals were sedated by intramuscular administration of ketamine  using a phased-array 6-channel body matrix coil together with a spine matrix coil. Subjects were investigated in a single session during free breathing in the supine position with electrodes for electrocardiographic (ECG) gating positioned on the chest. After assessment of cardiac and myocardial function, blood flow and LV T1 times at rest, measurements were repeated during stress, which was induced by intravenous infusion of dobutamine  at rates of 2–5 μg · kg3) were obtained in the LV two-chamber, three- and four-chamber views  cine images  with image orientation adjusted perpendicular to the course of the proximal ascending aorta 2 cm above the aortic valve. Time-resolved three-directional phase-contrast imaging (4D flow) data were acquired to measure mitral annular tissue velocity and blood flow in the left heart, the pulmonary veins and the coronary sinus; the structures were covered by gapless slices with a retrospectively ECG-gated, two-dimensional spoiled gradient-echo-based three-directional velocity-encoded cine phase-contrast sequence .Aortic cross section, vessel wall thickness, and aortic peak blood velocity were evaluated on retrospectively ECG-gated, two-dimensional spoiled gradient-echo-based through-plane velocity encoded cine phase-contrast images .To study myocardial strain, tagged cine images Fig.  were acq3) was used to acquire myocardial T1 maps in end-diastole  prototype sequence with single-shot balanced steady-state free precession (bSSFP) readout, motion correction and automatic T1 map generation (MOLLI protocol 5(5)5(5)5; echo spacing, 2.6 ms; echo time, 1.1 ms; flip angle, 35°; voxel size, 2.1 × 1.4 × 8.0 mmShort-axis cine images were analyzed by syngo.via software . To assess LV volume vs. time curves, LV epicardial and endocardial borders, excluding papillary muscles from myocardium, were traced manually in end-diastole and end-systole and semi-automatically adjusted to all cardiac phases Fig. . To defi2) = 0.0734 × weight (kg)0.656 [diastasis) was derived from LV volume vs. time curves as the time interval starting after rapid LV filling and atrial contraction. Derivative of LV volume-time curves provided assessment of normalized peak ejection rate (PER), peak ejection time (PET), as well as early  and active  normalized peak filling rates and times , end-systolic volume (ESV), stroke volume, cardiac output, and LV ejection fraction were evaluated from end-diastolic and end-systolic cardiac phases with the body surface area (BSA) estimated according to BSA (mkg)0.656 . The durmes Fig. .ED) and end-systolic (WTES) wall thickness, as well as LV wall thickening, were calculated as averages of segmental values.Papillary muscles were included when measuring normalized left ventricular mass (LVMM), but they were excluded in determining normalized mean end-diastolic and end-systolic wall thickness and thickening at the basal, mid-myocardial and apical levels; 16 myocardial segments were evaluated, according to the American Heart Association (AHA) segmentation scheme . Global Times of mitral valve opening (MVO), mitral valve closure (MVC), and aortic valve opening (AVO)/closure (AVC) were derived from cine 3-chamber images by visual analysis Fig. ; in turnMitral annular plane systolic excursion (MAPSE) was measured as the difference between the end-diastolic and the end-systolic distance from apex to lateral mitral annulus in 4-chamber view Fig. .max) and before mitral valve closure after LA contraction (minimum volume LAVmin). For consistency, the plane of the mitral annulus was defined as the LA inferior border. Areas of recesses of the mitral valve, pulmonary veins and left atrial appendage were excluded by drawing a straight line across these structures to adjacent atrial borders. LA length was measured from the center of the mitral plane to the superior margin of the LA in 4- and 2-chamber views. Normalized LA volumes were estimated using the bi-planar area-length method [2CH × A4CH/(L × BSA), where A2CH and A4CH are the LA areas in 2-chamber and 4-chamber views, respectively, and L is the length of the LA from either the 2- or the 4-chamber view (whichever is shorter). LA total ejection fraction (LATEF) was calculated according to LATEF = 100 × (LAVmax-LAVmin)/LAVmax.Left atrial (LA) volumes were evaluated from manually tracing the LA area and length in 4- and 2-chamber views Fig. . Normalih method  from LAVAortic peak velocity and cross-sectional area were evaluated from through-plane phase-contrast images by syngo.via . Aortic vessel wall thickness (AWT) was assessed from aortic cross section diameter measured from outer and inner vessel borders at the cardiac phases with maximum vessel cross section area at rest according to AWT = (outer diameter – inner diameter)/2.Transmitral early (E) and late (A) diastolic, pulmonary venous systolic  and early diastolic (D) velocities, coronary sinus net forward blood flow volume Fig. , and earGlobal myocardial perfusion (GMP) was derived as quotient of coronary sinus net forward blood flow volume multiplied by heart rate (which represents the time-averaged coronary sinus blood flow) and the left ventricular muscle mass . MyocardTagged images were evaluated semi-automatically by using prototype software . A grid was aligned to the myocardial tags at end-diastole and propagated throughout all images of the cardiac cycle ), volume fraction of cardiomyocytes  = 1- VV(int/lv)), of blood vessels (VV(ves/lv)) and of collagen fibrils either related to the left ventricle (VV(coll/lv)) or to the interstitium (VV(coll/int)) as reference volume. The total myocyte ), collagen V and blood vessel V content in the myocardium were obtained by multiplying LVMM with VV(myo/lv), VV(coll/lv), and VV(ves/lv), respectively.After in vivo measurements were completed, thoracotomy was performed and a bolus of 100 mmol potassium was administered intracoronarily to sacrifice each animal. For electron microscopic analysis of the myocardium, transmural biopsies were then collected from the lateral left ventricular wall and fixed in 1.5 % glutaraldehyde, 1.5 % paraformaldehyde in 0.15 M Hepes buffer. The samples were then stored in the fixative at 4 °C until further processing. The processing steps included post-fixation in 1 % osmium tetroxide solution, overnight staining in half-saturated uranyl acetate solution, dehydration in an ascending acetone series and finally embedding in epoxy resin. From the embedded samples, semi- and ultrathin sections were obtained for stereological analysis. The following parameters were assessed by the method of point counting : volume t-test. Significances of differences of rest and stress indices or absolute and relative stress reserves were tested by 1-sample t-test.Mean values are given together with standard deviations. Statistical analysis was performed using NCSS  NCSS, LLC. Kaysville, Utah). A significance level of 0.05 was employed for statistical tests. Rest and stress indices of left heart and myocardial function as well as morphological and stereological parameters assessed in DOCA and control animals were compared by 2-sample −1 · min−1; DOCA, 3.8 ± 1.0 μg · kg−1 · min−1). During stress, blood pressure in DOCA subjects failed to increase, and thus differences in blood pressures and rate-pressure product disappeared between groups.DOCA pigs developed hypertension within 12 weeks. At rest, arterial blood pressure  and the rate-pressure product were higher in DOCA pigs than in controls; the groups did not differ significantly in weight, BSA and heart rate  and the decrease in EDV, which was higher in DOCA animals . IVRT was longer in DOCA at rest but did not differ significantly between groups during stress /The main differences in the pulmonary venous flow patterns between the two groups were that DOCA pigs showed significantly higher D-wave velocities at rest, while controls displayed a significant stress-induced increase in pulmonary venous peak velocities that was not observed in the DOCA group. Accordingly, the S/D ratio differed significantly between the two groups at rest but not during stress.Under rest coronary sinus net forward blood volume was larger in the DOCA pigs than in the control pigs, but equalized under stress condition. Although global myocardial perfusion did not differ significantly between groups and increased with stress in both groups, myocardial perfusion reserve was significantly smaller in the DOCA group Table .Circumferential, radial and longitudinal strains and strain rates as well as torsion and torsion rates were evaluated in all DOCA and 6 controls pigs. One control pig was excluded from evaluation due to poor tagged imaging quality.min did not differ between groups at rest, it was smaller in DOCA animals during stress. Systolic torsion ratemax showed the opposite behavior, being significantly higher in DOCA pigs at rest but not during stress. Diastolic torsion ratemin did differ significantly between groups; however, the torsion ratemin failed to properly increase during stress in the DOCA group .Differences between DOCA and control pigs were found in the longitudinal strain and torsion rates Table . Whereasp = 0.094).Native myocardial T1 relaxation times were evaluated in 5 DOCA and 6 control animals. One control pig was excluded from evaluation due to poor image quality. No differences between groups were found in global native T1 times : controls, 21 ± 2 %; DOCA, 19 ± 2 %; p = 0.283) and of blood vessels (VV(ves/lv): controls, 4.4 ± 1.3 %; DOCA, 5.5 ± 0.4 %; p = 0.092) did not differ between the DOCA-salt treated and control group. Volume fractions of collagen with respect to both the LV (VV(coll/lv): controls, 1.7 ± 0.5 %; DOCA, 3.0 ± 0.7 %; p = 0.044) and the interstitium (VV(coll/int): controls, 8 ± 3 %; DOCA, 16 ± 4 %; p = 0.025) were significantly increased in the DOCA group , total collagen volume (V(col/lv): controls, 1.5 ± 0.4 ml; DOCA, 3.8 ± 1.0 ml; p = 0.011) and total blood vessel volume (V(ves/lv): controls, 3.7 ± 1.1 ml; DOCA, 7.1 ± 1.2 ml; p = 0.012) were higher in the DOCA pigs than in the control pigs.Samples of five DOCA and three control pigs were evaluated. Volume fractions of the interstitium  in stereological analysis) of hypertrophied cardiomyocytes rather than the elevated levels of collagen in the slightly smaller compartment of interstitium (VV(int/lv) in stereological analysis) in a voxel.Native T1 times did not resolve the higher myocardial collagen content documented by the stereological evaluation , 34. ThiIn accordance with other investigations of vessel wall thickening in DOCA pigs , 14, ourAlthough the exact cause for the altered myocardial vessel volume compartment in DOCA pigs cannot be specified on the basis of stereological data, structural changes in the myocardial microvasculature are in line with the impairment of the myocardial perfusion reserve observed in DOCA pigs: Coronary sinus net forward volume was higher in DOCA pigs at rest maintaining normal global myocardial perfusion of the hypertrophic myocardium. During stress, myocardial perfusion in DOCA pigs increased mainly due to a rise in heart rate (indicated by the insignificant stress-to-rest difference of coronary sinus net forward volume), resulting in a significantly reduced myocardial perfusion reserve in DOCA pigs compared to controls. These findings at rest and stress were also reported in patients with HFpEF  and wereDuring β-adrenergic stress, an adequate increase in LV ejection fraction and a decrease in ESV accounted for normal contractility reserves in both groups. However, EDV significantly decreased during stress in DOCA pigs, inhibiting stroke volume and cardiac index from properly increasing with the heart rate. A similar response was reported in patients with HFpEF during dynamic exercise , 39.min compared to control animals; thus systolic function – though normal at rest – showed signs of impairment during stress. These results are in accordance with findings in hypertensive patients with LV hypertrophy, where authors showed that systolic dysfunction may develop in parallel to diastolic dysfunction [Echocardiographic studies in HFpEF patients document reduced longitudinal and circumferential strain  as well function , 42, 43.function , MAPSE wE’ was significantly decreased in the DOCA group at rest and during dobutamine stress, and accordingly E/E’ and (E/E’)/EDV ratios were higher in DOCA than in control animals. It was previously shown in HFPEF patients that E/E’ correlates well with the LV end-diastolic pressure . The sigDue to increased D-wave peak velocities in the DOCA group, the pulmonary venous S/D ratio was significantly lower at rest in DOCA pigs than in control pigs. As LV relaxation is the main determinant of pulmonary venous flow , 50, obsmax were higher in DOCA than control pigs. Diastolic torsion ratemin significantly increased during β-adrenergic stimulation in the control group, indicating that intraventricular pressure gradients appropriately increased [min between stress and rest failed to properly increase; this failure relates to reduced intraventricular pressure gradients and impairment of LV relaxation [LV torsion is known to be dependent on LV shape, and in LV concentric hypertrophy increased torsion is due to an increased lever arm for epicardial fibers . Accordincreased . In DOCAlaxation .DOCA pigs showed significantly increased LA volumes at rest; this could be interpreted as a marker of altered diastolic function, LA pressure and early diastolic filling . During Several limitations of the present study need to be acknowledged. The study had a small sample size; therefore it was not possible to assess correlations between studied parameters. Moreover, samples for stereological analysis were collected in a sub-group of animals only from the LV lateral wall and, for reasons of feasibility, not by a random sampling scheme.Cardiac, myocardial and vascular CMR parameters were obtained for comparison from DOCA-treated and non-DOCA treated landrace pigs in anesthesia. As there were no obvious cardiovascular malformations and no outliers in the studied parameters, it is quite likely that all the non-DOCA treated pigs represent a normal collective. Neither the effect of the subcutaneous implantation procedure nor the overall effects of anesthesia were controlled in the current study.All CMR measurements were performed under mechanical ventilation, which reduced limitations on temporal resolution of cine acquisitions, in particular. The fact that diastolic functional parameters like E’, E/A or IVRT compare well with echocardiographic normal values in pigs , 56 mighThe comprehensive imaging protocol allowed the investigation of only one stress level. In accordance with results found in HFpEF patients , chronotFinally, invasive intra-cardiac hemodynamic measurements were not performed during CMR examinations, as appropriate MR-compatible equipment was not available.The present study documents numerous alterations in CMR-derived indices of cardiac and myocardial function at rest and during stress in pigs with DOCA-salt induced early-stage HFpEF. LA enlargement, metrics of myocardial tissue velocity, pulmonary venous and transmitral blood flow velocities presented as potential CMR markers of early-stage HFpEF at rest, highlighting the important role of LA impairment in the development of HFpEF. Inadequate increases in myocardial perfusion reserve and cardiac index during dobutamine stress may prove to be useful new CMR metrics for the diagnosis of HFpEF, and could probably account for exercise intolerance in early stages of disease. Myocardial T1 mapping, however, could not detect elevated levels of myocardial collagen found by stereology in DOCA pigs.


Structural examination of human heart specimens at the microscopic level is a prerequisite for understanding congenital heart diseases. It is desirable not to destroy or alter the properties of such specimens because of their scarcity. However, many of the currently available imaging techniques either destroy the specimen through sectioning or alter the chemical and mechanical properties of the specimen through staining and contrast agent injection. As a result, subsequent studies may not be possible. X-ray phase-contrast tomography is an imaging modality for biological soft tissues that does not destroy or alter the properties of the specimen. The feasibility of X-ray phase-contrast tomography for the structural examination of heart specimens was tested using infantile and fetal heart specimens without congenital diseases. X-ray phase-contrast tomography was carried out at the SPring-8 synchrotron radiation facility using the Talbot grating interferometer at the bending magnet beamline BL20B2 to visualize the structure of five non-pretreated whole heart specimens obtained by autopsy. High-resolution, three-dimensional images were obtained for all specimens. The images clearly showed the myocardial structure, coronary vessels, and conduction bundle. X-ray phase-contrast tomography allows high-resolution, three-dimensional imaging of human heart specimens. Intact imaging using X-ray phase-contrast tomography can contribute to further structural investigation of heart specimens with congenital heart diseases.The online version of this article (doi:10.1007/s00246-016-1527-z) contains supplementary material, which is available to authorized users. Examination of the structure of human heart specimens at the microscopic level is a prerequisite for understanding congenital heart diseases. Due to the scarcity of heart specimens with complex heart diseases, it is important not to destroy or alter the property of the specimens during examination.X-ray phase-contrast tomography (XPCT) is an imaging modality for soft tissue specimens that does not destroy or alter the properties of the specimen –3. The sHuman heart specimens obtained by autopsy from two fetuses and three infants were used. Absence of congenital heart disease was confirmed at autopsy. The ethical committees of the participating hospitals approved the study and waived the requirement of consent from the family of the deceased.3; density resolution, 1 mg/cm3; exposure time, 1 s/image; number of projections, 900; radiation dose 75 Gy. The horizontal field of view, namely the field of view in the reconstructed slice image, can be extended up to 40 mm by employing an offset tomographic scan.The XPCT was carried out at the SPring-8 synchrotron radiation facility using the Talbot grating interferometer at the bending magnet beamline BL20B2 to visualize the structure of heart specimens. The XPCT system has been described in detail elsewhere , 5. BrieHigh-resolution images were obtained for all specimens. A two-dimensional slice showing the ventricular septum of a fetal heart is shown in Fig. We have shown that XPCT provides high-resolution, three-dimensional images without destroying or altering the property of human heart specimens.Other modalities for visualizing the structures of soft tissues include histological observation, absorption contrast computed tomography (ACCT), and magnetic resonance imaging (MRI). However, each of these modalities has its shortcomings.Histological light microscopic observation is the standard technique for structural investigation, but a major limitation of this technique is the destruction of the specimen by sectioning, which renders the specimen unusable for other studies. ACCT has a high spatial resolution of up to 0.7 μm ; howeverSeveral XPCT imaging techniques have been developed, including the propagation-based technique, analyzer-based technique, crystal interferometry, grating interferometry, and the grating non-interferometric technique. We adopted grating interferometry because it provides adequate field of view, as well as high-density sensitivity and a dynamic range, and does not suffer from interferometer drift. A comprehensive review of each technique can be found elsewhere .In our current XPCT imaging system, investigation of the whole adult human heart is not possible due to two limitations. The first limitation is unsatisfactory spatial resolution. The size of the specimen is limited by the XPCT system’s effective field of view, which is defined by the X-ray detector, grating area size, and X-ray beam size. Since the X-ray detector is composed of a digital camera with pixel array, the field of view is defined by the pixel size and number of pixels. This means that a larger pixel size makes it possible to obtain a larger field of view. However, a larger pixel size decreases the quality of spatial resolution. The height of the specimen is limited to 30 mm due to the X-ray beam size of our current XPCT imaging system. It may be possible to measure a larger specimen up to 100 mm in width using an X-ray detector with a larger field of view and a larger grating area size at the expense of spatial resolution. The second limitation is increased X-ray absorption. A larger specimen must be placed in a larger saline-filled canister for XPCT imaging. If we use a canister of 100 mm in thickness, the X-rays irradiated into the canister are mostly absorbed by saline and the specimen. It would be necessary to use higher X-ray energy for X-ray transmission when imaging a specimen as large as 100 mm in diameter.In spite of extensive research, the structure of hearts with congenital heart diseases has not been elucidated. For example, a heart with isomerism may have anterior atrioventricular bundle, posterior atrioventricular bundle, or twin atrioventricular sling. The location of the conduction bundle cannot be definitively predicted in an isomeric heart . FurtherClinical application of XPCT is gaining traction ; howeverDue to the scarcity of heart specimens with congenital diseases, imaging modalities that allow examination without destroying or altering the property of the specimens are desirable. XPCT was feasible for high-resolution, three-dimensional imaging of human heart specimens. Intact imaging using XPCT will expand our understanding of congenital heart diseases.Supplementary video 1Video containing the slice shown in Fig. 1 (MP4 16049 kb)Supplementary video 2Video containing the slice shown in Fig. 2 (MP4 9816 kb)Supplementary video 3Three-dimensional volume-rendered reconstruction of the same heart. The heart valves, subvalvular apparatus, ventricular septum, and myocardial morphology are clearly depicted. The conduction bundle is visualized at the elapsed time of 41 s (MP4 20223 kb)Below is the link to the electronic supplementary material.


Left ventricular outflow tract obstruction (LVOTO) occurs from not only obstructive hypertrophic cardiomyopathy but also other conditions such as sigmoid septum or post mitral valve repair. However, the changes of the LVOT pressure gradient (LVOT PG) in LVOTO with various conditions remain unclear.The clinical characteristics and echocardiographic parameters of 73 patients with LVOT PG ≥50 mmHg at rest on Doppler ultrasound were retrospectively investigated.In these patients , high prevalences of hypertension (66%) and anemia (43%) were observed. The most frequent clinical disease causing LVOTO was hypertrophic obstructive cardiomyopathy (HOCM) (74%). There were other conditions, including hypertensive left ventricular hypertrophy (9%), post-open heart surgery (7%), sigmoid septum (4%), hyperkinetic LV (3%), takotsubo cardiomyopathy (1.5%), and discrete subaortic membrane (1.5%). Significant improvement or reduction of LVOTO was observed in 93% of cases at follow-up (mean 44 months) echocardiography compared with the initial one with the use of medications and transcatheter procedures.The causes of LVOTO are diverse. However, the occurrence of LVOTO might depend on the coexistence of primary morphological LV characteristics and hemodynamic LV status. Specific factors causing LVOTO need to be investigated, and efforts for improvement of each individual status by the appropriate approach are required. A left ventricular outflow tract pressure gradient (LVOT PG) ≥50 mmHg at rest in hypertrophic cardiomyopathy (HCM) is a predictor of heart failure and cardiovascular death , 2. The The present study was retrospectively designed. The study population consisted of 73 patients from the echocardiographic database of Cedars-Sinai Medical Center, Los Angeles, CA, with LVOT PG ≥50 mmHg at rest on Doppler ultrasound between January 2011 and February 2016. The study protocol was approved by the Cedars-Sinai Medical Center Institutional Review Board. Clinical data included age, sex, documented diagnosis of hypertension (HT), dyslipidemia (DL), diabetes mellitus (DM), anemia  and others are shown in Table E), peak late diastolic velocity (A), E/A ratio and deceleration time (DT) were derived from Doppler recordings of transmitral flow (TMF). Peak early diastolic mitral annular velocity (e′) of the lateral site of the mitral annulus was measured by tissue Doppler imaging. The ratio between E and e′ (E/e′) was calculated to estimate LV filling pressure. Systolic pulmonary artery pressure (SPAP) was estimated using the modified Bernoulli equation, measuring the peak systolic pressure gradient of the tricuspid valve regurgitant jet by continuous wave Doppler, and adding it to the estimated right atrial pressure. The severity of aortic valve regurgitation (AR), mitral valve regurgitation (MR), and tricuspid valve regurgitation (TR) was defined by the ACC/AHA task force on practice guidelines [LVOT PG ≥50 mmHg at rest was defined as LVOTO, and it was compared between initial and follow-up transthoracic echocardiography (TTE). TTE was performed by two-dimensional (2D) Doppler echocardiography using a Philips iE33 ultrasound system  in clinically stable patients. LVOT PG was determined with quantitative Doppler using Bernoulli’s equation. The end-diastolic left ventricular diameter (LVDd), end-systolic LV diameter (LVDs), LV septal wall thickness, LV posterior wall thickness, and left atrial dimension (LAD) were measured in the parasternal long-axis view. Left atrial area was measured during LV end-systole using the biplane area-length method in the apical four-chamber view and indexed for body surface area. Left ventricular ejection fraction (LVEF) was calculated using the biplane method of discs (modified Simpson rule). To assess diastolic function, peak early diastolic velocity  was used for statistical analysis. Continuous variables are expressed as mean ± SD. Differences between continuous variables were assessed using Student’s Table The most frequent clinical disease causing LVOTO was hypertrophic obstructive cardiomyopathy 74%) Table . However% Table .Forty-nine patients with HOCM were treated with combinations of negative inotropic agents such as disopyramide or antiarrhythmic agents such as amiodarone, or antiplatelet agents with and without β-blockers Table . AlcoholE/e′ and SPAP were the highest in patients with LVH.Echocardiographic parameters classified by clinical diagnosis are shown in Table p < 0.001) in HOCM with drug therapy, from 62 ± 14 to 24 ± 13 mmHg (p = 0.01) in HOCM with alcohol septal ablation and from 90 ± 20 to 33 ± 35 mmHg (p = 0.016) in LVH compared with initial TTE. However, in post-surgery patients, there was no significant reduction of LVOT PG. Changes of LVOT PG could not be analyzed statistically in patients with hyperkinetic LV, takotsubo cardiomyopathy and subaortic membrane because of the small number of cases, but LVOT PG decreased in all of them. Examples of LV outflow by continuous wave Doppler echocardiography at initial TTE and follow-up TTE are displayed in Fig. Seventy patients (96%) were followed by TTE. Results are shown with comparisons of initial and follow-up TTE results Fig. . LVOT PGThe most frequent cause of LVOTO has been well known to be HOCM. Maron et al.  reportedE/e′ (mean 18.8 ± 9.1) were observed. It was reported that LVOTO occurs if LV systolic function becomes hyperdynamic due to other factors in a patient with basal septal hypertrophy, and this tends to happen in elderly patients who have a history of HT [n = 9) and LVH (n = 6) showed an LVOT PG of 74.4 ± 55.2 mmHg. A reduction in LVOT PG of 40.9 mmHg on β-blockers, with a further reduction of 24.2 ± 25.8 mmHg with added disopyramide, was observed. Other investigators [Among other etiologies causing LVOTO, LVH was most frequently observed (9% of the study population) in the present series. In this group, multiple clinical characteristics including old age (mean age 81 ± 14 years), hypertension (100%), anemia (mean hemoglobin level 10.0 ± 1.8 mg/dl), small LV cavity (mean LVDd 37 ± 6 mm), hyperkinetic LV function (mean LVEF 79 ± 8%) and high ry of HT . Elderlyry of HT  reportedry of HT . Patientry of HT  reportedtigators . The mectigators . TherefoFrom the above, LVOTO is diverse. LVOTO could occur when two or more structural LV characteristics and hemodynamic status were present together. The degree of involvement of these factors may vary by the disease and/or its status and LVOT PG may also show different changes by pharmacological or surgical interventions.In the present study, 43% of patients had anemia at the time of the initial TTE. It has been reported that moderate-to-severe anemia increases stroke volume and heart rate  and anemThe present results showed that LVOTO might depend on the coexistence of primary morphological LV characteristics and hemodynamic LV status. To improve LVOTO, we should investigate clinically relevant factors and manage the LVOTO accordingly.


Right Atrial Volume Index (RAVI) measured by echocardiography is an independent predictor of morbidity in patients with heart failure (HF) with reduced ejection fraction (HFrEF). The aim of this study is to evaluate the predictive value of RAVI assessed by cardiac magnetic resonance (CMR) for all-cause mortality in patients with HFrEF and to assess its additive contribution to the validated Meta-Analysis Global Group in Chronic heart failure (MAGGIC) score.2; significantly larger in patients with than without an event . RAVI (per ml/m2) was an independent predictor of mortality . RAVI has a greater discriminatory ability than LVEF, left atrial volume index and right ventricular ejection fraction (RVEF) . The addition of RAVI to the MAGGIC score significantly improves risk stratification .We identified 243 patients  with left ventricular ejection fraction (LVEF) ≤ 35% measured by CMR. Right atrial volume was calculated based on area in two- and four -chamber views using validated equation, followed by indexing to body surface area. MAGGIC score was calculated using online calculator. During mean period of 2.4 years 33 patients (14%) died. The mean RAVI was 53 ± 26 ml/mRAVI by CMR is an independent predictor of mortality in patients with HFrEF. The addition of RAVI to MAGGIC score improves mortality risk stratification. An estimated 5.7 million Americans have heart failure (HF). The foreseen increase in the prevalence of HF will top 8 million by 2030. Approximately 870, 000 new cases of HF are diagnosed annually . Pocock Right atrium volume index (RAVI) measured by trans-thoracic echocardiography (TTE) was identified as an independent predictor of adverse outcome in patients with HF with reduced ejection fraction (HFrEF) . These fThis study is part of an ongoing outcomes registry of patients undergoing CMR imaging at the New York Methodist Hospital. Our study was approved by the institutional review board. Every patient enrolled in this study provided written informed consent for inclusion of CMR, demographic, and outcomes data to the registry. There was no external funding used to support this work. The authors are fully responsible for the design and conduct of this study, all data analysis, drafting, editing of the paper and its final content.We systematically obtained clinical, demographic, electrocardiographic  and laboratory data  via direct patient interview at the time of enrollment in the registry, and review of notes from referring physicians and electronic medical record at the time of CMR scan. Vital status was followed at regular intervals after initial CMR. Data were collected at regular intervals by cardiovascular research associates blinded to the CMR results through either standardized telephone interview with the patients or, if deceased, with family members or contact with the referring physician; review of inpatient and outpatient medical records. Vital status and date of death was additionally confirmed using Social Security Death Index. The primary outcome was all-cause mortality. Cause of death was adjudicated using electronic health records, death certificate, telephone interview with a relative or with a physician involved in care. We defined cause of death as cardiac or non-cardiac.Patients referred for CMR from June 2006 to December 2014, older than 18 years of age, with severely reduced left ventricular systolic function defined as an ejection fraction (EF) ≤ 35% at index CMR exam were and enrolled in the registry were enrolled in this study. We excluded patients with complex congenital heart disease, prior valve surgery, severe valvular disease by CMR, those scheduled for open heart surgery or patients with inadequate/incomplete imaging.CMR studies were performed with a 1.5-T CMR system Magnetom Avanto™ using standard pulse sequences. Before contrast administration, short-axis steady-state free precession (SSFP) images covering the left ventricle (LV) from the mitral valve annulus to the apex were obtained for the evaluation of LV function. Three chamber, four chamber and two (both right and left sided) chamber SSFP images were also acquired according to the American Heart Association imaging recommendation . All patTwo physicians blinded to patient clinical data performed area measurements of the right atrium in the 4-chamber view and right-sided-2 -chamber view at the end systole utilizing retrospectively gated SSFP images. Precession™ software (Heart Imaging Technologies®) was used for area measurements. The right atrial appendage was not included in the measurements. The equation(RA volume = 3.08*(2C area) +3.36*(4C area) -44.4) published elsewhere was utilized to calculate right atrial volume (7). Body surface area (BSA) was calculated according to the Mosteller formula. Right aBoth left and right ventricular volumes were measured during diastole and systole using PRECESSION software™. For the volumetric assessment of the both ventricles and calculation of ejection fraction we utilized technique described by Bourantas et al . Left Athttp://www.heartfailurerisk.org/). In order to calculate the MAGGIC score data regarding age, sex, history of diabetes, history of chronic obstructive pulmonary disease, duration of heart failure (more or less than 18 months), smoking status, current HF NYHA class[MAGGIC score was calculated using an on-line calculator  for normally distributed variables, or median and interquartile range (IQR) for non-gaussian distributed variables. We present categorical data as frequencies. Continuous variables were compared using Mann-Whitney test. The Wilcoxon rank sum-test test was performed for categorical variables. Spearman’s and Pearsons correlation coefficients were used to show a correlation between RAVI and tested variables. The primary outcome was assessed using Cox proportional hazard model controlled for MAGGIC (Integer) risk and right ventricular ejection fraction (RVEF). The proportional hazard assumption was confirmed using Schoenfeld residuals  and by the scaled Schoenfeld residuals plot. Kaplan–Meier curves with the log-rank statistic were used to illustrate outcome. Receiver operating characteristic (ROC) analysis was used to compare area under the curve (AUC) of RAVI to volumetric CMR parameters  and MAGGIC risk for the prediction of mortality . We thenWe included 243 patients in our analysis . Baselin2 vs 48 ml/m 2, p<0.001.  over a median follow-up of 2.2 years (interquartile range 1–3.4), and mean follow-up of 2.4 ± 2 years. Of those, seventeen patients (51%) had a cardiac, eight patients (24.5%)- non-cardiac and eight patients (24.5%) had unknown cause of death. There were four and 29 deaths in patients with RAVI ≤ than median and > than median, respectively, P<0.001, corresponding to annual mortality of 9.2% vs. 1.4%, respectively .On averaFig A in .MultivarFig A in , and eveFig A in .Further analysis showed that RAVI has greater C statistics (AUC 0.79 (CI 0.72–0.88) than RVEF (AUC 0.62 (CI 0.50–0.73), p<0.03), LAVI (AUC 0.68 (CI 0.57–0.78), p<0.02) and LVEF (AUC 0.55 (CI 0.45–0.66), p<0.01). There was no difference in C statistic between RAVI and MAGGIC score (AUC 0.77 (CI 0.67–0.86), p = 0.61, Twenty-five RAVI measurements were randomly selected and measured separately by two operators blinded to each other’s results (A.I. and A.M.).There was a excellent reproducibility of RAVI measurements for both observers: 1.9 ml/m2 (95% CI (-3.2)-4.9) for observer I and 1.5 ml/m2 ((-1.9)- 4.1) for observer II, with mean interobserver RAVI difference assessed by Bland-Altman method was -1.7 ml/m2 (95% CI -2.5–4.4).Spearman correlation coefficient for interobserver variability of RAVI was 0.951 (p<0.001)We report several findings emphasizing the prognostic importance of RAVI in our cohort of patients with severely reduced left ventricular systolic function referred for CMR imaging. First, RAVI was an independent predictor of mortality even after adjusting for MAGGIC score and right ventricular ejection fraction. Second, RAVI is a stronger predictor of mortality than LVEF and LAVI. Thirdly, addition of RAVI increased mortality discrimination of MAGGIC score when assessed by IDI and NRI. These findings make RAVI an important predictor of mortality in patients with HFrEF and suggest that it provides more important prognostic data when compared to conventionally used predictors.MAGGIC score is a result of a patient level meta-analysis of 39,372 patients from 30 studies with a median follow-up of 2.5 years—the largest dataset published for score derivation (2). Median and mean integer score were 19 and 20, respectively, in our cohort, consistent with the observed mortality of 12.8% at a median follow-up of 1.6 years. RAVI and MAGGIC score were predictors of mortality in the multivariable analysis, independent of each other. MAGGIC score has excellent discrimination ability for mortality, with AUC of 0.76± 0.1. The addition of RAVI to this well calibrated risk score leads to significant risk reclassification when assessed by IDI and NRI.Sallach et al.(3) assessed RAVI as a surrogate of right ventricular function by TTE in 192 patients. They found RAVI to be an independent predictor of primary outcome that consisted of mortality, cardiac transplant and repeated hospitalization for HF. In our analysis RVEF was a predictor of mortality only in univariate analysis, and RAVI remains to be an independent predictor in multivariate analysis even after controlling for RVEF and other variables. Moreover RAVI has a greater C statistics than RVEF for predicting mortality. These findings suggest that right atrial volume is a more sensitive parameter than right ventricular systolic function to identify higher risk patients. Their reported mean RAVI was 28 ml/m2, much smaller than in our analysis (53 ml/m2). This difference might be explained by several factors: CMR has a greater precision with higher spatial resolution of endocardial borders, use of slightly different anatomical views to measure right atrium by echocardiography and absence of geometric assumptions in retrospectively gated CMR images in contrast to echocardiography. Whitlock et al. reported a significant underestimation of RA volume by echocardiographic area length method when compared to CMR imaging.There are only a few peer-review articles addressing right atrial dimensions by CMR , 19–21. The underlying pathophysiologic mechanism linking RAVI with mortality is not fully understood. One of the possible explanation maybe the fact that right atrium has thin walls, and it is influenced by the same factors that are affecting right ventricular diastolic filling and increase in RAVI may serve as an early manifestation of right ventricular diastolic dysfunction even when RVEF is still relatively preserved. Further investigation of RAVI’s dynamic changes in patients with heart failure and their correlation with adverse clinical outcomes is warranted.There were multiple publication elaborating on utilization of transthoracic echocardiography for an evaluation of right ventricular function in patients with HfrEF –25. In oWe recognize several limitations of our study. There is, probably, some degree of referral and selection bias for patients undergoing cardiac MRI in our center. However the external validity of our findings was supported by MAGGIC score—predicted mortality rate is in line with observed one. The total number of patients was significantly affected by our inclusion/exclusion criteria, however our sample size is comparable or exceeding number of patients with LVEF<35% in recent publications–29. MoreDespite these limitations, we conclude that right atrium volume index measured by cardiac magnetic resonance imaging is an independent predictor of mortality in patients with heart failure with reduced ejection fraction. The addition of right atrium volume to the MAGGIC score significantly improves mortality risk stratification.S1 FileContains Fig A, Fig B.(DOCX)Click here for additional data file.


This study evaluated hemodynamic feasibility and reproducibility of a new method for in vivo simulation of human atrial fibrillation (AF). The method was tested during sinus rhythm in 10 patients undergoing catheter ablation for AF. A simple electronic device was assembled that allowed triggering a cardiac stimulator by predefined series of RR intervals. Irregular RR interval sequences with a mean heart rate of 90/min and 130/min were obtained from ECG recordings of another patients with AF. Simultaneous atrioventricular pacing was delivered by catheters placed inside the coronary sinus and at the His bundle region. Hemodynamic effect of the simulated AF was assessed by invasive measurement of the left ventricular (LV) pressure, dP/dt, and Tau. Compared to regular pacing at the same mean heart rate, the simulated AF significantly impaired the LV both systolic and diastolic function. Repeated AF pacing in the same patients generated similar LV hemodynamics. The proposed method provides a realistic and reproducible in-vivo model of AF. It can be exploited for investigation of the hemodynamic consequences of AF in various patient populations. Atrial fibrillation (AF) is a common clinical arrhythmia. It can have various hemodynamic consequences. Excessive heart rate, loss of left atrial kick, and heart rhythm irregularity can all impair the cardiac performance –3. MoreoWe developed a new pacing-based method for simulation of the hemodynamics in AF in vivo. Compared to previously used methods –4, 7, 8,In this study we evaluated the feasibility of the method by assessment of the left ventricular (LV) performance during the simulated AF and during regular pacing with matched heart rate. We also evaluated reproducibility of the method by repeated assessment of the LV performance in the same patients.Feasibility of the AF simulation was tested in 10 patients without structural heart disease who were undergoing catheter ablation for paroxysmal AF . The samOur method of AF ablation and the catheter setup were described in detail elsewhere . Using tThe region of the His bundle was first systematically probed by pace-mapping, to ensure stable capture of the conduction system. The catheter position was adjusted to produce QRS complexes that morphologically best resembled the patient’s native QRS complexes. The selected pacing site was tagged on the 3D electroanatomic map  to ensure finding of the identical site in case of incidental catheter dislodgement. The catheter-tissue contact was monitored continuously by inspection of local electrograms and QRS morphology.PhysioNet.org [The patients were paced at a mean heart rate of 90/min and 130/min in three 2-minute sequences: regular atrial pacing, regular simultaneous AV pacing, and simulated AF which was represented by the irregular simultaneous AV pacing. The irregular RR-interval sequences were obtained as text files from an open database of processed ECG recordings of patients with AF (The Long-Term AF Database at oNet.org ). The tewww.R-project.org). Data are reported as mean ± standard deviation. P-values <0.05 were regarded significant.The LV pressure signal and standard ECG leads were recorded at 1 kHz using a data acquisition device . The recordings were analyzed offline in a dedicated software . Statistical analyses were conducted in R  but it did not significantly affect the LV enddiastolic pressure or Tau. The irregularity of the heart rhythm itself significantly impaired both the systolic and diastolic LV function, though the dP/dt Max decreased only at the higher heart rates. The fast heart rate caused significant decrease in the systolic pressure but it did not affect any other LV hemodynamic characteristics. Compared to atrial pacing, the simulated AF significantly impaired all the LV hemodynamic characteristic at both heart rates. At last, We have demonstrated hemodynamic feasibility and reproducibility of a novel method for simulation of acute hemodynamics during human AF. The method has three key features: First, custom series of RR intervals—which can be obtained by processing of a real AF recording or created from scratch in a text editor—can be accurately reproduced by pacing in other individuals. This enables a rigorous study of the individual hemodynamic effects of an excessive heart rate and ventricular rhythm irregularity. Second, the His bundle pacing approximates an intrinsic ventricular depolarization, thereby allowing an unbiased study of the cardiac performance . FinallyPrevious studies investigated the hemodynamics in AF mainly by performing measurements before and after a cardioversion to sinus rhythm , 16. ThiDaud et al. studied the effects of the irregularity of the ventricular rhythm on the cardiac output by comparing regular pacing with pacing by a loop of five extrastimuli with variable cycle length . Of noteMelenovsky et al. used a specially designed external stimulator which delivered random pulses through exposed electrodes of a biventricular pacemaker . The bivOur method was in part inspired by the studies of Clark et al. and Segerson et al. , 4. The Our method does not require any special hardware but a low-cost (~40 USD) microcomputer based on the wide-spread Arduino platform. Setting-up of the device does not require a deep knowledge of engineering. The series of RR intervals can be adjusted according to the needs in a common text editor and subsequently simply copy-pasted as a string array to the control software. This feature provides a particularly productive framework when combined with a database of annotated ECG recordings such as the PhysioNet . Our metOur hemodynamic model of AF provides broad opportunities for studying various hemodynamic consequences of AF. For example, it can be exploited to identify which of the attributes of an AF—the fast heart rate, the irregularity of the rhythm or the loss of the atrial contraction—has the dominant role in a particular clinical setting. Such studies could provide data for future tailoring of patient-specific therapeutic strategies: rhythm control by drugs or catheter ablation versus rate control by drugs or AV junction ablation. Also, the hemodynamic model can be used to investigate whether the beat-to-beat variations in the blood pressure that occur in AF can disturb baroreflex or perfusion of organs, in which the blood flow is closely auto-regulated in response to the instantaneous perfusion pressure–particularly in the brain, kidney and myocardium.An inherent limitation of our method is that the paced sequence must be always faster than the patients’s intrinsic heart rate. Although all study participants fulfilled this condition, some other individuals might need bradycardizing medication during the experiment. Our model of AF would have been even more realistic by pacing independently from the His bundle and at the same time rapid random pacing from the coronary sinus in order to completely abolish the atrial contraction. However, such setup would require a complete AV block or significant bradycardia induced by administration of AV-nodal blocking agents. Moreover, rapid pacing from the coronary sinus could induce AF and thereby compromise the experiment.The hemodynamic feasibility of our AF model could be even better demonstrated if we reproduced LV hemodynamics assessed during the patient’s own AF. This would require additional measurements and on-line processing of the ECG recordings. Regrettably, such study protocol was not permitted by our institution’s Ethical Committee as it would significantly prolong the whole ablation procedure. On the other hand, the advantage of our protocol was that it allowed to evaluate the inter-individual reproducibility of the LV hemodynamics and, more importantly, it allowed to control the heart rate and heart rhythm irregularity. At last, our model of AF enabled to study only acute changes in the hemodynamics.