Accidental hypothermia is a condition associated with significant morbidity and mortality. Hypothermia has been reported to affect left ventricular systolic and diastolic function. However, most of the data come from animal experimental studies.The purpose of the present study was to assess the impact of severe accidental hypothermia on systolic and diastolic ventricular function in patients treated using veno-arterial extracorporeal membrane oxygenation (ECMO).We prospectively assessed nine hypothermic patients  who were transferred to the Severe Accidental Hypothermia Center and treated with ECMO. Transthoracic echocardiography was performed on admission (in patients without cardiac arrest) and on discharge from ICU after achieving cardiovascular stability. Cardiorespiratory stability and full neurologic recovery was achieved in all patients.Biomarkers of myocardial damage  were significantly elevated in all study patients. Admission echocardiography performed in patients in sinus rhythm, revealed moderate-severe bi-ventricular systolic dysfunction and moderate bi-ventricular diastolic dysfunction. Discharge echocardiography showed persistent mild bi-ventricular diastolic dysfunction, although systolic function of both ventricles returned to normal. Discharge echocardiography in patients admitted with cardiac arrest showed normal (5 patients) or moderately impaired (1 patient) global LV systolic function on discharge. However, mild or moderate LV diastolic dysfunction was observed in all 6 patients. Discharge RV systolic function was normal, whereas mild RV diastolic dysfunction was present in these patients.After severe accidental hypothermia bi-ventricular diastolic dysfunction persists despite systolic function recovery in survivors treated with ECMO. Accidental hypothermia is a condition associated with significant morbidity and mortality . In the There are several reports describing the effects of mild therapeutic hypothermia on the circulatory system , 5 and oThe purpose of the present study was to assess the impact of severe accidental hypothermia on systolic and diastolic ventricular function in patients treated by means of veno-arterial extracorporeal membrane oxygenation (ECMO).The observational case-series study was approved by the Local Ethical Committee of the John Paul II Hospital in Cracow. After full recovery, all the study subjects gave informed consent to the use of their medical data.We prospectively assessed 9 patients  who were reported to the Severe Accidental Hypothermia Center due to severe accidental hypothermia . All patients fulfilled the extracorporeal rewarming criteria and were admitted to our hospital for the implementation of invasive rewarming procedures.A standard 12-lead electrocardiogram (ECG) was performed within 5 min after their admission to the Emergency Room in patients without cardiac arrest. Blood samples were collected for the plasma levels of biomarkers for myocardial damage: creatine kinase (CK), cardiac isoenzyme of creatine kinase (CKMB) and high sensitivity troponin T (hsTnT) at the time of admission and after 6 h. Blood tests were assayed by routine automated laboratory techniques . All biochemical analyses were performed in the central hospital laboratory, certified with a cardiac and clinical chemistry program by RIQAS . Transthoracic echocardiography was performed on admission (in patients without cardiac arrest) and on discharge from the Intensive Care Unit (ICU) after achieving cardiovascular stability . The examinations were conducted by means of a portable ultrasound system equipped with a 1–5 MHz transthoracic phased-array transducer . Comprehensive echocardiographic examination was performed by experienced cardiologists-echocardiographers. All the studies were recorded as digital 10-s video clips and independently analyzed by 2 echocardiographers. In each case a consensus was achieved, no ambiguous echocardiographic examinations were recorded and all results were clearly stated in a dichotomic manner.The left ventricular ejection fraction (LVEF) was measured with the bi-plane Simpson method. Tricuspid annular plane systolic excursion (TAPSE) by M-mode was used to assess the global right ventricular systolic function. Pulsed-wave (PW) Doppler was performed in the apical 4-chamber view to obtain mitral and tricuspid inflow velocities. A sample volume was placed between the mitral or tricuspid leaflet tips during diastole. Spectral mitral and tricuspid velocity recordings were obtained at a sweep speed of 50 mm/s. PW tissue Doppler imaging (TDI) was performed in the apical views to acquire mitral and tricuspid annular velocities. The sample volume was positioned 1 cm within the septal and lateral insertion sites of the mitral leaflets and 1 cm within the lateral insertion site of the tricuspid leaflet. Spectral recordings were obtained at a sweep speed of 50 mm/s at end-expiration. Measurements obtained at 3 consecutive cardiac cycles were averaged , 13.The following spectral Doppler  parameters were taken into final analysis of left and right ventricular function: peak early filling (E-wave) velocity, late diastolic filling (A-wave) velocity, the E/A ratio, deceleration time (DT), early diastolic annular velocity (e’), the E/e’ ratio. Left ventricular (LV) diastolic dysfunction was diagnosed by the following TDI mitral parameters: septal e’ < 8 and lateral e’ < 10 cm/s . In patiThe study population consisted of nine patients , admitted to our hospital in severe accidental hypothermia . Six patients were admitted in hypothermic cardiac arrest  whereas 3 patients were in cardiogenic shock and their sinus rhythm was observed. Demographic and clinical data are presented in Table Biomarkers of myocardial damage  were significantly elevated in all the study patients Table .Table 2LAdmission echocardiography performed in patients in sinus rhythm, revealed moderate-severe bi-ventricular systolic dysfunction and moderate bi-ventricular diastolic dysfunction Table . DischarFive patients admitted with cardiac arrest had normal global LV systolic function on discharge. One patient (M/78 years) had moderately depressed LV systolic function LVEF 40 %). All the patients had mild (50 % of patients) or moderate LV diastolic dysfunction. Discharge RV systolic function was normal, whereas mild RV diastolic dysfunction was present in all the six patients from this group  were observed in all patients both on admission and 6 h later, indicating severe myocardial damage.Baseline echocardiography (prior to ECMO implantation) was conducted in 3 patients in sinus rhythm on admission. The examination revealed moderate-severe bi-ventricular systolic dysfunction requiring inotropic support and moderate bi-ventricular diastolic dysfunction in these patients. Systolic LV and RV function returned to normal, whereas mild bi-ventricular diastolic dysfunction was still present on discharge. Discharge echocardiography in patients admitted with cardiac arrest showed normal (5 patients) or moderately impaired (1 patient) global LV systolic function. However, in all the 6 patients mild or moderate LV diastolic dysfunction was observed. Discharge RV systolic function was normal, whereas mild RV diastolic dysfunction was present in these patients.There is no pre-hypothermia echocardiographic data to compare, because none of our patients had an echocardiographic study conducted before the incident. The follow-up period covered only ICU stay. However, despite these doubtless limitations, the data obtained allow the diagnosis of bi-ventricular diastolic cardiomyopathy in hypothermia survivors after ECMO rewarming persistent on discharge from the ICU. Mild LV diastolic dysfunction was present on discharge in six patients. Moderate LV diastolic dysfunction was observed in 3 patients: a 29-year-old male, a 25-year-old female and a 78-year-old male. Since age is a primary consideration when defining normal values of mitral inflow , all LV 2+  [2+ after 4-h hypothermia in an animal model. Schwarzl et al. [Transient cardiac systolic and diastolic dysfunction was observed in subjects with mild therapeutic hypothermia  and in pxchange) –11. Tveixchange) , 11 repoxchange)  found seBi-ventricular diastolic dysfunction after severe accidental hypothermia persists despite the recovery of systolic function in survivors after ECMO rewarming.Severe accidental hypothermia causes significant bi-ventricular systolic and diastolic dysfunction.Ventricular systolic function returns to normal after ECMO rewarming.Bi-ventricular diastolic dysfunction after severe accidental hypothermia persists despite the recovery of systolic function after ECMO rewarming.


Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by heterogeneous clinical symptoms and able to be visualized by cardiac magnetic resonance (CMR). Late gadolinium enhancement (LGE) represents myocardial fibrosis. There is still a challenge for HCM patients with contrast agent contraindication. T1rho CMR has been reported to determine fibrosis in myocardial infarction. To our best knowledge, no T1rho studies in HCM patients reported. We aimed to explore whether T1rho CMR detects fibrosis in HCM patients.Twelve HCM patients underwent T1rho imaging using an ECG-gated T1rho prepared gradient echo(GRE) sequence before LGE imaging. Three short-axis views of T1rho and LGE sequences were obtained from base to apex. T1rho imaging parameters were: TR 3.2 ms, TE 1.52 ms, flip angle 15°. FOV 172 mm × 232 mm, voxel size 1.8 mm × 1.8 mm × 8.0 mm, time of spin locking = 10, 30, 50 ms, and spin locking frequency = 340.6 Hz. Images were analyzed by a custom-made software written in Matlab 7.1. for T1rho relaxation time map. T1rho relaxation times in LGE area and remote area were measured by ImageJ.LGE was present in 8 patients. T1rho relaxation times of scarring and remote myocardium were summarized in Table LGE area showed hyper-T1rho values, suggesting T1rho has a potential to detect myocardial fibrosis in HCM patients. Hyper-T1rho values in area without LGE might be explained by diffuse fibrosis. In conclusion, non-contrast T1rho might visualize myocardial fibrosis includes diffuse fibrosis regardless contrast agent contraindication.http://www.973.gov.cn/English/Index.aspx].This work was supported by the National Natural Science Foundation of China [http://www.nsfc.gov.cn/Portal0/default166.htm], and the National Basic Research Program of China  [


Echocardiography has become a critical tool in the evaluation of patients presenting to the emergency department (ED) with acute cardiovascular diseases and undifferentiated cardiopulmonary symptoms. New technological advances allow clinicians to accurately measure left ventricular (LV) strain, a superior marker of LV systolic function compared to traditional measures such as ejection fraction, but most emergency physicians (EPs) are unfamiliar with this method of echocardiographic assessment.This article discusses the application of LV longitudinal strain in the ED and reviews how it has been used in various disease states including acute heart failure, acute coronary syndromes (ACS) and pulmonary embolism.It is important for EPs to understand the utility of technological and software advances in ultrasound and how new methods can build on traditional two-dimensional and Doppler techniques of standard echocardiography. The next step in competency development for EP-performed focused echocardiography is to adopt novel approaches such as strain using speckle-tracking software in the management of patients with acute cardiovascular disease. With the advent of speckle tracking, strain image acquisition and interpretation has become semi-automated making it something that could be routinely added to the sonographic evaluation of patients presenting to the ED with cardiovascular disease. Once strain imaging is adopted by skilled EPs, focused echocardiography can be expanded and more direct, phenotype-driven care may be achievable for ED patients with a variety of conditions including heart failure, ACS and shock. According to data from the Centers for Disease Control, cardiovascular disease remains the leading cause of death in the United States4In 2010, the American College of EPs and the American Society of Echocardiography formed a joint committee and released a position paper entitled Focused Cardiac Ultrasound in the Emergent Setting, establishing standards for ED-based POC echocardiography.0) from the distance between them at the end of the movement (L1), and dividing it by the starting length (ɛ=ΔL/L0 where ΔL=L1–L0). Speckle tracking is a recent development in image processing that uses proprietary software to automatically determine the movements of these points, which appear sonographically as “speckles,” within the myocardium over time. Tracking the movements of these speckles throughout the cardiac cycle yields positive values when the two points are moving away from one another, as in diastole, and negative values when they are moving towards one another, as in systole. Speckle tracking obviates the need for Doppler-based imaging technologies to calculate strain, which have proved to be challenging due to their angle-dependency.8In the past decade, a new echocardiographic technique known as strain has been used to make more accurate assessments of the contractile state of the heart.Strain can be measured in any of the longitudinal, circumferential or radial planes of the left ventricle (LV); however, the longitudinal orientation is likely the most useful, as impairment in this plane occurs earlier than in the other planes.LV strain on 2D echocardiography has been well studied in patients with ACS. For patients with ST-segment elevation MI (STEMI), LV GLS has been shown to be an important predictor of post-discharge adverse outcomes.15For the EP, strain echocardiography may offer a rapid and sensitive tool to determine which patients with NSTEMI would benefit from urgent revascularization. With the advent and implementation of increasingly sensitive troponin assays,The mechanics of the LV are much more complex than basic 2D imaging is able to depict. As mentioned previously, deformation of the LV occurs in three planes: longitudinal, circumferential, and radial. These vectors of motion are the result of natural myocardial fiber orientation, with subendocardial and subepicardial fibers being arranged in a longitudinal fashion, while mid-wall fibers are arranged in a circular orientation.24Of particular importance for the ED patient with acute HF, strain is acutely sensitive to the loading conditions of the heart,Although traditional measurements of LV systolic function do not change appreciably during treatment of acute HF, diastolic parameters do demonstrate improvements as patients undergo treatment.29A recent case from our ED highlights some of these important concepts. A 61-year-old female presented with two days of dyspnea, which had acutely worsened in the last several hours. Her initial blood pressure was 269/181mmHg, heart rate 105 beats per minute, respiratory rate 28 breaths per minute and pulse oximetry 89% on room air. Her lung examination revealed diffuse rates and a focused lung ultrasound showed diffuse B lines consistent with acute cardiogenic pulmonary edema. She was started on non-invasive positive pressure ventilation (NIPPV) and received multiple 2mg boluses of IV nitroglycerin, which were followed by a continuous nitroglycerin infusion. Shortly after the initiation of NIPPV a POC echocardiogram was performed showing mild LV hypokinesis on the standard 2D images, but significantly impaired anteroseptal myocardial strain with a GLS of −7.1% . ApproxiOne of the newer applications of strain is the assessment of the right ventricular (RV) free wall.Strain analysis using speckle-tracking technology does have several limitations that need to be understood prior to the adoption of this as a part of POC echocardiography in the ED. Speckle tracking relies on the ability of the ultrasound system to track specific acoustic markers in the myocardium over time, and is thus dependent on achieving high frame rates. Increasing depth and increasing sector width (among other variables related to the transducer) will decrease the frame rate. Frame rates less than 40 frames per second (Hz), result in large frame-to-frame changes, resulting in poor tracking of acoustic speckles. One strategy we have found useful to improve the frame rate is to decrease depth in the apical windows such that the left atrium is out of the field of view. Another limitation with speckle tracking is found in patients with heart rates >120 beats per minute; as fewer data points from a single cardiac cycle are available, strain data becomes less reliable. Also, if the heart rate is dramatically different from one clip to the next  the algorithm will be unable to calculate GLS because of the differences in tracking. As a consequence of beat-to-beat variability, conditions such as atrial fibrillation and multifocal atrial tachycardia are considered a relative contraindication to strain imaging. Fortunately, in our experience, the heart rate and rhythm-related limitations occur infrequently in patients being evaluated for ACS or acute HF.Beyond technical issues, a lack of standardization of left ventricular (LV) strain between the various ultrasound manufacturers has led to hesitancy in the adoption of strain as a routine measure of cardiac function in some echocardiography labs.Echocardiography in the ED has evolved from a single institution borrowing their cardiology department’s equipment during off-hours to standards of training that have been endorsed by the Accreditation Council for Graduate Medical Education and adopted by all emergency medicine residencies. A core curriculum currently exists for focused echocardiography in the ED with assessment of the pericardium, LV systolic function, the right heart, preload determination, and procedural guidance. We believe that the next step in the development of the field is to adopt novel approaches such as strain echocardiography using speckle-tracking software to assist in the management of patients with HF, MI and PE. The advent of portable ultrasound systems and semi-automated software to calculate strain makes this a real possibility for ED POC echocardiography in the near-term. Once EPs gain familiarity with the speckle-tracking software, a research agenda will need to follow, with a goal of studying the utility of strain imaging at the POC. The net result will be a more individualized, image-based understanding of conditions such as ACS, acute HF, and PE with the potential to develop treatment protocols directed at echocardiographic phenotypes.


Heart failure (HF) is a public health concern with an immense effect on the utilization of precious and finite health care resources. More than 23 million people worldwide have been affected by symptoms of HF . The equThe changing demographic profiles of the present-day HF populations underscore the importance of pursuing a multidisciplinary approach that will vigilantly address not only the cardiac pathology, but also the numerous accompanying comorbidities. Mortality burden of heart failure is comparable to or greater than that of malignancies such as melanoma, breast, kidney, and colorectal cancers . PromotiThe convergence of increasingly more effective acute HF therapies and the adverse downstream effects of modern-day lifestyles will likely lead to a further increase in chronic HF patients.Croatian Medical Journal, multiple venues of advanced HF treatment are reviewed (In this issue of the reviewed -10. The CMJ contains a comprehensive report on pre-transplant and perioperative predictors of heart transplantation outcomes (Although heart transplantation has marginal epidemiological gain, it remains the benchmark against which all other modes of management must be compared. The excellent 10-year survival of heart transplantation recipients reinforces its position as the gold standard in the management of end-stage HF . While ooutcomes . Multiploutcomes The principal limitation of heart transplantation, however, stems from a continued lack of available organs. This discrepancy between limited donor availability and high organ demand provides a fertile ground for the implementation of implantable ventricular assist devices. High-quality survival supremacy of mechanical circulatory assistance over optimal medical therapy among patients with end-stage HF is juxtaposed to the high incidence of complications that accompany this technology . Device-Among many challenges that exert an important impact on outcomes in this patient population, clearly pivotal is the management of the failing right ventricle. Options for long-term support of the failing right ventricle have not followed the rapid evolution seen in the LVAD arena. A common clinical scenario is unmasking of incipient right ventricular failure after LVAD implantation. Multiple laboratory, clinical, and echocardiographic parameters have been employed in the effort to identify LVAD patients in whom right ventricular failure will occur, but an effective predictive system is still not available. Nonetheless, this issue presents a summary of available scoring systems and their individual performances .CMJ provides a roadmap that aims to expedite this process.While the focus of the medical and industrial communities on long-term implantable devices is certainly warranted, one should also appreciate the progress made in the domain of supporting the circulation during acute cardiac failure ,7,8. TheThe revolutionary paradigm shift from pulsatile to continuous-flow ventricular assist devices allowed for their miniaturization, thereby rapidly expanding their clinical applicability. Also, the benefits of this technology have been underscored by improved durability and a lower incidence of adverse events. The clear advantages of novel devices notwithstanding, adverse sequelae of long-term exposure to non-pulsatile blood flow have been identified. An article in this issue  reviews Reducing the high thromboembolic burden associated with HF may become another therapeutic goal in this challenging patient population. The current evidence on antithrombotic agents in HF patients, however, does not support their wider implementation .In order to put the contemporary management strategies for advanced HF into perspective one has to contrast their unequivocal efficacy with their high financial and logistical burdens. Balancing these opposing forces requires finesse, and is pivotal to optimizing the results of HF management programs. Furthermore, equality of opportunity for all patients in the utilization of these finite resources should be fostered. The purpose of this theme issue is to disseminate information related to heart failure management, while providing insight into the associated challenges.


Echocardiography was performed at baseline and at days 2, 7, 14, and 28 post-MI to assess 3D LV volumes and cardiac function, as compared to infarcted controls (n = 9). Early infarct zone dyskinesia was measured on day 2 post-MI using a novel metric, the dyskinesia index. End-systolic volume was significantly reduced in the ECM-treated group compared to controls by day 14. Ejection fraction and stroke volume were also significantly improved in the ECM-treated group. ECM-treated hearts showed a significant (P < 0.005) reduction in dyskinetic motion on day 2. Thus, using high-frequency ultrasound, it was shown that treatment with a cardiac-derived ECM preparation reduced early infarct zone dyskinesia and post-MI LV remodeling in a mouse model of reperfused MI.Extracellular matrix (ECM) degradation after myocardial infarction (MI) leaves the myocardium structurally weakened and, as a result, susceptible to early infarct zone dyskinesia and left ventricular (LV) remodeling. While various cellular and biomaterial preparations have been transplanted into the infarct zone in hopes of improving post-MI LV remodeling, an allogeneic cardiac muscle-derived ECM extract has yet to be developed and tested in the setting of reperfused MI. We sought to determine the effects of injecting a novel cardiac muscle-derived ECM into the infarct zone on early dyskinesia and LV remodeling in a mouse model of MI. Cardiac muscle ECM was extracted from frozen mouse heart tissue by a protocol that enriches for basement membrane constituents. The extract was injected into the infarct zone immediately after ischemia/reperfusion injury ( In its acute stage, myocardial infarction (MI) results in immediate death to cardiomyocytes and contractile dysfunction. Immune cells infiltrate the area of infarction to phagocytize necrotic cells and apoptotic cell debris, and release enzymes that degrade the cardiac extracellular matrix (ECM)  of the transplanted cells , a reconstituted basement membrane-rich extract, has been widely used as a cell culture substrate for over 30 years  could improve cardiac function after reperfused MI. In order to test the therapeutic potential of cMatrix, high-frequency 3D echocardiography was used to serially assess cardiac function over 28 days in a mouse model of reperfused MI. Cardiac magnetic resonance (CMR) was performed in a subset of mice on day 2 post-MI to determine infarct size, and on day 28 post-MI to confirm volumetric results. A new metric, here termed the “dyskinesia index” (DI), was developed to measure early infarct zone dyskinesia, based on wall-motion data acquired from ultrasound image processing.The experimental design of this study is summarized in FigureThis study was conducted under protocols approved by the University of Virginia Animal Care and Use Committee. Adult male C57Bl/6 mice purchased from The Jackson Laboratory  were killed with an overdose of anesthetic and death was confirmed by cervical dislocation. Hearts were then immediately excised, rinsed in chilled phosphate-buffered saline (PBS), and atria were removed. The tissue was then snap-frozen in liquid nitrogen and stored at −80°C.3 pieces using a scalpel and homogenized. The homogenized mixture was then spun at 10,000 × g for 30 min. The pellet was washed with a 2 M buffered urea solution at a volume equal to the original weight of the tissue. The sample was then stirred overnight. Next, the sample was centrifuged at 18,000 × g for 30 min and the supernatant was decanted and stored at 4°C. Two additional extractions were performed by resuspending and homogenizing the pellet in urea buffer of one-half the volume of the original tissue weight, and repeating the centrifugation. Aliquots from each sample were evaluated for total protein and collagen content to assess quality. The combined supernatants were then dialyzed against 0.5% chloroform in tris-buffered saline (TBS) overnight to sterilize the extract. The extract was further dialyzed twice against TBS to remove the urea and chloroform, and once against PBS to enhance physiological compatibility. The final extract was aliquoted and stored at −20°C.All procedures were performed at 4°C. Frozen mouse tissue was thawed from −70°C in buffered 3.4 M NaCl with added protease inhibitors , in a volume six times the weight of the sample. The sample was minced into approximately 1 mmTotal protein concentration was measured using an assay kit based on the Lowry method . Collagen content was determined using the Sircol soluble collagen assay kit .μL were injected directly into the anterolateral sector of apical myocardium using a 31-gauge insulin syringe.Male C57Bl/6 mice were purchased from Jackson Laboratories (Bar Harbor). The mice were between 10–12 weeks old and weighed 24–29 g at the time of surgery. A standard myocardial ischemia/reperfusion protocol was employed, as described previously . Prior to scanning, fur was shaved from the chest followed by depilation with Nair cream. Ultrasound B-mode cardiac image sequences were acquired using a Vevo 2100 high-resolution ultrasound scanner . Anesthesia, body temperature, and EKG monitoring were performed as described previously  short-axis (SA) views were captured from the apex to the base of the LV in 1.0 mm intervals using a custom-built hardware/software system that controlled the motor stage holding the transducer and triggered the scanner to acquire and store image data. This system allowed for the full SA stack to be captured in less than one minute. Sequences averaged 30–40 frames per cardiac cycle. The total ultrasound imaging session duration was under 30 min per mouse.Left ventricular short-axis cross-sectional areas were calculated by manually delineating endocardial contours at end-diastole (ED) and end-systole (ES) at 1 mm intervals from apex to base. ED and ES were determined on a per slice basis. The LV end-systolic volumes (LVESV) and LV end-diastolic volumes (LVEDV) were then computed, and ejection fraction (EF) and stroke volume (SV) were derived from these values.The extent of wall thinning was calculated from endocardial and epicardial traces of long-axis images at end-diastole. Significant wall thinning was defined as a left ventricular wall thickness of 0.5 mm or thinner, relative to the normal end-diastolic wall thickness of 1 mm. The extent was defined as the percentage of the LV wall in which significant wall thinning occurred.A novel metric, here termed the “dyskinesia index” (DI) was employed to quantify early infarct zone dyskinetic motion, based on contraction index, as described by Li et al. . DI is ad” is the mean radial displacement of each segment “i”, at time point “t”. DI is defined as the average value of eff(t) over the course of the cardiac cycle (from t0 to tN): In this equation, “Thus, DI represents the average ratio of the sum of the signed radial displacements to the sum of the unsigned radial displacements, over the cardiac cycle. DI yields possible values ranging from 0 to 1, where a DI of unity represents completely polar radial displacement, and smaller values indicate increasing dyskinetic motion.In order to measure early infarct zone dyskinetic motion, DI was calculated on D2 post-MI using a short-axis slice 2 mm superior to the apex of the LV. VevoStrain speckle tracking software was used to obtain endocardial radial displacements for 96 segments over the course of a cardiac cycle  and DI was calculated as defined by Equation n = 3) and infarcted control (n = 6) mice on day 2 post-MI using late gadolinium-enhanced (LGE) CMR images. Infarct size was expressed as a percentage of the total LV mass. Post-MI day 28 volumetrics were determined for 3 cMatrix-treated mice using a black blood gradient echo pulse sequence imaging as described previously  . Tissue sections were then stained with picrosirius red to detect collagen.At D29 post-MI, the mice were killed with an overdose of anesthetic, and death was confirmed by cervical dislocation. Hearts were immediately excised and washed in PBS. The apical half of each heart was fixed in paraformaldehyde overnight at 4°C. The tissue was then washed three times in PBS and stored in 70% ethanol prior to embedding in paraffin and sectioning (5 P < 0.05. Data collected during reperfusion (days 2–28) were analyzed using two-way ANOVA for repeated measures. Post hoc analyses (Bonferroni post tests) were performed where appropriate. All other statistical analysis was performed using a two-tailed, unpaired Student's t-test. Statistical analyses were performed using Minitab version 16.1.1 .All data are presented as mean ± SEM. Statistical significance was presumed where Using the Lowry total protein assay, cMatrix had a total protein concentration of 5.1 mg/mL. Concentration of collagen types I through V was 240 mg per mg of total protein. Both the total protein and collagen concentration of cMatrix are comparable to the levels reported in similar extracts, such as Myogel and Matrigel  between groups.P = NS). Relative LV end-systolic volume (LVESV) was found to be significantly reduced in cMatrix-treated mice by day 14 and remained significant (P < 0.05) at day 28 . On day 28, EF was 35.0 ± 3.0% in cMatrix-treated mice versus 27.1 ± 2.0% in controls. By day 28, relative stroke volume (SV) was 22% greater in cMatrix vs. control mice . A CMR follow-up study was performed to confirm these results in a subset of animals on day 28 (n = 3). No statistically significant differences in LVESV or LVEDV were found between the two modalities.Serial 3D echocardiography performed at baseline and on days 2, 7, 14, and 28 post-MI showed improved LV remodeling and cardiac function in cMatrix-treated mice  over the course of the cardiac cycle (x-axis) to be viewed for each SA segment in an uninfarcted mouse .To determine the effect of cMatrix on early infarct dyskinesia, displacement data were obtained from speckle tracking analysis of echocardiographic image series collected on day 2 post-MI from short-axis (SA) image planes positioned 2 mm superior to the apex of the heart. Figureouse Fig.E, an untouse Fig.F, and a ouse Fig.G. Note tP < 0.05).The presence of cMatrix in the LV was confirmed by observing a thickening of the anterior septum surrounding the site of injection using CMR imaging on day 28 Fig.A and by This study describes the preparation and evaluation of cMatrix, a novel cardiac muscle-derived biomaterial. After reperfused MI, cMatrix-treated mice were shown to have reduced LV remodeling compared to infarcted control mice. Furthermore, speckle tracking displacement data were used to show that cMatrix reduced early infarct zone dyskinesia, as quantified using the novel dyskinesia index (DI).The cMatrix extract provides a number of advantages over previously tested scaffolds. Unlike solid scaffolds or patches, liquid scaffolds are not limited to superficial placement on the epicardial surface of the heart and have the ability to fully adapt to their environment , thus finding its way to the bedside more rapidly than many medical devices or drugs.A potential limitation of this study is the use of untreated, infarcted mice as the negative controls, as opposed to infarcted mice injected with an equal volume of vehicle. However, we elected to use untreated, infarcted animals to better approximate the clinical situation in which patients either would receive treatment or would be left untreated. Furthermore, trauma from the needle and injected fluid might worsen cardiac function in such a vehicle control group, thus potentially overestimating the efficacy of cMatrix treatment relative to uninjected controls. A second potential limitation of this study is that it did not directly compare the injection of cMatrix to Matrigel or an alternative mixture of ECM proteins. While such alternative tissue extracts or ECM mixtures might well be successful in attenuating LV remodeling  may also have therapeutic effects on LV remodeling. This strategy would become particularly attractive with the advent of new gene delivery systems that rapidly and selectively target the infarct border zone after reperfused MI (Konkalmatt et al. The nonstructural effects of cMatrix on LV remodeling remain to be examined, but are likely to be highly complex, in part because ECM composition over the course of LV remodeling is a dynamic and delicate balance of degradation and synthesis (French and Kramer Much of the complex role of ECM in LV remodeling remains to be defined. Analysis and selective modification of cMatrix content may yield insight into the factors and mechanisms contributing to cardiac regeneration, paving the way for other novel strategies for curtailing LV remodeling post-MI.


To characterize and directly quantify regurgitant jets of left atrioventricular valve (LAVV) in patients with corrected atrioventricular septal defect (AVSD) by four-dimensional (4D)Flow Cardiovascular Magnetic Resonance (CMR), streamline visualization and retrospective valve tracking.Medical ethical committee approval and informed consent from all patients or their parents were obtained. In 32 corrected AVSD patients (age 26 ± 12 years), echocardiography and whole-heart 4DFlow CMR were performed. Using streamline visualization on 2- and 4-chamber views, the angle between regurgitation and annulus was followed throughout systole. On through-plane velocity-encoded images reformatted perpendicular to the regurgitation jet the cross-sectional jet circularity index was assessed and regurgitant volume and fraction were calculated. Correlation and agreement between different techniques was performed with Pearson’s r and Spearman’s rho correlation and Bland-Altman analysis.In 8 patients, multiple regurgitant jets over the LAVV were identified. Median variation in regurgitant jet angle within patients was 36°(IQR 18–64°) on the 2-chamber and 30°(IQR 20–40°) on the 4-chamber. Regurgitant jets had a circularity index of 0.61 ± 0.16. Quantification of the regurgitation volume was feasible with 4DFlow CMR with excellent correlation between LAVV effective forward flow and aortic flow  for internal validation and moderate correlation with planimetry derived regurgitant volume  and echocardiographic grading .4DFlow CMR with streamline visualization revealed multiple, dynamic and eccentric regurgitant jets with non-circular cross-sectional shape in patients after AVSD correction. 4DFlow with retrospective valve tracking allows direct and accurate quantification of the regurgitation of these complex jets. After correction of an atrioventricular septal defect (AVSD), regurgitation of the left atrioventricular valve  is common. Up to 11% of the patients with corrected AVSD require surgery of the LAVV during follow-up . TherefoEchocardiography is most commonly used to evaluate mitral valve or LAVV regurgitation, but echocardiographic quantification of the regurgitation is based on several assumption, such as a cross-sectional circular shape of the jet and a static occurrence and position of the jet throughout systole. In adult patients with mitral regurgitation of diverse origin, poor inter-observer agreement has been described for echocardiographic grading of mitral valve regurgitation using proximal isovelocity surface area (PISA) and vena contracta methods in cases of eccentric regurgitant jets, with non-circular cross-sectional shape or for non-pansystolic regurgitation ,4. Also Four-dimensional (4D) Flow Cardiovascular Magnetic Resonance (CMR) allows visualization and reliable, validated and direct quantification of mitral valve flow using retrospective valve tracking perpendicular to inflow and regurgitant jet with the opportunity to perform internal validation ,9. HowevWe hypothesize that the dynamics and shape of the regurgitant jets after AVSD correction are not in accordance with the assumptions on which the currently available direct quantification techniques are based. Furthermore, we hypothesize that 4DFlow CMR with retrospective valve tracking allows direct and accurate quantification of LAVV regurgitation by tracing the regurgitant jet(s) throughout systole with good internal validation of LAVV flow with aortic flow. Therefore, the purpose of the current study was to characterize and directly quantify regurgitant jets of the LAVV in patients with corrected AVSD by 4DFlow CMR, streamline visualization and retrospective valve tracking.The study protocol was approved by the The medical ethical committee of the Leiden University Medical Center and informed consent was obtained from participants or their parents. Inclusion criteria were a history of AVSD correction and compatibility for CMR . Thirty-four patients were prospectively enrolled between October 2012 and October 2013 from an available surgical database . This pr3, flip angle 10°, echo time (TE) 3.2 ms, repetition time (TR) 7.7 ms, true temporal resolution 31 ms, SENSE factor 2 in anterior-posterior direction and Echo Planar Imaging with a factor 5. No segmented k-space acquisition was used. Mean acquisition time of the 4DFlow scan was 8 minutes (range 5–11 minutes). Commercially-available concomitant gradient correction and local phase correction filter were applied, from the software available on CMR system (Ingenia 3 T with Software Stream 4.1.3.0). Just prior to the 4DFlow acquisition, in the majority (26 out of 32) of patients Gadolinium contrast agent  was administrated, for other clinical evaluations. Furthermore prior to contrast administration, cine 2D left 2-chamber, 4-chamber, coronal and sagittal aorta views and a cine muli-2D short-axis stack of slices were acquired, using steady-state free-precession sequences with TE/TR 1.5/3.0, 350 mm field-of-view, 45° flip angle, acquisition resolution 1.0 × 1.0 × 8.0 mm3. Retrospective gating was used with 30 phases reconstructed to represent one cardiac cycle. Free breathing was allowed without using motion suppression, three signal averages were taken to suppress effects of breathing motion.A 3 T CMR system  with maximal amplitude of 45 mT/m for each axis and a slew rate of 200 T/m/sec, with a combination of FlexCoverage Posterior coil in the table top with a dStream Torso coil, providing up to 32 coil elements for signal reception was used. Whole-heart 4DFlow was performed with velocity-encoding of 150 cm/s in all three directions, spatial resolution 2.3 × 2.3 × 3.0 mmImage analysis was performed by one observer (EC) with two years of experience in CMR and verified by two observers (JW and LK) with over 15 years of experience in CMR. Image analysis was done using in-house developed Mass software (Leiden).Left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were calculated by planimetry: the endocardial border was manually traced at end-diastole and end-systole in short-axis slices, the enclosed areas were calculated, multiplied by slice thickness and summed over all slices. Ejection fraction (EF) was calculated as EF = (LVEDV ‐ LVESV)/LVEDV. The maximum left atrial volume (LAV) was calculated according to the biplane area-length from the 2- and 4-chamber view using the formula (8/3π × Area(4 ‐ chamber) × Area(2 ‐ chamber))/. Atrial and ventricular volume were indexed for body surface area (BSA) according to Du Bois formula .To assess the dynamics of the LAVV regurgitant jet, the jet direction was visualized using streamlines , in two Prior to velocity mapping, the source velocity images were checked for aliasing artefacts. In case aliasing occurred in the region of interest, phase unwrapping was performed in the source images. Retrospective valve tracking was performed to assess trans-LAVV and aortic flow volumes as described previously . In the For comparison of 4DFlow derived regurgitation volume with a conventional CMR quantification technique, regurgitation volume was also quantified as the difference between planimetry-derived stroke volume (LVEDV-LVESV), based on short-axis images minus aortic flow, derived from the 4DFlow acquisition.Variables were tested for normal distribution using the Shapiro-Wilk test. Continuous variables are expressed as mean ± standard deviation (SD) and as median with inter-quartile range (IQR) where appropriate. Linear regression analysis and Bland-Altman analysis  were perPatient characteristics are presented in Table Of 32 included patients, 2 patients underwent re-operation of the LAVV due to regurgitation. Mean left ventricular EF was 55±5% and 30 (94%) patients were categorized NYHA class I and two (6%) patients NYHA class II. Using echocardiography, LAVV regurgitation was classified based on visual grading as none to trace in 3 patients, mild in 15 patients and moderate in 14 patients.In 26 patients, the LAVV regurgitant jet(s) were visualized with streamlines. The six patients (including the 3 patients with none to trace LAVV regurgitation) in whom the LAVV regurgitant could not be visualized with streamlines were quantified with a low regurgitation volume 1–4ml). In 8 (31%) patients, multiple LAVV regurgitant jets were observed  and agreement  was observed between aortic flow and LAVV effective forward flow (Figure The key findings of current study were as follows: First, in patients with corrected AVSD, LAVV regurgitant jets are dynamic, eccentric and have a non-circular cross-sectional shape. Second, it is feasible to directly quantify LAVV regurgitation using 4DFlow CMR and retrospective valve tracking with excellent internal validation with aortic flow and this approach showed moderate correlation with the planimetry-based CMR approach.In patients after AVSD correction, recurrent regurgitation of the LAVV is common and associated with leaflet prolapse, rest cleft, large annular area and a different position of the papillary muscle ,17. ReopLittle is known about the natural history or predictive value of chronic LAVV regurgitation in patients with corrected AVSD. Adult patients with asymptomatic chronic organic mitral valve regurgitation show a high likelihood to develop cardiac symptoms and high incidence of mortality -22. ChroCurrently data on long-term outcome of patients with LAVV regurgitation after AVSD correction is lacking. This is complicated by the lack of a gold standard to quantify LAVV regurgitation in these patients. Furthermore, not all suggested quantification techniques have been evaluated in children with LAVV regurgitation after AVSD correction and no established cut-off values are available for LAVV/mitral valve regurgitation severity in children . The obsCMR offers non-invasive evaluation of atrial and ventricular dimensions and function with excellent reproducibly  within oIn this study, we have to acknowledge some limitations. Although 4DFlow CMR data was acquired, jet angles were measured in 2D planes based on streamline projections. Streamlines represent velocity direction at each location at one instant in time only. By measuring jet angles at different phases of systole, however, we provide useful information on jet direction at specific time points. Analysis of 4DFlow CMR, with manual positioning of the measurement plane, is time-consuming and might be affected by observer variation. However, compared to conventional 2D one-directional velocity-encoded CMR, additional analysis time consists only of time required for the valve tracking, which amounts to approximately 1–2 minutes per valve. Future technical advances may allow full automatic valve tracking and placement of the measurement plane perpendicular to the flow direction, which will reduce analysis time and also eliminate observer variation. Another limitation is that none of the studied patients were classified with severe regurgitation. Although we do not expect different results in these patients, future studies are needed to confirm regurgitant jet characteristics and the feasibility of quantification in patients with severe LAVV regurgitation after AVSD correction.4DFlow CMR with streamline visualization revealed multiple, dynamic, non-circular, eccentric regurgitant jets of the LAVV in corrected AVSD patients and enables direct quantification of the regurgitation of these challenging jets. The observed complex jets explicate the limitations of qualitative and quantitative assessment of LAVV in these patients with echocardiography ,18. Quan


Pulmonary hypertension (PH) can be a rapidly progressive and fatal disease. Although right heart catheterization remains the gold standard in evaluation of PH, echocardiography remains an important tool in screening, diagnosing, evaluating, and following these patients. In this article, we will review the important echocardiographic parameters of the right heart in evaluating its anatomy, hemodynamic assessment, systolic, and diastolic function in children with PH. Pulmonary hypertension (PH) is a progressive disease that carries high morbidity and mortality. Although cardiac catheterization is used to define PH, echocardiography is the most important non-invasive tool that is used to detect PH . It provTwo-dimensional (2D) echocardiography provides qualitative and quantitative evaluation of the severity of PH.The right atrium (RA) dilates over time in patients with PH and it represents decreased right ventricular (RV) compliance and RV diastolic dysfunction. It is a reservoir for systemic venous return when the tricuspid valve is closed, a passive conduit during early diastole, and active conduit in late diastole . ImagingThe inferior vena cava (IVC) can be dilated in patients with PH because of rising RA pressure. It is measured in the subcostal longitudinal view with IVC entering the RA. RA pressure can be estimated by IVC diameter and the presence of inspiratory collapse –9. IVC dWith chronic pressure overload, there is progressive hypertrophy and dilation of the RV. The complex morphology of the RV makes 2D imaging of the RV difficult and frequently requires multiple views in the parasternal, apical, and subcostal views to completely evaluate the RV dimensions. However, current recommendation from the guidelines in assessing the right heart includes a standardized imaging of the RV linear dimensions to evaluate for RV dilation . The RV Right ventricular pressure overload causes flattening of the interventricular septum (IVS) in end-systole into the left ventricle (LV), resulting a “D-shaped” LV in parasternal short axis view. Eccentricity index has been derived from the ratio between the LV anteroposterior dimension and the septolateral dimension at the level of the papillary muscle Figure  15). LV. LV15). The LV outflow tract (LVOT) is obtained from the parasternal long axis view and the velocity time integral (VTI) of LVOT is obtained by spectral Doppler from an apical four chamber view tilted anterior to view the LVOT. Cardiac index is calculated by dividing the CO by the body surface area.An atrial level shunt is important in patients with PH as it provides relief to symptoms of severe PH by increasing systemic flow, reducing RV preload, and increasing CO. Atrial level shunts can be assessed in the parasternal short axis, subcostal short axis, and subcostal long axis views. Contrast echocardiography may also help in the assessment of atrial level shunt. In adults with PH, functional class has been shown to improve with the creation of an atrial level shunt, which leads to cyanosis from right to left shunting but can increase CO. Some series advocate use of an atrial septostomy to prolong survival , 23.The presence of pericardial effusion is associated with increased risk of poor outcome in adults with PH but has not been a prognostic indicator for children . The sizThe normal TR jet has a maximal velocity of <2.5 m/s . The nore Figure , 29. In e Figure  In a rece Figure . NeverthV(VSD)max. Flow direction and velocity across the VSD may help in the diagnosis of PH. Right to left shunt across the VSD and low velocity left to right shunt may suggest the presence of elevated pulmonary pressure . This can only be done if there is no RV or LV outflow tract obstruction. This method has been shown to correlate well with invasive measurements obtained by cardiac catheterization , 33. Thee Figure . ParasteV(PDA) max. This method has been validated by invasive measurements in cardiac catheterization  in the following equation:rization . The florization . Left torization .The diastolic pulmonary artery pressure (DPAP) can be estimated from the velocity of the end-diastolic pulmonary regurgitant velocity using the modified Bernoulli equation Figure :DPAP=4VThe mean pulmonary artery pressure (mPAP) can be estimated from the following equation Figure :mPAP=4Vchildren , 36. Anochildren . Recentlchildren , 39. SomPulmonary vascular resistance is calculated from cardiac catheterization as pressure gradient across the pulmonary bed divided by the pulmonary blood flow. PVR is important in the evaluation of PH and can be estimated using the following equation:rization . In anotrization . Howeverrization . In chilrization . Howeverrization . The TRVrization . AlthougS/D) ratio  is a measure of RV systolic function and can be calculated by the following equation:S Figure . RV FAC S Figure . In adulS Figure .By eliminating the need for geometric assumptions, three-dimensional (3D) echocardiography provides more accurate and reproducible measurements of RV volume and EF in adults with PH Figure  50). Di. Di50). The myocardial performance index (MPI) is a non-invasive measurement of global ventricular function (systolic and diastolic) independent of geometric assumptions . It can ) Figure . RV MPI ) Figure  Another ) Figure . RV MPI z-scores established  is a method to measure the distance of systolic excursion of the RV annular segment along its longitudinal plane from the apical four chamber view in millimeters from end-diastole to end-systole. The greater the descent of the basal annulus in systole, the better RV systolic function is. TAPSE is usually acquired by placing the M-mode cursor through the lateral tricuspid annulus and measuring the amount of longitudinal motion of the annulus in peak systole Figure . TAPSE <ablished . Childreor death . The advS′) measures the systolic longitudinal function of the RV or the LV and two diastolic waves: early diastolic (E′) and late diastolic (A′) denote the diastolic function of the ventricles. Pulse-wave TDI measures the peak myocardial velocities and color TDI measures the mean velocities, which is lower than the pulse-wave TDI measurements by 20%. Acquisition of the TDI velocities must be as parallel as possible to avoid underestimation of the velocities. In adults with idiopathic PH, tricuspid and septal peak systolic (S′) velocities relate to the RV dysfunction and cut-off values have been described to predict RV systolic dysfunction  and are excluded from most studies. The evaluation of RV diastolic function includes tricuspid inflow velocities , TDI of the tricuspid annulus , deceleration time, and IVRT. The tricuspid E/E′ ratio, RA area, and diastolic strain rate have shown promise in the evaluation of RV diastolic function. In adult studies with chronic heart failure and PH, the presence of RV diastolic dysfunction is associated with worse functional class and is an independent predictor of mortality  using echocardiography but more data are needed in the pediatric population in the reproducibility of these new techniques.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. The Review Editor Jeffrey Feinstein declares that despite having collaborated with author Dr D. Dunbar Ivy, the review process was handled objectively and no conflict of interest exists.


There is a need to expand knowledge on cardio-pulmonary pathophysiology of bronchiolitis obliterans syndrome (BOS) following lung transplantation (LTx).The purpose of this study was to assess MRI-derived biventricular cardiac mass and function parameters as well as flow hemodynamics in patients with and without BOS after LTx.Using 1.5T cardiac MRI, measurements of myocardial structure and function as well as measurements of flow in the main pulmonary artery and ascending aorta were performed in 56 lung transplant patients. The patients were dichotomized into two gender matched groups of comparable age range: one with BOS (BOS stages 1–3) and one without BOS (BOS 0/0p).Significantly lower biventricular cardiac mass, right and left ventricular end-diastolic volume, biventricular stroke volume, flow hemodynamics and significant higher heart rate but preserved cardiac output were observed in patients with BOS 1–3 compared to the BOS 0/0p group (p<0.05). In a stepwise logistic regression analysis global cardiac mass (p = 0.046) and days after LTx (p = 0.0001) remained independent parameters to predict BOS. In a second model an indicator for the physical fitness level - walking number of stairs - was added to the logistic regression model. In this second model, time after LTx (p = 0.005) and physical fitness (p = 0.01) remained independent predictors for BOS.The observed changes in biventricular cardiac mass and function as well as changes in hemodynamic flow parameters in the pulmonary trunk and ascending aorta are likely attributed to the physical fitness level of patients after lung transplantation, which in turn is strongly related to lung function. Lung transplantation is an established treatment to improve the quality of life and the prognosis of patients with various causes of end-stage lung disease This study aimed to determine if there are differences in functional and morphological cardiac MRI-derived parameters and flow hemodynamics in the main pulmonary artery and ascending aorta in a gender-matched cohort with a comparable age range of double lung transplant patients without BOS and with BOS.In this study MRI scans were performed in sixty-nine patients during their regular visit in our lung transplant outpatient clinic between Dec. 2011 and Sept. 2013. Double lung transplanted patients during this period were included in this study. Local ethics committee  approval was obtained and all patients gave written informed consent. Exclusion criteria were single lung transplantation, heart and lung transplantation, non-ischemic cardiomyopathy, ischemic cardiomyopathy with left ventricular ejection fraction (LVEF) <50%, and acute pulmonary infections at the time of the MRI scan. Coronary artery disease was present in 6 included patients (4 BOS 1–3), all with an LV EF of ≥50%. Four male patients without BOS (BOS 0) were excluded to achieve gender matching and a comparable age distribution in the two study groups . In our 1), forced expiratory flow (FEF25–75)) as previously described Bronchiolitis obliterans syndrome was diagnosed using lung function test (forced expiratory volume after 1 second (FEV2) was measured. The indicator for the physical fitness level - the ability of walking stairs - was dichotomized in patients being able to walk 0–1 stairways and patients being able to walk 2 or more stairways for the linear regression model analysis.Glomerular filtration rate was calculated by use of the chronic kidney disease epidemiology collaboration (CKD-EPI) method as previously described 2, matrix size  = 208×256, temporal resolution  = 35 ms, in-plane resolution  = 1.4×1.4 mm2, bandwidth/pixel  = 540 Hz/pixel, 30 reconstructed phases. Additionally, phase-contrast flow measurements were performed in the ascending aorta (AO) and the main pulmonary artery (PA) using a through plane retrospectively ECG gated spoiled gradient echo sequence (FLASH) during free breathing. The following acquisition parameters were used: TE  = 2 ms, TR  = 20 ms, flip angle  = 30°, slice thickness  = 5 mm, field of view  = 345×460 mm2, matrix size  = 192×256, number of averages  = 3, in-plane resolution  = 1.8×1.8 mm2, bandwidth/pixel  = 930 Hz/pixel, 75 reconstructed phases.All examinations were performed on a commercially available 1.5T MR system  equipped with an eight-channel torso phased array coil  in supine position. Retrospectively ECG-gated cine balanced steady-state free precession (bSSFP) sequences were acquired during short inspiratory breath holds in standard short-axis views covering the whole heart using the following parameters: TE  = 1 ms, TR  = 43 ms, flip angle  = 75°, slice thickness  = 8 mm, field of view  = 290×360 mm2) and cardiac output (l/min) were calculated. Concerning the phase-contrast flow measurements, the following parameters were determined in the AO and the PA: acceleration volume (ml), acceleration time (msec), distensibility (%), maximal systolic flow (ml/sec) and mean systolic velocity (cm/sec) as previously described Short axis cine MR images and phase-contrast flow measurements were analyzed on a separate workstation with dedicated cardiac software . Cine images were analyzed by semi-automated contour detection for left and right ventricular endo- and epicardial contours in end-diastole and end-systole by one experienced radiologist. Papillary muscles as well as myocardial trabeculations were included in the blood pool. For calculation of the phase-contrast parameters the same radiologist contoured the border of the AO and the PA on the magnitude images and a semi-automated CVI42 software algorithm completed the contours on the remaining images. Manual corrections were performed if necessary. Using short axis cine images the following parameters were evaluated for the left and the right ventricle: stroke volume (ml), ejection fraction (%), end-diastolic volume (EDV) and end-systolic volume (ESV) (ml), myocardial mass (g) and ventricular mass index (VMI  =  right ventricular (RV) mass/left ventricular (LV) mass) The Shapiro-Wilk test was used to test for normality of distribution for the MRI-parameters and patient population variables. Summary statistical data for outcome and predictor variables were calculated for the BOS 0/0p and BOS 1–3 groups with results expressed as the mean [95% confidence interval] for normally distributed continuous variables, the median (interquartile range) for non-normally distributed continuous variables. Differences in mean values between the two study groups were compared using t-tests and ANOVA between multiple groups. Not normally distributed values were compared using the Wilcoxon test. Differences in proportions for the categorical variables were compared using the Fisher's exact test. Pearson correlation was used to correlate continuous normally distributed parameters. In all tests, a 2-tailed value of 0.05 was defined as the level of statistical significance. Multivariate linear regression was then used to adjust the MRI-derived cardiac function parameters for pertinent demographic and cardiovascular risk factors , diabetes and age).Furthermore, a forward-backward stepwise logistic regression analysis  was performed to analyze if biventricular cardiac mass and MRI-derived function parameters are associated with BOS. In a final logistic regression model (model 1) odds ratios (OR) and respective confidence intervals (CI), as well as p-values were calculated. The detected changes in cardiac mass and function were quite similar to reported changes due to inactivity, thus an additional logistic regression analysis (model 2) was performed with the same parameters from model 1 adding a parameter indicating the physical fitness level  to evaluate the influence of the physical status of the patients to cardiac mass and BOS stadium.Statistical analysis was performed using commercially available software .2 was significantly different in both groups (p = 0.006), but all patients had normal peripheral oxygen saturation  years) and 30 women  years), participated in this study . The results of the respective logistic regression model (model 1) are shown in In the proximal ascending aorta and in the main PA maximal systolic flow, acceleration volume, acceleration time and mean systolic velocity were lower in the BOS 1–3 group compared to the BOS 0/0p group p<0.05, . NeitherIn this study, a significantly lower biventricular cardiac mass, RV and LV EDV, biventricular stroke volume, a significantly higher heart rate and a preserved cardiac output was observed in patients with BOS 1–3 compared to a gender matched BOS 0/0p group of comparable age range. Similarly, a reduction in flow hemodynamics was observed in the ascending aorta and pulmonary trunk in the BOS 1–3 group. These changes have the footprint of changes due to physical inactivity as previously shown in the community setting in the MESA cohort The 20% difference in biventricular SV mainly resulted from lower biventricular EDVs suggesting a decreased biventricular filling/preload in patients with BOS after lung transplantation likely due to impaired physical fitness in the BOS group. The reverse process of improved ventricular filling with increased LV EDV could be shown in untrained men after short-term endurance training secondary to the Frank-Starling effect In a non-athletic population of 4,992 ethnically diverse participants free from clinically apparent cardiovascular disease (aged 45–84 years) Turkbey et al. observed that left ventricular mass and end-diastolic volume were positively associated with physical activity with a preserved cardiac output and ejection fraction and with a decreased heart rate In recent work from our center, we included 53 patients with advanced lung allograft dysfunction and divided them in two groups according to their exercise capacity Phase-contrast flow measurement in the pulmonary trunk and ascending aorta is a well established technique to study flow hemodynamics and flow profiles 1% and fitness level was present in our patient cohort as well.Airflow obstruction is also present in patients with chronic obstructive airways disease (COPD). In a population-based study, a greater extent of emphysema on CT scanning and more severe airflow obstruction were linearly related to impaired left ventricular filling, reduced stroke volume, and lower cardiac output without changes in ejection fraction after adjusting for covariates except for the level of physical activity in the main analysis All of the patients had a time since transplantation of at least 6 month. Kasimir et al. perioperatively investigated changes in right and left ventricular parameters by echocardiography in a group of single lung transplanted patients with pulmonary hypertension early after transplantation 2We observed a difference of about 8% in biventricular cardiac mass for male and female patients with a higher cardiac mass in patients without BOS. Cardiac mass is known to increase with the level of exercise as shown in several studies Also age and gender are important factors influencing cardiac mass and function 2 values compared to patients without BOS. Overall, all patients had a normal peripheral SpO2, thus a theoretically possible hypoxia-related pulmonary arterial hypertension seems unlikely in our cohort.Patients with BOS had a significantly lower SpOCalcineurin inhibitors are known to effect cardiac hypertrophy in mice positively Left ventricular mass is a well-known predictor for incident cardiovascular events e.g. heart failure, sudden death and cardiovascular mortality representing a minor factor of death after LTx (about 5%) We conclude that the observed changes in biventricular cardiac mass and function as well as changes in hemodynamic flow parameters in the pulmonary trunk and ascending aorta are likely attributed to the physical fitness level of patients after double lung transplantation, which in turn is strongly related to lung function.


Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation.Anthrax lethal toxin (LT) is an A-B type toxin secreted by  Bacillus anthracis, consists of two polypeptides: the cellular binding moiety protective antigen  and the enzymatic moiety lethal factor 220), which allows for oligomerization of the activated, membrane-bound 63-kDa fragment PA63 into a heptamer or octamer. The PA oligomers have binding sites for LF that span adjacent monomers5710Anthrax lethal toxin (LT), secreted by gen PA, 8 kDa and ctor LF, 0 kDa220)63 monomers, we generated intermolecular complementing PA variants that require the presence of both MMPs and uPA for activation5616Since proteolytic activation of PA on the cell-surface is absolutely required for the action of the toxin, modification of the cleavage site amino acid sequence can create tumor-selective PA variants that are only activated in the presence of tumor-associated proteases. Two such PA variants are PA-L1, which is activated by matrix metalloproteinases (MMPs)1214151718B. anthracis BH480 strains. Out of a theoretical library of 95 mutants, 79 were successfully expressed and supernatants containing the secreted PA variants were prepared for initial testing. PA proteins were usually expressed at high levels by the correspondingly transformed BH480 strains, often reaching levels above 50% of the total protein in the culture supernatant. Of the sixteen proteins that were not expressed, nine were mutants of K214 , four were mutants at I207 , two were mutants of I210 , and one was an R178 mutant (P). K214 lies at the end of a α-helix and the side chain makes no close contact with any other residues on PA. It is not clear why so many mutants at this position could not be expressed.To select PA variants with activity strictly dependent on intermolecular complementation for LF-binding, we constructed a PA variant library with NNS  codons at LF-binding subsite residue R200 on the counterclockwise-side monomer, and the residues R178, I207, I210, and K214 on the clockwise-side monomer  also re in6. WhiThe sterilized supernatants containing PA proteins were screened for activity relative to the original versions of the intermolecular complementing, PA-R200A or PA-I210A, as appropriate using cytotoxicity assays. In the screen of counterclockwise-side monomer mutants, ten mutant PA-R200X proteins  were initially identified as being less intrinsically toxic than PA-R200A. However, later detailed characterization found that these PA variants also showed reduced intermolecular complementation with PA-I210, resulting in less LF-induced cytotoxicity. As an example, although PA-R200C was slightly less toxic than PA-R200A, it was also slightly less effective in complementing with PA-I210A to promote killing of RAW264.7 cells (data not shown). Therefore, none of the counterclockwise-side monomer PA variants were found to be significantly superior to the original PA-R200A.In screens for the clockwise-side monomer variants PA-R178X, PA-I207X, PA-I210X, and PA-K214X, eight proteins with greatly reduced inherent cytotoxicity to RAW264.7 macrophages were identified: I207R, I207W, I210D, I210E, I210K, I210Q, I210R, and I210S . These w50 = 200 ng/mL) in these assays ) ) .To further evaluate the anti-tumor activity of the new PA variant, we performed a side-by-side comparison of the new combination, i.e., PA-L1-I207R plus PA-U2-R200A, and the original combination of PA-L1-I210A and PA-U2-R200A in treatment of B16-BL6 syngeneic tumors in C57BL/6J mice. The tumor-bearing mice were treated with either PBS, low doses of the new combination or the original combination of the PA variants plus LF (7.5 μg/7.5 μg/5 μg), or high doses of each combination plus LF (22.5 μg/22.5 μg/15 μg). All of the toxin-treated groups showed significant anti-tumor activities compared to the PBS control group  P < 0.0  is mostly resultant from PA-L1-I210A16210 to Ala mutation, making the protein more susceptible to protease degradation during purification. The new combination of the PA variants also seemed to have lower non-specific toxicity when used in the mouse tumor model. However, because of the low mortality rates (<30%) and the population sizes in this study (n = 10–14 for each group), this trend was not shown to be statistically significant.The new pairing of PA-L1-I207R and PA-U2-R200A had a cytotoxic potency similar to that of the original combination toward B16-BL6 melanoma cells . InteresIn summary, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, warranting further clinical development in conjunction with strategies to suppress an adaptive immune response as described recently29B. anthracis expression vector pYS5NNSGACAATGATGGAATCCCTA-3′ (R178X mut), 5′-CTGGAACCGTAGGTCCAGCACTTG-3′ (R178X rev), 5′-CGAGGTAGAAGGATATACGGTTGATGTCAAAAATAAANNSACCTTTCTTTCACCATGG-3′ (R200X mut), 5′-TGACATCAACCGTATATCCTTCTACCTCG-3′ (R200X rev), 5′-CAAAAATAAAAGGACCTTTCTTTCAACATGGNNSTCTAATATTCATGAAAAGAAAGGG-3′ (I207X mut), 5′-CATGGTGAAAGAAAGGTCCTTTTATTTTTG-3′ (I207/210X rev), 5′-CAAAAATAAAAGGACCTTTCTTTCACCATGGATATCTAATNNSCATGAAAAGAAAGGG-3′ (I210X mut), 5′-CTTTCACCATGGATATCTAATATTCATGAAAAGNNSGGGTTAACCAAATATAAATCATC-3′ (K214X mut), and 5′-CTTTTCATGAATATTAGATATCCATGGTGAAAG-3′ (K214X rev). Mutagenic primers were combined with PA-SwaI-Rev and reverse primers with PA-250 for the first round of PCR. The products were gel purified and the complementary PCR fragments were extended to full length by 10 cycles of PCR before addition of PA-250 and PA-SwaI-rev and an additional 35 cycles. These mutagenic inserts were gel purified and the inserts, along with pYS52, were digested with PstI and HindIII. The digestion products were ligated overnight and transformed into chemically competent E. coli MC1061, which were plated on LB-agar plates containing 100 μg/mL ampicillin. Colony PCR using PA-250 and PA-SwaI-Rev primers was performed to identify positive clones. Colonies were screened using primer PA-250 to sequence the plasmid and identify clones having all possible substitutions. Selected clones were then grown overnight in LB broth containing 100 μg/mL ampicillin and the plasmids were extracted by mini-scale preparation.Mutations were introduced by overlap PCR with NNS codons into the plasmid pYS52  supplemented with fetal bovine serum to 10% (Invitrogen) and gentamycin at 50 μg/mL (Invitrogen) at 37 °C in a tissue culture incubator with 5% CO5 cells/well and grown overnight. The next day, the PA variants were added at a concentration of 500 ng/mL and LF at a concentration of 100 ng/mL. The cells were incubated for 20 h and MTT -2,5-diphenyl-2H-tetrazolium bromide, Sigma, St. Louis, MO) was added at 500 μg/mL for the final hour. The medium was aspirated and the oxidized MTT was solubilized in 91% isopropanol containing 0.5% SDS and 0.038 M hydrochloric acid, then read at 570 nm using a SpectraMax 190 plate reader . The absorbance was used to determine percent survival compared to an untreated control. PA variants that showed less toxicity than the original constructs PA-R200A and PA-I210A were subjected to a double-agent gain-of-function test. RAW 264.7 macrophages were seeded as above and respective PA variants (250 ng/mL) combined with PA-R200A or PA-I210A (250 ng/mL) and 100 ng/mL LF were added. The cells were incubated for 6 h and viability was measured as above.To test the PA variants for a loss of function, RAW 264.7 macrophages were plated in a 96-well plate at 10CGTTCTAATATTCATGAAAAGAAAGGATTA-3′ (I207R sense), 5′- TAATCCTTTCTTTTCATGAATATTAGAACGCCATGGTGAAAGAAAAGTTCTTTTATTTT-3′ (I207R antisense). Sense and anti-sense primers were used with the Quikchange Lightning Kit  according to manufacturer’s recommendations and transformed into chemically competent E. coli XL-10 Gold. The plasmids were mini-prepared, sequenced, and transformed into E. coli SCS110 before transformation into BH480 for expression and purification. Cytotoxicities of PA-L1-I207R and PA-L1-I210A were tested singly and in combination with PA-U2-R200A  was replaced with a MMP substrate sequence GPLGMLSQB. anthracis BH480 strain. The recombinant proteins secreted into culture supernatants were purified as described previously32PA-L1-I210A, PA-L1-I207R, PA-U2-R200A, LF and FP59 were expressed using pYS5-based expression plasmids from C57BL/6J male and female mice (10 to 12-week-old) were injected intraperitoneally with 20 μg PA-L1-I207R or PA-LI-I210A along with 10 μg FP59 at 0, 24, and 48 hours and checked twice daily for signs of malaise and mortality for two weeks following the first injection. Mice were euthanized at the end of experiment. All animal studies were carried out in accordance with protocols approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee.5 B16-BL6 cells in the mid-scapular subcutis. B16-BL6 melanoma cells were a kind gift from Dr. Isaiah J. Fidler , and were authenticated by continual assessment of cellular morphology at both low and high magnifications. Eight days after injection, established tumors were measured with digital calipers . Tumor weights were estimated with the longest and shortest tumor dimensions in the formula: tumor weight (mg) = (length in mm × width in mm2) × 0.5Twelve-week-old female C57BL/6J mice  were injected with 5 × 10Statistical analysis was done with unpaired student’s t test using Excel. Survival curves were analyzed using log-rank test (Mantel-Cox) using GraphPad Prism software.How to cite this article: Wein, A. N. et al. An anthrax toxin variant with an improved activity in tumor targeting. Sci. Rep.5, 16267; doi: 10.1038/srep16267 (2015).


Sarcoidosis is a multisystem disease. Involvement of the left ventricle (LV) in Sarcoidosis detected by CMR in the form of early (EGE) and late gadolinium enhancement (LGE) is well validated. Autopsy studies have shown abnormalities in the right ventricle (RV) as well, albeit less frequently than the LV. There have also been several case reports of sarcoidosis mimicking ARVD suggesting predominant RV involvement in those cases Objective: We sought to determine if there was evidence of RV involvement in patients with Sarcoidosis that could be detected via early and late gadolinium enhancement on CMRA retrospective review was performed on a database of 122 patients  referred for evaluation of cardiac sarcoidosis between 2002 and 2013 at a single center. All scans were performed using a 1.5T scanner . Via that database, we found 37 patients demonstrated phenotypic cardiac sarcoidosis in the form of early and late gadolinium enhancement in the LV myocardium that was corroborated by the clinical presentation. The CMR images of these 37 patients  were then reviewed by a single experienced operator to assess for the presence of EGE or LGE in the RV by standard visual analysis, using the SA, 3- and 4-chamber views obtained at 2 and 10 minutes post contrast administration . In this study T2 was not uniformly performed. LVEDV, LVESV and LVEF measurements were obtained using semi-automated analysis software(Medis Qmass 7.4.26.0) and were available in 31 patients (81%). Ventricular dimensions were available in all 37 patients. Patient characteristics were compared between the groups that demonstrated RV involvement with those that did not. Presence of LGE in the RV was also correlated with volumetric data where available and ventricular dimensions. Categorical data was presented as counts and percentages and analyzed using the chi-square test. The independent samples t-test or Mann-Whitney test was used to assess the difference between patients with respect to continuous variables. Data was analyzed using PASW Statistics, version 18.0 (IBM SPSS Inc.).Out of the 37 patients that showed the presence of cardiac sarcoidosis in the LV, RV enhancement was seen in 18 (49%). There were no significant difference in LV or RV metrics between patients that exhibited RV enhancement versus those that did not. RV enhancement did not correlate with differences in LV volumes, LVEF, LV or RV dimensions.Our proof of concept study demonstrates that RV involvement does occur in a significant number of patients along with LV involvement in cardiac sarcoidosis and this can be detected via early and late gadolinium enhancement supporting a less myopic view of sarcoid. As to whether RV involvement portends worse outcome is ongoing work.Internal.


According to current guidelines, an adequate timeframe for revascularisation in the setting of acute non-ST-elevation myocardial infarction (NSTEMI) is considered by current guidelines as being up to 72 hours after symptom onset. Soon after myocardial infarction, changes take place in the affected myocardium that can be visualized and measured by cardiac magnetic resonance imaging (CMR). Aim of the present study was to compare tissue characteristics in patients with NSTEMI that underwent early vs. delayed revascularisation. Therefore, myocardial tissue characteristics were evaluated in the acute and chronic phase of reperfused NSTEMI patients and the findings were correlated to the particular symptom-to-reperfusion time.In 53 patients presenting with NSTEMI, CMR imaging was performed in addition to invasive coronary angiography. The patients were examined on a 1.5 T whole-body scanner using a 32-channel phased-array surface coil. Left-ventricular volumes were assessed by a standard steady-state free-precession sequence. Myocardial edema was evaluated using a 3D T2-weighted black-blood fat-saturated spin-echo sequence. Microvascular obstruction and late gadolinium enhancement were measured by a 3D phase-sensitive inversion-recovery gradient echo sequence about 12 minutes after intravenous administration of gadolinium based contrast agent. CMR imaging was conducted shortly after coronary revascularisation and about 90 days later at follow-up. Results were gained by two experienced readers in consensus.Patients with early (< 12 hours) vs. delayed (> 12 hours) symptom-to-reperfusion time did not differ significantly concerning CMR characteristics at baseline or follow up. In all patients left ventricular myocardial mass (p < 0.0001), infarct size (p = 0.007) and microvascular obstruction (p = 0.02) were significantly reduced at the follow-up in comparison to baseline. In patients with observed microvascular obstruction, infarct size was significantly larger (p = 0.003). Infarct size was negative correlated to the myocardial salvage index .Myocardial tissue characteristics in patients with NSTEMI in the acute and chronic phase can be assessed by CMR imaging. There were no significant differences in patients with early in comparison to delayed revascularisation. Based on these results, CMR-based parameters in NSTEMI patients may serve as a surrogate endpoint in therapeutic studies.This study was partially funded by a research grant of the German Heart Research Foundation (F/02/10).


Left ventricular rotation is an important part of myocardial mechanics during the cardiac cycle. Understanding the mechanisms of LV rotation in different cardiac diseases could play an important role for diagnosis, risk stratification and prediction of heart failure. We sought to analyze LV rotation using the feature tracking technique in patients with dilated cardiomyopathy (DCM) included in the European CMR Registry.82 Patients diagnosed with DCM and a control group consisting of 30 healthy volunteers were analyzed using dedicated feature tracking imaging software . An apical, midventricular and basal slice in short axis orientation were tracked to analyze the peak rotation of each slice. LV twist  and LV torsion (defined as the LV twist per ventricular length) were calculated.DCM patients and controls with normally directed rotation  were compared to each other table : DCM patAmongst the DCM patients an inversed rotational pattern was frequently observed Table : 57.3% oDCM is associated with an inverted direction of rotation in a significant amount of cases, predominantly affecting the LV apex. Patients with a normal direction of rotation exhibit significant lower torsion. These findings warrant further investigation including clinical follow-up data in order to analyze their impact on clinical outcome in patients with DCM.N/A.