We considered LV septum width as a surrogate of myocardial remodeling in our population, and this is the reason why we can consider the EML score as a possible tool to evaluate myocardial remodeling. A CCT-based radiomic approach for the identification of LV remodeling is possible in patients with past medical history of arterial hypertension.The aim of the study is to verify the feasibility of a radiomics based approach for the detection of LV remodeling in patients with arterial hypertension. Cardiac Computed Tomography (CCT) and clinical data of patients with and without history of arterial hypertension were collected. In one image per patient, on a 4-chamber view, left ventricle (LV) was segmented using a polygonal region of interest by two radiologists in consensus. A total of 377 radiomics features per region of interest were extracted. After dataset splitting (70:30 ratio), eleven classification models were tested for the discrimination of patients with and without arterial hypertension based on radiomics data. An Ensemble Machine Learning (EML) score was calculated from models with an accuracy >60%. Boruta algorithm was used to extract radiomic features discriminating between patients with and without history of hypertension. Pearson correlation coefficient was used to assess correlation between EML score and septum width in patients included in the test set. EML showed an accuracy, sensitivity and specificity of 0.7. Correlation between EML score and LV septum width was 0.53 ( Radiomics is defined as the quantitative extraction and analysis of data from medical images, that are translated into a high-dimensional mineable feature space using dedicated automatized feature extraction algorithm . This prCardiac Computed Tomography (CCT) has proven to be an optimal modality on which to perform TA. It has a rapid acquisition time with images of suitable quality resolution and has frequent use in clinical practice resulting in large datasets of readily available CCT images . RadiomiThe most common clinical use of CCT is the assessment of coronary artery disease (CAD). This is a multi-factorial disease with many patients having multiple co-morbidities such as hypertension, hypercholesterolemia or metabolic syndrome.Hypertension is the leading risk factor for cardiovascular complications and commonly is associated with adverse structural heart remodeling, leading LV disfunction and hypertensive cardiomyopathy . AdvanceThe aim of our research was to test the feasibility of extracting CCT-derived radiomics features for the detection of LV remodeling in patients with arterial hypertension.In this retrospective study, images of patients undergoing CCT between January to September 2020 at San Carlo di Nancy Hospital  were analyzed. For the purpose of this study, images of patients with and without prior diagnosis of arterial hypertension meeting the inclusion criteria were considered. Arterial hypertension was defined as per the European Society of Hypertension guidelines . The incDemographic and clinical characteristics see  were colCCT images were obtained using a 512-slice scanner , with prospective gating, setting the slice thickness at 0.625 mm, 120 kV, automatic mA, rotation time of 0.28 s, DFOV: 25 cm, Detector Coverage: 160 mm. All patients were subjected to administration of contrast material  at a rate of 5 mL/s followed by a saline chaser. The image acquisition was triggered after a threshold of 80 HU was reached in a region of interest placed in the left ventricle (bolus-tracking technique).In one image per patient, on a 4-chamber view and at 75% of R-R interval, LV was segmented using a polygonal region of interest (ROI) by two radiologists in consensus. Care was taken to not include blood in LV cavity, epicardial fat, myocardial trabeculations or major coronary arteries in the ROI .Gray-level normalization was performed by rescaling the histogram data within μ-gray-level mean ±3 σ (σ-gray-level standard deviation) to reduce contrast and brightness variations that might impair texture feature quantification .A total of 377 features per ROI were extracted using MaZDa ver. 4.6 software  . Texturet-test was used to determine the p-value assessing the significance of the difference between the investigated classes . p-value was corrected according to the false discovery rate (FDR) using the Benjamini–Hochberg procedure. Categorical variables were compared using the χ2-test. Radiomics data were scaled by an autoscaling process (mean-centered and divided by standard deviation of each variable). Pearson correlation coefficient was used to assess correlation between continuous variables.Clinical data distribution was analyzed using the D’Agostino–Pearson test. Student’s The auto-scaled dataset containing the texture analysis results, was first divided in two parts (70:30 ratio), one used to train the classification models and one to test them and to evaluate the model performances. In each dataset, the classes were quite evenly represented. Eleven classification models were trained and included: Partial Least Square Discriminant Analysis (PLS-DA), Naïve Bayes (NB), Generalized Linear Model (GLM), Logistic Regression (LR), Fast Large Margin (FML), Deep Learning (DL), Decision Tree (DT), Random Forest (RF), Gradient Boosted Trees (GBT), artificial Neural Network (aNN) and Support Vector Machine (SVM). Hyperparameter tuning was also applied searching the setting able to maximize each model’s accuracy. Overfittings were evaluated using a cross-validation procedure based on training dataset. Furthermore, models were trained using many features subsets. Features were first screened using 3 criteria: (a) correlation   stability  , (c) missing . Features that did not fulfil the screening criteria were deemed unsuitable and removed from the dataset to avoid overfitting and maximize the model’s accuracy.Receiver operating characteristic (ROC) curves, sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, and accuracy were measured to assess the ability of the EML score to correctly classify the images. A cut-off point was evaluated as the score maximizing the Youden’s Index (sensitivity + specificity − 1). DeLong non-parametric approach was used to compare the area under the curve receiver operating characteristic (AUCROC).Models showing an accuracy higher of 60% were selected and ensembled using a voting scheme assuming both cross-validation accuracy and confidence  as a vote weight. Ensembling was performed according to Cavallo et al. . BrieflyThe overall diagnostic performance of the proposed score was investigated using a confusion matrix to summarize the results obtained using samples in the test set.Statistical analyses were performed using Rapid Miner Studio v9.8.001 , and R v4.0.4 .y axis and the log of the fold change (FC) on the x axis. This resulted in data points with low p-values (highly significant) appearing towards the higher end of the plot and data points with higher FC at the plot margins. The log of the FC was used so that any change appeared equidistant from the center in both directions. By plotting the data in such a manner two regions of interest in the plot were delineated: at the higher end of the plot and at the left- or right margins of the plot. Any point in both locations on the plot represented values that displayed large magnitude FCs (located at the left or right plot margins) as well as high statistical significance (hence being toward the higher end of the plot).Radiomic features were investigated by means of smile plot obtained by plotting for each feature the negative log of the p-value on the Boruta algorithm was also used to evaluate the relevance of each radiomics feature. From the radiomics’ dataset (DSet) a new dataset (shadow dataset ShDSet) was created randomly shuffling each feature. ShDSet was attached to DSet to obtain a new dataset (Boruta dataset BaDSet), which has twice the number of columns of DSet.A Random Forest algorithm was trained using BaDSet, and the information gain was evaluated for all features. A threshold level was defined as the higher information gain among the features derived from ShDSet. All the features with a lower information gain were excluded. The whole process was repeated 2000 times.CCT images from 158 patients, 83 with history of arterial hypertension  and 75 without  were used to perform image analysis.Age, BMI, history of diabetes and LV septum thickness were higher in HTN patients, as was male sex . The difExploratory analysis of the 377 radiomic features showed that 43 (11.4%), showed a fold change higher of ±2 and an FDR corrected p-value in patients with HTN . There wClassification performances evaluated on test set are reported in p-value < 0.001) . Moreover, the EML score was well correlated with the LV septum width   ranged from 66.4% to 80.0%. Ensemble models showed 70.0% accuracy. Sensitivity and specificity resulted balanced for all models, ranging between 54–87% and 45–77%, respectively . Youden’< 0.001) A. Moreov 0.0001) C.p = 0.006). Moreover, BMI did not affect the EML score, with no difference (p > 0.05) among normal weight (BMI < 25 kg/m2), overweight (25 < BMI < 30) and obese (BMI > 30 kg/m2) subjects  only  feature parameters, which are a measure of intrinsic randomness/variability in neighborhood intensity value differences were selected by the Boruta algorithm. The Boruta algorithm ruled out four different RLN features , that also showed a statistically significant difference between groups. RLN is a measure of the similarity of run lengths throughout the image. The lower the RLN value, the more homogeneity among run lengths in the image is present. Patients with arterial hypertension showed higher RLN values compared to patients without arterial HTN , indicatOur results encourage an approach where a set of distinct radiomic features may be used to train an ensemble machine learning model with an overall accuracy of 70%, and relatively good sensitivity, specificity, positive and negative predictive values.The practical application of such an approach may lie in its ability to discriminate patients with LV remodeling from hypertension even without a past medical history of arterial hypertension using CT instead of cardiac MRI (CMR), which is currently the method of choice for assessing myocardial fibrosis through measures such as myocardial extracellular volume fraction (ECV) which quantifies the interstitial space. The latter is, however, an indirect measure that relies on the extravasation of gadolinium, a pure extravascular contrast agent, into the interstitial space and measurements of T1 relaxation before and after contrast administration. In the absence of myocardial edema or amyloidosis, ECV is an adequate measurement of diffuse myocardial fibrosis . CCT couRecent advances suggest that LV fibrosis is the end-stage of a number of myocardial processes including obesity, diabetes mellitus, sex, age, environmental exposures and genetic factors which collectively, through pathways of inflammatory stress, may eventually lead to fibrosis and LV dysfunction and hypertensive cardiomyopathy ,15. Indep-value < 0.0001), independent of diabetes, BMI and age in those above the age of 50 years old. The significantly low EML score in the diabetic vs. non-diabetic cohort suggests that either cardiac remodeling by arterial hypertension is a long-term disease process or that the effect of diabetes on the myocardium is more significant in younger patients. This finding adds additional information on the potential role of machine learning and texture analysis in detecting myocardial modifications in CCT images.Our analysis showed a significant correlation between EML score and septum width  the absence of a reference standard (CMR) for the quantification of LV remodeling and fibrosis; (2) retrospective nature of analysis; (3) patients with hypertension were on antihypertensive medication for a variable period of time (ranging from 1 to >20 years) which may affect cardiac remodeling; (4) the possibility of systematic differences related to the size of segmented volumes must be considered, especially for higher order features; (5) blood pressure control, type of antihypertensive medications, as well as control of comorbidities, were not assessed; (6) Clinical data of HTN and NC patients showed some statistically significant difference that might affect outcomes; for the EML we’ve taken into account only radiomics data in order to minimize possible biases.In summary, our study has identified a possible radiomic-based approach for the identification of LV remodeling in patients with arterial hypertension and potential radiomic features associated with LV remodeling in patients with this disease.We considered LV septum width as a surrogate of myocardial remodeling in our population, and this is the reason why we can consider the EML score as a possible tool to evaluate myocardial remodeling.Further confirmation of our findings will be needed also for other conditions associated with LV fibrosis, and in comparison with imaging modalities such as echocardiography and cardiac MRI.


Cardiac MR is widely used to diagnose cardiac amyloid, but cannot differentiate AL and ATTR subtypes: an important distinction given their differing treatments and prognoses. We used PET/MR imaging to quantify myocardial uptake of 18F-fluoride in ATTR and AL amyloid patients, as well as participants with aortic stenosis and age/sex-matched controls.MEAN) after correction for blood pool activity in the right atrium.In this prospective multicenter study, patients were recruited in Edinburgh and New York and underwent 18F-fluoride PET/MR imaging. Standardized volumes of interest were drawn in the septum and areas of late gadolinium enhancement to derive myocardial standardized uptake values (SUV) and tissue-to-background ratio  were higher than controls , aortic stenosis , and those with AL amyloid . TBRMEAN values within areas of late gadolinium enhancement provided discrimination between patients with ATTR (1.36 ± 0.23) and all other groups . A TBRMEAN threshold >1.14 in areas of LGE demonstrated 100% sensitivity (CI 72.25 to 100%) and 100% specificity (CI 67.56 to 100%) for ATTR compared to AL amyloid .53 patients were scanned: 18 with cardiac amyloid (10 ATTR and 8 AL), 13 controls, and 22 with aortic stenosis. No differences in myocardial TBR values were observed between participants scanned in Edinburgh and New York. Mean myocardial TBRQuantitative 18F-fluoride PET/MR imaging can distinguish ATTR amyloid from other similar phenotypes and holds promise in improving the diagnosis of this condition.The online version of this article (10.1007/s12350-020-02356-1) contains supplementary material, which is available to authorized users. Systemic amyloidosis represents a spectrum of conditions characterized by disordered protein folding and fibrils formation. The two predominant forms affecting the heart are amyloid light chain (AL) and amyloidosis transthyretin .4The diagnosis of cardiac amyloidosis is challenging. Echocardiography is frequently unable to differentiate amyloid from other forms of left ventricular hypertrophy such as that from aortic stenosis. Furthermore, some studies have suggested that cardiac amyloidosis is present in 6% to 29% of patients with significant aortic stenosis.99mTc-DPD, 99mTc-HMDP and 99mTc-PYP) can provide this discrimination, demonstrating increased cardiac uptake in ATTR compared to AL amyloid and control subjects, but these approaches only provide a semi-quantitative assessment of activity.Endomyocardial biopsy is still the gold standard to make a diagnosis of cardiac amyloid but it is invasive, may be prone to sampling error, and is associated with procedure-related risk. Alongside histology, cardiovascular magnetic resonance (CMR) is increasingly being used to aid in the diagnosis of amyloidosis based largely upon the characteristic pattern of late gadolinium enhancement and T1 mappingIn this novel prospective multicenter study, we aim to build on the initial findings by Trivieri et al,This multicenter hybrid imaging study was conducted between December 2015 and June 2018 at two sites: The British Heart Foundation centre for Cardiovascular Science at the University of Edinburgh, UK and the Icahn School of Medicine in New York, USA. All participants were older than 50 years. Patients with cardiac amyloid were recruited from outpatient clinics and inpatient wards over both sites. The diagnosis of AL or TTR amyloid was established on histological analysis of biopsy samples in all patients bar one who had Multiple Myeloma. This patient had CMR features of amyloid (included in the AL group) but did not have a tissue biopsy. Age/sex-matched subjects were recruited as a negative control group as were patients with aortic stenosis .2) or women of child-bearing potential.Exclusion criteria for all cohorts included inability to receive iodinated contrast, renal impairment  Administration of Radiation Substances Advisory Committee. It was performed in accordance with the Declaration of Helsinki and all patients provided written informed consent prior to any study procedures. Subjects recruited in New York had ethical and Institutional Review Board approval for the study (GCO#01-1032). All participants provided written informed consent. The study was registered on Clinicaltrials.gov (NCT03626584). We did not directly include patient and public involvement (PPI) in this study, but the patient information sheet used in the study was developed with PPI and was reviewed by a committee that includes patient representatives.This was a multicenter PET/MR study, but where possible protocols were standardized between centers with all patients undergoing simultaneous PET and MR imaging using the same hybrid PET/MR system . The MR protocol at each site was the same including long axis cine imaging , a short axis cine stack  coronary magnetic resonance angiography (CMRA for accurate co-registration) performed with 0.2 mmol/kg of intravenous gadobutrol contrast  and late gadolinium enhancement imaging 10 to 15 minute post-contrast administration. Importantly, the same radial gradient recalled echo  sequences was acquired for MR attenuation correction at both sites as previously described,18F-fluoride activity in the blood pool and ascending aorta with the corresponding anatomical structures on the CMRA.MEAN and SUVMAX) were calculated for these septal VOIs and corrected for blood pool activity (measured in the right atrium)MEAN and TBRMAX). Second, in patients with cardiac amyloid I8F-fluoride uptake was assessed in areas of myocardial LGE. Equal sized volumes of interest (VOI) were placed within areas of LGE demonstrating the greatest visual uptake on short axis slices with SUVMEAN and TBRMEAN values recorded . Accurate co-registration was achieved by aligning MEAN values for both healthy volunteers and amyloid patients imaged in Edinburgh to the equivalent subjects imaged in New York.Throughout the study TBR values were used in preference for comparison. In principle the correction of tissue uptake for blood pool measurements made on the same scan adjusts for potential differences in PET values acquired at different scanners. In order to test this principle, we compared TBRP < 0.05 was considered statistically significant. The distribution of all continuous variables was assessed using the Shapiro-Wilk test, which were presented using mean ± standard deviation or median [interquartile range]. Comparisons between groups were performed using the two-sample t test, Mann-Whitney test or ordinary one-way ANOVA as appropriate. Receiver operating characteristic (ROC) curves were generated to determine area under curve (AUC) and perform sensitivity and specificity analyses. We presented all categorical variables as percentages.All statistical analyses were performed using GraphPad Prism Version 8.0. A two-sided 53 patients (33 in Edinburgh and 20 in New York) were scanned and completed the study protocol without complication: 13 controls, 18 with cardiac amyloid (10 ATTR and 8 AL), and 22 aortic stenosis , allowing the cavity of the blood pool in the left and right ventricles to be delineated and facilitating accurate co-registration of the MR angiogram and PET datasets in 3 dimensions. Patients with aortic stenosis had left ventricular hypertrophy with elevated LV mass index (100 ± 26 g/m2) but normal ejection fraction (69 ± 17 %). 18F-flouride uptake was again lower than blood pool .CMR was normal in all controls Table . Myocard2 with mean ejection fraction of 60 ± 18%. 15/18 (83%) patients had a pattern of diffuse generalized pattern of LGE on CMR (8 with ATTR and 7 with AL). Two patients (1 TTR and 1 AL) had focal LGE, while 1 (TTR) had no appreciable LGE on CMR.18 patients with cardiac amyloidosis were scanned: 10 (56%) with ATTR and 8 (44%) with AL cardiac amyloid. Mean myocardial mass was elevated at 101 ± 37g/mP = .0002) and patients with both AL amyloid  and aortic stenosis .Increased septal 18F-fluoride activity was observed in patients with ATTR amyloid 1.13 ± 0.16) compared to healthy volunteers . Moreover 18F-fluoride TBR values in areas of LGE provided excellent discrimination between patients with TTR and AL amyloid  and 100% specificity (CI 67.56 to 100%) for TTR amyloid .In the ATTR patients the most intense myocardial 18F-fluoride uptake was observed in areas of LGE Figure . Indeed,d Figure B with a MEAN threshold of 1.14 provided perfect discrimination between patients with ATTR and these other conditions. Our findings are in keeping with our own and recently published work by other groups.24In the first multicenter study of its kind, we have used hybrid PET/MR to investigate myocardial 18F-fluoride activity in patients with TTR amyloid, demonstrating increased uptake compared to age- and sex-matched controls and patients with both aortic stenosis and AL amyloid Table A. IndeedThe diagnosis and treatment of cardiac amyloidosis has advanced markedly over the last 10 years. AL amyloid can now be treated with disease modifying novel chemotherapeutic agents18F-fluoride PET/MR appears to provide a solution to some of these problems and, therefore, holds promise in the diagnosis and monitoring of patients with TTR amyloid. This approach combines the accurate diagnosis of cardiac amyloid on the CMR with clear discrimination of biopsy proven AL and TTR amyloid on the 18F-fluoride PET in a single scan. Hybrid imaging also facilitates accurate co-registration and comparison of the CMR and PET images. This is an important advantage as it allows regions of interest to be accurately drawn in specific regions of the myocardium including areas of late gadolinium enhancement wherein TBR values provide the greatest discriminatory information. The value of hybrid imaging will be further explored in the upcoming ‘I-CARE’ study which will evaluate the incremental benefit of tissue imaging with CMR to 18F-fluoride PET/CT. Finally, although theoretical at present, the fully quantitative nature of PET could potentially provide an assessment of TTR burden that could be used to track disease progression and/or treatment responses to the array of new therapies being developed for this condition. Indeed, PET imaging has been used in a similar fashion in other contexts assessing treatments responses to both atherosclerosis and aortic stenosis .While patterns of late gadolinium enhancement tend to differ between patients with ATTR and aortic stenosis, we have also demonstrated that 18F-fluoride PET/MR can too provide excellent discrimination between ATTR and aortic stenosis. This too is becoming an increasingly important and challenging clinical distinction. Recent studies have suggested that up to 12% patients with significant aortic stenosis undergoing surgical AVR have co-existent cardiac amyloid.Our observations in the healthy control group are also of importance. A key advantage of 18F-fluoride imaging in the heart is the low myocardial activity, with uptake values 80% of that observed in the blood pool. This has several implications that facilitate the wider assessment of 18F-fluoride PET as a marker of disease activity in the coronary arteries and heart valves.To our knowledge, this is the largest multicenter study investigating myocardial 18F-fluoride uptake in cardiac amyloid and while there is an important need for multicenter cardiovascular PET studies, this approach comes with inherent challenges and limitations. These include variation in the injected activity of 18F-fluoride, reconstruction parameters, and between scanner differences in uptake measurement values. Despite these potential problems we have demonstrated that TBR values appear to correct for many of these between center variations with no difference in TBR values measured in equivalent patients imaged at the two centers. Future multicenter cardiovascular PET studies should, therefore, be encouraged in the knowledge that many between center differences can be overcome.We have demonstrated that myocardial 18F-fluoride PET uptake is increased in patients with TTR amyloid, with TBR values providing quantification and clear discrimination from other similar conditions such as AL amyloid and aortic stenosis. This hybrid imaging technique may, therefore, help in the diagnosis of ATTR amyloid.We identified that ATTR binds 18F-fluoride more avidly than AL and the negative control groupsWe present 18F-fluoride PET/MR as a hybrid imaging technique able to distinguish between amyloid subtypes, phenotypically similar patients with aortic stenosis and age/sex-matched controls.This may improve our ability as clinicians to make the correct diagnosis and offer appropriate disease specific treatment.Electronic supplementary material 1 (DOCX 16 kb)Electronic supplementary material 2 (PPTX 2118 kb)Electronic supplementary material 3 (M4A 12773 kb)Below is the link to the electronic supplementary material.


In swine models, there are well-established protocols for creating a closed-chest myocardial infarction (MI) as well as protocols for characterization of cardiac function with cardiac magnetic resonance (CMR). This methods manuscript outlines a novel technique in CMR data acquisition utilizing smart-signal gradient recalled echo (GRE)-based array sequences in a free-breathing swine heart failure model allowing for both high spatial and temporal resolution imaging. Nine male Yucatan mini swine weighing 48.7 ± 1.6 kg at 58.2 ± 3.1 weeks old underwent the outlined imaging protocol before and 1-month after undergoing closed chest left anterior descending coronary artery (LAD) occlusion/reperfusion. The left ventricular ejection fraction (LVEF) at baseline was 59.3 ± 2.4% and decreased to 48.1 ± 3.7% 1-month post MI (P = 0.029). The average end-diastolic volume (EDV) at baseline was 55.2 ± 1.7 ml and increased to 74.2 ± 4.2 ml at 1-month post MI (P = 0.001). The resulting images from this novel technique and post-imaging analysis are presented and discussed. In a Yucatan swine model of heart failure via closed chest left anterior descending coronary artery (LAD) occlusion/reperfusion, we found that CMR with GRE-based array sequences produced clinical-grade images with high spatial and temporal resolution in the free-breathing setting. CHF is the leading cause of deaths attributable to cardiovascular disease and it remains the leading cause of death in the modernized world2. CHF is a clinical syndrome often characterized clinically by dyspnea, fatigue, and progressive pulmonary and lower extremity edema3. Although currently approved therapies have provided a clear benefit to those suffering from CHF, the 5-year mortality rate remains approximately 40–50%5. Thus, an opportunity is present for additional basic research to discover novel pharmacologic and biologic therapies, with the aim of improving the outcomes in patients with CHF.Nearly half the population in the United States is projected to have some form of cardiovascular disease by 2035 with the annual total healthcare cost expected to reach over $1 trillion[7. Small animal models, such as murine and rat models, provide important insights into the mechanisms of CHF. However, significant physiologic differences exist between small animals and humans including heart rate, oxygen consumption, adrenergic receptor ratios, myocardial contractile protein expression, cardiomyocyte ion channel phenotype, cellular response to heart failure, and phenotypic differences between stem cells limiting the interpretation and translation of results from small animal models to humans11. Large animal models, and particularly porcine models, more closely approximate human physiology, anatomy, and immunology which makes them useful in the further development of preclinical therapies for heart failure14. Furthermore, porcine coronary vasculature is similar to that of humans13. The left anterior descending artery (LAD) provides approximately half of the blood supplied to the left ventricle (LV) and its occlusion results in ischemia of the anteroapical, lateral, and septal walls of the LV which correlate in size and distribution to humans that have LAD occlusion16. Furthermore, post-infarct LV myocardial remodeling of the heart in swine models closely resembles what occurs in humans18.The translation of experimental therapies into effective and approved treatment options requires validation in large animal models that mimic human cardiac pathophysiology20. Furthermore, CMR provides quantitative assessments of both myocardial mass and infarction mass as well as estimation of geometric remodeling and scar reduction22.Imaging plays a critical role in the characterization of baseline and post-infarct cardiac function making it necessary for the evaluation of potential heart failure therapies. CMR is particularly useful for heart failure models as it allows for noninvasive assessments of multiple reference standard cardiac parameters to assess systolic and diastolic function including, ventricle volumes, ejection fraction, and filling rates15. However, since its publication, advancements have been made in CMR technology and techniques, allowing for research-grade imaging to be comparable or better than clinical-grade. The previously described CMR methods utilize breath-holding or ventilator gating techniques which have been found to distort anatomy because of breath to breath variation, and cause problems with vital sign stability and end tidal CO2 increasing the risk for arrhythmia26.In swine models, there are well-established protocols for creating closed-chest MI utilizing cardiac catheterization with coronary angiography. Such a protocol published in 2012 outlined each component of the process, providing a comprehensive outline for fluoroscopy, surgery, electrocardiography, and CMR27 and calculation of diastolic and systolic left ventricular wall stress28. This new technique will be widely valuable in cardiovascular disease research in animal models as it outlines state-of-the-art CMR methods allowing for improved evaluation of cardiac function and anatomy. This methodology will be particularly useful for the evaluation of pre-clinical therapies for cardiovascular disease.This protocol outlines the novel use of smart-signal averaging GRE-based array of pulse sequences in nine Yucatan mini swine before and 1 month after 90 min of ischemia reperfusion of the LAD providing both high spatial and temporal resolution imaging in the free-breathing setting. Finally, we outline post-imaging analysis utilizing CMR acquired data including our use of the American Heart Association 17-segment model of infarct sizing standardizationResearch oversight and approval was conducted by University of Arizona’s Institutional Animal Care and Use Committee. University Animal Care provided veterinary care, husbandry and surgical oversight within their facilities that are accredited and registered by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALACi Number (accredited since 1969): 000163, Continuing Full Accreditation ), Public Health Service (NIH/OLAW), Effective August 8, 2019, Expires August 31, 2023) and the United States Department of Agriculture . All experiments and methods were performed in accordance with the National Institute of Health’s “Guide for the Care and Use of Laboratory Animals” and in compliance with Institutional Animal Care and Use Committee-approved -protocols at the University of Arizona Animal Care Program . The methods and results of this study are reported in accordance with ARRIVE guidelines.Nine male Yucatan mini swine weighing 48.7 ± 1.6 kg at 58.2 ± 3.1 weeks old underwent 90 min of occlusion/reperfusion of the left anterior descending coronary artery and were evaluated with the below detailed CMR methods at baseline and 1-month post MI.nil per os (NPO) 12 h prior to surgery with water ad libitum and were delivered awake to the MRI preparation suite. In brief, the animals were sedated with ketamine 11–33 mg/kg and/or Midazolam 0.1–0.5 mg/kg delivered intramuscularly and sedated with 5% Isoflurane and 2–4 l/min of Oxygen via a nose cone. After the anesthetic plane is reached, the animal is intubated, and isoflurane was reduced to a maintenance level. Ophthalmic ointment was applied to prevent eye dehydration and an intravenous catheter was placed in an ear vein to deliver isotonic fluids at 5–10 ml/kg/h. the respiration rate, patter, mucus membrane condition, pulse oximetry, heart rate, and temperature were monitored during preparation. A ventilator was not used to prevent extreme diaphragm movement and image quality disruption.The animals were Once anesthetized, the animals were transported to the CMR suite and all metal and electronic monitoring devices were removed. The swine was positioned in the dorsal recumbent position in a Siemens Skrya Magnetom 3 Tesla MRI system  with towel rolls to maintain stability while the head was kept accessible for extended ventilation tubes supplied by an MRI compatible ventilator. Intravenous contrast and saline were connected to Bracco Empower MR injector  for dynamic perfusion required later in the study. Physiological monitoring, End Tidal CO2, SPO2, EKG, respirations and blood pressures were monitored by veterinary staff and used by the MRI scanner for gating purposes by a Philips Expression IP5 . Finally, a standard 18 channel phased array flexible body coil is conformed around the chest, being centered on the heart region, and the animal is carefully fed to isocenter with care given to not dislodge any of the apparatus.After the imaging study, the animals were removed from the MRI unit, returned to the preparation suite, recovered to consciousness, and extubated. Observation continues until the animal can safely stand unsupported in the transport cart for transport back to University Animal Care where they were monitored visually for stability and activity .Nine standard two-dimensional turbo flash (TFL2D) scout images were acquired in 3 orthogonal planes to begin localization with a large 400 mm × 400 mm field of view (FOV), a thick 8 mm slice with 50% gap, resulting in 2.1 mm × 2.1 mm × 8 mm voxels. A second series, to acquire a 2 chamber view of refined orientation scout images were placed in the LV parallel to the septal wall with a smaller rectangular FOV of 274 mm × 300 mm and contiguous 8 mm slices for positioning 2 pseudo–short Axis (SA) sequences. One TFL2D with 15, 5 mm contiguous slices was acquired for further refining of the subsequent functional 2 chamber, 3 chamber and 4 chamber views. Additionally, a 2.3 s True Fast Imaging was obtained with steady-state free precession (SSFP) sequence with Cardiac B0 shim box adjusted tightly to the heart which was an inherited property of the later fast perfusion sequences. While the time to acquire this image set was not optimized for first pass myocardial perfusion images, it was duplicated for later use.The SAx stack, 2 chamber, 3 chamber, 4 chamber, and left ventricular outflow track (LVOT) functional scans were collected with 25 calculated phases of standard retroactively reconstructed cine TFL2D sequences with radio frequency (RF) spoiling and Phase Encode (PE) rewinding in segments of 4–7 based on heart rate acquired at each slice location with 0.8 mm × 0.8 mm × 4 mm voxels using 45% phase oversampling, a repetition time (TR) of 23.53 ms, an echo time (TE) of 2.88 ms, flip angle (FA) of 15° and 3 averages to mitigate respiratory motion were uniquely acquired during free breathing.Dark Blood Soft Tissue Inversion Recovery Turbo Spin Echo (STIR DB) sequences were performed in a similar orientation to the functional sets and were utilized to evaluate increased water content in myocardial tissue which is indicative of inflammation and is often not obvious in the cine image sets. TR is dependent on heart rate, and the target recovery time is approximately 3 s, with TE of 48 ms. These sequences are achieved with cardiac gating using 2–4 trigger pulses, depending on average heart rate. A slice selective inversion pulse of 160 m was used to achieve fat suppression and 10 long-term averages were used to mitigate respiratory motion. Scan time is just over 3 min based on heart rate with integrated parallel imaging technique (iPAT) generalized auto-calibrating partially parallel acquisitions (GRAPPA) set to 2 times (a factor of 2).Quantitative T1 and T2 mapping was also acquired using the proprietary Myo-Maps sequences  in SAx and 2 chamber views, shown in Fig. After a single measurement test scan of the dynamic TFL perfusion sequence verifies positioning with no phase wrap in any of the orientations, 0.2 mmol/kg of MultiHance gadobenate contrast (Bracco) at 2.0 ml/s was injected as 10 of 150 measurements were completed for visualization of the contrast uptake and myocardial filling. The perfusion sequences were comprised of 3 slices positioned at the best 2, 3 and 4 chamber cine locations. Data collection was adjusted so that all three gated slices fit within one RR. The voxels were interpolated to 1.6 mm × 1.6 mm with a 9 mm slice thickness using a TR controlled by heart rate but approximately 123 ms, TE of 1.02 ms achieved with a 2.1 ms echo spacing by a bandwidth set at 1008 Hz/Pixel.While waiting for Delay Enhanced contrast uptake standard flow quantitative BEAT_FQ FLASH were acquired at the mitral valve, pulmonary artery, and aortic root using one 4 mm slice positioned to measure through-plane flow. These were retroactively reconstructed with 144 mm phase FOV 210 read FOV and 118 × 192 acquisition matrix TR approx. 21 ms and TE of 3.02 ms with PAT 2 at approximately 2 min each depending on HR.With swine’s metabolism of contrast being faster than humans, an Inversion recovery Time scout (TI) was collected at roughly 6 min post injection to plan the delay needed to null the myocardium. This sequence runs 20 measurements and sweeps the TI from 100 to 645 ms. The TI was chosen anticipating an increase needed every several minutes. Delay enhancement scans are collected with the previously defined orientations, SAx stack, and 2–4 chamber views. These were 1.35 mm × 1.35 mm × 4 mm voxels collected with a gated TFL with a non-selective inversion pulse and 10-degree FA.The ICM was placed 1 month prior to the MI procedure. After completing the animal preparation process outlined above, the left scapular area was clipped and scrubbed for aseptic surgery. A 1″ incision was made laterally along the left scapula and gentle dissection is used to widen the subcutaneous tissue enough to accommodate the 44.8 mm sterile ICM device. A 2-0 absorbable suture was used to close subcutaneous layers and a 3-0 absorbable suture was used for subcuticular closure with surgical glue. Bupivacaine (2 mg/kg) and/or lidocaine (4–6 mg/kg) local and regional nerve block (inverted L block) were used at incision sites after closure. ICM data is quickly collected using a reader placed over the device.After completing the animal preparation process outlined above animals were connected to a monitor for real-time respiration rate, pulse oximetry, heart rate and temperature monitoring during the procedure. Groin areas were clipped and scrubbed for aseptic surgery and the animals were placed in dorsal recumbency with front legs folded and tied caudally along ribs, with rear legs retracted and tied caudally.The percutaneous groin access site was prepared by nicking the skin with a 10 or 15 blade scalpel. A 17–19 g percutaneous needle was then inserted into the femoral artery or femoral vein until blood returned was observed. A 0.35 J-wire is inserted through the needle, advanced into the vessel and the needle removed. An 8-11F introducer dilator and sheath were advanced over the wire. Blood reflux was confirmed, the dilator and guide wire were removed, and the introducer was flushed with heparinized saline then secured with 2-0 suture. A bolus of heparin (100–200 USP/kg) was used to prevent thromboembolism.Lidocaine (2–4 mg/kg) was administered and repeated every 20–60 min as needed to prevent arrythmia during the procedure. Additionally, an IV pump 20–60 mcg/kg/min of lidocaine was started. If needed, amiodarone was administered by bolus, up to 2 times using 150–300 mg first dose and 75–150 mg second dose with infusion using a standard IV pump at 1 mg/min.An AL 0.75 or AL 1.0 guide catheter was advanced to the left coronary artery. An angiogram with contrast was performed Fig. A, using The LAD was occluded for 90 min. Contrast boluses were performed again at 45 min and 90 min to confirm continuous occlusion. The balloon was then deflated, and another contrast bolus performed to confirm reperfusion of the LAD. All catheters, guide wires and sheaths were then removed.Pressure was applied to the percutaneous access sites as the sheaths were removed, holding heavy pressure for 10 min, medium pressure for 5 min and light pressure for 5 min to help prevent hematoma formation. The tissue layers and the skin were closed with absorbable suture and surgical glue followed by local Bupivacaine (2 mg/kg) and/or Lidocaine (4–6 mg/kg) injection at incision sites after closure.Research oversight and approval was conducted by University of Arizona’s Institutional Animal Care and Use Committee. University Animal Care provided veterinary care, husbandry and surgical oversight within their facilities that are accredited and registered by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALACi Number (accredited since 1969): 000163, Continuing Full Accreditation ), Public Health Service (NIH/OLAW), Effective August 8, 2019, Expires August 31, 2023) and the United States Department of Agriculture .The methods and results of this study are reported in accordance with ARRIVE guidelines.All authors give consent for publication.Nine male Yucatan mini swine weighing 48.7 ± 1.6 kg at 58.2 ± 3.1 weeks old underwent the procedure described. Table Figure 2) × 1000 and increased to 15.5 ± 3.6 (dyne/cm2) × 1000 at 1-month post MI while systolic wall stress at baseline was 31.6 ± 3.5 (dyne/cm2) × 1000 and increased to 36.2 ± 4.0 (dyne/cm2) × 1000 at 1-month post MI  stack evaluating the bot ventricles took between 8 and 12 min. Breath holding has been noted to result in additional issues with variations in cardiac volume and contraction resulting in decreased in resolution as well as vital sign stability, end tidal CO2 variation, and heart rate variabilityWhile the protocol provides a robust evaluation of myocardial size, function, and tissue characterization with superior temporal and spatial resolution that can be performed on anesthetized swine, it does have limitations. Particularly, while cardiac MRI is an ever-growing technique, its use is often limited to academic centers given the infrastructure needed for maintaining an MRI that can be used for both clinical and research purposes along with the computer hardware and software requirements to perform quantitative image analysis. Furthermore, swine models require facilities, staff, and procedures to comply with all federal and state guidelines concerning the use of animals in research and teaching, as defined by the Guide for the Care and Use of Laboratory Animals. Another important limitation of this study is that the results obtained utilizing this novel protocol were unable to be directly compared to conventional MRI protocols thus the degree of improvement from previous methods is unable to be objectively quantified in this manuscript.This CMR protocol is widely applicable to cardiovascular disease research in animal models as it outlines state-of-the-art CMR methods allowing for improved evaluation of cardiac function and anatomy. It will be particularly useful for the evaluation of pre-clinical therapies for cardiovascular disease including but not limited to cell therapy, drug therapy, and device therapy. Additionally, this protocol will be useful for research aimed at better understanding cardiovascular disease pathophysiology, biophysics, and computer modeling.27. Of note, the American Heart Association 17-segment model does tend to overestimate infarct sizing as there is no way to represent a partial infarct in one segment. This was demonstrated in this study as the CMR average measured LV percent scar was 23.2 ± 3.7% compared to the 17-segment computed LV infarct percent of 29 ± 3.0%.The American Heart Association 17-segment model analysis provides a means of standardizing and comparing infarct measurements obtained from various modalities such as echocardiography, CMR, cardiac CT, and positron emission computed tomography29 and heart failure30 patients. It is provided here to demonstrate another important metric that can be obtained through analysis of CMR data acquired using GRE based array sequences in the free-breathing setting.Wall stress has been shown to be an important predictor of mortality in myocardial infarctionIn a Yucatan swine model of heart failure with closed chest LAD occlusion/reperfusion, we find that this novel method of CMR with GRE based array sequences produced clinical-quality images with high spatial and temporal resolution in the free-breathing setting.Supplementary Information.


Method. This is a progressive observational study on 53 hospitalized patients with low ejection fraction heart failure, who were followed up for 12 months. The aetiology of heart failure was ischemic heart disease and dilated cardiomyopathy. The patients were clinically and biochemically evaluated by EKG (echocardiography) on admission and at 6 and 12 months. The biomarkers included in the present study were angiotensin-converting enzyme type 2 (ACE2), apelin-13, NT-proBNP (biomarkers involved in the counterregulation of RAAS), interleukin 17 (IL-17), hsCRP (inflammatory biomarkers), and urinary 8-iso-PGF2α (oxidative stress biomarker). The evolution was considered unfavourable if the patients presented complications during hospitalization, were readmitted for decompensated heart failure, or died. Results. From the study group, 14 patients (24.52%) presented an unfavourable clinical evolution. The biomarkers that were associated with the evolution of patients during hospitalization were ACE2, apelin-13, NT-proBNP, and hsCRP. Multivariate logistic regression analysis identified ACE2 and apelin-13 as independent, predictive biomarkers for the unfavourable evolution of patients over the study period. Values of ACE2 above 4000.75 pg/mL and of apelin-13 less than 402.5 pg/mL were associated with an unfavourable evolution . Conclusion. The serum values of ACE2 and apelin-13 correlate with the unfavourable evolution of patients with reduced ejection fraction heart failure.The progression of heart failure is the result of the interaction of several pathogenetic processes that involve the activation of biomarkers belonging to the renin angiotensin aldosterone system (RAAS), to its counterregulatory mechanisms, to the sympathetic nervous system and inflammation, and to oxidative stress. This study is aimed at determining the prognostic role of biomarkers in the evolution of patients with heart failure. These biomarkers are representative of different pathogenetic pathways involved in the progression of heart failure and the possible interrelationships between them and heart remodelling.  In patients with heart failure, congestion is the dominant clinical profile and the main pathophysiological mechanism that causes organic dysfunction .The therapeutic reduction of the congestion leads to improved organ function and improved patient prognosis .The identification of blood and urine biomarkers, which are closely linked to the functional damage of the organs affected in congestive heart failure, is important for a better stratification of the prognosis of patients and for the development of new therapeutic methods , 3.The renin angiotensin aldosterone system (RAAS) is a key pathophysiological mechanism in the development and progression of heart failure. RAAS is activated together with the sympathetic system in patients with heart failure as a compensatory mechanism to decrease cardiac output. It contributes to maintaining cardiac output through water and salt retention, peripheral vasoconstriction, increased cardiac contractility, and myocardial hypertrophy .However, RAAS also determines the progression of heart failure, causing diastolic and subsequently systolic dysfunction and progression to the final stage, namely, that of congestive heart failure. This occurs as a result of cardiac remodelling which involves increased myocardial stiffness secondary to the process of hypertrophy and myocardial fibrosis. The activation of the mediators of inflammation and oxidative stress by RAAS causes endothelial dysfunction and consequently altered microcirculation, subendocardial ischemia, and exacerbation of myocardial fibrosis and heart stiffness , 6.Angiotensin II (Ang II) is the main molecule in RAAS causing vasoconstriction, myocardial hypertrophy, and increased oxidative stress .ACE2 opposes the effects of Ang II by catabolizing and transforming it into Ang 1-7 which has a protective role in the cardiovascular system . The serThe apelin/APJ system is also an endogenous counterregulator of RAAS by antagonizing the Ang II activity .Apelin is a peptide encoded by the APLN gene, located on the long arm of the X chromosome. It is synthesized as a pre-pro-apelin consisting of 77 amino acids. Apelin is subsequently cleaved into smaller C-terminal fragments with biological activity  . From a There is evidence that ACE2 is the enzyme that fragments apelin by removing the C-terminus, the main area through which it exerts its effect on the APJ receptor , 14, 15.Natriuretic peptides (BNP and NT-proBNP) are now widely used in the diagnosis and follow-up of patients with heart failure. Natriuretic peptides are protein molecules that are secreted by the ventricular muscles in response to volume or pressure overload. These peptides act as a counterregulator of RAAS causing natriuresis, diuresis, vasodilation, and improved myocardial relaxation. Their role is similar in predicting the unfavourable evolution of patients with heart failure .Interleukin 17 (IL-17) is a proinflammatory cytokine, which is synthesized by macrophages, helper T cells, dendritic cells, and natural killer cells. Studies in mouse models have shown that IL-17 is involved in cardiac remodelling following myocarditis and in its progression to dilative cardiomyopathy. IL-17 causes cardiac fibrosis by producing several cytokines with a role in the recruitment of neutrophils and monocytes to the inflammation site, in the activation of the matrix metalloproteinases, and in the apoptosis of heart cells .α (synonymous with 15-F2t-IsoP) belongs to the class of F2-isoprostans. They are isomers of prostaglandin PGF2α and are accurate markers of oxidative stress in vivo due to their stability (they are not influenced by self-oxidation and are relatively easy to measure). 8-Iso-PGF2α is involved in cardiac remodelling causing proliferation of fibroblasts and cardiomyocytes [Oxidative stress has high values in heart failure. 8-Iso-PGF2myocytes .The aim of the study was to determine the prognostic role of biomarkers in the evolution of patients with heart failure and the possible interrelationships between biomarkers and heart remodelling. These biomarkers are representative of different pathogenetic pathways involved in the progression of heart failure.Our research is a prospective observational cohort single-centre study on 53 patients diagnosed with heart failure with reduced ejection fraction, hospitalized in the Cardiology Ward of the Emergency County Hospital during a 12-month period of time, with the approval of the Bio-ethics Commission of “Iuliu Hațieganu” University of Medicine and Pharmacy, Cluj-Napoca (No. 377/03NOV2014).The diagnosis was based on clinical evaluation, biomarker determination, and echocardiography. A control group was used; it was formed of 13 patients with arterial hypertension, which was well controlled with betablockers and/or ACEI, according to the European Society of Cardiology guidelines. The inclusion criteria were heart failure with reduced ejection fraction due to dilative cardiomyopathy or ischemic heart disease. We excluded the patients with significant valvular diseases other than secondary mitral and tricuspid regurgitation, acute myocardial ischemia, diabetes mellitus, severe renal impairment (creatinine clearance < 35 mL/min), untreated arterial hypertension, neoplasia (either current or in their history) , acute focal infections (over the previous 6 months) or chronic focal infections, and collagen diseases (with or without immunosuppressive treatment).All patients included in the study gave their written consent regarding their participation, and the presented study was approved by the Bio-ethics Commission of “Iuliu Hațieganu” University of Medicine and Pharmacy, Cluj-Napoca .All the patients were evaluated twice, once during the first admission and the second time after a period of 6-12 months. On both evaluations, the same parameters were recorded, namely, the clinical, biological, and echocardiographic ones. On admission, the NYHA class was recorded.2). A sample of serum was stored at -80°C, from which the determinations of apelin-13, ACE2, IL-17, and NT-proBNP were made, using ELISA commercial kits. Urine samples were stored in Eppendorf vials at -80°C after adding 10 μL of hydroxytoluene butylate per each mL of urine as antioxidant. The 8-iso-PGF2α was assessed by the immunoenzymatic method using a commercially available kit from Cayman Chemical Company, Ann Arbor, Michigan, USA, with IC50 = 10 pg/mL and a detection threshold of 2.7 pg/mL. 8-Iso-PGF2α levels were adjusted to the urine creatinine level assessed from the same urine sample using the reaction between urinary creatinine and alkaline picrate .For biological investigation purposes, blood was collected via venous puncture, under sterile conditions and with minimal venous compression. Biological dosing was performed with the aid of the KONELAB 30 I analyser, and it was as follows: blood sugar, HDL-cholesterol, LDL-cholesterol, uric acid, triglycerides, C reactive protein, creatinine clearance (mL/min), and corrected creatinine clearance  structural and performance parameters.The echocardiography was used to track the dimensions of the left ventricle and its systolic and diastolic functions, the presence of wall motion abnormalities, significant valvulopathies , and a pericardial collection. The left ventricular ejection fraction was calculated using Simpson's rule in which normal adult levels were considered to be 52-78%. All parameters were calculated as the arithmetic mean of 3 determinations, in the case of patients experiencing atrial fibrillation. The relative wall thickness (RWT) was calculated after measurements of end-diastolic volume and cardiac wall thickness, a value > 0.42 meaning eccentric hypertrophy , 22.The evolution was considered unfavourable when the patients presented complications during hospitalization , were readmitted for cardiac decompensation, or died.The statistical analysis was performed with the IBM SPSS 26 software package. The data were analysed descriptively and prescriptively. Because the data were not normally distributed, nonparametric tests were performed.The Kruskal-Wallis test was used if statistically significant differences between patients in more than two categories were detected. A Dunn-Bonferroni post hoc test was performed to determine the categories between which the difference was significant. The significance was adjusted by the sample size.The Spearman correlation was used to assess if there were significant associations between variables as the variables were measured on a continuous scale but were not normally distributed.The receiver-operating characteristic (ROC) curve was used to assess the accuracy of tests using the considered predicting variables. The predictive ability of each variable was determined by analysing the area under the curve (AUC).A binary logistic regression is a technique used to determine the impact of multiple independent variables on one dichotomous dependent variable. It uses the maximum likelihood method to correctly predict the category of the outcome variable .n = 53) and for the control group (n = 13) are shown in Baseline clinical and demographic characteristics of patients (The unfavourable evolution was recorded for 14 (24.52%) of the 53 patients followed up over a 12-month period.α, NT-proBNP, and the ejection fraction. Analysing the value of the area under the curve (AUC), it was observed that only ACE2, apelin-13, NT-proBNP, and hsCRP presented statistically significant values indicating that they have an ability to predict evolution . The area under the curve (AUC) was 0.796 (95% CI 0.644–0.947) indicating that the variable can be used to predict the evolution of the patient . The AUC was 0.731 (95% CI 0.581–0.881)  .p = 0.006). The AUC was 0.754 (95% CI 0.615–0.894)  with a sensitivity of 41% and a specificity of 100% (5–0.894) .p = 0.042). The AUC was 0.707 (95% CI 0.556–0.857)  with a sensitivity of 48.5% and a specificity of 90.9% (6–0.857) .The variables that had significant predicative power were used in a multivariate analysis. The variable NT-proBNP was removed from the analysis because it was highly correlated with the apelin-13 variable.χ2(3) = 22.090, p < 0.001. The model explained 50.7% (Nagelkerke R2) of the variance in the evolution of the patient and correctly classified 84.9% of the patients. An increase in ACE2 values is associated with a slight increase in the likelihood of patients evolving unfavourably. Decreasing values of apelin-13 were also associated with a slight negative evolution of the patients. The regression coefficients (B) and their associated significance (Sig.) can be found in A binary logistic regression was performed to ascertain the effect of ACE2, apelin-13, and CRP on the evolution of the patient. The logistic regression model was statistically significant, α. A strong negative correlation was identified between the serum values of apelin-13 and NT-proBNP .An analysis was performed using the Spearman correlation to test whether there is an association between the indicators ACE2, IL-17, apelin-13, NT-proBNP, the ejection fraction, and 8-iso-PGF2H (2) = 13,497, p = 0.001). Applying a Dunn-Bonferroni post hoc test, it was observed that NYHA IV class patients had significantly lower apelin-13 values than those in class II  and class III .A Kruskal-Wallis test was performed to determine whether there was a significant difference between patients in different NYHA classes of apelin-13. A significant difference was found between patients from different NYHA classes (H (2) = 15.163, p = 0.001). Applying a Dunn-Bonferroni post hoc test, it was observed that NYHA class IV patients had significantly higher hsCRP values than class II patients .A Kruskal-Wallis test was performed to determine whether there was a significant difference between patients in different NYHA classes of hsCRP values. A significant difference was found between patients from different NYHA classes .All heart failure patients had high values of LV mass , relatively stable during the follow-up .A significant negative correlation was found between end-systolic volume and apelin-13 .In the case of NYHA class IV, a significant positive correlation was found between the serum value of apelin-13 and the RWT .In NYHA class II patients, a negative correlation was identified between 8-iso-PGF2p = 0.034).In the case of NYHA class III, significant negative correlations were found between the ejection fraction and the end-systolic diameter . Also, a level of apelin-13 lower than 402.5 pg/mL had a high predictability capacity for an unfavourable outcome, with an AUC of 0.731, a sensitivity of 61.5%, and a specificity of 76.9% (p = 0.013). These results confirmed the importance of these biomarkers for an accurate evaluation of the prognosis of a patient with congestive heart failure. This dynamics is in concordance to other reports, both experimental and clinical studies [In our study, the levels of ACE2 higher than 4000.75 pg/mL had a high predictability capacity for an unfavourable outcome, with an AUC of 0.796, a sensitivity of 87.5%, and a specificity of 66.7%  and angiotensin I (Ang I) to angiotensin 1-7 (Ang 1-7) and angiotensin (Ang 1-9), respectively, two nonaggressive peptides on the failing myocardium, protective for the cardiovascular system, in contrast to the proinflammatory and profibrotic Ang I and II –29. In eThese observations are in line with the new reports about the increased levels of ACE2 in advanced cardiovascular disease and with the negative prognostic value of high levels of ACE2, both for cardiovascular disease and death of all causes .p < 0.01). Basically, as the heart failure aggravates and the congestion increases, the level of NT-proBNP rises and those of apelin-13 lowers [The relationship between apelin and NT-proBNP was analysed in several studies of heart failure patients, as is the research of Chandrasekaran et al. or Miettinen et al. –33. Alth3 lowers .The patients with NYHA IV heart failure and lower ejection fraction had significantly higher levels of NT-proBNP and ACE2 and significantly lower levels of apelin-13 compared to the patients with NYHA II heart failure. In the multivariate analysis, the decrease of apelin-13 and the increase in ACE2 were associated with a slight but significant increase of adverse events.p = 0.032). The relative wall thickness has a negative correlation with the level of urinary 8-iso-PGF2α in the group of NYHA II patients  and a positive correlation with the apelin-13 level in the group of NYHA IV patients . The failing heart undergoes a remodelling process that leads to its dilation, with or without associated LV hypertrophy, and to the alteration of the global geometry of the cavity. LV diameters increase, and the cavity becomes spherical, changing the distribution of the cavitary and wall stress, raising the oxygen demand of the myocardium. All of our heart failure patients had very high LV mass, so the RWT showed concentric (RWT > 0.42) or eccentric (RWT ≤ 0.42) LV hypertrophy. The higher values of RWT (concentric LV hypertrophy) were correlated with higher values of apelin-13 and lower levels of urinary 8-iso-PGF2α, meaning the eccentric hypertrophy is associated with opposites values of these biomarkers. In our observation, the lower levels of apelin-13 and higher levels of oxidative stress are associated with higher NT-proBNP levels, more severe heart failure, and unfavourable outcome during the follow-up.In our analysis, we observed a significant negative correlation between left ventricle end-systolic volume and the level of apelin-13 , and the level of apelin was significantly lower in NYHA class IV than in NYHA class III also . These observations are concordant to other studies that showed higher levels of CRP in heart failure patients [The heart failure is a chronic, constantly progressive disease, which promotes inflammation and also is aggravated by inflammation. It induces inflammation inside the myocardium due to the wall stress, hemodynamic factors, and the overstimulation of the RAAS. The patients with more severe heart failure (NYHA class IV) had higher levels of CRP and lower levels of apelin-13 than the patients in NYHA class II (patients  and raisThe cardiac remodelling by itself is an independent factor for the progression of heart failure, leading to congestion; the biomarkers studied here are parts of a molecular network with strong interconnections. The overstimulation of RAAS is a constant aggression for the failing myocardium that apelin and ACE2 systems are trying to overcome, the alteration of their serum levels, as described above, being the signs for the adverse outcome that is to come.The serum values of ACE2 and apelin-13 correlate with the unfavourable outcome in patients with reduced ejection fraction heart failure and may be useful biomarkers in determining the prognosis of these patients.


In hypertrophic cardiomyopathy (HCM), myocardial fibrosis is routinely shown by late gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR) imaging. We evaluated the efficacy of 2 novel contrast-free CMR methods, namely, diffusion-weighted imaging (DWI) and feature-tracking (FT) method, in detecting myocardial fibrosis.  This cross-sectional study was conducted on 26 patients with HCM. Visual and quantitative comparisons were made between DWI and LGE images. Regional longitudinal, circumferential, and radial strains were compared between LGE-positive and LGE-negative segments. Moreover, global strains were compared between LGE-positive and LGE-negative patients as well as between patients with mild and marked LGE. P < 0.001). The regional longitudinal and circumferential strain parameters showed significant associations with LGE (P < 0.001), while regional circumferential strain was the only independent predictor of LGE in logistic regression models . A comparison of global strains between patients with LGE percentages of below 15% and above 15% demonstrated that global circumferential strain was the only parameter to show impairment in the group with marked myocardial fibrosis, with borderline significance (P=0.09). A review of 212 segments demonstrated a qualitative visual agreement between DWI and LGE in 193 segments (91%). The mean apparent diffusion coefficient was comparable between LGE-positive and LGE-negative segments (P=0.51).  All 3 strains showed significant differences between LGE-positive and LGE-negative segments ( FT-CMR, especially regional circumferential strain, can reliably show fibrosis-containing segments in HCM. Further, DWI can function as an efficient qualitative method for the estimation of the fibrosis extent in HCM. Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disorder with autosomal dominant inheritance and heterogeneous patterns of penetration and expression , 2. The The results of a study on infarcted swine hearts indicated comparable accuracy between DWI, LGE, and histology in detecting and delineating densely scarred areas, border zone areas, and healthy tissues . A few sIn the present study, we studied 26 patients with HCM to evaluate the efficacy of the 2 novel techniques of DWI and FT-CMR in demonstrating areas with myocardial fibrosis.The current investigation enrolled 30 consecutive patients with HCM who were referred to our center for CMR between July 2019 and December 2019. All these patients provided informed written consent for participation in the project. The study protocol was approved by the Medical Research Ethics Board of Iran University of Medical Sciences.2), and the presence of cardiac devices such as internal cardioverter defibrillators and pacemakers; coronary artery disease; systemic hypertension; severe valvular disease; and systemic diseases affecting the myocardium such as amyloidosis.The exclusion criteria were a history of previous cardiac surgeries, including myectomy; contraindications for CMR such as severe contrast allergy, severe renal failure  sequences were performed on all the patients with an interactive ECG-gating regime to collect all signals at the freezing point of myocardial contraction .All the patients underwent a comprehensive study in keeping with a prespecific routine CMR imaging protocol using a 1.5 T MRI machine  and a vendor-supplied body surface coil. The imaging protocol consisted of the acquisition of 2-, 3-, and 4-chamber views, short-axis cine  images, and LGE images 10 minutes after contrast injection. Also, breath-hold low The presence of LGE in each patient and each different segment was detected by an expert with more than 5 years of experience in cardiac imaging. With the aid of CVI42 software, the LGE percentage was calculated for each patient as the sum of hyperenhanced regions with +5SD signal intensity above the normal remote myocardium divided by the total LV myocardial mass, expressed as the percentage of the enhanced myocardial mass. The patients were classified into 2 groups: marked fibrosis (fibrosis percentage ≥15%) and mild fibrosis (fibrosis percentage <15%).Circle CVI's imaging platform, cvi42 , was used to measure 3D longitudinal, circumferential, and radial strain parameters from cine images. First, brightness was adjusted to reach the optimal discrimination of the endocardium and blood pool. Next, endocardial and epicardial borders were defined on 3 long-axis views  and short-axis views . The nexDWI images were taken from the middle and apex of the heart for the evaluation of 6 middle and 4 apical AHA cardiac segments. The DWI images were evaluated via both qualitative and quantitative methods. For the qualitative evaluation, a radiologist and a cardiologist, who were both experienced in the field and blinded to the patients' history and other CMR sequences, independently observed the DWI images and classified the segments as fibrosis-positive and fibrosis-negative (intraobserver reproducibility: 0.89–0.92). In the case of disagreement between the 2 interpreters, the final decision was made by the senior radiologist in the ward. These visual assay findings were thereafter compared with those of segmental LGE. In the next step, ADC maps were calculated from the DWI dataset using MATLAB software, and the mean ADC value for each AHA segment was extracted .t-test. The predictive power of regional strain parameters for regional LGE was tested using logistic regression models. A 2-tailed P value of less than 0.05 was considered statistically significant.SPSS software, version 22.00, was used for the statistical analyses. Continuous variables with normal distributions were described as the mean ± the standard deviation (SD), while categorical variables were expressed as frequencies and percentages. Intergroup comparisons between LGE-positive and LGE-negative cases and between LGE < 15% and ≥15% were performed in terms of the quantitative variables by using an independent-samples n = 9), followed by pulmonary insufficiency (n = 4). LGE was present in 20 patients (76.9%: LGE-positive cases) and 52 out of 212 available segments (24.5%). P=0.024) and cardiac output , as well as significantly higher LV end-systolic volume index  and maximal wall thickness . The LV mass index and LV end-diastolic volume index did not show a significant difference between the two groups (P > 0.1 for both).The current study recruited 30 patients, of whom 4 were excluded due to severe motion artifacts. Twenty-six patients  at a mean (±SD) age of 46.5 (±14 y) were studied. The systolic anterior motion of mitral valve leaflets was seen in 13 patients (50%). Mild-to-moderate valvular abnormalities were seen in 11 patients (42.3%), with mitral regurgitation being the most common abnormality . According to their LGE percentage, the patients were divided into 2 groups of LGE of less than 15% and LGE of 15% or greater so that global strain values could be compared between the groups. The intergroup analysis showed no significant difference in the absolute global strain values . Thereafter, a comparison of segmental longitudinal, circumferential, and radial strain values revealed significant differences concerning all 3 strain parameters between LGE-positive and LGE-negative segments . As is shown in P < 0.001 for both), and circumferential strain was the only independent predictor of segmental LGE .The comparison of global strain values between LGE-positive and LGE-negative patients demonstrated no significant difference in global longitudinal strain (GLS), global radial strain (GRS), and global circumferential strain (GCS) .In 12 patients, the DWI images of cardiac apical segments were excluded due to severe motion artifacts. In the total of 212/260 reviewed segments, a visual agreement between DWI and LGE images was reported in 193 segments (91%). In the remaining 19 segments, 5 LGE-positive segments were classified as fibrosis-negative by DWI images  and 14 LGE-negative segments were classified as fibrosis-positive by DWI images . In 11 out of the 19 discordant segments (57%), the timing of the cardiac cycle was dissimilar between LGE and DWI images. The mean ADC was 0.64 ± 0.25 for LGE-positive and 0.61 ± 0.23 for LGE-negative segments. The difference between the ADC values of LGE-positive and LGE-negative segments failed to constitute statistical significance (All regional strain values showed significant deterioration in fibrosis-containing segmentsAmong the 3 regional strain values, circumferential strain was an independent predictor of fibrosisIn patients with notable amounts of fibrosis, GCS showed relative impairmentThe qualitative assessment of DWI images revealed an excellent agreement with LGE sequencesThe quantitative assessment of ADC showed no difference between segments with or without LGEThe main findings of our investigation are as follows:Contractile dysfunction in HCM and its relationship with tissue characteristics have been the focus of some previous studies. Using speckle-tracking echocardiography, Popovic et al  demonstrIn our study, global strains exhibited no significant difference between LGE-positive and LGE-negative cases. The regional strains, however, showed significant impairment in LGE-positive segments, and segmental circumferential strain proved to be an independent predictor of LGE. This might be due to the fact that, in HCM, myocardial fibrosis is typically midwall , 22, 23,b-value DWI is not an efficient quantitative technique for the evaluation of myocardial changes. This finding does not chime in with that reported by a few investigations that demonstrated significantly increased ADC measures in fibrosis-positive segments defined by either LGE or ECV images [b-value diffusion also contains data regarding the perfusion characteristics of the myocardium [A few previous studies have utilized DWI to detect fibrosis in HCM hearts –12. Our V images –12. One ocardium , 26. As ocardium . We, theThe current study has some notable limitations, first and foremost among which is our small sample size, possibly explaining the indeterminate significance of some findings. In our view, similar studies with larger samples should be considered. Second, DWI sequences were based on segmented spin-echo EPI, which added to the susceptibility of the lung tissue over the LV lateral wall and caused distortion in some images. Third, EPI-based sequences usually have a low signal-to-noise ratio, adding to the complexity of image and data analysis. Thus, we recommend the evaluation of balanced steady-state free precession for DWI in larger studies on HCM.b-value DWI is not a reliable method to quantify the scar extent. Further large-scale multicentric studies are warranted to reliably propose these 2 novel noncontrast CMR methods for fibrosis prediction in patients suffering from HCM.FT-CMR can reliably show fibrosis-containing segments in HCM. Circumferential strain is probably the first strain parameter to exhibit impairment in LGE-positive patients. This is of paramount importance since this novel postprocessing technique can serve as an available, easy, and fast method to show myocardial fibrosis in clinical practice and obviate the need for any additional CMR sequences or contrast injections. DWI can function as an efficient qualitative method for the estimation of the fibrosis extent in HCM, whereas low


The primary aim was to observe the predictive value of P-wave ECG index and left atrial appendage volume (LLAV) for atrial fibrillation recurrence after first radiofrequency catheter ablation.A total of 196 patients with paroxysmal atrial fibrillation were enrolled. The preoperative LLAV was measured by cardiac enhanced CT. The P-wave ECG index including minimum P-wave duration (P-min), maximum P-wave duration (P-max), mean P-wave duration (mPWD), P-wave dispersion (PWD), P-wave terminal force in lead V1 (PtfV1), PR interval prolongation, and interatrial block (IAB) were analyzed and recorded in 12-lead ECG of sinus rhythm.According to the follow-up results, the patients were divided into two groups: the non-recurrence group and the recurrence group. P-min, PWD, P-max, PtfV1 ≥ 0.04 mV·s, PR interval prolongation, and the ratio of first and third-degree IAB in the recurrence group were higher than those in the non-recurrence group, with significant statistical differences (P < 0.05). Kaplan–Meier curve analysis was performed on time to atrial fibrillation recurrence after catheter ablation when PtfV1 ≥ 0.04 mv s by comparison between groups . Kaplan–Meier curve analysis showed that the survival rate without recurrence of atrial fibrillation after catheter ablation was lower when the LLAV exceeded 8.0 mL (log-rank test P < 0.001).PWD, P-max, PtfV1, PR interval prolongation, first and third-degree IAB, and LLAV can effectively predict atrial fibrillation recurrence after radiofrequency catheter ablation. The combination might be a valid and alternative independent predictor of recurrence. Atrial fibrillation is a common arrhythmia in clinical practice. Based on the duration of symptom, atrial fibrillation can be categorized into permanent, persistent and paroxysmal atrial fibrillation. Atrial fibrillation often causes several symptoms, including shortness of breath, chest discomfort, vertigo, palpitation, and other symptoms, with an increased risk of stroke and heart failure. The condition poses a serious threat and adverse impact on the quality of life and the lives of patients , 2. RadiTotally 196 patients with paroxysmal atrial fibrillation underwent the first successful radiofrequency catheter ablation of atrial fibrillation in our hospital between November 2017 to November 2018. They were followed up for over 12 months after discharge. The symptoms and ECG information of the patients were closely monitored. Recurrence was defined as at least one occurrence of rapid atrial arrhythmia for more than 30 s in 12 months after Coronary according to the follow-up results, and the patients were divided into two groups: the non-recurrence group (n = 155) and the recurrence group (n = 41). There were 109 males and 46 females, aged 48.97–65.09 years, with an average age of (55.87 ± 3.98) years in the non-recurrence group. The non-recurrence group included 27 males and 14 females, aged 47.027–65.13 years, with an average age of (55.28 ± 4.01) years. The sex and age of patients in the two groups were comparable (P > 0.05). To be included in the present study, the following inclusion criteria should be met: (1) patients with paroxysmal atrial fibrillation who were treated successfully after radiofrequency catheter ablation of atrial fibrillation for the first time; (2) patients whose left atrial thrombus was not found by 64-slice spiral CT before the operation, and the left atrium and left atrial auricle were well filled with a contrast agent, and the boundary of them could be distinguished by CT scan; (3) informed consent form was required before radiofrequency catheter ablation of atrial fibrillation; (4) clinical data were complete; (5) there were no serious postoperative complications. Exclusion criteria were as follows: (1) poor quality of cardiac enhanced CT images, unclear and unmeasurable volume boundaries of the left atrium and the left atrial appendage; (2) hyperthyroidism; (3) incomplete electrical isolation of the circumferential pulmonary vein; (4) cardiac function grade 4 (New York cardiac function class); (5) severe hepatorenal insufficiency; (6) patients who were complicated with valvular heart disease or congenital heart disease; (7) patients who were complicated with severe chronic obstructive pulmonary disease. (8) patients who were unconscious and mentally abnormal; (9) patients had surgically treated cerebral infarction or cerebral hemorrhage; (10) disagreement, loss to follow-up, or withdrawal of consent; (11) familial hypercholesterolemia.N-terminal B-type natriuretic peptide precursor, uric acid, blood urea nitrogen, creatinine, C-reactive protein, and cardiac troponin I were detected after centrifugation. Cobase411 produced by Roche Co. was used to detect the precursor of N-terminal B-type brain natriuretic peptide and cardiac troponin I by electrochemiluminescence. C-reactive protein was detected by immune turbidimetry, and creatinine, blood urea nitrogen, and uric acid were detected through the enzymatic method with C8000 (Abbott Co.).The basic clinical data of patients were collected through the electronic medical record system, including gender, age, BMI, basic heart disease, concomitant diseases, and the use of drugs for treatment. Fasting venous blood (3–5 mL) was taken before the operation, and the laboratory indexes such as The 12-lead ECG of sinus rhythm was recorded and analyzed in all patients before operation. P-wave dispersion (PWD), minimum P-wave duration (P-min), maximum P-wave duration (P-max), and P-wave terminal force in lead V1 (PtfV1) were measured and the mean values were calculated Figs. . PR is tThe results of preoperative cardiac enhanced CT in all patients were recorded and analyzed, and the volume of the left atrial appendage was measured accurately. CT scan was done by 64-row spiral CT (Siemens). Patients were injected with 90 mL ioproamine from the anterior elbow vein to maintain the speed of 4–5 mL/s, followed by 40 mL 0.9% saline injected at 4–5 mL/s. The retrospective ECG current regulation technique was used to collect image data, and the maximum tube current (450–500 mA) was used in the systole of the cardiac cycle, while in other periods, the appropriate current could be selected to reduce the current properly. The images were analyzed by Intuition platform software . The largest left atrium and left atrial appendage volume at the end of the systole was measured before mitral valve opening, so the left ventricle volume was the smallest and the left atrium volume was the largest. The left atrium and left atrial appendage margin were drawn manually on the axial section, and the left atrium and left atrial appendage orifice were accurately distinguished by the orthogonal view, in which the left atrial appendage cavity, including the left atrial appendage muscle trabecula, and the three-dimensional volume of the left atrial appendage and the left atrium were calculated by Simpson method  and compared by Age, gender, and history of diabetes were comparable between the two groups (P > 0.05), as shown in Table P-min, PWD, P-max, PtfV1 ≥ 0.04 mV s, PR interval prolongation, and the ratio of first and third-degree IAB in the recurrence group were higher than those in the non-recurrence group, and the difference was statistically significant Table .Table 2CThe ROC curves of PWD, P-max, and PtfV1 are shown in Figs. In the CT image features of the two groups, the left atrial appendage volume and left atrial volume in the recurrence group were significantly higher than those in the non-recurrence group (P < 0.05). Based on the echocardiographic features, the diameter of the left atrial appendage and left atrial volume in the recurrence group were higher than those in the non-recurrence group, but without significant difference (P > 0.05), and there was no significant difference in the left ventricular ejection fraction between the two groups (P > 0.05).When the volume of the left atrial appendage was > 8.0 mL, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of atrial fibrillation recurrence after atrial fibrillation catheter ablation were 95.12%, 70.73%, 76.47%, 93.55%, and 82.93%, respectively. Kaplan–Meier curve analysis showed that the survival rate without recurrence of atrial fibrillation after catheter ablation was lower when the volume of the left atrial appendage exceeded 8.0 mL . The ROC curve showed that PWD was 0.771 and P-max was 0.747, suggesting that P-min, PWD, P-max, PR interval prolongation, and first and third-degree IAB might have great predictive value for atrial fibrillation recurrence after catheter ablation. Besides, when PWD was 105.6 ms, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of atrial fibrillation recurrence after catheter ablation were 74.18%, 71.9%, 41.27%, 90.89%, and 72.76%, respectively. When P-max was 117.3, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of atrial fibrillation recurrence after catheter ablation were 93.2%, 62.1%, 55.6%, 97.6%, and 74.2%, respectively. The results indicated that PWD of 105.6 ms and PWD max > 117.5 ms were of high value in predicting recurrence after catheter ablation.Salah et al.  found thThe anterior initial vector of P-wave in ECG is right atrial activation, and the posterior terminal vector is left atrial activation; hence, it is called left and right atrial comprehensive depolarization wave. PtfV1 represents the area of transverse left atrial depolarization vector, and the increase of PtfV1 represents larger left atrial depolarization vector, indicating abnormal left atrial interatrial conduction . PtfV1 aThe left atrial appendage is namely an appendage of the left atrium, and there are complex and staggered myocardial fibers at the junction. The anisotropic myocardial fibers at the junction can accelerate atrial beats at this site, resulting in abnormal electrophysiological characteristics , 19. In PWD, P-max, PtfV1, PR interval prolongation, first and third-degree IAB, and the left atrial appendage volume are considered to be effective predictors of recurrence after first radiofrequency catheter ablation. Admittedly, some limitations should be acknowledged. During the follow-up, outpatient follow-up twice a week, ordinary ECG, and dynamic ECG once a week were performed to detect the recurrence of atrial fibrillation, while a small number of asymptomatic patients with atrial fibrillation could not be detected in time or ignored. Besides, given the limited time and sample size of our study, further studies are warranted to confirm the current findings (Additional file Additional file 1: ROC data for PWD, P-max, PtfV1 and prediction of recurrence.