MEK signature was predictive of response of selumetinib in GC cell lines regardless of KRAS mutation status. The proportion of high MEK signature by nanostring assay was 6.9% and the proportion of high MEK signature was significantly higher in KRAS altered group in a Korean cohort. None of PIK3CA altered cases belonged to high MEK signature group. MEK high signature was more prevalent in intestinal type by Lauren classification. The correlation between MEK signature, KRAS alteration and treatment response to selumetinib should be validated in prospective clinical trials.MEK inhibitor (selumetinib) is a potent, orally active inhibitor of MAPK/ERK pathway. It is important to develop an accurate and robust method indicative of RAS pathway activity to stratify potential patients who can benefit from selumetinib treatment in gastric cancer (GC). First, we surveyed the sensitivity to selumetinib in a panel of 22 GC cell lines and correlated with MEK signature to selumetinib sensitivity. Next, we analyzed MEK signature via nanostring assay in two Asian cohorts using clinical samples ( RAS and RAF family members [In vitro studies demonstrated a tendency toward sensitivity to MEK inhibitors in tumor cell lines harboring KRAS or BRAF mutations [KRAS-mutant lung cancer patients and colorectal cancer [Selumetinib  is a potent, orally active inhibitor of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)-1/2 that suppresses the pleiotropic output of the RAF/MEK/ERK pathway [ members , 4. In vutations , 5, 6. Bl cancer , 8.KRAS mutation and the RAS pathway signature is superior to KRAS mutation status for the prediction of response to RAS pathway inhibitor [On the other hand, there was no absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity to MEK inhibitor response in large tumor cell panels of diverse cancer types . An 18-gnhibitor .KRAS status, of gastric cancer (GC) patients according to MEK signature in two Asian cohorts using clinical samples. In this study, we first surveyed the sensitivity to MEK inhibitor in a panel of GC cell lines and correlated with, RAS alteration, MEK signature to MEK inhibitor sensitivity. Next, we analyzed MEK signature via nanostring assay in FFPET  samples from advanced GC patients and then performed a correlative analysis with MEK signature status and KRAS genotype in GC.The aim of this study was to investigate the clinicopathologic and genomic status, especially p < 0.00054) , and the combination (MEK score – Cres score) further separated sensitivity form resistance (ANOVA p < 0.00064)  Figure . Further) Figure . InteresKRAS amplification and mutation status in two large cohorts from previous study [KRAS amplification was 1.5% (3/191) in the ACRG cohort  and 7.5% (36/477) in the TCGA cohort  in the ACRG cohort and 8.8% (28/317) in the TCGA cohort.First, we surveyed the incidence of us study , 11. Thet Figure . The incKRAS alteration (17 (13.6%) with KRAS mutation and 10 (8.0%) with KRAS amplification) in a Korean cohort. Detected KRAS mutations were as follows; G12C (n = 2), G12D (n = 9), G12V (n = 1), A146P (n = 1), A146T (n = 1), F156L (n = 1), Q61H (n = 1), and Q61R (n = 1). Of the 125 patients, 15 (12%), 71 (56.9%), and 39 (31.2%) patients were classified as high, intermediate, and low respectively for the MEK gene expression signature score by nanostring. Of 125 patients, 87 patients were prospectively enrolled onto the VIKTORY screening program. In this patient cohort, 6 (6.9%), 50 (57.5%), and 31 (35.6%) patients had high, intermediate, and low MEK signature, respectively.In total in the Korean cohort, 27 out of 125 patients (21.6%) showed KRAS status (P = 0.001) in the Korean cohort , 35 (35.7%), 57 (58.2%), and 6 (6.1%) cases were classified into low, intermediate, and high MEK signature, respectively. For KRAS altered group (n = 27), 4 (14.8%), 14 (51.9%), and 9 (33.3%) cases were classified into low, intermediate, and high MEK signature, respectively. The proportion of high MEK signature was significantly higher in the KRAS altered group  , 7.62 (7.28–7.97), and 8.05 (7.77–8.33) (P = 0.01)  Figure . The mea) Figure .KRAS altered group (18.1%) compared to KRAS wild group (8.5%) although there was no statistical significance (P = 0.48)  Figure .BRAF and PIK3CA in each MEK signature group is summarized in Table PIK3CA mutant and 1 (1.15%) PIK3CA amplified cases. None of 8 PIK3CA altered cases belonged to high MEK signature group. The mean MEK signature score of PIK3CA wild and altered group was 7.27 , 7.40 (7.22–7.58) . BRAF mutation was not identified in any of the cases.The status of ) Figure . There wPIK3CA mutation was detected in 13 (14.0%) out of 93 patients in a Vietnamese cohort. Likewise, none of these patients belonged to the MEK high group. In terms of BRAF mutation, 7 (7.5%) BRAF mutated patients were identified and 1 out of 7 BRAF mutated patients belonged to the MEK high group. There was no association between MEK signature score and BRAF status (data not shown).On the other hand, P = 0.034). In addition, there was a higher frequency of intestinal type of Lauren classification (66.7%) in high MEK group compared to low MEK (20.5%) and intermediate MEK group (36.6%) (P = 0.012). There was no significant difference of age, sex and tumor stage. Long-term follow up data (more than 5 years) was available for the patients of the discovery set (n = 38). Disease-related death rate was 37% in low, 61.1% in intermediate, and 66.7% in high group.Table KRAS mutant lung cancer and biliary tract cancer [MEK inhibition in combination with chemotherapy has shown beneficial effects in t cancer , 12. Thit cancer . TherefoRAF-MEK-ERK pathway has profound effects on proliferative, apoptotic, and differentiation pathways . Mutatioin vivo and in vitro studies demonstrated a tendency toward sensitivity to selumetinib in tumors harboring KRAS or BRAF mutations compared with those without those mutations [BRAF, RAS, or PIK3CA [Selumetinib  is a potent, orally active inhibitor of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase [KRAS-mutant non-small-cell lung cancer or BRAF-mutant melanoma, it is not clear that MEK inhibitors are specific for KRAS-mutant cancers [KRAS G12C or G12V mutations showed better response for selumetinib plus docetaxel compared with other KRAS mutations [KRAS mutations [KRAS mutant and KRAS wild-type advanced lung cancer [Although the development and clinical trial of selumetinib has focused on  cancers . The resutations . On the g cancer .in vivo and in vitro studies have suggested that gene expression signature would provide a better measure of RAS activity in cancer cells than mutation analysis [KRAS mutations had high signature scores [KRAS wild-type cell lines and tumors exhibited high RAS pathway signature scores, suggesting that these samples have upregulated RAS signaling through another mechanism [DUSP4, DUSP6, ETV4, ETV5, PHLDA1, and SPRY2, in advanced GC patients. Our results showed that KRAS altered tumors have a tendency to have high MEK signature. However, the relationship between MEK signature and KRAS status was not absolute in our study. There is a subset of KRAS wild type GCs which revealed high MEK signature which is in agreement with previous findings that the RAS pathway signature is a high sensitivity but low specificity predictor of KRAS mutation status [PIK3CA is known to mediate resistance to MEK inhibitor [PIK3CA mutation had high MEK signature. A phase II selumetinib/docetaxel trial is currently ongoing for second-line metastatic GC patients as part of an umbrella trial with patient selection based on high vs low MEK signature or RAS gene alterations .Clinically, it is important to develop an accurate and robust method indicative of RAS pathway activity to stratify potential patients who can benefit from selumetinib treatment. Previous analysis . In a pre scores . Howeverechanism . In our n status . PIK3CA nhibitor . In our KRAS amplification, the activation of KRAS signaling pathway, and cell growth in GC [KRAS mutation was more frequently found in intestinal type, according to the Lauren classification [KRAS mutation, MEK1 S72G mutation [GC is the second most common cause of cancer-related deaths worldwide, and the prognosis of advanced GC is still poor . To dateth in GC . KRAS mufication , 23. Ourmutation , which aKRAS genomic alterations and intestinal type tumors by Lauren classification. The correlation between MEK signature, KRAS alteration and treatment response to selumetinib should be validated in prospective clinical trials.In conclusion, 6.9% of metastatic GC score high for expression of the MEK signature assessed by nanostring assay and have tendency to be enriched for 2. Cells were treated with concentrations of selumetinib ranging from 30 to 0.003 μmol/L for 72 hours. For the MTS endpoint, cell proliferation was measured by the CellTiter AQueous Non-Radioactive Cell Proliferation Assay (Promega) reagent as per manufacturer's instructions. Absorbance was measured with a Tecan Ultra instrument. Expression of MEK and Cres signature genes was determined by RNA sequencing. Total RNA was extracted from cell lines and mRNA was enriched using the oligo dT magnetic beads. cDNA was synthesized from fragmented and size selected mRNA (~200 bp). Sequencing adaptors were ligated to the fragments and PCR amplified before sequenced using Illumina HiSeq 2000's pair-end sequencing technology (2 × 90 bp). Roughly ~120 million reads were obtained for each cell line.MTS was used to determine cell proliferation. Cells were seeded in 96-well plates at a density to allow for logarithmic growth during the 72-hour assay and then incubated overnight at 37°C, 5% COSensitivity to MEK inhibition with selumetinib was defined as a GI50 under 3 μM, and further divided into partial (TGI not reached within 30 mM concentration range tested) and extreme (TGI< 5 μM) sensitivity. Resistant cell lines showed GI50> 20 μM and did not reach TGI within 30 μM concentration range tested. MEK signature was scored from RNAseq as the mean of gene centric RPKM for the 18 MEK signature genes [n = 125) and a Vietnamese cohort (n = 93). For the training set in the Korean cohort, positive control  and negative control (KRAS wild) cases were selected from 312 surgically resected GC samples that had been sequenced as previously reported [n = 17) consisted of 8 KRAS amplified tumors and 9 KRAS mutated tumors by target sequencing. Negative control group (n = 21) was confirmed to have no KRAS mutation and amplification. The remaining 87 of 125 specimens in the Korean cohort were from the GC molecular screening program, the VIKTORY molecular screening study (NCT# 02299648). Briefly, 87 patients had MEK signature scoring results from nanostring assay, in addition to the cancer panel sequencing results (either from Ion torrent or Illumina HiSeq). Clinicopathological information of Korean GC cohort, including age, sex, WHO histologic type, Lauren classification, clinical stage, and GC-associated survival data was collected. The study was approved by the institutional review board of Samsung Medical Center (IRB no. 2014-1-136). Written informed consent form was obtained from all patients before the study. All experimental procedures were carried out in accordance with guidelines approved by Samsung Medical Center.This study was conducted in two cohorts of Asian GC patients, which consisted of a Korean cohort (reported . PositivHematoxylin and Eosin stain was performed on one tumor section per patient and tumors were reviewed by a pathologist for tumor purity. Samples with < 50% tumor was discarded from the study. The tumor component was macro-dissected from 2 × 5 μm FFPET sections either from FFPET or fresh frozen samples and RNA was extracted using RNeasy FFPE Extraction kit (Qiagen) or QIAamp DNA Mini Kit  according to the manufacturer's instructions. Sample RNA was quantified using Qubit 2.0 Flourometer with the Broad Range RNA kit using the standard protocol. Samples that had < 20 ng/μl total RNA were not tested in the nanostring assay. Where available, more tissue for these samples were ordered, re-extracted, and those 20 ng/ul or greater were tested in the nanostring assay.Nanostring assays were performed by following the standard protocol ‘Setting up 12 nCounter Assays . Hybridization incubations were performed between 17–18hrs. Cartridges were either read immediately or stored dark  at 4°C until ready to be read. All cartridges were read within 2 days of preparation, on the AZ GEN2 Digital Analyzer station with high resolution selected.To enable assessment of sample MEK signature scores against previously defined cut-points, three reference standards, representing high, medium and low MEK signature scores were run in every assay. These reference samples were selected from a previous assessment of MEK signature scores using Nanostring in 197 commercially sourced Vietnamese Gastric cancer samples (unpublished data). Up to eight test samples and a background control sample (RNase free water instead of RNA in the hybridisation reaction) using the same reagents were ran alongside the reference samples. MEK signature score adjustment was undertaken by applying a linear transformation to the calculated MEK signatures for each sample. The transformation coefficients were calculated as the linear regression coefficients calculated from the fit of the reference sample MEK signature score values generated by Samsung Medical Center against those previously generated by AstraZeneca.in vivo models and fresh/fixed tissue samples, crossing tumor type and gene expression platforms . For each patient sample, the 6-gene MEK signature score was calculated by taking the arithmetic average of the normalized gene expression value  of the 6 MEK genes; DUSP4, DUSP6, ETV4, ETV5, PHLDA1, and SPRY2 . For each patient sample, the 6-gene MEK signature score was calculated by taking the arithmetic average of the normalized gene expression value  of the 6 MEK genes; DUSP4, DUSP6, ETV4, ETV5, PHLDA1, and SPRY2. Then, the adjusted MEK signature score was compared against the following two cut-off values; ‘MEK-low’ (25th percentile), 7.17 and ‘MEK-high’ (90th percentile), 8.08. Where adjusted MEK score for a patient was less than or equal to the MEK-low cut-off, the patient was considered as part of the MEK-low. Where adjusted MEK score for a patient was greater than or equal to the MEK-high cut-off, the patient was considered as part of the MEK-high.Each sample was normalized individually with the three reference samples using nSolver Analysis Software version 2.5. The normalized data was exported as Log2 data and then adjusted using the method described previously. Previously we published an 18-gene mRNA expression signature reproducibly predictive of MEK pathway output and response to MEK inhibition with selumetinib and subsequently demonstrated that a. 6-gene sub-signature shows enhanced reproducibility in cell lines, primary tumor explant KRAS status and MEK signature results. One way Anova test was done to compare the distribution of MEK score according to KRAS and PIK3CA status. Contingency tables and Fisher's exact tests were also used to correlate MEK signature with KRAS status, age, sex, WHO tumor type, Lauren classification, and tumor stage. A P-value <0.05 was considered significant.Statistical analysis was performed using SPSS version 18.0 . Contingency tables and Fisher's exact tests were used to correlate


To investigate acute stress response in residents playing nurse and physician roles during emergency simulations.Sixteen second-year internal medicine residents participated in teams of four (two playing physician roles and two playing nurse roles). Stress markers were assessed in 24 simulations at baseline (T1) and immediately after the scenario (T2), using heart rate, systolic and diastolic blood pressure, salivary α-amylase, salivary cortisol and salivary interleukin-1β. The State-Trait Anxiety Inventory was applied at T2. Continuous data were summarized for the median (1st-3rd interquartile ranges), and the Mann-Whitney U Test was used to compare the groups.The percent variations of the stress markers in the physician and nurse roles, respectively, were the following: heart rate: 70.5% (46.0-136.5) versus 53.0% (29.5-117.0), U=89.00, p=0.35; systolic blood pressure: 3.0% (0.0-10.0) versus 2.0% (-2.0-9.0), U=59.50, p=0.46; diastolic blood pressure: 5.5% (0.0-13.5) versus 0.0% (0.0-11.5), U=91.50, p=0.27; α-amylase: -5.35% (-62.70-73.90) versus 42.3% (12.4-133.8), U=23.00, p=0.08; cortisol: 35.3% (22.2-83.5) versus 42.3% (12.4-133.8), U=64.00, p=0.08); and interleukin-1β: 54.4% (21.9-109.3) versus 112.55% (29.7-263.3), U= 24.00, p=0.277. For the physician and nurse roles, respectively, the average heart rate was 101.5 (92.0-104.0) versus 91.0 (83.0-99.5) beats per minute, U=96.50, p=0.160; and the state anxiety inventory score was 44.0 (40.0-50.0) versus 42.0 (37.50-48.0) points, U= 89.50, p=0.319.Different roles during emergency simulations evoked similar participants’ engagement, as indicated by acute stress levels. Role-play strategies can provide high psychological fidelity for simulation-based training, and these results reinforce the potential of role-play methodologies in medical education.  Assessing the acute stress levels evoked by different roles during simulations can provide relevant understanding regarding training immersion and engagement. The hypothesis of the present study is that acute stress levels induced in residents during an emergency simulation are equivalent between nurse and physician roles, indicating that both roles lead to similar engagement.This was a cross-sectional study carried out at the Simulation Center of University of São Paulo Medical School in São Paulo, Brazil. This study was approved by the Ethics and Research Committee of the University of São Paulo, and a written informed consent was obtained from all subjects. All second-year internal medicine residents rotating in an internal medicine ward were invited to voluntarily participate. Exclusion criteria were the following: pregnancy, infections of any kind, disease of the immune system, endocrine or metabolic diseases, use of any kind of medication  and inadequate salivary samples. The participation of the same resident in both simulated scenarios was allowed on different days. A total of 16 unique residents were enrolled in this study, and 8 of these participated in both scenarios. Therefore, a total of 24 observations (6 simulations) were analyzed. The median age of residents was 26.0 (IQR: 25.0-26.0) years, and 13 (54.2%) of them were female. At the time of the study enrollment, the participants had completed 15.5 (IQR: 15.0-16.0) months of residency. The emergency scenarios had a median duration of 25.0 (IQR: 23.0-27.0) minutes.In the simulation center, the scenarios were performed using a high-fidelity computer-based mannequin simulator, with the possibility of remote control of vital signs. All medications and equipment required during the scenarios were available, and the simulation room was set up similar to a real-life emergency room. The mannequin had a peripheral venous catheter already placed at the beginning of each scenario. Standardized physiologic responses to anticipated management steps were programmed into the scenario and activated by a physician facilitator. During the scenario, the facilitator remained in a one-way mirrored glass control room.The scenarios used in this study were elaborated by three experienced simulation facilitators of the Clinical Emergency Discipline of University of São Paulo Medical School and were tested for two years prior to this study. The following emergency scenarios were applied:         -  A fifty-five-year-old patient was admitted to the ER with precordial pain, with evidence of right ventricular myocardial infarction. During care, the patient develops cardiogenic shock and third-degree atrioventricular block, requiring volemic expansion, vasoactive drug and a transcutaneous cardiac pacing.         -  A twenty-six-year-old patient was admitted to the ER with signs and symptoms of cocaine intoxication and develops unstable ventricular tachycardia. The patient presents with hypotension and acute pulmonary edema, requiring electrical cardioversion and invasive ventilation.The researcher collected the following demographic variables after stress marker collection during T2: age, gender, time of residency and scenario duration. Acute stress levels were assessed using the following parameters:Heart rate: This parameter was continuously measured using a heart rate monitor. The maximum between T1 and T2 was recorded from the watch system. Heart rate response was calculated as the percentage of variation from baseline to T2. For heart rate response, the maximum heart rate value was considered as T2. Average heart rate during overall simulation was also recorded.Systolic blood pressure (SBP): This measure was obtained using an aneroid sphygmomanometer, and the mean of three measures was considered. Systolic blood pressure response was calculated as the percentage of variation from baseline to T2.Diastolic blood pressure (DBP): This measure was obtained using an aneroid sphygmomanometer, and the mean of three measures was considered. Diastolic blood pressure response was calculated as the percentage of variation from baseline to T2.Salivary analyses: Saliva samples were obtained by placing a specific swab under the tongue for 2 minutes. The samples were centrifuged at 1500 x g for 15 minutes at 4 °C and stored in a freezer at – 80 °C. The participants were required to abstain from eating, drinking (except water) and brushing teeth 1 hour before the material collection. Acute stress responses were calculated as the percentage of variation from baseline to T2, relating to the following markers:   •   Salivary α-amylase (AA): measured using a kinetic colorimetric kit   •   Salivary interleukin-1 β (IL-1β): measured using an immunoenzymatic kit   •   Salivary cortisol (cortisol): measured using an immunoenzymatic kitState component of State-Trait Anxiety Inventory: this widely used self-report inventory was applied to participants immediately after the end of the simulated scenario (T2), and its value score was considered as an acute stress indicator.Each emergency team was formed by four residents, two playing the role of a nurse and two playing the role of a physician. The roles and scenarios were randomly chosen at the beginning of the simulation. The facilitator asked residents playing the nurse role to perform in the same way as nurses do in a real-life setting. The facilitator also stated that all decisions about task management should be defined by the team, without facilitator interference. Residents equally participated in both simulated scenarios (12 participants in each scenario). During simulated scenarios, all of the following procedures were performed exclusively by residents playing the physician role: orotracheal intubation, transcutaneous pacing and synchronized cardioversion. On the other hand, residents playing the nurse role performed exclusively the following procedures: medication preparation and infusion, infusion pump preparation and intubation material preparation. Residents playing the physician role took the leadership position in all simulations.Residents were placed in a sitting position at rest for 5 minutes, and their baseline (T1) stress levels were measured between 1:30 pm and 2:00 pm. Before starting the scenario, all participants were oriented for 15 minutes by a physician facilitator about the simulation room setup, mannequin features and simulation methodology. Immediately after the end of the simulated emergency scenario (T2), stress levels were measured again. During the period between T1 and T2, participants remained with a heart rate monitor on. The end of the emergency care situation (T2) was considered when a) the mean arterial pressure was greater than 65 mmHg; b) oxygen saturation by pulse oximetry was greater than 90% after confirmed endotracheal tube position; c) there was return of spontaneous circulation maintained for at least 5 minutes or when terminated efforts and death was confirmed; or d) the patient was hemodynamically stable after therapeutic measures. Regardless of the clinical management, the scenario was finalized 30 minutes after its start.The sample size was calculated based on the nomogram proposed by AltmanThe Shapiro-Wilk test was used to assess normality. Data were expressed as median and interquartile ranges (1st-3rd IQR) for continuous variables and as number and percentage for categorical variables. The Mann-Whitney U test was used to compare acute stress levels between the different roles. The U statistic value, difference between medians and 95% confidence interval (CI) for this difference were provided. Acute stress levels were defined as the percentage of variation from baseline (T1) to T2 regarding each parameter measuring stress. State component of State-Trait Anxiety Inventory was measured only during T2, and the average heart rate was measured during the entire period between T1 and T2. The level of statistical significance for this study was set at 0.05 (two-sided). All analyses were conducted using the SPSS Statistics software.Comparing the group of residents playing the physician role with those playing the nurse role, we observed that the average heart rate during simulation was 101.5 (IQR: 92.0-104.0) versus 91.0 (IQR: 83.0-99.5) beats per minute, respectively, and this difference did not present statistical significance, diff: 10.5 (95%CI -2.0 to 23.0), U = 96.5, p = 0.16. Residents presented a State Anxiety Inventory score of 44.0 (IQR: 40.0-50.0) in the physician role versus 42.0 (IQR: 37.5-48.0) points in the nurse role, and this difference was not statistically significant, diff: 2.0 (95%CI -5.7 to 9.7), U = 89.5, p = 0.32.In relation to the percent variation of the stress markers between baseline (T1) and T2, residents playing physician and nurse roles, respectively, presented the following: heart rate: 70.5% (IQR: 46.0-136.5) versus 53.0% (IQR: 29.5-117.0), diff: 17.50 (95%CI -50.9 to 85.9), U = 89.0, p=0.35; systolic blood pressure: 3.0% (IQR: 0.0-10.0) versus 2.0% (IQR: -2.0-9.0), diff: 1.0 (95%CI -6.8 to 8.8), U = 59.5, p=0.46; diastolic blood pressure: 5.5% (IQR: 0.0-13.5) versus 0.0% (IQR: 0.0-11.5), diff: 5.5 (95%CI -5.9 to 16.9), U = 91.5, p=0.27; cortisol: 35.3% (IQR: 22.2-83.5) versus 42.3% (IQR: 12.4-133.8), diff: -7.0 (95%CI -80.5 to 66.5), U = 64.0, p=0.08; α-amylase: -5.4% (IQR: -62.70-73.90) versus 37.5% (IQR: 11.0-186.8), diff: -42.9 (95%CI -186.3 to 100.6), U = 23.0, p=0.08; and interleukin-1 β: 54.4% (IQR: 21.9-109.3) versus 112.5% (IQR: 29.7-263.3), diff: -58.1 (95%CI -216.6 to 100.4), U = 24.0, p=0.27 .The present study assessed acute stress levels in residents playing physician and nurse roles during two different simulated emergency scenarios. Since acute stress measurement presents an intricate complexity and there is no gold standard parameter to measure stress response,-,-,A growing number of studies have reported that simulated scenarios may provoke substantial acute stress in doctors, nurses and other allied professionals.Despite this evidence, few studies have investigated stress levels in physicians playing different roles in simulated scenarios. Girzadas and colleagues,--,,In our experience using simulation in medical education, we have incorporated role-play and role reversal strategies for two different reasons. First, because the relationship between doctors and nurses in acute care settings has long been known to present many conflicts, misunderstandings and potential negative impact on quality of care.The second reason for using role-play is to enhance learner engagement in simulated scenarios. In fact, some studies have suggested that for teamwork training, the physical and environmental fidelities are less important than the psychological fidelity.,,,The relationship between realism or fidelity and training effectiveness, as well as the factors that lead to a more realistic learning environment, are highly controversial.This study presents important limitations. First, the volunteer nature of inclusion may have favored the occurrence of selection bias. Second, since this study was carried out in a single center and included only second-year internal medicine residents, the findings may not necessarily generalize to other settings. In addition, the sample size of this study was relatively small. In future studies, larger samples may identify differences between groups that this research could not find.The findings of the present study suggest that different roles during emergency simulations evoke similar participants’ engagement, as indicated by acute stress levels. Role-play strategies can provide high psychological fidelity for simulation-based training, and these results reinforce the potential of role-play methodologies in medical education. Future studies can address the impact of acute stress on knowledge and skills retention as well as performance of residents.This research was funded by FAPESP (São Paulo Research Foundation).The authors declare that they have no conflict of interest.


In patients with heart failure (HF) and left bundle branch block (LBBB) cardiac resynchronization therapy (CRT) improves left ventricular (LV) function and leads to reverse LV remodeling. A major concern with CRT remains that using current clinical indicators and implementation methods, 30-40% of patients are non-responders. CRT response is highly dependent on the presence of delayed mechanical activation and absence of scar at the LV lead implantation site . The aimShort axis images were acquired on a 1.5T MR system from 6 healthy subjects and 50 HF-LBBB patients  with established clinical criteria for CRT. Late gadolinium enhancement (LGE) was used to detect the scar location. DENSE circumferential strain was computed using previously described methods . Spatiotemporal strain data were arranged into a 2D matrix over space (LV segment) and time (cardiac phase) and singular value decomposition (SVD) was used to denoise the strain data. An active contour method automatically detected regions of delayed mechanical activation, defined by the time-of-onset of contraction, as per Figure Figure In HF patients selected for CRT, mechanical activation times measured with cine DENSE correlate well with electrical activation times. Imaging of mechanical activation demonstrates heterogeneity of the site of latest activation. Based on the heterogeneity and the suboptimal lead locations achieved using current CRT methods, pre-procedure MRI of mechanical activation shows potential for improving the implementation of CRT.


A 58 years old male with a long-standing history of HLA-B27 positive ankylosing spondylitis presented with increasing fatigue and dyspnea on exertion. He had left ventricular dysfunction and enlargement, flail right coronary leaflet of aortic valve with severe eccentric aortic insufficiency along with left ventricular non-compaction in echocardiography. The most common cardiac manifestations of ankylosing spondylitis are aortic insufficiency and conduction disturbances. Involvement of myocardium, in the form of dilated cardiomyopathy and restrictive cardiomyopathy, has also been reported. This case presents a very rare association of ankylosing spondylitis with non-compaction cardiomyopathy. More than half of patients also have peripheral joint involvement or enthesitis.+ ankylosing spondylitis presented with increasing dyspnea on moderate exertion and easy sense of fatigue. His joint pain was controlled effectively with non-steroidal anti-inflammatory drugs. He never smoked and the remaining of his medical history was unremarkable. Family history for cardiac or rheumatologic disorders was negative. On physical exam, he appeared tachycardic with a blood pressure of 135/60 mm Hg. Apical impulse was displaced to left and there was a diastolic murmur and S3 gallop on cardiac auscultation. In transthoracic echocardiography there was left ventricular enlargement with left ventricular end diastolic diameter of 7.2 cm and left ventricular end systolic diameter of 6.1 cm. Left ventricular ejection fraction was 32% (Supplementary Video 1). Right coronary cusp (RCC) of aortic valve (AV) was thick and fibrotic. RCC was flail causing significant defect in coaptation that yielded severe eccentric turbulent jet towards anterior leaflet of mitral valve (Supplementary Video 2). There was also biventricular non-compaction (A 58 years old male with a 16-year history of HLA-B27mpaction . Descend5 Cardiac involvement has long been recognized as an extra-articular manifestation of the disease and presents most commonly with aortic insufficiency and conduction abnormalities.5 Involvement of myocardium also reported, yet is not frequently encountered and remains less well recognized.7 Recently presence of left ventricular asynchrony has been shown in patients with ankylosing spondylitis.6 Cardiac involvement occurs more frequently in long-standing disease however they can precede skeletal symptoms.7 The prevalence of dilated cardiomyopathy in patients with ankylosing spondylitis is under-reported because it symptoms such as dyspnea on exertion may be attributed to the underlying disease. Restrictive cardiomyopathy secondary to amyloidosis has also been rarely been reported in patients with ankylosing spondylitis. Inflammatory processes and ensuing fibrosis have been implicated in the valvular dysfunction and conduction disturbances that associate with ankylosing spondylitis.8 In a recent study on patients with ankylosing spondylitis and no apparent cardiac involvement, increased aortic stiffness and decreased global myocardial performance have been reported which signifies the subclinical cardiac involvement in this group of patients.3Ankylosing spondylitis is a chronic inflammatory disease of the axial skeleton at large that predominantly affects young men.9 It is speculated that left ventricular non-compaction may happen as an adaptive mechanism to certain hemodynamic conditions. Chronic left ventricular volume overload may be a potential trigger for increased trabeculation of myocardium. It is unclear whether left ventricular non-compaction cardiomyopathy has occurred secondary to aortic insufficiency or independently in this patient. The association of non-compaction cardiomyopathy with ankylosing spondylitis remains exceedingly rare.Left ventricular non-compaction may arise from the arrest of trabecular remodeling that happens during early fetal life. It is an uncommon entity that may present in isolated form or in association with other cardiac or systemic conditions. Though it is commonly considered a congenital disorder, the acquired cases have also reported. Right ventricle is involved in less than 50% of cases. Three major clinical manifestations are heart failure, arrhythmias, and systemic embolic events.The local ethics committee approved the study.None.videos 1Click here for additional data file.videos 2Click here for additional data file.videos 3Supplementary file consists of videos 1, 2, and 3.Click here for additional data file.


Quality of life is as important as survival in heart failure (HF) patients. Controversies exist with regards to echocardiographic determinants of exercise capacity in HF, particularly in patients with preserved ejection fraction (HFpEF). The aim of this study was to prospectively examine echocardiographic parameters that correlate and predict functional exercise capacity assessed by 6 min walk test (6-MWT) in patients with HFpEF.In 111 HF patients , an echo-Doppler study and a 6-MWT were performed in the same day. Patients were divided into two groups based on the 6-MWT distance (Group I: ≤ 300 m and Group II: >300 m).p = 0.008), had higher prevalence of diabetes (p = 0.027), higher baseline heart rate (p = 0.004), larger left atrium - LA (p = 0.001), longer LV filling time - FT (p = 0.019), shorter isovolumic relaxation time (p = 0.037), shorter pulmonary artery acceleration time - PA acceleration time (p = 0.006), lower left atrial lateral wall myocardial velocity (a’) (p = 0.018) and lower septal systolic myocardial velocity (s’) (p = 0.023), compared with Group II.Group I were older (p = 0.007), higher baseline heart rate (p = 0.005), higher NT-ProBNP (p = 0.001), larger LA (p = 0.004), lower septal s’, e’, a’ waves, and septal mitral annular plane systolic excursion (MAPSE), shorter PA acceleration time , lower lateral MAPSE, higher E/A & E/e’, and shorter LVFT , lower lateral e’ (p = 0.009), s’ (p = 0.006), right ventricular e’ and LA emptying fraction (p = 0.012 for both), compared with HFpEF patients.Patients with HF and reduced EF (HFrEF) had lower hemoglobin , p = 0.033] independently predicted poor 6-MWT performance in the group as a whole. In HFrEF, age  and LA diameter , but in HFpEF, lateral s’ , and hemoglobin level  independently predicted poor 6-MWT performance.In multivariate analysis, only LA diameter [2.676 (1.242–5.766), In HF patients determinants of exercise capacity differ according to severity of overall LV systolic function, with left atrial enlargement in HFrEF and longitudinal systolic shortening in HFpEF as the the main determinants. Despite advances in the diagnosis and treatment of heart failure (HF), it still presents a major public health problem , with inWe studied 111 patients , with clinical diagnosis of HF, and New York Heart Association (NYHA) functional class I-III, secondary to ischemic or non-ischemic etiology. Patients were referred to the Clinic of Cardiology, University Clinical Centre of Kosova, between May 2013 and June 2016. At the time of the study all patients were on optimum HF medications, optimized at least 2 weeks prior to enrollment, based on patient’s symptoms and renal function: 82% were receiving ACE inhibitors or ARB, 78% beta-blockers, 12% calcium-blockers, 10% digoxin, 52% spironolactone, 62% diuretics. Patients with HFrEF had ischemic aetiology in 45%, hypertensive in 38%, and unknown aetiology in 17%. Patients with HFpEF had ischemic aetiology in 41% and hypertensive in 59%. All patients were in sinus rhythm. Patients with clinical evidence for cardiac decompensation, limited physical activity due to factors other than cardiac symptoms (e.g. arthritis), more than moderate mitral regurgitation, more than mild renal failure, chronic obstructive pulmonary disease or those with recent acute coronary syndrome, stroke or anemia were excluded. Patients gave a written informed consent to participate in the study, which was approved by the local Ethics Committee.Detailed history and clinical assessment were obtained in all patients, in whom routine biochemical tests were also performed including hemoglobin, lipid profile, blood glucose level, and kidney function tests. Estimated body mass index (BMI) was calculated from weight and height measurements. Waist, hip measurements were also made and waist/hip ratio was calculated.A single operator performed all echocardiographic examinations using a Philips Intelligent E-33 system with a multi-frequency transducer, and harmonic imaging as appropriate. Images were obtained with the patient in the left lateral decubitus position and during quiet expiration. Measurements of interventricular septal thickness, posterior wall thickness, and LV dimensions were made at end-diastole and end-systole, as recommended by the American Society of Echocardiography . LV massLV volumes and EF were calculated from the apical 2 and 4 chamber views using the modified Simpson’s method. Ventricular long axis motion was studied by placing the M-mode cursor at the lateral and septal angles of the mitral ring and the lateral angle of the tricuspid ring. Total amplitude of long axis motion was measured as previously described  from peaLeft atrial diameter was measured from aortic root recordings with the M-mode cursor positioned at the level of the aortic valve leaflets. LA volumes were measured using area-length method from the apical four chamber views, according to the guidelines of the American Society of Echocardiography and European Association of Echocardiography . Left atDiastolic LV and RV function was assessed from filling velocities using spectral pulsed wave Doppler with the sample volume positioned at the tips of the mitral and tricuspid valve leaflets, respectively, during a brief apnea. Peak LV and RV early (E wave) and late (A wave) diastolic velocities were measured and E/A ratios were calculated. E wave deceleration time (DT) was also measured from peak E wave to the end of its deceleration in all study patients. The E/e’ ratio was calculated from the transmitral E wave and the mean lateral and septal segments e’ wave velocities. The isovolumic relaxation time was also measured from aortic valve closure to mitral valve opening, on the pulsed wave Doppler recording. LV filling pattern was considered ‘restrictive’ when E/A ratio was >2.0, E wave deceleration time < 140 ms and the left atrium dilated of more than 40 mm in transverse diameter . Total LMitral regurgitation severity was assessed by colour and continuous wave Doppler and was graded as mild, moderate, or severe according to the relative jet area to that of the left atrium as well as the flow velocity profile, in line with the recommendations of the American and European Society of Echocardiography , 26. LikFasting venous blood was collected from study participants after they had rested in a supine position for 20 min. Samples were placed in disposable EDTA containers (1 g/L of plasma), and N-terminal proBNP was measured by a Cobas Elecsys E 411 analyzer (measuring range 5–35000 pg/mL) using a chemiluminescent immunoassay kit .Within 24 h of the echocardiographic examination a 6-MWT was performed on a level hallway surface, administered by a specialized nurse who was blinded to the results of the echocardiogram. According to the method of Gyatt et al.  patients∗ height (cm) − 5.32 ∗ age (years) −1.8 ∗ weight (kg) + 51.31 ∗ sex .Using the norm-reference equation developed by Troosters  for the t test and discrete data with Chi-square test. Correlations were tested with Pearson coefficients. Determinants of 6 MWT distance were identified with univariate analysis and multivariate logistic regression was performed using the step-wise method. A significant difference was defined as p < 0.05 (2-tailed). Patients were divided according to their ability to walk >300 m into good and limited exercise performance groups , only enlarged LA diameter , and diabetes , independently predicted the limited 6-MWT distance.High baseline heart rate (p = 0.012), low hemoglobin level (p = 0.031), diabetes (p = 0.037), and NYHA class > 1 (p = 0.049), increased LVMI (p = 0.025), low lateral s’ (p = 0.018) and a’ (p = 0.039) predicted limited 6-MWT distance. In multivariate analysis, lateral s’ , independently predicted the limited 6-MWT distance.In univariate analysis, body surface area - BSA p = 0.01, low hemp = 0.02) and enlarged LA (p = 0.009) predicted limited exercise distance, which also remained as independent determinants in multivariate analysis: age  and LA diameter [3.685 (1.348–10.071), p = 0.011, Fig. In univariate analysis, age (The results of this study show that in general HF patients with limited exercise capacity are older and have worse left ventricular function and raised filling pressures than those with satisfactory exercise performance. However, determinants of exercise capacity differed significantly according to ejection fraction. While patients with reduced ejection fraction have the enlarged left atrium and advanced age as the independent determinants of exercise capacity, it was the low hemoglobin and reduced lateral s’ the respective determinants in those with preserved ejection fraction.Patients with heart failure due to reduced ejection fraction are known to have worse segmental and overall ventricular function, with additional signs of myocardial stiffness and raised filling pressures in many of them . These pThe main limitation of our study is that we did not investigate the response of echocardiographic measurements to exercise, at the time of symptoms development. However the objective of this study was to determine determinants of ordinary walking exercise limitation rather than heavy exercise in HF patients. The other limitation was the lack of invasive measurements of left atrial pressures, but the study was based on Doppler measurements which have been shown to be reproducible and correlate closely with invasive pressure measurements . The smaPatients with HF have significantly limited exercise tolerance. Although ejection fraction is considered as the most useful index of LV function and the corner stone for recruiting patients for various treatment modalities, the other echo parameters should be considered as part of the conventional protocol of the follow-up of such patients, depending on overall LV systolic function: enlarged left atrium in HFrEF and impaired longitudinal systolic shortening and reduced hemoglobin in those with HFpEF. While management of patients with HFrEF could be standardized, and follow one protocol, that of patients with HFpEF is likely to be individualized.In HF patients determinants of exercise capacity differ according to severity of overall LV systolic function, with left atrial enlargement in HFrEF and longitudinal systolic shortening and low hemoglobin in HFpEF as the the main determinants.


E/e’ decreased with uCIII by 0.168 . Based on age-specific echocardiographic criteria, 182 participants (23.3%) had subclinical diastolic LV dysfunction. Partial least squares discriminant analysis contrasting normal vs. diastolic LV dysfunction confirmed the aforementioned associations with the uCI and uCIII fragments. PICP and TIMP-1 increased in relation to uCI (p<0.0001), whereas these serum markers decreased with uCIII (p≤0.0006). Diastolic LV dysfunction was associated with higher levels of TIMP-1 . In a general population, the non-invasively assessed diastolic LV function correlated inversely with uCI and serum markers of collagen I deposition, but positively with uCIII. These observations generalise previous studies in patients to randomly recruited people, in whom diastolic LV function ranged from normal to subclinical impairment, but did not encompass overt diastolic heart failure.Current knowledge on the pathogenesis of diastolic heart failure predominantly rests on case-control studies involving symptomatic patients with preserved ejection fraction and relying on invasive diagnostic procedures including endomyocardial biopsy. Our objective was to gain insight in serum and urinary biomarkers reflecting collagen turnover and associated with asymptomatic diastolic LV dysfunction. We randomly recruited 782 Flemish . We assessed diastolic LV function from the early and late diastolic peak velocities of the transmitral blood flow and of the mitral annulus. By sequencing urinary peptides, we identified 70 urinary collagen fragments. In serum, we measured carboxyterminal propeptide of procollagen type 1 (PICP) as marker of collagen I synthesis and tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1), an inhibitor of collagen-degrading enzymes. In multivariable-adjusted analyses with Bonferroni correction, we expressed effect sizes per 1-SD in urinary collagen I (uCI) or collagen III (uCIII) fragments. In relation to uCI fragments, e’ decreased by 0.183 cm/s (95% confidence interval, 0.017 to 0.350;  Diastolic heart failure, also known as heart failure with preserved ejection fraction (HFpEF) represents half of all heart failure cases. It has a prognosis as dire as heart failure with reduced ejection fraction with a rate of cardiovascular mortality of over 30% within one year of the first hospital admission. In patients with diastolic heart failure, the left ventricular (LV) wall undergoes fibrosis characterised by increased interstitial deposition  and crosCapillary electrophoresis coupled with high-resolution mass spectrometry (CE-MS) enables detection of over 5000 distinct peptide fragments in urine samples . Our preFLEMENGHO complies with the Helsinki declaration  for reseThe acquisition, offline analysis, staging of diastolic LV function and quality control are extensively described in previous publications ,7,18 andOne observer (TKu) did the ultrasound examination , using aOne reader (TKu) analysed the digitally stored images, averaging three heart cycles, using a workstation running EchoPac software, version 4.0.4 . LV internal diameter and interventricular septal and posterior wall thickness were measured at end-diastole from the 2-dimensionally guided M-mode tracing. When optimal orientation of M-mode ultrasound beam could not be obtained, the reader performed linear measurements on correctly oriented 2-dimensional images. End-diastolic LV dimensions were used to calculate LV mass . Left atIntra-observer reproducibility was the 2-SD interval about the mean of the relative differences across pairwise readings. The intra-observer reproducibility for the tissue Doppler peak velocities across the four sampling sites ranged from 4.5% to 5.3% for e’ and from 4.0% to 4.5% for a’ . ReproduParticipants collected 24-h urine samples within one week of the echocardiographic examinations. Aliquots (0.7 mL) were stored at –80°C and thawed immediately before analysis. Previous publications provide a detailed account of the methods applied for preparation of the urine samples and running the CE-MS analysis ,23. For We identified 88 sequenced peptides with mean signal amplitude different from undetectable in over 95% of participants. Proteomics and peptidomics data usually display missing values, which result from either the absence of a particular peptide in certain datasets or from the fact that this particular peptide may be below of the limit of detection of the technological platform. For the current analysis, the values of peptides undetectable in less than 5% of randomly varying study participants were set to the minimum of the distribution of each sequenced peptide. This strategy has been validated by other research groups ,27. FurtOn the day of echocardiography, venous blood samples were drawn after at least 8 hours of fasting. Serum was analysed for carboxyterminal propeptide of procollagen I (PICP), a marker of collagen I synthesis; carboxyterminal telopeptide of collagen I (CITP) and tissue inhibitor of the matrix metalloproteinase type 1 (TIMP-1) as markers of degradation of collagen I; and amino terminal propeptide of procollagen type III (PIIINP), a marker of synthesis and degradation of collagen III . Serum mBlood pressure was the average of five consecutive auscultatory readings obtained according to European guidelines  with a sFor database management and statistical analysis, we used the SAS system, version 9.4 . Means were compared using the large-sample z-test and proportions by Fisher’s exact test. We normalised the distributions of γ-glutamyltransferase, insulin and PIIINP by a logarithmic transformation.p-values for covariables to enter and stay in the models set at 0.15. In continuous analyses, we standardised diastolic LV function for the average in the whole study population (mean or ratio) of the covariables identified by stepwise regression. While accounting for covariables, we regressed the indexes of diastolic LV function on the urinary peptide markers and constructed–log10 probability plots. Based on the number of parent collagen proteins , we adjusted significance levels by the Bonferroni method. In the next step of our analyses, we applied partial least squares discriminant analysis (PLS-DA). PLS-DA is a statistical technique that constructs predictive models for categorical outcomes in relation to correlated high dimensional predictors. PLS-DA allowed us to identify a set of independent latent factors that were linear combinations of the urinary peptides and that maximised the covariance between the urinary peptides and the variables describing diastolic LV function. We studied normal vs. diastolic dysfunction in relation to the latent factors. We retained the smallest number of latent factors for which the predicted residual sums of squares  did not differ significantly (p>0.10) from the model with the minimum PRESS value as assessed by the van der Voet T2 statistic. The importance of each urinary peptide in the construction of the PLS factors and in the association of diastolic function was assessed from the Variable Importance in Projection (VIP) scores of Wold. Using principal component analysis, we summarized the urinary peptides with a VIP score higher than 1.0 and a correlation coefficient of less than –0.04 or higher than 0.04 into a single factor. Finally, we constructed Receiver Operating Characteristic (ROC) plots and evaluated the area under the ROC curve (AUC).We identified covariables to be retained in the analyses by a stepwise regression procedure with Of 782 participants, 401 (51.3%) were women. All were White Europeans. Mean values (SD) in the 782 participants were 50.5 (15.5) years for age, 26.4 (4.2) kg/m2 for body mass index, 129.1 (17.5) mm Hg and 79.6 (9.5) mm Hg for systolic and diastolic blood pressure, and 5.25 (0.97) mmol/L for total cholesterol. Among all participants, 327 (41.8%) had hypertension, of whom 199 (60.9%) were on antihypertensive drug treatment, and 9 (1.2%) had diabetes.vs. 69.0%, p = 0.29). p≤0.003) used medications: any diuretic (5.3% vs. 25.3%), loop diuretics (0.2% vs. 2.2%), spironolactone (1.0% vs. 7.7%), β-blockers (9.0% vs. 36.3%), angiotensin-converting enzyme inhibitors (3.0% vs. 9.3%), angiotensin I type-1 receptor blockers (2.2% vs. 10.4%) and vasodilators including calcium-channel blockers and α-blockers (2.8% vs. 11.0%).The prevalence of diastolic LV dysfunction amounted to 182 (23.3%). Two participants had tricuspid regurgitation and 10 had an E/e’ ratio exceeding 14. Among participants with normal or impaired diastolic LV function only two and seven had an ejection fraction of less than 50%. There was no difference in the ejection fraction between these two groups . The correlation between the two collagen III fragments, p107460 and p112106, was 0.19 (p<0.0001). There was a weak inverse correlation  between p70635 (collagen I fragment) and p107460 (collagen III fragment).In general, urinary peptides derived from collagen I correlated with one another. For instance, the correlation coefficients relating p70635 to the other collagen I fragments ranged from 0.14 to 0.30 (p≥0.082).Among all 782 participants, eGFR averaged 80.7 (16.4) mL/min/1.73 m2. The prevalence of renal function stages 1, 2, 3A, 3B, 4 and 5 amounted to 175 (22.4%), 552 (70.8%), 49 (6.3%), 5 (0.6%), 1 (0.1%) and 0, respectively. With adjustments applied for mean arterial pressure, waist-to-hip ratio, smoking, γ-glutamyltransferase, the total-to-HDL cholesterol ratio, plasma glucose, and use of antihypertensive medications by drug class, none of the associations of eGFR with the urinary collagen fragments reached significance (p) probability plot of the multivariable-adjusted associations of various indexes of diastolic LV function with the urinary peptides. With Bonferroni correction applied, the urinary peptides that remained significantly associated with the Doppler indexes of diastolic LV function included six fragments of collagen I  and two fragments of collagen III (p107460 and p112106). Focusing on collagen I (p = 0.025) and p77952 , whereas the E/e’ ratio increased with p72896 , p77018  and p115491 . The a’ peak velocity declined with p72896  and p73697 . In relation to collagen III fragments, A peak velocity  and the E/e’ ratio  declined with p107460 and the A peak also with p112106 . Sensitivity analyses with additional adjustment for HDL cholesterol and insulin produced confirmatory results  yielding a sensitivity, specificity and positive and negative predictive values of 67.6%, 93.3%, 75.5% and 90.5%, respectively.To evaluate diagnostic accuracy, we combined the urinary peptides with a VIP score higher than 1.5 and a correlation coefficient lower than –0.04 or higher than 0.04 into a single factor , using principal component analysis. The AUC for diagnosing diastolic LV dysfunction was 0.92 , TIMP-1 (696 vs. 653 ng/mL) and PIIINP (562 vs. 447 pg/mL) were significantly higher (p≤0.020) in patients with diastolic LV dysfunction (n = 175) than in people with normal function (n = 565), with no between-group differences in serum PICP.With adjustments applied for metabolic confounders  and concurrent treatment by drug class , serum lp≤0.0016) by 14.9 μg/L, 0.31 μg/L and 4.63% in relation to p73697. TIMP-1 increased by 21.4 ng/mL (p = 0.0013) and by 39.4 ng/mL (p<0.0001) in relation to p77018 and p77763, respectively. The association sizes of PICP, CITP and TIMP-1 with collagen III fragments amounted to –5.39 μg/L, –0.62 μg/L and –30.9 ng/mL for p107460 (p≤0.0006) and to –0.44 μg/L for CITP.vs. diastolic LV dysfunction confirmed the associations with urinary collagen I and III fragments  the correlations between urinary collagen I and III fragments were inverse; (ii) in continuous analyses, e’ peak and e’/a’ decreased and E/e’ increased with urinary collagen I fragments, whereas the A peak and E/e’ decreased with urinary collagen III fragments ; (iii) tragments ; (iv) PIragments ; and (v)ragments .vs. 6.4). Our current findings are novel, because they show in a general population that echocardiographic indexes of diastolic LV function are associated with sequenced urinary collagen I and III fragments Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S1 Fig(DOC)Click here for additional data file.S2 Fig(DOC)Click here for additional data file.


Conventional echocardiographic technique for assessment of volume status and cardiac contractility utilizes left ventricular end-diastolic area (LVEDA) and fractional area of change (FAC), respectively. Our goal was to find a technically reliable yet faster technique to evaluate volume status and contractility by measuring left ventricular end-diastolic diameter (LVEDD) and fractional shortening (FS) in a cohort of mechanically ventilated trauma and burn patients using hemodynamic transesophageal echocardiographic (hTEE) monitoring.Retrospective chart review performed at trauma/burn intensive care unit (TBICU). Data on 88 mechanically ventilated surgical intensive care patients cared for between July 2013 and July 2015 were reviewed. Initial measurements of LVEDA, left ventricular end-systolic area (LVESA) and FAC were collected. Post-processing left ventricular end-systolic (LVESD) and end-diastolic diameters (LVEDD) were measured and fractional shortening (FS) was calculated. Two orthogonal measurements of LV diameter were obtained in transverse (Tr) and posteroanterior (PA) orientation.r = 0.80, p < 0.01 for LVEDD-Tr , and LVEDD-PA diameters . Left ventricular area (LVEDA) also significantly correlated with left ventricular diameter LVEDD-Tr  and LVEDD-PA . Both transverse and PA measurements of fractional shortening were significantly (p < 0.0001) and similarly correlated with systolic function as measured by FAC. Bland–Altman analyses also indicated that the assessment of fractional shortening using left ventricular posteroanterior diameter measurement shows agreement with FAC.There was a significant correlation between transverse and posteroanterior left ventricular diameter measurements in both systole and diastole. In systole, r = 0.92, p < 0.01 for LVESD-Tr  and LVESD-PA . In diastole, Left ventricular diameter measurements are a reliable and technically feasible alternative to left ventricular area measurements in the assessment of cardiac filling and systolic function. Historically, the assessment of volume status and cardiac function with the goal of achieving appropriate resuscitation targets has been an area of ongoing interest to intensivists worldwide. The pulmonary artery catheter has long been used in the intensive care unit (ICU) to evaluate volume status, cardiac function, and to guide resuscitation. Recently, multiple studies have questioned the benefits of pulmonary artery catheter use and increased the awareness of associated complications, resulting in a decline in its use , 2.Recently, the debate between static and dynamic indices of volume responsiveness was resolved in favor of the latter with multiple studies demonstrating that central venous pressure lacks predictability as a measure of volume responsiveness  comparedOver the last few years, the use of bedside ultrasonography and echocardiography has expanded both in trauma and ICU settings; surgeons, intensivists, and emergency care physicians have developed training protocols to facilitate the ability to detect free fluid in the abdomen with high sensitivity and specificity . The BEAMonoplane hemodynamic transesophageal echocardiography  is a relatively new diagnostic tool, allowing the intensivist to directly assess both the contractility and filling status of both the right and left ventricles at the bedside in real-time. Unlike conventional transesophageal echocardiography (TEE) probes, the hTEE probe is smaller (5.5 mm in diameter), disposable, and can remain in place for up to 72 h, permitting continuous visual quantitative estimation of cardiac contractility and cardiac filling. The probe can be placed safely  in intubEchocardiographically estimated fractional area of change (FAC) calculated as the percentage change between left ventricular end-systolic and end-diastolic areas  has been used as a surrogate marker for left ventricular ejection fraction . Similarly, LV preload can also be assessed using LVEDA with consistent accuracy . DespiteIn this study, the authors used hTEE to obtain LV diameter measurements to estimate left ventricular systolic function , and filling status (using left ventricular end-diastolic diameter) as a technically feasible, less time-consuming alternative to area-based measurements.This is a retrospective chart review performed at the trauma and burn intensive care unit (TBICU) at the University of Alabama at Birmingham. After Institutional Review Board (IRB) approval, medical records and hTEE video clips of patients admitted to our TBICU between July 1, 2013 and July 1, 2015 were reviewed. Inclusion criteria were all mechanically ventilated trauma and burn patients admitted to our TBICU during the study period who underwent hTEE exam to evaluate volume status and hemodynamic stability, or with persistent hypotension (systolic blood pressure < 90 mmHg after a 1 L fluid bolus). Patients with poor-quality echocardiographic windows or with known or recognized wall motion abnormalities as agreed upon by two hTEE-trained examiners (DY and JR) or with incomplete data were excluded from the study.hTEE exams were performed by an intensivist, resident staff, or a trained mid-level provider (physician assistant). We used all echocardiographic views available by hTEE to perform our evaluation of cardiac function and filling. We did not endorse a specific protocol for resuscitation. Personnel who performed the hTEE measurements were all previously trained in performing focused transthoracic echocardiography (TTE). Personnel who performed the hTEE measurements also attended a dedicated 6-hour hTEE training session. A total of ten hTEE exams performed independently were deemed sufficient to achieve competence. Exams were performed using monoplane hTEE . hTEE cineloops were stored and analyzed on the Imacor Zura imaging system . The hTEE system used in this study was not equipped with automated border detection technology.We obtained two perpendicular LV diameter measurements from video clips of the standard transgastric mid-papillary short-axis view. Diameter 1 (transverse–Tr) was obtained by measuring between the papillary muscles from the lateral wall to the septal wall in a transverse orientation  while Diameter 2 (postero-anterior–PA) was obtained by measuring from the inferior wall to the anterior wall (corresponding to an inner diameter drawn between the mid-inferior and mid-anterior segments in the standard 17-segment echocardiographic model) , medial Maximum end-diastolic diameters were determined by electrocardiography and echocardiography. All measurements were agreed upon by two hTEE-trained examiners (DY and JR). Evaluation of cardiac filling and contractility were obtained by using LVEDA and LVESA Figs. , 4 measuLeft ventricular endocardial fractional shortening (FS), a surrogate marker for LV systolic function, was calculated using the formula, FS = (LVEDD − LVESD)/LVEDD, corresponding to the FAC formula based on LVEDA and LVESA measurements.r = 0.277 (r2 = 0.073) or larger at a 5% level. The relationship between area- and diameter-based measurements and FAC were also examined using the Bland–Altman technique, which compares the difference in two measures to their mean. Specifically, the bias in measurement is reflected in the mean difference in measures, which was tested with a paired t test. Given the strong correlations among these measures, we had 80% statistical power to detect a difference of 0.16 standard deviation units at a 5% significance level. The covariance (correlation) between the difference in the mean is used to assess agreement, in that a significant correlation indicates poor agreement. The limits of agreement are defined as bias ± 1.96 (standard deviation of bias). All statistical analyses were performed using SAS 9.3 .Pearson correlations were used to assess the relationships between area- and diameter-based measurements and FAC and FS ratios. With a sample size of approximately 100, we had 80% statistical power to detect correlation coefficients of A total of 107 echocardiographic studies were performed during the study period. Eight patients had incomplete data and were excluded from the study, resulting in a total of 88 patients with 99 measurements.t test indicated that this difference in means was significant (p < 0.0001). There was a significant correlation between LVEDD-Tr and LVEDD-PA diameters . The intraclass correlation coefficient (ICC) for LVEDD measures was 0.83.Table t test indicated that this difference in means was significant (t (98) = 4.10, p < 0.0001). Pearson correlation indicated that the transverse and PA LVESD measures were significantly correlated . The ICC for these LVESD measures was 0.93.The mean LVESD-Tr was 23.47 mm (SD ± 6.77); the mean LVESD-PA was 24.84 mm (SD ± 8.23); however, a paired r = 0.84, p < 0.0001) and LVEDD-PA . Similarly, LVESA significantly correlated with LVESD-Tr  and LVESD-PA , respectively, suggesting that diameter-based and area-based measurements provide a similar assessment of LV preload.We also compared area-based (LVEDA) with diameter-based measurements (both transverse and PA LVEDD) as surrogates for LV preload. The LVEDA significantly correlated with LVEDD-Tr (p < 0.0001), indicating that each of these measurements has the potential to serve as proxy measure for contractility. The Bland–Altman technique tests the relationship between the mean of the two measures  and their difference. A statistically significant relationship between the mean and the difference indicates that the two measures are not in close agreement. The results in Table We compared area-based FAC with diameter-based FS in both PA and transverse planes to evaluate contractility. Table This study demonstrates a correlation between area-based and diameter-based measurements of left ventricular contractility and preload. Despite this correlation, hTEE-based posterior–anterior and transverse measurements used in diameter-based calculations significantly differed in a cohort of mechanically ventilated trauma and burn patients. This difference may be explained by ventricular shape and geometry.Our study is in line with previous studies that have used end-diastolic diameter as a surrogate marker for preload and in the assessment of contractility. Inoue et al.  in a cohIn our study, both transverse and PA FS measurements correlated with area-based FAC measurements. The higher correlation of transverse diameter compared with PA diameter with FAC measurements can likely be explained by the relatively easier and hence more consistent measurements of LV end-diastolic transverse diameter compared with LV end-diastolic PA diameter. It is important to note that both PA and transverse diameters are linear measurements and may vary from area measurement based on LV geometry in health and in disease. Equally important, LV geometry should only be measured in the context of accurate and adequate image acquisition, since imaging errors such as foreshortening may be misinterpreted as a change in LV geometry.Additionally, diameter-based measurements, due to their linear nature, may not be applicable for patients with regional wall motion abnormalities such as the elderly or those with known ischemic heart disease. It is also important to note that the use of either area or diameter measurement does not preclude other methods of assessment and that the purpose of this study is to highlight a more feasible way of assessing cardiac filling and contractility for clinicians who are not formally trained in echocardiography. Whether a difference in clinical outcomes can be detected by either measurement remains to be determined in future studies.As of now, there is no consensus on a formal training protocol for the use of hTEE as compared with conventional TEE. As a result, trainees are exposed most commonly to training protocols performed by the company representatives or by a trained echocardiographer at the bedside. Although hTEE training protocols provide significant utility, they may vary between institutions and currently lack accreditation. Likewise, hTEE-guided resuscitation protocols also vary between institutions and have not yet been validated in multicenter trials. A consensus on the appropriate level of mastery for basic hTEE use and goal-directed, hTEE-guided therapies is an important topic for future exploration in adequately powered studies.Our study suffers from several limitations. First, diameters are linear rather than volume measurements. As such, they are more prone to missing LV pathology not incorporated in the area of measurement. It should be noted, however, that our study demonstrated a significant agreement between LV diameter and area. Second, an LV with greater mass may require higher filling pressures to manifest a change in diameter compared with an LV with smaller mass. Finally, our study suffers from the limitations of retrospective studies such as missing data, lack of a gold standard against which our measurements were made, reliance on qualitative rather than quantitative agreements between hTEE-trained individuals, and the absence of concomitant conventional transthoracic echocardiography to rule out cardiac abnormalities undetected by hTEE. However, we would like to emphasize that the goal of this paper was to test the correlation between two TEE-based methods of assessing cardiac filling and contractility. We did not attempt to explore the adequacy of either measurement in terms of outcomes or in terms of comparison to a gold standard.Further prospective, adequately powered studies are required to compare diameter-based measurements with previously validated volumetric measurements of left ventricular preload and contractility using a more standardized level of operator training. Similarly, the utility of diameter-based echocardiographic assessment for the evaluation of volume responsiveness and to guide resuscitation must be validated before its role in the resuscitation of critically ill or injured patients can be clearly established.In conclusion, left ventricular diameter-based measurements are reliable, less technically challenging alternatives to area-based measurements in the assessment of cardiac filling and LV contractility. More studies are needed to compare the use of diameter-based measurements to left ventricular volumes as a gold standard for assessing left ventricular systolic function and preload. More studies are needed to examine the feasibility of performing LVEDD by less-experienced caregivers.


We report a case of infra-nodal complete heart block and cardiogenic shock in a previously healthy 64-year-old man after administration of 180 mg of intravenous Propofol. Although bradycardia, hypotension, and heart block are commonly seen with propofol administration, such findings are transient and respond quickly to administration of vagolytic or sympathomimetic agents suggesting an AV nodal mechanism of heart block. Sustained left ventricular systolic dysfunction and cardiogenic shock by an alternative, non-autonomic mechanism has also been described in the setting of Propofol administration. Our case is the first to note sustained complete infra-nodal heart block in this setting. Early recognition of such a complication, restoration of atrio-ventricular (A-V) synchrony with dual chamber pacing, and aggressive circulatory support is essential in bridging such patients to recovery. Propofol, a commonly utilized anesthetic agent used for conscious sedation and general anesthesia, is well known to induce bradycardia, hypotension, and heart block during its administration. Such effects are most often transient and respond quickly to the administration of vagolytic or sympathomimetic agents. Sustained left ventricular systolic dysfunction and hypotension by an alternative, non-autonomic mechanism has also been described. We report a unique case of sustained complete infra-nodal heart block and cardiogenic shock in the setting of anesthesia induction with Propofol.A 64-year-old man with type II non insulin dependent diabetes mellitus presented to an outpatient surgery center for elective cataract extraction. His pre-operative ECG demonstrated sinus rhythm with a right bundle branch block (PR 190 msec and QRS duration 150 msec) . IntravePropofol, 2, 6-diisopropylphenol, is an intravenous anesthetic that has become increasingly popular over recent years for both induction and maintenance of general anesthesia in children and adults. It has both a rapid onset of action  and a fast recovery from clinical effect (< 30 min) . In addiThe mechanism of these hemodynamic changes appears to be a result of decreased sympathetic tone. Ebert et al demonstrated that progressive infusions of Propofol to achieve moderate and deep sedation in healthy volunteers ages 21 - 37 resulted in significantly reduced sympathetic nerve activity, norepinephrine levels, and forearm vascular resistance . This stAt higher concentrations, Propofol has been shown to directly depress myocardial contractility. Sprung et al demonstrated that Propofol caused a concentration-dependent decrease in maximal myocardial tension. However such changes in myocardial contractility only became significant P < 0.05) at supra-physiologic concentrations (> or = 56 µM). Interestingly, isoproterenol infusion restored contractility to the level prior to Propofol exposure, again suggesting a sympatholytic mechanism [ at supraIn addition to bradycardia and hypotension, Propofol has been commonly reported to induce transient complete heart block at clinical concentrations -9. The sIn the early 1990’s a series of Propofol related deaths came to light in children who were administered high doses (7 - 10 mg/kg/hour) for prolonged periods of time (> 50 hours) . The ProThe majority of patients in published literature with confirmed PRIS died from heart failure. However, despite a high mortality rate, PRIS has been demonstrated to be completely reversible with the cessation of Propofol and supportive measures. A report by Culp KE et al, described a case report of a 13 year old male diagnosed with PRIS after being placed on a high dose Propofol infusion (140 µg/kg/min) for cerebral edema associated with an emergent intracranial AVM repair . At 72 hMore rarely, acute heart failure has been seen with a shorter course of low dose Propfol or an isolated bolus. Gonzalez et al described a case of a 32-year-old woman who received Propofol (1 mg/kg) and Fentanyl (100 µg) intravenously for induction of anesthesia prior to a Bartholin cyst removal . Within Our current report describes a unique case of sudden onset, long lasting complete heart block unresponsive to atropine and sympathomimetic agents accompanying cardiogenic shock with the administration of a low dose bolus of Propofol. The presence of underlying sinus tachycardia and the lack of response to vagolytic and sympathomimetic agents suggests an alternative mechanism to the atropine responsive transient heart block seen in the aforementioned case series and animal studies. Native AV conduction resumed during DDD pacing at 100 bpm  which is inconsistent with tachycardia dependent A-V block. The development of transient LBBB, intermittent Mobitz type 2 AVB, and ultimate transition to the patient’s baseline RBBB also occurred at a constant atrial rate -5 atria.In conclusion, Propfol appears to have a predictable effect on the cardiovascular system resulting in a usually well tolerated and transient bradycardia, mild hypotension, and heart block at low doses. These autonomically mediated changes promptly resolve with vagolytic or sympathomimetic agents. Prolonged administration of high doses (> 100 µg/kg/hr) may impair mitochondrial function and can lead to a syndrome of a metabolic acidosis, rhabdomyolysis and severe heart failure known as the Propofol infusion syndrome. Rarely, a rapid cardiovascular decompensation with severe left ventricular systolic dysfunction, pulmonary edema, and infra-hisian complete heart block can occur with even an initial low dose bolus. Whether this represents a more rapid uncoupling of electron transport in cardiac myocyte and his-purkinje mitochondria as seen in PRIS or represents an alternative undefined mechanism remains unclear. Regardless, if Propofol is to be utilized as an anesthetic agent, a pre-operative ECG should be obtained to ascertain the presence of underlying his-purkinje disease, and if present placement of peri-operative trans-cutaneous pads and monitoring in a facility capable of rapid placement of hemodynamic support devices including an A-V sequential pacing system seems warranted. Fortunately, LV systolic and electrophysiologic recovery appears to be universal as long as such hemodynamic support is achieved.


Myocardial T1 mapping, particularly ECV values, reliably and non-invasively detected early cardiotoxicity, allowing serial monitoring of chemotherapy-induced cardiotoxicity.A reliable, non-invasive diagnostic method is needed for early detection and serial monitoring of cardiotoxicity, a well-known side effect of chemotherapy. This study aimed to assess the feasibility of T1-mapping cardiac magnetic resonance imaging (CMR) for evaluating subclinical myocardial changes in a doxorubicin-induced cardiotoxicity rabbit model. Adult male New Zealand White rabbits were injected twice-weekly with doxorubicin and subjected to CMR on a clinical 3T MR system before and every 2–4 weeks post-drug administration. Native T1 and extracellular volume (ECV) values were measured at six mid-left ventricle (LV) and specific LV lesions. Histological assessments evaluated myocardial injury and fibrosis. Three pre-model and 11 post-model animals were included. Myocardial injury was observed from 3 weeks. Mean LV myocardium ECV values increased significantly from week 3 before LV ejection fraction decreases (week 6), and ECVs of the RV upper/lower insertion sites and papillary muscle exceeded those of the LV. The mean native T1 value in the mid-LV increased significantly increased from week 6, and LV myocardium ECV correlated strongly with the degree of fibrosis (r = 0.979,  Notoriously, the anthracycline class of cytotoxic agents, which are highly effective against many cancers, can lead to irreversible myocardial damage3. In addition, considerable myocardial damage can occur below the known threshold level5. Accordingly, the early detection of myocardial dysfunction and prevention of overt heart failure are important3. Several guidelines and recommendations for cardiotoxicity monitoring have been published7, including the American Heart Association (AHA)/American College of Cardiology (ACC) guidelines for anthracycline-induced cardiotoxicity, which recommend the use of radionuclide imaging, including multi-gated acquisition (MUGA) scans, and echocardiography for ejection fraction monitoring9. However, no other imaging modalities have been specified for cardiac function monitoring during anthracycline therapy. Accordingly, a reliable, non-invasive method is needed.Cardiotoxicity is a well-recognized adverse effect of chemotherapy11. Accordingly, the purpose of this study was to assess the diagnostic value of T1-mapping cardiac magnetic resonance imaging (CMR) for evaluating early myocardial changes, to evaluate histopathology, and correlate the quantitative values obtained via CMR T1 mapping and histology of a rabbit model of cardiotoxicity.Myocardial T1 mapping is a promising technique that could improve the detection of diffuse myocardial changes and quantitatively evaluate such changes using parameters such as T1 and extracellular volume (ECV) values. By measuring T1-mapping parameters, this novel technique confers the additional benefit of detecting myocardial changes caused by chemotherapy-induced cardiotoxicity at an earlier time point than would be available with other modalitiesA total of 20 rabbits were included in this study. After 3 weeks of modelling, three rabbits were sacrificed for histological evaluation after CMR scanning. The remaining rabbits remained subject to modelling and CMR scanning, and two and four additional rabbits were sacrificed for histological evaluation after 6 and 12 weeks, respectively. Modelling continued in the remaining rabbits, which were subjected to CMR scans until the end of the 16-week modelling period, followed by sacrifice for histological evaluation. We note that four rabbits died of severe anaemia (n = 1) or infection (n = 3) during the modelling period. Figure p < 0.001; Table Table p = 0.002; Table p < 0.001; Table The ECV and native T1 values changed as the modelling period increased. The mean ECV values of the mid-LV increased relative to those of the baseline group and significantly increased beginning at week 3  (Table p < 0.001), the native T1 value did not correlate with the histologic CVF . The RV insertion site had higher CVF values than the LV myocardium (p < 0.001), and the ECV of the RV insertion site correlated well with the CVF . Additionally, a correlation was observed between the myocardial injury score and degree of fibrosis according to the modelling time .Myocardial injury was noted at an early modelling time point  Table . Althougp = 0.001) and native T1 (p < 0.001), whereas the AUCs of the native T1 and EF did not differ significantly (p = 0.051). In the second analysis, which set the disease onset at 12 weeks of modelling after observing a significant increase in CVF at this time point, the AUCs of the ECV, native T1, and LVEF were 0.981 , 0.882 , and 0.857 (0.801–0.906), respectively  and native T1 (p < 0.001); again, the AUCs of the native T1 and EF did not differ significantly (p = 0.821).In the first analysis, post-modelling subjects  were considered disease-positive and used to confirm the ability to detect early anthracycline-induced cardiotoxicity. The areas under the receiver operating characteristic (ROC) curves (AUCs) of the ECV, native T1, and LVEF were 0.974 , 0.893 (0.848–0.930), and 0.916 (0.874–0.951), respectively Fig. . The AUCely Fig. . Here, tThe present study aimed to assess the diagnostic feasibility of T1-mapping CMR for the evaluation of early myocardial changes in a rabbit model of doxorubicin-induced cardiotoxicity. Notably, in this model, the ECV changed significantly beginning at week 3 of modelling, or earlier than the observed changes in the native T1 and LVEF. Accordingly, the ECV had a higher diagnostic value than either the native T1 value or LVEF, and correlated well with the histology findings.12. Given its status as the most commonly used chemotherapeutic agent in cancer patients and the increasing cancer survival rate, the clinical significance of anthracycline-induced cardiotoxicity is expanding12.Doxorubicin, a representative anthracycline, is highly effective against a broad spectrum of malignancies, including lymphoma, sarcoma, and breast cancer, as well as other malignancies that occur in young female patients13. The early detection and treatment of cardiotoxicity is critical for the recovery of cardiac function and reduction in the incidence of associated adverse cardiac events14. Although the need for cardiac monitoring of asymptomatic anthracycline-treated adult patients is generally acknowledged, the existing guidelines offer no clear consensus regarding the timing or duration of such surveillance15.Anthracycline-induced cardiotoxicity is considered a continuum that begins with subclinical myocardial cell injury and, leads to an early asymptomatic decline in LVEF that can develop into symptomatic heart failure17, these methods are associated with a low sensitivity and specificity17; in addition, functional approaches cannot detect subclinical cardiac damage before an apparent and irreversible decrease in cardiac function. Accordingly, additional strategies for the early detection of cardiac damage and monitoring of treatment-induced cardiotoxicity are needed16. In this light, T1 mapping has emerged as a promising quantitative method for the detection of subclinical myocardial changes in various cardiomyopathies19. Furthermore, T1 mapping and ECV measurement have been considered promising tools for the detection and quantification of myocardial damage resulting from chemotherapy-induced cardiotoxicity11.Currently, LVEF identification is most commonly used to screen for cardiotoxicity. Although MUGA scans and echocardiography are widely used to monitor the EF in cancer patientsIn our study, we observed changes in myocardial ECV without detectable changes in LVEF in early models; specifically, the ECV changed significantly at 3 weeks after model induction, whereas significant changes in the LVEF and native T1 were not observed until after 6 weeks. Furthermore, the mean CVFs of the LV were significantly higher than the baseline at 12 weeks. Small differences in native T1 between the normal and abnormal myocardium may be a cause of a later elevation of native T1.21. Previous studies demonstrated that at the tissue level, early anthracycline toxicity is associated with myocardial inflammation, vacuolization, cell swelling, and oedema, and that these changes occur before the onset of myocardial dysfunction23. Our study also demonstrated tissue changes in early anthracycline toxicity models , particularly degenerative myocardial changes, vacuolization, and interstitial oedema. Notably, all of these types of damage, other than fibrosis, would cause an increase in the ECV in early models23. The process of myocardial injury, which was quantitatively evaluated using Billingham scores, was also found to begin at week 3 along with the elevation of the ECV, thus corresponding to the subclinical stage before the LVEF and CVF changed significantly. In other words, myocardial injury preceded the development of fibrosis, and the myocardial ECV might reflect subclinical myocardial injury resulting from anthracycline-induced cardiotoxicity. An ECV based on a T1 mapping sequence could serve as an early marker of myocardial damage.Anthracycline-induced chemotoxicity results from the production of free radicals in both normal and malignant cells; these radicals react with oxygen to produce superoxide anion radicals, which damage the collagen network and cardiomyocytes25. A previous study reported that because of this higher collagen concentration, the RV has a significantly higher myocardial native T1 value than the LV24. Notably, the RV and LV myocardium are combined at the RV insertion site, and accordingly this site had a higher CVF when compared with other parts of the myocardium. Our results also demonstrated that the papillary muscle had the highest ECV among all LV areas. An increase in the amount of abnormal collagen in the papillary muscle in response to chemotherapy-induced cardiotoxicity would greatly impair the contractile capacity and electrical conductance, thus severely compromising cardiac function26. However, even analyses of RV insertion sites and papillary muscles are not critical to a diagnosis of cardiotoxicity. Such a diagnosis might be facilitated by an additional focus on subclinical cardiotoxicity in cases without apparent myocardial fibrosis.In our study, the CVF was higher in the RV insertion sites than in the LV throughout the modelling period. The RV and right atria are known to have significantly higher collagen concentrations than the LV and left atriaThe strengths of our study included the availability of serial data based on the modeling time and a histologic evaluation of the myocardial structure. Our results also demonstrated that T1-mapping CMR could detect early myocardial damage.Additionally, native T1 and the ECV might facilitate the detection of anthracycline responders before LV functional impairment becomes evident, and thus allow therapeutic adjustments and consideration of other treatment regimens. Further studies are required.We also note several limitations of our study. First, the sample size was small, and our inclusion of extreme injury models led to a high incidence of severe cardiotoxicity, which could be considered a potential weakness. Further study is required to evaluate the incidence of cardiotoxicity in patients who exhibit early increases in ECV while receiving clinical doses of chemotherapy, as well as the prognostic value of an early increase of ECV in the clinical patient group.Second, in the statistical analysis, we did not correct the significance level  for multiple comparisons in the post hoc analysis. Some results with p-values near 5% might therefore be false positives. A further confirmatory study involving a larger sample size is needed to confirm the ability of our method to detect anthracycline-induced cardiotoxicity. Third, the small size of the papillary muscle precluded an evaluation of CVF. Finally, the control group was limited and did not include animals subjected to placebo treatment; rather, this group comprised all animals that underwent pre-modelling CMR. However, the use of the same animals in both groups allowed us to compare the baseline and serial data from each modelling time point in the same subject.In conclusion, myocardial T1 mapping is a reliable and non-invasive method for the detection and serial monitoring of cardiotoxicity. This technique could form the basis of an appropriate strategy for the early detection of subclinical cardiotoxicity, even before a conventional critical doxorubicin dose has been reached.27. Twenty rabbits  were included in this study. Three rabbits were sacrificed after initial scanning to provide baseline histological findings. The remaining 17 rabbits received 1.0-mg/kg injections of doxorubicin  twice weekly until the time of sacrifice for histological evaluation. We followed a protocol that involved the lowest mortality rate in our previous study28. Although doxorubicin is known to be particularly venotoxic29, we did not experience difficulties with preserving vein integrity and did not encounter problems such as vein or skin necrosis during the maximum study period of 16 weeks.All experiments were approved by animal care and use committee (IACUC) of Yonsei University Health System  and were performed according to National Institutes of Health guidelinesRabbits underwent MR examinations prior to drug administration to provide baseline data, followed by MR examinations 3 weeks after the first doxorubicin injection and every 2 weeks thereafter until the end of the specific post-modelling time period. Before each MR examination, rabbits were anesthetized with an intra-muscular injection of tiletamine  and xylazine . The auricular veins of each rabbit were prepared for contrast injection. Venous sampling was performed to determine the haematocrit (Hct) values of all rabbits immediately before the MR examinations. After anaesthesia induction, the animals were intubated and mechanically ventilated  using a mixture of oxygen and isoflurane.®; GE Healthcare, Princeton, NJ, USA). LGE MR imaging was obtained 15 min after contrast agent injection using a magnitude- and phase-sensitive inversion recovery-prepared steady-state free-precession sequence, with the inversion time adjusted to nullify the normal myocardium .CMR was performed using a 3-T MR scanner  with a six-channel anterior body matrix coil and posterior part of a 12-channel head matrix coil. Cardiac localization was achieved using a steady-state free-precession sequence under electrocardiogram (ECG) gating. Pre- and post-contrast T1 mapping, cine, and late gadolinium enhancement (LGE) images were acquired. T1 mapping was performed using a prototype modified Look–Locker inversion recovery (MOLLI) sequence during the end-expiratory period in the mid-ventricular short-axis view. Pre-contrast T1 mapping images were acquired before contrast injection. Post-contrast T1 mapping images were acquired 13 min after the intravenous injection of a 0.2-mmol/kg dose of gadolinium contrast agent  with 10 years of experience in cardiovascular image interpretation analysed all MR images.All MR cine images were transferred to cvi42 image analysis software . The LV function was assessed on short-axis cine MR images according to Simpson’s method. The endocardial and epicardial borders of the LV wall were delineated semi-automatically on end-diastolic and end-systolic images. The LV end-diastolic volume and LV end-systolic volume were automatically measured, and the LV ejection fraction  and LV mass/weight (g/kg) were calculated.-and post-contrast T1 images obtained in a short-axis plane at the mid-LV level and divided into six segments based on AHA recommendations. A round, <5 mm2 region of interest (ROI) that avoided the papillary muscle was drawn in the LV cavity  were drawn, and the pre- and post-contrast T1 and ECV values of specific lesions and the LV cavity were measured automatically . The heart was extracted immediately after euthanasia for histologic evaluation. Each heart was fixed in 10% phosphate-buffered paraformaldehyde. After 1 week of fixation, the heart was sectioned serially along the short-axis plane using a rabbit heart slicer , and the entire section of the heart at the mid-ventricle level, wherein papillary muscles were visible, was embedded in paraffin. Four-micrometre-thick slices of this section were obtained, stained with haematoxylin and eosin (H&E) and picrosirius red, and subsequently examined by a pathologist (H.S.P) blinded to the MR results.et al.22. Twelve sets of LV myocardium micrographs of  were analysed per animal. The grading system was as follows: 0 = no damage; 0.5 = not completely normal; 1, 1.5, 2, 2.5, and 3 = damage to 1–5%, 6–15%, 16–25%, 26–35% and >35% of all cells, respectively.H&E staining was used to evaluate myocardial injury. The degree of myocardial injury was evaluated using a light microscope  and scored according to a seven-point scale described by Billingham 18 .Picrosirius red staining was used to evaluate the CVF in the extracellular space. Twelve sets of LV myocardium micrographs  and six fields from the RV upper and lower insertion sites  were acquired per animal and transferred to a computer for further analysis. The mean percentage of fibrosis was also determined. Perivascular fibrosis was not included in this analysisAll continuous data are expressed as means ± standard deviations, and categorical variables are presented as numbers or percentages. The Shapiro–Wilk test was performed to evaluate data distributions.A linear mixed model with restricted maximum-likelihood estimation was used to evaluate the time point of each variable  and the native T1 and ECV values of the whole LV and specific LV lesions  in each experimental group that exhibited a significant change from the baseline group according to the modelling time. The MIXED protocol in SAS  was used to generate this linear mixed model, which included fixed effects for time and specific lesions, interactions between time and specific lesions, and random intercepts for each animal. Time was considered a categorical variable in this model, and equal covariance was assumed between all-time points.30. Post-modelling groups were considered positive for disease. A bootstrap method with 1,000 replications was used for an evaluation that yielded a 95% confidence interval (CI) and p value of the AUC. Spearman’s correlation was used to compare the ECV values of the LV myocardium and RV insertion site (mean ECV of the RV upper and lower insertion sites), as well as the myocardial injury scores and histologic CVFs. The CVFs of the LV myocardium and RV insertion site were compared using a paired t test. Inter-observer agreement regarding the measured native T1 and ECV values of the LV and specific lesions was assessed using ICCs, as well as a two-way random effects model that included the observer and animal as random effects. All statistical analyses were performed using SAS, and a p value < 0.05 was considered statistically significant.To compare the diagnostic performances of ECV, native T1, and LVEF, a ROC curve was constructedSupplementary Information