Background: Cardiac magnetic resonance perfusion imaging during vasodilator stress is an established modality in patients with suspected and known coronary artery disease (CAD).Aim: This study aimed to evaluate the performance of fast-Strain-Encoded-MRI (fast-SENC) for the diagnostic classification and risk stratification of patients with ischemic heart disease.Methods: Perfusion and fast-SENC cardiac magnetic resonance (CMR) images were retrospectively analyzed in 111 patients who underwent stress CMR. The average myocardial perfusion score index, global and segmental longitudinal and circumferential strain , were measured at rest and during stress. The combination of SLS and SCS was referred to as segmental aggregate strain (SAS). Segments exhibiting perfusion defects or SAS impairment during stress were defined as “ischemic.” All-cause mortality, non-fatal infarction, and urgent revascularization were deemed as our combined clinical endpoint.Results: During adenosine stress testing, 44 of 111 (39.6%) patients exhibited inducible perfusion abnormalities. During a mean follow-up of 1.94 ± 0.65 years, 25 (22.5%) patients reached the combined endpoint . Inducible perfusion defects were associated with higher number of segments with inducible SAS reduction ≥6.5% . In addition, patients with inducible perfusion defects or SAS impairment exhibited poorer outcomes .Conclusion: Purely quantitative strain analysis by fast-SENC during vasodilator stress was related to the diagnosis of ischemia by first-pass perfusion and is non-inferior for the risk stratification of patients with ischemic heart disease. This may bear clinical implications, especially in patients with contraindications for contrast agent administration. Cardiovascular diseases are the main cause of morbidity and death in the world, annually claiming more lives than all forms of cancer together . Non-invCurrently, the detection of inducible ischemia during vasodilator stress CMR is mostly based on the visual assessment of perfusion defects –8, whichTherefore, we sought to determine the diagnostic and prognostic value of SENC during vasodilator stress in patients with known or suspected CAD. Global LS (GLS) and CS (GCS) and average global strain (GS), as well as segmental LS (SLS) and CS (SCS) and combined information from SLS or SCS, referred to as segmental aggregate strain (SAS) were compared with perfusion data.Our patient cohort consisted of patients who underwent adenosine stress CMR between September 2017 and July 2019 in the Marien Hospital Hamburg, Hamburg, Germany and had verified follow-up outcomes. Patients were referred for stress CMR due to known or suspected CAD based on current guidelines  equipped with cardiac phased-array receiver coils. Data acquisition was triggered on the R-wave using a 4-lead vector ECG. Cine images were obtained using a breath-hold segmented-k-space balanced fast-field echo sequence (SSFP), employing retrospective ECG gating in long-axis planes  and contiguous short-axis slices covering the whole ventricles, with typically reconstructed 30 phases per cardiac cycle with 50 and 67% acquired heart phases, respectively, for short and long-axis images.®- gadoterate meglumine in a dosage of 0.05 mmol/kg). Three short-axis slices were acquired using a single-shot saturation recovery gradient echo sequence . Fast-SENC acquisitions were performed at baseline, and repeated acquisitions were performed in identical planes during infusion of adenosine after meeting the criteria mentioned above and before the administration of gadolinium for the acquisitions of first-pass perfusion scans. The rest perfusion was performed after 10 min using identical planes and the same dosage of the contrast agent. Afterward, late gadolinium enhancement (LGE) acquisitions were performed in three long-axis and multiple short axes, covering the entire left ventricle.A standard protocol for adenosine stress CMR was used, which is described in detail elsewhere . In shorAll analyses were performed on a commercially available workstation . Results for ventricular volumes, left- and right-ventricular (LV and RV) ejection fraction (%), and myocardial mass were derived from short-axis slices. The presence of myocardial perfusion defects was performed visually in three short-axis images. A perfusion defect was defined as a region in the myocardium exhibiting hypo-enhancement by visual criteria that persists after peak myocardium enhancement for at least 4 RR intervals and corresponds to a coronary territory . In addi1 = Normal Perfusion,2 = Perfusion Deficits With <50% Transmurality and3 = Perfusion Deficits With ≥50% Transmurality During Adenosine StressCorresponding images in a patient with normal perfusion during stress, with a subendocardial and with a transmural perfusion defect during vasodilator stress are provided in our As described previously, fast-SENC is based on the acquisition of high- and low-tuning image sequences with different frequency modulation. Fast-SENC image sequences were analyzed using the MyoStrain software , as described previously .With fast-SENC, bright regions in the two frequency modulation images represented static and fully contracted tissues, respectively. Circumferential and longitudinal strain within a range from 5 to −30% were encoded, with negative values translating into active myocardial contraction. In our study, a single heartbeat, a fast-SENC variant with single-shot spiral readouts was employed. Typical imaging parameters were as follows: field-of-view = 256 × 256 mm, slice thickness = 10 mm, voxel size = 4 × 4 × 10 mm, reconstructed resolution = 1 × 1 × 10 mm, single-shot spiral readout with acquisition time TA = 10 ms, flip angle = 30°, effective echo time (TE) = 0.7 ms, repetition time (TR) = 12 ms, temporal resolution = 36 ms, the typical number of acquired heart phases = 22, spectrally selective fat suppression (SPIR), and total acquisition time per slice < 1 s. Data were acquired in three long-axis  views, and three short-axis views of the LV .Global circumferential strain by fast-SENC is extracted from 3 long-axis views, whereas GLS is extracted from the 3 short-axis images. The endocardial and epicardial borders were drawn at the end-systolic cardiac phase and are traced throughout the cardiac cycle, using an automatic tissue tracking algorithm. Tracking is then verified and manually corrected if necessary. A 16-segment model was used for the GLS and a 21-segment model for the GCS. For the analysis, GLS and GCS were expressed as the average value of all 16 and 21 segments, respectively. In addition, a GS was calculated by averaging LS and CS in all available segments , 20.Global circumferential strain, GLS, and GS were measured both at baseline and during adenosine stress. In addition, segments with relevant segmental LS and/or CS strain (SAS) impairment during stress were defined as ischemic. The number of such “ischemic” segments was calculated for SLS, SCS, and SLS or SCS which was referred to as SAS in each patient.Because myocardial shortening occurred in both longitudinal and circumferential directions during systole, the strain values were consequently negative and were so reported. However, throughout the text, and in keeping with most of the literature on the subject, we refer to the absolute values, i.e., higher strain values meaning more deformation and consequently more “negative” values.Personnel unaware of the CMR results contacted all patients or relatives of patients who underwent stress CMR studies during follow-up. All-cause mortality, the occurrence of non-fatal myocardial infarction, and urgent coronary revascularization by PCI or CABG were selected as the combined primary endpoint of our study. We used all-cause and not only cardiovascular mortality as an endpoint since the prior is free of potential subjectivity, clinically compelling, and therefore most relevant to the patients . In addiM ± SD for continuous variables and as absolute values and percentages for categorical values. A paired t-test was used to compare two groups of normally distributed values. The ANOVA test was used for comparing three or more normally distributed groups with the Scheffé test for post-hoc analysis , we calculated that a minimum of 70 patients would be necessary for our study for a comparison between strain and conventional first-pass perfusion imaging. The MedCalc software version 20.009  was used throughout. All p < 0.05 were considered statistically significant.Data were presented as analysis . The Mananalysis . SurvivaComplete CMR and follow-up data were available in 111 individuals who underwent vasodilator stress perfusion CMR for clinical reasons. Demographic, clinical, and CMR data are provided in During the adenosine stress test, 44 of 111 (39.6%) patients exhibited inducible perfusion abnormalities. Patients with inducible perfusion abnormalities had similar demographic and baseline CMR data to those with negative stress results, except for LV-mass, which was increased in patients with inducible perfusion abnormalities. However, significant differences were observed with the average perfusion score index and for global strain parameters during stress in patients with vs. without perfusion abnormalities .p < 0.05 for all) compared with patients without perfusion defects, whereas all baseline strain values were similar between the 2 groups  reduction during vasodilator stress was selected as best indicative for the presence of segmental myocardial ischemia by perfusion analysis. Thus, the number of ischemic segments with inducible SLS, SCS, or SAS decrease ≥6.5% were all indicative for the presence of inducible perfusion defects, with SAS exhibiting the highest accuracy, followed by SCS and SLS  and significantly higher absolute SAS decrease during adenosine stress (p < 0.001)  . In addin = 15) or CABG (n = 5).During a mean follow-up duration of 1.94 ± 0.65 years, 2 patients died, 3 had a non-fatal myocardial infarction and 20 underwent urgent coronary revascularization by PCI . The perfusion score index was significantly higher in patients with vs. without cardiac endpoints . Similarly, the perfusion score index performed as well as the number of ischemic segments by SAS, predicting the combined endpoint  per patient.A 58-year-old female patient with arterial hypertension, hyperlipidemia and suspected CAD due to atypical angina underwent adenosine stress CMR, which did not show abnormal findings (not shown). Normal strain response was seen by fast-SENC in the short axis views . HoweverIn this analysis of 111 patients with complete perfusion, segmental strain analysis, and follow-up outcomes, we found that:Myocardial perfusion abnormalities during vasodilator administration were associated with clinical outcomes.Evaluation of segmental myocardial strain during baseline and vasodilator stress using fast-SENC was feasible with high reproducibility and within reasonable time-spent.An inducible absolute SAS decrease ≥6.5% during adenosine stress in one or more myocardial segments was associated with inducible perfusion abnormalities during first-pass perfusion imaging.Perfusion and strain abnormalities during vasodilator stress predicted future cardiac outcomes in patients with ischemic heart disease. Overall, perfusion defects showed numerically higher predictive values for future events, which was an expected finding since perfusion is a well-established marker for the prediction of future events , 7. HoweWe and others had previously described the incremental value of SENC both for the diagnostic classification and for the risk stratification of patients with suspected and known CAD , 12. QuaFunctional stress testing has been the non-invasive gold standard for the diagnostic work-up of patients with suspected and known CAD within the last decades, whereas its role in the diagnostic classification and risk stratification is widely accepted in patients with chronic coronary syndromes (CCS) based on current guidelines . ParticuFrom a pathophysiologic point of view, myocardial strain is more sensitive to disturbances of the myocardial metabolism or perfusion, which may be seen in the early stages of many cardiovascular disorders, including CAD and heart failure . In thisa priori ROC analysis, as best indicative for the presence of myocardial ischemia by perfusion analysis, which may overestimate the association between perfusion defects and strain. However, the same cut-off was then independently applied for the prediction of cardiac events, exhibiting significant prognostic value. In addition, a certain overlap was observed for strain values in patients with and without perfusion abnormalities, so that average strain values may not be useful on an individual scale. However, by a selection of a cut-off value for the strain on a segmental level, clinically acceptable accuracy rates could be achieved for the estimation of perfusion abnormalities and clinical endpoints on a patient-by-patient level. Furthermore, we cannot evaluate the accuracy of strain or perfusion abnormalities for CAD detection by comparing these variables to invasive data in conjunction with FFR measures. However, most of our patients had negative stress test results, which helped defer invasive angiography, and this is clinically meaningful.Our cohort was relatively small and heterogeneous with a high percentage of patients with known CAD. This limits the extrapolation of our findings to lower risk cohorts. In addition, the follow-up duration was quite short and only 25 endpoints were recorded. Especially the small number of hard endpoints such as death and non-fatal myocardial infarction and the inclusion of only patients with CMR and complete follow-up data are limitations, that need to be accounted for when interpreting our results. In addition, patients who underwent elective revascularization within the first 90 days after abnormal stress perfusion by CMR were excluded from analysis, and no repeated CMR was performed after the revascularization procedures, which would have helped define the impact of residual ischemia on clinical outcomes. Furthermore, the cut-off value of SS decrease ≥6.5% during vasodilator stress was selected by Fast-SENC during vasodilator stress was non-inferior compared with standard visual perfusion analysis for the diagnosis of ischemia and the risk stratification of patients with ischemic heart disease. This may bear clinical implications since fast-SENC relies on purely quantitative analysis, which is reproducible and can be performed within reasonable time-spent. Thus, fast-SENC can obviate the need for contrast agent injections in this regard, in the interest of time and costs and potentially patient safety. Now prospective larger-scale trials are warranted to test the ability of fast-SENC to predict hard cardiac outcomes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the General Medical Council Hamburg. The patients/participants provided their written informed consent to participate in this study.GK and HS designed the study, performed the analysis, wrote and reviewed the manuscript, and provided important intellectual input. MM performed the acquisitions, reviewed the manuscript, and provided important intellectual input. SK, SE, AS, and SG reviewed the manuscript and provided important intellectual input. All authors contributed to the article and approved the submitted version.HS, SK, and GK received research grants from Myocardial Solutions. SK owns stock options of Myocardial Solutions. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless they tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays using samples from patients with chronic inflammatory diseases collected prior to April 2019, thus defined as negative. Samples from patients with multiple sclerosis , rheumatoid arthritis  with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and systemic lupus erythematosus  with or without RF, were analyzed for SARS-CoV-2 antibodies using 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed IgG multiplex bead-based assay. Six LFA and the in-house validated IgG assay correctly produced negative results for all samples. However, the majority of assays (n=13), gave false positive signal for samples from patients with RA and SLE. This was most notable in samples from RF positive RA patients. No false positive samples were detected in any assay using samples from patients with MS. Poor specificity of commercial serological assays could possibly be, at least partly, due to interfering antibodies in samples from patients with chronic inflammatory diseases. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19), which emerged as a pandemic late 2019 . As a reSerological tests are useful for determining past infection and present immunity. The presence of IgM antibodies indicates a recent infection, whereas presence of IgG antibodies indicates possible long-lasting immunity . ImportaDue to the substantial global demand, SARS-CoV-2 serological testing has been rapidly developed and released to the market. The assays are validated before release and also often independently verified before being approved , 16. HowThe aim of this pilot study was to verify the specificity in a number of the commercially available SARS-CoV-2 serological tests, using a panel of samples from patients with different chronic inflammatory diseases collected before the SARS-CoV-2 outbreak as negative controls, to get an indication of the extent of the issue for further developments, validations and verifications of serology assays.To evaluate specificity of SARS-CoV-2 serological assays in patients with chronic inflammatory diseases, a selection of negative control samples was retrieved from the biobank (n=68). To exclude individuals with risk of previous exposure to SARS-CoV-2 infection, only samples collected before April 2019,  at the Centre for Molecular Medicine, Karolinska Institutet in Stockholm and had been treated with interferon beta (IFNβ). Three MS samples were ADA positive. Of the RA samples, 40 were from the Swedish population-based case control study Epidemiological Investigation of RA (EIRA) and had not been treated with any disease modifying anti-rheumatic drug (DMARD) . Of thesThis retrospective cohort study was approved by the Ethics Review Authority in Stockholm and Gothenburg . Samples and data were collected with informed consent in compliance with the Helsinki Declaration.Analysis IgA, IgG and IgM isotypes of RA samles from the EIRA cohort and SLE samples was performed using the EliA immunoassay on the Phadia 2500 instrument and the cutoff values as stated in the manufacturer’s instructions  . Serum s® ELISA , in accordance with the manufacturer´s instructions.Anti-CCP2 IgG in EIRA was previously determined using the Immunoscan CCPlusA total of 19 commercially available serological assays were evaluated in this study and compared to an in-house assay. Two Enzyme-Linked Immunosorbent Assays (ELISA) and 17 rapid diagnostic lateral flow assays (LFA) were included. These tests were assigned a letter from A – S Table 2 LFAs are designed to enable point of care analyses and can generate immediate results with read-outs as bands in small cartridges. These rapid lateral flow tests are developed for whole blood, serum and plasma. At time of testing, the appropriate volume of serum was applied to the designated well and then the buffer was added. After the recommended incubation period, the presence and intensity of the bands were investigated and graded from negative to four levels of positivity by the same operator.The results were compared to an in-house multiplex bead-based and validated SARS-CoV-2 serological assay developed at SciLifeLab and KTH Royal Institute of Technology as previously described . In brieMycoplasma pneumoniae, Chlamydia pneumoniae). In addition, they also tested specificity using samples from people with conditions that present with atypical immune system activity including EBV infection, pregnancy, ANA and RF-positive subjects. The cut-off for positivity was calculated according to the manufacturer’s instructions.The two included ELISAs were performed according to the manufacturers’ instructions. The first ELISA used to detect IgG against SARS-CoV-2 test A, Table 2 The second ELISA test was the EDI Novel Coronavirus COVID-19 IgG Elisa Kit  to detect IgG test B, Table 2.Rate of false positive signals were determined as the number of positive samples divided by the total number of samples tested for each assay. Statistical analyses and figures were generated using GraphPad Prism (version 8.2.1). The statistical difference between RF positive and RF negative RA subsets were calculated with Fishers exact test. The other groups were too small to make any meaningful statistical evaluations and thus these results are only presented as descriptive analyses.n=67). Notably, all samples from MS patients (n=10) were negative for both IgM and IgG in all 20 assays.Serum samples from 47 RA patients (with two samples from one of the patients), 10 SLE and 10 MS patients were evaluated using 19 SARS-CoV-2 commercial serological assays and compared to an in-house developed multiplex bead-based assay . The oven= 23), with five detecting IgM and one detecting IgG   that were positive for RF, 10 assays had unspecific signal detected for at least one immunoglobulin isotype 3. Five n=7, samples n=8) (When using IFX treated-RA patients as SARS-CoV-2 serology negative controls (patients les n=8) Figure 2False positive signal were detected in one ELISA test for IgG, five IgM LFAs and four IgG LFAs using the 10 SLE samples. All of these were ANA positive and four were anti-dsDNA positive Table S1The levels of IgG, IgM and IgA RF were very high in the RF positive RA samples (n=20), as these had been selected as such. Thus, associations between specific RF isotypes and false positive IgM/IgG anti-SARS-CoV-2 response could not be analyzed. Some indications could be retrieved from the SLE samples (n=10) who had a diversity of RF isotypes, i.e. none of the patients had IgM RF, three had IgA RF and one was positive for IgG RF. No associations were identified between RF isotypes and false positive anti-SARS-CoV-2 IgM or IgG signal in the SLE samples. However, there was a higher level of IgA RF and absence of false positive IgM/IgG anti-SARS-CoV-2 in two samples  , since iIt could be argued that the unspecific signals detected in this study might actually be due to some underlying immunity, if there are mechanisms such as molecular mimicry behind the triggering of autoimmunity , 43 and It should also be noted that samples used to determine the specificity of SARS-CoV-2 serological assays will be highly variable between manufacturers and often not reported in detail in the assay labels or information inserts. However, some manufacturers and vendors are aware of the issue and have included this in the information about the assay. Encode (test O) for example, report that specimen containing higher titers of heterophobic antibodies or RF may affect the results. With the RF positive RA samples used in the present study, 100% reacted in the IgM assay and 5% in the IgG assay, but also both RF positive and RF negative SLE samples gave signal. The Easy Diagnosis (test N) have tested RF, human anti mouse antibody (HAMA), and ANA and claim that they do not interfere with the kit, but in the present study we show that 5-30% of the samples gave a false positive signal. Innovita (test S) reported that samples positive for RF, ANA, HAMA have been analyzed, and they do not report cross reactivity in their test, as could also be independently verified by us.Mikrogen Diagnostik (test A) reported cross-reactivity with RF, but we could only identify cross-reactivity in the IgG assay with an RF negative SLE sample. Abbott (test L) reported that they tested samples positive for RF (3/3), HAMA (3/3), and ANA (3/3), and that these did not affect the performance of the test. However, we see two false positive signals in the IgG test for RF positive RA and SLE samples. Sugentech (test I) reported no cross-reactivity with anti-human IgG, IgM, IgA and IgE, but here we report false positive signal in three samples from an IFX treated patients. Notably, for Sienna (test E), two samples from an IFX treated patient were tested, one sample taken before IFX2, Table S1It should be noted that several serology tests did not give any false positive results with these complicated sera and thus there are methods to avoid unwanted interference. A suggested method to resolve the RF issue, at least in ELISA tests, is to use urea for dissociation of the interfering signals .There are some limitations in this study. Firstly, there are over 400 SARS-CoV-2 serology assays available on the market and with the limited set of stored samples, we could only analyze a fraction of these. For reliable use of serological assays for patients with chronic inflammatory diseases, each assay would need to be individually analyzed with SARS-CoV-2 negative serum from that patient population, before one starts to screen that patient group. Here we can only report the specificity in relation to MS, RA and SLE. Due to the limited availability of sample material, only one test result per sample, per assay, was retrieved and it was not possible to further elucidate the molecular mechanism behind the positive signals. To delineate the molecular explanation of the false positive signal, several more factors would need to be known about the samples, including more extensive information on RF, biological treatments and ADA. Ideally, a well characterized common set of serum samples should be made available to verify several additional serological tests in parallel. Secondly, the LFA’s are primary made for whole blood, to enable individual to do a rapid test with a drop a blood from the fingertip, but here we only had stored serum to use for testing. However, all of the assays also indicate that they work with serum and plasma. Given that the serum from MS patients did not give any signal in any assay, the false positive signals detected in this study are most probably not an issue of having a different matrix, but more likely the unspecific antibody contents of the serum.In conclusion, serological assays could be sensitive to interfering antibodies, as shown in sera from persons with autoimmune diseases. There is a trade-off between requiring extensive screening for unspecific binding in these assays and the harm of delaying the process of making these assays available for emergency use during a pandemic. A cost benefit analysis including all these aspects has to be made on both national and global level. However, if persons with autoimmune disease, health care providers and decision makers are aware about this issue, they could adapt the testing strategy and selecting kits only passing these specificity requirements. To enable such informed decisions, it would be helpful if information about which types and number of samples have been used for validation of specificity is clearly stated in the label of all tests.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the Ethics Review Authority in Stockholm and Gothenburg . Samples and data were collected with informed consent in compliance with the Helsinki Declaration. The ethics committee waived the requirement of written informed consent for participation.All authors contributed to conceptualization, execution, writing, review, and editing of the manuscript. All authors approved the final version of the manuscript.This work was supported by grants from the Dr. Margaretha Nilsson’s Foundation for Medical Research, Vinnova (grant number 2020-02865) and the PCORI [Patient-Centered Outcomes Research Institute] contract MS-1511-33196 for Funded Research Project Standard CR8 with Karolinska Institutet for the project: “Rituximab in Multiple Sclerosis: A Comparative Study on Effectiveness, Safety, and Patient-Reported Outcomes”. Autoantibody analyses in EIRA was supported by grants from the Swedish Research Council, King Gustav V:s 80-year foundation, and the EU/EFPIA IMI project RTCure (grant n° 777357). Autoantibody analyses in Bio-ADA cohort were supported by the grants from Professor Nanna Svartz Foundation, the Gothenburg Medical Society (GLS-889421), and the Regional agreement on medical training, clinical research between the Western Götaland county council and the University of Gothenburg (ALFGBG-926621) and Aina (Ann) Wallströms och Mary-Ann Sjöbloms Foundation for Medical Research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Early structured mobilization has become a key element of Enhanced Recovery After Surgery programs to improve patient outcomes and decrease length of hospital stay. With the intention to assess and improve early mobilization levels, the 8-point ordinal John Hopkins Highest Level of Mobility (JH-HLM) scale was implemented at two gastrointestinal and oncological surgery wards in the Netherlands. After the implementation, however, healthcare professionals perceived a ceiling effect in assessing mobilization after gastrointestinal and oncological surgery. This study aimed to quantify this perceived ceiling effect, and aimed to determine if extending the JH-HLM scale with four additional response categories into the AMsterdam UMC EXtension of the JOhn HOpkins Highest Level of mObility (AMEXO) scale reduced this ceiling effect.All patients who underwent gastrointestinal and oncological surgery and had a mobility score on the first postoperative day before (July–December 2018) or after (July–December 2019) extending the JH-HLM into the AMEXO scale were included. The primary outcome was the before-after difference in the percentage of ceiling effects on the first three postoperative days. Furthermore, the before-after changes and distributions in mobility scores were evaluated. Univariable and multivariable logistic regression analysis were used to assess these differences.p < 0.001). During the first three postoperative days, 118 (87.4%) patients scored the highest possible mobility score before compared to 40 (16.8%) patients after . A change in mobility was observed in 88 (65.2%) patients before as compared to 225 (94.5%) patients after . Of these 225 patients, the four additional response categories were used in 165 (73.3%) patients.Overall, 373 patients were included . On the first postoperative day, 61 (45.2%) patients scored the highest possible mobility score before extending the JH-HLM into the AMEXO as compared to 4 (1.7%) patients after (OR = 0.021, CI = 0.007–0.059, A substantial ceiling effect was present in assessing early mobilization in patients after gastrointestinal and oncological surgery using the JH-HLM. Extending the JH-HLM into the AMEXO scale decreased the ceiling effect significantly, making the tool more appropriate to assess early mobilization and set daily mobilization goals after gastrointestinal and oncological surgery.The online version contains supplementary material available at 10.1186/s12893-021-01445-3. Annually, over 300 million patients undergo surgery worldwide . ProlongIn order to facilitate postoperative recovery, the enhanced recovery after surgery (ERAS) program has been well established worldwide –7. The iWith the intention to create a culture of safe and early mobilization, the John Hopkins University Hospital developed and implemented the Activity Measure for Post-Acute Care (AM-PAC) “6-clicks” Basic Mobility Short Form to assess limitations in functional mobility  and the John Hopkins Highest Level of Mobility scale (JH-HLM) to assess mobilization , set mobilization goals and discuss mobilization success during inter-professional meetings –14. SpecAfter the implementation of the JH-HLM scale at our tertiary university medical center, however, healthcare professionals perceived a ceiling effect in patients after gastrointestinal and oncological surgery as they noticed that at least half of the patients scored the highest possible score on the first day postoperative. Floor and ceiling effects are considered to be present if respectively more than 15% of the patients achieved the lowest or highest possible score, limiting the usability, reliability and responsiveness of the tool [Here, in our effort to provide the multidisciplinary team with a tool that can adequately be used after gastrointestinal and oncological surgery, we extended the JH-HLM scale by adding four additional response categories into the AMsterdam UMC EXtension of the JOhn HOpkins Highest Level of mObility (AMEXO) scale. The AMEXO scale was subsequently implemented in routine clinical practice. If the ceiling effects were reduced by extending the JH-HLM into the AMEXO scale, the multidisciplinary team may be better able to assess mobilization, set mobilization goals and discuss mobilization success in patients after gastrointestinal and oncological surgery. Therefore, the aim of this study was two-folded. First, to quantify the perceived ceiling effect of the JH-HLM scale when the multidisciplinary team used it to assess mobility in patients after gastrointestinal and oncological surgery. Second, to determine if extending the JH-HLM scale with four additional response categories into the AMEXO scale reduced this ceiling effect.This is an uncontrolled before-after study performed at two surgical wards. First, the JH-HLM scale was implemented at both surgical wards between March and June 2018, after which the JH-HLM scale was used during routine clinical care from July 2018 until January 2019. Second, the JH-HLM was extended into the AMEXO scale where after the AMEXO scale was implemented in routine care between February 2019 and June 2019 to assess mobilization and set daily mobilization goals during routine care instead of the JH-HLM scale. Data were extracted from the electronic medical records in January 2019  and January 2020  to quantify and compare both tools when used to assess mobilization in gastrointestinal surgical patients.This study has been conducted according to the principles of the Declaration of Helsinki. The Medical Ethical Review Committee of the Amsterdam UMC, location Academic Medical Center, assessed and approved this study (reference number W19_034 # 19.053). As the dataset was supplied by the medical center and included only de-identified (anonymous) data; the Medical Ethical Review Committee waived the need for individual informed consents. The study was reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for cohort studies .All adult patients who were admitted to one of the two surgical wards between July and December 2018 (before) or July and December 2019 , underwent gastrointestinal and oncological surgery and had a JH-HLM or AMEXO score on the first postoperative day were included. Moreover, every patient was only included once, meaning that all subsequent hospital admissions were excluded from the analysis.95) was 0.6, and evidence was provided that the JH-HLM measured constructs of the ICF domain ‘mobility’ [The JH-HLM scale is a 1-item ordinal scale with eight response categories and is used by healthcare professionals to assess mobilization, set mobilization goals and discuss mobilization success during inter-professional meetings . Each caobility’ , 23. To At first, the JH-HLM was implemented at our hospital and we placed meter markings on the walls to facilitate healthcare professionals in estimating the achieved JH-HLM score and in setting mobilization goals together with hospitalized adult patients. The highest JH-HLM score  represents a functional household ambulation distance and is estimated as 4 metabolic equivalents . In threIn summary, the AMEXO scale is an extended version of the JH-HLM scale, in which four additional categories (category 9–12) have been added on top of the already existing eight ordinal response categories. Each of the four additional categories presents an incremental increase in mobilization using the highest possible JH-HLM score  as a starting point  over a fixed observation period  . Nurses The following patient characteristics were collected: age, sex, surgical area, acute admission and hospital length of stay. Furthermore, the Katz Activities of Daily Living (Katz-ADL) score and the t-tests, Mann–Whitney U tests or Fisher’s Exact tests, depending on normality and type of data. Due to low number of patients having Katz-ADL score 1 to 6, we recoded this variable to a binary variable .All analysis were conducted using IBM-SPSS Statistics version 26  and R . A two-tailed p-value of 0.05 was considered statistically significant. Normality of data was evaluated by visually inspecting continuous and ordinal data using Q-Q plots. Patient characteristics were described descriptively and differences in patient characteristics before-after extending the JH-HLM into the AMEXO scale were assessed using independent Due to the fact that > 15% of the mobility scores on the second and third postoperative days were missing a multiple-imputation model with 10 imputed sets was applied to both variables and pooled using Rubin’s rules , 29. MisOnly the highest mobility score on each postoperative day was used for analysis, as has been in previous studies using the JH-HLM scale to assess mobilization . First, Overall, 933 surgical patients were assessed for eligibility , of whom 560 were excluded (60%). Main reasons for exclusion were no gastrointestinal and oncological surgery  or no mobility score on the first postoperative day . Consequently, a total of 373 patients  were included for analysis  patients had a JH-HLM mobility score on each of the first three postoperative days. In 35 25.9%) patients the JH-HLM mobility score was missing on the second postoperative day, and in 60 (44.4%) patients the JH-HLM mobility score was missing on the third postoperative day. Of the 238 patients whose mobilization was assessed after extending the JH-HLM into the AMEXO scale, 73 (30.7%) patients had a AMEXO mobility score on each of the first three postoperative days. In 99 (41.6%) patients the AMEXO mobility score was missing on the second postoperative day, and in 134 (56.3%) patients the AMEXO mobility score was missing on the third postoperative day. The distribution of mobility scores on all three postoperative days before and after extending the JH-HLM into the AMEXO scale can be found in Additional file 5.9% patip > 0.05), except for the number of elective hospital admissions (p = 0.024).Patient characteristics are presented in Table p < 0.001). Furthermore, 118/135 (87.4%) patients scored the highest possible mobility score on one of the first three postoperative days before, compared to 40/238 (16.8%) patients after extending the JH-HLM into the AMEXO scale . A change in mobility score was observed in 88/135 (65.2%) patients before, compared to 225/238 (94.5%) patients after extending the JH-HLM into the AMEXO scale . Of these 225 patients, 165 (73.3%) patients showed a change in mobility score and scored AMEXO scale response category 9 to 12 on one of the first three postoperative days. The number of elective hospital admissions did not significantly affect the logistic regression models. A complete case analysis using the before imputation dataset can be found in Additional file Sixty-one of the 135 (45.2%) patients scored the highest possible mobility score on the first postoperative day before extending the JH-HLM into the AMEXO scale . When divided into subgroups, 20/29 (68.9%) patients after upper gastrointestinal surgery, 17/46 (36.9%) patients after hepato-pancreato-biliary surgery, and 24/60 (40%) patients after colorectal surgery scored the highest possible mobility score on the first operative day before extending the JH-HLM into the AMEXO scale. In contrast, 4/238 (1.7%) patients scored the highest possible mobility score on the first postoperative day after extending the JH-HLM into the AMEXO scale  , Hierarchical Assessment of Balance and Mobility (HABAM), Short Physical Performance Battery (SPPB), Performance Oriented Mobility Assessment (POMA), Elderly Mobility Scale (EMS) and the AM-PAC “6-clicks” Basic Mobility short form , 33, 34.A strength of this study is the fact that all patients after gastrointestinal and oncological surgery who were admitted to two wards in a university medical center were included. Although the population in non-university hospitals may differ, most hospitals have implemented ERAS program and early mobilization has become universal in surgical practices worldwide , 3. Ther95 of 0.6 for the JH-HLM, the MDC95 might have increased over the 1.0 after extending the JH-HLM into the AMEXO scale. Fifth, the scientific limitations that come with single-item measures—such as the JH-HLM and AMEXO—require healthcare professionals, researchers, and policymakers to carefully consider what they intend to use these scales for [This study also has to be interpreted in light of some limitations. First, our nonrandomized, uncontrolled before and after study design does not allow us to conclude a direct causation between extending the JH-HLM into the AMEXO scale and the reduction in ceiling effect . Second,ales for . While tAlthough local healthcare professionals found the AMEXO scale to be a suitable tool to assess mobilization after gastrointestinal and oncological surgery in current clinical care, further psychometric evaluation is warranted. Regarding the validity of the AMEXO scale we would suggest using another tool measuring mobility performance  to determine whether the response categories of the AMEXO scale are validly assessed after gastrointestinal and oncological surgery . Furthermore, the inter-rater reliability, test–retest reliability and the responsiveness of the AMEXO scale in patients after gastrointestinal and oncological surgery should be assessed using established methods as described by COSMIN  , 38.Furthermore, involving patients in improving and sustaining postoperative mobilization has the potential to impact adherence to the ERAS program which is central to effectiveness , 40. TheLastly, early mobilization entails the incremental increase in activity ranging from passive range-of-motion exercises to active ambulation, depending on the physical capabilities of the patient, from the first day after surgery to reach predetermined targets using a standardized and structured approach , 7, 9. THealthcare professionals who use the JH-HLM scale to assess early mobilization in patients after gastrointestinal and oncological surgery were frequently hampered by a ceiling effect. Extending the JH-HLM into the AMEXO scale decreased this ceiling effect significantly, making the AMEXO scale more appropriate to assess early mobilization and set daily mobilization goals after gastrointestinal and oncological surgery. Furthermore, the use of the AMEXO scale in patients after gastrointestinal and oncological surgery may provide healthcare professionals with an opportunity to involve patients in creating a culture of safe and improved postoperative mobilization.Additional file 1. Content of the JH-HLM scale and AMEXO scale.Additional file 2. Distribution of mobility scores using the JH-HLM and AMEXO scales during the first three postoperative days (not imputed data).Additional file 3. Comparing the JH-HLM scale with the AMEXO scale (not imputed data).


Doxorubicin belongs to the class of anthracycline antibiotics that is widely used in the treatment protocols of a wide range of malignancies. The major deleterious effect of doxorubicin use is the possible occurrence of cardiotoxicity. This study aimed to delineate the possible effects of targeting oxidative stress, NLRP3 inflammasome, and autophagy by fraxetin on doxorubicin-induced cardiac dysfunction in rats. In a model of doxorubicin-induced cardiotoxicity, the effects of different doses of fraxetin were assessed by determination of biochemical, histopathological, immunohistochemical, and electron microscopic changes. Fraxetin, in a dose-dependent manner, was found to have the ability to mitigate the harmful effects of oxidative stress and inflammation on myocardial muscles with significant decrease in NLRP3 inflammasome, augmentation of autophagy, and amelioration of the apoptotic signaling pathways. In addition, fraxetin, in a dose-dependent manner, had the ability to combat the echocardiographic, histopathological, immunohistochemical, and electron microscopic changes induced by doxorubicin in cardiomyocytes. As a result, fraxetin may be put into consideration as a new adjuvant line of therapy on the way to mitigate doxorubicin-induced cardiotoxicity. Doxoruber agent . Over ther agent . In addier agent . Moreoveer agent .Recent reports paid attention to the salient role of the interaction between the Nucleotide-Binding Domain-Like Receptor Protein 3 (NLRP3) inflammasome, autophagy and apoptosis in the pathogenesis of doxorubicin-induced myocardial dysfunction . DoxorubFraxetin is a simple coumarin that is extracted from Cortex Fraxini . The ongDoxorubicin hydrochloride was supplied by Bio-Techne Co., Minneapolis, MN 55413, USA (CAS # 25316-40-9). Fraxetin was purchased from Molnova, Ann Arbor, MI 48104, USA (CAS # 574-84-5). All other reagents and chemicals were provided as a kind gift from Sigma Pharmaceutical industry, Quesna, Egypt, and were of analytical grade. Both doxorubicin and fraxetin were dissolved in normal saline.This study was conducted on thirty-two male adult Wistar rats weighing about 160–240 g purchased from the animal house of the Faculty of Science, Tanta University, Egypt. The animals were given two weeks to acclimatize to the surrounding circumstances before starting the experiments. Rats were kept at a constant temperature of 24 ± 3 °C with relative humidity of 57 ± 10%, and were subjected to 12 h light/dark cycles. The protocol of this study was conducted following the Helsinki declaration of animal ethics and was approved by the Research Ethics Committee of Faculty of Medicine, Tanta University, Egypt . A laboratory technician who was blinded of the identity of the experimental groups randomly divided the animals into four equal groups as follows: (I) The control group was injected intraperitoneally with 0.5 mL normal saline twice weekly for two weeks; (II) the untreated doxorubicin group was injected intraperitoneally with doxorubicin (4 mg/kg) twice weekly for two weeks ; (III) tAt the end of the experimental period, pentobarbital sodium was used by intraperitoneal injection   to facilRats were euthanized and cardiac tissues were extracted. A portion of the extracted tissues was homogenized by a Teflon homogenizer  and the homogenate was centrifuged at 3000 rpm for 15 min. The resulting supernatant was utilized for exploration of the levels of the biochemical parameters in the specimens of cardiac tissues. The other portion of the cardiac tissue was processed for further histopathological and electron microscopic examination and immunohistochemical staining.Cardiac total antioxidant capacity was quantified using ELISA kits purchased from Abcam, Waltham, MA 02453, USA (CAT # ab65329) following the vendor’s instructions. Kits obtained from KyvoBio, Evere, Belgium (CAT #MBS268427-96) were utilized for assay of malondialdehyde (MDA) levels in cardiac tissues. Nrf2 content of the cardiac tissues was quantified using kits supplied by Elabscience, Houston, TX 77079, USA (CAT # E-EL-R1052) following the manufacturer’s instructions.Kits purchased from Abcam, Waltham, USA (CAT # ab119558 and ab214566), were utilized for assessment of tissue TGF-β1 and IL10 levels, respectively. IFN-α levels were quantified using kits supplied by CUSABIO, Houston, TX 77054, USA (CAT #CSB-E08637r). Assay of NLRP3 levels was executed using kits provided by Aviva Systems Biology Co., San Diego, CA 92121, USA (CAT # OKCD04232). Determination of the fore-mentioned parameters was carried out following the vendor’s protocol.Kits provided by RayBiotech, Peachtree Corners, GA 30092, USA (CAT #CBEL-P38-2-RB and PEL-AKT-S473-1) were employed for assay of p-MAP kinase and p-AKT, respectively, according to the vendor’s instructions. Phospho-mTOR levels were quantified using kits purchased from Boster Biological Technology, Pleasanton, CA 94566, USA (CAT #EKC2466) following the provider’s protocol.Beclin-1 levels were determined in cardiac tissues using kits supplied by Elabscience Biotechnology, Houston, TX 77079, USA (CAT # E-EL-R1122-ELS). Cardiac tissue NGF-β levels were quantified using kits provided by Biomatik, Wilmington, Delaware 19809, USA (CAT # EKF58099). Kits purchased from Abcam, Waltham, MA 02453, USA (CAT # ab273417) were employed to assay JNK activity in the cardiac tissues. Determination of the fore-mentioned parameters was executed following the vendor’s protocol.In vivo murine echocardiography was performed to evaluate cardiac functions in awake rats. Briefly, rats were imaged in the M-mode using a linear array ultrasound probe  both at the start and end of the study. These images were utilized to determine dimensions and thickness of the left ventricle and to calculate indices of the cardiac functions. The determined parameters included heart rate, left ventricle end-diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), and myocardial performance index (MPI) .Specimens of the cardiac tissues were fixed in 10% formaldehyde solution, then put in paraffin blocks. After that, these specimens were deparaffinized by xylene, hydrated in alcohol, stained with hematoxylin for 10 min, and then counterstained with 1% eosin solution. These sections were examined by using light microscope  to assess the histopathological changes.The cardiac sections were stained with the primary antibody polyclonal IgG to caspase-3  and the slides were examined under light microscope . The percentage of the positive immunostaining for caspase-3 was calculated using IHC profiler tool in image J software (1.49v)  and was graded as follows: (+) refers to mild immunoexpression, (++) denotes moderate immunoexpression, and (+++) means marked immunoexpression.After fixation of the cardiac specimens in 2.5–4% glutaraldehyde solution (pH 7.4) for 48 h at 4 °C, these specimens were cut into small particles and washed with distilled water. After that, they were fixed in 1% osmium tetraoxide with 15 mg/mL of potassium ferrocyanide for 1–2 h at 4 °C. The tissue specimens thereafter were cut with an ultramicrotome to sections of 0.5–1 μm thickness and stained with uranyl acetate and lead. After that, these sections were examined and photomicrographs captured using a JEOL, JEM 1010 electron microscope .p-value of less than 0.05.The obtained data were analyzed and statistically evaluated using Graph Pad Prism version 7.0. Comparisons between the different studied groups were executed using one way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Data were referred to as mean ± standard deviation (SD) and the significance was considered at Doxorubicin-treated rats exhibited significant increase in serum CK-MB, LDH, troponin Im and NT-pro BNP, relative to the control group. Fraxetin was found to have the ability to elicit significant decrease in these parameters, when compared to rats treated with doxorubicin alone. The improvement in the cardiac function tests was more pronounced in rats that received 80 mg/kg fraxetin, compared to the group that received 40 mg/kg fraxetin .The group that was injected with doxorubicin alone exhibited significant increase in MDA levels and significant decrease in total antioxidant capacity and Nrf2 content in cardiac tissues, compared to the control group. Administration of fraxetin elicited significant decrease in MDA levels with significant increase in the total antioxidant capacity and Nrf2 content of cardiac tissues relative to rats treated with doxorubicin alone. These changes were more evidenced with 80 mg/kg fraxetin, compared to the group that received 40 mg/kg fraxetin .The injected doxorubicin elicited significant increase in cardiac tissue TGF-β1 and NLRP3 inflammasome with significant decrease in IL-10 and IFN-α levels when compared with the control group. Fraxetin, in a dose-dependent manner, had detrimental effects on TGF-β1 and NLRP3 inflammasome levels with significant increase in IL-10 and IFN-α levels resulting in amelioration of the inflammatory processes compared to rats treated with doxorubicin alone .Animals injected with doxorubicin exhibited significant detriment of autophagy evidenced by significant decline in cardiac tissue p-MAPK and beclin-l with significant inhibition of JNK activity relative to the control group. This was accompanied with significant elevation of cardiac tissue p-AKT and p-mTOR in doxorubicin-treated group compared to the control animals. Treatment with different doses of fraxetin was able to significantly ameliorate these changes with significant increase of p-MAPK, JNK activity and beclin-l levels associated with significant mitigation of p-AKT and p-mTOR levels relative to rats treated with doxorubicin alone but the maximal enhancement of autophagy was encountered with the high dose of fraxetin (80 mg/kg) .Apoptosis was significantly enhanced in doxorubicin-treated group, manifested by significant decrease in NGF-β  and signAt the baseline of the study, the echocardiographic indices, including heart rate, LVEDD, LVESD, LVFS, LVEF, and MPI were within the normal range for all rats. At the end of the study, the group that was injected with doxorubicin alone exhibited significant increase in LVESD with significant decline in LVEF, LVFS, and MPI, compared to the control group. These changes were reversed with administration of the different doses of fraxetin but maximal improvement was pronounced in rats that received 80 mg/kg fraxetin. No significant differences were noticed regarding heart rate or LVEDD at the end of the study between the different groups .Massive infiltration of cardiac tissues with different types of inflammatory cells with fragmentation of the myocardial fibers were observed in rats treated with doxorubicin alone c–e. FraxAdministration of doxorubicin-induced marked disruption of the normal cardiac architecture with fragmentation and wide separation of the myofibrils and depletion of the mitochondria C. Also, Anthracycline antibiotics represent a large group of drugs that inhibit topoisomerase II enzyme, and thus have been used effectively in the treatment of a large scale of malignancies worldwide . Among tThe pathways that regulate Nrf2 signaling were thought to play a fundamental role in the pathogenesis of doxorubicin-induced cardiotoxicity . DoxorubIn the current study, administration of fraxetin to doxorubicin-treated rats induced a dose-dependent significant increase in Nrf2 content and total antioxidant capacity with significant decline in MDA levels in cardiac tissue, when compared to rats treated with doxorubicin alone. This was in accordance with the findings of Najmanová et al.  who threRecent studies threw light on the interesting role of NLRP3 inflammasome in the pathophysiology of anthracycline-induced myocardial dysfunction . NLRP3 iCoinciding with the results of the present work, Chen et al.  reportedThe interplay between autophagy and the apoptotic pathways was reported to play a fundamental role in the pathogenesis of doxorubicin-induced myocardial toxicity and serves as an important platform for any treatment strategy for this condition . In the In the present study, fraxetin administration was associated with dose-dependent enhancement of the molecular events related to autophagy including downregulation of mTOR/AKT signaling together with enhancement of MAP kinase activity and JNK activity and increased beclin-1 levels compared to rats treated with doxorubicin alone. This was in accordance with the results of Sánchez-Reus et al. , who estFraxetin may be considered as a new adjuvant line of therapy on the way to abrogate doxorubicin-induced cardiotoxicity. This might be due to its effects on ROS production with subsequent affection of NLRP3 inflammasome activity, which is the center point of the pathogenic events that occur in the myocardium of doxorubicin-treated animals. In addition, modulation of autophagy/apoptosis balance may be another mechanism by which fraxetin might restore cardiac functions . Further


At present, the approaches aimed at increasing myocardial regeneration after infarction are not available. The key question is the identity of cells capable of producing functional cardiac myocytes (CMs), replenishing those lost during ischemia. With identification of resident cardiac stem cells (CSCs), it has been supposed that this cell population may be crucial for myocardial self-renewal and regeneration. In the last few years, the focus has been shifted towards another concept, implying that new CMs are produced by dedifferentiation and proliferation of mature CMs. The observation that CSCs can undergo development inside immature cardiac cells by formation of “cell-in-cell structures” (CICSs) has enabled us to conclude that encapsulated CICSs are implicated in mammalian cardiomyogenesis over the entire lifespan. Earlier we demonstrated that new CMs are produced through formation of CSC-derived transitory amplifying cells (TACs) either in the CM colonies or inside encapsulated CICSs. In this study, we described the phenomenon of CSC penetration into mature CMs, resulting in the formation of vacuole-like CICSs (or non-encapsulated CICSs) containing proliferating CSCs with subsequent differentiation of CSC progeny into TACs and their release. In addition, we compared the phenotypes of TACs derived from encapsulated and non-encapsulated CICSs developing in immature and mature CMs, respectively. However, identification of resident cardiac stem cells (CSCs) of c-kit1+ types ,3 and th1+ types ,5. This 1+ types ,7. This  [+ CSCs . Togethe [+ CSCs  on the c [+ CSCs ,11. It sinto CMs ,13. In pinto CMs ; and (2)into CMs ,16. As finto CMs . The keyEthical Statement. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals  and the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The Institutional Animal Care and Use Committee at Almazov Centre approved the study protocol.Animals. Male, 260–320 g, SPF Wistar rats  were used throughout the experiments. The animals were maintained under a 12/12-h light/dark cycle with free access to food and water.Experimental Design. The animals were randomly allocated to 3 groups: myocardial ischemia–reperfusion , permanent coronary artery occlusion , and sham-operated animals .Myocardial Ischemia–Reperfusion and Permanent Ischemia Model. Anesthesia was induced by placing the animal in a chamber filled with 5% isoflurane, using low-flow gas apparatus . After induction, the animals were tracheotomized and ventilated , using gas containing 2% isoflurane, 35% oxygen, and 63% room air, with a tidal volume of 2 mL/100 g and an approximately 60 breaths per minute rate. Body temperature was maintained at 37.0 ± 0.5 °C by a feedback-controlled heating pad . The left carotid artery and right femoral vein were cannulated for measuring mean arterial pressure (MAP) and drug injection, respectively. Lead II of the electrocardiogram was monitored for registering heart rate (HR) and arrhythmias. After 10 min of stabilization, left thoracotomy was performed. A 6.0 polypropylene thread was placed around the left coronary artery, and the ends passed through a polyethylene tube as an occluder. After surgical preparation, the animals were subjected either to 30 min coronary artery occlusion followed by 180 min reperfusion (MIRI group) or permanent coronary occlusion for 1–5, 7, or 14 days (PCAO group). Exclusion criteria were MAP < 50 mmHg and/or HR < 300 at any time point during the experiment. The animals were then euthanized by the overdose of isoflurane, followed by heart excision and preparation for cell experiments.Isolation of cardiac cells for ex vivo experiments. Dissected myocardial fragments of hearts were enzymatically dissociated into a single cell and small fragment suspension as previously described [g for 10 min. Gentle pipetting and centrifugation were used to preserve the integrity of myocardial CSC-derived colonies and CICSs with TACs. The enzyme-free cell suspension was stained, using antibodies, followed by suspending the cells between the slide and cover slip.escribed . BrieflyThe number of encapsulated TAC-containing CICSs was estimated in the suspension of myocardial cells. Encapsulated CICSs were quantified in each of three groups in triplicate. It seems important to note that the methodology used is our know-how based on quantification of encapsulated CICSs in the suspension of immunocytochemically labeled cells. Since the weight of initial myocardial samples varied from 50 to 120 mg, the number of CICSs is expressed per 100 mg of tissue.This particular technique of cell isolation was used because we aimed to maintain the integrity of specific cellular structures, including CSC-derived colonies and CICSs containing TACs, which are not identifiable with other approaches. Given rare occurrence of CSC-derived colonies and CICSs , we were as yet unable to identify and describe these structures by using FACS analysis, or confocal or electron microscopy of myocardial tissue specimens. Taking this into account, the used technique of ex vivo characterization of freshly isolated mammalian myocardial cells seems to be optimal for identification of previously unknown CICSs with TACs and CSC-derived colonies of different size and maturity.Immunocytochemistry. After rinsing with PBS and fixation for 20 min in 2.5% paraformaldehyde at room temperature, the cells were permeabilized with 0.25% Triton-X100 for 10 min. For immunostaining, we used primary mouse anti-Islet-1 antibodies  and mouse antibodies to sarcomeric alpha-actinin (Abcam), alpha-sarcomeric actin (Sigma-Aldrich), and cardiac troponin T (Abcam) pre-conjugated with Alexa 532, 546, 568, 594, or 647 according to Zenon technology (Invitrogen). Commercially available FITC-conjugated anti-c-kit (Abcam) and anti-Sca-1 (Abcam) antibodies were also used at 1:100 dilution. Filamentous actin was detected by using rhodamine phalloidin. Hoechst  staining at 1:1000 dilution was used for the detection of cell nuclei. The cells were stained with antibodies at room temperature.Visualization. A microscope “Axio Observer.Z1” (Carl Zeiss) with ×10, ×20 and ×40 (oil) lens was used to fluorescent microscopy. Confocal microscopy was performed by using the Leica TCS SP5 microscope with ×20 and ×40 (glycerol) magnification. Intensity of the corresponding fluorochrome served to establish a type of CSC, which generated a colony or CICS. In addition, it enabled one, for the first time, not only to reveal c-kit+- and Sca-1+- CSCs but also to identify an antigen phenotype of the host cell when CICSs are formed. Negative responses to Islet-1 antibodies indicate that CSCs of this type do not form CICSs in the myocardium of adult mammals. Furthermore, the lack of positive staining with antibodies to smooth muscle, endothelial cells, and fibroblasts demonstrated that intracellular development of c-kit+- and Sca-1+- CSCs occurs only in variously matured CMs.Time-lapse microscopy. The images of the living cardiomyocytes were recorded at a magnification of ×40 (glycerol) for 7 days, at a rate of 1 frame per min .Statistical analysis. All of the data are expressed as the mean ± standard deviation. The statistical analyses were performed by using the SPSS 13.0 software package .+ CSC-derived colonies , we observed remnants of ruptured encapsulated pre-existing CICSs A, newly colonies C. In thecolonies E and c-kcolonies F were focolonies D. In thecolonies G, young colonies H, and rucolonies I were idThe data on the number of intact and ruptured encapsulated CICSs at various time points in all groups studied in three areas of the myocardium  are given in +-type  of CSCs inside CMs of newborn (A), 20-day-old (B), and 40-day-old (C) rats. Here, it should be noted that CSCs can either be positioned close to the nucleus (or between nuclei) of the host cell, being in this case inside an individual vacuole  stimulated the intracellular development of CSCs, enhancing the numbers of CICSs by 5–10 times in cell culture compared to the control [+ CSCs is mediated by increased expression of Pim-1 kinase and concomitant reduction in pro-apoptotic factors [Moreover, our previous study demonstrated that increased CO control . These d control . It is a control . Hypoxic control . Molecul factors . TherefoWe suppose that the data presented are essential for developing new strategies for myocardial regeneration after irreversible injury. Although it is not easy to envisage the potential clinical applications of the phenomena described, we did our best to provide certain relevant speculations. According to our findings, it is felt that TACs might become the crucial cellular target for cardioregenerative therapy in the future. There are several potential ways to harness regenerative potential of TACs. First, one cannot exclude the possibility that TACs will be directly transplanted in the damaged heart by using 3D NOGA endocardial mapping system and electromagnetically guided percutaneous intramyocardial therapy. It is also conceivable that TACs will be subjected to genetic modification aimed at boosting of their cardiomyogenic potency and/or enhancing their survival in the harsh ischemic environment prior to intramyocardial administration. Second, the endogenous pool of TACs could be activated by means of their pharmacological stimulation with growth factors, microRNAs, and low-molecular-weight drugs. These stimulating factors could be packed into membrane vesicles or bound with other transporting vehicles, which might increase their targeted delivery into injured myocardium after systemic administration. Third, there might be an opportunity to “switch” from one pathway of cardiomyogenesis to another, thereby providing optimal contribution of colony-derived cardiomyocytes and those produced within encapsulated and non-encapsulated cell-in-cell-structures. For instance, within the first days after infarction, the main cellular target for myocardial regeneration would be the population of immature TACs released from encapsulated CICSs. The approaches aimed at enhanced survival of these cells, as well as at their augmented proliferation and differentiation, could potentially contribute to formation of new functionally competent CMs. During the more protracted stage of infarction, namely starting from the 4th day, the most promising endogenous cell population for heart regeneration would be TACs developing inside mature CMs, that is, in non-encapsulated CICSs. Future studies, no doubt, will shed light on the molecular mechanisms of CSC-mediated myocardial self-renewal and regeneration.


Evidence has indicated that depression and obesity were associated with functional disability, independently. However, little is known about the detrimental impact of comorbid depression and obesity, as well as its transition on functional disability. This study investigated the association of baseline depression-obesity status and its dynamic change with incident functional disability among middle-aged and older Chinese.2. Participants were cross-classified by depression and obesity status at baseline, and its change during follow-up. Logistic regression models were constructed to evaluate the association of baseline depression-obesity status and its transition with incident functional disability defined by the Katz index of activities of daily living scale.This cohort study included 5507 participants aged ≥45 years from the 2011 and 2015 waves of China Health and Retirement Longitudinal Study. Depression was defined with a score ≥ 10 using the 10-item Centre for Epidemiologic Studies Depression Scale. Obesity was defined as body mass index ≥28 kg/mOver four-year follow-up, 510 (9.3%) participants developed functional disability. Individuals with baseline comorbid depression and obesity had the highest risk of functional disability  than non-depressive participants without obesity, or those with depression or obesity alone. When investigating the dynamic changes of depression-obesity status on functional disability incidence, those with stable comorbidity throughout two surveys had the greatest risk of functional disability . Progression of depression-obesity status was associated with increased risk of functional disability, while regression from baseline to follow-up was linked to attenuated risk estimates.Among middle-aged and older Chinese adults, the risk of functional disability was exaggerated with comorbid depression and obesity. Our data further suggest that transitions of depression and obesity over time are associated with the risk of developing functional disability.The online version contains supplementary material available at 10.1186/s12877-022-02972-1. With ageing population growing worldwide, most countries have to face the increasing burden of ageing-related health issues. One of such concerns is functional disability, which refers to the impairment of the capacity that enable people to be and to do what they have reason to value . A crossDepression and obesity are common in older people nowadays. Both conditions have been found to be associated with increased risk of functional disability –8. AdditTherefore, this cohort study aimed to investigate (1) the association between baseline depression-obesity status and the risk of incident functional disability among middle-aged and older adults in China, and (2) whether transition of depression-obesity status over time can affect the risk of incident functional disability. Our findings may provide insight about whether depressive symptom should also be screened in addition to body mass index (BMI) among middle-aged and older adults in clinical practice. Furthermore, the results can help identify high-risk individuals of developing functional disability who may benefit from health intervention.Participants of this cohort study were recruited from the China Health and Retirement Longitudinal Study (CHARLS), a nationally representative survey of Chinese aged 45 years and older –17. The The ethics of CHARLS study was approved by the Institutional Review Board (IRB) at Peking University  [Functional disability was evaluated using the Katz index of independence in ADLs, which includes six items of dressing, bathing, eating, transferring, toileting, and continence . Each itDepression was evaluated over the previous 7 days using the Chinese version of the 10-item Centre for Epidemiological Studies Depression Scale (CESD-10) , which h2). According to the recommendations of Working Group on Obesity in China, obesity was defined as BMI ≥ 28 kg/m2 in Chinese adults [The standing height and weight were measured by a standardized stadiometer  and a validated scale , respectively. BMI was calculated as weight (kg) divided by the square of height (me adults .All participants were divided into four groups according to the baseline depression and obesity status: neither depression nor obesity (neither condition); depression without obesity ; obesity without depression ; with both depression and obesity (comorbidity).Face-to-face interviews were used to collect information of socio-demographic and lifestyle factors, including age, gender, education level , current marital status , area of residence , smoking status (never and ever smoker), and drinking status (never and ever drinker).Systolic blood pressure (BP) and diastolic BP were measured three times with at least 45-s intervals by an electronic sphygmomanometer . Hypertension was confirmed if the participant met any of the following criteria: systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg, and/or with self-reported physician-diagnosed hypertension, and/or on anti-hypertensive drugs . GlycateTo describe the baseline characteristics, mean ± standard deviation (SD) was used for continuous variables, and frequency (percentage) was applied for categorical variables. Analysis of variance (ANOVA) and Chi-square tests were conducted to compare differences of continuous variables and categorical variables across the four depression-obesity groups at baseline, respectively. Comparisons of baseline characteristics between the included and excluded individuals were further conducted with independent student t-test or Chi-square tests, where appropriate.In the first analysis, logistic regression models were established to evaluate the associations between baseline depression-obesity status and incident functional disability. Model 1 was a crude model. Model 2 was adjusted for baseline age, gender, education level, current marital status, area of residence, smoking and drinking status. Model 3 was additionally adjusted for multimorbidity status, as previous research has indicated its detrimental impact on functional disability . Odds raIn the second analysis regarding the transition of depression and obesity status, a Sankey diagram was depicted to directly visualize the number of participants transitioning across different groups from baseline to follow-up. To determine the association between changes in depression-obesity status and the risk of developing functional disability, participants were divided into three categories: (1) stability (3 groups); (2) progression (2 groups); and (3) regression (2 groups). The stability category was further divided into 3 groups, including participants with neither condition, single condition , and comorbidity throughout the baseline and follow-up surveys. Participants with depression alone or obesity alone were combined together in the analysis as the risk of functional disability were similar in these two groups according to our results. Additionally, the number of individuals changed from depression alone to obesity alone or vice versa was relatively small, while combination of these individuals into a single group enabled us to perform analyses with sufficient statistical power. The progression category included 2 groups, i.e., participants progressed from neither condition at baseline to single condition at follow-up, and participants progressed from 0 or 1 condition at baseline to comorbidity at follow-up. The regression category also included 2 groups, one group with participants regressed from comorbidity to single condition, and the other group with participants regressed from comorbidity or single condition at baseline to neither condition at follow-up. Three logistic regression models with the same covariates adjusted in the first analysis were constructed, with the group of neither condition throughout the baseline and follow-up surveys treated as the reference.P < 0.05.All analyses were conducted with Stata/SE 16.0 . Statistical significance was determined as two-tailed Of the 5507 participants enrolled in the first analysis, the mean age was 60.6 ± 9.4 years and 62.4% were females Table . The numAfter 4 years of follow-up, 510 (9.3%) participants developed functional disability. The incidence of functional disability was the highest among participants with comorbid depression and obesity (15.7%), followed by those with depression alone (10.6%) and obesity alone (9.6%), and was the lowest among individuals with neither condition (7.1%). Similar patterns were observed for the incidence of functional disability in the dressing, bathing, eating, transferring, and toileting subscales, except for the incidence of continence, which was comparable across different depression-obesity groups  or vice versa (N = 29) was relatively small in our study, thus we were unable to explore their risk of developing functional disability. Future studies are needed to further confirm the findings.The strengths of this study include the longitudinal design, the relatively large study sample, the repeated measurements, and the validated questionnaires used to measure depression and functional disability. A further strength is that we also investigated how the transition of depression-obesity status affected the onset of functional disability, which provide important clinical implication for prevention and intervention of functional disability in middle-aged and older Chinese adults. However, some limitations merit discussion. First, a number of individuals were excluded from this study due to missing information or loss to follow-up. In addition, several baseline characteristics were statistically different between the included and excluded individuals, imposing concerns on the generalizability of our study. Nevertheless, results obtained from the imputed datasets also demonstrated increased risk of functional disability in those with comorbid depression and obesity (data not shown), indicating the robustness of our findings. Second, depression was ascertained by CESD-10 instead of the gold-standard clinical diagnoses, which might partially cause misclassification bias. However, CESD-10 has been demonstrated to be reliable and valid in epidemiology research among older adults in China . Third, In conclusion, our study shows that comorbid depression and obesity could exert greater detrimental effects on the risk of functional disability among middle-aged and older adults in China. Importantly, our data suggest that depression and obesity status is transient, with the highest functional disability risk for those who remain comorbidity. The risk of functional disability was increased with deterioration in depression-obesity status and reduced with regression of depression and obesity. Apart from body weight monitoring, our findings highlight the need of screening depression among middle-aged and older adults, especially for obese adults, which may help identify the high-risk population of developing functional disability.Additional file 1: Supplementary Table 1. Comparison of baseline characteristics between included and excluded individuals. Supplementary Figure 1. Incidence of functional disability in 2015 in different groups according to baseline depression-obesity status.


Hypertrophic cardiomyopathy (HCM) is associated with adverse left ventricular (LV) remodeling causing dysfunction and malignant arrhythmias. Severely affected patients present with disease onset during childhood and sudden cardiac death risk (SCD) stratification is of the highest importance in this cohort. This study aimed to investigate genotype–phenotype association regarding clinical outcome and disease progression in pediatric onset HCM. Medical charts from forty-nine patients with pediatric HCM who had undergone genetic testing were reviewed for retrospective analysis. Demographic, clinical, transthoracic echocardiographic, electrocardiographic, long-term electrocardiogram, cardiopulmonary exercise test, cardiac magnetic resonance, and medication data were recorded. Childhood onset HCM was diagnosed in 29 males and 20 females. Median age at last follow-up was 18.7 years (range 2.6–51.7 years) with a median follow-up time since diagnosis of 8.5 years (range 0.2–38.0 years). Comparison of patients carrying mutations in distinct genes and comparison of genotype-negative with genotype-positive individuals, revealed no differences in functional classification, LV morphology, hypertrophy, systolic and diastolic function, fibrosis and cardiac medication. Patients with compound mutations had a significantly higher risk for major arrhythmic events than a single-mutation carrier. No association between affected genes and disease severity or progression was identified in this cohort. Hypertrophic cardiomyopathy (HCM) is the most common genetically inherited heart disease with a prevalence of about 0.2% ,2. It isDisease-causing mutations inherited in an autosomal dominant manner are currently identified in about 60% of HCM patients . They arThe wide range of genes that can be affected by mutations makes HCM a genetically heterogeneous disease . GeneticSpecifically, in pediatric onset HCM, precise knowledge about disease progression, depending on different mutations, is still lacking. Insight of HCM genotype–phenotype association would facilitate counseling and management of affected patients.This study aimed to investigate genotype–phenotype association of disease severity and progression in patients with childhood onset HCM.A total of 49 patients diagnosed with pediatric onset HCM between November 1981 and November 2019 at the outpatient clinic of the German Heart Center Munich were included in the study. Pediatric onset HCM was defined as either positive molecular-genetic testing or evidence of disease phenotype before the age of 18 years. Phenotype-positive was defined by the presence of isolated hypertrophied left ventricle with a z-score of ≥2 on transthoracic echocardiographic evaluation based on the guidelines of the European Society of Cardiology (ESC)  and guidData collection was performed retrospectively by medical chart review including demographic and clinical status, transthoracic echocardiography (TTE), electrocardiogram (ECG), 24-h Holter ECG, cardiopulmonary exercise testing (CPET), and cardiac magnetic resonance imaging (CMR). To assess disease progression, patient-related information was obtained at first presentation at the outpatient clinic and compared to information available from last follow-up in those patients, where information for both timepoints was available. CMR data were obtained from 31 patients (63.3%). Since CMR was performed at only one time point, data could not be used for the analysis of disease progression.Clinical and imaging parameters available to assess the presence of pathological myocardial remodeling included end-diastolic and end-systolic LV diameters on CMR and TTE for general cardiac morphology, CMR mass (g/m²) and TTE end-diastolic LV wall thicknesses (z-score) for myocardial hypertrophy, CMR late gadolinium enhancement (LGE) for patchy and CMR T1 map for interstitial myocardial fibrosis, echocardiographic pulse wave mitral valve (MV) E/A ratio and septal/lateral MV tissue Doppler measurements for LV diastolic function, TTE EF and strain analysis for LV systolic function, presence of arrhythmias on ECG, CPET, and 24-h Holter ECG, and clinical functional status assessed by the New York Heart Association (NYHA) class or modified age-adjusted Ross classification , medicatRoutine molecular genetic testing was performed for the identification of causative genes ,15. One p-value of <0.05 was considered statistically significant.Statistical analysis was performed with the SPSS software program version 25.0.0 . Differences between all groups defined at latest follow-up were analyzed by the Kruskal–Wallis test. For comparison of two individual selected groups, the Mann–Whitney U test was utilized. Categorical variables were analyzed by Pearson Chi-square test. With the help of the Kaplan–Meier calculation, the survival probability was estimated and compared between the groups by log-rank test. Continuous variables were expressed as median (minimum–maximum). For assessment of disease progression, the delta of respective parameters between the date of first presentation and last follow-up was compared between groups using the dependent non-parametric Kruskal–Wallis test. Statistical tests were two sided and a The study population consisted of 29 male (59.2%) and 20 female (40.8%) patients with a diagnosis of pediatric onset HCM based on either clinical findings and/or positive molecular genetic testing. Genetic testing identified 35 consecutive patients as genotype-positive/phenotype-positive (71.4%), four patients as genotype-positive/phenotype-negative (8.2%), and 10 patients as genotype-negative/phenotype-positive (20.4%) at first presentation. Distribution of the detected mutations are depicted in p-value 0.982). Family history was negative in 15 patients (30.6%), positive for the presence of HCM in 23 patients (46.9%) and for SCD in 11 patients (22.4%) (Patient characteristics are depicted in  (22.4%) .Age at first diagnosis and at last follow-up was not different between the distinct patient groups, separated by the respective results of moleculargenetic diagnosis . MortaliThere was no difference in the need for hospitalizations and surgery, medication use was similar between groups, and the majority of patients were in NYHA/Ross class I or II at latest follow-up without a difference between groups. Additionally, no differences were found with regard to LV morphology (end-diastolic and end-systolic diameters or presence of LVOTO), myocardial hypertrophy and fibrosis, LV systolic and diastolic function parameters, and the occurrence of arrhythmias on ECG, 24-h Holter ECG or CPET, when comparing patients grouped according to the affected gene .n = 20) and male (n = 29) patients was performed. Analysis of clinical outcome and imaging parameter did not reach significance, which might be due to the small sample size. Furthermore, investigation of whether there are sex-differences between females and males carrying mutations in the same gene did not yield significant results.To evaluate gender-difference in disease presentation of pediatric onset HCM patients, a comparison of female . Additionally, there was a difference in LGE localization measured by CMR . Patients with single mutations were LGE-positive mainly in the septal area, whereas LGE was positive in the entire myocardium in compound-mutation carrier. There was a trend toward increased left atrial parameters and decreased EF in TTE in patients with compound mutations compared to single-mutation carriers, but the findings did not reach statistical significance, possibly due to small patient numbers . Detailed descriptive information regarding clinical characteristics, outcome, and imaging parameter of patients with compound mutation are displayed in Since carriers with compound mutations were identified at increased risk for MAEs, a subgroup analysis was carried out comparing clinical and imaging findings between patients with one compared to patients with more than one pathogenic/likely pathogenic mutation. Patients with compound mutations had increased maximal wall thickness with higher end-diastolic interventricular septal z-scores at latest follow-up compared to single-mutation carrier . Less patients with troponin mutation displayed LVOTO compared to patients with non-troponin mutations .All multiple-mutation carriers comprised one mutation in the troponin gene in addition to a MYH7 mutation . To assep-value 0.018).In order to differentiate between the effect of a single troponin mutation compared to a troponin mutation in addition to a second mutation, we compared the patients with a single troponin mutation with those carrying multiple mutations. Only patients with compound mutations including a troponin mutation and no patient with a single troponin mutation experienced MAEs  differed depending on mutation type and count. For this, differences between clinical and imaging parameters at follow-up and first presentation were calculated in those patients, where data were present for both timepoints. Based on the results of the statistical analysis, no evidence of statistically significant differences in disease progression of different patient groups could be found .The purpose of this study was to investigate genotype–phenotype association in patients with childhood onset HCM, depending on mutation type and count. Furthermore, we examined disease progression in these individuals, especially the dependency of different mutations on disease severity. Genotype–phenotype relationship and predictors for disease progression in adult patients have been previously described, but precise data of children with HCM are rare. This study adds to the body of evidence about genotype–phenotype association in patients diagnosed with HCM during childhood.The current study revealed no differences with regard to clinical disease course and myocardial pathology comparing patients carrying a single mutation in distinct genes. These findings are in line with other studies mostly including adult HCM patients stating that mutation type is negligible for clinical phenotype and prognosis prediction ,23,24. VA major finding of the current study is that pediatric patients carrying more than one disease-causing mutation, one of which affects the troponin proteins, are at substantially increased risk for experiencing MAEs. This finding is in line with a large body of evidence derived mostly from adult studies, where HCM patients carrying compound mutations or mutations in the cardiac troponin T are expected to be at higher risk for life-threating arrythmias and SCD ,30,31,32Patients carrying single-gene mutations encoding troponins in the absence of a second mutation did not display a higher risk for SCD and MAEs in the current study. In addition, troponin-mutation carriers were not affected by a worse myocardial phenotype or more severe disease progression compared to patients with other mutations. One reason for this could be the dilemma with a limited number of troponin T mutation carriers, similar to other studies , given tThe cause of potentially lethal arrhythmic events such as sustained and non-sustained ventricular tachycardia is still unclear . AdverseThe major limitation of the present study is the small number of studied individuals. A general low prevalence of patients with pediatric onset HCM makes it difficult to obtain a large study cohort and to achieve an adequate informative value. A single-center design was chosen given the heterogeneity in imaging modalities, inter-observer variability in imaging data analysis, and the lack of moleculargenetic diagnosis in a multicenter setting. Additionally, the limitations of a retrospective study in general apply. Comparison to other studies was hampered by the fact that most studies investigating genotype–phenotype relationship in HCM to date have been conducted in adult patients who were not diagnosed with HCM until the age of 18. This affects the comparability of individual study results between childhood and adulthood onset of HCM. Finally, the study results cannot simply be transferred to other centers as this was a single-center study at a tertiary care university hospital.Despite the limitations, the current study enhances a better understanding of the multifactorial nature of HCM in pediatric patients. Clinical phenotypes of patients with childhood onset HCM is heterogenous and mainly independent of distinct single mutations. As mutation-specific risk stratification still remains a challenge, further research is needed to identify predictors for severe arrhythmias, causing SCD in pediatric patients with HCM.In this single center observational study, pediatric patients carrying compound mutations were at increased risk of suffering from MAEs, which drastically increased morbidity and mortality. Molecular genetic testing during childhood can identify those high-risk children and allows for early initiation of preventive measures to avoid the occurrence of life-threatening arrhythmias and SCD.