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In the WHO 1996 classification of cardiomyopathies, myocarditis is defined as an “inflammatory disease of the myocardium associated with cardiac dysfunction” and is listed among “specific cardiomyopathies”. Myocarditis is diagnosed on endomyocardial biopsy (EMB) by established histological, immunological, and immunohistochemical criteria, and molecular techniques are recommended to identify viral etiology. Infectious, autoimmune, and idiopathic forms of inflammatory cardiomyopathy are recognized that may lead to dilated cardiomyopathy. According to Dallas criteria, myocarditis is diagnosed in the setting of an “inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes, not typical of ischemic damage associated with coronary artery disease”. The majority of experts in the field agree that an actual increase in sensitivity of EMB has now been reached by using immunohistochemistry together with histology. A value of >14 leukocytes/mm 2 with the presence of T lymphocytes >7 cells/mm 2 has been considered a realistic cut off to reach a diagnosis of myocarditis. The development of molecular biological techniques, particularly amplification methods like polymerase chain reaction (PCR) or nested-PCR, allows the detection of low copy viral genomes even from an extremely small amount of tissue such as in EMB specimens. Positive PCR results obtained on EMB should always be accompanied by a parallel investigation on blood samples collected at the time of the EMB. According to the recent Association for European Cardiovascular Pathology guidelines, optimal specimen procurement and triage indicates at least three, preferably four, EMB fragments, each 1–2 mm in size, that should immediately be fixed in 10 % buffered formalin at room temperature for light microscopic examination. In expected focal myocardial lesions, additional sampling is recommended. Moreover, one or two specimens should be snap-frozen in liquid nitrogen and stored at −80 °C or alternatively stored in RNA-later for possible molecular tests or specific stains. A sample of peripheral blood (5–10 ml) in EDTA or citrate from patients with suspected myocarditis allows molecular testing for the same viral genomes sought in the myocardial tissue.

The mature left ventricular myocardium is arranged in a complex three-dimensional network of fibers that form a counterclockwise helix in the endocardial layer and a clockwise helix in the epicardial layer. There are no data in the literature on the development of left ventricular myocardium during the fetal life. The aims of this paper were to study the physiological maturation steps of the LV myocardium in fetuses from 17 to 40 gestational weeks, by means of speckle tracking applied to the endocardial and epicardial aspect of the left ventricle, and, to confirm our finds, through the histologic study of the myocardium of demised fetuses. We studied longitudinal endocardial and epicardial strain by echocardiography in 105 fetuses. Twenty non-diseased fetal hearts from autopsies were selected to assess the layer thickness and cardiac fiber orientation in relation to gestational age. Echocardiography showed a progressive increasing of epicardial/endocardial longitudinal strain ratio with gestational age (r=0.51; p<0.0001). The strain rate E/A ratio increased over time (r=0.27; p=0.018). Histological data revealed that during the same gestational period, the proportion of the epicardial layer increased fourfold, the mesocardiac layer decreased and the endocardial layer remained stable. We found an excellent correlation between the epicardial to endocardial strain ratio and epicardial to endocardial wall thickness (r=0.950, p<0.001). Left ventricular myocardium maturation begins early during fetal life. As the fetus develops, both the relative tissue volume and peak systolic strain rates shift together from the endocardium towards the epicardium. It is a slow process, completed late in fetal life.

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation.

Antibody-mediated rejection (AMR) is a serious complication affecting the survival of patients receiving transplantation. Human cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are common viral infections that occur after transplantation, frequently emerging as viral reactivation in donor grafts or transplant recipients. The present study aimed to investigate the association between CMV and EBV infections and early-onset AMR. This study was conducted at the Heart Transplantation Center of Padova General Hospital and included a cohort of 47 heart transplant recipients (HTxs), including 24 HTxs diagnosed with AMR and 23 control HTxs with no episodes of AMR. Only early cases of CMV and/or EBV infections (1-90 days after transplantation) were considered. Fisher's exact test and logistic regression analysis were used to statistically analyze the correlation and association between AMR and CMV or EBV infection. We observed a positive statistical association between CMV and EBV infections (two-sided Fisher's exact test, p = 0.0136) and between EBV infection and AMR (two-sided Fisher's exact test, p = 0.0034). Logistic regression analysis revealed a direct statistical association between CMV and EBV infections and AMR risk (p = 0.037 and 0.006 and odds ratio = 1.72 and 2.19, respectively). AMR occurrence was associated with increased viral loads of both CMV and EBV early after transplantation. These findings suggest the role of CMV and EBV infections as relevant risk factors for AMR in HTxs for the first time.

The utilization of kidneys from donors with acute kidney injury (AKI) is often limited by unpredictable post-transplantation outcomes. The aim of our study was to identify protein mediators implicated in either recovery or failure of these organs. Forty kidney biopsies from donors with (20) and without AKI (20) were selected and then subdivided according to the post-transplant outcome defined as a threshold of 45 ml/min for the eGFR at 1 year from transplantation. Tissue homogenates were analysed by western blot to assess how the levels of 17 pre-selected proteins varied across the four groups. Samples from AKI kidneys with a poor outcome showed a fourfold increase in the levels of PPARg and twofold reduction of STAT1 compared to the other groups (p < 0.05). On the contrary, antioxidant enzymes including TRX1 and PRX3 were increased in the AKI kidneys with a good outcome (p < 0.05). An opposite trend was observed for the detoxifying enzyme GSTp which was significantly increased in the AKI group with poor versus good outcome (p < 0.05). The importance of lipid metabolism (PPARg) and inflammatory signals (STAT1) in the function recovery of these kidneys hints to the therapeutical targeting of the involved pathways in the setting of organ reconditioning.

ObjectivesR2* cardiac magnetic resonance (CMR) allows the non-invasive measurement of myocardial iron. We calibrated cardiac R2* values against myocardial tissue–measured iron concentration by using a segmental approach and we assessed the iron distribution.MethodsFive hearts of thalassemia patients were donated after death/transplantation to the CoreLab of the Myocardial Iron Overload in Thalassemia Network. A multislice multiecho R2* approach was adopted. After CMR, used as guidance, the heart was cut in three short-axis slices and each slice was cut into different equiangular segments according to AHA segmentation and differentiated into endocardial and epicardial layers. Tissue iron concentration was measured by atomic absorption spectrometer technique.ResultsFifty-five samples were used since only for two hearts all the 16 samples were analyzed. Mean iron concentration was 4.71 ± 4.67 mg/g dw. Segmental iron levels ranged from 0.24 to 13.78 mg/g dw. The coefficient of variability of iron for myocardial segments ranged from 8.08 to 24.54% (mean 13.49 ± 6.93%). Iron concentration was significantly higher in the epicardial than in the endocardial layer (5.99 ± 6.01 vs 4.84 ± 4.87 mg/g dw; p = 0.042). Four different circumferential regions (anterior, septal, inferior, and lateral) were defined. A circumferential heterogeneity was noted, with more iron in the anterior region, followed by the inferior region. The direct nonlinear fitting of R2* and [Fe] data led to the calibration curve: [Fe] = 0.0022 ∙ (R2*-ROI)1.462 (R-square = 0.956).ConclusionsOur data further validate R2* CMR using a segmental approach as a sensitive and early technique for quantifying iron distribution in the current clinical practice.Key Points• Calibration in humans for cardiovascular magnetic resonance R2* against myocardial iron concentration was provided.• A circumferential heterogeneity in cardiac iron distribution was detected: more iron was observed in the anterior region, followed by the inferior region. This finding corroborates the use of a segmental T2* CMR approach in the clinical practice to detect a heterogeneous iron distribution.• The comparison between the cardiac T2* values obtained with the region-based and the pixel-wise approaches showed a significant correlation and no significant difference but, in presence of significant iron load, the region-based approach resulted in significantly higher T2* values.

Cytomegalovirus (CMV) infection is a relevant threat to heart-transplanted patients during the first year after surgery, leading to increased morbidity and, in some cases, mortality. This study aims to assess the transcriptomic profile of CMV infection in cardiac transplanted patients as a new diagnostic approach to discriminate infection and Acute Cellular Rejection (ACR) on EMB specimens. We performed a microarray-based messenger RNA (mRNA) and micro-RNA (miRNA) profiling. We analyzed three patient groups in the setting of CMV viremia and inflammatory infiltrate: a control group (n=5), an ACR group (n=5), and an infection group (n=6). Differentially expressed mRNA and miRNA were further investigated through bioinformatic pathway analysis. Focusing on infection vs rejection comparison, we investigated the role of the 18 differentially expressed mRNAs and the 12 miRNAs with the most significative p-value (gene level fold change, FC <-2 or >2, p-value <0.05). Based on the bioinformatic analysis, we explored the regulatory effects of these miRNAs on the mRNA pathways independently identified in the same samples. The results showed that two genes, IL7R and GZMK (-38.63 and -3.15 FC, respectively), and two miRNAs, mir-93-5p and mir-345-5p (-2.63 and -2.18 FC, respectively), are differentially expressed in infection and can be exploited to differentiate CMV-positive from ACR-positive EMB specimens, reaching an AUC of 0.87 and an accuracy of 91% at cross-validation. We have identified a distinctive combined molecular profile of mRNAs and miRNAs for infection in post-cardiac transplant follow-up. Based on IL7R, GZMK, mir-93-5p, and mir-345-5p we suggest a novel possible workflow to distinguish infection, where those markers are downregulated, from rejection, where they are overexpressed, on EMB specimens. This analysis showed good accuracy and promising predictive performance. The future combined analysis of these genes and these miRNAs through user-friendly techniques, such as quantitative PCR, could reduce turn-around time and improve our diagnostic power for distinguishing CMV infection from ACR in EMB specimens.

Background: In this study, we assessed the mid-term outcomes of patients who received a heart donation from a marginal donor (MD), and compared them with those who received an organ from a standard donor (SD). Methods: All patients who underwent HTx between January 2012 and December 2020 were enrolled at a single institution. The primary endpoints were early and long-term survival of MD recipients. Risk factors for primary graft failure (PGF) and mortality in MD recipients were also analyzed. The secondary endpoint was the comparison of survival of MD versus SD recipients. Results: In total, 238 patients underwent HTx, 64 (26.9%) of whom received an organ from an MD. Hospital mortality in the MD recipient cohort was 23%, with an estimated 1 and 5-year survival of 70% (59.2–82.7) and 68.1% (57.1–81), respectively. A multivariate analysis in MD recipients showed that decreased renal function and increased inotropic support of recipients were associated with higher mortality (p = 0.04 and p = 0.03). Cold ischemic time (p = 0.03) and increased donor inotropic support (p = 0.04) were independent risk factors for PGF. Overall survival was higher in SD than MD (85% vs. 68% at 5 years, log-rank = 0.008). However, risk-adjusted mortality (p = 0.2) and 5-year conditional survival (log-rank = 0.6) were comparable. Conclusions: Selected MDs are a valuable resource for expanding the cardiac donor pool, showing promising results. The use of MDs after prolonged ischemic times, increased inotropic support of the MD or the recipient and decreased renal function are associated with worse outcomes.