Endomyocardial Biopsy Specimens Research Articles

Myocardial DNA damage is responsible for the relationship between genotype and treatment response in patients with dilated cardiomyopathy

Abstract Background Dilated cardiomyopathy (DCM) is one of the leading causes of heart transplantation. Our group and others have previously reported that DNA damage accumulation in myocardial tissue and certain genetic variants such as those in LMNA are associated with treatment response and prognosis (ref 1-5). However, the potential association between myocardial DNA damage and genotype, and their contribution when combined to predicting treatment response, remains unclear. Methods We identified the deleterious variants in cardiomyopathy-related genes by analyzing the whole exome sequencing data from 89 patients with DCM who were recruited from two participating facilities. Immunostaining of γ-H2A.X, a DNA damage marker, was performed on endomyocardial biopsy specimens from the same patients, from which the percentage of nuclei positive for γ-H2A.X signals (%γ-H2A.X) was calculated. Left ventricular reverse remodeling (LVRR) was assessed by transthoracic echocardiography at 1 year after biopsy. We examined the associations among genotype, myocardial DNA damage, and the achievement of LVRR. Results Among the 89 patients with DCM, 11 (12.4%) carried pathogenic or likely pathogenic (P/LP) variants in sarcomere genes such as TTN, and 21 (23.6%) carried P/LP variants in non-sarcomere genes such as LMNA, while the rest had no P/LP variants in cardiomyopathy-related genes (Figure A). Patients with P/LP variants in sarcomere genes had a lower %γ-H2A.X [21.2 (10.7–29.0)% vs. 7.0 (4.3–12.4)%, P = 0.002] (Figure B), and a higher probability to achieve LVRR at 1-year (81.8% vs. 23.8%, P = 0.003) (Figure C) when compared to those with P/LP variants in non-sarcomere genes. The odds ratio for carriers of P/LP variants in sarcomere genes to achieve LVRR was 14.40 (95%CI 2.31–89.94) compared to that for carriers of P/LP variants in non-sarcomere genes. However, this effect was markedly attenuated after adjusting for the proportion of γ-H2A.X-positive nuclei [adjusted odds ratio 6.05 (95%CI 0.86–42.53)] (Table). Conclusions Among patients with DCM, carriers of P/LP variants in sarcomere genes showed less DNA damage and a favorable treatment response, when compared with carriers of P/LP variants in non-sarcomere genes. Our results suggest that myocardial DNA damage was partially responsible for the relationship between genotype and treatment response in patients with DCM.Figure, panels A-C

Arrhythmias and conduction disorders in patients with viral heart disease.

Viral heart disease comprises of two cardiovascular entities being evoked by viral infection: acute viral myocarditis and viral cardiomyopathy. Viral myocarditis may completely resolve leaving no traceable sign or cause ongoing inflammation with subsequent development of hypokinetic dilated/non-dilated cardiomyopathy. The exact epidemiology of viral myocarditis remains unknown due to its sometimes asymptomatic course, but according to the Global Burden of Disease Study 2019, the prevalence of myocarditis in young adults is estimated to range between 6.1 per 100,000 in men and 4.4 per 100,000 in women, with the most common viral etiology. According to the literature viral genome can be found in considerable percentage (up to 67,4%) of endomyocardial biopsy specimens obtained from patients with idiopathic left ventricular dysfunction- suggesting viral etiology of the cardiomyopathy. In this review we would like to enlighten most common types of arrhythmias and conduction disorders as well as their prevalence in patients with viral heart disease. Moreover, our paper depicts probable pathological mechanisms in which viruses induce arrhythmias and cardiac conduction system disease in both, acute viral infection and chronic viral disease. We would also like to highlight unresolved problem of sudden death protection in the course of acute myocarditis.

Interleukin-17 and interferon-gamma in patients with different forms of non-ischemic cardiomyopathies

Abstract Introduction Dilated cardiomyopathy (DCM) characterized by left ventricular dilation and impaired left ventricular function represents a common and severe cause of heart failure (HF). Predominantly affecting younger patients, it is considered a leading cause for sudden cardiac death and heart transplantation. Several studies identified myocarditis as a substantive precursor and potential cause of DCM. Inflammatory cardiomyopathy (DCMi) can lead to cardiomyocytal damage and exposure of cardiac myosin and other heart proteins. This cascade can in susceptible individuals trigger an altered immune response consecutively leading to autoimmunity and chronic inflammation. A variety of inflammatory and immunologic agents are discussed to be involved in the pathophysiology of myocardial inflammation. Several studies suggested a role of Interleukin -17 (IL-17) and Interferon-γ (IFN-γ) in chronic HF, myocardial infarction and other cardiovascular diseases. Underlying pathomechanisms, however, are poorly understood. Methods In this study we measured IL-17 and IFN-γ levels in serum by ELISA in patients in which an endomyocardial biopsy (EMB) was performed due to chronic unexplained heart failure symptoms after exclusion of ischemic or valvular heart disease at our center. All patients underwent transthoracic echocardiography prior to EMB sampling. Patients were classified according to the histological presence of inflammation into DCMi, DCM and patients with preserved left ventricular ejection fraction (LVEF) without inflammation. EMB specimens were analyzed including histology, immunohistochemistry and molecular virology. Results A total of 289 patients were included into the final analysis. Mean age was 47 years and 71% were male. Mean LVEF was 50% and mean left ventricular end-diastolic diameter (LVEDD) was 57 mm. Based on EMB results, 71 (25%) samples were categorized as DCMi and 113 (39%) as DCM. 105 (36%) samples were assigned to the group without myocardial inflammation and normal LVEF. The groups differed significantly in their inflammatory profiles. Levels of IL-17 (p < 0.001) and IFN-γ (p < 0.01) were significantly higher in patients with DCMi compared to the DCM as well as the normal LVEF group (Figure 1, Figure 2). Therefore, this effect is not specific for heart failure, but seems to be an important mediator in inflammatory cardiomyopathy. Conclusion Our data showed a significant elevation of IL-17 and IFN-γ in patients with inflammatory dilated cardiomyopathy. These findings suggest that these agents play an important role in the disease entity development and progression that remains poorly understood. So far treatment strategies in DCMi are unspecific. Therefore, blocking IL-17 expression could be an interesting potential therapeutic target in future (personalized) treatment for a wide group of patients with heart failure generated by inflammatory causes.Serum IL-17 levels.t-SNE analysis of the different groups.

Abstract 12653: Plasma LTBP-2 is Associated With Myocardial LTBP-2 and Poor Prognosis in Dilated Cardiomyopathy

Introduction: Cardiac extracellular matrix proteins provide structural support to the heart function and are associated with fibrosis in dilated cardiomyopathy (DCM). Latent transforming growth factor beta-binding protein 2 (LTBP-2) is a type of extracellular matrix protein, but the significance of LTBP-2 in patients with dilated cardiomyopathy is still unclear. Objective: To elucidate the significance of LTBP-2 in dilated cardiomyopathy. Methods: Plasma LTBP-2 levels were measured by Enzyme-linked immunosorbent assay in 131 DCM patients who underwent endomyocardial biopsy and 44 controls who had no history of cardiac diseases. Immunohistochemistry for LTBP-2, LTBP-1 (another LTBP family member), and Elastica-Masson staining were performed in endomyocardial biopsy specimens. DCM patients were followed up for cardiac death or ventricular assist device (VAD) implantation. Results: DCM patients had elevated plasma LTBP-2 levels compared to controls (104 vs. 57 ng/mL, P < 0.001). Plasma LTBP-2 levels were positively correlated with LTBP-2-positive fraction in the myocardium from the biopsy specimen (R = 0.311, P < 0.001). Myocardial LTBP-2-positive fraction was positively correlated with myocardial LTBP-1-positive fraction (R = 0.444, P = 0.014) and fibrosis fraction estimated by Elastica-Masson staining (R = 0.387, P < 0.001). In the Kaplan-Meier analysis, the high plasma LTBP-2 concentration group and the high myocardial LTBP-2-positive fraction group had more cardiac deaths or VAD implantations than low groups (P = 0.008 and P < 0.001, respectively). Multivariable Cox proportional hazard analysis showed that plasma LTBP-2 and myocardial LTBP-2-positive fraction were independently associated with cardiac death or VAD implantation. Conclusion: Plasma LTBP-2 reflects myocardial LTBP-2 and can be a biomarker to predict adverse outcomes in DCM patients.

Myocardial DNA Damage Predicts Heart Failure Outcome in Various Underlying Diseases

BackgroundReliable predictors of treatment efficacy in heart failure have been long awaited. DNA damage has been implicated as a cause of heart failure. ObjectivesThe purpose of this study was to investigate the association of DNA damage in myocardial tissue with treatment response and prognosis of heart failure. MethodsThe authors performed immunostaining of DNA damage markers poly(ADP-ribose) (PAR) and γ-H2A.X in endomyocardial biopsy specimens from 175 patients with heart failure with reduced ejection fraction (HFrEF) of various underlying etiologies. They calculated the percentage of nuclei positive for each DNA damage marker (%PAR and %γ-H2A.X). The primary outcome was left ventricular reverse remodeling (LVRR) at 1 year, and the secondary outcome was a composite of cardiovascular death, heart transplantation, and ventricular assist device implantation. ResultsPatients who did not achieve LVRR after the optimization of medical therapies presented with significantly higher %PAR and %γ-H2A.X. The ROC analysis demonstrated good performance of both %PAR and %γ-H2A.X for predicting LVRR (AUCs: 0.867 and 0.855, respectively). There was a negative correlation between the mean proportion of DNA damage marker–positive nuclei and the probability of LVRR across different underlying diseases. In addition, patients with higher %PAR or %γ-H2A.X had more long-term clinical events (PAR HR: 1.63 [95% CI: 1.31-2.01]; P < 0.001; γ-H2A.X HR: 1.48 [95% CI: 1.27-1.72]; P < 0.001). ConclusionsDNA damage determines the consequences of human heart failure. Assessment of DNA damage is useful to predict treatment efficacy and prognosis of heart failure patients with various underlying etiologies.

Prognostic Role of Circulating LTBP-2 in Patients With Dilated Cardiomyopathy: A Novel Biomarker Reflecting Extracellular Matrix LTBP-2 Accumulation.

Dilated cardiomyopathy (DCM) is a life-threatening disease related to heart failure. Extracellular matrix proteins have an important role in the pathogenesis of DCM. Latent transforming growth factor beta-binding protein 2 (LTBP-2), a type of extracellular matrix protein, has not been investigated in DCM. First, we compared plasma LTBP-2 levels in 131 patients with DCM who underwent endomyocardial biopsy and 44 controls who were matched for age and sex and had no cardiac abnormalities. Next, we performed immunohistochemistry for LTBP-2 on endomyocardial biopsy specimens and followed the DCM patients for ventricular assist device (VAD) implantation, cardiac death, and all-cause death. Patients with DCM had elevated plasma LTBP-2 levels compared with controls (P < 0.001). Plasma LTBP-2 levels were positively correlated with LTBP-2-positive fraction in the myocardium from the biopsy specimen. When patients with DCM were divided into 2 groups according to LTBP-2 levels, Kaplan-Meier analysis demonstrated that patients with high plasma LTBP-2 were associated with increased incidences of cardiac death/VAD and all-cause death/VAD. In addition, patients with high myocardial LTBP-2-positive fractions were associated with increased incidences of these adverse outcomes. Multivariable Cox proportional hazard analysis showed that plasma LTBP-2 and myocardial LTBP-2-positive fraction were independently associated with adverse outcomes. Circulating LTBP-2 can serve as a biomarker to predict adverse outcomes, reflecting extracellular matrix LTBP-2 accumulation in the myocardium in DCM.

Digital Pathology for Analyzing Endomyocardial Biopsy Specimens

Introduction: Visual assessment of endomyocardial biopsy (EMB) specimens may result in errors due to the human factor and inability to obtain quantitative data on the intensity of immunohistochemical (IHC) reactions and severity of pathological changes in the heart transplant. Detailed digital characterization of EMB specimens based on automated or semi-automated computer-assisted morphometry improves the diagnostic accuracy of EMB. Objective: To develop a method of and algorithms for digital computer-assisted analysis of EMB specimens to quantify pathomorphological and immunophenotypic changes in the myocardium of patients with various types and grades of heart transplant rejection. Materials and methods: We studied 257 EMB specimens from 56 heart transplant recipients at Scientific Research Institute – Ochapovsky Regional Clinical Hospital No. 1 (Krasnodar, Russian Federation). Sections were stained with hematoxylin and eosin. We used the streptavidin-biotin method to determine the CD68 expression and computer-assisted morphometry of digital images to measure the area of pathological changes and CD68 expression. Results: When developing a method for computer-assisted analysis of EMB specimens, we proposed a new evaluation criterion: staining area coefficient (%) that is a ratio of the total stained area to the total section area. We created and field-tested algorithms for digital morphometric analysis to assess the intensity of IHC reactions and to identify the relative area of pathological changes in the EBM specimens. Conclusions: New digital criteria for diagnosing heart transplant rejections are to improve the accuracy of EMB results interpretation, but can also be used for analyzing other biopsy specimens.

Relationship Between Results of Pathological Evaluation of Endomyocardial Biopsy and Echocardiographic Indices in Patients With Non-Ischemic Cardiomyopathy.

Background: Endomyocardial biopsy (EMB) is a useful modality in diagnosing the origin of cardiomyopathy and the condition of the impaired myocardium. However, the usefulness of obtaining an EMB from the right and left ventricles (RV and LV, respectively), and its associations with echocardiographic parameters, have not been explored. Methods and Results: Ninety-five consecutive patients with non-ischemic cardiomyopathy excluding myocarditis who underwent EMB between July 2017 and May 2019 were studied. Seventy-nine RV and 93 LV biopsy specimens were pathologically analyzed. The relationships among echocardiographic data before EMB and pathologically measured cardiomyocyte diameter (CMD) and interstitial fibrosis (IF) were evaluated. CMD in both LV and RV specimens correlated with echocardiographic LV morphology, but only CMD in the LV was significantly correlated with cardiac function evaluation, including LV ejection fraction, E' and E/E'. In contrast, there were no significant correlations between IF in either the LV or RV and any echocardiographic parameters measured. Furthermore, CMD of both ventricles was significantly correlated with B-type natriuretic peptide (BNP) concentration at EMB, whereas IF of the LV was barely related and IF of the RV was not significantly correlated with BNP concentrations. Conclusions: Pathologically evaluated CMD of EMB specimens of the LV may be more related to functional parameters for heart failure status and LV geometry on echocardiographic examination, than IF.

Anything But a Biopsy: The Quest for Noninvasive Alternatives in Heart Transplantation.

Anything But a Biopsy: The Quest for Noninvasive Alternatives in Heart Transplantation.

216 ARRHYTHMOGENIC CARDIOMYOPATHY: IS THERE EVIDENCE OF AN INFLAMMATORY MYOCARDIAL DISEASE?

Abstract Background and aim Arrhythmogenic cardiomyopathy (ACM) is a heredo-familiar primary heart muscle disease, mainly due to mutations of desmosomal genes and characterized by progressive myocardial atrophy with fibro-fatty replacement, causing electrical instability at risk of sudden cardiac death (SCD). Since the first anatomopathological descriptions, the hypothesis that myocardial inflammation could have an important role in the onset and evolution of ACM has been advanced. With this work we aim to describe the prevalence of inflammatory cells in ACM and their characterization through histological and immunohistochemical (IHC) analysis in endomyocardial biopsy (EMB) and heart specimens coming from either SCD or heart transplantation (HTx). Material and methods We analysed EMB and whole heart specimens (from juvenile SCD registry – below 40 years of age at the time of death – and HTx) with a diagnosis of ACM, focusing on the presence of inflammatory infiltrates. For the whole hearts, in a subgroup of cases the inflammatory infiltrate was evaluated on 9 myocardial sections by applying a semi-quantitative score according to the number of sections involved (0 = none; 1 = 1 section involved; 2 = 2-5 sections; 3 = &amp;gt;5 sections) and an IHC panel for myocarditis on 1 right ventricular and 1 left ventricular section (CD45, CD3, CD8, CD4, CD68, CD20, CD138 and vimentin- as a control for fibroblast). Results Among the 63 analysed EMB (41 M, mean age 45 years, range 18-71 years), 26 cases (41%) showed foci of inflammation with CD3+ &amp;gt; 7/mm2. In all cases viral genome (DNA and RNA virus) was absent. A total of 106 heart specimens with a diagnosis of ACM were enrolled, including 73 juvenile SCD (49 M, mean age 26 years) and 33 HTx (18 M, mean age 45 years). Inflammation was reported in 95% of SCD an d 67% of HTx. The inflammatory infiltrate was focal or multifocal in all, except one case of juvenile SCD with diffuse myocarditis in the postero-lateral wall of the left ventricle. In the 24 reviewed cases, the inflammation was focal with a mean inflammatory score of 1.75 ±0.9 in the HTx group and 1.5±0.9 in the SD group (p = NS). By IHC we showed that: a) there is no significant difference in the inflammatory score between SD and HTx; b) the inflammation is mostly near-scarring (13 cases); c) inflammatory cells consist prevalently of cytotoxic T lymphocytes CD3+CD8+ (with CD4+&amp;lt;CD8+ both in SD and HTx, p &amp;lt; 0.05). Conclusions Our pathological data confirm that ACM is an inflammatory cardiomyopathy with a higher prevalence of inflammation in SCD cases. The inflammatory infiltrate is mainly composed of cytotoxic T lymphocytes with a variable presence of macrophages. These data, combined with the recent experimental studies demonstrating the role of immune mediators in the evolution of the disease, support the potential application of immunosuppressive therapy in ACM.

183 GLOBAL LONGITUDINAL STRAIN BY SPECKLE TRACKING ECHOCARDIOGRAPHY IN SUBCLINICAL ORTHOTOPIC HEART TRANSPLANT REJECTION

Abstract Objective heart transplant (HT) patients need to be strictly monitored, especially within the first months after surgery by advanced and sensitive techniques, in order to rule out possible acute or subacute cellular rejection (CR) and hinder poor outcomes. This includes frequently repeated endomyocardial biopsy (EMB) and biomarkers. Echocardiography has emerged as an easily available and powered technique able to detect morphofunctional changes in various clinical settings. More recently, speckle tracking ultrasound and left ventricular (LV) strain have been documented as more accurate tools for detecting early myocardial dysfunction in most cardiac diseases. This study aimed at recognizing whether strain echocardiography can improve early diagnosis of subclinical CR. Methods We studied 32 patients, mean aged 52±4 years, who had undergone heart transplantation over the last 3 years at IRCCS ISMETT-UPMC (Palermo, Italy). They were scheduled for EMBs on a regular basis, and multisite biopsy specimens served to make diagnosis of CR. Conventional and advanced ultrasound measurements were performed on the same day of EMB. LV ejection fraction (LVEF) was assessed by using the Simpson rule biplane method and global longitudinal strain (GLS) by speckle tracking. Diastolic function was achieved by PW-Doppler/TDI sampling, as the E/E’ ratio. Echocardiographic findings were also related to anti-HLA antibodies (HAb). Results Multiple EMBs and ultrasound studies were performed in each patient. Echocardiographic findings from those likely to be healthy (CR- group) or with possible CR (CR+ group) are summarized in Table underneath (numbers are the average values of the various appraisals). Overall, 300 EMBs were negative (score 0-1) and 94 were positive (score 2-3). Global longitudinal strain and E/E’ ratio were significantly impaired in the first group, despite preserved LVEF. Tacrolimus therapeutic levels were similar between the groups, whereas a higher title of HAb was recognized in the CR+ group (13.3% vs 10.8%, p=0.012). Conclusions Cardiac ultrasound was confirmed to be a useful technique for monitoring HT patients over time. In those likely to have CR as by EMB specimens and HAb title, GLS and diastolic indices were significantly impaired irrespective of LVEF, suggesting speckle tracking echocardiography as a choice modality for detecting subclinical LV dysfunction in patients at risk of CR.

Soluble tumor endothelial marker 1 in heart failure with reduced ejection fraction: A pilot study.

Tumor endothelial marker 1 (TEM1/CD248) is a transmembrane protein that expresses in mesenchymal lineage derived cells during embryogenesis and becomes undetectable in normal adults after birth. Re-expression of TEM1 is found in organ fibrosis, wound healing and cardiac remodeling indicating its potential role in heart failure (HF). The purpose of this study is to explore the role of soluble TEM1 (sTEM1) in patients with HF with reduced ejection fraction. We examined endomyocardial biopsy specimens from three HF patients and blood samples from 48 patients admitted for acute decompensated HF (age 72 years, men 61.7%). The expression of TEM1 in cardiac tissue and concentrations of sTEM1 in plasma were evaluated. Cultured rat cardiomyocytes (H9c2) and human cardiac fibroblasts (HCF) were stimulated with hypoxia or transforming growth factor beta (TGF-β) to observe the release of sTEM1 into culture media. The conditioned media of hypoxia-stimulated H9c2 cells was harvested and added into cultured cardiac fibroblast to evaluate its biological effect. Immunofluorescence study of biopsy specimens from three HF patients showed TEM1 expression in cardiomyocytes and cardiac fibroblasts. The plasma level of sTEM1 was significantly higher in patients (0.90 ± 0.23 vs. 0.33 ± 0.10 ng/mL, p = 0.032) with LVEF ≤ 35% compared with those with LVEF 36-49%. The sTEM1 levels had correlations with HF biomarkers of cardiac fibrosis, including growth differentiation factor-15 (GDF-15) and galectin-3. There was a significant increase in sTEM1 levels in the cultured media of H9c2 and HCF after being stressed with hypoxia or TGF-β. The conditioned media derived from hypoxia-stimulated H9c2 cells significantly increased cell proliferation of cardiac fibroblasts. This effect was partially reversed by anti-TEM1 antibody. This pilot study demonstrated that cardiac TEM1 expression was upregulated in HF. The levels of sTEM1 were significantly higher in HF patients with LVEF ≤ 35% and correlated with other biomarkers of cardiac fibrosis. In vitro study proved that functional sTEM1 was released into cultured media after stressing cardiomyocytes and HCF.

Abstract 14265: Proteomic Discovery of Molecular Pathways in Patients With Biopsy-Proven Myocarditis

Introduction: Myocarditis is an inflammatory disease of the myocardium that results in injury to the cardiac myocytes. However, there is a lack of research focusing on its immune reaction. Also, most studies have been done through analysis of peripheral blood. Here, we aimed to identify proteome-based immunopathogenesis in patients with biopsy-proven myocarditis. Methods: Comparative proteomic analysis of right ventricle biopsy specimens was performed from six patients with myocarditis and four controls (normal endomyocardial biopsy specimens from one-year surveillance of heart transplant recipients) by tandem mass tag combined with liquid chromatography-mass spectrometry. Based on the results of protein expression, cell fractions were calculated using CIBERSORTx and Gene ontology (GO), and ingenuity pathway analysis was conducted to address immune features and related molecular mechanisms in myocarditis. Results: In total, 720 up-regulated proteins were screened. The top 10 GO terms in up-regulated proteins were mostly related to immune response. Also, the T follicular helper (Tfh) cells and plasma cell fractions were significantly increased in myocarditis. The ingenuity pathways analysis showed that the pathway for activation and immune response of immune cells included signaling of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) with other mediators including interferon regulatory factor (IRF) 1,5, 7 and signal transducer and activator of transcription 1 (STAT1). Conclusions: From mass spectrometry protein analysis, we revealed that Tfh cells and plasma cells play a major role in the development of immune response in myocarditis. Also, TNF-driven interferon response via STAT1 and IRFs can be the main pathways for B cell activation by Tfh cells. Further studies are necessary to validate whether these proteins correlate with the clinical characteristics or can be used as biomarkers for myocarditis.

Diagnosing myocarditis in endomyocardial biopsies: survey of current practice

BackgroundDallas criteria (DC) and European Society of Cardiology criteria (ESCC) have provided valuable frameworks for the histologic diagnosis and classification of myocarditis in endomyocardial biopsy (EMB) specimens. However, the adaptation and the usage of these criteria are variable and depend on local practice settings and regions/countries. Moreover, several ancillary tests that are not included in the current criteria, such as immunohistochemistry (IHC) or viral polymerase chain reaction (PCR), have proven useful for the diagnosis of myocarditis. MethodAs a joint effort from the Association for European Cardiovascular Pathology (AECVP) and the Society for Cardiovascular Pathology (SCVP), we conducted an online survey to understand the current practice of diagnosing myocarditis. ResultA total of 100 pathologists from 23 countries responded to the survey with the majority practicing in North America (45%) and Europe (45%). Most of the pathologists reported to examine less than 200 native heart biopsies per year (85%), and to routinely receive 3-5 fragments of tissue per case (90%). The number of hematoxylin-eosin-stained levels for each case varies from 1 to more than 9 levels, with 20% of pathologists routinely asking for more than 9 levels per case. Among the 100 pathologists, 52 reported to use the DC alone, 12 the ESCC alone, 28 both DC and ESCC and 8 reported to use neither the DC nor the ESCC. Overall, 80 pathologists reported to use the DC and 40 the ESCC. Use of DC alone is more common among North American pathologists compared to European ones (80% vs 32.6%) while use of ESCC alone is more common in Europe (20.9% vs 2.5%). IHC is utilized in either every case or selected cases by 79% of participants, and viral PCR is performed by 35% of participants. Variable terminologies are used in reporting, including both histological and clinical terms. The diagnosis of myocarditis is rendered even in the absence of myocyte injury (e.g., in cases of borderline or inactive/chronic myocarditis) by 46% respondents. The majority of the participants think it is time to update the current criteria (83%). ConclusionsThe survey data demonstrated that pathologists who render a myocarditis diagnosis practice with variable tissue preparation methods, use of ancillary studies, guideline usage, and reporting. This result highlights the clinically unmet need to update and standardize the current diagnostic criteria for myocarditis on EMB. Additional studies are warranted to establish standard of practice.

Role of immunosuppression in patients with lymphocytic myocarditis and myocardial parvovirus B19 with or without human herpesvirus 6 co-presence

Abstract Background Parvovirus B19 (B19V) and Human Herpesvirus 6 (HHV6) are commonly detected in endomyocardial biopsy (EMB) specimens of patients with myocarditis symptoms. Whether B19V- and HHV6-DNA belong to the cardiac bio-portfolio remains unclear [1,2]. Until today, the role of B19V-/HHV6-DNA presence in myocarditis is doubtful. Both viruses have been detected in the myocardium, even independent of cardiac inflammation. Yet, their contribution to myocarditis remains controversial. The European Society of Cardiology guidelines exclude the use of immunosuppression in patients with virus-associated myocarditis [3]. Whether myocarditis patients with the presence of B19V-DNA alone or with HHV6-DNA in EMB findings can be treated using immunosuppression, remains a delicate question for clinicians. Methods 931 patients with unexplained heart failure symptoms underwent EMB investigation to determine the underlying cause. Patients with low-levels (&amp;lt;1000 copies/μg DNA) of B19V-DNA and HHV6-DNA were identified. A sub-cohort of 28 patients who suffered from chronic-persistent lymphocytic myocarditis with ongoing symptoms was treated with azathioprine 100 mg once daily and prednisolone 1 mg/kg/day tapered down by 10 mg every two weeks followed by a second EMB. Twenty out of 28 patients had B19V-DNA only (mean LVEF=38%, age=47±15) and eight patients had B19V-/HHV6-DNA copresence (mean LVEF=39%, age=42±10). Patients with systemic infections were excluded. Both cohorts received standard heart failure medications. Continuous variables are expressed as mean±SD. Results B19V-DNA alone and in the presence of HHV6-DNA was detectable in the EMB of 377 and 63 patients, respectively. Following the immunosuppression course, the patients with B19V-DNA only and those with B19V-/HHV6-DNA showed complete resolution of inflammation in 12/20 and 5/8 patients, New York Heart Association (NYHA) functional class improvement in 9/20 and 4/8 patients, LVEF improvement by 8.0±13.8% (p&amp;lt;0.05) and 8.4±9.6% (p&amp;lt;0.05), and a reduction of LVEDD by 3.8±6.1mm (p&amp;lt;0.05) and 1.0±7.7mm (p&amp;gt;0.05), respectively. Importantly, following immunosuppression B19V and HHV6-DNA copy numbers went down from 186±266 to 130±186 copies/μg DNA and from 71±141 to 58±143 copies/μg DNA, respectively. Conclusion Chronic lymphocytic myocarditis patients with persistent B19V-DNA even in co-presence of HHV6-DNA may benefit from combined immunosuppression therapy. The therapy is clinically effective and safe to reduce cardiac inflammation independent of B19V- and HHV6-DNA copy numbers. In conclusion, we show for the first time that cases with chronic lymphocytic myocarditis can be principally treated with immunosuppression, despite B19V-/HHV6-DNA EMB presence. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Berlin Institute of Health-Center for Regenerative Therapies

Characterization of myocardial proteomics in biopsy proven cardiomyopathies

Abstract Background Difference in proteomic expression according to the etiology of cardiomyopathies is not well known. Purpose We aimed to identify proteome based pathogenesis in patients with biopsy proven cardiomyopathies. Methods Comparative proteomic analysis of biopsy specimens were performed from 9 patients with cardiomyopathy (3 dilated cardiomyopathy (DCM), 2 hypertrophic cardiomyopathy (HCM) and 4 myocarditis) as well as 5 controls (normal endomyocardial biopsy specimen from one-year surveillance of heart transplant recipients) by tandem mass tag combined with liquid chromatography-mass spectrometry. Differential expression protein analysis, gene ontology analysis and Ingenuity pathway analysis were done to discover molecular mechanism for the differentially expressed proteins in each cardiomyopathy compared to the control. Results Differential expression protein analysis showed higher proportion of significantly increased proteins (Log2 fold change ≥1) in HCM and myocarditis, whereas higher proportion of significantly decreased proteins (Log2 fold change ≤−1) in DCM compared to controls (Figure). According to the gene ontology analysis, upregulation of neutrophil degranulation, and down-regulation of mitochondrial translation protein was noted in patients with DCM. In patients with HCM, platelet degranulation protein was increased, and mitochondrial ATP synthesis coupled electron transport, was decreased. In patients with myocarditis, neutrophil related proteins and calcium ion binding protein was increased, but muscle cell development, protein was decreased. Ingenuity pathway analysis revealed downregulation of oxidative phosphorylation and upregulation of sirtuin signalling pathway both in DCM and HCM. In myocarditis, various pathways related to inflammation were upregulated with only RHO GDP dissociation inhibitors downregulated. Conclusions This study showed that each cardiomyopathy exhibited different proteomic expression compared to normal heart. Further large detailed study is needed to understand the association between proteomic expression and disease pathophysiology. Funding Acknowledgement Type of funding sources: None.

246.3: MicroRNA Tests for Complex Diagnostic of Acute Rejection And Bacterial Infection After Heart Transplantation

Introduction: The acute rejection and bacterial infections are the main risk factors of primary graft dysfunction and high mortality after heart transplantation (HTx). The search for new minimally invasive diagnostic methods and biomarkers of graft damage is extremely relevant. MicroRNAs are widely known as small molecules, regulating gene expression. Some of them (miR-27, -101, and -424) are involved in the mechanisms of cardiovascular diseases. The aim of the study is to determine the diagnostic value of miR-27, miR-101 and miR-424 levels for early post-transplant complications – heart graft acute rejection and gram-negative bacterial infections. Methods: The study enrolled 83 heart transplant recipients, aged 16 to 70 (48.6±10.9) years. The expression levels of miR-27, -101, and -424 were measured by PCR (Qiagen, USA) in plasma after HTx. Graft rejection was verified through morphological analysis of endomyocardial biopsy specimens; infection – through microbiological identification in blood culture. Results: The miR-424 level didn’t differ (p=0.47) in recipients with (n=39) and without acute graft rejection (n=44), however miR-27 and miR-101 levels are significantly lower in recipients with acute graft rejection than in recipients without (p=0.01 and p=0.02 resp.). By the way the diagnostic value of miR-27 and -101 and its’ threshold value for heart transplant acute rejection was found: when miR-27 expression level is below threshold value the relative risk of acute rejection is RR=1.8 [95% CI 1.13–3.01]; miR-101 – below threshold value RR=1.9 [95% CI 1.13–3.37]. For miR-27 the sensitivity (Se) and specificity (Sp) were 53.6% and 79.2%; for miR-101 – 64.3% and Sp=70.8% resp. (Fig. 1). MiR-27, -101 and -424 levels were determined in heart transplant recipients with bacterial infections after HTx (n=20). More than 50% of bacteria spectrum were gram-negative pathogenes: Аcinetobacter baumannii (55%), and Klebsiella pneumoniae (40%). MiR-27 and -101 didn’t differ in recipients with and without infection (p>0.05), but miR-424 level was significantly higher in heart transplant recipients with gram-negative bacteremia (p=0.02). When miR-424 level is above -5.72 fold change, the relative risk of bacteremia is RR=3.9 [95% CI 1.94–7.61]; Se=60.0%; Sp=89.2% (Fig. 2). Conclusion: The measurement of miR-27, -101, and -424 expression levels can be used as complex tests for monitoring the risk of acute rejection and bacterial infections after HTx.

Impact of Autophagy on Prognosis of Patients With Dilated Cardiomyopathy

BackgroundAutophagy is a cellular process that degrades a cell’s own cytoplasmic components for energy provision and to maintain a proper intracellular environment. Left ventricular reverse remodeling (LVRR) promises a better prognosis for patients with dilated cardiomyopathy (DCM). ObjectivesThe authors tested the hypothesis that autophagy is involved in LVRR and has prognostic value in the human failing heart. MethodsUsing left ventricular endomyocardial biopsy specimens from 42 patients with DCM (21 LVRR-positive and 21 LVRR-negative) and 7 patients with normal cardiac function (control), the authors performed immunohistochemistry and immunofluorescent labeling of LC3 and cathepsin D and electron microscopic observation in addition to general morphometry under light microscopy. ResultsThe clinical characteristics of LVRR-positive patients were similar to those of the LVRR-negative patients, except for pulmonary artery pressure and left atrial dimension. Morphometry under light microscopy did not differ among specimens from DCM patients, regardless of their LVRR status. Electron microscopy revealed that autophagic vacuoles (autophagosomes and autolysosomes) and lysosomes were abundant within cardiomyocytes from DCM patients. Moreover, cardiomyocytes from LVRR-positive patients contained significantly more autophagic vacuoles with higher autolysosome ratios and cathepsin D expression levels than cardiomyocytes from LVRR-negative patients. Logistic regression analysis adjusted for age showed that increases in autophagic vacuole number and cathepsin D expression were predictive of LVRR. DCM patients who achieved LVRR experienced fewer cardiovascular events during the follow-up period. ConclusionsThe authors show that autophagy is a useful marker predictive of LVRR in DCM patients. This provides novel pathologic insight into a strategy for treating the failing DCM heart.

Serial histopathologic assessment of fulminant myocarditis after the first mRNA COVID-19 vaccine dose.

A 77-year-old man with hypertension presented to the emergency department with fever and cardiogenic shock 8 days after receiving the first dose of the BNT16B2b2 mRNA COVID-19 vaccine (Pfizer-BioNTech). Nucleic acid amplification via polymerase chain reaction with a nasopharyngeal swab sample was negative for SARS-CoV-2. Electrocardiography showed atrial fibrillation with poor R progression and ST depressions in the precordial leads (Panel A). Chest radiography showed cardiomegaly and pulmonary congestion (Panel B). Echocardiography revealed severe left ventricular (LV) dysfunction (LV ejection fraction, 24%) (Panel C; Supplementary material online, Video S1). Laboratory tests showed marked elevation of troponin I (>50 ng/mL) and NT-proBNP levels (59 239 pg/mL). Coronary angiography identified no significant stenosis. Histopathologic analysis of endomyocardial biopsy specimens showed infiltration of numerous CD3-positive lymphocytes with myocyte degeneration and myocytolysis (Panel D). Tests for parvovirus, influenza virus, and other viruses were all...

Ultrastructural Changes in Mitochondria in Patients with Dilated Cardiomyopathy and Parvovirus B19 Detected in Heart Tissue without Myocarditis.

Understanding the meaning of parvovirus B19 (PB19V) in an etiology of dilated cardiomyopathy (DCM) is difficult. Viruses change the dynamics of the mitochondria by interfering with the mitochondrial process/function, causing the alteration of mitochondrial morphology. In this study, the ultrastructural changes in the mitochondria in endomyocardial biopsy (EMB) samples from patients with DCM and PB19V were determined. Methods: The PB19V evaluation was performed in EMB specimens by real-time PCR in 20 patients (age: 28 ± 6 years). The biopsy specimens were examined by histo- and immunohistochemistry to detect the inflammatory response. The ultrastructural features of the mitochondria were evaluated by electron microscopy. Results: The presence of PB19V in the heart tissue without the presence of inflammatory process, defined according to Dallas and immunohistochemical criteria, was associated with ultrastructural changes in the mitochondria. Distinctive ultrastructural pathologies were indicated, such as the presence of mitochondria in the vicinity of the expanded sarcoplasmic reticulum with amorphous material, blurred structure of mitochondria, interrupted outer mitochondrial membrane and mitophagy. Conclusions: Extending diagnostics with ultrastructural analysis of biopsy samples provides new knowledge of the changes associated with the presence of PB19V in the heart tissue. The observed changes can be a basis for searching for the damage mechanisms, as well as for new therapeutic solutions.