Endomyocardial Biopsy Samples Research Articles

Four cardiomyopathy patients with a heterozygous DSG2 p.Arg119Ter variant

DSG2, encoding desmoglein-2, is one of the causative genes of arrhythmogenic cardiomyopathy. We previously identified a homozygous DSG2 p.Arg119Ter stop-gain variant in a patient with juvenile-onset cardiomyopathy and advanced biventricular heart failure. However, the pathological significance and prevalence of the heterozygous DSG2 p.Arg119Ter variant remains uncertain. Here, we identified four unrelated patients with cardiomyopathy with heterozygous DSG2 p.Arg119Ter variants among 808 patients with nonischemic cardiomyopathy; the allele frequency was 0.0037, which is more than 50-fold greater than that reported in the general Japanese population. These patients were clinically diagnosed with arrhythmogenic right ventricular cardiomyopathy (Pt-1), dilated cardiomyopathy (DCM) after ventricular septum defect closure surgery (Pt-2), DCM (Pt-3), and end-stage hypertrophic cardiomyopathy (Pt-4). The patients also exhibited reduced left ventricular contractile function and varying clinical courses. Genetic analysis identified additional possible causative variants, DSG2 p.Arg292Cys in Pt-1 and BAG3 p.His166SerfsTer6 in Pt-3. Immunohistochemical analysis of endomyocardial biopsy samples revealed that the expression of not only desmoglein-2 but also desmoplakin was markedly reduced. Transmission electron microscopy revealed pale and fragmented desmosomes and widened gaps between intercalated discs in the myocardium. A microforce test using human cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) demonstrated reduced contractility in iPSC-CMs carrying a heterozygous truncating variant in DSG2. These data suggest that the DSG2 p.Arg119Ter variant is concealed in patients with cardiomyopathy with heart failure, and desmosome impairment may be a latent exacerbating factor of contractile dysfunction and disease progression.

In-depth morphological and functional analysis of ATTR-CA myocardial cells and niche by electron microscopic 3D reconstruction

Abstract Background Cell-cell interactions and interplay with extracellular matrix are known to be important in the steady state and pathological conditions, including wild-type transthyretin cardiac amyloidosis (wtATTR-CA). However, alterations of the microstructure of cardiomyocytes (CMs) and cardiac niches have not been well analyzed. Normal histological staining is usually limited in resolution, and 2D electron microscopy cannot capture stereoscopic images or the distribution of target particles and organelles in intra- and extracellular 3D space, so novel methods are being attempted. Methods and Results Using Serial Block Face-Scanning Electron Microscopy (SBF-SEM) and a 3D structural reconstruction system, we for the first time investigated the microstructure of human myocardial tissue from control hypertrophied heart (hypertensive heart disease) and wtATTR-CA patients. The microstructure of myocardial niche, cell morphology/numbers, and the distribution of intracellular organelles such as intercalated discs (ICDs) were compared to control endomyocardial biopsy (EMB) samples (2 controls vs 5 wtATTR-CA patients). In all wtATTR-CA samples, amyloid fibers were deposited originating around the capillary structures with various electron densities, compressing myocardial cells not only from the periphery but also dividing them from the center of the cell body, and CMs showed degeneration and dwarfing according to the severity of amyloid fiber deposition (Figure 1). The capillary structures showed degeneration and narrowing with stratified basement membrane, and rarefaction (3378±216.5 vs 1448±116.8/mm2, p<0.0001). Notably, the stromal cells (pericytes, fibroblasts, macrophages) were more likely buried by amyloid fibers than CMs, and their numbers were also reduced, indicating a loss of adjacent and intercellular interaction with capillaries and CMs (Fig. 2, 1892±194.4 vs 1189±107.4 mm2, p=0.0043). This method can also be used to analyze intracellular organelles and microstructures in CMs. In ATTR-CA, morphological abnormalities were observed in ICDs (Figure 3) and mitochondria. Conclusion In the ATTR-CM patients, there was a significant decrease in the number of capillaries in accordance with the deposition of myocardial amyloid fibers, as well as a decrease in stromal cells. These results support the hypothesis that amyloid fibril deposition not only causes chronic myocardial ischemia, but also reduces the number of niche cells, which play a supportive role for CM function and entirely exacerbate the damage. The SBF-SEM is an innovative tool for 3D imaging and quantitative analysis of myocardial ultrastructure and functions.Methods & Figure 1Figure 2 and Figure 3

Ser194Leu DSG2 mutation, associated with arrhythmogenic left ventricular cardiomyopathy and ventricular tachycardia.

Arrhythmogenic cardiomyopathy (AC) is an inherited cardiomyopathy characterized by fibro-fatty replacement of cardiomyocytes, leading to life-threatening ventricular arrhythmia and heart failure. Pathogenic variants of desmoglein2 gene (DSG2) have been reported as genetic etiologies of AC. In contrast, many reported DSG2 variants are benign or variants of uncertain significance. Correct genetic variant classification is crucial for determining the best medical therapy for the patient and family members. Pathogenicity of the DSG2 Ser194Leu variant that was identified by whole exome sequencing in a patient, who presented with ventricular tachycardia and was diagnosed with AC, was investigated by electron microscopy and immunohistochemical staining of endomyocardial biopsy sample. Electron microscopy demonstrated a widened gap in the adhering junction and a less well-organized intercalated disk region in the mutated cardiomyocytes compared to the control. Immunohistochemical staining in the proband diagnosed with AC showed reduced expression of desmoglein 2 and connexin 43 and intercalated disc distortion. Reduced expression of DSG2 and Connexin 43 were observed in cellular cytoplasm and gap junctions. Additionally, we detected perinuclear accumulation of DSG2 and Connexin 43 in the proband sample. Ser194Leu is a missense pathogenic mutation of DSG2 gene associated with arrhythmogenic left ventricular cardiomyopathy.

Failing to Make the Grade: Conventional Cardiac Allograft Rejection Grading Criteria Are Inadequate for Predicting Rejection Severity.

Cardiac allograft rejection is the leading cause of early graft failure and is a major focus of postheart transplant patient care. While histological grading of endomyocardial biopsy samples remains the diagnostic standard for acute rejection, this standard has limited diagnostic accuracy. Discordance between biopsy rejection grade and patient clinical trajectory frequently leads to both overtreatment of indolent processes and delayed treatment of aggressive ones, spurring the need to investigate the adequacy of the current histological criteria for assessing clinically important rejection outcomes. N=2900 endomyocardial biopsy images were assigned a rejection grade label (high versus low grade) and a clinical trajectory label (evident versus silent rejection). Using an image analysis approach, n=370 quantitative morphology features describing the lymphocytes and stroma were extracted from each slide. Two models were constructed to compare the subset of features associated with rejection grades versus those associated with clinical trajectories. A proof-of-principle machine learning pipeline-the cardiac allograft rejection evaluator-was then developed to test the feasibility of identifying the clinical severity of a rejection event. The histopathologic findings associated with conventional rejection grades differ substantially from those associated with clinically evident allograft injury. Quantitative assessment of a small set of well-defined morphological features can be leveraged to more accurately reflect the severity of rejection compared with that achieved by the International Society of Heart and Lung Transplantation grades. Conventional endomyocardial samples contain morphological information that enables accurate identification of clinically evident rejection events, and this information is incompletely captured by the current, guideline-endorsed, rejection grading criteria.

X-ray phase contrast imaging of endomyocardial biopsy samples preserved in formalin and embedded in paraffin – a comparison of tissue preparation methods

X-ray phase contrast imaging of endomyocardial biopsy samples preserved in formalin and embedded in paraffin – a comparison of tissue preparation methods

Selection and Interpretation of Molecular Diagnostics in Heart Transplantation.

The number of heart transplants performed annually in the United States and worldwide continues to increase, but there has been little change in graft longevity and patient survival over the past 2 decades. The reference standard for diagnosis of acute cellular and antibody-mediated rejection includes histologic and immunofluorescence evaluation of endomyocardial biopsy samples, despite invasiveness and high interrater variability for grading histologic rejection. Circulating biomarkers and molecular diagnostics have shown substantial predictive value in rejection monitoring, and emerging data support their use in diagnosing other posttransplant complications. The use of genomic (cell-free DNA), transcriptomic (mRNA and microRNA profiling), and proteomic (protein expression quantitation) methodologies in diagnosis of these posttransplant outcomes has been evaluated with varying levels of evidence. In parallel, growing knowledge about the genetically mediated immune response leading to rejection (immunogenetics) has enhanced understanding of antibody-mediated rejection, associated graft dysfunction, and death. Antibodies to donor human leukocyte antigens and the technology available to evaluate these antibodies continues to evolve. This review aims to provide an overview of biomarker and immunologic tests used to diagnose posttransplant complications. This includes a discussion of pediatric heart transplantation and the disparate rates of rejection and death experienced by Black patients receiving a heart transplant. This review describes diagnostic modalities that are available and used after transplant and the landscape of future investigations needed to enhance patient outcomes after heart transplantation.

18F-FDG/13N-ammonia cardiac PET findings in ATTR cardiac amyloidosis

18F-flurodeoxyglycose (FDG)/13N-ammonia positron emission tomography/computed tomography (PET/CT) is frequently utilized to evaluate cardiac sarcoidosis (CS) but findings can reflect other forms of myocardial inflammation or altered myocardial metabolic activity. Herein, we present five cases where cardiac PET findings suggested CS, but right ventricular endomyocardial biopsy samples revealed ATTR-type cardiac amyloidosis.

Evaluation of contemporary echocardiographic and histomorphology parameters in predicting mortality in patients with endomyocardial biopsy-proven cardiac AL amyloidosis.

This study examined the role of echocardiographic and cardiac histomorphology parameters in predicting mortality in patients with cardiac AL amyloidosis. Patients with endomyocardial biopsy-proven cardiac AL amyloidosis treated at MD Anderson Cancer Center between 6/2011 and 6/2020 were identified. Stored echocardiographic images and endomyocardial biopsy samples were processed for myocardial strain analysis and a detailed histomorphology characterization. Of 43 patients; 44% were women and 63% white. Median age was 65 years; 51% underwent stem cell transplantation (SCT). Thirty patients (70%) died during follow up (median follow up: 4.1 years). Lower LA strain (<13.5%) and absence of SCT as a time-varying covariate were significantly associated with increased risk of death in the multivariate cox regression analysis. Higher LV mass and lower RV tricuspid annular plane systolic excursion were associated with increased odds of having ≥5% interstitial amyloid deposition on biopsy in the multivariate logistic regression analysis. Lower LA strain independently predicted mortality in our cohort, and its performance in the routine assessment of AL amyloidosis may be beneficial. Furthermore, SCT for cardiac AL amyloidosis was associated with improved OS. These findings need to be confirmed by larger studies in the era of contemporary systemic therapies.

Persistent viral infections and their role in heart disease.

Viral infections are the culprit of many diseases, including inflammation of the heart muscle, known as myocarditis. Acute myocarditis cases have been described in scientific literature, and viruses, such as parvovirus B19, coxsackievirus B3, or more recently, SARS-CoV-2, were the direct cause of cardiac inflammation. If not treated, myocarditis could progress to dilated cardiomyopathy, which permanently impairs the heart and limits a person's lifespan. Accumulated evidence suggests that certain viruses may persist in cardiac tissue after the initial infection, which could open up the door to reactivation under favorable conditions. Whether this chronic infection contributes to, or initiates, cardiac damage over time, remains a pressing issue in the field of virus-induced heart pathology, and it is directly tied to patients' treatment. Previously, large case studies found that a few viruses: parvovirus B19, coxsackievirus, adenovirus, human herpesvirus 6, cytomegalovirus and Epstein-Barr virus, are most commonly found in human endomyocardial biopsy samples derived from patients experiencing cardiac inflammation, or dilated cardiomyopathy. SARS-CoV-2 infection has also been shown to have cardiovascular consequences. This review examines the role of viral persistence in cardiac inflammation and heart disease, and discusses its implications for patients' outcomes.

Abstract 12664: Comprehensive Histological Analysis of Endomyocardial Biopsy and T1 Mapping for Predicting Left Ventricular Reverse Remodeling in Patients with Recent-Onset Dilated Cardiomyopathy

Background: Cardiomyocyte injury is a key pathological pathway in the progression of heart failure that predicts a poor prognosis; therefore, combined assessment of myocardial fibrosis and cardiomyocyte DNA damage from endomyocardial biopsy (EMB) samples may be of great importance. Meanwhile, cardiovascular magnetic resonance (CMR) native T1 reflects a composite of both intra- and extracellular compartments in the whole myocardium. We sought to assess the performance of native T1 mapping and EMB results to predict left ventricular reverse remodeling (LVRR) in recent-onset dilated cardiomyopathy (DCM). Methods: A total of 29 patients with DCM underwent cine CMR and triple-slice T1 mapping using the MOLLI sequence at 3T, and EMB before receiving guideline-directed medical therapy. Poly ADP-ribose (PAR) nuclear and sirius-red staining were used for quantification of DNA damage and collagen volume fraction (CVF), respectively. LVRR was defined as an increase in LV ejection fraction of ≥10% to the final value of &gt;35%, accompanied by a decrease in LV end-diastolic volume ≥10% at the follow-up CMR. Results: Fifteen patients (52%) achieved LVRR. The mean native T1, histological CVF, and proportion of PAR-positive nuclei were 1376 ± 55 ms, 0.13 ± 0.05, 25 ± 15%, respectively. Native T1 correlated well with CVF (p=0.004) and trended to be higher in DCM patients with increased DNA damage (p=0.17). There was no significant association between CVF and %PAR-positive nuclei. Native T1 &lt;1400 ms provided 86% sensitivity and 67% specificity, with the C-statistic of 0.82 (95% CI 0.67-0.98) for predicting LVRR. By contrast, C-statistic of %PAR nuclei, CVF, and combined assessment of %PAR nuclei and CVF were 0.59, 0.61 and 0.66, respectively. Conclusion: CMR T1 mapping may be a promising strategy for noninvasive biopsy of the whole heart and provide a better prediction of LVRR in recent-onset DCM than comprehensive histological analysis of EMB.

Abstract 12022: Predictors of Mortality in Patients With Biopsy-Proven Cardiac AL Amyloidosis

Introduction: Cardiac involvement occurs in ~50% of patients with light-chain (AL) amyloidosis and conveys a poor prognosis. This study examined the role of histopathologic and echocardiographic parameters in predicting mortality. Methods: Patients with endomyocardial biopsy-proven cardiac AL amyloidosis treated at MD Anderson Cancer Center between 6/2011 and 6/2020 were identified. Stored echocardiographic images were processed for longitudinal strain (global [GLS], apical, mid, basal) as well as radial and circumferential strain of the left ventricle (LV), right ventricle (RV) free wall, left atrium (LA), and right atrium, using the Epsilon EchoInsight Software. A detailed histomorphologic characterization of stored endomyocardial biopsy samples was performed by a cardiac pathologist and scored as &lt; or &gt; 5% AL deposits. Results: We identified 43 patients; 44% were women and 63% white. Median age was 65 years (IQR 59-70). Twenty-two (51%) underwent stem cell transplantation (SCT). A total of 29 patients (67%) died during follow up period (median follow up: 4.2 years (95% CI, 2.8-8.6)). Median overall survival (OS) was 18 months (95% CI, 8.1-37). Lower LA GLS and absence of SCT as a time-varying covariate were significantly and independently associated with increased risk of death in the multivariable cox regression analysis (HR 3.8, 95% CI 1.4-10.2 for LA GLS &lt; 13.5 vs &gt;13.5, p=0.008 and 0.20, 0.06-0.65 for SCT vs no SCT, p&lt;0.001, respectively). Higher LV mass and lower RV TAPSE were associated with increased odds of having &gt;5% of interstitial amyloid deposition on biopsy in the multivariable logistic regression analysis (OR 5.5, 95% CI 1.1-26.8 for &gt;195.6g vs ≤195.6g, p=0.036 and 8.1, 1.2-54.3 for RV TAPSE&lt;1.6cm vs ≥1.6cm, p=0.032, respectively). Conclusions: LA GLS &lt; 13.5 was an independent predictor of mortality in our cohort, and its performance in the routine assessment of AL amyloidosis may be justified. Furthermore, SCT for cardiac AL amyloidosis was associated with improved OS. This finding needs to be confirmed by larger studies in the era of contemporary systemic therapies. Although LV mass and RV TAPSE were associated with higher interstitial amyloid deposition, histopathologic parameters did not predict mortality in this cohort.

Downregulation of Mannose-6-Phosphate Receptors in Fabry Disease Cardiomyopathy: A Potential Target for Enzyme Therapy Enhancement.

Background: The efficacy of enzyme replacement therapy (ERT) in mobilizing globotryaosylceramide (GB-3) from Fabry cardiomyocytes is limited. The mechanism involved is still obscure. Methods: Assessment of M6Pr, M6Pr-mRNA, and Ubiquitin has been obtained by Western blot analysis and real-time PCR of frozen endomyocardial biopsy samples, from 17 pts with FD, various degree of left ventricular hypertrophy, and maximal wall thickening (MWT) from 11.5 and 20 mm. The diagnosis and severity of FDCM followed definitions of GLA mutation, α-galactosidase A enzyme activity, cardiac magnetic resonance, and left ventricular endomyocardial biopsy with the quantification of myocyte hypertrophy and the extent of Gb-3 accumulation. All patients have received alpha or beta agalsidase for ≥3 years without a reduction in LV mass nor an increase in T1 mapping at CMR. Controls were surgical biopsies from 15 patients undergoing mitral valve replacement. Results: Protein analysis showed mean M6Pr in FDCM to be 5.4-fold lower than in a normal heart (4289 ± 6595 vs. 23,581 ± 4074, p = 0.0996) (p < 0.001): specifically, 9-fold lower in males, p = 0.009, (p < 0.001) and 3-fold lower in females, p = 0.5799, (p < 0.001) showing, at histology, a mosaic of normal and diseased cells. M6Pr-mRNA expression was normal, while ubiquitin showed an increase of 4.6 fold vs. controls (13,284 ± 1723 vs. 2870 ± 690, p = 0.001) suggesting that ubiquitin-dependent post-translational degradation is likely responsible for the reduction of M6Pr in FDCM. Conclusion: M6Pr expression is remarkably reduced in FDCM as a likely result of post-translational degradation. This may explain the reduced efficacy of ERT and be a therapeutic target for the enhancement of ERT activity.

Transthyretin Amyloid Cardiomyopathy: Impact of Transthyretin Amyloid Deposition in Myocardium on Cardiac Morphology and Function.

Background: Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is increasingly being recognized as a cause of left ventricular (LV) hypertrophy (LVH) and progressive heart failure in elderly patients. However, little is known about the cardiac morphology of ATTR-CM and the association between the degree of TTR amyloid deposition and cardiac dysfunction in these patients. Methods: We studied 28 consecutive patients with ATTR-CM and analyzed the relationship between echocardiographic parameters and pathological features using endomyocardial biopsy samples. Results: The cardiac geometries of patients with ATTR-CM were mainly classified as concentric LVH (96.4%). The relative wall thickness, a marker of LVH, tended to be positively correlated with the degree of non-cardiomyocyte area. The extent of TTR deposition was positively correlated with enlargement of the non-cardiomyocyte area, and these were positively correlated with LV diastolic dysfunction. Additionally, the extent of the area containing TTR was positively correlated with the percentage of cardiomyocyte nuclei stained for 8-hydroxy-2′deoxyguanosine, a marker of reactive oxygen species (ROS). ROS accumulation in cardiomyocytes was positively correlated with LV systolic dysfunction. Conclusion: Patients with ATTR-CM mainly displayed concentric LVH geometry. TTR amyloid deposition was associated with cardiac dysfunction via increased non-cardiomyocyte area and ROS accumulation in cardiomyocytes.

Anti-Thymocyte Globulin Protects Against Ischemia Reperfusion Injury in the Immediate Post Heart Transplant Period

<h3>Purpose</h3> Ischemia reperfusion injury after heart transplant is not uncommon. This may lead to rejection and endothelial cell dysfunction with subsequent development of cardiac allograft vasculopathy (CAV). It has been reported that the use of anti-thymocyte globulin (ATG) as induction therapy post-transplant may lessen the ischemia reperfusion injury that occurs to donor hearts. This has not been confirmed. <h3>Methods</h3> Between 2010 and 2020, we assessed 1082 heart transplant patients and divided them into those who received induction with ATG versus those who did not. In our program, ATG is given to patients who are sensitized or have baseline serum creatinine >2.0 mg/dL to delay initiation of tacrolimus. Weekly endomyocardial biopsy samples were reviewed for the first month after heart transplantation for ischemia reperfusion injury. First year outcomes included survival and freedom from CAV (stenosis ≥30%). <h3>Results</h3> Patients who underwent induction therapy with ATG had significantly greater freedom from any ischemia reperfusion injury noted on weekly endomyocardial biopsies in the first month post-transplant. There was no difference between the two groups in first-year survival and freedom from CAV. <h3>Conclusion</h3> ATG as induction therapy appears to provide protection against ischemia reperfusion injury in donor hearts. Longer follow-up is needed to show potential outcomes benefit. This finding should be confirmed in a randomized clinical trial.

Single-Nuclear RNA-Sequencing Identifies Cell-Specific Transcriptional Programs in Cardiac Allograft Vasculopathy

<h3>Purpose</h3> Cardiac allograft vasculopathy (CAV) is the leading cause of late graft failure and mortality after heart transplantation (HT). Current therapies have limited efficacy in preventing or stalling the progression of CAV. Improved understanding of CAV biology is needed to identify novel therapeutics. <h3>Methods</h3> We performed single-nuclear RNA sequencing (snRNA-seq) on human cardiac tissue obtained by endomyocardial biopsy from the right ventricle of 3 HT recipients without CAV and at the time of heart explant in 3 HT recipients with severe CAV. Differentially expressed genes (DEGs) between cohorts were identified with DESeq2 using a 5% false discovery rate threshold for significant genes. <h3>Results</h3> Unsupervised clustering of 38,211 nuclei identified 15 major cell types (Figure A). Relative to cells from non-CAV hearts, endothelial cells and fibroblasts from hearts with severe CAV showed enrichment for genes and pathways associated with inflammation, angiogenesis, and extracellular matrix organization (Figure B and C; endothelial cells). Intersecting these DEGs with the druggable genome identified novel cell-specific drug targets. <h3>Conclusion</h3> We show feasibility of performing snRNA-seq from human endomyocardial biopsy samples and identify cell-specific transcriptional programs that are unique to patients with severe CAV. These data lay the groundwork for larger-scale studies that can accelerate mechanistic understanding of CAV development.

Utility of Intracardiac Echocardiography to Biopsy Cardiac Tumors

<h3>Introduction</h3> Primary cardiac tumors are extremely rare and associated with poor prognosis, mainly due to its nonspecific symptoms and delayed diagnosis. Tissue diagnosis is the gold standard which often requires open-chest procedures, which itself has a high rate of complications. Alternatives include emerging utilization of intracardiac echocardiography (ICE) - guided biopsy which commonly used for atrial septal closure and advanced electrophysiology procedures, but can be ideally suited for imaging right heart structures. <h3>Case Report</h3> A 62-year-old female with history of left forearm peripheral sheath sarcoma with transhumeral amputation, initially presented for post-MVA care where the CT chest revealed two lung nodules of 1 cm. She was discharged in stable condition and presented a month later for palpitations and diffuse abdominal pain. A repeat chest CT revealed a 5 × 4 c.m. right atrial mass with large pericardial effusion. Pericardiocentesis of 500 cc fluid had negative cytology and TTE showed right atrial free wall mass extending into the tricuspid annulus and into the right atrial cavity. Patient was referred to us for further diagnostic management given the complexity of the cardiac mass. She underwent intracardiac echocardiography with right femoral vein access under local anesthesia. The ICE catheter was advanced through the 8 French sheaths and key images were obtained of the right atrial mass to guide bioptome targeting. A 1.85mm/2.46 mm3 Procure bioptome was advanced under direct fluoroscopic guidance into the right atrium and 3 right ventricular endomyocardial biopsy samples were obtained. Patient tolerated the procedure well without any immediate postoperative complications. The biopsy results showed tissue composed of spindle cells with hyperchromatic pleomorphic nuclei and high mitotic ratio (score 3), changes that are consistent with unclassified high-grade sarcoma. However, patient passed away due to disease progression over the course of the next few months. <h3>Summary</h3> In our study, we demonstrated the feasibility of utilizing ICE for intracardiac mass biopsy on the right side of the heart. It is an innovative and well-described procedure for intracardiac mass that avoids usage of general anesthesia, avoids radiation exposure to the patients and the physicians, and decreases the complications rate as compared to alternate options such as open-chest procedures.

Bioinformatics Identification of Candidate Biomarkers in Endomyocardial Biopsy and Peripheral Blood for Cardiac Allograft Rejection.

BackgroundCardiac allograft rejection is still a crucial barrier to achieving satisfactory outcomes after surgery. In this study, we propose to find candidate biomarkers from endomyocardial biopsy (EMB) and peripheral blood (PB) samples for efficient diagnosis and treatment of cardiac allograft rejection.Material/MethodsMicroarray datasets were obtained from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) of cardiac allograft rejection patients and control subjects from EMB and PB samples were screened using the online tool GEO2R. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of all samples’ DEGs were performed with the DAVID online tool. Protein–protein interaction (PPI) networks were constructed and visualized using Cytoscape and the top 10 hub genes were selected. Finally, the most highly enriched GO and KEGG pathways of the top 10 hub genes were determined.ResultsA total of 57 502 genes from EMB samples and 131 624 genes from PB samples were identified. Gene characteristics and enrichment analysis indicated that both EMB and PB samples contained DEGs involved in antigen presentation, immune cells activation, inflammatory process, and cellular injuries. In EMB samples, there were some DEGs related to heart tissue injury and cardiac malfunction. Moreover, DEGs that regulates hypoxia-induced factors and erythrocyte function in response of ischemia and hypoxia stress were present in PB samples but were absent in EMB samples.ConclusionsThe screened differentially expressed genes (DEGs) from EMB and PB samples of patients with cardiac graft rejection are potential candidate biomarkers of diagnosis and treatment.

Serum autotaxin as a novel prognostic marker in patients with non-ischaemic dilated cardiomyopathy.

AimsAutotaxin (ATX) promotes myocardial inflammation, fibrosis, and the subsequent cardiac remodelling through lysophosphatidic acid production. However, the prognostic impact of serum ATX in non‐ischaemic dilated cardiomyopathy (NIDCM) has not been clarified. We investigated the prognostic impact of serum ATX in patients with NIDCM.Methods and resultsWe enrolled 104 patients with NIDCM (49.8 ± 13.4 years, 76 men). We divided the patients into two groups using different cutoffs of median serum ATX levels for men and women: high‐ATX group and low‐ATX group. Cardiac events were defined as a composite of cardiac death and heart failure resulting in hospitalization. Median ATX level was 203.5 ng/mL for men and 257.0 ng/mL for women. Brain natriuretic peptide levels [224.0 (59.6–689.5) pg/mL vs. 96.5 (40.8–191.5) pg/mL, P = 0.010] were higher in the high‐ATX group than low‐ATX group, whereas high‐sensitivity C‐reactive protein and collagen volume fraction levels in endomyocardial biopsy samples were not significantly different between the two groups. Kaplan–Meier survival analysis revealed that the event‐free survival rate was significantly lower in the high‐ATX group than low‐ATX group (log‐rank; P = 0.007). Cox proportional hazard analysis revealed that high‐ATX was an independent determinant of composite cardiac events. In both sexes, serum ATX levels did not correlate with high‐sensitivity C‐reactive protein levels and collagen volume fraction but had a weak correlation with brain natriuretic peptide levels (men; spearman's rank: 0.274, P = 0.017, women; spearman's rank: 0.378, P = 0.048).ConclusionHigh serum ATX levels can be associated with increasing adverse clinical outcomes in patients with NIDCM. These results indicate serum ATX may be a novel biomarker or therapeutic target in NIDCM.

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.

Immunofluorescent Localization of Plakoglobin Is Altered in Endomyocardial Biopsy Samples from Dogs with Clinically Relevant Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC)

Diagnosing the early stages of canine Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is complicated by day-to-day arrhythmia variability, and absence of reliable, transthoracic echocardiographic features. Definitive diagnosis requires histopathologic identification of transmural fibrofatty replacement of the right ventricle. Reduction of immunofluorescent signal for plakoglobin (PG) at the intercalated disc (ID) is reported in ARVC-affected humans and boxers. Our objective was to determine whether reduced immunofluorescent signal for PG in endomyocardial biopsy samples (EMBs) correspond with a histopathologic diagnosis of ARVC. Here, 49 dogs were evaluated: 43 with advanced cardiac disease and 6 non-clinical boxers with mild to moderate ventricular arrhythmia (VA) burden. EMBs were obtained from all dogs; samples were prepared with antibodies recognizing cadherin (PC) and PG and evaluated with confocal microscopy. Investigators were blinded to breed and clinical status. ARVC was histopathologically diagnosed in 8 out of 49 dogs. Of these, three out of eight had clinical signs consistent with ARVC (two boxers, one English bulldog) and reduced PG signal at ID; five out of eight were non-clinical boxers with moderate VA and no reduction in PG. A total of 41 out of 49 dogs were histopathologically diagnosed with non-ARVC cardiac disease; 1 out of 41 showed reduction of PG at ID, while 40 out of 41 had no PG reduction. These results suggest that EMB PG signal is reduced in dogs with advanced ARVC, but not in the occult phase of the disease. Additionally, presence of PG at ID supports a diagnosis of non-ARVC cardiac disease in dogs with clinical signs. These results may offer an additional test that helps differentiate advanced ARVC from other myocardial diseases.