Invasive Endomyocardial Biopsy Research Articles

Development of a Flexible Sensor-Integrated Tissue Patch to Monitor Early Organ Rejection Processes Using Impedance Spectroscopy.

Heart failure represents a primary cause of hospitalization and mortality in both developed and developing countries, often necessitating heart transplantation as the only viable recovery path. Despite advances in transplantation medicine, organ rejection remains a significant post-operative challenge, traditionally monitored through invasive endomyocardial biopsies (EMB). This study introduces a rapid prototyping approach to organ rejection monitoring via a sensor-integrated flexible patch, employing electrical impedance spectroscopy (EIS) for the non-invasive, continuous assessment of resistive and capacitive changes indicative of tissue rejection processes. Utilizing titanium-dioxide-coated electrodes for contactless impedance sensing, this method aims to mitigate the limitations associated with EMB, including procedural risks and the psychological burden on patients. The biosensor's design features, including electrode passivation and three-dimensional microelectrode protrusions, facilitate effective monitoring of cardiac rejection by aligning with the heart's curvature and responding to muscle contractions. Evaluation of sensor performance utilized SPICE simulations, scanning electron microscopy, and cyclic voltammetry, alongside experimental validation using chicken heart tissue to simulate healthy and rejected states. The study highlights the potential of EIS in reducing the need for invasive biopsy procedures and offering a promising avenue for early detection and monitoring of organ rejection, with implications for patient care and healthcare resource utilization.

Outcome-based Risk Assessment of Non-HLA Antibodies in Heart Transplantation: A Systematic Review

Current monitoring after heart transplantation (HT) employs repeated invasive endomyocardial biopsies (EMB). Although positive EMB confirms rejection, EMB fails to predict impending, subclinical, or EMB-negative rejection events. While non-human leukocyte antigen (non-HLA) antibodies have emerged as important risk factors for antibody-mediated rejection after HT, their use in clinical risk stratification has been limited. A systematic review of the role of non-HLA antibodies in rejection pathologies has the potential to guide efforts to overcome deficiencies of EMB in rejection monitoring. Databases were searched to include studies on non-HLA antibodies in HT recipients. Data collected included the number of patients, type of rejection, non-HLA antigen studied, association of non-HLA antibodies with rejection, and evidence for synergistic interaction between non-HLA antibodies and donor-specific anti-human leukocyte antigen antibody (HLA-DSA) responses. A total of 56 studies met the inclusion criteria. Strength of evidence for each non-HLA antibody was evaluated based on the number of articles and patients in support versus against their role in mediating rejection. Importantly, despite previous intense focus on the role of anti-major histocompatibility complex class I chain-related gene A (MICA) and anti-angiotensin II type I receptor antibodies (AT1R) in HT rejection, evidence for their involvement was equivocal. Conversely, the strength of evidence for other non-HLA antibodies supports that differing rejection pathologies are driven by differing non-HLA antibodies. This systematic review underscores the importance of identifying peri-HT non-HLA antibodies. Current evidence supports the role of non-HLA antibodies in all forms of HT rejection. Further investigations are required to define the mechanisms of action of non-HLA antibodies in HT rejection.

Abstract 16450: Increased Monocyte Count and Neutrophil-to-Lymphocyte Ratio With ICI Myocarditis

Introduction: Diagnosing immune checkpoint inhibitor (ICI) myocarditis remains complex in practice, often requiring advanced imaging or invasive endomyocardial biopsy. We constructed an institutional cohort of patients who received at least 1 dose of ICI and developed probable or definite ICI myocarditis. Hypothesis: We hypothesized that routine serum biomarkers, including troponin I, neutrophil-to-lymphocyte ratio (NLR), monocytes, and AST/ALT, would be elevated and peak at diagnosis. Methods: This retrospective cohort study used EHR data from patients on ICI at a single academic institution who developed probable or definite myocarditis from 7/2015 - 1/2023. Inclusion criteria and diagnosis date were defined using Bonaca criteria and/or documented clinical consensus, e.g. initiation of steroids for presumed myocarditis. Primary outcomes were levels of troponin I/high sensitivity troponin I, monocytes, NLR, and AST/ALT. LOESS regression with default smoothing parameter α = 0.75 was used to evaluate biomarker patterns 7 days pre- and post- diagnosis. Results: We included 16 cases of myocarditis. At diagnosis, most patients had at least 1 significant elevation in troponin I (100%), AST (81%), ALT (63%), or monocytes (69%) while on ICI (Figure 1). After diagnosis, absolute monocyte and lymphocyte count initially decreased before rebounding. Serum NLR rose prior to and briefly after diagnosis (Figure 2). Conclusions: Peripheral cardiac and non-cardiac biomarkers change with ICI myocarditis. Further research on routine non-cardiac biomarkers associated with myocarditis and other ICI toxicities may help prevent high-grade adverse events.

Abstract 15922: Integrative Analysis of Extracellular Vesicles in Heart Transplant Rejection: From Biomarker Discovery to Novel Immune Mechanisms

Introduction: At the forefront of post-heart transplant care is early detection of transplant rejection. However, the current gold standard for detecting acute cellular transplant rejection is recurrent invasive endomyocardial biopsies (EMB). We aimed at developing a non-invasive biomarker for acute cellular heart transplant rejection (ACR) using a plasma extracellular vesicle (EV) platform. Methods: Peripheral and coronary sinus plasma samples were prospectively collected from 183 (31.7% female) patients from 2020 to 2023 during routine surveillance EMB. A discovery cohort comprised five patients who displayed ISHLT 0-1R biopsy followed by an ISHLT 2R grade rejection event. Plasma EVs were isolated from the baseline (0-1R) and rejection (2R) time points. Subsequently, integrative analysis of plasma EV proteomic, miRNomic, and metabolomics was performed pairwise with the samples. Results: The study collected 943 sample time points from 183 patients. Of these sample collections, 43 (4.5%) cases were 2R rejections. Our proteomic analysis of the plasma EVs identified 28 proteins found explicitly during a grade 2R rejection and 87 differentially expressed proteins during a 2R rejection event. An Ingenuity Pathway Analysis (IPA) analysis of the upregulated proteins highlighted EVs mediated mTOR signaling and antigen presentation as central to rejection events. Although all patients in the discovery cohort were negative for antibody-mediated rejection (AMR), the proteomic findings also revealed increased B-cell receptor signaling, implying a mechanistic intersection of ACR and AMR. Conclusions: A distinct set of proteins were found in plasma EVs during ACR events, providing potentially specific and sensitive biomarkers to reduce the need for EMB that could be exploited for ACR diagnosis. The proteins identified formed an immune signaling network that suggests a novel role for EVs in ACR pathogenesis.

Linear discriminant analysis on electrocardiogram achieved classification of cardiac involvement status in amyloid light-chain amyloidosis

ObjectivesCardiac amyloidosis (CA) is the most crucial determinant of amyloid light-chain (AL) amyloidosis patients' prognosis. We attempted cardiac involvement prediction by 12‑lead electrocardiograph (ECG) and echocardiography (UCG) in AL amyloidosis patients. Materials and methodsFifty patients with histologically confirmed AL amyloidosis underwent gadolinium-enhanced magnetic resonance imaging (Gd-MRI), and CA was assessed using late gadolinium enhancement. ECG and UCG parameters were measured on admission. Fisher's linear discriminant analysis was used to create a model for predicting CA using the ECG and UCG parameters. ResultsPrediction by five ECG parameters [QTc(B), QRS-T-angle, III-QRS, aVF-QRS, and V3-R] showed the best performance. Average sensitivity and specificity in the modeling sets, utilizing a linear discriminator based on these five variables, were 99.2 % and 96.8 % and in validation sets, 94.2 % and 90.3 %, respectively. In addition, we tested this model on an additional 26-patient cohort and survival analysis using the Kaplan-Meier method, and significant differences between CA positively predicted and negatively predicted patients were observed. ConclusionHere, we suggest the application of a condensed classical multivariate statistical technique for the diagnosis of CA. It can be used as a guide to invasive endomyocardial biopsy for those in whom Gd-MRI is contraindicated and as a guide for repeat Gd-MRI in follow-up of AL amyloidosis.

Use of Donor Derived Cell Free DNA for Long Term Rejection Surveillance in OHT Recipients

IntroductionThe advent of donor-derived cell free DNA (dd cf DNA) testing has transformed care for orthotopic heart transplant (OHT) recipients by reducing the number of invasive endomyocardial biopsies (EMB) for surveillance of acute rejection (AR). The dd cf DNA is elevated in any form of graft injury ranging from AR to ischemic injury from cardiac allograft vasculopathy (CAV). When used in combination with gene expression profiling, the AUC for the combination to rule out acute rejection is about 80.1% However, all the data about using dd cf DNA for AR surveillance is from studies done in the first few years after OHT. There is no data or recommendation on using dd cf DNA in long-term OHT survivors (>5 years). Here we present an interesting case of acute rejection detected by dd cf DNA in a patient who is 8 years post OHT.Case Report55 yo African American male with a history of OHT for non ischemic cardiomyopathy in 2013. He had no previous history of AR or CAV. As part of his routine post-transplant annual work-up, dd cf DNA was checked and it came back elevated at 0.95 % (Ref: >0.25% - significantly elevated, >1% - high probability of AR). Donor specific antibodies were negative. Echocardiogram revealed an ejection fraction of 55-60%. He had a right heart catheterization, which showed normal to low filling pressures and preserved cardiac index. EMB revealed Grade 1R cellular rejection as well as swollen endothelial cells, and >50% capillary positive staining with C4d consistent with acute antibody-mediated rejection (pAMR2). He was successfully treated with plasmapheresis/IVIG x 5 cycles and Rituximab. On further probing, the patient admitted to missing a few doses of his immunosuppression (IS) medications.SummaryThis case highlights the successful use of dd cf DNA for allograft rejection surveillance several years (>5 years) after OHT. However, there is sparse data in this population. Further studies are needed in large patient groups to assess the validity and reproducibility of dd cf DNA in long-term surveillance of rejection. The advent of donor-derived cell free DNA (dd cf DNA) testing has transformed care for orthotopic heart transplant (OHT) recipients by reducing the number of invasive endomyocardial biopsies (EMB) for surveillance of acute rejection (AR). The dd cf DNA is elevated in any form of graft injury ranging from AR to ischemic injury from cardiac allograft vasculopathy (CAV). When used in combination with gene expression profiling, the AUC for the combination to rule out acute rejection is about 80.1% However, all the data about using dd cf DNA for AR surveillance is from studies done in the first few years after OHT. There is no data or recommendation on using dd cf DNA in long-term OHT survivors (>5 years). Here we present an interesting case of acute rejection detected by dd cf DNA in a patient who is 8 years post OHT. 55 yo African American male with a history of OHT for non ischemic cardiomyopathy in 2013. He had no previous history of AR or CAV. As part of his routine post-transplant annual work-up, dd cf DNA was checked and it came back elevated at 0.95 % (Ref: >0.25% - significantly elevated, >1% - high probability of AR). Donor specific antibodies were negative. Echocardiogram revealed an ejection fraction of 55-60%. He had a right heart catheterization, which showed normal to low filling pressures and preserved cardiac index. EMB revealed Grade 1R cellular rejection as well as swollen endothelial cells, and >50% capillary positive staining with C4d consistent with acute antibody-mediated rejection (pAMR2). He was successfully treated with plasmapheresis/IVIG x 5 cycles and Rituximab. On further probing, the patient admitted to missing a few doses of his immunosuppression (IS) medications. This case highlights the successful use of dd cf DNA for allograft rejection surveillance several years (>5 years) after OHT. However, there is sparse data in this population. Further studies are needed in large patient groups to assess the validity and reproducibility of dd cf DNA in long-term surveillance of rejection.

Diagnosis of Cardiac Involvement in Amyloid A Amyloidosis by Cardiovascular Magnetic Resonance Imaging.

Background: Diagnosis of cardiac involvement in amyloid A (AA) amyloidosis is challenging since AA amyloidosis is a rare disease and cardiac involvement even less frequent. The diagnostic yield of currently available non-invasive imaging methods is not well-studied and rather limited, and invasive endomyocardial biopsy (EMB) is rarely performed due to the potential risk of this procedure. Cardiovascular magnetic resonance (CMR)-based myocardial tissue characterization by late-gadolinium-enhancement (LGE) imaging and novel-mapping approaches may increase the diagnostic yield in AA amyloidosis.Methods: Two patients with AA amyloidosis in whom cardiac involvement was suspected based on CMR findings and subsequently proven by biopsy work-up are presented. CMR studies were performed on a 1.5-T system and comprised a cine steady-state free precession pulse sequence for ventricular function and a late-gadolinium-enhancement (LGE) sequence for detection of myocardial pathology. Moreover, a modified Look-Locker inversion recovery (MOLLI) T1-mapping sequence was applied in basal, mid and apical short-axes prior to contrast agent administration and ~20 min thereafter to determine native T1 and ECV values.Results: Both patients showed slightly dilated left ventricles (LV) with mild to moderate LV hypertrophy and preserved systolic function. Only a very subtle pattern of LGE was observed in both patients with AA amyloidosis. However, markedly elevated native T1 (max. 1,108 and 1,112 ms, respectively) and extracellular volume fraction (ECV) values (max. 39 and 48%, respectively) were measured in the myocardium suggesting the presence of cardiac involvement - with subsequent EMB-based proof of AA amyloidosis.Conclusion: We recommend a multi-parametric CMR approach in patients with AA amyloidosis comprising both LGE-based contrast-imaging and T1-mapping-based ECV measurement of the myocardium for non-invasive work-up of suspected cardiac involvement. The respective CMR findings may be used as gatekeeper for additional invasive procedures (such as EMB) and as a non-invasive monitoring tool regarding assessment and modification of ongoing treatments.

Cardiac Amyloidosis: Diagnostic Tools for a Challenging Disease

Amyloidosis is a group of diseases in which amyloid fibrils build up in tissues, leading to organ dysfunction. Cardiac involvement is observed in immunoglobulin light chain amyloidosis (AL) and transthyretin amyloidosis (ATTR) and, when it occurs, the prognosis worsens. Cardiac tissue infiltration can lead to restrictive cardiomyopathy with clinical signs of diastolic heart failure, without reduction of ejection fraction (HFpEF). The aim of multiple and less invasive diagnostic tests is to discern peculiar characteristics and reach the diagnosis without performing an invasive endomyocardial biopsy. These diagnostic tools allow early diagnosis, and they are crucial to best benefit from target therapy. In this review article, we describe the mechanism behind amyloid fibril formation, infiltration of tissues, and consequent clinical signs, focusing on the diagnostic tools and the red flags to obtain a diagnosis.

The combination of carboxy-terminal propeptide of procollagen type I blood levels and late gadolinium enhancement at cardiac magnetic resonance provides additional prognostic information in idiopathic dilated cardiomyopathy-A multilevel assessment of myocardial fibrosis in dilated cardiomyopathy.

AimsTo determine the prognostic value of multilevel assessment of fibrosis in dilated cardiomyopathy (DCM) patients.Methods and resultsWe quantified fibrosis in 209 DCM patients at three levels: (i) non‐invasive late gadolinium enhancement (LGE) at cardiac magnetic resonance (CMR); (ii) blood biomarkers [amino‐terminal propeptide of procollagen type III (PIIINP) and carboxy‐terminal propeptide of procollagen type I (PICP)], (iii) invasive endomyocardial biopsy (EMB) (collagen volume fraction, CVF). Both LGE and elevated blood PICP levels, but neither PIIINP nor CVF predicted a worse outcome defined as death, heart transplantation, heart failure hospitalization, or life‐threatening arrhythmias, after adjusting for known clinical predictors [adjusted hazard ratios: LGE 3.54, 95% confidence interval (CI) 1.90–6.60; P < 0.001 and PICP 1.02, 95% CI 1.01–1.03; P = 0.001]. The combination of LGE and PICP provided the highest prognostic benefit in prediction (likelihood ratio test P = 0.007) and reclassification (net reclassification index: 0.28, P = 0.02; and integrated discrimination improvement index: 0.139, P = 0.01) when added to the clinical prediction model. Moreover, patients with a combination of LGE and elevated PICP (LGE+/PICP+) had the worst prognosis (log‐rank P < 0.001). RNA‐sequencing and gene enrichment analysis of EMB showed an increased expression of pro‐fibrotic and pro‐inflammatory pathways in patients with high levels of fibrosis (LGE+/PICP+) compared to patients with low levels of fibrosis (LGE‐/PICP‐). This would suggest the validity of myocardial fibrosis detection by LGE and PICP, as the subsequent generated fibrotic risk profiles are associated with distinct cardiac transcriptomic profiles.ConclusionThe combination of myocardial fibrosis at CMR and circulating PICP levels provides additive prognostic value accompanied by a pro‐fibrotic and pro‐inflammatory transcriptomic profile in DCM patients with LGE and elevated PICP.

The Safety of a Non-Invasive Monitoring Protocol in Heart Transplant Recipients

<h3>Purpose</h3> Heart transplant (HT) recipients require monitoring for allograft rejection with frequent endomyocardial biopsies (EMB). The aim of this study was to evaluate the safety of transitioning heart transplant recipients to a non-invasive monitoring protocol utilizing HeartCare (combination of gene expression profiling [Allomap] and donor derived cell free DNA [Allosure]). <h3>Methods</h3> This is a prospective single-center cohort study evaluating the utilization of HeartCare from as early as one month post-HT. Positive Allomap test was defined as ≥32 in patients ≤6 months and ≥34 in patients > 6 months post-HT. Positive Allosure test was defined as ≥0.12%. Positive biopsy included ISHLT Grades ≥1B. <h3>Results</h3> 320 heart transplant recipients enrolled to this study. Mean age was 57.5±14.0, 231 were males (72.0%). There was a total of 523 HeartCare blood tests drawn and 148 biopsies were performed (Figure). Of 82 patients with elevated Allomap and Allosure, only 7 (8.5%) of them had a confirmed rejection episode. Among 71 patients with elevated Allosure with normal Allomap, only 2 (2.8%) patients had confirmed rejection. 2 patients (4.3%) with negative Allomap and Allosure had rejection. During the study period 55 patients (17.2%) were admitted to the hospital for a total of 67 admissions. 17 patients were admitted for rejection (5.3%). None of the patients died during the study period. <h3>Conclusion</h3> Transitioning from invasive endomyocardial biopsy monitoring to a non-invasive HeartCare protocol in HT recipients seems feasible based on the high negative value of the combined Allomap and Allosure tests. However, further study is needed to follow the long-term outcomes and presence of rejection in patients post-transplant with this protocol.

Longitudinal profiling of circulating miRNA during cardiac allograft rejection: a proof-of-concept study.

AimsAllograft rejection following heart transplantation (HTx) is a serious complication even in the era of modern immunosuppressive regimens and causes up to a third of early deaths after HTx. Allograft rejection is mediated by a cascade of immune mechanisms leading to acute cellular rejection (ACR) and/or antibody‐mediated rejection (AMR). The gold standard for monitoring allograft rejection is invasive endomyocardial biopsy that exposes patients to complications. Little is known about the potential of circulating miRNAs as biomarkers to detect cardiac allograft rejection. We here present a systematic analysis of circulating miRNAs as biomarkers and predictors for allograft rejection after HTx using next‐generation small RNA sequencing.Methods and resultsWe used next‐generation small RNA sequencing to investigate circulating miRNAs among HTx recipients (10 healthy controls, 10 heart failure patients, 13 ACR, and 10 AMR). MiRNA profiling was performed at different time points before, during, and after resolution of the rejection episode. We found three miRNAs with significantly increased serum levels in patients with biopsy‐proven cardiac rejection when compared with patients without rejection: hsa‐miR‐139‐5p, hsa‐miR‐151a‐5p, and hsa‐miR‐186‐5p. We identified miRNAs that may serve as potential predictors for the subsequent development of ACR: hsa‐miR‐29c‐3p (ACR) and hsa‐miR‐486‐5p (AMR). Overall, hsa‐miR‐486‐5p was most strongly associated with acute rejection episodes.ConclusionsMonitoring cardiac allograft rejection using circulating miRNAs might represent an alternative strategy to invasive endomyocardial biopsy.

Eosinophilic myocarditis: Is endomyocardial biopsy essential for diagnosis in the era of advanced cardiac imaging?

A 60-year-old female presented with dyspnea and chest pressure. Clinical presentation, laboratory data, echocardiography, and cardiac magnetic resonance (CMR) imaging findings confirmed diagnosis of eosinophilic myocarditis and obviated unnecessary invasive endomyocardial biopsy. She was treated with oral steroid and oral anticoagulation. Follow-up CMR imaging showed resolution of the left ventricle thrombus with improvement in endomyocardial inflammation.

Induction of Nrg1 and ErbB4 Is Specific to Virus Induced Injury of the Myocardium and May be Detected during Pathogenesis of Viral Myocarditis as Blood‐Based Biomarkers for Diagnosis

BackgroundMyocarditis, inflammation of the myocardium, globally affects &gt;3 million people annually. Heterogeneous histological findings and clinical presentation ranging from flu‐like illness to acute cardiogenic shock make diagnosis exceedingly difficult. The current gold standard for diagnosis requires histological examination of invasive endomyocardial biopsies, which provides a sensitivity of &lt;30% in independently published studies. Although etiologies are diverse, viruses are the most prominent causes of myocarditis. Our studies demonstrated that the coxsackievirus B3 (CVB3), a common pathogen associated with viral myocarditis, manipulates the expression of Neuregulin 1 (Nrg1) and its ligand, receptor tyrosine‐protein kinase, ErbB4. Moreover, previous studies in other viruses have demonstrated preferential expression of the higher affinity Nrg1β versus the less potent Nrg1α isoform. The goal of this research is to determine the specificity of Nrg1 and ErbB4 induction to viral myocarditis as compared to other forms of acute myocardial injury, particularly myocardial infarction (m.i.). Additionally, we aim to analyze expression, tissue and isoform specificity of Nrg1 and ErbB4 in blood and heart tissue during the pathogenesis of viral myocarditis towards developing a non‐invasive blood‐based diagnostic assay via novel biomarkers.Methods4‐week old male A/J mice were sham or CVB3 infected. Blood and tissue were harvested at time points corresponding to the acute, sub‐acute and chronic phases of disease. Specimens were analyzed for mRNA and protein expression and sub‐cellular localization using RT‐qPCR, Western blotting and confocal microscopy, respectively. Expression of Nrg1 and ErbB4 in viral myocarditis models was compared to murine myocardial infarction models via confocal microscopy.ResultsUpregulation (p&lt;0.05) of Nrg1 and ErbB4 protein fragments were observed in murine heart tissue at different phases of viral pathogenesis as compared to m.i. and non‐infected controls. The observed upregulation was specific to the heart and not observed in the infected pancreas or lung. In addition, ~35 kDa and ~26 kDa fragments of Nrg1 and ErbB4 respectively were detected in the plasma of infected mice, while absent in the non‐infected controls. Confocal microscopy revealed that Nrg1 localized to the nuclear periphery while a diffuse increase in cellular ErbB4 expression was observed during infection. mRNA levels of Nrg1β were significantly upregulated (~3‐fold) at the acute phase (6–7 dpi) of viral myocarditis, while the differences in expression of Nrg1α were not significant.ConclusionTissue specific upregulation and fragments detected in the plasma of both ErbB4 and Nrg1, with preferential expression of Nrg1β, were observed as a result of CVB3 infection. Moreover, upregulation was not observed in an ischemic model of acute myocardial injury, indicating a viral specific process. These composite observations support the concept that these biomarkers may ultimately yield a non‐invasive blood‐based diagnostic assay for viral myocarditis.Support or Funding InformationThis research is supported by the Myocarditis Foundation and the Michael Smith Foundation for Health Research.

Differential Expression of NRG1‐ERBB4‐PSEN1‐NUP98 (NEPN) Signaling Axis as Potential Blood‐Based Biomarkers for Viral Myocarditis

BackgroundMyocarditis, inflammation of the myocardium, is a leading cause of unexpected heart failure in young adults, commonly attributable to viral infections. Myocarditis manifests in a wide range of clinical presentations, from asymptomatic to acute heart failure. The current gold standard for diagnosis relies upon invasive endomyocardial biopsy, requiring histopathological demonstration of inflammation with/without associated myocyte damage. Under these guidelines, diagnostic sensitivity in independent published studies is &lt;30%. We have previously demonstrated that detection of biomarkers within the NEPN pathway (implicated in immune response and heart failure) significantly improved diagnostic sensitivity to ~80%, however, this approach still relies upon biopsy. Thus, in the present study, our aim is to develop a non‐invasive, blood‐based diagnostic assay for viral myocarditis by examining the NEPN signaling axis and their cleavage fragments during the evolution of myocarditis.DesignHeLa cells and human induced pluripotent stem cell (iPSC)‐derived cardiomyocytes (CM) were CVB3‐ or sham‐infected. Western blot analysis and confocal microscopy were performed to determine the expression and subcellular localization of NEPN signaling axis proteins in tissue culture. In addition, tissue and blood from murine models of viral myocarditis (A/J and C57BL/6 mice) were collected at 4 dpi (viremic phase) and 5 to 8 dpi (acute phase), and were analyzed by Western blot analysis, IHC and IF to detect protein expression.ResultsNEPN proteins all show changes in expression and subcellular localization throughout the infection time course. NUP98 is upregulated and cleaved during infection. NRG1 is upregulated in both lysates and supernatants. ERBB4 co‐localizes at the cell membrane with the protease PSEN1. ERBB4 and NRG1 cleavage fragments are secreted and detected in both tissue culture supernatant and mouse plasma. In the plasma of the less susceptible C57BL/6 mice, ERBB4 and NRG1 cleavage fragments increase throughout the viremic and acute phases. However, in the highly susceptible A/J mice, ERBB4 and NRG1 cleavage fragments were not detected in the plasma until 6 dpi (acute phase), when they are highly upregulated as compared to C57BL/6 mice (p=0.001).ConclusionDifferential expression, subcellular localization and cleavage fragments of NEPN signaling axis proteins were detected during the pathogenesis of viral myocarditis. These composite changes may be useful in the development of a blood‐based diagnostic.Support or Funding InformationDr. Hanson's research is supported by the Myocarditis Foundation and Michael Smith Foundation for Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiac Magnetic Resonance Imaging in Infiltrative Cardiomyopathy

Infiltrative cardiomyopathies are a wide spectrum of disorders characterized by deposition of abnormal substances in the myocardium. These have a varied etiology and can be idiopathic, familial, or secondary to systemic disorders. The infiltrative process primarily causes a diastolic dysfunction resulting in heart failure with preserved ejection fraction. Common infiltrative cardiomyopathies encountered are cardiac amyloidosis, sarcoidosis, Fabry disease, iron overload cardiomyopathy, endomyocardial fibrosis, and idiopathic restrictive cardiomyopathy. Early and accurate detection of cause of infiltration is very important to improve outcomes through disease-specific therapies. Cardiac magnetic resonance (CMR) plays an important role in the diagnosis, avoiding the need of invasive endomyocardial biopsy in many cases. The use of postcontrast late gadolinium enhancement and T1 and T2 mapping sequences in CMR is improving diagnosis of infiltrative cardiomyopathy. In this review, we highlight the role of CMR in detection of different types of infiltrative cardiomyopathy.

Utility of Echocardiogram and Electrocardiogram in the Prediction of TTR Amyloid Diagnosed by Tc-Pyrophosphate SPECT Imaging

Introduction Cardiac transthyretin amyloidosis (ATTR) results from myocardial deposition of insoluble TTR fibril proteins. In contrast to AL cardiac amyloidosis, there are no blood or urine tests to aid in the diagnosis. In clinical practice, ATTR is most commonly suspected when there is unexplained heart failure with preserved ejection fraction (HFpEF), left ventricular hypertrophy (LVH) on transthoracic echocardiogram (TTE), along with low voltage criteria on electrocardiogram (ECG). Definitive diagnosis historically required invasive endomyocardial biopsy, however technetium pyrophosphate (Tc-PYP) scans have recently emerged as a highly useful non-invasive test in the diagnosis of ATTR. We sought to examine the predictive value of multiple ECG and TTE parameters in the diagnosis of Tc-PYP positive ATTR. Methods We performed a retrospective chart review of 34 consecutive patients during January 2017-April 2018 at Scripps Clinic who underwent Tc-PYP scanning for clinically suspected ATTR. Patients were then separated into Tc-PYP positive and negative groups, and ECG and TTE obtained closest to the scan were reviewed. Statistical significance was determined by Mann-Whitney U and Fischer Exact test. Results Twenty patients had Tc-PYP positive scans versus 11 patients with Tc-PYP negative scans. Three patients with “equivocal” reads were excluded from the analysis. Overall no ECG parameters were significantly predictive of Tc-PYP+ scans. Rates of “low voltage” ECGs were similar between both groups (3/11 vs. 1/8, p-NS), and while there was a trend towards lower occurrence of sinus rhythm (5/20 vs. 5/11, p-NS) and increased ventricular-paced rhythms in patients with Tc-PYP+ scans (9/20 vs. 3/11, p-NS), these were not statistically significant. Comparing TTE variables, we found that Tc-PYP+ patients had significantly higher LV mass index values (171 vs. 123, p = 0.026) compared to Tc-PYP- patients. Additional TTE parameters (listed in Table) did not demonstrate any statistical significance. Conclusion In this single center study, we demonstrated that routinely used ECG and TTE parameters in clinical practice such as low voltage ECG and Tissue Doppler velocities lack sensitivity in the prediction of TTR Amyloid by Tc-PYP scans. Of various echocardiographic parameters, only LV mass index was statistically significant in predicting Tc-PYP positive scans. With promising new therapies on the horizon, and the emergence of Tc-PYP SPECT imaging as a highly specific noninvasive imaging modality for the diagnosis of ATTR, clinicians should have a high index of suspicion and should not stop with a TTE or ECG as both modalities lack sensitivity.

Ultrasound molecular imaging of acute cardiac transplantation rejection using nanobubbles targeted to T lymphocytes

Clinical surveillance of acute heart transplantation rejection requires repeated invasive endomyocardial biopsies and noninvasive diagnostic techniques are desperately needed. It is acknowledged that T lymphocyte infiltration is the central process of acute rejection. We hypothesized that ultrasound molecular imaging with T lymphocyte-targeted nanobubbles could be used to detect acute rejection in heart transplantation. In this study, nanobubbles bearing anti-CD3 antibody (NBCD3) or isotype antibody (NBcon) were prepared and characterized. There was significant adhesion of NBCD3 to T lymphocytes compared with NBconin vitro. The signal intensity of the adherent NBCD3 was significantly higher than that of the NBcon in allograft rats, but not significantly different in isograft rats. Furthermore, the signal intensity of NBCD3 in allograft rats was significantly higher than that in isograft rats, indicating more T lymphocyte infiltration in allograft rats compared with isograft rats. These results were further confirmed by immunohistochemistry examination, and the signal intensity of NBCD3 was positively correlated with the number of T lymphocytes in allograft rats. In summary, ultrasound molecular imaging with T lymphocyte-targeted nanobubbles can detect T lymphocyte infiltration in acute rejection and could be used as a noninvasive method in acute rejection detection after cardiac transplantation.

The AlloMap™ genomic biomarker story: 10 years after.

Over the last >20 years, we have co-developed the rationale for the first diagnostic and prognostic leukocyte gene expression profiling (GEP) biomarker test in transplantation medicine that gained US-FDA-regulatory clearance and international evidence-based medicine guideline acceptance to rule out moderate/severe acute cellular cardiac allograft rejection without invasive endomyocardial biopsies (EMB). Based on this test, a non-invasive clinical algorithm was implemented since 2005. After clinical implementation, this GEP-based monitoring in direct comparison with an EMB-based strategy was non-inferior with respect to detection of clinical rejection, defined as new onset allograft dysfunction with/without histology of ACR, re-transplantation or death, and at the same time improved patient satisfaction. Subsequently, we demonstrated the test's capacity when used as serial monitoring tool to predict these clinical rejection events. In this Personal Viewpoint article, I will discuss the various decision-making branching points that were made in the AlloMap biomarker test development to inform future genomic biomarker test development projects.

Magnetic Resonance Imaging Applications on Infiltrative Cardiomyopathies.

Infiltrative cardiomyopathies are a heterogenous group of diseases that typically lead to restrictive cardiac dysfunction. Due to similar phenotypes, accurate diagnosis is challenging without invasive endomyocardial biopsy which has historically been considered mandatory. Cardiac magnetic resonance (CMR) has been well established in the diagnostic workup of patients with suspected cardiomyopathies due to its unique capability for tissue differentiation and its unsurpassed accuracy in defining cardiac morphology and function. The increasing variety of CMR techniques has generated both excitement and uncertainty with regard to their potential clinical use and its role vis-à-vis conventional noninvasive imaging techniques. The purpose of this review is to give an overview of established and emerging CMR techniques and typical image characteristics of the most commonly encountered infiltrative cardiomyopathies.

Assessment of Suspected Cardiac Mass with Multi-Parametric Cardiac Magnetic Resonance: Do we Really Need Invasive Endomyocardial Biopsy in Everyone?

We present the case of a 44-year-old woman with a cardiac mass. This case highlights the role of cardiac magnetic resonance (CMR) in assessing non-invasively a myocardial mass and delineating its anatomy and tissue characterization, thus potentially avoiding endomyocardial biopsy (EMB).