Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation.

BACKGROUND: Beyond the observed alterations in cellular structure and mitochondria, the mechanisms linking rare genetic mutations to the development of heart failure in patients affected by desmin mutations remain unclear due in part, to the lack of relevant human cardiomyocyte models. METHODS: To shed light on the role of mitochondria in these mechanisms, we investigated cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DESE439K mutation that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cardiomyocytes were either cultured on an anisotropic micropatterned surface to obtain elongated and aligned cardiomyocytes, or as a cardiac spheroid to create a micro-tissue. Moreover, when applicable, results from cardiomyocytes were confirmed with heart biopsies of suddenly died patient of the same family harboring DESE439K mutation, and post-mortem heart samples from five control healthy donors. RESULTS: The heterozygous DESE439K mutation leads to dramatic changes in the overall cytoarchitecture of cardiomyocytes, including cell size and morphology. Most importantly, mutant cardiomyocytes display altered mitochondrial architecture, mitochondrial respiratory capacity and metabolic activity reminiscent of defects observed in patient's heart tissue. Finally, to challenge the pathological mechanism, we transferred normal mitochondria inside the mutant cardiomyocytes and demonstrated that this treatment was able to restore mitochondrial and contractile functions of cardiomyocytes. CONCLUSIONS: This work highlights the deleterious effects of DESE439K mutation, demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens up new potential therapeutic perspectives for this disease.

Replication Activities of Major 5' Terminally Deleted Group-B Coxsackievirus RNA Forms Decrease PCSK2 mRNA Expression Impairing Insulin Maturation in Pancreatic Beta Cells.

Emergence of 5' terminally deleted coxsackievirus-B RNA forms (CVB-TD) have been associated with the development of human diseases. These CVB-TD RNA forms have been detected in mouse pancreas during acute or persistent experimental infections. To date, the impact of the replication activities of CVB-TD RNA forms on insulin metabolism remains unexplored. Using an immunocompetent mouse model of CVB3/28 infection, acute and persistent infections of major CVB-TD populations were evidenced in the pancreas. The inoculation of mice with homogenized pancreases containing major CVB-TD populations induced acute and chronic pancreatic infections with pancreatitis. In the mouse pancreas, viral capsid protein 1 (VP1) expression colocalized with a decrease in beta cells insulin content. Moreover, in infected mouse pancreases, we showed a decrease in pro-hormone convertase 2 (PCSK2) mRNA, associated with a decrease in insulin plasmatic concentration. Finally, transfection of synthetic CVB-TD50 RNA forms into cultured rodent pancreatic beta cells demonstrated that viral replication with protein synthesis activities decreased the PCSK2 mRNA expression levels, impairing insulin secretion. In conclusion, our results show that the emergence and maintenance of major CVB-TD RNA replicative forms in pancreatic beta cells can play a direct, key role in the pathophysiological mechanisms leading to the development of type 1 diabetes.

Early Emergence of 5' Terminally Deleted Coxsackievirus-B3 RNA Forms Is Associated with Acute and Persistent Infections in Mouse Target Tissues.

Major EV-B populations characterized by 5' terminal deletions (5'TD) have been shown to be associated with the development of myocarditis and type 1 diabetes in mice or humans. To date, the dynamics of EV-B 5'TD-RNA forms' emergence during the course of infection and their impact on cellular functions remain unclear. Using a RACE-PCR approach in CVB3/28-infected mouse organs, we showed an early (3 days post infection, DPI) emergence of major 5'TD populations associated with minor full-length RNA forms. Viral replication activities with infectious particle production were associated with heart, liver, and pancreas acute inflammatory lesions, whereas clearance of viral RNA without organ lesions was observed in the brain, lung, intestines, and muscles from 3 to 7 DPI. At 28 DPI, low viral RNA levels, +/-RNA ratios < 5 associated with viral protein 1 expression revealed a persistent infection in the heart and pancreas. This persistent infection was characterized by molecular detection of only 5'TD RNA forms that were associated with dystrophin cleavage in the heart and insulin production impairment in beta-pancreatic cells. These results demonstrated that major EV-B 5'TD RNA forms can be early selected during systemic infection and that their maintenance may drive EV-induced acute and persistent infections with target cell dysfunctions.

Fulminant Myocardial Involvement in Neonatal Echovirus-induced Sepsis. Two Autopsy Cases.

Group-B Enteroviruses, such as Echoviruses, are a common cause of infections in neonates but fatal myocarditis during Echovirus-induced sepsis have been rarely reported. We report on 2 cases of neonatal Echovirus-related sepsis with myocarditis. Fatal cardiorespiratory failure occurred in both cases. Autopsies and thorough histologic and microbiologic investigations evidenced Echoviruses 5- and 11-induced myocarditis as the cause of death.

Abdominal metastases from Merkel cell carcinoma: Prevalence and presentation on CT examination in 111 patients.

PURPOSE: The purpose of this study was to report the prevalence and imaging features of abdominal metastases from Merkel cell carcinoma (MCC) on computed tomography (CT) examinations. MATERIALS AND METHODS: A total of 111 patients with MCC from two institutions were initially identified. Of these, 27 patients (27/111; 24.3%) had abdominal metastases from MCC present on CT examination. There were 19 men and 8 women with a mean age of 75  ± 10.8 (SD) years (age range: 46-92 years). CT examinations were retrospectively reviewed by two radiologists and analyzed quantitatively for the number and dimensions of abdominal metastases from MCC and qualitatively in terms of location, margins, contours, homogeneity, patterns of enhancement, vascular involvement and extension of metastases from MCC. RESULTS: Fifteen patients (15/27; 56%) had abdominal metastatic disease at initial diagnosis and twelve (12/27; 44%) developed abdominal metastases during the course of the disease. The mean number of locations of abdominal metastases was 2.1 ± 1.12 (SD) (range: 1-5). Abdominal metastases involved abdominal lymph nodes (16/27; 59%), adrenal glands/kidneys/retroperitoneum (14/27; 52%), mesentery/peritoneum (13/27; 48%), liver (7/27; 26%) and pancreas (7/27; 26%). Vascular involvement was found in association with peritoneal/mesenteric metastases in 6/13 (46%) patients with intraperitoneal metastases or in association with abdominal lymph nodes in 4/16 (25%) patients. Ureteral encasement and/or dilatation was found in 4/14 (28%) patients with retroperitoneal metastases and 3/16 (19%) patients with abdominal lymph nodes. Metastases to the liver, pancreas, peritoneum, retroperitoneum and adrenal glands displayed internal enhancement during the arterial phase in 1/2 (50%), 4/5 (80%), 4/7 (57%) and 5/8 (62%) patients for whom arterial phase was available, respectively. CONCLUSION: Metastases from MCC have a prevalence of 24.3% on CT examination and may involve a variety of abdominal organs, mainly lymph nodes, peritoneal and retroperitoneal structures, but also the liver and pancreas. CT features of abdominal metastases from MCC include hypervascularity during the arterial phase of enhancement and eventually vascular and ureteral involvement.

Coinfection of Parvovirus B19 with Influenza A/H1N1 Causes Fulminant Myocarditis and Pneumonia. An Autopsy Case Report.

Parvovirus-B19 (PVB19) is a frequent causative agent of myocarditis. For unclear reasons, viral reactivation can cause acute myocarditis, a leading cause of sudden death in the young. Influenza A/H1N1(2009) virus (IAV/H1N1) is known for causing flu/pneumonia, but the heart is rarely involved. Co-infections of cardiotropic viruses are rarely reported and the mechanisms of viral interactions remain unknown. A 5-year old girl had a flu-like syndrome, when she suddenly presented with a respiratory distress and cardiac arrest. At autopsy, the lungs were found haemorrhagic. Lungs' histology showed severe bronchiolitis, diffuse haemorrhagic necrosis, and mononuclear inflammation. In the heart, a moderate inflammation was found with no necrosis. IAV/H1N1 was detected in nasal and tracheal swabs, lungs, and the heart. The viral load was high in the lungs, but low in the heart. PVB19 was detected in the heart with a high viral load. Viral co-infection increases the risk of severe outcome but the mechanisms of interaction between viruses are poorly understood. In our case, viral loads suggested a reactivated PVB19-induced acute myocarditis during an IAV/H1N1 pneumonia. Viral interactions may involve an IAV/H1N1-induced cytokine storm, with a fulminant fatal outcome. Clinically, our case shows the importance of investigating inflammatory pathways as therapeutic targets.

[Autopsies are essential for a better knowledge and prevention of sudden cardiac death]. / L'autopsie : indispensable pour la connaissance et la prévention de la mort subite cardiaque.

In France, the incidence of sudden cardiac deaths (SCD) is approximately 30,000-50,000 per year. In the whole population, their cause is an atherosclerotic coronary disease in more than 80 % of cases, but in the young (<35 years old), causes are various, including genetic, infectious, toxic, congenital anomalies, immune…Therefore a multidisciplinary approach is required for a better knowledge and prevention of SCD. In this article, we examine different aspects of autopsies and complementary investigations: histopathology, toxicology, biochemistry, genetics and virology. Six cases illustrate the importance of a multidisciplinary approach. There are two categories of autopsies: medicolegal or medical. Medicolegal autopsies are requested by a judicial authority when a death is considered suspicious. These autopsies are performed by forensic doctors. Most of them are not pathologists. During the autopsies, blood and tissue samples are taken, but analyses are done only at the request of the judicial authority if the analyses are useful for the truth. Consequently, the cause of death can remain uncertain. Medical autopsies are performed by a pathologist at the request of a clinician. The family consent is required. Useful analyses are performed, which is essential for the determination of a precise cause of death. In the young, "molecular autopsy", in addition to histology and other analyses, is essential in preventing genetic causes of SCD.

Clinical impact of post-mortem genetic testing in cardiac death and cardiomyopathy.

Post-mortem genetic analyses may help to elucidate the cause of cardiac death. The added value is however unclear when a cardiac disease is already suspected or affirmed. Our aim was to study the feasibility and medical impact of post-mortem genetic analyses in suspected cardiomyopathy. We studied 35 patients with cardiac death and suspected cardiomyopathy based on autopsy or clinical data. After targeted sequencing, we identified 15 causal variants in 15 patients (yield 43%) in sarcomeric (n = 8), desmosomal (n = 3), lamin A/C (n = 3) and transthyretin (n = 1) genes. The results had various impacts on families, i.e. allowed predictive genetic testing in relatives (15 families), planned early therapeutics based on the specific underlying gene (5 families), rectified the suspected cardiomyopathy subtype (2 families), assessed the genetic origin of cardiomyopathy that usually has an acquired cause (1 family), assessed the diagnosis in a patient with uncertain borderline cardiomyopathy (1 family), reassured the siblings because of a de novo mutation (2 families) and allowed prenatal testing (1 family). Our findings suggest that post-mortem molecular testing should be included in the strategy of family care after cardiac death and suspected cardiomyopathy, since genetic findings provide additional information useful for relatives, which are beyond conventional autopsy.

Enterovirus Persistence in Cardiac Cells of Patients With Idiopathic Dilated Cardiomyopathy Is Linked to 5' Terminal Genomic RNA-Deleted Viral Populations With Viral-Encoded Proteinase Activities.

BACKGROUND: Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear. METHODS: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture. RESULTS: Major enterovirus B populations characterized by 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes. CONCLUSIONS: We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.

Enterovirus but not Parvovirus B19 is associated with idiopathic dilated cardiomyopathy and endomyocardial CD3, CD68, or HLA-DR expression.

We assessed Enterovirus (EV) &Parvovirus B19 (PVB19) genomes and CD3, CD68&HLA-DR detection in dilated cardiomyopathies (DCM). EV&PVB19 genomes and CD3, CD68&HLA-DR were detected by PCR and immunohistochemistry assays in 115 endomyocardial biopsies obtained in 13 idiopathic DCM (iDCM) and 10 explained DCM (eDCM) patients. Results were compared with those of 47 atrial surgical samples (47 surgery controls) and 22 autoptic cardiac samples (11 healthy heart controls) (2008-2014, Reims, France). EV was detected in 23.1% of iDCM patients but not in eDCM and controls (P = 0.003) (viral load 803 copies/µg). PVB19 was detected in 76.9%, 80.0%, 63.6% and 78.2% of iDCM, eDCM, healthy heart and surgery controls (P = 0.99) with a mean viral load of 413, 346, 1,428, and 71 copies/µg. CD3, CD68 or HLA-DR were detected in 100 and 50% of EV and PVB19 "mono-infected" iDCM patients. EV was exclusively detected in iDCM cases in association with CD3, CD68, or HLA-DR indicating that EV could be an etiological cause in a subset of iDCM cases. By contrast the equal frequent detection of PVB19 in iDCM cases and controls without association with CD3, CD68, or HLA-DR suggested that PVB19 could be a bystander in many DCM cases. J. Med. Virol. 89:55-63, 2017. © 2016 Wiley Periodicals, Inc.

Major Persistent 5' Terminally Deleted Coxsackievirus B3 Populations in Human Endomyocardial Tissues.

We performed deep sequencing analysis of the enterovirus 5' noncoding region in cardiac biopsies from a patient with dilated cardiomyopathy. Results displayed a mix of deleted and full-length coxsackievirus B3, characterized by a low viral RNA load (8.10(2) copies/µg of nucleic acids) and a low viral RNA positive-sense to RNA negative-sense ratio of 4.8.

Protein carbamylation is a hallmark of aging.

Aging is a progressive process determined by genetic and acquired factors. Among the latter are the chemical reactions referred to as nonenzymatic posttranslational modifications (NEPTMs), such as glycoxidation, which are responsible for protein molecular aging. Carbamylation is a more recently described NEPTM that is caused by the nonenzymatic binding of isocyanate derived from urea dissociation or myeloperoxidase-mediated catabolism of thiocyanate to free amino groups of proteins. This modification is considered an adverse reaction, because it induces alterations of protein and cell properties. It has been shown that carbamylated proteins increase in plasma and tissues during chronic kidney disease and are associated with deleterious clinical outcomes, but nothing is known to date about tissue protein carbamylation during aging. To address this issue, we evaluated homocitrulline rate, the most characteristic carbamylation-derived product (CDP), over time in skin of mammalian species with different life expectancies. Our results show that carbamylation occurs throughout the whole lifespan and leads to tissue accumulation of carbamylated proteins. Because of their remarkably long half-life, matrix proteins, like type I collagen and elastin, are preferential targets. Interestingly, the accumulation rate of CDPs is inversely correlated with longevity, suggesting the occurrence of still unidentified protective mechanisms. In addition, homocitrulline accumulates more intensely than carboxymethyl-lysine, one of the major advanced glycation end products, suggesting the prominent role of carbamylation over glycoxidation reactions in age-related tissue alterations. Thus, protein carbamylation may be considered a hallmark of aging in mammalian species that may significantly contribute in the structural and functional tissue damages encountered during aging.

Diffusion-weighted MR imaging of the rectum: clinical applications.

Dramatic advances in image quality over the past few years have made diffusion-weighted magnetic resonance imaging (DW-MRI) a promising tool for rectal lesion evaluation. DW-MRI derives its image contrast from differences in the motion of water molecules between tissues. Such imaging can be performed quickly without the need for the administration of exogenous contrast medium. The technique yields qualitative and quantitative information that reflects changes at a cellular level and provides information about tumor cellularity and the integrity of cell membranes. The sensitivity to diffusion is obtained by applying two bipolar diffusion-sensitizing gradients to a standard T2-weighted spin echo sequence. The diffusion-sensitivity can be varied by adjusting the "b-factor", which represents the gradient duration, gradient amplitude and the time interval between the two gradients. The higher the b-value, the greater the signal attenuation from moving water protons. In this review, technical considerations relatively to image acquisition and to quantification methods applied to rectal DW-MRI are discussed. The current clinical applications of DW-MRI, either in the field of inflammatory or neoplastic rectal disease are reviewed. Also, limitations, mainly in terms of persistent lack of standardization or evaluation of tumoral response, and future directions of rectal DW-MRI are discussed. The potential utility of DW-MRI for the evaluation of rectal tumor response is on its way to being admitted but future well-designed and multicenter studies, as well as standardization of DW-MRI, are still required before a consensus can be reached upon how and when to use DW-MRI.

Desmosomal cadherins are decreased in explanted arrhythmogenic right ventricular dysplasia/cardiomyopathy patient hearts.

AIMS: Arrhythmogenic right ventricular Dysplasia/cardiomyopathy (ARVD/C) is an autosomal dominant inherited cardiomyopathy associated with ventricular arrhythmia, heart failure and sudden death. Genetic studies have demonstrated the central role of desmosomal proteins in this disease, where 50% of patients harbor a mutation in a desmosmal gene. However, clinical diagnosis of the disease remains difficult and molecular mechanisms appears heterogeneous and poorly understood. The aim of this study was to characterize the expression profile of desmosomal proteins in explanted ARVD/C heart samples, in order to identify common features of the disease. METHODS AND RESULTS: We examined plakophilin-2, desmoglein-2, desmocollin-2, plakoglobin and ß-catenin protein expression levels from seven independent ARVD/C heart samples compared to two ischemic, five dilated cardiomyopathy and one healthy heart sample as controls. Ventricular and septum sections were examined by immunoblot analysis of total heart protein extracts and by immunostaining. Immunoblots indicated significant decreases in desmoglein-2 and desmocollin-2, independent of any known underlying mutations, whereas immune-histochemical analysis showed normal localization of all desmosomal proteins. Quantitative RT-PCR revealed normal DSG2 and DSC2 mRNA transcript levels, suggesting increased protein turn-over rather than transcriptional down regulation. CONCLUSION: Reduced cardiac desmoglein-2 and desmocollin-2 levels appear to be specifically associated with ARVD/C, independent of underlying mutations. These findings highlight a key role of desmosomal cadherins in the pathophysiology of ARVD/C. Whether these reductions could be considered as specific markers for ARVD/C requires replication analysis.

Post-mortem diagnosis, of cytomegalovirus and varicella zoster virus co-infection by combined histology and tissue molecular biology, in a sudden unexplained infant death.

BACKGROUND: An autopsy case of a two-month-old male infant who suddenly and unexpectedly died during his sleep, eight days after the onset of benign varicella. OBJECTIVES: To describe post-mortem combined histological and tissue molecular biological techniques for the diagnosis of cytomegalovirus and varicella zoster virus co-infection as a cause of death. STUDY DESIGN: Real-time quantitative PCR and RT-PCR assays for Herpesviruses, respiratory viruses, Adenovirus, Enterovirus and Parvovirus B19 were performed on multi-organ frozen samples and paraffin-embedded tissues in combination with histology. RESULTS: Cytomegalovirus and varicella zoster virus were detected by molecular biology with highest viral loads detected in the lungs (4.6×10(7) and 1.9×10(5) genome copies per million of cells, respectively). Pulmonary extensive necrotizing inflammation and immunohistochemistry correlated to virological data. Virological molecular biology was negative on paraffin-embedded tissues. CONCLUSIONS: This case shows that thorough quantitative virological investigations on frozen tissues must be performed in combination with histology and immunohistochemistry for the determination of the cause of a sudden unexplained infant death.

[Investigation of the sudden infant death syndrome: a multidisciplinary approach is required]. / Exploration d'une mort inattendue du nourrisson : nécessité d'une approche multidisciplinaire.

The concept of sudden infant death syndrome (SIDS) is defined as the sudden, unexpected death of an infant less than a year old which remains unexplained after in-depth investigations comprising a complete autopsy, biological analyses, and a clinical examination of the circumstances surrounding the death. This definition underlines the importance of finding the cause of this disease in order to improve preventative measures to reduce the number of deaths due to sudden infant death syndrome. Among the causes of SIDS, pediatric infectious diseases may be neglected and must be systematically sought after. We report upon a SIDS death case of a four and a half month-old that occurred during his sleep. Following the absence of an evident cause of death a scientific autopsy was performed. The histological examination of pulmonary tissue revealed broncolitic lesions associated with numerous micro-abscesses. The post mortem microbiological analyses revealed evidence of an infection by the respiratory syncytial virus complicated by a bacterial infection due to Haemophilus influenzae. The case underlines the necessity of a multidisciplinary approach to researching SIDS, involving both clinicians and biologists, in order to determine the causes of these deaths.

Virus detection and semiquantitation in explanted heart tissues of idiopathic dilated cardiomyopathy adult patients by use of PCR coupled with mass spectrometry analysis.

Viral detection in heart tissues has become a central issue for the diagnosis and exploration of the pathogenesis of idiopathic dilated cardiomyopathy (IDCM). In the present study, common cardiotropic viruses in 67 explanted heart samples of 31 IDCM adult patients were detected and semiquantified by using for the first time a new technology based on PCR assay coupled to electrospray ionization-time of flight mass spectrometry analysis (PCR-MS), with comparison to reference quantitative real-time PCR (RT-qPCR) assay. PCR-MS identified single or mixed enterovirus (EV) and parvovirus B19 (PVB19) infections in 27 (40.2%) of 67 samples, corresponding to 15 (48.3%) of the 31 patients, whereas RT-qPCR identified viral infections in 26 (38.8%) samples, corresponding to 16 (51.6%) of the patients. The PCR-MS results correlated well with EV and PVB19 detection by RT-qPCR (kappa = 0.85 [95% confidence interval {CI}, 0.72 to 1.00] and kappa = 0.82 [95% CI, 0.66 to 0.99], respectively). The levels of EV RNA (median, 550 [range, 178 to 3,200] copies/µg of total extracted nucleic acids) and of PVB19 DNA (median, 486 [range, 80 to 1,157] copies/µg of total extracted nucleic acids) were measured using PCR-MS and correlated with those obtained by RT-qPCR (r(2) = 0.57, P = 0.002 and r(2) = 0.64, P < 0.001 for EV and PVB19, respectively). No viruses other than EV and PVB19 strains were detected using the new PCR-MS technology, which is capable of simultaneously identifying 84 known human viruses in one assay. In conclusion, we identified single or mixed EV and PVB19 cardiac infections as potential causes of IDCM. The PCR-MS analysis appeared to be a valuable tool to rapidly detect and semiquantify common viruses in cardiac tissues and may be of major interest to better understand the role of viruses in unexplained cardiomyopathies.

MR imaging features of nongynaecologic cystic lesions of the pelvis.

OBJECTIVE: The purpose of our article is to review the magnetic resonance imaging (MRI) features of nongynaecologic cystic lesions of the pelvis. CONCLUSION: The rising use of MRI for pelvic exploration will result in an increase in incidental detection of pelvic cystic cysts. Pelvic cysts of non gynecologic origin are less frequent than gynecologic cysts. However, they account for a wide range of abnormalities, and radiologists must be aware of their features and characteristics.