Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by Involving an Integrin-αVß6/TGF-ß Signaling Cascade.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVß6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVß6 and subsequent transforming growth factor-ß signaling as driver of cardiac fibrosis. Blocking integrin-αVß6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVß6 and transforming growth factor-ß signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

Telemedical monitoring in patients with inborn cardiac disease - experience of a tertiary care centre.

BACKGROUND: The number of cardiologically relevant genetic findings will continue to increase. This is due to the use of high-throughput sequencing techniques and the critical role of incidental findings in cardiac disease genes. Telemedicine can be a useful diagnostic tool to monitor the heart rhythm of patients with inborn cardiac diseases. METHODS: Patients were screened once they had been referred to our outpatient department for rare cardiac diseases between January 2020 and May 2022. Those patients who underwent genetic testing and were consequently diagnosed with a genetic disorder were included in this study. Their medical records were evaluated regarding implanted cardiac electronic devices and findings in the telemedical monitoring. RESULTS: 304 patients were seen in our outpatient department for rare cardiac diseases in the mentioned period. In 100 cases, genetic testing was performed. 10 patients (10%) with an identified inborn cardiac disease were monitored via telemedicine until the end of May 2022. 4 patients were monitored by implantable loop recorders (ILR), 4 patients were monitored by Implantable Cardioverter Defibrillators (ICD), and 2 patients received both devices. Clinical relevant arrhythmias making medical intervention necessary were identified in 4 cases. In two cases, data interpretation was hampered by sinus tachycardia caused by physical exercise. DISCUSSION: Telemonitoring of the heart rhythm by medical devices is beneficial for patients with monogenic heart diseases. Especially, when the indication for an ICD is not clear, implantation of a telemonitored ILR can be a suitable choice. However, rhythm analysis can be challenging in young patients who are physically active.

Functional Characterization of a Spectrum of Novel Romano-Ward Syndrome KCNQ1 Variants.

The KCNQ1 gene encodes the α-subunit of the cardiac voltage-gated potassium (Kv) channel KCNQ1, also denoted as Kv7.1 or KvLQT1. The channel assembles with the ß-subunit KCNE1, also known as minK, to generate the slowly activating cardiac delayed rectifier current IKs, a key regulator of the heart rate dependent adaptation of the cardiac action potential duration (APD). Loss-of-function variants in KCNQ1 cause the congenital Long QT1 (LQT1) syndrome, characterized by delayed cardiac repolarization and a QT interval prolongation in the surface electrocardiogram (ECG). Autosomal dominant loss-of-function variants in KCNQ1 result in the LQT syndrome called Romano-Ward syndrome (RWS), while autosomal recessive variants affecting function, lead to Jervell and Lange-Nielsen syndrome (JLNS), associated with deafness. The aim of this study was the characterization of novel KCNQ1 variants identified in patients with RWS to widen the spectrum of known LQT1 variants, and improve the interpretation of the clinical relevance of variants in the KCNQ1 gene. We functionally characterized nine human KCNQ1 variants using the voltage-clamp technique in Xenopus laevis oocytes, from which we report seven novel variants. The functional data was taken as input to model surface ECGs, to subsequently compare the functional changes with the clinically observed QTc times, allowing a further interpretation of the severity of the different LQTS variants. We found that the electrophysiological properties of the variants correlate with the severity of the clinically diagnosed phenotype in most cases, however, not in all. Electrophysiological studies combined with in silico modelling approaches are valuable components for the interpretation of the pathogenicity of KCNQ1 variants, but assessing the clinical severity demands the consideration of other factors that are included, for example in the Schwartz score.

Variant interpretation in molecular autopsy: a useful dilemma.

Sudden cardiac death (SCD) in adolescents and young adults may be the first manifestation of an inherited arrhythmic syndrome. Thus identification of a genetic origin in sudden death cases deemed inconclusive after a comprehensive autopsy and may help to reduce the risk of lethal episodes in the remaining family. Using next-generation sequencing (NGS), a large number of variants of unknown significance (VUS) are detected. In the majority of cases, there is insufficient evidence of pathogenicity, representing a huge dilemma in current genetic investigations. Misinterpretation of such variants may lead to inaccurate genetic diagnoses and/or the adoption of unnecessary and/or inappropriate therapeutic approaches. In our study, we applied current (ACMG) recommendations for variant classification in post-mortem genetic screening of a cohort of 56 SCD victims. We identified a total 53 rare protein-altering variants (MAF < 0.2%) classified as VUS or worse. Twelve percent of the cases exhibited a clinically actionable variant (pathogenic, likely pathogenic or VUS - potentially pathogenic) that would warrant cascade genetic screening in relatives. Most of the variants detected by means of the post-mortem genetic investigations were VUS. Thus, genetic testing by itself might be fairly meaningless without supporting background data. This data reinforces the need for an experienced multidisciplinary team for obtaining reliable and accountable interpretations of variant significance for elucidating potential causes for SCDs in the young. This enables the early identification of relatives at risk or excludes family members as genetic carriers. Also, development of adequate forensic guidelines to enable appropriate interpretation of rare genetic variants is fundamental.

Post-mortem genetic investigation of cardiac disease-associated genes in sudden infant death syndrome (SIDS) cases.

The sudden infant death syndrome (SIDS) is one of the leading causes of postneonatal infant death. It has been shown that there exists a complex relationship between SIDS and inherited cardiac disease. Next-generation sequencing and surveillance of cardiac channelopathy and cardiomyopathy genes represent an important tool for investigating the cause of death in SIDS cases. In the present study, targeted sequencing of 80 genes associated with genetic heart diseases in a cohort of 31 SIDS cases was performed. To determine the spectrum and prevalence of genetic heart disease associated mutations as a potential monogenic basis for SIDS, a stringent variant classification was applied and the percentage of rare (minor allele frequency ≤ 0.2%) and ultra-rare variants (minor allele frequency ≤ 0.005%) in these genes was assessed. With a minor allele frequency of ≤ 0.005%, about 20% of the SIDS cases exhibited a variant of uncertain significance (VUS), but in only 6% of these cases, gene variants proved to be "potentially informative." The present study shows the importance of careful variant interpretation. Applying stringent criteria misinterpretations are avoided, as the results of genetic analyses may have an important impact of the family members involved.

Characterization of an N-terminal Nav1.5 channel variant - a potential risk factor for arrhythmias and sudden death?

BACKGROUND: Alterations in the SCN5A gene encoding the cardiac sodium channel Nav1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. METHODS: Mutant as well as wild type GFP tagged Nav1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. RESULTS: Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Nav1.5 current over the entire voltage window. CONCLUSION: The results support the assumption that the detected sequence aberration alters Nav1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death.

Changes in gene expression patterns in postmortem human myocardial infarction.

In murine models, the expression of inducible nitric oxide synthase (iNOS) in myocardial infarction (MI) has been reported to be the result of tissue injury and inflammation. In the present study, mRNA expression of iNOS, hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) was investigated in postmortem human infarction hearts. Since HIF-1α is the inducible subunit of the transcription factor HIF-1, which regulates transcription of iNOS and VEGF, the interrelation between the three genes was observed, to examine the molecular processes during the emergence of MI. iNOS and VEGF mRNAs were found to be significantly upregulated in the affected regions of MI hearts in comparison to healthy controls. Upregulation of HIF-1α was also present but not significant. Correlation analysis of the three genes indicated a stronger and significant correlation between HIF-1α and iNOS mRNAs than between HIF-1α and VEGF. The results of the study revealed differences in the expression patterns of HIF-1 downstream targets. The stronger transcription of iNOS by HIF-1 in the affected regions of MI hearts may represent a pathological process, since no correlation of iNOS and HIF-1α mRNA was found in non-affected areas of MI hearts. Oxidative stress is considered to cause molecular changes in MI, leading to increased iNOS expression. Therefore, it may also represent a forensic marker for detection of early changes in heart tissue.

Increased inducible nitric oxide synthase (iNOS) expression in human myocardial infarction.

Increased inducible nitric oxide synthase (iNOS) expression has been reported in heart failure, cardiomyopathies, and arteriosclerosis. iNOS is expressed in the heart upon inflammatory stimuli and produces excessive amounts of nitric oxide (NO). The overproduction of NO is cytotoxic and involved in cardiovascular diseases. Furthermore, iNOS produces superoxide anion which proceeds with NO to the harmful oxidant peroxynitrite, causing oxidative stress in the heart. The aim of the study was to gain new insights into the role of iNOS in human myocardial infarction (MI) and its contribution to oxidative stress in the heart. Furthermore, we investigated the unaffected myocardium of the infarction hearts, to study if iNOS expression is increased, probably as an indicator for oxidative stress. Our results show a significant increase (p = 0.013) of the iNOS expression in the affected regions of MI hearts (n = 9) in comparison with healthy control hearts (n = 4). In the unaffected regions of MI hearts, an increase in the iNOS expression in some samples was found as well. Our study demonstrated the direct detection of iNOS mRNA in human myocardial tissue. The balance between beneficial and deleterious effects of iNOS may be particularly influenced by the presence or absence of concurrent oxidative stress.

Identification of a cono-RFamide from the venom of Conus textile that targets ASIC3 and enhances muscle pain.

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels that are expressed throughout the nervous system. ASICs have been implicated in several neuronal disorders, like ischemic stroke, neuronal inflammation, and pathological pain. Several toxins from venomous animals have been identified that target ASICs with high specificity and potency. These toxins are extremely useful in providing protein pharmacophores and to characterize function and structure of ASICs. Marine cone snails contain a high diversity of toxins in their venom such as conotoxins, which are short polypeptides stabilized by disulfide bonds, and conopeptides, which have no or only one disulfide bond. Whereas conotoxins selectively target specific neuronal proteins, mainly ion channels, the targets of conopeptides are less well known. Here, we perform an in vitro screen of venoms from 18 cone snail species to identify toxins targeting ASICs. We identified a small conopeptide of only four amino acids from the venom of Conus textile that strongly potentiated currents of ASIC3, which has a specific role in the pain pathway. This peptide, RPRFamide, belongs to the subgroup of cono-RFamides. Electrophysiological characterization of isolated dorsal root ganglion (DRG) neurons revealed that RPRFamide increases their excitability. Moreover, injection of the peptide into the gastrocnemius muscle strongly enhanced acid-induced muscle pain in mice that was abolished by genetic inactivation of ASIC3. In summary, we identified a conopeptide that targets the nociceptor-specific ion channel ASIC3.

Influence of genetic modifiers on sudden cardiac death cases.

Sequence variants in the ion channel genes KCNH2 and SCN5A may cause the cardiac disorder long QT syndrome (LQTS). This disorder is associated with incomplete penetrance and variable expression in KCNH2- or SCN5A-mutation carriers. Common genetic variants, if associated with a mutation, may affect the severity of this cardiac disorder. This study identified rare mutations in the cardiac ion channel genes KCNH2 and SCN5A in a SCD case, as well as in a LQTS-affected family with a history of SCD. Moreover, common variants were found to occur together within the same genes. These findings support the concept that common single-nucleotide polymorphisms (SNPs) in genes encoding cardiac ion channels can directly modulate the functional effect of mutations and therefore enhance or weaken the risk of cardiac events.

Characterization of a novel KCNJ2 sequence variant detected in Andersen-Tawil syndrome patients.

BACKGROUND: Mutations in the KCNJ2 gene encoding the ion channel Kir2.1 have been linked to the Andersen-Tawil syndrome (ATS). Molecular genetic screening performed in a family exhibiting clinical ATS phenotypes unmasked a novel sequence variant (c.434A > G, p.Y145C) in this gene. The aim of this study was to investigate the effect of this variant on Kir2.1 ion channel functionality. METHODS: Mutant as well as wild type GFP tagged Kir2.1 channels were expressed in HEK293 cells. In order to examine the effect of the new variant, electrophysiological measurements were performed using patch clamp technique. Cellular localization of the mutant in comparison to the wild type ion channel was analyzed by confocal laser scanning microscopy. RESULTS: The currents of cells expressing only mutant channels or a mixture of wild type and mutant were significantly reduced compared to those expressing wild type (WT) channels (p < 0.01). Whereas WT expressing cells exhibited at -120 mV an averaged current of -4.5 ± 1.9 nA, the mutant generates only a current of -0.17 ± 0.07 nA. A co-expression of mutant and WT channel generates only a partial rescue of the WT current. Confocal laser scanning microscopy indicated that the novel variant is not interfering with synthesis and/or protein trafficking. CONCLUSIONS: The detected sequence variant causes loss-of-function of the Kir2.1 channel and explains the clinical phenotypes observed in Andersen-Tawil syndrome patients.

Case report on the feasibility of determining the compatibility between mandible and skull by morphological examination.

When human bones are found it is important to establish whether they belong to one or more individuals. Osteological examinations assessing morphological characteristics provide an important tool, in particular for a first appraisal. In the reported case, an isolated, toothless mandible that had been found in a river appeared to be too large to match a skull found in the same context. Moreover, shortly before and after the mandible had been found, two headless corpses had been recovered from the same river. The initial conclusion was, therefore, that the mandible and the skull belonged to different individuals. Forensic DNA-testing confirmed, however, that the mandible, the skull, and one of the bodies were from the same individual, who could later be identified through DNA comparison with two biological children. So far, current knowledge has only been that mandibles may erroneously be considered too small by morphological assessment to be compatible with a found skull, as reported in a paper by Reichs (1989). The reported case establishes that the opposite phenomenon may also occur. The findings show that more extensive studies investigating the correlation between mandible and cranial base in a moden population seem to be necessary.

Mutation in S6 domain of HCN4 channel in patient with suspected Brugada syndrome modifies channel function.

Diseases such as the sick sinus and the Brugada syndrome are cardiac abnormalities, which can be caused by a number of genetic aberrances. Among them are mutations in HCN4, a gene, which encodes the hyperpolarization-activated, cyclic nucleotide-gated ion channel 4; this pacemaker channel is responsible for the spontaneous activity of the sinoatrial node. The present genetic screening of patients with suspected or diagnosed Brugada or sick sinus syndrome identified in 1 out of 62 samples the novel mutation V492F. It is located in a highly conserved site of hyperpolarization-activated cyclic nucleotide-gated (HCN)4 channel downstream of the filter at the start of the last transmembrane domain S6. Functional expression of mutant channels in HEK293 cells uncovered a profoundly reduced channel function but no appreciable impact on channel synthesis and trafficking compared to the wild type. The inward rectifying HCN4 current could be partially rescued by an expression of heteromeric channels comprising wt and mutant monomers. These heteromeric channels were responsive to cAMP but they required a more negative voltage for activation and they exhibited a lower current density than the wt channel. This suggests a dominant negative effect of the mutation in patients, which carry this heterozygous mutation. Such a modulation of HCN4 activity could be the cause of the diagnosed cardiac abnormality.

[Postmortem genetic analysis following sudden cardiac death : Background, approach, and future]. / Postmortale Genetik nach einem plötzlichen Herztod : Hintergründe, Vorgehen und Zukunft.

BACKGROUND: Sudden cardiac death (SCD) is defined as an unexpected, nontraumatic death with a possible cardiac or unknown cause. The lowest incidence is observed in infancy and childhood (1 per 100,000), and the incidence is approximately 50 per 100,000 in the middle-aged population, reaching a plateau around the age of 80 (200 per 100,000). While most SCD cases occur in older people with coronary artery disease, there is a predominance of monogenetic and polygenetic diseases in the young. METHODS: Postmortem genetic analysis (molecular autopsy) using next-generation sequencing reveals a definite pathogenic genetic alteration, which can explain SCD of young patients in near 20% of the cases. Hence, postmortem genetic analysis has become an important tool to unravel the inheritable cause of death. Furthermore, early identification of a pathogenic genetic sequence variant in the deceased is crucial to reduce risk in relatives due to preventive personalized measures. RESULTS AND CONCLUSION: Postmortem genetic analysis forms together with the clinical assessment the basis for early identification of at-risk relatives. A new guideline for the management of ventricular arrhythmias and prevention of sudden death was recently published by the European Society of Cardiology. The new recommendations give genetic testing, also in deceased patients a much higher priority reflecting increasing relevance of genetic testing for diagnostic evaluation, risk stratification and prevention.

Influence of microRNAs on iNOS expression in postmortem human infarction hearts.

MicroRNAs (miRNAs) are important post-transcriptional regulators in several diseases, including cancer, immunologic and cardiovascular diseases. A growing list of miRNAs are dysregulated in cardiac arrhythmias, contractility diseases, myocardial infarction (MI), sudden cardiac death (SCD), chronic heart failure and hypertrophy. However, the exact regulatory pathways, through which miRNAs exert their effects are often unclear. In this study, we measured the expression patterns of miR-21, miR-939 and miR-30e in postmortem human MI. The aim of the study was to examine the influence of these miRNAs on cardiac inducible nitric oxide synthase (iNOS) mRNA levels. We measured iNOS mRNA and miRNA expression patterns by means of qPCR. Further we used correlation analyses to determine causality between miRNA expression and cardiac iNOS levels. iNOS mRNA, miR-21, miR-939 and miR-30e were significantly upregulated in infarcted and non-infarcted regions of postmortem human MI hearts in comparison to healthy controls. While miR-21 and miR-939 showed their strongest expression in infarcted regions, miR-30e peaked in the non-infarcted myocardium. Further, we found a significant correlation between miR-939 and iNOS expression levels in controls and infarcted regions. The results indicate, that miR-939 is a regulator of cardiac iNOS expression. However, a massive iNOS activation might exceed the capability of miR-939 to keep its expression in balance. miR-21 and miR-30e do not seem to influence cardiac iNOS levels in MI. Further studies are needed to evaluate downstream targets of these miRNAs and their signaling pathways to clarify their role in human MI.

From rare events to systematic data collection: the RESCUED registry for sudden cardiac death in the young in Germany.

BACKGROUND: Approximately one-third of sudden cardiac deaths in the young (SCDY) occur due to a structural cardiac disease. Forty to fifty percent of SCDY cases remain unexplained after autopsy (including microscopic and forensic-toxicological analyses), suggesting arrhythmia syndromes as a possible cause of death. Due to the possible inheritability of these diseases, blood relatives of the deceased may equally be carriers of the causative genetic variations and therefore may have an increased cardiac risk profile. A better understanding of the forensic, clinical, and genetic data might help identify a subset of the general population that is at increased risk of sudden cardiac death. STUDY DESIGN: The German registry RESCUED (REgistry for Sudden Cardiac and UnExpected Death) comprises information about SCDY fatalities and clinical and genetic data of both the deceased and their biological relatives. The datasets collected in the RESCUED registry will allow for the identification of leading causes of SCDY in Germany and offer unique possibilities of scientific analyses with the aim of detecting unrecognized trends, risk factors, and clinical warning signs of SCDY. In a pilot phase of 24 months, approximately 180 SCDY cases (< 50 years of age) and 500 family members and clinical patients will be included. CONCLUSION: RESCUED is the first registry in Germany collecting comprehensive data of SCDY cases and clinical data of the biological relatives reviewed by cardiac experts. RESCUED aims to improve individual risk assessment and public health approaches by directing resources towards early diagnosis and evidence-based, personalized therapy and prevention in affected families. Trial registration number (TRN): DRKS00033543.

Cardiac channelopathy causing sudden death as revealed by molecular autopsy.

BACKGROUND: Mutations in cardiac ion channel genes have been identified to cause sudden unexplained deaths (SUD), and polymorphisms have also been suggested to be risk factors. Therefore, postmortem genetic testing has become an important tool in elucidating the cause of death. METHODS AND RESULTS: In a sudden death case, a LQT-3-associated mutation (Il768V) in the cardiac sodium channel gene SCN5A was detected beside the common polymorphism H558R which is known to mitigate the effect of mutations in the gene. Both sequence variations were heterozygous. Large number of intervening base pairs make it impossible to identify whether they were located in cis or trans. Functional consequences of both variants were characterized after expressing different cRNAs in Xenopus oocytes by voltage clamp measurements. Western blot analysis indicates that the cis configuration of both variants may lead to a null allele. Since the woman had received an injection of Ultracain®, the potential effect of this drug was tested. In a trans configuration of both variants, the mutant channel exhibited an increase susceptibility of at least 10% for blocking with the drug articaine. Another novel finding is that the midpoint of activation in the case of the mutant channel is leftward shifted of at least -10 mV. CONCLUSION: The results of the study suggest that postmortem molecular screening is an important tool to elucidate the cause of SUD and that the administration of a drug and a functional interaction between polymorphisms and ion channel mutations may trigger the risk for sudden death.

Biological activities of ethanolic extracts from deep-sea Antarctic marine sponges.

We report on the screening of ethanolic extracts from 33 deep-sea Antarctic marine sponges for different biological activities. We monitored hemolysis, inhibition of acetylcholinesterase, cytotoxicity towards normal and transformed cells and growth inhibition of laboratory, commensal and clinically and ecologically relevant bacteria. The most prominent activities were associated with the extracts from sponges belonging to the genus Latrunculia, which show all of these activities. While most of these activities are associated to already known secondary metabolites, the extremely strong acetylcholinesterase inhibitory potential appears to be related to a compound unknown to date. Extracts from Tetilla leptoderma, Bathydorus cf. spinosus, Xestospongia sp., Rossella sp., Rossella cf. racovitzae and Halichondria osculum were hemolytic, with the last two also showing moderate cytotoxic potential. The antibacterial tests showed significantly greater activities of the extracts of these Antarctic sponges towards ecologically relevant bacteria from sea water and from Arctic ice. This indicates their ecological relevance for inhibition of bacterial microfouling.

iNOS expressing macrophages co-localize with nitrotyrosine staining after myocardial infarction in humans.

Introduction: Inducible nitric oxide synthase (iNOS) produces micromolar amounts of nitric oxide (NO) upon the right stimuli, whose further reactions can lead to oxidative stress. In murine models of myocardial infarction (MI), iNOS is known to be expressed in infiltrating macrophages, which at early onset enter the infarcted zone and are associated with inflammation. In contrast cardiac tissue resident macrophages are thought to enhance regeneration of tissue injury and re-establish homeostasis. Both detrimental and beneficial effects of iNOS have been described, still the role of iNOS in MI is not fully understood. Our aim was to examine cell expression patterns of iNOS and nitrotyrosine (NT) production in human MI. Material and Methods: We examined in postmortem human MI hearts the iNOS mRNA expression by means of qPCR. Further we performed immunohistochemical stainings for cell type identification. Afterwards a distance analysis between iNOS and NT was carried out to determine causality between iNOS and NT production. Results: iNOS mRNA expression was significantly increased in infarcted regions of human MI hearts and iNOS protein expression was detected in resident macrophages in infarcted human hearts as well as in controls hearts, being higher in resident macrophages in MI hearts compared to control. Furthermore in MI and in healthy human hearts cells showing signs of NT production peaked within 10-15 µm proximity of iNOS+ cells. Discussion: These results indicate that, unexpectedly, resident macrophages are the main source of iNOS expression in postmortem human MI hearts. The peak of NT positive cells within 10-15 µm of iNOS+ cells suggest an iNOS dependent level of NT and therefore iNOS dependent oxidative stress. Our results contribute to understanding the role of iNOS in human MI.

Suitable biomarkers for post-mortem differentiation of cardiac death causes: Quantitative analysis of miR-1, miR-133a and miR-26a in heart tissue and whole blood.

Cardiovascular diseases are the most common causes of death worldwide. Cardiac death can occur as reaction to myocardial infarction (MI). A diagnostic challenge arises for sudden unexpected death (SUD) cases with structural abnormalities (SA) or without any structural abnormalities (without SA). Therefore, the identification of reliable biomarkers to differentiate cardiac cases from each other is necessary. In the current study, the potential of different microRNAs (miRNAs) as biomarkers in tissue and blood samples of cardiac death cases was analyzed. Blood and tissue samples of 24 MI, 21 SUD and 5 control (C) cases were collected during autopsy. Testing for significance and receiver operating characteristic analysis (ROC) were performed. The results show that miR-1, miR-133a and miR-26a possess a high diagnostic power to discriminate between different cardiac death causes in whole blood and in tissue.