Profiling Reduced Expression of Contractile and Mitochondrial mRNAs in the Human Sinoatrial Node vs. Right Atrium and Predicting Their Suppressed Expression by Transcription Factors and/or microRNAs.

(1) Background: The sinus node (SN) is the main pacemaker of the heart. It is characterized by pacemaker cells that lack mitochondria and contractile elements. We investigated the possibility that transcription factors (TFs) and microRNAs (miRs) present in the SN can regulate gene expression that affects SN morphology and function. (2) Methods: From human next-generation sequencing data, a list of mRNAs that are expressed at lower levels in the SN compared with the right atrium (RA) was compiled. The mRNAs were then classified into contractile, mitochondrial or glycogen mRNAs using bioinformatic software, RStudio and Ingenuity Pathway Analysis. The mRNAs were combined with TFs and miRs to predict their interactions. (3) Results: From a compilation of the 1357 mRNAs, 280 contractile mRNAs and 198 mitochondrial mRNAs were identified to be expressed at lower levels in the SN compared with RA. TFs and miRs were shown to interact with contractile and mitochondrial function-related mRNAs. (4) Conclusions: In human SN, TFs (MYCN, SOX2, NUPR1 and PRDM16) mainly regulate mitochondrial mRNAs (COX5A, SLC25A11 and NDUFA8), while miRs (miR-153-3p, miR-654-5p, miR-10a-5p and miR-215-5p) mainly regulate contractile mRNAs (RYR2, CAMK2A and PRKAR1A). TF and miR-mRNA interactions provide a further understanding of the complex molecular makeup of the SN and potential therapeutic targets for cardiovascular treatments.

Further insights into the molecular complexity of the human sinus node - The role of 'novel' transcription factors and microRNAs.

RESEARCH PURPOSE: The sinus node (SN) is the heart's primary pacemaker. Key ion channels (mainly the funny channel, HCN4) and Ca2+-handling proteins in the SN are responsible for its function. Transcription factors (TFs) regulate gene expression through inhibition or activation and microRNAs (miRs) do this through inhibition. There is high expression of macrophages and mast cells within the SN connective tissue. 'Novel'/unexplored TFs and miRs in the regulation of ion channels and immune cells in the SN are not well understood. Using RNAseq and bioinformatics, the expression profile and predicted interaction of key TFs and cell markers with key miRs in the adult human SN vs. right atrial tissue (RA) were determined. PRINCIPAL RESULTS: 68 and 60 TFs significantly more or less expressed in the SN vs. RA respectively. Among those more expressed were ISL1 and TBX3 (involved in embryonic development of the SN) and 'novel' RUNX1-2, CEBPA, GLI1-2 and SOX2. These TFs were predicted to regulate HCN4 expression in the SN. Markers for different cells: fibroblasts (COL1A1), fat (FABP4), macrophages (CSF1R and CD209), natural killer (GZMA) and mast (TPSAB1) were significantly more expressed in the SN vs. RA. Interestingly, RUNX1-3, CEBPA and GLI1 also regulate expression of these cells. MiR-486-3p inhibits HCN4 and markers involved in immune response. MAJOR CONCLUSIONS: In conclusion, RUNX1-2, CSF1R, TPSAB1, COL1A1 and HCN4 are highly expressed in the SN but not miR-486-3p. Their complex interactions can be used to treat SN dysfunction such as bradycardia. Interestingly, another research group recently reported miR-486-3p is upregulated in blood samples from severe COVID-19 patients who suffer from bradycardia.

Post-Mortem Diagnosis and Autopsy Findings in SARS-CoV-2 Infection: Forensic Case Series.

Towards the end of 2019, a novel coronavirus was identified as the culprit for a cluster of pneumonia cases in Wuhan, China. Since then, it has rapidly spread worldwide, affecting more than 43 million people, and in March 2020, the World Health Organization (WHO) declared it a pandemic. The purpose of the study is to present the findings of 15 forensic autopsies performed in Romania, on SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) positive subjects, and to present the case of one SARS-CoV-2 infected patient who experienced a violent death, as established during their autopsy. A total of 11 male and 4 female patients were autopsied, and SARS-CoV-2 infection was diagnosed post-mortem in two cases. The most frequent symptoms before death were dry cough, dyspnoea, and fever. Hypertension, ischemic cardiac disease, and a history of stroke were the most frequent associated diseases. The mean duration from the symptoms' debut to a RT-PCR positive SARS-CoV-2 test was 3.7 days, while the mean survival time from the RT-PCR positive test was 4.2 days. A histological examination was performed in seven cases and revealed, in most of them, hyaline membranes, and mixed inflammatory cell infiltration of the interstitium, alveoli, and perivascular areas. In addition, all of the examined cases developed small vessel thrombosis. A case of violent death was also reported, regarding a 87-year-old male subject who suffered a femur fracture (domestic fall) and was diagnosed with SARS-CoV-2 infection the following day after surgery. After transfer to a COVID-19 (coronavirus disease-19) support hospital, during an episode of behavioral disorder, the patient jumped from the first floor window. Death occurred a few days later, and the cause was established as bronchopneumonia superimposed on SARS-CoV-2 infection. In conclusion, autopsies should be conducted while providing a safe environment for professionals to perform them, because they are crucial procedures that can help gain a better understanding of the role of SARS-CoV-2 infection in thanatogenesis.

Identification of Key Small Non-Coding MicroRNAs Controlling Pacemaker Mechanisms in the Human Sinus Node.

Background The sinus node (SN) is the primary pacemaker of the heart. SN myocytes possess distinctive action potential morphology with spontaneous diastolic depolarization because of a unique expression of ion channels and Ca2+-handling proteins. MicroRNAs (miRs) inhibit gene expression. The role of miRs in controlling the expression of genes responsible for human SN pacemaking and conduction has not been explored. The aim of this study was to determine miR expression profile of the human SN as compared with that of non-pacemaker atrial muscle. Methods and Results SN and atrial muscle biopsies were obtained from donor or post-mortem hearts (n=10), histology/immunolabeling were used to characterize the tissues, TaqMan Human MicroRNA Arrays were used to measure 754 miRs, Ingenuity Pathway Analysis was used to identify miRs controlling SN pacemaker gene expression. Eighteen miRs were significantly more and 48 significantly less abundant in the SN than atrial muscle. The most interesting miR was miR-486-3p predicted to inhibit expression of pacemaking channels: HCN1 (hyperpolarization-activated cyclic nucleotide-gated 1), HCN4, voltage-gated calcium channel (Cav)1.3, and Cav3.1. A luciferase reporter gene assay confirmed that miR-486-3p can control HCN4 expression via its 3' untranslated region. In ex vivo SN preparations, transfection with miR-486-3p reduced the beating rate by ≈35±5% (P<0.05) and HCN4 expression (P<0.05). Conclusions The human SN possesses a unique pattern of expression of miRs predicted to target functionally important genes. miR-486-3p has an important role in SN pacemaker activity by targeting HCN4, making it a potential target for therapeutic treatment of SN disease such as sinus tachycardia.

High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling.

Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.