Single-cell analysis of adult human heart across healthy and cardiovascular disease patients reveals the cellular landscape underlying SARS-CoV-2 invasion of myocardial tissue through ACE2.

BACKGROUND: The distribution of ACE2 and accessory proteases (ANAD17 and CTSL) in cardiovascular tissue and the host cell receptor binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial to understanding the virus's cell invasion, which may play a significant role in determining the viral tropism and its clinical manifestations. METHODS: We conducted a comprehensive analysis of the cell type-specific expression of ACE2, ADAM17, and CTSL in myocardial tissue from 10 patients using RNA sequencing. Our study included a meta-analysis of 2 heart single-cell RNA-sequencing studies with a total of 90,024 cells from 250 heart samples of 10 individuals. We used co-expression analysis to locate specific cell types that SARS-CoV-2 may invade. RESULTS: Our results revealed cell-type specific associations between male gender and the expression levels of ACE2, ADAM17, and CTSL, including pericytes and fibroblasts. AGT, CALM3, PCSK5, NRP1, and LMAN were identified as potential accessory proteases that might facilitate viral invasion. Enrichment analysis highlighted the extracellular matrix interaction pathway, adherent plaque pathway, vascular smooth muscle contraction inflammatory response, and oxidative stress as potential immune pathways involved in viral infection, providing potential molecular targets for therapeutic intervention. We also found specific high expression of IFITM3 and AGT in pericytes and differences in the IFN-II signaling pathway and PAR signaling pathway in fibroblasts from different cardiovascular comorbidities. CONCLUSIONS: Our data indicated possible high-risk groups for COVID-19 and provided emerging avenues for future investigations of its pathogenesis. TRIAL REGISTRATION: (Not applicable).

Vitamin C Regulates the Profibrotic Activity of Fibroblasts in In Vitro Replica Settings of Myocardial Infarction.

Extracellular collagen remodeling is one of the central mechanisms responsible for the structural and compositional coherence of myocardium in patients undergoing myocardial infarction (MI). Activated primary cardiac fibroblasts following myocardial infarction are extensively investigated to establish anti-fibrotic therapies to improve left ventricular remodeling. To systematically assess vitamin C functions as a potential modulator involved in collagen fibrillogenesis in an in vitro model mimicking heart tissue healing after MI. Mouse primary cardiac fibroblasts were isolated from wild-type C57BL/6 mice and cultured under normal and profibrotic (hypoxic + transforming growth factor beta 1) conditions on freshly prepared coatings mimicking extracellular matrix (ECM) remodeling during healing after an MI. At 10 µg/mL, vitamin C reprogramed the respiratory mitochondrial metabolism, which is effectively associated with a more increased accumulation of intracellular reactive oxygen species (iROS) than the number of those generated by mitochondrial reactive oxygen species (mROS). The mRNA/protein expression of subtypes I, III collagen, and fibroblasts differentiations markers were upregulated over time, particularly in the presence of vitamin C. The collagen substrate potentiated the modulator role of vitamin C in reinforcing the structure of types I and III collagen synthesis by reducing collagen V expression in a timely manner, which is important in the initiation of fibrillogenesis. Altogether, our study evidenced the synergistic function of vitamin C at an optimum dose on maintaining the equilibrium functionality of radical scavenger and gene transcription, which are important in the initial phases after healing after an MI, while modulating the synthesis of de novo collagen fibrils, which is important in the final stage of tissue healing.

Protection of zero-valent iron nanoparticles against sepsis and septic heart failure.

BACKGROUND: Septic heart failure accounts for high mortality rates globally. With a strong reducing capacity, zero-valent iron nanoparticles (nanoFe) have been applied in many fields. However, the precise roles and mechanisms of nanoFe in septic cardiomyopathy remain unknown. RESULTS: NanoFe was prepared via the liquid-phase reduction method and functionalized with the biocompatible polymer sodium carboxymethylcellulose (CMC). We then successfully constructed a mouse model of septic myocardial injury by challenging with cecal ligation and puncture (CLP). Our findings demonstrated that nanoFe has a significant protective effect on CLP-induced septic myocardial injury. This may be achieved by attenuating inflammation and oxidative stress, improving mitochondrial function, regulating endoplasmic reticulum stress, and activating the AMPK pathway. The RNA-seq results supported the role of nanoFe treatment in regulating a transcriptional profile consistent with its role in response to sepsis. CONCLUSIONS: The results provide a theoretical basis for the application strategy and combination of nanoFe in sepsis and septic myocardial injury.

Arrhythmogenic Cardiomyopathy: Exercise Pitfalls, Role of Connexin-43, and Moving beyond Antiarrhythmics.

Arrhythmogenic Cardiomyopathy (ACM), a Mendelian disorder that can affect both left and right ventricles, is most often associated with pathogenic desmosomal variants that can lead to fibrofatty replacement of the myocardium, a pathological hallmark of this disease. Current therapies are aimed to prevent the worsening of disease phenotypes and sudden cardiac death (SCD). Despite the use of implantable cardioverter defibrillators (ICDs) there is no present therapy that would mitigate the loss in electrical signal and propagation by these fibrofatty barriers. Recent studies have shown the influence of forced vs. voluntary exercise in a variety of healthy and diseased mice; more specifically, that exercised mice show increased Connexin-43 (Cx43) expression levels. Fascinatingly, increased Cx43 expression ameliorated the abnormal electrical signal conduction in the myocardium of diseased mice. These findings point to a major translational pitfall in current therapeutics for ACM patients, who are advised to completely cease exercising and already demonstrate reduced Cx43 levels at the myocyte intercalated disc. Considering cardiac dysfunction in ACM arises from the loss of cardiomyocytes and electrical signal conduction abnormalities, an increase in Cx43 expression-promoted by low to moderate intensity exercise and/or gene therapy-could very well improve cardiac function in ACM patients.

Development and diseases of the coronary microvasculature and its communication with the myocardium.

We review the current understanding of formation and development of the coronary microvasculature which supplies oxygen and nutrients to the heart myocardium and removes waste. We emphasize the close relationship, mutual development, and communication between microvasculature endothelial cells and surrounding cardiomyocytes. The first part of the review is focused on formation of microvasculature during embryonic development. We summarize knowledge about establishing the heart microvasculature density based on diffusion distance. Then signaling mechanisms which are involved in forming the microvasculature are discussed. This includes details of cardiomyocyte-endothelial cell interactions involving hypoxia, VEGF, NOTCH, angiopoietin, PDGF, and other signaling factors. The microvasculature is understudied due to difficulties in its visualization. Therefore, currently available imaging methods to delineate the coronary microvasculature in development and in adults are discussed. The second part of the review is dedicated to the importance of the coronary vasculature in disease. Coronary microvasculature pathologies are present in many congenital heart diseases (CHD), especially in pulmonary atresia, and worsen outcomes. In CHDs, where the development of the myocardium is impaired, microvasculature is also affected. In adult patients coronary microvascular disease is one of the main causes of sudden cardiac death, especially in women. Coronary microvasculature pathologies affect myocardial ischemia and vice versa; myocardial pathologies such as cardiomyopathies are closely connected with coronary microvasculature dysfunction. Microvasculature inflammation also worsens the outcomes of COVID-19 disease. Our review stresses the importance of coronary microvasculature and provides an overview of its formation and signaling mechanisms and the importance of coronary vasculature pathologies in CHDs and adult diseases. This article is categorized under: Cardiovascular Diseases > Stem Cells and Development Congenital Diseases > Molecular and Cellular Physiology Cardiovascular Diseases > Molecular and Cellular Physiology.

CVD and COVID-19: Emerging Roles of Cardiac Fibroblasts and Myofibroblasts.

Cardiovascular disease (CVD) is the leading cause of death worldwide. Current data suggest that patients with cardiovascular diseases experience more serious complications with coronavirus disease-19 (COVID-19) than those without CVD. In addition, severe COVID-19 appears to cause acute cardiac injury, as well as long-term adverse remodeling of heart tissue. Cardiac fibroblasts and myofibroblasts, being crucial in response to injury, may play a pivotal role in both contributing to and healing COVID-19-induced cardiac injury. The role of cardiac myofibroblasts in cardiac fibrosis has been well-established in the literature for decades. However, with the emergence of the novel coronavirus SARS-CoV-2, new cardiac complications are arising. Bursts of inflammatory cytokines and upregulation of TGF-ß1 and angiotensin (AngII) are common in severe COVID-19 patients. Cytokines, TGF-ß1, and Ang II can induce cardiac fibroblast differentiation, potentially leading to fibrosis. This review details the key information concerning the role of cardiac myofibroblasts in CVD and COVID-19 complications. Additionally, new factors including controlling ACE2 expression and microRNA regulation are explored as promising treatments for both COVID-19 and CVD. Further understanding of this topic may provide insight into the long-term cardiac manifestations of the COVID-19 pandemic and ways to mitigate its negative effects.

Immunohistochemical Expression of VDR in Myocardium: Postmortem Evaluation of COVID-19 Patients.

Morphological data on heart damage and its mechanisms due to extremely severe course of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection are limited, as well as data on the correlation of damage and expression of vitamin D receptors (VDRs). In this study, we analyzed a series of myocardial samples obtained during postmortem autopsy of 48 critically ill patients with COVID-19 who died with SARS-CoV-2-associated pneumonia. The purpose of the study was to evaluate immunohistochemical VDR expression in the myocardium. The results showed the only minimal or no immunohistochemical expression of VDR in the nuclei of cardiomyocytes in most cases, along with the persisted strong expression in lymphoid cells. To the best of our knowledge, it is the first study and data provided were regarding myocardial VDR expression in COVID-19 patients. The results are of interest in terms of further study of the effects of ligand-associated VDR activation on the cardiovascular system.

Identification of Crucial Genes and Key Functions in Type 2 Diabetic Hearts by Bioinformatic Analysis.

Type 2 diabetes (T2D) patients with SARS-CoV-2 infection hospitalized develop an acute cardiovascular syndrome. It is urgent to elucidate underlying mechanisms associated with the acute cardiac injury in T2D hearts. We performed bioinformatic analysis on the expression profiles of public datasets to identify the pathogenic and prognostic genes in T2D hearts. Cardiac RNA-sequencing datasets from db/db or BKS mice (GSE161931) were updated to NCBI-Gene Expression Omnibus (NCBI-GEO), and used for the transcriptomics analyses with public datasets from NCBI-GEO of autopsy heart specimens with COVID-19 (5/6 with T2D, GSE150316), or dead healthy persons (GSE133054). Differentially expressed genes (DEGs) and overlapping homologous DEGs among the three datasets were identified using DESeq2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses were conducted for event enrichment through clusterProfile. The protein-protein interaction (PPI) network of DEGs was established and visualized by Cytoscape. The transcriptions and functions of crucial genes were further validated in db/db hearts. In total, 542 up-regulated and 485 down-regulated DEGs in mice, and 811 up-regulated and 1399 down-regulated DEGs in human were identified, respectively. There were 74 overlapping homologous DEGs among all datasets. Mitochondria inner membrane and serine-type endopeptidase activity were further identified as the top-10 GO events for overlapping DEGs. Cardiac CAPNS1 (calpain small subunit 1) was the unique crucial gene shared by both enriched events. Its transcriptional level significantly increased in T2D mice, but surprisingly decreased in T2D patients with SARS-CoV-2 infection. PPI network was constructed with 30 interactions in overlapping DEGs, including CAPNS1. The substrates Junctophilin2 (Jp2), Tnni3, and Mybpc3 in cardiac calpain/CAPNS1 pathway showed less transcriptional change, although Capns1 increased in transcription in db/db mice. Instead, cytoplasmic JP2 significantly reduced and its hydrolyzed product JP2NT exhibited nuclear translocation in myocardium. This study suggests CAPNS1 is a crucial gene in T2D hearts. Its transcriptional upregulation leads to calpain/CAPNS1-associated JP2 hydrolysis and JP2NT nuclear translocation. Therefore, attenuated cardiac CAPNS1 transcription in T2D patients with SARS-CoV-2 infection highlights a novel target in adverse prognostics and comprehensive therapy. CAPNS1 can also be explored for the molecular signaling involving the onset, progression and prognostic in T2D patients with SARS-CoV-2 infection.

Activation of Immune System May Cause Pathophysiological Changes in the Myocardium of SARS-CoV-2 Infected Monkey Model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is an extremely contagious disease whereby the virus damages the host's respiratory tract via entering through the ACE2 receptor. Cardiovascular disorder is being recognized in the majority of COVID-19 patients; yet, the relationship between SARS-CoV-2 and heart failure has not been established. In the present study, SARS-CoV-2 infection was induced in the monkey model. Thereafter, heart tissue samples were collected, and pathological changes were analyzed in the left ventricular tissue by hematoxylin and eosin, trichrome, and immunohistochemical staining specific to T lymphocytes and macrophages. The findings revealed that SARS-CoV-2 infection induces several pathological changes in the heart, which cause cardiomyocyte disarray, mononuclear infiltrates of inflammatory cells, and hypertrophy. Furthermore, collagen-specific staining showed the development of cardiac fibrosis in the interstitial and perivascular regions in the hearts of infected primates. Moreover, the myocardial tissue samples displayed multiple foci of inflammatory cells positive for T lymphocytes and macrophages within the myocardium. These findings suggest the progression of the disease, which can lead to the development of severe complications, including heart failure. Additionally, SARS-CoV-2 antigen staining detected the presence of virus particles in the myocardium. Thus, we found that SARS-CoV-2 infection is characterized by an exaggerated inflammatory immune response in the heart, which possibly contributes to myocardial remodeling and subsequent fibrosis.

Mechanism of Blood-Heart-Barrier Leakage: Implications for COVID-19 Induced Cardiovascular Injury.

Although blood-heart-barrier (BHB) leakage is the hallmark of congestive (cardio-pulmonary) heart failure (CHF), the primary cause of death in elderly, and during viral myocarditis resulting from the novel coronavirus variants such as the severe acute respiratory syndrome novel corona virus 2 (SARS-CoV-2) known as COVID-19, the mechanism is unclear. The goal of this project is to determine the mechanism of the BHB in CHF. Endocardial endothelium (EE) is the BHB against leakage of blood from endocardium to the interstitium; however, this BHB is broken during CHF. Previous studies from our laboratory, and others have shown a robust activation of matrix metalloproteinase-9 (MMP-9) during CHF. MMP-9 degrades the connexins leading to EE dysfunction. We demonstrated juxtacrine coupling of EE with myocyte and mitochondria (Mito) but how it works still remains at large. To test whether activation of MMP-9 causes EE barrier dysfunction, we hypothesized that if that were the case then treatment with hydroxychloroquine (HCQ) could, in fact, inhibit MMP-9, and thus preserve the EE barrier/juxtacrine signaling, and synchronous endothelial-myocyte coupling. To determine this, CHF was created by aorta-vena cava fistula (AVF) employing the mouse as a model system. The sham, and AVF mice were treated with HCQ. Cardiac hypertrophy, tissue remodeling-induced mitochondrial-myocyte, and endothelial-myocyte contractions were measured. Microvascular leakage was measured using FITC-albumin conjugate. The cardiac function was measured by echocardiography (Echo). Results suggest that MMP-9 activation, endocardial endothelial leakage, endothelial-myocyte (E-M) uncoupling, dyssynchronous mitochondrial fusion-fission (Mfn2/Drp1 ratio), and mito-myocyte uncoupling in the AVF heart failure were found to be rampant; however, treatment with HCQ successfully mitigated some of the deleterious cardiac alterations during CHF. The findings have direct relevance to the gamut of cardiac manifestations, and the resultant phenotypes arising from the ongoing complications of COVID-19 in human subjects.

Clinico-histopathologic and single-nuclei RNA-sequencing insights into cardiac injury and microthrombi in critical COVID-19.

Acute cardiac injury is prevalent in critical COVID-19 and associated with increased mortality. Its etiology remains debated, as initially presumed causes - myocarditis and cardiac necrosis - have proved uncommon. To elucidate the pathophysiology of COVID-19-associated cardiac injury, we conducted a prospective study of the first 69 consecutive COVID-19 decedents at CUIMC in New York City. Of 6 acute cardiac histopathologic features, presence of microthrombi was the most commonly detected among our cohort. We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak erythrocyte sedimentation rate and C-reactive protein were independently associated with increased odds of microthrombi, supporting an immunothrombotic etiology. Using single-nuclei RNA-sequencing analysis on 3 patients with and 4 patients without cardiac microthrombi, we discovered an enrichment of prothrombotic/antifibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling among cardiac fibroblasts in microthrombi-positive, relative to microthrombi-negative, COVID-19 hearts. Non-COVID-19, nonfailing hearts were used as reference controls. Our study identifies a specific transcriptomic signature in cardiac fibroblasts as a salient feature of microthrombi-positive COVID-19 hearts. Our findings warrant further mechanistic study as cardiac fibroblasts may represent a potential therapeutic target for COVID-19-associated cardiac microthrombi.

Interaction of SARS-CoV-2 with cardiomyocytes: Insight into the underlying molecular mechanisms of cardiac injury and pharmacotherapy.

SARS-CoV-2 causes respiratory illness with a spectrum of systemic complications. However, the mechanism for cardiac infection and cardiomyocyte injury in COVID-19 patients remains unclear. The current literature supports the notion that SARS-CoV-2 particles access the heart either by the circulating blood cells or by extracellular vesicles, originating from the inflamed lungs, and encapsulating the virus along with its receptor (ACE2). Both cardiomyocytes and pericytes (coronary arteries) express the necessary accessory proteins for access of SARS-CoV-2 particles (i.e. ACE2, NRP-1, TMPRSS2, CD147, integrin α5ß1, and CTSB/L). These proteins facilitate the SARS-CoV-2 interaction and entry into the pericytes and cardiomyocytes thus leading to cardiac manifestations. Subsequently, various signaling pathways are altered in the infected cardiomyocytes (i.e. increased ROS production, reduced contraction, impaired calcium homeostasis), causing cardiac dysfunction. The currently adopted pharmacotherapy in severe COVID-19 subjects exhibited side effects on the heart, often manifested by electrical abnormalities. Nonetheless, cardiovascular adverse repercussions have been associated with the advent of some of the SARS-CoV-2 vaccines with no clear mechanisms underlining these complications. We provide herein an overview of the pathways involved with cardiomyocyte in COVID-19 subjects to help promoting pharmacotherapies that can protect against SARS-CoV-2-induced cardiac injuries.

The Pathogenesis of COVID-19 Myocardial Injury: An Immunohistochemical Study of Postmortem Biopsies.

Rationale: Myocardial injury associates significantly and independently with mortality in COVID-19 patients. However, the pathogenesis of myocardial injury in COVID-19 remains unclear, and cardiac involvement by SARS-CoV-2 presents a major challenge worldwide. Objective: This histological and immunohistochemical study sought to clarify the pathogenesis and propose a mechanism with pathways involved in COVID-19 myocardial injury. Methods and Results: Postmortem minimally invasive autopsies were performed in six patients who died from COVID-19, and the myocardium samples were compared to a control group (n=11). Histological analysis was performed using hematoxylin-eosin and toluidine blue staining. Immunohistochemical (IHC) staining was performed using monoclonal antibodies against targets: caspase-1, caspase-9, gasdermin-d, ICAM-1, IL-1ß, IL-4, IL-6, CD163, TNF-α, TGF-ß, MMP-9, type 1 and type 3 collagen. The samples were also assessed for apoptotic cells by TUNEL. Histological analysis showed severe pericardiocyte interstitial edema and higher mast cells counts per high-power field in all COVID-19 myocardium samples. The IHC analysis showed increased expression of caspase-1, ICAM-1, IL-1ß, IL-6, MMP-9, TNF-α, and other markers in the hearts of COVID-19 patients. Expression of caspase-9 did not differ from the controls, while gasdermin-d expression was less. The TUNEL assay was positive in all the COVID-19 samples supporting endothelial apoptosis. Conclusions: The pathogenesis of COVID-19 myocardial injury does not seem to relate to primary myocardiocyte involvement but to local inflammation with associated interstitial edema. We found heightened TGF-ß and interstitial collagen expression in COVID-affected hearts, a potential harbinger of chronic myocardial fibrosis. These results suggest a need for continued clinical surveillance of patients for myocardial dysfunction and arrythmias after recovery from the acute phase of COVID-19.

Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor-like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Cardiac Fibrosis Is a Risk Factor for Severe COVID-19.

Increased left ventricular fibrosis has been reported in patients hospitalized with coronavirus disease 2019 (COVID-19). It is unclear whether this fibrosis is a consequence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection or a risk factor for severe disease progression. We observed increased fibrosis in the left ventricular myocardium of deceased COVID-19 patients, compared with matched controls. We also detected increased mRNA levels of soluble interleukin-1 receptor-like 1 (sIL1-RL1) and transforming growth factor ß1 (TGF-ß1) in the left ventricular myocardium of deceased COVID-19 patients. Biochemical analysis of blood sampled from patients admitted to the emergency department (ED) with COVID-19 revealed highly elevated levels of TGF-ß1 mRNA in these patients compared to controls. Left ventricular strain measured by echocardiography as a marker of pre-existing cardiac fibrosis correlated strongly with blood TGF-ß1 mRNA levels and predicted disease severity in COVID-19 patients. In the left ventricular myocardium and lungs of COVID-19 patients, we found increased neuropilin-1 (NRP-1) RNA levels, which correlated strongly with the prevalence of pulmonary SARS-CoV-2 nucleocapsid. Cardiac and pulmonary fibrosis may therefore predispose these patients to increased cellular viral entry in the lung, which may explain the worse clinical outcome observed in our cohort. Our study demonstrates that patients at risk of clinical deterioration can be identified early by echocardiographic strain analysis and quantification of blood TGF-ß1 mRNA performed at the time of first medical contact.

Coronavirus Disease 2019 (COVID-19) Coronary Vascular Thrombosis: Correlation with Neutrophil but Not Endothelial Activation.

Severe coronavirus disease 2019 (COVID-19) increases the risk of myocardial injury that contributes to mortality. This study used multiparameter immunofluorescence to extensively examine heart autopsy tissue of 7 patients who died of COVID-19 compared to 12 control specimens, with or without cardiovascular disease. Consistent with prior reports, no evidence of viral infection or lymphocytic infiltration indicative of myocarditis was found. However, frequent and extensive thrombosis was observed in large and small vessels in the hearts of the COVID-19 cohort, findings that were infrequent in controls. The endothelial lining of thrombosed vessels typically lacked evidence of cytokine-mediated endothelial activation, assessed as nuclear expression of transcription factors p65 (RelA), pSTAT1, or pSTAT3, or evidence of inflammatory activation assessed by expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tissue factor, or von Willebrand factor (VWF). Intimal EC lining was also generally preserved with little evidence of cell death or desquamation. In contrast, there were frequent markers of neutrophil activation within myocardial thrombi in patients with COVID-19, including neutrophil-platelet aggregates, neutrophil-rich clusters within macrothrombi, and evidence of neutrophil extracellular trap (NET) formation. These findings point to alterations in circulating neutrophils rather than in the endothelium as contributors to the increased thrombotic diathesis in the hearts of COVID-19 patients.

Marked Up-Regulation of ACE2 in Hearts of Patients With Obstructive Hypertrophic Cardiomyopathy: Implications for SARS-CoV-2-Mediated COVID-19.

OBJECTIVE: To explore the transcriptomic differences between patients with hypertrophic cardiomyopathy (HCM) and controls. PATIENTS AND METHODS: RNA was extracted from cardiac tissue flash frozen at therapeutic surgical septal myectomy for 106 patients with HCM and 39 healthy donor hearts. Expression profiling of 37,846 genes was performed using the Illumina Human HT-12v3 Expression BeadChip. All patients with HCM were genotyped for pathogenic variants causing HCM. Technical validation was performed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. This study was started on January 1, 1999, and final analysis was completed on April 20, 2020. RESULTS: Overall, 22% of the transcriptome (8443 of 37,846 genes) was expressed differentially between HCM and control tissues. Analysis by genotype revealed that gene expression changes were similar among genotypic subgroups of HCM, with only 4% (1502 of 37,846) to 6% (2336 of 37,846) of the transcriptome exhibiting differential expression between genotypic subgroups. The qRT-PCR confirmed differential expression in 92% (11 of 12 genes) of tested transcripts. Notably, in the context of coronavirus disease 2019 (COVID-19), the transcript for angiotensin I converting enzyme 2 (ACE2), a negative regulator of the angiotensin system, was the single most up-regulated gene in HCM (fold-change, 3.53; q-value =1.30×10-23), which was confirmed by qRT-PCR in triplicate (fold change, 3.78; P=5.22×10-4), and Western blot confirmed greater than 5-fold overexpression of ACE2 protein (fold change, 5.34; P=1.66×10-6). CONCLUSION: More than 20% of the transcriptome is expressed differentially between HCM and control tissues. Importantly, ACE2 was the most up-regulated gene in HCM, indicating perhaps the heart's compensatory effort to mount an antihypertrophic, antifibrotic response. However, given that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses ACE2 for viral entry, this 5-fold increase in ACE2 protein may confer increased risk for COVID-19 manifestations and outcomes in patients with increased ACE2 transcript expression and protein levels in the heart.

Heart-rate-variability (HRV), predicts outcomes in COVID-19.

BACKGROUND: Patients with COVID-19 present with a variety of clinical manifestations, ranging from mild or asymptomatic disease to severe illness and death. Whilst previous studies have clarified these and several other aspects of COVID-19, one of the ongoing challenges regarding COVID-19 is to determine which patients are at risk of adverse outcomes of COVID-19 infection. It is hypothesized that this is the result of insufficient inhibition of the immune response, with the vagus nerve being an important neuro-immuno-modulator of inflammation. Vagus nerve activity can be non-invasively indexed by heart-rate-variability (HRV). Therefore, we aimed to assess the prognostic value of HRV, as a surrogate marker for vagus nerve activity, in predicting mortality and intensive care unit (ICU) referral, in patients hospitalized with COVID-19. METHODS: A retrospective cohort study including all consecutive patients (n = 271) diagnosed and hospitalized with COVID-19 between March 2020 and May 2020, without a history of cardiac arrhythmias (including atrial and ventricular premature contractions), pacemaker, or current bradycardia (heart rate <50 bpm) or tachycardia (heart rate >110 bpm). HRV was based on one 10s ECG recorded at admission. 3-week survival and ICU referral were examined. RESULTS: HRV indexed as standard deviation of normal to normal heartbeat intervals (SDNN) predicted survival (H.R. = 0.53 95%CI: 0.31-0.92). This protective role was observed only in patients aged 70 years and older, not in younger patients. HRV below median value also predicted ICU referral within the first week of hospitalization (H.R = 0.51, 95%CI: 0.29-0.90, P = 0.021). CONCLUSION: Higher HRV predicts greater chances of survival, especially in patients aged 70 years and older with COVID-19, independent of major prognostic factors. Low HRV predicts ICU indication and admission in the first week after hospitalization.

Cardiac SARS-CoV-2 infection is associated with pro-inflammatory transcriptomic alterations within the heart.

AIMS: Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell-specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry, and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19. METHODS AND RESULTS: In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). Massive analysis of cDNA ends (MACE)-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset 'Heart Cell Atlas' and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis. MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The Gene Ontology (GO) term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response. CONCLUSION: This study reveals that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.

The cardiac molecular setting of metabolic syndrome in pigs reveals disease susceptibility and suggests mechanisms that exacerbate COVID-19 outcomes in patients.

Although metabolic syndrome (MetS) is linked to an elevated risk of cardiovascular disease (CVD), the cardiac-specific risk mechanism is unknown. Obesity, hypertension, and diabetes (all MetS components) are the most common form of CVD and represent risk factors for worse COVID-19 outcomes compared to their non MetS peers. Here, we use obese Yorkshire pigs as a highly relevant animal model of human MetS, where pigs develop the hallmarks of human MetS and reproducibly mimics the myocardial pathophysiology in patients. Myocardium-specific mass spectroscopy-derived metabolomics, proteomics, and transcriptomics enabled the identity and quality of proteins and metabolites to be investigated in the myocardium to greater depth. Myocardium-specific deregulation of pro-inflammatory markers, propensity for arterial thrombosis, and platelet aggregation was revealed by computational analysis of differentially enriched pathways between MetS and control animals. While key components of the complement pathway and the immune response to viruses are under expressed, key N6-methyladenosin RNA methylation enzymes are largely overexpressed in MetS. Blood tests do not capture the entirety of metabolic changes that the myocardium undergoes, making this analysis of greater value than blood component analysis alone. Our findings create data associations to further characterize the MetS myocardium and disease vulnerability, emphasize the need for a multimodal therapeutic approach, and suggests a mechanism for observed worse outcomes in MetS patients with COVID-19 comorbidity.