Characterization of COVID-19-associated cardiac injury: evidence for a multifactorial disease in an autopsy cohort.

As the coronavirus disease 2019 (COVID-19) pandemic evolves, much evidence implicates the heart as a critical target of injury in patients. The mechanism(s) of cardiac involvement has not been fully elucidated, although evidence of direct virus-mediated injury, thromboembolism with ischemic complications, and cytokine storm has been reported. We examined suggested mechanisms of COVID-19-associated heart failure in 21 COVID-19-positive decedents, obtained through standard autopsy procedure, compared to clinically matched controls and patients with various etiologies of viral myocarditis. We developed a custom tissue microarray using regions of pathological interest and interrogated tissues via immunohistochemistry and in situ hybridization. Severe acute respiratory syndrome coronavirus 2 was detected in 16/21 patients, in cardiomyocytes, the endothelium, interstitial spaces, and percolating adipocytes within the myocardium. Virus detection typically corresponded with troponin depletion and increased cleaved caspase-3. Indirect mechanisms of injury-venous and arterial thromboses with associated vasculitis including a mixed inflammatory infiltrate-were also observed. Neutrophil extracellular traps (NETs) were present in the myocardium of all COVID-19 patients, regardless of injury degree. Borderline myocarditis (inflammation without associated myocyte injury) was observed in 19/21 patients, characterized by a predominantly mononuclear inflammatory infiltrate. Edema, inflammation of percolating adipocytes, lymphocytic aggregates, and large septal masses of inflammatory cells and platelets were observed as defining features, and myofibrillar damage was evident in all patients. Collectively, COVID-19-associated cardiac injury was multifactorial, with elevated levels of NETs and von Willebrand factor as defining features of direct and indirect viral injury.

Advanced detection strategies for cardiotropic virus infection in a cohort study of heart failure patients.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein-Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches.

Cleavage and Sub-Cellular Redistribution of Nuclear Pore Protein 98 by Coxsackievirus B3 Protease 2A Impairs Cardioprotection.

Myocarditis, inflammation of the heart muscle, affects all demographics and is a major cause of sudden and unexpected death in young people. It is most commonly caused by viral infections of the heart, with coxsackievirus B3 (CVB3) being among the most prevalent pathogens. To understand the molecular pathogenesis of CVB3 infection and provide strategies for developing treatments, we examined the role of a key nuclear pore protein 98 (NUP98) in the setting of viral myocarditis. NUP98 was cleaved as early as 2 h post-CVB3 infection. This cleavage was further verified through both the ectopic expression of viral proteases and in vitro using purified recombinant CVB3 proteases (2A and 3C), which demonstrated that CVB3 2A but not 3C is responsible for this cleavage. By immunostaining and confocal imaging, we observed that cleavage resulted in the redistribution of NUP98 to punctate structures in the cytoplasm. Targeted siRNA knockdown of NUP98 during infection further increased viral protein expression and viral titer, and reduced cell viability, suggesting a potential antiviral role of NUP98. Moreover, we discovered that expression levels of neuregulin-1 (NRG1), a cardioprotective gene, and presenilin-1 (PSEN1), a cellular protease processing the tyrosine kinase receptor ERBB4 of NRG1, were reliant upon NUP98 and were downregulated during CVB3 infection. In addition, expression of these NUP98 target genes in myocardium tissue not only occurred at an earlier phase of infection, but also appeared in areas away from the initial inflammatory regions. Collectively, CVB3-induced cleavage of NUP98 and subsequent impairment of the cardioprotective NRG1-ERBB4/PSEN1 signaling cascade may contribute to increased myocardial damage in the context of CVB3-induced myocarditis. To our knowledge, this is the first study to demonstrate the link between NUP98 and the NRG1 signaling pathway in viral myocarditis.