ABSTRACT
Introduction: Recent evidences suggested that SARS-CoV-2 virus may directly infect heart cells, eventually driving the insurgence of cardiac complications in COVID19 patients, including ischemic heart disease and atrial fibrillation. Angiotensin-converting enzyme 2 (ACE2), which serves as gateway for coronavirus entry, is expressed in cardiomyocytes (CM). It has been reported that its expression may be upregulated in stress conditions, as in CM during dilated and hypertrophic cardiomyopathy. We aimed to explore the susceptibility of human CM derived from induced pluripotent stem-cells (hiPS-CM) in presence of doxorubicin-induced stress (Dx-CM). Methods: hiPS-CM were treated with a sub-apoptotic concentration of doxorubicin (200nM). Pseudo-typed lentiviral particles expressing different genotypes of SARS-CoV-2 Spike or mock, encoding a fluorescent reporter mCherry was used to evaluate susceptibility to virus entry in treated and control CM. Real time PCR and flow cytometry were used to evaluate respectively integration and expression of exogenous construct. The proximity ligation assay (PLA) was performed to directly visualize Spike-ACE2 interaction on the surface of CM. Results: Oxidative and metabolic stress occured in Dx-CM as a result of exposure to doxorubicin, as demonstrated by a significant increase of number of DNA damage-associated γ-H2AX-positive foci, increased ROS levels and significant depolarization of mitochondria membrane potential (ΔΨm). Dx-CM showed a higher expression of the host-cell surface ACE2 as compared to control, thus suggesting an enhanced SARS-CoV-2 tropism. Indeed, PLA assay showed increased efficiency in virions binding onto surface of Dx-CM as compared to control. The integration of viral genome in host cells was increased by 2-folds in Dx-CM versus untreated CM as assessed by PCR. Data were further confirmed by flow cytometry analysis showing the expression of mCherry in infected cells. Conclusion: Our preliminary results suggest that human stressed cardiomyocytes are more susceptible to SARS-CoV-2 infection then their control counterpart, suggesting a direct mechanism beyond cardiac comorbidities and COVID-19 disease.
ABSTRACT
Introduction: Growing evidences suggest that cardiac complications in COVID19 patients, including ischemic heart disease and atrial fibrillation, may involve direct mechanism of heart infection and damage by the circulating SARS-CoV-2 virus. Angiotensin-converting enzyme 2 (ACE2), which serves as gateway for coronavirus entry, is expressed in cardiomyocytes (CM). Its expression can increase in failing hearts at both mRNA and protein levels and it is specifically upregulated in CM during dilated and hypertrophic cardiomyopathy. Very recently, RNA-sequencing analysis performed on isolated CM reveled that ACE2 is highly expressed in CM of aged individuals as compared to young ones. Methods: We used human induced pluripotent stem-cells derived cardiomyocytes (hiPS-CM) to develop a model of human senescent CM by sub-lethal concentration of doxorubicin (Doxo) exposure. Pseudotype lentiviral particles with SARS-CoV-2 Spike and encoding the mCherry reporter was used to evaluate CM susceptibility to virus entry. Real time PCR and flow cytometry were used to evaluate expression of exogenous construct. The proximity ligation assay (PLA) was performed to directly visualize Spike-ACE2 interaction on the surface of CM. Results: Cellular senescence occurs in CM as a result of exposure to Doxo, as cells with senescence-associated β-galactosidase activity were significantly more over untreated ones (control). Senescent profile in CM was associated with overexpression of the host-cell surface ACE2 protein, suggesting an enhanced SARS-CoV-2 pseudovirions tropism for aged CM. Indeed, PLA assay showed increased efficiency in virions binding (fluorescent signal) onto surface of senescent-like CM. PCR evaluation of mCherry mRNA revealed augmented viral transduction in senescent-like hiPS CM by 2-fold increase compared to control group;FACS analysis further confirmed fluorescent reporter expression in infected cells. Conclusion: Our preliminary results suggest that human aged cardiomyocytes are more susceptible to SARS-CoV-2 infection then their young counterpart, with PLA assay being shown a suitable tool to directly visualize occurred interaction between spike and ACE2 proteins on the surface of CM.
ABSTRACT
In recognition of the increasing health burden of cardiovascular disease, the Dutch CardioVascular Alliance (DCVA) was founded with the ambition to lower the cardiovascular disease burden by 25% in 2030. To achieve this, the DCVA is a platform for all stakeholders in the cardiovascular field to align policies, agendas and research. An important goal of the DCVA is to guide and encourage young researchers at an early stage of their careers in order to help them overcome challenges and reach their full potential. Young@Heart is part of the DCVA that supports the young cardiovascular research community. This article illustrates the challenges and opportunities encountered by young cardiovascular researchers in the Netherlands and highlights Young@Heart's vision to benefit from these opportunities and optimise collaborations to contribute to lowering the cardiovascular disease burden together as soon as possible.