Doxorubicin-induced cardiotoxicity enhance SARS-CoV-2 infection susceptibility in human cardiomyocytes

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.