Human Aged Cardiomyocytes Are Susceptible to SARS-CoV-2 Infection

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