SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition.

Cardiac involvement in patients infected with COVID-19, in terms of myocarditis and troponin release, is associated with higher mortality. However, the underlying mechanisms are poorly understood. Infection of cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) with a wild-type variant of SARS-CoV-2 exhibited a cardiotoxic effect. We examined whether elevated intramitochondrial calcium causes opening of the mitochondrial permeability transition pore (mPTP) leading to cell death. The mPTP inhibitor Cyclosporine A (CsA) did not improve viability, and phosphorylation levels of pyruvate dehydrogenase (PDH) remained similar pre- and post-infection, likely suggesting no substantial alteration of the intramitochondrial calcium level. Also, the protein expression of mitochondrial respiratory complexes did not change after SARS-CoV-2 infection. Next, we examined whether cell death is related to necroptosis or pyroptosis upregulation. The phosphorylation level of receptor-interacting protein kinase 3 (RIP3) was elevated post-infection with SARS-CoV-2 while phosphorylation of mixed lineage kinase domain (MLKL)-S358 remained unaltered. This pattern may point toward an alternative regulation of necroptosis. Chemical inhibition of necroptosis (Necrostatin-1) and pyroptosis (MCC950) did not confer any protection. Notably, the phosphorylation of RIP3 under Necrostatin-1 was still elevated, suggesting that autophosphorylation of RIP3 may be a possible confounder. Our data suggest that SARS-CoV-2 compromises cell viability in iPSC-CMs and may engage in non-canonical signaling via RIP3 phosphorylation. The lack of MLKL activation and the absence of protective effects from CsA indicate that neither classical necroptosis nor mitochondrial permeability transition are likely to be central regulators of cell death.