Abstract 14877: Spike Protein of SARS-CoV-2 Virus Induces Mitochondrial Dysfunction in Swine Heart via Redox Impairment of Heme Proteins and Increasing Superoxide Generation

Abstract

The cellular entry of the SARS-CoV-2 virus depends on the binding of spike (S) protein to its biological ligand, ACE2. Although the virus commonly causes respiratory distress, cardiac injury can occur in COVID-19 patients, which is consistent with the expression of ACE2 in myocytes. Previous reports indicate SARS-CoV-2 proteins can target cardiac mitochondria and suppress mitochondrial function via enhancing MPTP pore opening and perturbing cardiac bioenergetics. To explore the underlying mechanism of the effect of SARS-CoV-2 on cardiac mitochondria, we measured the interactions of SARS-CoV-2 proteins with swine heart mitochondria in vitro. Incubation of recombinant S protein with isolated mitochondria significantly decreased state-3 oxygen consumption rate (OCR, 95.10 vs 65.68 nmol/min/mg) and FCCP uncoupling OCR (82.94 vs 66.95 nmol/min/mg). We further detected that S protein impaired the enzymatic activities of electron transport chain (ETC) (by 15.62% to 34.44%). However, S protein had no effect on TCA cycle enzymes, indicating the involvement of mitochondrial membrane components in decreased OCR by S proteins. Recombinant nucleocapsid (N) protein of SAR-CoV-2 had no effect on the OCR and ETC activities of swine mitochondria. S proteins decreased the intensity of mitochondrial heme spectrum determined by dithionite reduction with UV/VIS spectroscopy (by 17.52% heme a , 15.82% heme c 1 ). The results were further assessed using isolated complex III (Cx3) and complex IV (Cx4). Treatment of isolated Cx3 and Cx4 with S protein decreased the spectral intensities of heme a and heme c 1 . The spectra of both Cx3 and Cx4 were not affected by N protein. The results suggested S protein downregulates redox potentials of ETC in swine mitochondria. Treatment of swine mitochondria with S proteins enhanced superoxide ( . O 2 – ) generation by Cx1 (by 43.7%) and by Cx3 (by 10.9-fold) assessed by EPR and cytochrome c reduction assays. However, we detected S proteins modestly decreased . O 2 – generation by swine mitochondria under state-2 condition (by 9.52%), indicating impairing pH gradient by S protein. In conclusion, the spike protein of SARS-CoV-2 virus mediates mitochondrial dysfunction of swine heart via impairing the redox function and increasing . O 2 – generation.