SARS-CoV-2 spike protein-mediated cardiomyocyte fusion may contribute to increased arrhythmic risk in COVID-19

Figures

• 

  Fig 1. SARS-CoV-2 spike protein induces formation of syncytia in hiPSC-CMs. (A) Light microscopy showing hiPSC-CMs following transfection with CoV-2 S-mEm. Red circles indicate clusters of nuclei which represent hiPSC-CM syncytia. Scale bar 400 μm. (B) Confocal immunofluorescence microscopy of both untransfected and CoV-2 S-mEm-transfected hiPSC-CMs displaying the cardiac marker α-Actinin and sarcoplasmic reticulum proteins calreticulin and SERCA2a. Scale bars 50 μm.

• 

  Fig 2. SARS-CoV-2 spike protein-induced hiPSC-CM syncytia exhibit reduced repolarization reserve with marked prolongation in action potential duration (APD). (A) Representative action potential traces derived from untransfected and CoV-2 S-mEm-transfected hiPSC-CMs. (B) Cell capacitance in untransfected (n = 10) and CoV-2 S-mEm-transfected (n = 10) hiPSC-CMs. (C) Action potential duration at 20% (APD20; left), 50% (APD50; middle) and 90% (APD90; right) in untransfected hiPSC-CMs (n = 10) paced at 1 Hz and CoV-2 S-mEm-transfected hiPSC-CMs paced at 1 Hz (8 cells) and 0.67 Hz normalized to 1 Hz (2 cells). Graphs are presented as mean±SEM. Unpaired two-tailed Student’s t-tests were conducted to determine significance.

• 

  Fig 3. SARS-CoV-2 spike protein-induced hiPSC-CM syncytia display cellular arrhythmias. (A) Representative action potential traces derived from untransfected hiPSC-CMs showing regular beating (black) and CoV-2 S-mEm-transfected hiPSC-CMs displaying erratic beating and delayed afterdepolarizations (DADs; red). Black arrows indicate individual DADs. (B) Quantification of the number of untransfected (n = 10) or CoV-2 S-mEm-transfected (n = 9) hiPSC-CMs showing irregular beating (left) or delayed afterdepolarizations (DADs; middle) as well as the rate at which DADs occur (DADs/episode; right). Graphs depicting categorical data are presented as percentage, and Fischer’s exact tests were used to determine significance. Graphs depicting continuous data are presented as mean±SEM, and unpaired two-tailed Student’s t-tests were used to determine significance.

• 

  Fig 4. SARS-CoV-2 spike protein-induced hiPSC-CM syncytia display abnormal calcium handling. (A) Representative Fluo-4 calcium imaging traces derived from untransfected hiPSC-CMs (black) showing normal calcium transients (left) and from CoV-2 S-transfected hiPSC-CMs (red) showing calcium sparks (middle) and tsunamis (right). (B) Still frame images of Fluo-4 calcium imaging in CoV-2 S-transfected hiPSC-CMs. Red circles indicates the calcium tsunami. (C) Representative Fluo-4 calcium imaging traces comparing the amplitude and duration of a calcium tsunami in CoV-2 S-transfected hiPSC-CMs to normal calcium transients in untransfected hiPSC-CMs. (D) Quantification of calcium sparks (left), calcium tsunamis (middle), and pacing (right) in untransfected (n = 39) and CoV-2 S-transfected (n = 20) hiPSC-CMs. (E) Confocal immunofluorescence microscopy showing colocalization of SERCA2a and CoV-2 S. Scale bars 10 μm. (F) Representative Fluo-4 calcium imaging traces and data summary graphs derived from untransfected hiPSC-CMs (black, n = 143) showing normal calcium transients and from CoV-2 S-transfected hiPSC-CMs (red, n = 189) showing increased amplitude, upstroke velocity and Vmax of calcium…

• 

  Fig 5. Biochemical and genetic interference prevents formation of hiPSC-CM syncytia and restores calcium handling. (A) Light microscopy (left) and representative Fluo-4 calcium imaging trace (right) from CoV-2 S-transfected hiPSC-CMs treated with furin inhibitor (FI). Scale bar 400 μm. (B) Quantification of calcium sparks (left), calcium tsunamis (middle), and pacing (right) in CoV-2 S-transfected (n = 7) and CoV-2 S + FI (n = 12) hiPSC-CMs. (C) Quantification of calcium sparks (left), calcium tsunamis (middle), and pacing (right) in a second line of hiPSC-CMs transfected with CoV-2 S (n = 28), CoV-2 S + FI (n = 25), and CoV-2 S R682S (n = 26). (D) Light microscopy (left) and representative Fluo-4 calcium imaging trace (right) from CoV-2 S R682S-transfected hiPSC-CMs. Scale bar 400 μm. Graphs are presented as mean±SEM. Unpaired two-tailed Student’s t-tests were conducted to determine significance between two groups and one-way ANOVAs with Tukey’s multiple comparisons tests were conducted to determine significance between three groups.