SARS-CoV-2 Spike Variants Differentially Rewire Cytoskeletal Architecture in Human Cardiomyocytes

Background/Objectives: The COVID-19 pandemic has resulted in more than 7.11 million deaths worldwide and has been associated with numerous extrapulmonary complications, including cardiovascular dysfunction. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily targets the respiratory system, increasing evidence suggests that viral components may directly affect cardiac cells and contribute to long-term cardiovascular sequelae. In this study, we investigated the effects of recombinant spike proteins from the Alpha, Delta, and Omicron SARS-CoV-2 variants on the cytoskeletal organization and proteomic landscape of human AC16 cardiomyocytes. Methods: Confocal microscopy and quantitative image analysis were employed to evaluate cytoskeletal organization, including F-actin filament count and length, phalloidin fluorescence intensity, and cell solidity, following spike-protein exposure at 24, 48, and 72 h, while quantitative tandem mass tag (TMT)-based proteomics at the 72 h exposure time point was used to characterize variant-associated changes in host protein abundance. Results: Quantitative analysis revealed time- and variant-dependent F-actin and morphological remodeling. Mean filament length was significantly reduced by all three spike variants at 48 h, whereas significant reductions in filament count emerged at 72 h across all variant-exposed groups. Phalloidin fluorescence intensity and cell solidity further revealed variant-specific responses, particularly at 72 h, with Delta and Omicron exhibiting distinct quantitative remodeling patterns. Morphologically, Delta spike-exposed cells exhibited the most pronounced disruption of overall cellular architecture at 72 h. Proteomic analyses revealed distinct abundance patterns among proteins involved in cytoskeletal regulation, cell adhesion, membrane dynamics, mechanotransduction, and intracellular trafficking. Notably, Omicron was associated with broader changes in protein abundance, whereas Delta exhibited fewer protein-abundance changes despite showing the most pronounced morphological alterations. Collectively, these findings demonstrate time- and variant-dependent patterns of cytoskeletal and morphological remodeling, together with distinct variant-associated proteomic abundance patterns at the 72 h exposure time point. The divergence between the pronounced morphological alterations associated with Delta and the broader proteomic response induced by Omicron suggests that cytoskeletal, morphological, and molecular remodeling represent distinct but interconnected dimensions of the cardiomyocyte response to SARS-CoV-2 spike proteins. Conclusions: These findings identify cytoskeletal regulation and associated signaling pathways as potential contributors to SARS-CoV-2-associated cardiac cellular dysfunction.