Introduction:
Heart failure involves structural remodeling, altered energetics, and metabolic reprogramming of cardiomyocytes. Reactive oxygen species (ROS) play a central role in these processes. Despite their link to many diseases, antioxidant therapies have been largely ineffective, suggesting a more nuanced role of ROS in signaling. The spatial and temporal diffusion of ROS within cells critically affects redox balance. This study investigates ROS dynamics across cardiomyocyte compartments, identifies ROS-related mitochondrial alterations, and examines their impact on cellular morphology and function.
Methods:
To control and monitor ROS generation, a chemogenetic D-amino acid oxidase (DAO) system coupled to the redox biosensor HyPer was used to produce hydrogen peroxide (H₂O₂) upon D-alanine (D-ala) stimulation. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were engineered via CRISPR-Cas9 to express HyPer-DAO specifically in the cytosol (NES), nucleus (NLS), or mitochondria (MLS). H₂O₂ diffusion between compartments was analyzed using a red HyPer variant targeted to adjacent regions. Real-time imaging of ROS production and mitochondrial or cytoskeletal changes was performed with confocal and stimulated emission depletion (STED) microscopy.
Results:
Compartment-specific HyPer-DAO expression revealed distinct H₂O₂ dynamics. Cytosolic and nuclear H₂O₂ readily diffused into neighboring compartments, whereas mitochondrial H₂O₂ showed limited spread to the cytoplasm. H₂O₂ generation increased dose-dependently with D-ala concentration. At 10 mM D-ala, mitochondrial ROS produced a gradient extending ~1.8 µm into the cytosol; 50 mM D-ala caused widespread H₂O₂ accumulation. All cell lines exhibited a peak ROS response at 6 minutes, followed by decay. Inhibition of Peroxiredoxin 2 (Prx2) with Conoidin A (2.5 µM) further enhanced H₂O₂ levels, particularly in NES and MLS cells. High cytosolic H₂O₂ (50 mM D-ala) transiently altered mitochondrial structure within seconds.
Conclusions:
ROS signaling in cardiomyocytes is highly compartmentalized and tightly regulated. Prx2 limits mitochondrial H₂O₂ diffusion, while elevated cytosolic ROS transiently remodel mitochondria. These findings highlight the importance of spatial ROS control in redox regulation and suggest that targeting ROS compartmentalization may offer new therapeutic strategies for heart failure.
