Oxygen Responsive Myosin Activation Governs Oscillatory Piezo1 Signaling in RBCs during Circulatory Transit

BACKGROUND: In mature red blood cells (RBCs), endothelial nitric oxide synthase (eNOS) migrates from cytosol to membrane in an O₂-dependent fashion (i.e., eNOS exhibits cyclic translocation/activation within RBCs during circulation). Upon RBC deoxygenation, RBC eNOS associates with the membrane and is activated, playing an important role in glycolytic metabolon disassembly and regulation of RBC energetics. We have previously demonstrated that eNOS translocation and activation is mechanistically linked to (1) deoxyHb docking on the Band 3 cytoplasmic domain (cdB3), followed by (2) Piezo1-based calcium influx and (3) eNOS phosphorylation, which results in (4) S-nitrosylation of multiple regulatory/structural proteins. Here, we investigated the role of non-muscle myosin (NMIIA) as a force-generating link between deoxyHb~cdB3 docking and activation of the mechanosensitive Piezo1 channel and additional details of RBC eNOS migration, activation, and functional consequences. METHODS: Heparinized human, or murine (eNOS(-/-), wt C57BL/6J) RBCs were imaged for analysis of cytoskeletal protein arrangement and eNOS migration. RBCs were subjected to oxygenation/deoxygenation, with/without pre-treatment with the NMIIA inhibitor Blebbistatin. RBCs were fixed (paraformaldehyde 4%/glutaraldehyde 0.08%), permeabilized (Triton X100 amount 0.1%) and incubated with primary antibodies (spectrin, actin, or eNOS), then with the secondary antibody prior to visualization. Interaction between NMII and Band3, was determined using a proximity ligation assay (Duolink). Intracellular RBC calcium levels were assessed in oxygenated/deoxygenated RBCs utilizing an environmentally controlled fluorescent plate reader (Biotek Neo2, USA), and the calcium probe Fluo 3. NO production in RBCs was assessed using DAF-FM. Quantification of murine RBC deformability (eNOS(-/-) or wt) was performed with Brillouin Microscopy, whilst O2 delivery homeostasis (eNOS(-/-) or wt) was assessed with measurement of oxygen dissociation curves (ODCs) at 2 fixed CO2 levels (thin film rotating tonometer in conjunction with a gas blender - samples measured by arterial blood gas machine; ABL90Flex). RESULTS: Upon RBC deoxygenation, phosphorylation of eNOS increased at serine 1177. PKCα inhibition (Goumi6983) reduced serine 1177 phosphorylation, while PKCα activation (PMA) increased it; these changes were associated with expected decrease and increase (respectively) in eNOS activation (DAF-FM). Upon RBC deoxygenation, both calcium flux (Fluo-3) and eNOS activation (DAF-FM) were associated with increased NMIIA phosphorylation and were attenuated by NMIIA inhibition (Blebbistatin). Functionally, O2 responsive eNOS activity was required for the described oscillation in RBC deformability that normally occurs during circulatory transit. Summary: We demonstrate that PKCα and myosin-mediated activation of Piezo1 contribute oxygen-responsive regulation of RBC eNOS, which modulates RBC phenotype during circulatory transit.