Abstract 1879: Novel Intracellular Sodium Nanosensors to Study Sodium Dynamics in Cardiomyocytes
Background: The use of fluorescent calcium-sensitive dyes such as Fura-2 has fundamentally advanced our knowledge of calcium signaling in excitable cells. Sodium flux through voltage-gated sodium channels is responsible for initiating action potentials in excitable cells. However, little is known about the compartmentalization and dynamics of sodium fluxes in cells with complex cyto-architecture such as cardiomycoytes (CMs). Here we describe novel nanosensors that can report sodium concentration with microsecond response time, high intensity, and minimal photobleaching. We demonstrate the feasibility of studying sodium fluxes in CMs.
Methods: We previously reported polymer nanosensors that accurately report sodium concentration. The sensors are ~ 100 nm in diameter and fluorescently report sodium concentration with a kD near 10 mM. The nanosensors were able to load though the seal of a whole cell patch clamp. Once loading was achieved, electrical control of the membrane channels was performed and sodium flux was simultaneously measured optically.
Results: Control experiments showed that loading the nanosensors through the patch pipette did not alter the electrical recording or control of the cell membrane voltage gated channels during IV curve generation and steady state recovery experiments. IV curve experiments were performed 20 minutes after whole cell patch was achieved to verify that channel function was not altered by the presence of the nanosensors. Changes in the fluorescent intensity were recorded when the cell was depolarized to open the voltage gated sodium channels. Results showed correlation between optical response of the nanosensors and electrical response from the whole cell patch clamp. Also, it was feasible to load neonatal rat CMs with these nanosensors using cold electroporation.
Conclusions: We have created sodium sensitive nanosensors that are exquisitely specific for sodium ions, have favorable dynamics for studying kinetics of sodium fluxes through voltage gated sodium channels and can be introduced into CMs. This tool will yield fundamental knowledge about CM biology.