Calibration of the calcium dissociation constant of Rhod₂ in the perfused mouse heart using manganese quenching

Both theoretical and experimental results are presented for in vivo calibration of the dissociation constant K_d^Ca of the calcium-sensitive fluorescent dye Rhod₂ in the perfused mouse heart, using manganese quenching of flourescence transients. An analytical model is derived, based on the biochemical equilibrium of manganese competition with calcium for Rhod₂ binding. Expressing the differential of the changes between systole and diastole in fluorescence transient (δΔF_sys-dia). δΔF_sys-dia in a beating heart as a function of the perfusate manganese concentration [Mn²⁺]_p allows correlation of the measured differential transient changes δΔF_sys-dia with the calcium dissociation constant K_d^Ca of Rhod₂ and the calcium concentration in the heart. Numerical modeling indicates that the K_d^Ca predominantly affects the asymptotic slope of the δΔF_sys-dia versus [Mn²⁺]_p curve at certain manganese concentrations, which suggests that the K_d^Ca can be inversely calculated by partially fitting the δΔF_sys-dia distribution as a function of the perfusate manganese concentration. The feasibility of this approach is confirmed by quenching of calcium transients by manganese infusion into isolated perfused beating mouse hearts. The resulting calculated dissociation constant K_d^Ca of Rhod₂ is 720 nM. Using the same approach, we are able to also estimate intracellular calcium concentrations of 700 nM at peak systole and 300 nM in diastole. This is in good agreement with values obtained by calibration of fluorescence values with a calcium saturation tetanization procedure in the same perfused mouse heart model.