Mathematical modeling of phasic calcium dynamics in HaCaT cells

Background: Connexin (Cx) channels are ubiquitous, providing pathways for movement of molecules between cells and for release of molecular effectors into the extracellular environment (plasma membrane hemichannels (HCs)). To maintain an adequate permeability barrier, HCs are tightly regulated by normal extracellular calcium ion (Ca2+) to be closed under most conditions. Cx mutations that disrupt HC regulation by Ca2+ cause human pathologies, due to aberrantly open HCs. These pathological conditions including cardiac infarct, stroke, deafness, cataracts, epilepsy, Alzheimer’s disease, and skin diseases. Therefore, Cx HCs have emerged as a valid therapeutic target. Cx HCs activity has been explored indirectly by the release of adenosine triphosphate (ATP) and other metabolite, as well as by electrophysiological methods and/or using HC-permeable dye uptake measurements. Recently, all-optical assay based on fluorometric measurements of Ca2+ uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s), that permits the optical tracking of cytosolic Ca2+ concentration changes with high sensitivity, was presented, the assay in stable pools of HaCaT cells overexpressing Cxs of interest, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on), for the characterization of new monoclonal antibodies targeting the extracellular domain of the HCs. Aim: Developing a mathematical model of Ca2+ dynamics with the aim of simulating the complex responses measured experimentally by fluorescence microscopy in HaCaT cells challenged by a sudden increase of the extracellular Ca2+ concentration. Moreover, the model aims to explain the observed damping effects caused by increasing concentrations of different HC blockers, including monoclonal antibodies targeted at the HC extracellular domain. Method: Open source ImageJ software as well as Vimmaging (a custom-made software developed under the MATLAB environment) were used to derive Ca2+ uptake traces in HaCaT-Cx26- GCaMP6s cell cultured bathed in extracellular medium and exposed to CaCl2 bolus concentrations to reach final extracellular Ca2+ concentrations of 2mM, 1mM, 560M, 260M, and 100M. A set of previously derived differential equations that successfully modeled epidermal Ca2+ dynamics in vivo was adapted to the peculiar toolset of ion channels and transporters expressed in HaCaT cells to generate an original model variant account quantitatively for the insurgence of a biphasic elevation of intracellular Ca2+ when the extracellular Ca2+ concentration overcomes a critical threshold. Conclusion: Our mathematical simulation of Ca2+ dynamic has revealed that the first elevation phase of Ca2+ observed experimentally is due to the release of Ca2+ from endoplasmic reticulum to cytosol, whereas the second peak is the result of influx of Ca2+ from extracellular milieu to cytosol through HC.