Methodological analysis of dual voltage-calcium whole-heart optical signals during restitution pacing under different thermal states

Dual voltage-calcium fluorescence optical recordings are increasingly appealing to characterize complex spatiotemporal cardiac dynamics within ex-vivo whole-heart experimental preparations. Synchrony among voltage and calcium signals allows us to unveil novel multi-scale and multi-physics couplings at the ventricular scale and quantify features that define the intrinsic nonlinearities of the observed phenomena. Within such a complex scenario, we propose a rigorous methodological analysis comparing and contrasting multiple cardiac alternans onset and evolution indicators for rabbit pacing-down restitution protocols. We introduce a novel integral index quantified upon voltage and calcium signals, validated against well-accepted post-processing analyses, and generalized in terms of statistical restitution curves obtained under four different thermal states. Our study suggests that such a novel indicator can further advance our predictability on alternans onset, linking the concurrent evolution to an innovative quantification of the characteristic length obtained for both voltage and calcium at different thermal states.