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 spa-tiotemporal cardiac dynamics within ex-vivo whole-heart ex-perimental 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 phenom-ena. 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 quan-tification of the characteristic length obtained for both voltage and calcium at different thermal states.