Abstract Body: Background: Isochoric supercooling of biological organs at sub-0°C temperatures offers an opportunity to extend preservation times by preventing ice formation in a metastable liquid preservation fluid without the addition of cryoprotectants. In this study, we aimed to assess the effects of supercooling on cardiac Ca²⁺ dynamics and functionality in murine hearts.

Objective: To evaluate cardiac Ca²⁺ dynamics in supercooled (-4°C) vs. traditional static cold storage (+4°C) murine hearts ex vivo. We hypothesized that isochoric supercooling would better preserve the functional Ca²⁺ dynamics in comparison to static cold storage due to the exponential dependence of metabolism on temperature.

Methods: Hearts were excised from 8-12 week old C57BL6/J mice, flushed of blood with chilled Belzer MPS solution, and evaluated immediately as a fresh control or stored for 24 hours in UW solution supplemented with polyethylene glycol (35 kDa) in conventional refrigeration (+4°C) or isochoric supercooling (-4°C) (n=6/group). A Langendorff perfusion system loaded with Rhod-2 AM for optical mapping of Ca²⁺ transient dynamics was used to assess cardiac function. Expression of cardiac Ca²⁺ handling proteins ryanodine receptor (RyR2) and sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA2a) were quantified using qPCR and Western Blotting.

Results: Control and supercooled hearts resumed regular ventricular function upon Langendorff perfusion. The duration of Ca²⁺ transients to 75% decay (CaD75) was similar between control (85.98 ± 3.27 ms) and 24h supercooled hearts (76.04 ± 2.85 ms; p=0.11). No changes were observed in the level of RyR2 protein expression or phosphorylation at its serine (S2808) site between supercooled and control hearts. However, significant reduction in the phosphorylation of RyR2 (S2808) was observed for +4°C hearts (0.60 ± 0.09) as compared to -4°C hearts (1.35 ± 0.21, p=0.03), and these hearts either failed to regain ventricular function entirely or regained only partial or irregular function.

Conclusions: Isochoric supercooling of murine hearts demonstrated superior Ca²⁺ dynamics and protein expression indicative of successful preservation, compared to traditional static cold storage. This study reveals new insights into the preservation of key cellular features critical for cardiac function and transplant.