# Cold storage of mouse hearts prior to cardiomyocyte isolation preserves electromechanical function, microstructure, and gene expression for 24 h

## TL;DR

This study shows that mouse hearts can be stored for 24 hours before isolating cardiomyocytes without affecting their function or gene expression.

## Contribution

The study challenges the assumption that cardiomyocytes must be isolated immediately after heart procurement.

## Key findings

- Cold storage of mouse hearts for 24 hours does not alter cardiomyocyte electromechanical function or microstructure.
- Only 128 genes were differentially expressed after cold storage, mostly related to immune and inflammatory processes.
- Mitochondrial function and calcium signaling remained equivalent between cold-stored and freshly isolated cardiomyocytes.

## Abstract

Isolation of myocytes from mouse hearts, especially of transgenic animals or disease models, is crucial in cardiac research. The presumption that cardiomyocytes must be isolated immediately after heart procurement to avoid deterioration implies that transgenic mouse lines must be present on site, causes schedule inflexibility, and hampers collaborations, thereby increasing the number, suffering, and costs of animals. This study challenges this presumption by investigating whether the cell isolation can be postponed for 24 h without affecting the results. Adult mouse hearts were subjected to enzymatic myocyte isolation immediately after excision (CTRL) or after 24 h of cold storage (CS). Sufficient numbers of viable cardiomyocytes were obtained in all groups. The transverse-axial tubular system was unchanged in CS versus CTRL. No significant changes were detected in cell capacitance, resting membrane potential, action potential shape and duration, amplitudes, and kinetics of the K+ currents Ito, IK1, and IK. Sarcomere length, contractility, and relaxation as well as Ca2+ signals were equivalent in CS and CTRL at pacing rates of 1–4 Hz. Mitochondrial function assays also yielded equivalence. RNA sequencing yielded only 128 differentially expressed genes, which were mainly related to immune cell function and inflammation. Key findings were reproduced in infarcted mouse hearts, which were shipped overnight as a proof of principle. This study demonstrates that the isolation of cardiomyocytes can be postponed up to 24 h after the procurement of the heart. This opens up new possibilities for collaboration between different laboratories, increases experimental flexibility, and allows to reduce the number of experimental animals by avoiding unnecessary propagation of transgenic lines.

The online version contains supplementary material available at 10.1007/s00395-025-01131-y.