Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w

Reducing the number of experimental animals by cold storage of hearts prior to cardiomyocyte isolation

B. Pfeilschifter1, P. Potue1, D. Fiegle1, T. Volk1, T. Seidel1

1Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen;

Background Cardiac myocytes isolated from experimental animal hearts are essential for studying a variety of important research questions. Unfortunately, the laboratory animal needs to be killed for this purpose. It is widely believed that cell isolation must occur immediately after heart removal to avoid loss of cell quality. For this reason, animal models, especially transgenic mice models are frequently and potentially unnecessarily repeated. In this study we investigated if hearts from laboratory mice can be preserved for at least 24h prior to myocyte isolation without affecting the subsequent experiments. Methods Hearts were removed from adult mice (age 8¬30 weeks) after anaesthesia and cervical dislocation. Left-ventricular myocytes were enzymatically isolated either immediately or after preserving the hearts for 24h at 4°C in a storage solution. Following the isolation, action potentials (APs), K+ and L-type Ca2+ currents were measured in the ruptured whole-cell patch-clamp configuration. Cell membranes were stained to investigate the density of the transverse tubular system (t-system) by confocal microscopy. Intracellular Ca2+ release and contractility were determined at 37˚ C with FURA-2 as a Ca2+ indicator and Fourier transform-based assessment of sarcomere shortening, respectively. Results Parameters of steady state APs at 1 Hz pacing showed no significant difference between control (n=27/9 cells/hearts) and preserved hearts  (n=20/11): resting membrane potential was  83.9±0.4 and ¬ 83.9±0.6 mV (p=0.97), AP overshoot 36.3±2.2 and 34.1±2.3 mV (p=0.49), APD90 68.2±7.0 and 70.1±8.3 ms (p=0.86), upstroke velocity 186.8±13.5 mVms-1 and 177.2±17.6 mVms-1 (p=0.67), respectively. Transient outward K+ current (Ito) at VPip = 60 mV was similar in myocytes from control (n=43/12) and preserved hearts (n=34/12), with 42.8±3.0 and 43.2±3.1 pApF-1 (p=0.76), respectively. Delayed rectifying K+ current (IK) was nearly identical in both groups: 18.8±1 and 18.9±1 pApF-1 (p=0.96). Inward rectification current (IK1) at VPip = -120mV was also comparable in control and preserved myocytes:  5.3±0.4 pApF-1 and -6.2±0.5 (n=24/10 and n=11/7, respectively, p=0.12). L-type Ca2+ current reached its highest density at VPip = 10 mV with -6.2±0.5 pApF-1 in control (n=6/1) and -7.1±0.3 pApF-1in preserved hearts (n=7/1, p=0.2). The density of the t-system appeared unchanged after preservation, as indicated by intracellular t-system distances (0.41±0.05 µm control vs 0.44±0.11 µm preserved, n=28/3 and 30/3, p=0.22). Intracellular Ca2+ transients and sarcomere length were measured in n=30/3 control and n=40/4 preserved heart cells at pacing between 0.5Hz and 5Hz. Both parameters showed no significant difference at any pacing rate. At 5 Hz, peak Ca2+ signals reached 0.11±0.01 AU and 0.12±0.01 AU (p=0.15), sarcomere shortening 0.19±0.02 µm and 0.19±0.03 µm (p =0.54), respectively. Conclusion We did not detect differences in transmembrane ionic currents, t-system density, Ca2+ signaling or contractility after 24h of heart preservation before isolation of myocytes. This allows much higher flexibility than previously thought and new opportunities for collaborations as well as saving costs and reducing the number of laboratory animals, for example via inter-institute exchange of mouse hearts.

https://dgk.org/kongress_programme/jt2023/aP2222.html