Background

With the establishment of long-term cultivation of beating human ventricular tissue slices, organotypic cardiac tissue culture has become an emerging tool in basic and translational cardiac research. However, whether human atrial myocardium can be cultivated with comparable stability, is unknown. Here, we present and evaluate an approach for long-term cultivation of beating human atrial trabeculae.

Methods

After written informed consent, atrial tissue that would have been discarded otherwise was obtained from patients with sinus rhythm undergoing cardiac bypass surgery with extracorporal circulation. Trabeculae (pectinate muscles) were prepared from the right-atrial appendage, installed into cultivation chambers at a diastolic preload of 500–1000 µN (0.25-0.5 mN/mm²) and kept at 37 °C. Trabeculae were constantly stimulated by field electrodes. After an initial adaptation period of 2 days with 0.5 Hz pacing, stimulation frequency was set to 1 Hz. Contractile force was monitored continuously. Beta-adrenergic response, refractory period and maximum captured frequency were assessed periodically. After cultivation, viability and electromechanical function were assessed as well as the expression of selected genes of proteins involved in excitation-contraction coupling and electrophysiology. Tissue microstructure was analyzed by confocal microscopy.

Results

We cultivated 22 trabeculae from 8 patient samples for 12 days. Of these, 5 trabeculae from 3 samples were kept in culture for 21 days. All trabeculae responded to electrical stimulation during these periods. Contractile force was 1024±116 µN (n=22) directly after installation (0d), 626±20 µN after 6d, 754±88 µN after 12d, and 1049±236 µN after 21d in culture. Transient addition of 100 nM isoprenaline at 7d increased the force from 668±100µN to 860±170µN (p<0.01, paired t-test, n=11/3 trabeculae/patient samples), shortened refractory period from 318±22ms to 229±14ms (p<0.01) and decreased relaxation time from 208±17ms to 139±11ms (p<0.01). Maximum captured stimulation frequency ranged from 3 to 6 Hz and did not change significantly during culture. MTT assays indicated preserved viability after 12d in culture (relative absorbance 0.17±0.03 at 0d vs. 0.49±0.39 at 12d, 3 samples each). mRNA expression of the L-Type Ca²⁺ channel alpha subunit (CACNA1c) and connexin-43 (GJA1) did not change significantly after 12d in culture when compared with non-cultivated trabeculae (n=8/8 trabeculae/samples). However, genes encoding SERCA (ATP2A) and Kir2.3 (KCNJ4) were downregulated (p<0.01), while Kir2.1 (KCNJ2) was upregulated (p<0.05). Simultaneous Ca²⁺imaging and force recording demonstrated preserved excitation-contraction coupling after 2 weeks of culture. Confocal microscopy indicated preserved microstructure of cardiomyocytes and tissues.

Conclusions

We established a method for stable long-term cultivation of beating human atrial trabeculae, which is indicated by conserved contractile force, response to beta-adrenergic stimulation and preserved microstructure. We expect that this research model will provide novel insights into the physiology and pathophysiology of human atrial myocardium, promoting basic and translational cardiac research as well as drug discovery.