Abstract
Organotypic culture of human ventricular myocardium is emerging in basic and translational cardiac research. However, few institutions have access to human ventricular tissue, whereas atrial tissue is more commonly available and important for studying atrial physiology. This study presents a method for long-term cultivation of beating human atrial myocardium. After written informed consent, tissues from the right-atrial appendage were obtained from patients with sinus rhythm undergoing open heart surgery with cardiopulmonary bypass. Trabeculae (pectinate muscles) prepared from the samples were installed into cultivation chambers at 37°C with a diastolic preload of 500 μN. After 2 days with 0.5 Hz pacing, stimulation frequency was set to 1 Hz. Contractile force was monitored continuously. Beta-adrenergic response, refractory period (RP) and maximum captured frequency (fmax) were assessed periodically. After cultivation, viability and electromechanical function were investigated, as well as the expression of several genes important for intracellular Ca2+ cycling and electrophysiology. Tissue microstructure was analyzed by confocal microscopy. We cultivated 19 constantly beating trabeculae from 8 patient samples for 12 days and 4 trabeculae from 3 specimen for 21 days. Functional parameters were compared directly after installation (0 d) with those after 12 d in culture. Contraction force was 384 ± 69 μN at 0 d and 255 ± 90 μN at 12 d (p = 0.8, n = 22), RP 480 ± 97 ms and 408 ± 78 ms (p = 0.3, n = 9), fmax 3.0 ± 0.5 Hz and 3.8 ± 0.5 Hz (p = 0.18, n = 9), respectively. Application of 100 nM isoprenaline to 11 trabeculae at 7 d increased contraction force from 168 ± 35 μN to 361 ± 60 μN (p < 0.01), fmax from 6.4 ± 0.6 Hz to 8.5 ± 0.4 Hz (p < 0.01) and lowered RP from 319 ± 22 ms to 223 ± 15 ms. CACNA1c (L-type Ca2+ channel subunit) and GJA1 (connexin-43) mRNA expressions were not significantly altered at 12 d vs 0 d, while ATP2A (SERCA) and KCNJ4 (Kir2.3) were downregulated, and KCNJ2 (Kir2.1) was upregulated. Simultaneous Ca2+ imaging and force recording showed preserved excitation-contraction coupling in cultivated trabeculae. Confocal microscopy indicated preserved cardiomyocyte structure, unaltered amounts of extracellular matrix and gap junctions. MTT assays confirmed viability at 12 d. We established a workflow that allows for stable cultivation and functional analysis of beating human atrial myocardium for up to 3 weeks. This method may lead to novel insights into the physiology and pathophysiology of human atrial myocardium.
Highlights
Organotypic long-term culture of adult human and animal cardiac tissue is emerging as a promising tool in basic and translational cardiac research (Fischer et al, 2019; Ou et al, 2019; Qiao et al, 2019; Watson et al, 2019; Perbellini and Thum, 2020)
Myocardial tissue culture prevents the quick degradation and dedifferentiation occurring during culture of isolated adult cardiomyocytes (Banyasz et al, 2008), but still provides a level of control over physical and chemical stimuli that cannot be achieved in vivo
F max was decreased after dofetilide application (Figures 5E vs. G). In the example it decreased from 5 to 2.5 Hz. These results demonstrate that refractory period (RP) and f max as assessed by the chosen pacing protocols and criteria may indirectly detect changes in action potential duration
Summary
Organotypic long-term culture of adult human and animal cardiac tissue is emerging as a promising tool in basic and translational cardiac research (Fischer et al, 2019; Ou et al, 2019; Qiao et al, 2019; Watson et al, 2019; Perbellini and Thum, 2020). Myocardial tissue culture prevents the quick degradation and dedifferentiation occurring during culture of isolated adult cardiomyocytes (Banyasz et al, 2008), but still provides a level of control over physical and chemical stimuli that cannot be achieved in vivo. Another important advantage is that the technique can be used to cultivate fully differentiated human myocardial tissue obtained from explanted hearts or accrued during open heart surgery. Because stem-cell derived engineered heart tissues have not yet reached the differentiation, multicellularity and physiological properties of adult myocardium, the “heart in a dish” approach of myocardial tissue culture can currently be considered the most realistic in vitro model
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