부산대학교 도서관

Purpose: Myocardial research is hampered by the lack of relevant experimental models. Isolated cardiomyocytes dedifferentiate during culture and do not represent an integral multicellular tissue environment of human myocardium. More complex tissue models (e.g. perfused heart) are not available from human origin, a fact that severely impairs translational research. To overcome these limitations, we established slice preparations from human ventricular myocardium as an experimental model, and optimized the conditions of long-term tissue culture.Methods: Myocardial tissue, which was removed during heart-valve replacement surgery, was cut into 300 μm slices under cardioplegic conditions. Slices were prepared within 3 hours after surgery and were either used in acute experiments or cultured for up to 28 days. Force development of slices during electrical stimulation (1 Hz) was determined under isometric conditions and expression as well as histologic analysis was used to characterize cardiomyocyte differentiation.Results: Acute slices show high viability and preservation of contractile apparatus, which was confirmed in force measurements. Contractions of fresh slices showed a clear preload dependency and reached optimum forces of 8 mN, corresponding to a tension of 5.3 mN/mm. Isoproterenol and calcium raised the developed force in a concentration dependent manner (EC50 of 0.3 μM and 5 mM, respectively). In case of isoproterenol a 2.0 fold increase in contractility was observed.Cardiomyocytes stayed viable in tissue culture for up to 28 days. Messenger RNA expression of myocyte specific genes (SERCA2, connexin 43, titin, phospholamban) and of cardiac ion channels and transporters (Cav 1.2, Kv 4.3, hERG1, NCX, Na+/K+-ATPase) was constant throughout the culture period. Transcription of sarcomeric components (myosin light chain 2, α-actin 1) was down-regulated within the first day in culture, and reached a steady-state thereafter. This differentiation was accompanied by restructuring of linear sarcomer organization which was reflected in a reduction of maximal developed force of contraction. However the inotropic response of cultured slices to β-adrenergic and calcium stimulation was maintained. Analogous to acute experiments, isoproterenol stimulation evoked a 2.0 fold increase in contraction force after 21 days of culture.Conclusion: Overall, slice preparations of human myocardium are well suited for functional research and drug development. The newly introduced method of myocardial tissue cultivation enables experimental long-term interventions and investigation of cellular differentiation in vitro.