Excitation of wave propagation on self-organized monolayers, formed by cardiomyocytes with pathological prolonged QT-interval.
Prolonged QT-interval is one of the main reasons to assign a person to the risk group of cardiovascular disease. Elongation of the QT-interval may be caused by several acquired pathologies or specific mutations. In our work, we used human induced pluripotent cells of two lines: if-31, which are reprogrammed cells from a patient with the prolonged QT-interval, as well as a control line isma6L, derived from the healthy patient. For getting iPSC lines, in the fibroblasts of patients with the syndrome of prolonged QT- interval and an elongated spinal muscular atrophy episomal vector expressing genes OCT4, SOX2, KLF4, c-MYC via nukleofektsii was delivered. These genes are responsible for maintaining the pluripotent state. For differentiation to the cardio cells GiWi protocol was used, the duration of which takes 12 days . However, for the mature cardiomyocytes in a number of protocols cells were kept in the post-differentiation conditions up to six months.
After differentiation, (day 11-12) cell monolayer formed visually, and about 30-50% of the layer surface contracted. Using flow cytometry, we counted the number of cells, which are positive for sarcomeric alpha-actinin. For if31-5 line (on day 16) it was 15.3%, for iSMA6L line (on day 20) - 14.6%. But it's worth noting that the cells did not survive for several months. As a control, cells were analyzed using immunocytochemistry. Staining was done on sarcomeric alpha-actinin (actinin), cardiac troponin T (cTnT) and myosin heavy chain (MHC). Figure 1 showed cells, differentiated from isma6L , with alpha-actinin (actinin) staining.
Also, on day 15 optical mapping of the differentiated cell monolayers was conducted. An excitation wave propagated over the entire surface of the monolayer, which allowed measuring critical stimulation frequencies for the tissue culture. The monolayers were removed and seeded as single cells to check for Patch Clamp. However, on a day 16 from starting differentiation procedure cells showed only potassium currents.
As the number of cells, which are positive for sarcomeric alpha-actinin for both cell lines were about 15%, it shows the stability of differentiation. In addition, it indicates the role of these cells in monolayer surface contractility function. However, from the other hand, the low presence of cardiac cells in the entire monolayer could correspond to notable inability of cells from both lines to survive for more than 50 days.
Interestingly, in contradistinction to other differentiation protocols, electro-mechanical syncytium formed already on the 15th day after differentiation. Considering patch-clamp results, it could show the main role of potassium channels to form the excitable tissue itself.