further information Further cultivation of iSABs for weeks o

Further cultivation of iSABs for 3 weeks on gelatin-coated dishes led to attached, highly synchronized cell layers beating at >350 bpm (Movie S4). The further information exhibited the typical shape of nodal cells (i.e., spindle and spider-shaped cells; Figure 3C; Movie S5). To address the electrophysiological parameters of iSAB-derived cardiomyocytes, we performed single-cell patch clamping again. Out of 65 cells analyzed, 53 cells (81.5%) now corresponded to the regular and fast-beating pacemaker subtype, and only 12 cells (18.5%) represented the irregular and slowly beating myocardial subtypes. Moreover, 43 of the 53 cells classified as the pacemaker-system subtype showed AP parameters of mature pacemaker cells, whereas ten of these spontaneously and regularly beating cells were still immature (Figures 4A and S2; Table S3). The latter exhibited a short plateau phase and a more negative MDP, which distinguishes them from fully mature pacemaker cells. Yet, they also showed typical pacemaker features, such as regular spontaneous AP generation with a fast DDR, and expressed the typical pacemaker current If comparably to the cells classified as mature pacemaker cells (Figures 4B–4E). Likewise, the iSAB-derived cells responded to isoproterenol and carbachol with an acceleration and deceleration, respectively, of AP rates (Figure 4F). Thus, the beating rates were able to reach >550 bpm. To further verify the pacemaker cell identity, we characterized the Ca2+ flux from the extracellular space and intracellular Ca2+ stores, which can be mediated by sarcolemmal Ca2+ channels or by Ca2+ release from the stores. Electrogenic features of the sarcolemmal channels also determine the frequency of the Ca2+ transients (Movie S6). As a characteristic of pacemaker cells, we confirmed that hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels modulated the rate of the Ca2+ transients. The application of the HCN channel blocker ZD 7288 time dependently reduced the frequency of Ca2+ transients in iSABs (Figure 5A). In addition, the frequencies of Ca2+ transients are also based on the activity of T-type and L-type voltage-sensitive Ca2+ channels. Correspondingly, the spontaneous frequencies of the Ca2+ transients were dramatically reduced after inhibition of L-type Ca2+ channels with nifedipine, and moderately by inhibition of T-type Ca2+ channels with mibefradil in iSAB-derived cells (Figure 5B). A functional sarcoplasmic reticulum (SR) characterizes the maturation state of cardiomyocytes. Therefore, in pacemaker cells, Ca2+ from SR plays an important role in spontaneous activity. The impact of Ca2+ from the SR on spontaneous Ca2+ transients becomes evident if a complete release of Ca2+ from SR with caffeine or an inhibition of SERCA with thapsigargin is induced. In iSAB-derived cells, caffeine-induced SR-Ca2+ release increased the diastolic Ca2+ level comparable to a Ca2+ peak, but with recognizable spontaneous Ca2+ transients in the time course of the Ca2+ peak and unchanged systolic Ca2+ values (Figure S3A). A similar caffeine-induced effect on the SR-Ca2+ release could not be detected in aCaB-derived cells. Blocking Ca2+ reuptake into SR by thapsigargin resulted in an increase of the diastolic Ca2+ level only in iSAB-derived cells (Figure S3B). The main proportion of Ca2+ underlying the Ca2+ transients is derived from the extracellular space. Therefore, replacement of extracellular Ca2+ leads to an abolishment of Ca2+ transients. When sarcolemmal Ca2+ flux was eliminated by blocking the Na+/Ca2+ exchanger and the Ca2+ channels, only intracellular Ca2+ cycling could be detected. In this case, the application of caffeine induced a Ca2+ peak in iSAB-derived cells that was 4-fold greater than in aCaB-derived cells (Figures 5C and S3C). Additional inhibition of SERCA under these conditions revealed a leaky SR in iSAB-derived cells. The rate of intracellular Ca2+ accumulation was increased 4-fold compared with no SERCA inhibition. After addition of caffeine 5 min after thapsigargin incubation, the Ca2+ peak was reduced by a factor of 4. In contrast, the caffeine-induced Ca2+ peak was identical with and without SERCA inhibition in control cells (Figures 5C and S3C), where an intracellular Ca2+ accumulation also could not be detected.