Figure 4.
SHOX2-EBs Exhibit Intensified Automaticity In Vitro and Function as Biological Pacemakers when Implanted In Vivo
(A) The percentages of spontaneously contracting EBs were measured at D6+4 and D6+14 (left) by direct visualization. More than one beating focus could be detected in some EBs, and thus, the number of spontaneously beating foci per EB was measured at the same time points (right).
(B) The spontaneous beating rates were measured by culturing the EBs in MEAs and measuring the extracellular FPs from the GFP-EBs and SHOX2-EBs. Shown is a six-well MEA (left) in which two to three EBs were cultured per well (right).
(C) The average beating rates were measured at five time points by recording the spontaneous FPs for 20 min at 37°C in normal Tyrode’s (n > 10 for each data point).
(D) Representative raw FP traces are shown for a GFP-EB and a SHOX2-EB at D6+14.
(E) Whole-heart optical mapping recordings were performed on the rat hearts injected with GFP-EBs or SHOX2-EBs in vivo 2–3 days prior to the heart harvest. Raw AP traces reported as di-8-ANNEPS signal from one photodiode (123) are shown before (baseline) and after complete heart block. Upon complete heart block, the hearts injected with SHOX2-EBs demonstrated significantly higher heart rate compared to control (right).
(F) Optically recorded isochronal maps superimposed on the whole-heart injected with GFP-EBs (left) or SHOX2-EBs (right). A white circle indicates the EB implantation site, and the aorta is positioned at the bottom of the image. The anterior side of the heart was positioned to face the CMOS camera. Note the differences in the time scale of the isochronal map; 16 and 27 ms for end-to-end AP propagation for GFP-heart and SHOX2-heart, respectively.
(G) Frame-by-frame analysis of the optical mapping exhibiting AP propagation. The data illustrate slower AP propagation upon complete heart block in the hearts injected with SHOX2-EBs compared with control. Each hexagonal frame, recorded with a photodiode array, measures to a small area of about 8 mm2 on the heart.
(H) The conduction velocity of SHOX2-EB injected hearts was significantly slower than that from the control heart upon complete heart block. Data are represented as mean ± SEM. All experiments were performed on three independent biological replicates.