The mechanism and location of the pacemaker in the embryonic heart is highly controversial due to the lack of physiological in vivo recordings. Here we report fluorescent macroscopic in vivo recordings of embryonic hearts (E12.5-E14.5) from mice with cardiac expression (a-MHC or Cx40 promotor) of the Ca²⁺ sensor GCaMP2.

Initial observations from the ventral surface showed regular uniform Ca²⁺ transients in the atrium, ventricle and a structure below/behind the right atrium that preceded every atrial activation. To better understand the origin of these Ca²⁺ transients we established a dorsal preparation leaving the heart and veins intact. Ca²⁺ transients activated in the region of the putative sinus node, propagated bidirectionally along the right superior and in a u-shaped pattern into the coronary sinus and left superior vena cava, and conducted faster (244 mm/sec) than the atria (152 mm/sec; n=5; p<0.02); we have termed this structure the "streak". In most hearts the streak fired before every atrial activation with a delay of 7910 ms (n=12); variations in this delay was not dependent on the heart rate (10415 bpm, n=12). Some hearts showed 2:1 coupling and in others only the streak fired at 17839 bpm (n=5). Staining against HCN4, an established marker for pacemaking cells, showed highly positive cardiomyocytes in the streak within the wall of the left and right superior vena cava. The streak contracted, could be electrically paced and spontaneous local field potentials were recorded as sharp spikes at the onset of Ca²⁺ transients. In some experiments we observed a diastolic Ca²⁺ increment just prior the field potential of the streak, in line with the proposed role of Ca²⁺ oscillations for the initiation of pacemaking in embryonic heart cells.

The spontaneous activity of the streak prior to atrial activation, higher intrinsic frequency of this region, and diastolic Ca²⁺ release establish the pacemaker function of the streak. Thus, cardiomyocytes within the vena cava walls are involved in murine embryonic heart pacemaker activity. Values: meanSEM.