Abstract 69: Diminished T-Tubule Density in Newborn Human Ventricular Cells Is Associated with Heterogeneity of Calcium Transients

For children with congenital heart disease (CHD), therapies developed for adults may have different effects on immature myocardium. Thus, it is critical to understand calcium handling in the young human ventricle. Methods: Human ventricular cells were isolated from tissue removed as part of the surgical repair for CHDs from two age groups: newborns (<1 week old) and infants (2-12 months old). Developmental changes in t-tubules were examined in live cells with Di-8 ANEPPS which highlights the cell membrane. We measured the T-index (percent of cell interior occupied by t-tubules), averaged for at least 3 confocal z-sections per cell. Changes in calcium transients were measured by confocal line scans in cells loaded with Fluo-4 and field stimulated (37°C). Results: Newborn human myocytes have very few t-tubules whereas infant myocytes have a range of t-tubule densities. The T-index in newborn myocytes was significantly less than in infant myocytes (4.4±0.2%, n=8 cells, 3 newborns vs. 9.1±0.9%, n=55 cells, 7 infants). Furthermore, there was sizable heterogeneity in the T-index for each patient in the infant group, in which some cells had few t-tubules and others had many t-tubules. We calculated the coefficient of variation (CV=SD/mean*100) for each patient and compared CV values for newborns vs. infants. CV was significantly greater in infants compared to newborns (46.5±7.4%, n=7, vs. 9.4±1.9%, n=3, p<0.02). In addition, calcium transients from newborn cells had a ‘U’ shaped wavefront with calcium first increasing at the outer edge of the cell then propagating toward the center. In contrast, infant cells had a homogenous calcium wavefront. The delay of peak calcium from the edge to center was significantly longer in newborns compared to infants (61.7±8.9 ms, n=8 cells, 3 newborns vs. 10.6±1.6 ms, n=6 cells, 4 infants, p<0.001). Conclusions: There are heterogeneous calcium transients in newborn human myocytes that correspond to a lack of t-tubules. Furthermore, t-tubule development occurs in a heterogeneous manner throughout the first year of life with cells from the same patient exhibiting different degrees of t-tubule development. This is the first study to examine t-tubule development and calcium transients in the very young human ventricular myocardium.