Subcellular [Ca2+](i) gradients during excitation-contraction coupling in newborn rabbit ventricular myocytes

The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctio ns in excitation-contraction (EC) coupling in adult (AD) ventricular myocyt es suggests that their absence in newborn (NB) cells may manifest as an alt ered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [ Ca2+](i) transients in the subsarcolemmal space and cell center of field-st imulated NE and AD rabbit ventricular myocytes. Peak systolic [Ca2+](i) occ urred sooner and was higher in the subsarcolemmal space compared with the c ell center in NE myocytes. In AD myocytes, [Ca2+](i) rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling th e SR (10 mu mol/L thapsigargin) slowed the rate of rise and decline of Ca2 in AD myocytes but did not alter Ca2+ transient kinetics in NE myocytes. I n contrast to adults, localized SR Ca2+ release events ("Ca2+ sparks") occu rred predominantly at the cell periphery of NE myocytes. Immunolabeling exp eriments demonstrated overlapping distributions of the Na+-Ca2+ exchanger a nd ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spati ally separated from the sarcolemma in NE myocytes. Confocal sarcolemmal ima ging of di-8-ANEPPS-treated myocytes confirmed an extensive T-tubule networ k in AD cells, and that T-tubules are absent in NE myocytes. A mathematical model of subcellular Ca2+ dynamics predicts that Ca2+ flux via the Na+-Ca2 + exchanger during an action potential can account for the subsarcolemmal C a2+ gradients in NE myocytes. Spatial separation of sarcolemmal Ca2+ entry from SR Ca2+ release channels may minimize the role of SR Ca2+ release duri ng normal EC coupling in NE ventricular myocytes.