Subcellular [Ca 2+ ] i Gradients During Excitation-Contraction Coupling in Newborn Rabbit Ventricular Myocytes

Abstract

Abstract —The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctions in excitation-contraction (EC) coupling in adult (AD) ventricular myocytes suggests that their absence in newborn (NB) cells may manifest as an altered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [Ca 2+ ] i transients in the subsarcolemmal space and cell center of field-stimulated NB and AD rabbit ventricular myocytes. Peak systolic [Ca 2+ ] i occurred sooner and was higher in the subsarcolemmal space compared with the cell center in NB myocytes. In AD myocytes, [Ca 2+ ] i rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling the SR (10 μmol/L thapsigargin) slowed the rate of rise and decline of Ca 2+ in AD myocytes but did not alter Ca 2+ transient kinetics in NB myocytes. In contrast to adults, localized SR Ca 2+ release events (“Ca 2+ sparks”) occurred predominantly at the cell periphery of NB myocytes. Immunolabeling experiments demonstrated overlapping distributions of the Na + -Ca 2+ exchanger and ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spatially separated from the sarcolemma in NB myocytes. Confocal sarcolemmal imaging of di-8-ANEPPS–treated myocytes confirmed an extensive T-tubule network in AD cells, and that T-tubules are absent in NB myocytes. A mathematical model of subcellular Ca 2+ dynamics predicts that Ca 2+ flux via the Na + -Ca 2+ exchanger during an action potential can account for the subsarcolemmal Ca 2+ gradients in NB myocytes. Spatial separation of sarcolemmal Ca 2+ entry from SR Ca 2+ release channels may minimize the role of SR Ca 2+ release during normal EC coupling in NB ventricular myocytes.