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Spatial organization of sodium calcium exchanger and caveolin-3 in developing mammalian ventricular cardiomyocytes Hung, Hsiao-Yu

Abstract

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures.

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