Int. J. Dev. Biol. 59: 313 - 325 (2015)
doi: 10.1387/ijdb.150206am
© UPV/EHU Press

Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca2+ signaling during myogenesis in intact zebrafish

Jeffrey J. Kelu1, Hayley L.H. Chan2, Sarah E. Webb1, Arthur H.H. Cheng1,#, Margarida Ruas2, John Parrington2, Antony Galione2 and Andrew L. Miller*,1,3

1Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, PRC, 2Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK and 3MBL, Woods Hole, MA, USA

ABSTRACT We have recently characterized essential inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR)-mediated Ca2+ signals generated during the differentiation of slow muscle cells (SMCs) in intact zebrafish embryos. Here, we show that the lysosomal two-pore channel 2 (TPC2) also plays a crucial role in generating, and perhaps triggering, these essential Ca2+ signals, and thus contributes to the regulation of skeletal muscle myogenesis. We used a transgenic line of zebrafish that expresses the bioluminescent Ca2+ reporter, aequorin, specifically in skeletal muscle, in conjunction with morpholino (MO)-based and pharmacological inhibition of TPC2, in both intact embryos and isolated SMCs. MO-based knock-down of TPC2 resulted in a dramatic attenuation of the Ca2+ signals, whereas the introduction of TPCN2-MO and TPCN2 mRNA together partially rescued the Ca2+ signaling signature. Embryos treated with trans-ned-19 or bafilomycin A1, a specific NAADP receptor inhibitor and vacuolar-type H+ ATPase inhibitor, respectively, also displayed a similar disruption of SMC Ca2+ signaling. TPC2 and lysosomes were shown via immunohistochemistry and confocal laser scanning microscopy to be localized in perinuclear and striated cytoplasmic domains of SMCs, coincident with patterns of IP3R and RyR expression. These data together imply that TPC2-mediated Ca2+ release from lysosomes acts upstream from RyR- and IP3R-mediated Ca2+ release, suggesting that the former might act as a sensitive trigger to initiate the SR-mediated Ca2+-induced-Ca2+-release essential for SMC myogenesis and function.


two-pore channel 2, Ca2+ signaling, slow muscle cell myogenesis, zebrafish, in vivo imaging

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