Int. J. Dev. Biol. 59: 313 - 325 (2015)

https://doi.org/10.1387/ijdb.150206am

Vol 59, Issue 7-8-9

Special Issue: Ionic Messengers in Development and Cancer

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

Published: 19 November 2015

Jeffrey J. Kelu¹, Hayley L.H. Chan², Sarah E. Webb¹, Arthur H.H. Cheng^1,#, 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.

Keywords

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