title: The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells.
abstract: The sarcoplasmic/endoplasmic reticulum Ca2+ATPases (SERCAs) are the main Ca2+ pumps which decrease the intracellular Ca2+ level by reaccumulating Ca2+ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca2+]i measured at the 20th and 40th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca2+-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca2+]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: Adrienn
authors.initials: A
authors.lastname: Tóth
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: János
authors.initials: J
authors.lastname: Fodor
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: János
authors.initials: J
authors.lastname: Vincze
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Oláh
authors.affiliation: Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Juhász
authors.affiliation: Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: Róza
authors.initials: R
authors.lastname: Zákány
authors.affiliation: Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Csernoch
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Ernő
authors.initials: E
authors.lastname: Zádor
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D015536:Down-Regulation; D020868:Gene Silencing; D006706:Homeostasis; D051379:Mice; D008954:Models, Biological; D032342:Muscle Cells / Q000378:metabolism*; D024510:Muscle Development; D018482:Muscle, Skeletal / Q000166:cytology* / Q000254:growth & development; D034741:RNA, Small Interfering / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014162:Transfection
chemical_list: C506944:Atp2a1 protein, mouse; D034741:RNA, Small Interfering; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium