Spontaneous and Repetitive Calcium Transients in C2C12 Mouse Myotubes during In Vitro Myogenesis

Lorenzon, Paola; Giovannelli, Alessandro; Ragozzino, Davide; Eusebi, Fabrizio; Ruzzier, Fabio

doi:10.1111/j.1460-9568.1997.tb01429.x

Cited by 49 publications

(57 citation statements)

References 44 publications

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“…This correlation strengthens the validity of our analysis method and shows that the SR filling state is reduced in CK Ϫ/Ϫ myotubes at higher stimulation frequencies. This reduced filling could have direct consequences for myotube maturation (which is associated with the occurrence of spontaneous Ca 2ϩ transients) (44) given the hypothesized role of Ca 2ϩ as an intra-SR messenger (45). The observation that Ca 2ϩ removal becomes progressively impaired in CK Ϫ/Ϫ myotubes as a function of stimulus intensity shows that the CK system is especially important under conditions of high ATP demand.…”

Section: Discussionmentioning

confidence: 96%

The Creatine Kinase System Is Essential for Optimal Refill of the Sarcoplasmic Reticulum Ca2+ Store in Skeletal Muscle

Groof¹,

Fransen²,

Errington³

et al. 2002

Journal of Biological Chemistry

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Muscle function depends on an adequate ATP supply to sustain the energy consumption associated with Ca 2؉ cycling and actomyosin sliding during contraction. In this regulation of energy homeostasis, the creatine kinase (CK) circuit for high energy phosphoryl transfer between ATP and phosphocreatine plays an important role. We earlier established a functional connection between the activity of the CK system and Ca 2؉ homeostasis during depolarization and contractile activity of muscle. Here, we show how CK activity is coupled to the kinetics of spontaneous and electrically induced Ca 2؉ transients in the sarcoplasm of myotubes. Using the UV ratiometric Ca 2؉ probe Indo-1 and video-rate confocal microscopy in CK-proficient and -deficient cultured cells, we found that spontaneous and electrically induced transients were dependent on ryanodine-sensitive Ca 2؉ release channels, sarcoplasmic/endoplasmic reticulum Ca 2؉ -ATPase pumps, extracellular calcium, and functional mitochondria in both cell types. However, at increasing sarcoplasmic Ca 2؉ load (induced by electrical stimulation at 0.1, 1, and 10 Hz), the Ca 2؉ removal rate and the amount of Ca 2؉ released per transient were gradually reduced in CK-deficient (but not wild-type) myotubes. We conclude that the CK/phosphocreatine circuit is essential for efficient delivery of ATP to the sarcoplasmic/endoplasmic reticulum Ca 2؉ -ATPase pumps and thereby directly influences sarcoplasmic reticulum refilling and the kinetics of the sarcoplasmic Ca 2؉ signals.

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Section: Discussionmentioning

confidence: 96%

The Creatine Kinase System Is Essential for Optimal Refill of the Sarcoplasmic Reticulum Ca2+ Store in Skeletal Muscle

Groof¹,

Fransen²,

Errington³

et al. 2002

Journal of Biological Chemistry

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“…Myoblasts express two classes of voltage-gated Ca 2ϩ channel: in the early stages of differentiation, T type (Ca V 3.2) channels are essential for fusion (18), and during differentiation, L type channel expression becomes prominent (12,19). The experiments of Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Replication-deficient adenoviruses expressing GFP as a marker protein and Rem were constructed as described (10). C2C12(A) cells were infected with Ad-Ctr or Ad-Rem adenoviruses, placed in differentiation media (DM) for 6 days to induce robust Ca 2ϩ channel expression (11)(12)(13), and then loaded with 1 M fura 2 acetoxymethyl ester (fura 2-AM) with 1:1 dilution of Pluronic F127 (Molecular Probes) for 5 min at 37°C in a 5% CO 2 humidified incubator. Deesterification and removal of unincorporated fura 2-AM was accomplished by rinsing cells for 20-30 min in imaging bath solution containing 140 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1 mM NaHPO 4 , 5 mM dextrose, and 10 mM Hepes, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases

Finlin

Crump²,

Satin³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

198

233

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Rem, Rem2, Rad, and Gem͞Kir (RGK) represent a distinct GTPase family with largely unknown physiological functions. We report here that both Rem and Rad bind directly to Ca 2؉ channel ␤-subunits ( R em, Rem2, Rad, and Gem͞Kir (RGK) are members of a Ras-related GTPase subfamily (RGK family), with many unique characteristics that distinguish them from other members of the Ras superfamily (1-5). The common structure for all RGK proteins consists of a conserved Ras-related core domain, a series of nonconservative amino acid substitutions within regions known to be involved in guanine nucleotide binding and hydrolysis, a non-CAAX-containing C-terminal extension, and large N-terminal extensions relative to other Ras family proteins. The conservation of structural features within the RGK proteins suggests shared mechanisms of regulation and the control of common cellular signal transduction networks. However, RGK subfamily members differ from each other and from other Ras-related proteins in their putative effector (G2) domains, suggesting that they interact with distinct regulatory and effector proteins, and each exhibits distinct tissue-specific expression patterns (1-5). Thus, despite their conserved structural and biochemical properties, functional evidence to suggest a unified mechanism of action has been limited.In this study, we investigated the ability of Rem and Rad to regulate Ca 2ϩ channel activity. Our results demonstrate that, although both proteins have distinct effector interaction domains, they each bind directly to Ca V ␤ subunits and inhibit detectable ionic current expression from the native cardiac L type, but not from T type, Ca 2ϩ channels when coexpressed in human embryonic kidney (HEK)293 cells. The Rem protein is expressed in striated muscle cells, and we demonstrate that Rem inhibits L type Ca 2ϩ channel activity in differentiated C2C12 myotubes. Deletion analysis demonstrates that the C terminus of Rem plays an important role in Ca V ␤ subunit association and is necessary for regulation of channel activity. Taken together, these studies indicate that Rem functions as a potent regulator of L type Ca 2ϩ channel function in muscle and suggest that a conserved physiological role for the RGK GTPase gene family is the control of Ca 2ϩ channel activity via modulation of Ca V ␤ subunit function. Experimental ProceduresRNase Protection Assays and Cell Culture. C2C12DS and C2C12(E) mouse muscle myoblast cell lines were from Robert Krauss (Mount Sinai School of Medicine, New York) and were cultured and induced to differentiate as described (6). Primary mouse muscle cultures and MM14 cells were provided by

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“…Post-natal myogenesis critically relies on Ca 2ϩ influx through the SOCE pathway (1,(33)(34)(35)(36)(37). We recently established that the initiation of myoblast differentiation requires, likely among other Ca 2ϩ sources, STIM1/Orai1-dependent cytosolic Ca 2ϩ signals, which are amplified by a plasma membrane hyperpolarization (1,33,38,39).…”

Section: Stim1 and Stim2 Are Endoplasmic Reticulum (Er)mentioning

confidence: 99%

Human Muscle Economy Myoblast Differentiation and Excitation-Contraction Coupling Use the Same Molecular Partners, STIM1 and STIM2

Darbellay

Arnaudeau

Ceroni

et al. 2010

Journal of Biological Chemistry

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Our recent work identified store-operated Ca 2؉ entry (SOCE) as the critical Ca 2؉ source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370 -5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca 2؉ mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca 2؉ content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca 2؉ -content during repeated KCl-induced Ca 2؉ releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitationcontraction coupling. STIM1 and STIM2 are endoplasmic reticulum (ER)2 transmembrane proteins that are activated by a drop in Ca 2ϩ content in the ER (2-5). Once activated, STIM1 and STIM2 trigger a Ca 2ϩ influx (also called store-operated Ca 2ϩ entry (SOCE)) through Ca 2ϩ -selective Orai channels located at the plasma membrane (6 -13). This Ca 2ϩ influx restores the ER Ca 2ϩ content (2-4, 10, 12, 14 -26).Several studies examined whether STIM2 had a specific role, distinct from STIM1, but no clear answer has emerged yet. A lower Ca 2ϩ -activation threshold of STIM2 as compared with STIM1 has been suggested (2), although N-terminal Ca 2ϩ -binding affinity seems to be similar for STIM1 and STIM2 (27-29). In STIM2 knock-out mice, and also in several cell types in which STIM2 has been silenced, SOCE amplitude is only slightly reduced. This could reflect either a smaller activation of Orai1 by the N-terminal part of STIM2 or a lower expression of STIM2 as compared with STIM1 (3,4,24,30). In other studies, overexpression of STIM2 has been reported to inhibit SOCE (31) or, on the contrary, to restore SOCE i...

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Spontaneous and Repetitive Calcium Transients in C2C12 Mouse Myotubes during In Vitro Myogenesis

Cited by 49 publications

References 44 publications

The Creatine Kinase System Is Essential for Optimal Refill of the Sarcoplasmic Reticulum Ca2+ Store in Skeletal Muscle

The Creatine Kinase System Is Essential for Optimal Refill of the Sarcoplasmic Reticulum Ca2+ Store in Skeletal Muscle

Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases

Human Muscle Economy Myoblast Differentiation and Excitation-Contraction Coupling Use the Same Molecular Partners, STIM1 and STIM2

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