Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability

Barnoy, Sivia; Supino-Rosin, Lia; Kosower, Nechama S.

doi:10.1042/bj3510413

Cited by 30 publications

(6 citation statements)

References 41 publications

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“…There is evidence that stable proteins require longer times of exposure to siRNAs in order to be knocked down compared to less stable ones. Both ubiquitous calpain isoforms have metabolic half-lives of approximately five days [26,27], which accords with the optimal calpain-2 downregulation after 48 h in our experiments. In line with 72 h. The highest calpain-2 downregulation was observed in response to 20 nM siRNA after 24 h and 48 h ( Figure 1).…”

Section: Discussionsupporting

confidence: 82%

Suppression of Calpain-2 by Small Interfering RNA Inhibits Hydrogen Peroxide-Induced Apoptosis in Rat Pancreatic Acinar Cells AR42J

Hiemer¹

2017

J Toxicol Risk Assess

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Apoptosis is defined as controlled programmed cell death that occurs as physiological process in the development and morphogenesis of multi-cellular organisms, but is also implicated in various diseases, including acute pancreatitis. Apoptosis can be induced by a wide range of stimuli. Using pharmacological inhibitors, our previous results suggest that the cytosolic calcium-dependent cysteine protease calpain-2 plays a crucial role in hydrogen peroxide (H 2 O 2)induced apoptosis in pancreatic AR42J cells by activating caspases-12,-8 and-3. The present study aimed to corroborate these results using the small interfering RNA (siRNA) technology to knockdown the calpain-2 gene. AR42J cells were incubated with or without calpain-2 siRNA for 48 h, followed by apoptosis induction using H 2 O 2 for 1 h. Calpain-2 downregulation was assessed by immunoblot and immunofluorescence analyses. The effect of calpain-2 downregulation was evaluated by the degree of caspase activation and cell damage. siRNA-induced calpain-2 gene silencing diminished caspase activation and cell damage, leading to an increased viability of the AR42J cells. Our results indicate that calpain-2 plays an important regulatory role in H 2 O 2-induced apoptosis by activating caspases, thus corroborating our previous findings. The molecular mechanisms proposed for H 2 O 2-induced apoptosis in the pancreatic acinar cell model could well also be of great importance in the pathogenesis of acute pancreatitis.

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Section: Discussionsupporting

confidence: 82%

Suppression of Calpain-2 by Small Interfering RNA Inhibits Hydrogen Peroxide-Induced Apoptosis in Rat Pancreatic Acinar Cells AR42J

Hiemer¹

2017

J Toxicol Risk Assess

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“…Calpains are calcium activated cysteine proteases regulated by myogenic factors like myoD and myogenin (Dedieu et al, 2003). Calpains have a relevant role in signal transduction (Glading et al, 2001; Sato and Kawashima, 2001), cell-cycle regulation, apoptosis (Atencio et al, 2000; Patel and Lane, 2000), cell spreading and migration (Dourdin et al, 1997; Huttenlocher et al, 1997; Potter et al, 1998), and myogenesis (Barnoy et al, 2000). Calpains are also involved with myofibrillar protein disassembly and degradation, contributing to loss of the Z disk (Busch et al, 1972).…”

Section: Resultsmentioning

confidence: 99%

Genome-Wide Association Study for Identifying Loci that Affect Fillet Yield, Carcass, and Body Weight Traits in Rainbow Trout (Oncorhynchus mykiss)

et al. 2016

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Fillet yield (FY, %) is an economically-important trait in rainbow trout aquaculture that affects production efficiency. Despite that, FY has received little attention in breeding programs because it is difficult to measure on a large number of fish and cannot be directly measured on breeding candidates. The recent development of a high-density SNP array for rainbow trout has provided the needed tool for studying the underlying genetic architecture of this trait. A genome-wide association study (GWAS) was conducted for FY, body weight at 10 (BW10) and 13 (BW13) months post-hatching, head-off carcass weight (CAR), and fillet weight (FW) in a pedigreed rainbow trout population selectively bred for improved growth performance. The GWAS analysis was performed using the weighted single-step GBLUP method (wssGWAS). Phenotypic records of 1447 fish (1.5 kg at harvest) from 299 full-sib families in three successive generations, of which 875 fish from 196 full-sib families were genotyped, were used in the GWAS analysis. A total of 38,107 polymorphic SNPs were analyzed in a univariate model with hatch year and harvest group as fixed effects, harvest weight as a continuous covariate, and animal and common environment as random effects. A new linkage map was developed to create windows of 20 adjacent SNPs for use in the GWAS. The two windows with largest effect for FY and FW were located on chromosome Omy9 and explained only 1.0–1.5% of genetic variance, thus suggesting a polygenic architecture affected by multiple loci with small effects in this population. One window on Omy5 explained 1.4 and 1.0% of the genetic variance for BW10 and BW13, respectively. Three windows located on Omy27, Omy17, and Omy9 (same window detected for FY) explained 1.7, 1.7, and 1.0%, respectively, of genetic variance for CAR. Among the detected 100 SNPs, 55% were located directly in genes (intron and exons). Nucleotide sequences of intragenic SNPs were blasted to the Mus musculus genome to create a putative gene network. The network suggests that differences in the ability to maintain a proliferative and renewable population of myogenic precursor cells may affect variation in growth and fillet yield in rainbow trout.

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“…Despite stable expressions ofand m-calpain (Figs. 1 and 2), both activities would be increased during the myoblast fusion, concomitantly with myotube formation, and restored after the fusion (8,11). Balances between -calpain and its specific inhibitor, calpastatin, or between m-calpain and calpastatin are assumed to determine their net proteolytic activities, when the proteases and inhibitors are freely accessible to one another.…”

Section: Discussionmentioning

confidence: 99%

Specific knockdown of m-calpain blocks myogenesis with cDNA deduced from the corresponding RNAi

Honda

Masui²,

Kanzawa³

et al. 2008

American Journal of Physiology-Cell Physiology

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Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both mu- and m-calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of mu-calpain) induced moderate platelet dysaggregation, preserving the normal development and growth, although knockout of calpain-2 (m-calpain) is lethal in mice. Therefore, there should be muscle-specific function of m-calpain per se. Previous methods lack direct evidence for the involvement of m-calpain, because the specific inhibitor to m-calpain has not been developed yet and the inhibition was less potent. Here, we show that screened RNA interference (RNAi) specifically blocked the m-calpain expression by 95% at both the protein and the activity levels. After transfection of adenovirus vector-mediated cDNA corresponding to the RNAi-induced short hairpin RNA, m-calpain in C(2)C(12) myoblasts was knocked down with no compensatory overexpression of mu-calpain or calpain-3. The specific knockdown strongly inhibited the fusion to multinucleated myotubes. In addition, the knockdown modestly blocked ubiquitous effects, including cell migration, cell spreading, and alignment of central stress fiberlike structures. These results may indicate that m-calpain requiring millimolar Ca(2+) level for the full activation plays specific roles in myogenesis, independent of mu-calpain, and leave us challenging problems in the future.

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Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability

Cited by 30 publications

References 41 publications

Suppression of Calpain-2 by Small Interfering RNA Inhibits Hydrogen Peroxide-Induced Apoptosis in Rat Pancreatic Acinar Cells AR42J

Suppression of Calpain-2 by Small Interfering RNA Inhibits Hydrogen Peroxide-Induced Apoptosis in Rat Pancreatic Acinar Cells AR42J

Genome-Wide Association Study for Identifying Loci that Affect Fillet Yield, Carcass, and Body Weight Traits in Rainbow Trout (Oncorhynchus mykiss)

Specific knockdown of m-calpain blocks myogenesis with cDNA deduced from the corresponding RNAi

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