Ferry AL, Vanderklish PW, Dupont-Versteegden EE. Enhanced survival of skeletal muscle myoblasts in response to overexpression of cold shock protein RBM3. Am J Physiol Cell Physiol 301: C392-C402, 2011. First published May 18, 2011; doi:10.1152/ajpcell.00098.2011 is suggested to be involved in the regulation of skeletal muscle mass. Cell death pathways are implicated in the loss of muscle mass and therefore the role of RBM3 in muscle apoptosis in C 2C12 myoblasts was investigated in this study. RBM3 overexpression was induced by either cold shock (32°C exposure for 6 h) or transient transfection with a myc-tagged RBM3 expression vector. Cell death was induced by H 2O2 (1,000 M) or staurosporine (StSp, 5 M), and it was shown that cold shock and RBM3 transfection were associated with attenuation of morphological changes and an increase in cell viability compared with normal temperature or empty vector, respectively. No changes in proliferation were observed with either cold shock or RBM3 transfection. DNA fragmentation was not increased in response to H 2O2, and a cell permeability assay indicated that cell death in response to H 2O2 is more similar to necrosis than apoptosis. RBM3 overexpression reduced apoptosis and the collapse of the membrane potential in response to StSp. Moreover, the increase in caspase-3, -8, and -9 activities in response to StSp was returned to control levels with RBM3 overexpression. These results indicate that increased RBM3 expression decreases muscle cell necrosis as well as apoptosis and therefore RBM3 could potentially serve as an intervention for the loss of muscle cell viability during muscle atrophy and muscle diseases. muscle atrophy; hibernation; RNA-binding proteins; apoptosis; necrosis SKELETAL MUSCLE HAS A REMARKABLE capacity to adapt to changes in mechanical and metabolic environment. Whereas increased use in the form of resistance exercise leads to hypertrophy, reduced muscle activity, as well as certain physiological and pathological processes, can lead to a severe loss in muscle mass (for review see Ref. 25). Skeletal muscle atrophy is often associated with diseases such as cancer, acquired immunodeficiency syndrome, diabetes, and congestive heart failure, and when present carries a poor prognosis (6, 44). In addition, age-related loss of muscle mass (sarcopenia) is an important predictor of mortality in the aged (40,48,49,63) and has been suggested as a main contributor to the loss of independence in elderly (40, 50). Muscle atrophy is also a common symptom in neuromuscular diseases, even though the etiology of the disorders is often quite different. Cell deathrelated signaling events are common in muscle cells undergoing atrophy, but the exact role of apoptosis, autophagy, and necrosis (three common forms of cell death) remains to be determined.In a previous study, we identified the cold-inducible RNAbinding motif protein 3 (RBM3) as a possible regulator of skeletal muscle mass under disuse-induced atrophy (23). RBM3 gene expression and protein abundance were increased ...

Changes in the structure and function of aging non-locomotor muscles remains understudied, despite their importance for daily living. Extraocular muscles (EOMs) have a high incidence of age-related mitochondrial defects possibly because of the metabolic stress resulting from their fast and constant activity. Apoptosis and autophagy (type I and II cell death, respectively) are lnked to defects in mitochondrial function and contribute to sarcopenia in hind limb muscles. Therefore, we hypothesized that apoptosis and autophagy are alered with age in the EOMs. Muscles from 6-, 18-, and 30-month old male Fisher 344-Brown Norway rats were used to investigate type I cell death, caspase-3, -8, -9, and -12 activity, and Type II cell death. Apoptosis, as measured by TUNEL positive nuclei, and mono-and oligonucleosomal content, did not change with age. Similarly, caspase-3, -8, -9, and -12 activity was not affected by aging. By contrast, autophagy, as estimated by gene expression of Atg5 and Atg7, and protein abundance of LC3 was lower in EOMs of aged rats. Based on these data, we suggest that the decrease in autophagy with age leads to the accumulation of damaged organelles, particularly mitochondria, which resuls in the decrease in function observed in EOM with age.

Skeletal muscle atrophy is associated with elevated apoptosis while muscle differentiation results in apoptosis resistance, indicating that the role of apoptosis in skeletal muscle is multifaceted. The objective of this study was to investigate mechanisms underlying apoptosis susceptibility in proliferating myoblasts compared to differentiated myotubes and we hypothesized that cell death-resistance in differentiated myotubes is mediated by enhanced anti-apoptotic pathways. C2C12 myoblasts and myotubes were treated with H2O2 or staurosporine (Stsp) to induce cell death. H2O2 and Stsp induced DNA fragmentation in more than 50% of myoblasts, but in myotubes less than 10% of nuclei showed apoptotic changes. Mitochondrial membrane potential dissipation was detected with H2O2 and Stsp in myoblasts, while this response was greatly diminished in myotubes. Caspase-3 activity was 10-fold higher in myotubes compared to myoblasts, and Stsp caused a significant caspase-3 induction in both. However, exposure to H2O2 did not lead to caspase-3 activation in myoblasts, and only to a modest induction in myotubes. A similar response was observed for caspase-2, -8 and -9. Abundance of caspase-inhibitors (apoptosis repressor with caspase recruitment domain (ARC), and heat shock protein (HSP) 70 and -25 was significantly higher in myotubes compared to myoblasts, and in addition ARC was suppressed in response to Stsp in myotubes. Moreover, increased expression of HSPs in myoblasts attenuated cell death in response to H2O2 and Stsp. Protein abundance of the pro-apoptotic protein endonuclease G (EndoG) and apoptosis-inducing factor (AIF) was higher in myotubes compared to myoblasts. These results show that resistance to apoptosis in myotubes is increased despite high levels of pro-apoptotic signaling mechanisms, and we suggest that this protective effect is mediated by enhanced anti-caspase mechanisms.

The POU-homeodomain transcription factor Pit-1 is required for the differentiation of the anterior pituitary cells and the expression of their hormone products. Pit-1 , an alternate splicing isoform, has diametrically different outcomes when it is expressed in different cell types. Pit-1 acts as a transcriptional repressor of prolactin (PRL) and growth hormone genes in pituitary cells, and as a transcriptional activator in non-pituitary cells. In order to explore these differences, we: (1) identified the transcriptional cofactors necessary for reconstitution of repression in non-pituitary cells; (2) tested the effect of the -domain on heterodimerization with Pit-1 and physical interaction with the co-activator CREB binding protein (CBP); and (3) determined the -domain sidechain chemistry requirements for repression. Co-expression of both Pit-1 isoforms reconstituted the repression of the PRL promoter in non-pituitary cells. The -domain allowed heterodimerization with Pit-1 but blocked physical interaction with CBP, and specific chemical properties of the -domain beyond hydrophobicity were dispensable. These data strongly suggest that Pit-1 represses hormone gene expression by heterodimerizing with Pit-1 and interfering with the assembly of the Pit-1-CBP complex required for PRL promoter activity in pituitary cells.

Many transcription factors are expressed as multiple isoforms with distinct effects on the regulation of gene expression, and the functional consequences of structural differences between transcription factor isoforms may allow for precise control of gene expression. The pituitary transcription factor isoforms Pit-1 and Pit-1 differentially regulate anterior pituitary hormone gene expression. Pit-1 is required for the development of and appropriate hormone expression by anterior pituitary somatotrophs and lactotrophs. Pit-1 differs structurally from Pit-1 by the splice-insertion of the 26-residue -domain in the transactivation domain, and it differs functionally from Pit-1 in that it represses expression of the prolactin promoter in a cell-type specific manner. In order to identify signal and promoter context requirements for repression by Pit-1 , we examined its function in the presence of physiological regulatory signals as well as wild-type and mutant Pit-1-dependent target promoters. Here, we demonstrate that Pit-1 impairs recruitment of cAMP response elementbinding protein (CREB)-binding protein to the promoters that it represses. In addition, we show that repression of target promoter activity, reduction in promoter histone acetylation, and decrease of CREB-binding protein recruitment all depend on promoter context. These findings provide a mechanism for promoter-specific repression by Pit-1 .