AB16503 Sigma-AldrichAnti-Apaf-1 Antibody, CT

Aging of skeletal muscle is often accompanied by muscle atrophy and it appears that apoptosis plays an important role in this process. The detailed mechanism(s) is not completely understood, however. In this study, we examined expression of the apoptosis regulatory proteins as well as the heat shock proteins, which have been shown to modulate the apoptotic process in certain cell types, in order to more completely elucidate apoptotic signaling in aged skeletal muscle. To more specifically identify alterations that are likely to be the result of aging, we compared 16-month-old middle-aged (MD) and 29-month-old senescent (SE) male Fischer 344 x Brown Norway rats in our study. Our results show that the degree of DNA laddering was higher in SE compared to MD rats. Using total tissue homogenates we examined the level of expression of several apoptosis-related proteins in two categories: mitochondria-associated proteins and caspases. Of the mitochondria-associated proteins, the levels of p53 showed a significant increase in SE compared to MD rats. There was also a significant increase in the expression of Bax, Bcl-2 and Apaf-1 in SE rats over that of MD rats; cytochrome c and AIF levels remained unchanged, however. Regarding the caspases, there were increases in the levels of pro-caspases-12 and -7 and cleaved caspase-9, although the levels of pro- and cleaved caspase-3 as well as cleaved caspase-12 remained unchanged. Furthermore, our results showed significant increases in HSP27, HSP60, and the inducible HSP70. These data show that in rat skeletal muscle increased apoptosis occurs between middle-age and senescence, indicating an aging-related increase in apoptosis in skeletal muscle. The involvement of different apoptotic pathways in the aging process is suggested by the selective alterations in the apoptosis regulatory proteins. The increased expression of the HSPs suggests a relationship between HSPs and the aging-related apoptotic process.

The influence of aging on skeletal myocyte apoptosis is not well understood. In this study we examined apoptosis and apoptotic regulatory factor responses to muscle atrophy induced via limb unloading following loading-induced hypertrophy. Muscle hypertrophy was induced by attaching a weight to one wing of young and aged Japanese quails for 14 days. Removing the weight for 7 or 14 days after the initial 14 days of loading induced muscle atrophy. The contralateral wing served as the intra-animal control. A time-released bromodeoxyuridine (BrdU) pellet was implanted subcutaneously with wing weighting to identify activated satellite cells/muscle precursor cells throughout the experimental period. Bcl-2 mRNA and protein levels decreased after 7 days of unloading, but they were unchanged after 14 days of unloading in young muscles. Bcl-2 protein level but not mRNA level decreased after 7 days of unloading in muscles of aged birds. Seven days of unloading increased the mRNA level of Bax in muscles from both young and aged birds. Fourteen days of unloading increased mRNA and protein levels of Bcl-2, decreased protein levels of Bax, and decreased nuclear apoptosis-inducing factor (AIF) protein level in muscles of aged birds. BrdU-positive nuclei were found in all unloaded muscles from both age groups, but the number of BrdU-positive nuclei relative to the total nuclei decreased after 14 days of unloading compared with 7 days of unloading. The TdT-mediated dUTP nick end labeling (TUNEL) index was higher after 7 days of unloading in both young and aged muscles and after 14 days of unloading in aged muscles. Immunofluorescent staining revealed that almost all of the TUNEL-positive nuclei were also BrdU immunopositive, suggesting that activated satellite cell nuclei (both fused and nonfused) underwent nuclear apoptosis during unloading. There were significant correlations among levels of Bcl-2, Bax, and AIF and TUNEL index. Our data are consistent with the hypothesis that apoptosis regulates, at least in part, unloading-induced muscle atrophy and loss of activated satellite cell nuclei in previously loaded muscles. Moreover, these data suggest that aging influences the apoptotic responses to prolonged unloading following hypertrophy in skeletal myocytes.

The effect of exercise on apoptosis in postmitotic tissues is not known. In this study, we investigated the effect of regular moderate physical activity (i.e., exercise training) on the extent of apoptosis in rat skeletal and cardiac muscles. Adult Sprague Dawley rats were trained (TR) 5 days weekly for 8 wk on treadmill. Sedentary rats served as controls (CON). An ELISA was used to detect mono- and oligonucleosome fragmentation as an indicator of apoptosis. Bcl-2, Bax, Apaf-1, AIF, cleaved PARP, cleaved caspase-3, cleaved/active caspase-9, heat shock protein (HSP)70, Cu/Zn-SOD, and Mn-SOD protein levels were determined by Western analyses. Bcl-2 and Bax transcript contents were estimated by RT-PCR. A spectrofluorometric assay was used to determine caspase-3 activity. DNA fragmentation in ventricles of the TR group decreased by 15% whereas that in soleus of the TR group tended to decrease (P=0.058) when compared with CON group. Protein contents of Bcl-2, HSP70, and Mn-SOD increased in both soleus and ventricle muscles of TR animals when compared with CON animals. Apaf-1 protein content in the soleus of TR animals was lower than that of CON animals. Bcl-2 mRNA levels increased in both ventricle and soleus muscles of TR animals, and Bax mRNA levels decreased in the soleus of TR animals when compared with CON animals. Furthermore, HSP70 protein content was negatively correlated to Bax mRNA content and was positively correlated to Bcl-2 protein and mRNA contents. Mn-SOD protein content was negatively correlated to the apoptotic index, and caspase-3 activity and was positively correlated to Bcl-2 transcript content and HSP70 protein content. These data suggest that exercise training attenuates the extent of apoptosis in cardiac and skeletal muscles.

The cytosolic protein APAF1, human homolog of C. elegans CED-4, participates in the CASPASE 9 (CASP9)-dependent activation of CASP3 in the general apoptotic pathway. We have generated by gene trap a null allele of the murine Apaf1. Homozygous mutants die at embryonic day 16.5. Their phenotype includes severe craniofacial malformations, brain overgrowth, persistence of the interdigital webs, and dramatic alterations of the lens and retina. Homozygous embryonic fibroblasts exhibit reduced response to various apoptotic stimuli. In situ immunodetection shows that the absence of Apaf1 protein prevents the activation of Casp3 in vivo. In agreement with the reported function of CED-4 in C. elegans, this phenotype can be correlated with a defect of apoptosis. Our findings suggest that Apaf1 is essential for Casp3 activation in embryonic brain and is a key regulator of developmental programmed cell death in mammals.

We report here the purification and cDNA cloning of Apaf-1, a novel 130 kd protein from HeLa cell cytosol that participates in the cytochrome c-dependent activation of caspase-3. The NH2-terminal 85 amino acids of Apaf-1 show 21% identity and 53% similarity to the NH2-terminal prodomain of the Caenorhabditis elegans caspase, CED-3. This is followed by 320 amino acids that show 22% identity and 48% similarity to CED-4, a protein that is believed to initiate apoptosis in C. elegans. The COOH-terminal region of Apaf-1 comprises multiple WD repeats, which are proposed to mediate protein-protein interactions. Cytochrome c binds to Apaf-1, an event that may trigger the activation of caspase-3, leading to apoptosis.