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Evaluation of whole muscle apoptotic susceptibility in young and old animals
Author(s) -
Kazak Lawrence,
Hood David A.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1163.16
Subject(s) - apoptosis , cytosol , in vitro , mitochondrion , dna fragmentation , fragmentation (computing) , biology , incubation , dna damage , phosphorylation , microbiology and biotechnology , programmed cell death , biochemistry , dna , ecology , enzyme
Apoptosis regulates muscle mass during disease and aging. However, the signaling pathways leading to apoptosis need to be established. We developed an in vitro muscle incubation and fractionation protocol to assess the activation and localization of apoptotic proteins in response to various stimuli. Soleus strips (∼10–15mg) from young (6 mo) and old (36 mo) F344XBN rats were used. These strips contain ∼30 fibers/mg weight with <1% fiber damage and they can be incubated in vitro for 4–6 hrs to assess their responsiveness to apoptotic agents. Following incubation, the strips were fractionated into mitochondrial and cytosolic fractions. Subsequently, indices of apoptotic susceptibility were measured. Incubation of muscle strips in vitro with the apoptosis‐inducing agent camptothecin for 4 hours resulted in an increase in mitochondrial Bax in both young and old animals. JNK phosphorylation and DNA fragmentation were elevated by 3‐fold in old, compared to young animals. Bax localization to mitochondria was increased by ∼40% in old animals, and there was a 30% increase in AIF localized to the cytosol. Surprisingly, we detected no differential localization of Cyto c to the cytosol between young and old animals. We conclude that this in vitro muscle strip protocol is useful for the assessment of apoptotic susceptibility in whole muscle.