Comparative proteomic profiling reveals a role for Cisd2 in skeletal muscle aging

Skeletal muscle has emerged as one of the most important tissues involved in regulating systemic metabolism. The gastrocnemius is a powerful skeletal muscle composed of predominantly glycolytic fast‐twitch fibers that are preferentially lost among old age. This decrease in gastrocnemius muscle mass is remarkable during aging; however, the underlying molecular mechanism is not fully understood. Strikingly, there is a ~70% decrease in Cisd2 protein, a key regulator of lifespan in mice and the disease gene for Wolfram syndrome 2 in humans, within the gastrocnemius after middle age among mice. A proteomics approach was used to investigate the gastrocnemius of naturally aged mice, and this was compared to the autonomous effect of Cisd2 on gastrocnemius aging using muscle‐specific Cisd2 knockout (mKO) mice as a premature aging model. Intriguingly, dysregulation of calcium signaling and activation of UPR/ER stress stand out as the top two pathways. Additionally, the activity of Serca1 was significantly impaired and this impairment is mainly attributable to irreversibly oxidative modifications of Serca. Our results reveal that the overall characteristics of the gastrocnemius are very similar when naturally aged mice and the Cisd2 mKO mice are compared in terms of pathological alterations, ultrastructural abnormalities, and proteomics profiling. This suggests that Cisd2 mKO mouse is a unique model for understanding the aging mechanism of skeletal muscle. Furthermore, this work substantiates the hypothesis that Cisd2 is crucial to the gastrocnemius muscle and suggests that Cisd2 is a potential therapeutic target for muscle aging.     

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.     

Transcriptional profiling of skeletal muscle tissue from two breeds of cattle

We used a 9.6 K cattle muscle/fat cDNA microarray to study gene expression differences between the longuissimus dorsi (LD) muscle of Japanese Black (JB) and Holstein (HOL) cattle. JB cattle exhibit an unusual ability to accumulate intramuscular adipose tissue with fat melting points lower than that in other breeds. The LD biopsies from three JB (Tajima strain) and three HOL animals were used in this breed comparison. Seventeen genes were identified as preferentially expressed in LD samples from JB and seven genes were found to be expressed more highly in HOL. The expression of six selected differentially expressed genes was confirmed by quantitative real-time PCR. The genes more highly expressed in JB are associated with unsaturated fatty acid synthesis, fat deposition, and the thyroid hormone pathway. These results are consistent with the increased amounts and proportions of monounsaturated fatty acids observed in the muscle of JB animals. By discovering as yet uncharacterized genes that are differentially regulated in this comparison, the work may lead us to a better understanding of the regulatory pathways involved in the development of intramuscular adipose tissue.     

The human muscle proteome in aging

The aim of the present study was to assess age-dependent changes of proteins in the vastus lateralis muscle of physically active elderly and young subjects by a combination of two-dimensional difference gel electrophoresis, SDS-PAGE and ESI-MS/MS. The differences observed in the elderly group included down-regulation of regulatory myosin light chains, particularly the phosphorylated isoforms, a higher proportion of myosin heavy chain isoforms 1 and 2A, and enhanced oxidative and reduced glycolytic capacity.     

Transcriptional profiling of initial differentiation events in human embryonic stem cells

Currently, there are no differentiation strategies for human embryonic stem cells (hESCs) that efficiently produce one specific cell type, possibly because of lack of understanding of the genes that control signaling events prior to overt differentiation. sed HepG2 cell conditioned medium (MEDII), which induces early differentiation in mouse ES cells while retaining pluripotent markers, to query gene expression in hESCs. Treatment of adherent hESCs with 50% MEDII medium effected differentiation to a cell type with gene expression similar to primitive streak stage cells of mouse embryos. MEDII treatment up-regulates TDGF1 (Cripto), a gene essential for anterior-posterior axis and mesoderm formation in mouse embryos and a key component of the TGFB1/NODAL signaling pathway. LEFTYA, an antagonist of NODAL/TDGF1 signaling expressed in anterior visceral endoderm, is down-regulated with MEDII treatment, as is FST, an inhibitor of mesoderm induction via the related INHBE1 pathway. In summary, the TGFB1/NODAL pathway is important for primitive-streak and mesoderm formation and in using MEDII, we present a means for generating an in vitro cell population that maintains pluripotent gene expression (POU5F1, NANOG) and SSEA-4 markers while regulating genes in the TGFB1/NODAL pathway, which may lead to more uniform formation of mesoderm in vitro.     

Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection

Using a novel cDNA microarray prepared from sources of actively responding immune system cells, we have investigated the changes in gene expression in the target tissue during the early stages of infection of neonatal chickens with infectious bursal disease virus. Infections of two lines of chickens previously documented as genetically resistant and sensitive to infection were compared in order to ascertain early differences in the response to infection that might provide clues to the mechanism of differential genetic resistance. In addition to major changes that could be explained by previously described changes in infected tissue, some differences in gene expression on infection, and differences between the two chicken lines, were observed that led to a model for resistance in which a more rapid inflammatory response and more-extensive p53-related induction of apoptosis in the target B cells might limit viral replication and consequent pathology. Ironically, the effect in the asymptomatic neonatal infection is that more-severe B-cell depletion is seen in the more genetically resistant chicken. Changes of expression of many chicken genes of unknown function, indicating possible roles in the response to infection, may aid in the functional annotation of these genes.