Kathleen O'Connell

Proteomic DIGE analysis of the mitochondria-enriched fraction from aged rat skeletal muscle

Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria‐enriched fraction from young adult versus aged muscle revealed an age‐related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age‐dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX‐III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate‐coenzyme A ligase, acyl‐coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol‐cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE‐identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic‐oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging.

Proteomics of skeletal muscle aging

Extended human longevity has resulted in increasing numbers of elderly persons in the general population. However, old age is also associated with a variety of serious physical disorders. Frailty among sedentary elderly patients is related to the impaired structure and function of contractile fibers. Biochemical research into cellular mechanisms that underlie sarcopenia promises to acquire the scientific basis of evidence to aid the development of new diagnostic and therapeutic strategies. The recent application of MS‐based proteomic methodology has identified a large cohort of disease‐specific markers of sarcopenia. This review critically examines the biomedical implications of the results obtained from the proteomic screening of both aged human muscle and established animal models of sarcopenia. Substantial alterations in proteins involved in key metabolic pathways, regulatory and contractile elements of the actomyosin apparatus, myofibrillar remodeling and the cellular stress response are discussed. A multi‐factorial etiology appears to be the basis for a slower‐twitching aged fiber population, which exhibits a shift to more aerobic‐oxidative metabolism. It is hoped that the detailed biomedical characterization of the newly identified biomarkers of sarcopenia will translate into better treatment options for reversing age‐dependent muscle degeneration, which could improve the standard of living for a large portion of society.

Proteomic profiling of animal models mimicking skeletal muscle disorders.

Over the last few decades of biomedical research, animal models of neuromuscular diseases have been widely used for determining pathological mechanisms and for testing new therapeutic strategies. With the emergence of high‐throughput proteomics technology, the identification of novel protein factors involved in disease processes has been decisively improved. This review outlines the usefulness of the proteomic profiling of animal disease models for the discovery of new reliable biomarkers, for the optimization of diagnostic procedures and the development of new treatment options for skeletal muscle disorders. Since inbred animal strains show genetically much less interindividual differences as compared to human patients, considerably lower experimental repeats are capable of producing meaningful proteomic data. Thus, animal model proteomics can be conveniently employed for both studying basic mechanisms of molecular pathogenesis and the effects of drugs, genetic modifications or cell‐based therapies on disease progression. Based on the results from comparative animal proteomics, a more informed decision on the design of clinical proteomics studies could be reached. Since no one animal model represents a perfect pathobiochemical replica of all of the symptoms seen in complex human disorders, the proteomic screening of novel animal models can also be employed for swift and enhanced protein biochemical phenotyping.

Reduced expression of sarcalumenin and related Ca2+ -regulatory proteins in aged rat skeletal muscle.

In skeletal muscle, Ca(2+)-cycling through the sarcoplasm regulates the excitation-contraction-relaxation cycle. Since uncoupling between sarcolemmal excitation and fibre contraction may play a key role in the functional decline of aged muscle, this study has evaluated the expression levels of key Ca(2+)-handling proteins in senescent preparations using immunoblotting and confocal microscopy. Sarcalumenin, a major luminal Ca(2+)-binding protein that mediates ion shuttling in the longitudinal sarcoplasmic reticulum, was found to be greatly reduced in aged rat tibialis anterior, gastrocnemius and soleus muscle as compared to adult specimens. Minor sarcolemmal components of Ca(2+)-extrusion, such as the surface Ca(2+)-ATPase and the Na(+)-Ca(2+)-exchanger, were also diminished in senescent fibres. No major changes were observed for calsequestrin, sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor Ca(2+)-release channel. In contrast, the age-dependent reduction in the alpha(1S)-subunit of the dihydropryridine receptor was confirmed. Hence, this report has shown that downstream from the well-established defect in coupling between the t-tubular voltage sensor and the junctional Ca(2+)-release channel complex, additional age-related alterations exist in the expression of essential Ca(2+)-handling proteins. This may trigger abnormal luminal Ca(2+)-buffering and/or decreased plasmalemmal Ca(2+)-removal, which could exacerbate impaired signaling or disturbed intracellular ion balance in aged fibres, thereby causing contractile weakness.

Lectin-based proteomic profiling of aged skeletal muscle: decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation.

Since various neuromuscular diseases are associated with abnormal glycosylation, it was of interest to determine whether this key post-translational modification is also altered in aged skeletal muscle. Lectins represent highly versatile carbohydrate-binding proteins that are routinely employed for the characterization of glycoproteins. Here, we used the lectin wheat germ agglutinin (WGA) for the proteomic profiling of senescent fibers. WGA labeling of the soluble proteome from 3-month- versus 30-month-old rat gastrocnemius muscle, following two-dimensional gel electrophoretic separation, resulted in the identification of 13 distinct protein species. Analysis of WGA binding levels, in conjunction with mass spectrometric fingerprinting, revealed that one isoform of a major metabolic muscle protein exhibited a drastic alteration in the content of sialic acid and N-acetylglucosaminyl sugar residues. Pyruvate kinase isoform M1 with protein accession number gi|16757994|, exhibiting a pI of 6.6 and an apparent molecular mass of 57.8 kDa, showed a six fold increase in N-glycosylation and a three fold decrease in protein expression. In contrast to comparable levels of N-glycosylated proteins in young adult versus senescent muscle, as judged by fluorescein-conjugated WGA labeling of transverse muscle cryosections, staining with antibodies to the M1 isoform of pyruvate kinase showed reduced expression of this cytosolic element. Furthermore, activity assays demonstrated a reduced activity of this glycolytic enzyme in senescent muscle. This agrees with the idea that abnormal post-translational modifications in key metabolic enzymes may be involved in the conversion of aged muscle to slower twitch patterns and a drastic shift to more aerobic-oxidative metabolism.

Proteomic Profiling of Mitochondrial Enzymes during Skeletal Muscle Aging.

Mitochondria are of central importance for energy generation in skeletal muscles. Expression changes or functional alterations in mitochondrial enzymes play a key role during myogenesis, fibre maturation, and various neuromuscular pathologies, as well as natural fibre aging. Mass spectrometry-based proteomics suggests itself as a convenient large-scale and high-throughput approach to catalogue the mitochondrial protein complement and determine global changes during health and disease. This paper gives a brief overview of the relatively new field of mitochondrial proteomics and discusses the findings from recent proteomic surveys of mitochondrial elements in aged skeletal muscles. Changes in the abundance, biochemical activity, subcellular localization, and/or posttranslational modifications in key mitochondrial enzymes might be useful as novel biomarkers of aging. In the long term, this may advance diagnostic procedures, improve the monitoring of disease progression, help in the testing of side effects due to new drug regimes, and enhance our molecular understanding of age-related muscle degeneration.

The use of LC‐MS to identify differentially expressed proteins in docetaxel‐resistant prostate cancer cell lines

Docetaxel is a taxane‐derived chemotherapy drug that has been approved for treatment of prostate cancer. While docetaxel is frequently used as a treatment for hormone‐refractory prostate cancer, a subset of patients either do not respond to this treatment or those that do respond eventually become resistant to the drug over time. Resistance to docetaxel is complex and multi‐factoral and further understanding of the cellular biochemistry underlying resistance is vital to improve treatment efficacy. To identify proteins altered in the resistant phenotype, three parental cell lines DU145, 22RV1 and PC‐3, as well as their docetaxel resistant sub‐lines, were subjected to quantitative label‐free LC‐MS proteomic profiling. A total of 189 significant (p < 0.05) protein abundance changes were identified in the DU145 resistant sub‐lines, 254 in the 22RV1 sub‐lines, and 51 and 72 in the 8 and 12 nM resistant PC‐3 sub‐lines, respectively. From these, 29 proteins demonstrated a significant (p < 0.05) fold change across two or more resistant variants. These included proteins indicative of an epithelial‐to‐mesenchemyl transition as well as altered heat shock response elements.