Philip Doran

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.

Drastic reduction of sarcalumenin in Dp427 (dystrophin of 427 kDa)-deficient fibres indicates that abnormal calcium handling plays a key role in muscular dystrophy.

Although the primary abnormality in dystrophin is the underlying cause for mdx (X-chromosome-linked muscular dystrophy), abnormal Ca2+ handling after sarcolemmal microrupturing appears to be the pathophysiological mechanism leading to muscle weakness. To develop novel pharmacological strategies for eliminating Ca2+-dependent proteolysis, it is crucial to determine the fate of Ca2+-handling proteins in dystrophin-deficient fibres. In the present study, we show that a key luminal Ca2+-binding protein SAR (sarcalumenin) is affected in mdx skeletal-muscle fibres. One- and two-dimensional immunoblot analyses revealed the relative expression of the 160 kDa SR (sarcoplasmic reticulum) protein to be approx. 70% lower in mdx fibres when compared with normal skeletal muscles. This drastic reduction in SAR was confirmed by immunofluorescence microscopy. Patchy internal labelling of SAR in dystrophic fibres suggests an abnormal formation of SAR domains. Differential co-immunoprecipitation experiments and chemical cross-linking demonstrated a tight linkage between SAR and the SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1) isoform of the SR Ca2+-ATPase. However, the relative expression of the fast Ca2+ pump was not decreased in dystrophic membrane preparations. This implies that the reduction in SAR and calsequestrin-like proteins plays a central role in the previously reported impairment of Ca2+ buffering in the dystrophic SR [Culligan, Banville, Dowling and Ohlendieck (2002) J. Appl. Physiol. 92, 435-445]. Impaired Ca2+ shuttling between the Ca2+-uptake SERCA units and calsequestrin clusters via SAR, as well as an overall decreased luminal ion-binding capacity, might indirectly amplify the Ca2+-leak-channel-induced increase in cytosolic Ca2+ levels. This confirms the idea that abnormal Ca2+ cycling is involved in Ca2+-induced myonecrosis. Hence, manipulating disturbed Ca2+ handling might represent new modes of abolishing proteolytic degradation in muscular dystrophy.

Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragm

The disintegration of the dystrophin–glycoprotein complex represents the initial pathobiochemical insult in Duchenne muscular dystrophy. However, secondary changes in signalling, energy metabolism and ion homeostasis are probably the main factors that eventually cause progressive muscle wasting. Thus, for the proper evaluation of novel therapeutic approaches, it is essential to analyse the reversal of both primary and secondary abnormalities in treated muscles. Antisense oligomer‐mediated exon skipping promises functional restoration of the primary deficiency in dystrophin. In this study, an established phosphorodiamidate morpholino oligomer coupled to a cell‐penetrating peptide was employed for the specific removal of exon 23 in the mutated mouse dystrophin gene transcript. Using DIGE analysis, we could show the reversal of secondary pathobiochemical abnormalities in the dystrophic diaphragm following exon‐23 skipping. In analogy to the restoration of dystrophin, β‐dystroglycan and neuronal nitric oxide synthase, the muscular dystrophy‐associated differential expression of calsequestrin, adenylate kinase, aldolase, mitochondrial creatine kinase and cvHsp was reversed in treated muscle fibres. Hence, the re‐establishment of Dp427 coded by the transcript missing exon 23 has counter‐acted dystrophic alterations in Ca2+‐handling, nucleotide metabolism, bioenergetic pathways and cellular stress response. This clearly establishes the exon‐skipping approach as a realistic treatment strategy for diminishing diverse downstream alterations in dystrophinopathy.

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

The age-dependent decline in skeletal muscle mass and function is believed to be due to a multi-factorial pathology and represents a major factor that blocks healthy aging by increasing physical disability, frailty and loss of independence in the elderly. This study has focused on the comparative proteomic analysis of contractile elements and revealed that the most striking age-related changes seem to occur in the protein family representing myosin light chains (MLCs). Comparative screening of total muscle extracts suggests a fast-to-slow transition in the aged MLC population. The mass spectrometric analysis of the myofibril-enriched fraction identified the MLC2 isoform of the slow-type MLC as the contractile protein with the most drastically changed expression during aging. Immunoblotting confirmed an increased abundance of slow MLC2, concomitant with a switch in fast versus slow myosin heavy chains. Staining of two-dimensional gels of crude extracts with the phospho-specific fluorescent dye ProQ-Diamond identified the increased MLC2 spot as a muscle protein with a drastically enhanced phosphorylation level in aged fibres. Comparative immunofluorescence microscopy, using antibodies to fast and slow myosin isoforms, confirmed a fast-to-slow transformation process during muscle aging. Interestingly, the dramatic increase in slow MLC2 expression was restricted to individual senescent fibres. These findings agree with the idea that aged skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower-twitching fibre population and suggest the slow MLC2 isoform as a potential biomarker for fibre type shifting in sarcopenia of old age.

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 Analysis of Dystrophic Muscle

Mass spectrometry-based proteomics had a major impact on the global characterization of skeletal muscles and has decisively enhanced the field of neuromuscular pathology. Proteomic profiling of x-linked muscular dystrophy has identified a large number of new signature molecules involved in fiber degeneration. Here, we describe the difference in-gel electrophoretic analysis of the dystrophic diaphragm muscle from the MDX mouse model of Duchenne muscular dystrophy. This chapter summarizes the various experimental steps involved in muscle proteomics, such as sample preparation, fluorescence labeling, isoelectric focusing, second-dimension slab gel electrophoresis, image analysis, in-gel digestion and electrospray ionization mass spectrometry.

Sample Preparation of Primary Astrocyte Cellular and Released Proteins for 2-D Gel Electrophoresis and Protein Identification by Mass Spectrometry

The neurotrauma field needs more accurate and sensitive tools to diagnose and evaluate damage to the brain or spinal cord. A controlled, in vitro mechanical trauma model to study protein changes during astrocyte activation after injury is used as a means to discover neurotrauma biomarkers. This model offers several advantages over analyzing cerebrospinal fluid (CSF) or microdialysates including increased sample amount, higher reproducibility between biological samples, enrichment of undegraded proteins due to fewer extracellular matrix proteases in the in vitro versus in vivo system and the ability to relate trauma changes to one cell type. This protocol describes an approach for preparing intact protein samples from conditioned medium (CM) and whole cell lysates of murine cortical astrocytes. The proteins from these samples are separated using 2-D gel electrophoresis, and the protein spot intensities are measured. Proteins showing statistically significant trauma induced changes are then identified using tandem mass spectrometry. This protocol contains optimization steps to enrich proteins of interest, to remove substances that interfere with isoelectric focusing (IEF) and to improve IEF conditions. With this procedure, the 2-D gels show high resolution spot separation and allow for high confidence protein identifications from 107 released and 22 whole cell lysates protein spots so far. Some proteins were identified from multiple spots, suggesting post-translational modification had occurred as a result of the mechanical trauma.

Proteomic and Biochemical Profiling of Aged Skeletal Muscle

Muscle proteomics is concerned with the large-scale profiling of the protein complement from contractile tissues in order to enhance our biochemical knowledge of fundamental physiological processes, as well as the pathophysiological mechanisms that underlie neuromuscular disorders. Since the loss of skeletal muscle mass and strength is one of the most striking features of the senescent body, a large number of proteomic studies have recently attempted the global analysis of age-related fibre degeneration. Although the large size of the muscle proteome and its broad range of expression levels complicates a comprehensive cataloguing of the entire muscle protein complement, mass spectrometry-based proteomic studies have succeeded in the identification of many novel sarcopenia-specific markers. Changes in the expression of affected muscle proteins, as well as altered post-translational modifications, can now be used to establish a reliable biomarker signature of age-dependent fibre wasting. Muscle proteins that are changed during aging belong to the regulatory and contractile elements of the actomyosin apparatus, key bioenergetic pathways, the myofibrillar remodeling machinery and the cellular stress response. The proteomic profiling of crude muscle extracts and distinct subcellular fractions agrees with the notion that sarcopenia of old age is due to a multi-factorial pathology. Changes in muscle markers of the contractile apparatus and energy metabolism strongly indicate a fast-to-slow fibre transition process and a shift to more aerobic-oxidative metabolism during aging. In the long-term, newly established biomarkers of sarcopenia might be useful for the design of improved diagnostic procedures and the identification of new therapeutic targets.