Fiber-Type Shifting in Sarcopenia of Old Age: Proteomic Profiling of the Contractile Apparatus of Skeletal Muscles

The progressive loss of skeletal muscle mass and concomitant reduction in contractile strength plays a central role in frailty syndrome. Age-related neuronal impairments are closely associated with sarcopenia in the elderly, which is characterized by severe muscular atrophy that can considerably lessen the overall quality of life at old age. Mass-spectrometry-based proteomic surveys of senescent human skeletal muscles, as well as animal models of sarcopenia, have decisively improved our understanding of the molecular and cellular consequences of muscular atrophy and associated fiber-type shifting during aging. This review outlines the mass spectrometric identification of proteome-wide changes in atrophying skeletal muscles, with a focus on contractile proteins as potential markers of changes in fiber-type distribution patterns. The observed trend of fast-to-slow transitions in individual human skeletal muscles during the aging process is most likely linked to a preferential susceptibility of fast-twitching muscle fibers to muscular atrophy. Studies with senescent animal models, including mostly aged rodent skeletal muscles, have confirmed fiber-type shifting. The proteomic analysis of fast versus slow isoforms of key contractile proteins, such as myosin heavy chains, myosin light chains, actins, troponins and tropomyosins, suggests them as suitable bioanalytical tools of fiber-type transitions during aging.


Introduction
The loss of skeletal muscle mass and contractile strength can be induced by the lack of suitable physical activity levels, extended periods of disuse or disease [1][2][3]. Acute forms of skeletal muscle wasting are often observed during physical trauma and sepsis [4]. Many chronic conditions are also associated with muscular atrophy, including cancer cachexia, congestive heart failure, diabetes mellitus, chronic obstructive pulmonary disease, glucocorticoid-induced Cushing syndrome, malnutrition, long-lasting infections, acquired immunodeficiency syndrome and kidney failure [5][6][7]. Chronic diseases triggering motor neuron abnormalities, such as amyotrophic lateral sclerosis, are a major clinical cause of muscular atrophy [8]. However, the most common form of contractile fiber wasting in association with muscular atrophy is represented by systemic changes during sarcopenia of old age [9][10][11].
Atrophying skeletal muscles are a major feature of the aging phenotype in humans [12], and often the degree of contractile weakness is even more pronounced than the extent of lost muscle mass [13][14][15]. Sarcopenia of old age is closely connected to frailty [16], as well as an increased frequency of falls and fractures [17][18][19], resulting in a drastically reduced quality of life in the elderly [20] that are affected by substantial skeletal muscle wasting [21]. Reduced skeletal muscle tissue mass in conjunction with low gait speed are typical indicators of sarcopenia [22], whereby the clinical definition of sarcopenia [23] Int. J. Mol. Sci. 2023, 24, 2415 3 of 47

Proteomic Analysis Platforms and Associated Biochemical and Cell Biological Methodology
Following the establishment of the concept of the proteome [53] and mass-spectrometrybased proteomics as a highly useful screening tool in the modern biosciences [54], there has been a steady improvement of sample preparation, mass spectrometric instrumentation and data analysis pipelines using both bottom-up [55][56][57] and top-down proteomic techniques [58][59][60]. Importantly, modern biochemical analyses focus on the unifying concept of dynamic proteoforms being the basic units of protein activity [61][62][63]. This has given unprecedented insights into protein diversity and the role of proteins in cellular functions [64], including skeletal muscle tissues [65][66][67][68]. Advances in the field of proteomics now allow researchers to comprehensively study proteins expressed by an organism or biological system associated with physiological and pathophysiological phenotypes [44]. High-throughput technologies and more precision-based methodologies are now available to identify proteins and their modifications in complex samples [69][70][71][72]. This wide-ranging approach provides a solid platform to understand protein function in a particular biological pathway, and when perturbed, how this affects the biological system [73]. Consequently, proteomics has major applications in medicine and drug development [45][46][47]. The international HUPO Project has made enormous progress in establishing and cataloguing the highly dynamic human proteome [74][75][76], which forms the scientific basis of understanding protein homeostasis at the level of systems biology [77][78][79].

Two-Dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis (2D-GE) is a classic and commonly used method for proteome analysis [80][81][82] and presents an ideal bioanalytical approach for optimum protein separation prior to the systematic mass spectrometric profiling of proteoforms [61]. Although current proteomic analyses use mostly gel-free systems for the initial protein separation step, 2D-GE has not been superseded by chromatographical techniques for specialized applications in top-down proteomics [80,82]. 2D-GE is still a highly useful protein separation method that plays a key role in many proteomics analysis pipelines that focus on the identification and characterization of isolated and intact proteoforms [52,61]. The 2D-GE-based separation step is especially beneficial in the field of applied myology for analyzing the highly diverse array of isoforms of contractile proteins [65][66][67]. The largescale survey of skeletal muscle proteins can be carried out under both native or denaturing conditions [66], including the thorough separation of key contractile proteins [52]. In the most frequently employed version of the 2D-GE technique, mixtures of proteins are separated by charge (based on the isoelectric point, pI, of individual proteins) in the first dimension, and by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE), which discriminates proteins based on their molecular weight, in the second dimension [83][84][85]. This approach can be used to separate several thousand different proteins on one 2D-gel [86][87][88]. Of note, the recently described micro-needling of the first-dimension gel can be used to considerably shorten the time requirements for the initial isoelectric focusing step in 2D-GE [89].
Most 2D-GE approaches are based on the usage of high concentrations of sodium dodecyl sulfate (SDS) for optimum solubilization of proteins in the second dimension [90,91], but 2D-GE can also be carried out with combinations of alternative detergents to increase the resolution of integral membrane proteins [92]. For example, a combination of the cationic detergent named benzyldimethyl-n-hexadecylammonium chloride (BAC) in the first dimension and SDS detergent in the second dimension is used for the BAC/SDS-PAGE technique [93]. Two-dimensional blue native polyacrylamide gel electrophoresis, usually referred to as BN-PAGE [94], separates proteins under native conditions [95] and is frequently used to characterize large protein assemblies in mitochondria [96][97][98]. Natural or modified differences between skeletal muscle protein species or protein complexes can be conveniently examined by diagonal non-reducing/reducing 2D-GE following chemical cross-linking [99][100][101]. Following 2D-GE, the next steps typically involve protein spot

Sample Preparation for Proteomic Analysis
The proteomic analysis of skeletal muscle samples is routinely performed with both crude total extracts or subcellular fractions [165]. Subsets of organelles or enriched protein complexes can be isolated by differential centrifugation, density gradient ultracentrifugation, affinity isolation methods or chemical crosslinking approaches [166][167][168]. Optimum protein extraction for subsequent digestion and MS analysis can be carried out by a variety of standardized sample preparation methods [169][170][171]. The filter-aided sample preparation (FASP) technique is ideal for efficient buffer exchange and the removal of MS-incompatible detergents [172]. For designing an optimized proteomic analysis pipeline, it is important to take into account the biological properties of the starting material, such as individual cells, complex tissues or biofluids, and whether a top-down or bottom-up proteomic approach is needed for studying the proteins of interest [58]. Total protein extracts from tough skeletal muscle tissue samples can be conveniently prepared by the FASP method [173]. An alternative methodology for sample preparations is the In-StageTip (iST) technique [174]. In addition, single-pot solid-phase-enhanced sample preparation (SP3) [175] and its variation, named universal solid-phase protein preparation (USP3) [176], can be employed in proteomic applications. If technical complications are encountered with cell or tissue lysis prior to MS analysis [177], these issues can be addressed with recently developed pressure cycling technology (PCT) [178]. In tissue proteomics, the quantification of hydrophobic proteins by MS analysis can be particularly difficult [179][180][181]. Of note, for the proteomic evaluation of large and highly complex protein assemblies, novel high-resolution native MS techniques have been developed [182][183][184].

Protein Digestion for Peptide Mass Spectrometry
The controlled and highly reproducible digestion of proteins for the production of a distinct peptide population is an essential requirement for the successful proteomic identification of specific proteoforms. Protein digestion can be carried out by various approaches that differ in the presentation of the proteins of interest in solution, in a gel matrix or on a membrane. One can therefore differentiate between in-solution [185,186], in-gel [187,188] and on-membrane [189][190][191] digestion protocols. The most frequently used protease in MS-based proteomics is trypsin [192], but alternative proteases can be used alone or in combination for protein digestion [193][194][195]. A rapid in-gel digestion protocol was recently designed for GeLC-MS/MS applications [196], which suits the systematic proteomic detection of very large proteins that do not properly move into the second dimension during 2D-GE [110]. An alternative method named BAC-gel dissolution to digest PAGE-resolved objective proteins, BAC-DROP [197], uses the above-described BAC detergent in gel systems, which enables swift solubilization by chemical reduction.

Mass Spectrometric Analysis
The standardized detection of individual proteins in complex mixtures can be routinely performed by MS-based peptide analysis using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) [69,198,199] or liquid chromatography tandem mass spectrometry (LC-MS/MS) [70][71][72]200]. A detailed protocol for LC-MS/MS analysis has been recently published that includes a description of all materials, chemicals, buffers, experimental steps, mass spectrometric parameters and bioinformatic software tools needed for a successful proteomic study [201]. Untargeted quantitative proteomics approaches using mass spectrometry are designed to provide a comprehensive unbiased quantitation of the global proteome using label-free and/or labelling techniques [70][71][72][73]. Label-free quantitation of proteins analyzed by MS uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), including the use of spectral counts. Using next-generation mass spectrometry instruments with high-resolution capabilities and enhanced sensitivity, peak intensity areas from selected parent ions in MS1 can be detected, quantitated and combined with other proteinassociated peptides when comparing expression levels between samples [44-46]. When using spectral counting, MS2 spectra, generated by peptide fragmentation, are summed with the number of spectra matched to peptides from a specific protein and are then used as a measure of protein abundance. In the field of sarcopenia research, as outlined in more detail below, the label-free MS technique was used by Théron et al. [202] to profile the proteome from vastus lateralis muscle samples obtained during surgery from mature and older women. The comparison of protein profiling between these two cohorts identified 35 differentially expressed proteins during skeletal muscle aging, mainly associated with energy metabolism and contractile functionality [202], showing the usefulness of employing label-free MS approaches in sarcopenia research.
A critical disadvantage of using a label-free approach is that all samples must be measured independently and require significant instrument time in order to achieve a comprehensive analysis of the proteome under investigation. Alternatively, quantitation can be performed using stable heavy isotopes incorporated into proteins by metabolic or chemical labelling protocols [203]. Tandem mass tags (TMT) [204], stable isotope labelling by amino acids in cell culture (SILAC) [205], isobaric tags for relative and absolute quantitation (iTRAQ) [206] and isotope-coded affinity tags (ICAT) [207] are labelling techniques that are routinely used in research studies investigating the proteome under different conditions. TMT labelling, an example of a chemical labelling methodology, is instrumental to quantitative proteomics, especially as the multiplexing approach allows for greater throughput. This enables quantitative analyses with a comprehensive proteome coverage [208].
Each mass-tagging reagent within a set (TMTpro enables multiplexing of up to 16 samples for protein identification and quantitation) has the same nominal mass and chemical structure composed of an amine-reactive NHS-ester group, a spacer arm, and an MS/MS reporter. The intensity of the unique MS/MS reporter ions (different m/z), detected using LC-MS/MS, is used to determine the amount that each peptide from the labelled samples contributes to the selected parent mass, facilitating relative quantitation. TMT-based proteomics has the advantage of higher quantitative accuracy, fewer missing quantitative values among samples, and reduced sample run times on MS instruments. TMT probes have been used in aging research to quantitate the proteome from young versus old rats [209]. The comparative analysis of slow-twitching soleus muscles versus fast-twitch extensor digitorum longus muscles revealed 78 and 174 proteins being differentially expressed during aging, respectively, and were shown to be generally associated with energy metabolism, oxidative stress, detoxification and transport [209].
SILAC is a quantitative proteomic approach using metabolic labels, which allows the comparison of cultured cells (lysates/secretome) under different conditions [204]. Using this approach, identification and quantitation of thousands of proteins can be performed in a single experiment by combining differently labelled samples prior to analysis by LC-MS/MS [210]. A standard SILAC experiment can be used to compare two or three samples by labelling with a light label (standard media), medium label (media containing 2 H 4 -lysine and 13 C 6 -arginine) and a heavy label (media containing 15 N 2 13 C 6 -lysine and 15 N 4 13 C 6 -arginine) [211]. The complete incorporation of heavy amino acids during protein turnover, in combination with the use of trypsin as the digestive enzyme, means that peptides from the differentially labelled samples can be accurately quantified relative to each other, based on the defined mass difference between the samples [212].
In skeletal muscle proteomics, SILAC was used to study differentiation, fiber damage and fiber typing [213][214][215]. An interesting application of SILAC in combination with an immunoaffinity protocol was the investigation of muscular atrophy in mice that were fed a SILAC diet containing 13 C 6 -lysine for 4, 7 or 11 days when comparing denervation-induced changes after sciatic nerve section in the gastrocnemius muscle as compared to control samples [216]. Ubiquitin remnant peptides (K-ε-GG) were profiled by immunoaffinity enrichment, with results showing that >2100 diglycine remnants were identified, providing an insight into the ubiquitination process during muscular atrophy [216].
Dynamic proteome profiling (DPP) with a deuterium label can be employed to determine time-dependent changes in peptide mass isotopomer abundances [217]. The DPP technique was recently applied to study the relative abundance and fractional synthesis rate of proteins in human muscle biopsy specimens [218], and during C2C12 myoblast differentiation [219] and cellular aging [220]. As listed below, a study by Murphy et al. [221] of obese and healthy men of old age, who underwent resistance training and caloric restriction, determined the amount of newly synthesized skeletal muscle proteins via deuterated water labeling.
Importantly, MS analyses combined with artificial intelligence (AI) are increasing the potential for research and analysis of proteins in the field of proteomics [222]. The MS approach has proven to be a pillar for quantitative studies in addition to the identification of PTMs. Higher-plexing labelling reagents, in combination with advanced data acquisition protocols using the next generation of instruments, provide data on hundreds of thousands of protein isoforms in large sample cohorts. As datasets are becoming more all-encompassing, the use of AI, along with Machine Learning (ML) and Deep Learning (DL) algorithms, will become common features for analyzing the complex spectral data to identify pathophysiological patterns for actionable biology.

Data Acquisition by Mass Spectrometry
Data acquisition by mass spectrometry can be performed using data-dependent acquisition (DDA) [223], data-independent acquisition (DIA) [224] and targeted data acquisition (TDA) [225]. The DDA analysis mode involves using the MS instrument to generate a full-scan mass spectra (MS1), where the N most intense peptide ions (i.e., top 15) are selected and MS/MS spectra acquired. This approach generates thousands of MS/MS spectra that can be used for protein identification and subsequent quantitation. However, as the most abundant peptide ions are selected in the full scan, lower abundant peptide ions are repeatedly excluded from selection, even when using filtering criteria such as dynamic exclusion.
The DIA analysis mode involves using the MS instrument to direct the analysis on a narrow mass window of precursors and acquiring MS/MS data from all precursors detected within that window [226]. By stepping across the defined mass range using specific mass windows, collected MS/MS data will be acquired from all detected precursors. This strategy then uses highly specific fragment ion maps in a spectral library for qualitatively and quantitatively analyzing DIA data sets [227]. Sequential window acquisition of all theoretical mass spectra (SWATH-MS), as described by Gillet et al. [228], is a common method to generate DIA data by dividing the mass range into small mass windows.
The verification phase of many proteomics investigations centers on confirming that the abundances of target peptides are significantly different between sample cohorts by using MS-derived quantitative measurements. Selected/Multiple-Reaction Monitoring (SRM/MRM) or Parallel-Reaction Monitoring (PRM) are examples of approaches that can be utilized, where precursor peptide ions are measured in predefined m/z and retention time [229][230][231]. Stable-isotope-labelled, synthetic peptides are often spiked into the samples of interest, a process that increases the overall accuracy of target peptide quantitation.

Single-Cell Proteomics
Within the last decade, single-cell RNA sequencing (scRNA-seq) has come to the fore as an informative approach to decode tissue composition at the single-cell level and to provide important mechanistic data about pathophysiological associated networks [232]. Protein abundance in single cells is often deduced from complementary analysis platforms (scRNA-seq), as the ability to quantitate the proteome at a single-cell level has remained challenging [233]. Initial approaches for quantitating proteins in single cells relied on antibodies. Hence, these methods depend heavily on the availability of high-quality antibodies, therefore limiting their impact in the analysis of many antigens [234]. However, the use of MS/MS combined with LC-based separation is gaining traction with respect to its application in the analysis of the single-cell proteome [235].
Notable breakthroughs in this area include the use of isobaric labelling for single-cell proteomics, called Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) [236], and the second-generation protocol called Single Cell ProtEomics (SCoPE2) [237]. Such protocols permit cells from heterogeneous populations to be adapted into single-cell suspensions by FACS or CellenONE [238,239]. CellenONE is a precision dispensing technology combined with advanced image processing that delivers real-time and high-accuracy single cell isolation and dispensing. The isolated single cells are lysed, proteins digested, and the resultant peptides labelled with TMTs [240]. The different steps of this protocol can be automated, allowing for reproducibility and scalability. Labelled peptides are mixed and analyzed by MS/MS combined with LC [241].
Label-free analysis of individual cells does not require the use of TMTs, but their throughput is lower than that of the labelling approach [242]. The use of TMTs and the ability to multiplex ultimately increase the amount of peptides detected and quantitated by MS, which is particularly important when analyzing small-diameter mono-nucleated cell populations. However, the analysis of skeletal muscle fibers has some advantages, given that these types of fibers are multi-nucleated single cells and relatively bulky compared to other cell types. Individual muscle fibers contain on average a few micrograms of protein, and their isolation by dissection is more straightforward than having to use FACS or Cel-lenONE approaches. A recent manuscript by Murgia et al. [243] demonstrated the utility of single-cell proteomics when comparing the proteome of type 2X fibers to that of type 1 and 2A fibers in young individuals. Their dataset contained more than 3800 proteins detected by single-fiber proteomics, with approximately 10% of the identified proteins displaying a statistically significant difference among the fiber types investigated. This approach has the potential to increase our understanding of musculoskeletal tissue development and disease within individual muscle fibers [244]. The application of single-cell proteomics in muscle aging is discussed below. Importantly, nanotechnology is increasingly used for optimum sample preparation in single-cell proteomics, as discussed by Arias-Hidalgo et al. [245].

Aptamer-Based Proteomics
Other proteomic-based platforms that are growing in popularity and number include aptamer-based approaches [246]. Aptamers are single strands of oligonucleotides (either ssDNA or ssRNA) that bind with high specificity and high affinity to preselected proteins [247]. The range of the preselected protein panels is ever increasing, with one of the leading aptamer-based proteomics platforms, SomaLogic, offering different protein panels ranging from 1300 to over 7000 targets in as little as 55 mL of plasma or serum. Hathout et al. [248] recently used the SomaLogic platform to identify 108 elevated and 70 decreased proteins in dystrophic patients who were not yet treated with glucocorticoids compared to age-matched healthy controls. High-throughput multiplexing techniques can be combined with TMT technology to detect serum biomarkers that have been released from damaged skeletal muscle fibers [249].

Proteomic Profiling of Fiber-Type Specification in Skeletal Muscles
Most individual skeletal muscles consist of a distinct mixture of fast-twitching, slowtwitching and hybrid fibers [250][251][252], and this fiber-type composition can undergo substantial alterations during progressive muscle wasting [50,51,253]. Fiber-type specification has traditionally been determined by histological, histochemical and immunohistological staining procedures [132,254,255]. Recently, Kallabis et al. [215] described a novel high-throughput proteomic workflow for myosin isoform profiling in single muscle fibers based on the usage of a capillary LC-MS gradient in a 96-well format. This is an excellent improvement of the fiber-type-specific screening of the skeletal muscle proteome. Over the last two decades, the steady improvement of protein separation methodology and mass spectrometric detection efficiency, in combination with enormous advances in bioinformatics, has resulted in the greatly enhanced coverage of the skeletal muscle protein constituents [65][66][67]256].
A large number of proteomic markers are now available for the comprehensive profiling of subcellular fractions from skeletal muscles [257]. Over 10,000 protein species belonging to the core proteome of human and animal skeletal muscles have been identified and characterized by mass spectrometry [258][259][260][261][262][263]. The proteomic profiling of differing skeletal muscles with specific fiber-type distribution patterns has especially focused on human vastus lateralis, deltoideus and trapezius muscles [264][265][266] and mouse gastrocnemius, soleus and diaphragm muscles [267][268][269]. Comparative MS-based studies of mouse extensor digitorum longus and soleus muscles [270][271][272], and tissue extracts from rodent gastrocnemius, extensor digitorum longus, tibialis anterior and soleus muscles [273][274][275][276], have given comprehensive insights into the biochemical complexity of fiber-type-specific protein expression patterns using single-fiber proteomics [277]. The study by Eggers et al. [276] utilized immunolabeling of individual skeletal muscle fibers with antibodies to specific myosin heavy chain isoforms followed by laser micro-dissection and MS analysis. The detailed biochemical characterization of mouse muscle fibers by single-cell proteomics revealed an in-depth profile of fiber-type-specific protein expression levels [276].

Composition of the Acto-Myosin Apparatus and Its Proteomic Profile
Skeletal muscle fibers are highly specialized cellular structures for the generation of force and movement [278]. The sarcomeric components of the acto-myosin apparatus [279] provide the molecular machinery for coordinated filament sliding during skeletal muscle contractions [280]. Contractile proteins exist in a large number of isoforms [281] and can be divided into groups of proteins that are mostly located in the thick myosin-containing filament [282], the thin actin-containing filament [283], the M-line [284] and the Z-disk [285], as well as auxiliary filamentous structures [286]. The sarcomere units have extensive intrinsic connections [287] and are embedded in the overall muscle structure by an extensive cytoskeletal system linking them to organelles for energy supply and signaling mechanisms, and to the costamers for force transmission [288]. Figure 1 provides an overview of the contractile acto-myosin apparatus within the sarcomeric structure of skeletal muscles.
Skeletal muscle fibers are highly specialized cellular structures for the generation of force and movement [278]. The sarcomeric components of the acto-myosin apparatus [279] provide the molecular machinery for coordinated filament sliding during skeletal muscle contractions [280]. Contractile proteins exist in a large number of isoforms [281] and can be divided into groups of proteins that are mostly located in the thick myosincontaining filament [282], the thin actin-containing filament [283], the M-line [284] and the Z-disk [285], as well as auxiliary filamentous structures [286]. The sarcomere units have extensive intrinsic connections [287] and are embedded in the overall muscle structure by an extensive cytoskeletal system linking them to organelles for energy supply and signaling mechanisms, and to the costamers for force transmission [288]. Figure 1 provides an overview of the contractile acto-myosin apparatus within the sarcomeric structure of skeletal muscles. In the thick filaments of skeletal muscles [282,287], the hexameric composition of the major motor protein myosin consists of two myosin heavy chains (MyHCs) [289] and four myosin light chains (MLCs) [290], which can be further subdivided into two phosphorylatable regulatory light chains and two non-phosphorylatable alkali light chains [291]. The main MyHC isoforms in human skeletal muscle are the slow type I isoform MyHC-1 (MYH7 gene), the fast type IIa isoform MyHC-2a (MYH2 gene) and the fast type IIx isoform MyHC-2x (MYH1 gene) [250,252,292]. Another fast isoform of type IIb is named MyHC-2b (MYH4 gene), and is present at high concentration only in small mammals, such as mice, rats and rabbits [110]. Type IIb fibers with high levels of MyHC-2b are extremely fast-contracting and quickly fatigable units that are usually not found in mature human skeletal muscles [251]. In addition, MyHC-emb (MYH3 gene), MyHC-neo (MYH8 gene) and six other MyHC isoforms, encoded by the genes MYH6, MYH7B, MYH13, MYH14, MYH15 and MYH16, respectively, exist in embryonic/fetal muscles [293] and specialized In the thick filaments of skeletal muscles [282,287], the hexameric composition of the major motor protein myosin consists of two myosin heavy chains (MyHCs) [289] and four myosin light chains (MLCs) [290], which can be further subdivided into two phosphorylatable regulatory light chains and two non-phosphorylatable alkali light chains [291]. The main MyHC isoforms in human skeletal muscle are the slow type I isoform MyHC-1 (MYH7 gene), the fast type IIa isoform MyHC-2a (MYH2 gene) and the fast type IIx isoform MyHC-2x (MYH1 gene) [250,252,292]. Another fast isoform of type IIb is named MyHC-2b (MYH4 gene), and is present at high concentration only in small mammals, such as mice, rats and rabbits [110]. Type IIb fibers with high levels of MyHC-2b are extremely fast-contracting and quickly fatigable units that are usually not found in mature human skeletal muscles [251]. In addition, MyHC-emb (MYH3 gene), MyHC-neo (MYH8 gene) and six other MyHC isoforms, encoded by the genes MYH6, MYH7B, MYH13, MYH14, MYH15 and MYH16, respectively, exist in embryonic/fetal muscles [293] and specialized adult muscles, including masticatory, extraocular and laryngeal muscles, as well as muscle spindles [294][295][296]. The recent proteomic profiling of extraocular muscles has also detected, besides the long-established MyHC-13 isoform, MyHC-14 and MyHC-15 being present in these highly specialized and mostly fast-twitching muscles [297]. The slow and fast isoforms of MLC proteins are represented by slow/cardiac regulatory light chain MLC-2s (MYL2 gene), fast regulatory light chain MLC-2f (MYL11 gene; with the previous HGNC gene symbol MYLPF), slow essential light chain MLC-1s (with isoform MLC-1sb encoded by the MYL3 gene; and MLC-1sa encoded by the MYL6B gene) and fast essen-tial light chain MLC-1/3f (with MLC-1 and MLC-3 being splicing products of the MYL1 gene) [289,291,298]. Myosin-binding proteins (MYBP) [299] are located at the thick filament interface and are present as slow and fast isoforms, i.e., MYBP-C1 (slow myosin-binding protein C1; encoded by the MYBPC1 gene) and MYBP-C2 (fast myosin-binding protein C2; encoded by the MYBPC2 gene) [300][301][302]. As a representative example of how proteomics can be employed to routinely detect and characterize a large number of specific isoforms of contractile proteins, Table 1 lists the mass spectrometric identification of major sarcomeric proteins that are associated with the thick myosin filament, thin actin filament, the titin filament, the Z-disc and the M-line in diaphragm muscle [269,333]. The information presented includes the protein names and abbreviations of particular isoforms, their accession number, the name of the coding gene, percentage of sequence coverage, number of peptides and calculated molecular mass. As listed in Table 1, diaphragm muscles are characterized by the presence of MyHC-1, MyHC-2x, MyHC-2b, MyHC-8, MLC-1/3, MLC-2 and MLC-3 in the thick filament, and muscle-type ACTA and various slow and fast isoforms of TPM, TNC, TNI and TNT in the thin filament. Abundant components in the Z-disc were established to include FLNC, TCAP, ACTN isoforms and MYOZ, and the M-line was shown to contain MYOM and OBSCN. The muscle protein that was recognized by the highest number of peptides is represented by the giant sarcomeric protein TTN [108]. A closely linked component of the titin filament was identified as the muscle ankyrin repeat protein MARP. In the thin filament [303], the basic units that form helical actin (ACT) filaments are alpha-actin-1 monomers of the skeletal muscle ACTA type (ACTA1 gene) or the cardiac muscle ACTC type (ACTC1 gene) [304,305]. The Ca 2+ -dependent process of regulating interactions between the MyHC heads and ACT filaments is provided by tropomyosin (TPM) and the troponin (TN) complex [306,307]. Sarcomeric TPM molecules are alpha-1tropomyosin (TPM-1; encoded by the TPM1 gene), slow beta-tropomyosin (TPM-2; encoded by the TPM2 gene) and muscle-type alpha-3-tropomyosin (TPM-3; encoded by the TPM3 gene) [308,309]. The alpha-4-tropomyosin isoform named TPM-4 (TPM4 gene) is a nonsarcomeric cytoskeletal component [310]. The TN complex consists of the Ca 2+ -binding subunit TNC, the TPM-interaction subunit TNT and the inhibitory subunit TNI [311]. All three subunits exist in fast and slow isoforms and exist in various combinations in matured skeletal muscles [312]. This includes TNC-1, the slow/cardiac troponin TnC isoform (TNNC1 gene), TNC-2, the skeletal muscle troponin TnC isoform (TNNC2 gene), TNT-1,  the slow muscle troponin TnT isoform (TNNT1 gene), TNT-3, the fast muscle troponin  TnT isoform (TNNT3 gene), TNI-1, the slow muscle troponin TnI isoform (TNNI1 gene) and TNI-2, the fast muscle troponin TnI isoform (TNNI2 gene) [313]. In addition, the cardiac isoforms TNNI3 and TNNT2 have been found in aged and denervated skeletal muscles [314].
As a representative example of how proteomics can be employed to routinely detect and characterize a large number of specific isoforms of contractile proteins, Table 1 lists the mass spectrometric identification of major sarcomeric proteins that are associated with the thick myosin filament, thin actin filament, the titin filament, the Z-disc and the M-line in diaphragm muscle [269,333]. The information presented includes the protein names and abbreviations of particular isoforms, their accession number, the name of the coding gene, percentage of sequence coverage, number of peptides and calculated molecular mass. As listed in Table 1, diaphragm muscles are characterized by the presence of MyHC-1, MyHC-2x, MyHC-2b, MyHC-8, MLC-1/3, MLC-2 and MLC-3 in the thick filament, and muscle-type ACTA and various slow and fast isoforms of TPM, TNC, TNI and TNT in the thin filament. Abundant components in the Z-disc were established to include FLNC, TCAP, ACTN isoforms and MYOZ, and the M-line was shown to contain MYOM and OBSCN. The muscle protein that was recognized by the highest number of peptides is represented by the giant sarcomeric protein TTN [108]. A closely linked component of the titin filament was identified as the muscle ankyrin repeat protein MARP.

Pathobiological Hallmarks of Sarcopenia of Old Age
Skeletal muscle aging can be considered a fundamental biological process that occurs in all humans of advanced age [334]. However, individual muscles in the same body age differently [28,335] and considerable inter-individual differences exist in the extent and time course of muscle tissue loss and decline in contractile strength [9,336]. Importantly, skeletal muscle degeneration can be accompanied by progressive deterioration of myocardial functions in the elderly, causing serious medical complications due to cardio-sarcopenia syndrome [337]. Although sarcopenia of old age is due to multi-factorial mechanisms, it is most likely that neurological changes during aging play a key role in the initiation of muscular atrophy. The loss of spinal motor neurons appears to be associated with the initial decline in the proper innervation of voluntary muscles. The damage of the neuronal systems is exacerbated by a diminished capacity for reinnervation or patterns of faulty reinnervation [338]. The tendency of fast-to-slow muscle fiber-type transitions in a large number of aged human muscles was shown to be linked to a higher susceptibility of faster-contracting fibers to muscular atrophy [50,51,339]. This higher vulnerability of faster-twitching and mostly glycolytic fibers under atrophic conditions is closely related to specific signaling pathways involving peroxisome proliferator-activated receptor gamma coactivator PGC1-alpha and transforming growth factor TGF-beta [340].
Epidemiological studies of sarcopenia, assessed by both cross-sectional and longitudinal investigations, indicate that everyday life of a large proportion of the population over 75 years of age is impaired by a certain degree of physical frailty and impaired skeletal muscle functioning [341][342][343][344][345][346][347]. Worsening cofactors of age-related muscle wasting include sarcopenia-independent chronic diseases and their extensive pharmacological therapy, as well as chronic low-grade inflammation, insulin resistance, poor nutrition, extended bedrest and the lack of appropriate physical activity levels [9,10,14,348,349]. Thus, to counteract the age-dependent decline in skeletal muscle performance, optimized rehabilitation [350] and appropriate physical exercise regimes, such as moderate resistance exercises [351][352][353], are crucial to minimize oxidative stress and inflammation in sarcopenia [354,355]. Since older adults exhibit a higher rate of protein turnover [356], and an apparent imbalance between accelerated muscle protein breakdown and impaired levels of protein re-synthesis exists in aged muscles [9][10][11], the resulting reduced levels of contractile components in older individuals should be addressed by avoiding a poor diet quality [357][358][359] and instead provide an adequate intake of high-quality protein in the elderly [360][361][362][363].
Recent publications have critically examined the diverse and multi-factorial aspects of aging and sarcopenia, including senescence-related changes linked to abnormal metabolic pathways [364], mitochondrial dysfunctions [365][366][367][368][369][370][371], the role of reactive oxygen species and disrupted redox signaling [372][373][374][375], abnormal calcium handling [376], functional changes in neuromuscular transmission [377], altered myokine and myomitokine signaling [369,378], the role of miRNAs in the decline of proteostasis [379,380], anabolic resistance and impaired muscle protein metabolism [381][382][383], adipocyte crosstalk in aged skeletal muscle and sarcopenic obesity [384,385], immune system alterations, chronic inflammation and immune-metabolic dysfunction associated with oxidative stress [33, 366,[386][387][388], the role of telomere length during aged fiber regeneration [389], the interplay between sarcopenia, frailty and cognitive impairments in the elderly [390], cardio-sarcopenia syndrome [337] and the influence of nutrition on the aging phenotype [391]. The finding that the satellite cell pool is preferentially affected in fast type II fibers in the elderly [392] has established the idea that stem cell exhaustion is majorly involved in sarcopenia and possibly even facilitates age-associated fast-to-slow transitions [393]. Thus, the reduction in muscle-specific stem cells appears to play a key role in the impaired regenerative capacity of aged fibers [394][395][396]. This phenomenon underlines the enormous complexity of the molecular and cellular mechanisms that are associated with skeletal muscle aging.

Proteomics of Aged Skeletal Muscle
Biomarker discoveries using omics-type surveys are crucial to improve the monitoring of impaired physiological functioning, altered energy metabolism and chronic inflammation in aged muscle [397], and to advance the diagnosis, prognosis and therapeutic monitoring of frailty syndrome and sarcopenia in the aging population [398][399][400], whereby proteomics plays a key role in detecting and characterizing novel marker candidates [48,49]. In the context of aging and alterations in contractile proteins, human skeletal muscles were extensively studied using both top-down/gel-based approaches versus bottom-up/peptide-centric analyses [202,221,[401][402][403][404][405][406][407][408][409][410][411][412][413][414]. Changes in particular isoforms of skeletal muscle proteins during the aging process can give detailed insights into molecular and cellular mechanisms that underlie sarcopenia of old age. Although individual skeletal muscles exhibit differing degrees of susceptibility to aging-induced muscular atrophy [28], proteomics has confirmed the previous findings from biochemical, cell-biological and histological studies that suggest a general trend of fast-to-slow transitions in senescent muscles [50, 415,416] and concomitant alterations in glycolytic and mitochondrial pathways [39,417]. This includes a stepwise transition from faster isoforms of MyHC, MLC, ACT, TPM, TNC, TNI and TNT to their slower counterparts. Of note, the recent proteomic profiling of single fibers from human vastus lateralis muscle of young adults has given a comprehensive overview of fiberrelated differences in protein isoform expression patterns [243]. These types of proteomic catalogs can be highly useful as reference databanks for studying proteome-wide changes during aging. Table 2 lists major MS-based investigations with a bioanalytical focus on protein changes in contractile proteins during human skeletal muscle aging [202,221,[401][402][403][404][405][406][407][408][409][410][411][412][413][414]. The listings of individual proteomic investigations summarize the analyzed muscle specimens, the age range of samples, the bioanalytical approach and the detected proteomewide alterations with a focus on the contractile apparatus. Since considerable physiological and biochemical differences exist between untrained versus trained skeletal muscles [68,[418][419][420], contractile fiber aging has also been studied in select master athletes [421,422] in addition to the below-listed studies on neuromuscular changes in the general and mostly untrained population. Major proteomics surveys of aged human muscles that did not focus on the contractile apparatus include investigations into the role of mitochondrial abnormalities [423] and molecular chaperones [424], as well as metabolic changes due to oxidatively modified proteins in satellite cells [425]. Table 2. List of major mass-spectrometry-based proteomic profiling studies focusing on contractile proteins in aged human skeletal muscle tissue.

Specimens Bioanalytical Approach Proteomic Changes References
Vastus lateralis  Top-down proteomics using routine 2D-GE or fluorescent 2D-DIGE is an ideal bioanalytical approach for the efficient separation of contractile proteins below 150 kDa [122], such as fast and slow isoforms of MLC, TPM, TNC, TNI, TNT and ACT [52,66]. Human skeletal muscles usually contain a mixture of slow-twitching fibers, which are characterized by high levels of oxidative metabolism, and faster-twitching fibers with glycolytic-oxidative or mostly glycolytic metabolism [250], in addition to various hybrid fibers [426]. Mass spectrometric analyses of separated 2D spots clearly confirmed shifts from fast protein isoforms to their slower protein counterparts [401][402][403][404][405]409,413], which agrees with the general tendency of fast-to-slow transitions during skeletal muscle aging [50,51, 253,339]. These findings could be complemented with bottom-up strategies and LC-MS/MS analyses to study contractile proteins of higher molecular mass, such as MyHC and TNN [202,221,[406][407][408][410][411][412].
As illustrated in the representative findings on abundance changes in Ca 2+ -regulatory components and contractile proteins in Figure 3, MS-based proteomics is an excellent bioanalytical tool to establish decreases in important Ca 2+ -handling proteins that are involved in cellular signaling pathways and the regulation of excitation-contraction coupling. This includes subunits of the dihydropyridine receptor L-type Ca 2+ -channel of the transverse tubules, the ryanodine receptor Ca 2+ -release channel of the triad junction, the luminal Ca 2+ -binding protein calsequestrin of the terminal cisternae region within the sarcoplasmic reticulum and the structural protein triadin [487][488][489]. Thus, a key mechanism involved in skeletal muscle aging appears to be a certain degree of pathophysiological uncoupling between sarcolemmal excitation and the initiation of acto-myosin sliding that mediates fiber contraction [376,[490][491][492], combined with a reduced association between Ca 2+ -release units and aged mitochondria [493]. Disturbed Ca 2+ -homeostasis may be involved in altered myocyte signaling in the context of fiber-type specification, which is supported by proteomic data that indicate a general tendency of fast-to-slow transitions at the level of isoform switching of contractile proteins [50].

Figure 3.
Representative example of the mass-spectrometry-based proteomic analysis of skeleta muscle aging. Shown are crucial regulatory proteins of excitation-contraction coupling and Ca 2+ homeostasis (dihydropyridine receptor L-type Ca 2+ -channel, ryanodine receptor Ca 2+ -release chan nel, calsequestrin and triadin) and sarcomeric proteins (titin, troponin, myosin light chain and my osin heavy chain). Mass spectrometric analyses of young versus aged wild-type mouse diaphragm muscle specimens were carried out as previously described in detail [201,269,333].

Mechanisms of Age-Related Muscular Atrophy
Research over the last few decades has clearly established that the molecular and cellular mechanisms of aging are highly complex [30-32] and specifically affect the skeletal musculature [9][10][11]. The multi-factorial processes that are associated with age-related muscular atrophy and sarcopenia of old age include:  Figure 4 summarizes crucial aspects of muscle aging, including the preferential susceptibility of fast type II fibers to age-related degeneration, which causes a general shift to slower-twitching fiber populations in most senescent skeletal muscles. Representative example of the mass-spectrometry-based proteomic analysis of skeletal muscle aging. Shown are crucial regulatory proteins of excitation-contraction coupling and Ca 2+homeostasis (dihydropyridine receptor L-type Ca 2+ -channel, ryanodine receptor Ca 2+ -release channel, calsequestrin and triadin) and sarcomeric proteins (titin, troponin, myosin light chain and myosin heavy chain). Mass spectrometric analyses of young versus aged wild-type mouse diaphragm muscle specimens were carried out as previously described in detail [201,269,333].

Mechanisms of Age-Related Muscular Atrophy
Research over the last few decades has clearly established that the molecular and cellular mechanisms of aging are highly complex [30-32] and specifically affect the skeletal musculature [9][10][11]. The multi-factorial processes that are associated with age-related muscular atrophy and sarcopenia of old age include:  Figure 4 summarizes crucial aspects of muscle aging, including the preferential susceptibility of fast type II fibers to age-related degeneration, which causes a general shift to slower-twitching fiber populations in most senescent skeletal muscles.

Biomarker Discovery for the Improved Evaluation of Sarcopenia of Old Age
In order to improve the differential diagnosis of pre-, mild or severe sarcopenia [9 11], the establishment of reliable and robust biomarkers of frailty and skeletal muscl wasting is crucial [29,49]. Suitable markers can be measured by physical performance as sessments [25][26][27], imaging technology [22,28] and/or biochemical assays [494]. A nove imaging marker system is the ultrasound sarcopenic index (USI), which can determine th loss of skeletal muscle mass in association with sarcopenia in a practical and relativel inexpensive way [495]. Ideally, abundance changes in protein biomarkers of sarcopeni should be easily measurable with high levels of specificity and sensitivity [496,497], a well as not being majorly affected by gender, ethnicity, co-morbidities, exposure to phar macological agents and unrelated therapeutic treatments [498]. To avoid potential com plications due to elaborate tissue biopsy procedures, the development of non-invasiv disease indicators is favorable [499]. A recent meta-analysis of proteomic studies by Stal mach et al. [500], using a gene ontology-driven approach, suggests that it is advantageou to integrate MS data sets from both muscle tissue samples and suitable biofluids to gai more comprehensive insights into atrophying changes in the human skeletal muscle pro teome.
This gives non-invasive biomarker investigations of biological fluids, such as serum saliva or urine, a central role in aging research [501][502][503]. The serum of both older human suffering from sarcopenia [504][505][506][507] and senescent mice [508] were shown to exhibit dif ferential changes in common markers that are associated with inflammation, remodelin of the extracellular matrix and mitochondrial functions [398]. This suggests the potentia usage of pro-inflammatory cytokines, growth factors, differentiation factors and leaked mitochondrial proteins as suitable biofluid markers to evaluate the degree of skeletal mus cle aging [400]. The regulatory factor myostatin and insulin growth factor IGF-1 show considerable potential to be useful as gender-specific markers of low skeletal muscle mas and frailty [509]. Ideally, proteomic findings are correlated to the results from systemati Overview of the multi-factorial changes during human skeletal muscle aging. The higher susceptibility of fast-twitching type II fibers causes a tendency of fast-to-slow transitions in senescent muscles. This is reflected by a switch from fast myosin heavy chain isoforms (MyHC-2a, MyHC-2x) to slower counterparts (MyHC-1) during skeletal muscle aging.

Biomarker Discovery for the Improved Evaluation of Sarcopenia of Old Age
In order to improve the differential diagnosis of pre-, mild or severe sarcopenia [9][10][11], the establishment of reliable and robust biomarkers of frailty and skeletal muscle wasting is crucial [29,49]. Suitable markers can be measured by physical performance assessments [25-27], imaging technology [22,28] and/or biochemical assays [494]. A novel imaging marker system is the ultrasound sarcopenic index (USI), which can determine the loss of skeletal muscle mass in association with sarcopenia in a practical and relatively inexpensive way [495]. Ideally, abundance changes in protein biomarkers of sarcopenia should be easily measurable with high levels of specificity and sensitivity [496,497], as well as not being majorly affected by gender, ethnicity, co-morbidities, exposure to pharmacological agents and unrelated therapeutic treatments [498]. To avoid potential complications due to elaborate tissue biopsy procedures, the development of non-invasive disease indicators is favorable [499]. A recent meta-analysis of proteomic studies by Stalmach et al. [500], using a gene ontology-driven approach, suggests that it is advantageous to integrate MS data sets from both muscle tissue samples and suitable biofluids to gain more comprehensive insights into atrophying changes in the human skeletal muscle proteome.
This gives non-invasive biomarker investigations of biological fluids, such as serum, saliva or urine, a central role in aging research [501][502][503]. The serum of both older humans suffering from sarcopenia [504][505][506][507] and senescent mice [508] were shown to exhibit differential changes in common markers that are associated with inflammation, remodeling of the extracellular matrix and mitochondrial functions [398]. This suggests the potential usage of pro-inflammatory cytokines, growth factors, differentiation factors and leaked mitochondrial proteins as suitable biofluid markers to evaluate the degree of skeletal muscle aging [400]. The regulatory factor myostatin and insulin growth factor IGF-1 show considerable potential to be useful as gender-specific markers of low skeletal muscle mass and frailty [509]. Ideally, proteomic findings are correlated to the results from systematic transcriptomic and metabolomic studies of sarcopenia [510][511][512].
Promising biofluid protein markers of sarcopenia are the carboxy-terminal fragment of agrin (CAF) [513][514][515][516] and the brain-derived neurotrophic factor [517][518][519]. The proteoglycan agrin is closely associated with the sarcolemmal dystrophin/utrophin-glycoprotein complex that is involved in the cytoskeletal stabilization of the neuromuscular junction [520]. The loss of neuromuscular junction integrity appears to play a key role in muscular atrophy [377,521] including sarcopenia of old age [522]. The activity of the synapse-specific protease neuro-trypsin [523], and agrin cleavage, are clearly related to the age-dependent degeneration of the neuromuscular junction [513]. The remodeling of aged motor units in turn is linked to the preferential denervation of fast-twitching and mostly glycolytic type II fibers, and faulty patterns of reinnervation by smaller motor neurons that establish slower-contracting type I motor units [524][525][526].
The age-related fiber-type shifting and accompanying changes in MyHC isoforms [527] can only generate lower maximum force levels in senescent skeletal muscles as compared to young and adult muscle systems. These alterations in the overall composition of motor units probably plays a central role in the gradual loss of skeletal muscle strength during aging [528]. This makes circulating CAF a potential biofluid biomarker of motor unit changes in sarcopenia, in conjunction with fast-to-slow fiber-type shifting in aged muscle tissues, as outlined in Figure 5. junction [520]. The loss of neuromuscular junction integrity appears to play a key role in muscular atrophy [377,521] including sarcopenia of old age [522]. The activity of the synapse-specific protease neuro-trypsin [523], and agrin cleavage, are clearly related to the age-dependent degeneration of the neuromuscular junction [513]. The remodeling of aged motor units in turn is linked to the preferential denervation of fast-twitching and mostly glycolytic type II fibers, and faulty patterns of reinnervation by smaller motor neurons that establish slower-contracting type I motor units [524][525][526].
The age-related fiber-type shifting and accompanying changes in MyHC isoforms [527] can only generate lower maximum force levels in senescent skeletal muscles as compared to young and adult muscle systems. These alterations in the overall composition of motor units probably plays a central role in the gradual loss of skeletal muscle strength during aging [528]. This makes circulating CAF a potential biofluid biomarker of motor unit changes in sarcopenia, in conjunction with fast-to-slow fiber-type shifting in aged muscle tissues, as outlined in Figure 5.

Figure 5.
Agrin as a potential serum biomarker of skeletal muscle aging. Shown is the linkage between the disintegration of the neuromuscular junction during skeletal muscle aging and resulting preferential loss of neurotransmission to fast type II fibers. A potential biomarker candidate of this process is the release of carboxy-terminal agrin fragments (CAF) that can be measured in the serum (sCAF) of patients suffering from sarcopenia of old age. At the neuromuscular junction, the proteoglycan agrin associates with the dystroglycan complex (alpha/beta-DG), which forms an integral part of the sub-sarcolemmal utrophin/dystrophin lattice and its associated proteins (DAPs) at the post-synaptic membrane. During skeletal muscle aging, the integrity of the neuromuscular junction is lost, and agrin is proteolytically cleaved by the enzyme neuro-trypsin. This results in the production of distinct agrin fragments that can be conveniently detected in a minimally invasive way in suitable biofluids.

Therapeutic Approaches to Counteract Age-Related Muscular Atrophy
Aging-associated processes lead to a general decline of health status, a higher risk of disease and drastically reduced physical fitness. It is crucial to take multi-system derangements into consideration when designing novel therapeutic approaches to treat individual age-related ailments, such as sarcopenia. Frailty syndrome can result in a diminished quality of life and even loss of independence in the case of severe and chronic muscle Figure 5. Agrin as a potential serum biomarker of skeletal muscle aging. Shown is the linkage between the disintegration of the neuromuscular junction during skeletal muscle aging and resulting preferential loss of neurotransmission to fast type II fibers. A potential biomarker candidate of this process is the release of carboxy-terminal agrin fragments (CAF) that can be measured in the serum (sCAF) of patients suffering from sarcopenia of old age. At the neuromuscular junction, the proteoglycan agrin associates with the dystroglycan complex (alpha/beta-DG), which forms an integral part of the sub-sarcolemmal utrophin/dystrophin lattice and its associated proteins (DAPs) at the post-synaptic membrane. During skeletal muscle aging, the integrity of the neuromuscular junction is lost, and agrin is proteolytically cleaved by the enzyme neuro-trypsin. This results in the production of distinct agrin fragments that can be conveniently detected in a minimally invasive way in suitable biofluids.

Therapeutic Approaches to Counteract Age-Related Muscular Atrophy
Aging-associated processes lead to a general decline of health status, a higher risk of disease and drastically reduced physical fitness. It is crucial to take multi-system derangements into consideration when designing novel therapeutic approaches to treat individual age-related ailments, such as sarcopenia. Frailty syndrome can result in a diminished quality of life and even loss of independence in the case of severe and chronic muscle wasting. General recommendations to support healthy aging include the positive influence of a healthy and balanced diet, sufficient sleep, regular relaxation, proper physical exercise, calm breathing patterns, regular social interactions and a positive view of life [529][530][531][532]. Thus, promoting a healthy lifestyle should include countermeasures against sarcopenia of old age to avoid the premature loss of physical strength and skeletal muscle mass. However, a crucial issue for the elderly is proper access to advanced strength training equipment and the realistic implementation of health-promoting support structures, especially during pandemics. During the current COVID-19 crisis, the aged population has only limited access to gyms, parks, recreational facilities and rehabilitation services, causing long-term negative effects on muscle health [533][534][535], and this situation has to be urgently addressed to promote healthy aging. In addition, the treatment of acute sarcopenia in patients with or without COVID-19 infection has been complicated by the restricted access to health services during the pandemic [536][537][538][539], and the increased application of mechanical ventilation and complications during ventilator weaning has caused considerable side effects, including skeletal muscle wasting [540].
Therapeutic approaches to attenuate the impact of age-related skeletal muscle degeneration include non-pharmacological interventions, such as lifestyle changes that incorporate regular and appropriate resistance training [351][352][353]541,542], and optimized dietary considerations, including a protein-rich diet and the frequent ingestion of small portions of high-quality food [358][359][360]. Nutritional combinations of Vitamin D, leucine-enriched protein supplements and whey protein were shown to have some effects on building skeletal muscle mass and improve physical functionality of the neuromuscular system [543][544][545][546]. The combination of mixed types of regular physical exercise with a balanced diet and nutritional supplementation appears to be the most suitable multi-component intervention strategy to minimize the effects of sarcopenia and avoid mobility disability in older adults [547,548]. A protein-rich diet, combined with high levels of physical activity, should both stimulate muscle protein synthesis and thus prevent impaired proteostasis in senescent fibers, and have generally positive effects on metabolism, bioenergetics and hormonal balance. At advanced age, combining a low-intensity form of home-based resistance exercise with proper nutrition and a multi-ingredient supplementation seems to be the most effective way to treat sarcopenia. Regular exercise has a profound effect on the skeletal muscle proteome [68,354,419] and muscle fiber-type diversification [549][550][551]. In particular, resistance exercise aimed at improving the contractile strength of aged skeletal muscles is generally associated with alterations in myofiber size, muscle re-innervation, fiber-type-specific myonuclear adaptations, mitochondrial remodeling and fiber-type shifting [552][553][554][555][556][557]. Since the agerelated loss of skeletal muscle mass is mostly due to a drastic reduction in the size of fast-twitching type II fibers [50,51,339,416,558], it is encouraging that resistance exercise specifically results in the hypertrophy of type II muscle fibers, although it does not appear to affect patterns of fiber-type grouping in aged muscles [559]. Distinct changes in MyHC isoform expression patterns are usually exemplified by reduced MyHC-1 and increased MyHC-2x levels [554].
Myostatin is a secreted myogenic factor that acts as a negative regulator of skeletal muscle growth. It belongs to the transforming growth factor TGF-beta family of proteins and functions by inhibiting the phosphorylation of Akt protein kinase within the insulin-like growth factor 1-phosphatidylinositol-3-kinase-serine/threonine protein kinase PKB-mammalian target of rapamycin (IGF-1/PI3K/Akt/mTOR) signaling pathway [569]. Consequently, the inhibition of a negative regulator might result in a positive effect on skeletal muscle growth. This can be supported by (i) natural mechanisms, such as physical exercise, (ii) dietary supplements and nutraceutical agents and/or (iii) pharmacological/biotechnological intervention with myostatin inhibitors [570][571][572], including antibody-based therapy [573]. The rebalancing of muscular atrophy versus hypertrophy by a growth-promoting process could modulate the aging process and have a positive effect on physical fitness and neuromuscular function [574].
Ideally, the above-described therapeutic approaches to improve general skeletal muscle strength would especially target the fast-twitching fiber population that is mostly susceptible to muscular atrophy in the elderly [50, 339,558]. Figure 6 provides a summary of current therapeutic options to treat sarcopenia of old age. For a critical assessment of current pharmacological strategies to halt or reverse age-related muscular atrophy, see the recent review articles by Cho et al. [15], Kim et al. [561] and Huang et al. [563]. and functions by inhibiting the phosphorylation of Akt protein kinase within the insulinlike growth factor 1-phosphatidylinositol-3-kinase-serine/threonine protein kinase PKBmammalian target of rapamycin (IGF-1/PI3K/Akt/mTOR) signaling pathway [569]. Consequently, the inhibition of a negative regulator might result in a positive effect on skeletal muscle growth. This can be supported by (i) natural mechanisms, such as physical exercise, (ii) dietary supplements and nutraceutical agents and/or (iii) pharmacological/biotechnological intervention with myostatin inhibitors [570][571][572], including antibody-based therapy [573]. The rebalancing of muscular atrophy versus hypertrophy by a growth-promoting process could modulate the aging process and have a positive effect on physical fitness and neuromuscular function [574]. Ideally, the above-described therapeutic approaches to improve general skeletal muscle strength would especially target the fast-twitching fiber population that is mostly susceptible to muscular atrophy in the elderly [50, 339,558]. Figure 6 provides a summary of current therapeutic options to treat sarcopenia of old age. For a critical assessment of current pharmacological strategies to halt or reverse age-related muscular atrophy, see the recent review articles by Cho et al. [15], Kim et al. [561] and Huang et al. [563]. Figure 6. Overview of therapeutic approaches to counteract age-related muscular atrophy.

Conclusions
The proteomic analysis of muscular atrophy in association with sarcopenia has detected distinct changes in a variety of protein families. Alterations in aging skeletal muscles include proteins involved in fiber contraction and relaxation, the regulation of excitation-contraction coupling, ion homeostasis, energy metabolism, maintenance of the cytoskeleton, the extracellular matrix and the cellular stress response. Skeletal muscle aging was shown to be linked to a tendency of fast-to-slow transitions and increased oxidative bioenergetics, as well as myofibrotic changes and a drastic increase in the expression of molecular chaperones. These proteomic findings support the concept of extensive degenerative and adaptive responses in the skeletal musculature due to sarcopenia of old age. Independently verified transcriptomic and proteomic markers of fiber-type shifting and metabolic modifications can now be used as indicators of molecular and cellular changes in both aging human skeletal muscles and animal models of sarcopenia. In the future, it will be of interest to study proteome-wide differences between age-related skeletal muscle wasting and other types of muscular atrophy caused by a variety of diverse triggering factors, such as denervation following motor nerve crush or spinal cord injury, prolonged bedrest in association with chronic disease, inappropriate levels of neuromuscular loading during plaster cast immobilization or prolonged exposure to microgravity. Since skeletal muscle performance deteriorates following extended periods of microgravity [3,575,576], which has been studied by proteomics [577], it has been suggested that certain Figure 6. Overview of therapeutic approaches to counteract age-related muscular atrophy.

Conclusions
The proteomic analysis of muscular atrophy in association with sarcopenia has detected distinct changes in a variety of protein families. Alterations in aging skeletal muscles include proteins involved in fiber contraction and relaxation, the regulation of excitationcontraction coupling, ion homeostasis, energy metabolism, maintenance of the cytoskeleton, the extracellular matrix and the cellular stress response. Skeletal muscle aging was shown to be linked to a tendency of fast-to-slow transitions and increased oxidative bioenergetics, as well as myofibrotic changes and a drastic increase in the expression of molecular chaperones. These proteomic findings support the concept of extensive degenerative and adaptive responses in the skeletal musculature due to sarcopenia of old age. Independently verified transcriptomic and proteomic markers of fiber-type shifting and metabolic modifications can now be used as indicators of molecular and cellular changes in both aging human skeletal muscles and animal models of sarcopenia. In the future, it will be of interest to study proteome-wide differences between age-related skeletal muscle wasting and other types of muscular atrophy caused by a variety of diverse triggering factors, such as denervation following motor nerve crush or spinal cord injury, prolonged bedrest in association with chronic disease, inappropriate levels of neuromuscular loading during plaster cast immobilization or prolonged exposure to microgravity. Since skeletal muscle performance deteriorates following extended periods of microgravity [3,575,576], which has been studied by proteomics [577], it has been suggested that certain aspects of neuromuscular alterations during prolonged spaceflights resemble changes in sarcopenia [578]. This opens new possibilities to study accelerated types of muscle-related stress and the molecular and cellular factors involved in muscular atrophy by the exposure of muscle cells to microgravity [579]. The detailed comparison of proteomic and systems bioinformatic data of different forms of muscular atrophy can be helpful to dissect the signaling mechanisms and disturbed biochemical, physiological and cellular processes that lead to diverse forms of muscle wasting.    Tandem mass tag  TNC-1  Troponin TnC, slow/cardiac  TNC-2  Troponin TnC, skeletal muscle  TNI-1  Troponin TnI, slow muscle  TNI-2  Troponin TnI, fast muscle  TNT-1  Troponin TnT, slow muscle  TNT-3  Troponin TnT, fast muscle  TOF  Time-of-flight  TPM-1  Alpha-1-tropomyosin  TPM-2  Beta-tropomyosin, slow muscle  TPM-3 Alpha-3-tropomyosin, muscle TPM-4 Alpha-4-tropomyosin, cytoskeletal TTN Titin USP3 Universal solid-phase protein preparation