3.1. Acto-Myosin Apparatus and Auxilary Components
In the longitudinal direction, mature skeletal muscle fibres exhibit a characteristic striated appearance due to the regular patterning of anisotropic A-bands and isotropic I-bands, which form sarcomeric units between two neighbouring Z-disks [
81]. A major feature of muscle genetics is the fact that the isoform-specific expression of a limited number of genes that encode contractile components leads to the production of the majority of the protein content of contractile cells [
61]. Within the sarcomere structure, the contractile apparatus of skeletal muscle fibres consists of two main contractile filaments forming the acto-myosin system [
82,
83,
84] and two auxiliary filamentous systems acting as myofibrillar gap structures [
85,
86,
87,
88]. The complex network of the sarcomere units consists mainly of myosin light and heavy chains, actin and the regulatory tropomyosin/troponin complex, the giant proteins titin and nebulin, as well as the structural and functional support units presented by the M-band zone and the Z-disk complex [
89].
The principal motor molecules of the contractile protein assembly, which produce force via cross-bridge/swinging lever-arm mechanisms [
90], are filamentous actin and the myosin heavy chains [
91]. The entire myosin complex exists as a hexameric structure of two myosin heavy chains (MyHC) and four myosin light chains (MLC) [
92]. The MyHC head structure mediates the reversible coupling and cross-bridging process between the thick filaments located in the A-band region and the actin filaments [
93]. The regulatory and catalytic light chains provide critical phosphorylation sites, which are essential for the movement of phosphorylated myosin cross-bridges away from the thick filament, as well as the fine tuning of myosin motor function by providing structural stability to the lever arm domain of the myosin head [
94]. Below listed are the main components of the thick myosin-containing filament, the thin actin-containing filament, the auxiliary titin filament, the auxiliary nebulin filament, the sarcomeric M-band complex and the sarcomeric Z-disk complex [
66,
95]:
Thick myosin-containing filament: hexameric myosin complex consisting of myosin heavy chains (MyHC I, IIa, IId/x, and IIb), myosin light chains (fast and slow MLC isoforms; regulatory and catalytic subunits) and myosin binding proteins
Thin actin-containing filament: α-actin, α/β-tropomyosin, troponin complex (fast and slow troponin-I, troponin-T and troponin-C isoforms)
Auxiliary titin filament: half-sarcomere spanning titin and muscle ankyrin repeat protein
Auxiliary nebulin filament: nebulin, in close contact to actin-containing thin filament
Sarcomeric M-band complex: myomesin and obscurin, linked to titin filament
Sarcomeric Z-disk complex: α-actinin, plectin, telethonin, desmin, myozenin, myotilin, synemin and filamin
The systematic 2DGE separation of protein isoforms from skeletal muscle subtypes has led, in combination with MS analysis, to the identification of distinct isoforms of myosins, actins, troponins and tropomyosins, as well as many associated proteins of the sarcomeric structure [
10,
42]. Building on the findings from initial gel-based studies of the skeletal muscle proteome [
96,
97,
98], high-resolution 2DGE was applied to the cataloguing of human
vastus lateralis,
deltoideus and laryngeal muscles [
99,
100,
101,
102] and a variety of animal species including rat, rabbit, cow and fish [
103,
104,
105,
106,
107,
108,
109,
110]. The differential expression patterns of contractile proteins in predominantly fast- versus slow-twitching skeletal muscles was established for various muscle subtypes, such as
gastrocnemius,
extensor digitorium longus,
longissimus dorsi,
semitendinosus and
soleus muscles, using 2DGE [
111,
112,
113,
114,
115]. The gel-based fibre specification maps agree with the distribution of fast versus slow muscle protein isoforms as determined by
in vivo stable isotope labelling with amino acids in the SILAC mouse model [
116].
Figure 3 shows representative 1DGE and 2DGE images of separated muscle protein populations. These approaches are routinely used for the proteomic identification of distinct proteoforms of contractile components.
3.2. Top-Down Proteomics of Myosins
Muscle fibre type classification can be conveniently carried out by determining the distribution of myosin light and heavy chain isoforms, making this large family of contractile proteins an essential class of fibre type markers [
92]. The expression pattern of MyHC molecules is often performed by immunofluorescence microscopy of transverse cryosections from individual skeletal muscles [
117]. Most adult skeletal muscles contain a mixture of the main fibre types I, IIa, IIx, IIb, as well as the mixed hybrid fibre types I/IIa, IIa/IIx and IIx/IIb. However, considerable species-specific differences exist in relation to the distribution of cellular subtypes within large fibre populations [
118]. Although at least 11 MyHC isoforms and nine MLC isoforms appear to exist in developing and matured skeletal muscles [
119,
120], the spectrum of slow-twitching oxidative fibres towards faster-twitching and more glycolytic fibres can be accurately evaluated by the abundance of the main human slow/cardiac MyHC-I-beta (P12883;
MYH7 gene), fast MyHC-2a (Q9UKX2;
MYH2 gene), fast MyHC-2x (P12882;
MYH1 gene) and fast MyHC-2b (Q9Y623;
MYH4 gene) isoforms [
117,
118]. In addition, developing and regenerating muscles contain MyHC-neo (P13535;
MYH8 gene) and MyHC-emb (P11055;
MYH3 gene), and the highly specialist extraocular muscle fibres are characterized by the presence of MyHC-EO (Q9UKX3;
MYH13 gene), MyHC-I-ton (A7E2Y1;
MYH7b gene) and MyHC-15 (Q9Y2K3;
MYH15 gene) [
92]. Human MLC molecules exist mainly as essential catalytic subunits and regulatory/phosphorylatable subunits, i.e., as fast MLC1/MLC3 (P05976;
MYL1 gene) and fast MLC2 (Q96A32;
MYLPF gene) or slow MLC1 (MLC3) (P08590;
MYL3 gene) and slow MLC2 (P10916;
MYL2 gene) isoforms. The combination of various MyHC and MLC subunits form a very large number of hexameric iso-myosins in the sarcomere [
92,
121,
122]. In contrast, human filamentous F-actin polymers in developing and mature fibres consist of only two G-actin isoforms, i.e., cardiac alpha-actin (P68032;
ACTC1 gene) and muscle alpha-actin (P68133;
ACTA1 gene). An overview of the systematic characterization of contractile proteins from skeletal muscle using top-down proteomics is provided in
Figure 4.
The detailed proteomic analysis of fast versus slow skeletal muscles has confirmed the complexity of myosin expression patterns. Due to their differing molecular mass and electrophoretic mobility, heavy chains and light chains of myosin complexes are routinely studied by 1DGE versus 2DGE, respectively. The 2DGE top-down proteomic analysis of rat
gastrocnemius versus
soleus muscle extracts has clearly shown the differential expression pattern of myosin light chains MLC1s and MLC2s in slow muscle versus MLC1f, MLC2f and MLC3f in fast muscle [
114]. Thus, myosin subunits represent reliable proteomic markers of fibre type specification and the expression of the main fast versus slow isoforms correlates well with major histological, physiological and biochemical parameters, such as tissue colour, fibre diameter, contraction time, power output, endurance type, aerobic versus anaerobic activities, degree of resistance to fatigue and capillary density, as well as glycolytic versus oxidative metabolism. During fibre type transitions [
123], triggered by graded exercise or chronic neuromuscular stimulation, myosin subunits show characteristic shifts in density and isoform expression patterns [
36,
124,
125]. Detailed adaptations of the acto-myosin apparatus have been examined in a large number of gel-based proteomic studies, which have employed different types of physical activity (interval training, endurance exercise, vibration exercise during long-term bed rest, repeated eccentric exercise, downhill running, bouts of exhaustive exercise) in skeletal muscles from human, pig, rat and mouse [
39,
126,
127,
128,
129,
130,
131,
132,
133,
134,
135,
136,
137,
138,
139,
140], as well as chronic low-frequency electro-stimulated rabbit muscle [
141,
142].
Fast-to-slow transitions are clearly associated with the up-regulation of the slow/cardiac isoform MyHC-I and MLC1s, and a concomitant decrease in MLC1f, MLC2f and MLC3f [
141] showing the usefulness of myosin changes for determining molecular adaptations during skeletal muscle transformation [
11,
36]. The specific effects of myostatin-related hypertrophy [
143,
144], hypoxia-induced skeletal muscle adaptations [
145,
146,
147,
148] and disuse-associated muscular atrophy (due to immobilization, denervation or neuromuscular unloading) [
149,
150,
151,
152,
153,
154,
155,
156,
157,
158] could also confirm distinct shifts in sarcomeric proteins due to fibre type transitions. The 2DGE top-down proteomic analysis of skeletal muscle development [
159,
160,
161,
162,
163] versus muscle aging [
164,
165,
166,
167,
168,
169,
170,
171,
172,
173] has revealed interesting alterations in key proteins of the contractile apparatus [
174,
175]. For example, the gel-based analysis of fast-to-slow fibre transitions during muscle aging demonstrated a drastic increase of slow myosin light chain MLC2 in senescent skeletal muscle tissue [
170], establishing this myosin subunit as an excellent marker of age-related fibre type shifting [
176]. PTMs, such as altered phosphorylation levels of MLCs, appear to play a crucial role during skeletal muscle adaptations and fibre aging [
177,
178].
A key 2DGE technique is represented by fluorescence difference in-gel electrophoresis (DIGE) [
179,
180,
181].
Figure 5 compares minimal protein labelling on lysine residues versus saturation protein labelling on cysteine residues prior to gel electrophoretic separation, and also illustrates the different CyDye labeling protocols used in fluorescent two-dye versus three-dye systems [
182,
183,
184].
The 2D-DIGE method was originally developed by Minden and co-workers [
185,
186,
187] and has been frequently modified as an efficient protein biochemical tool for studying large-scale changes in protein expression and interaction patterns [
182,
188,
189,
190]. The application of the 2D-DIGE technique for the comparative analysis of the skeletal muscle proteome from human and animal specimens has been described in detail in various methods papers [
191,
192,
193,
194,
195]. For the optimum pre-electrophoretic DIGE labelling of protein fractions from tissue extracts, 50 μg protein aliquots can be differentially labelled with Cy2, Cy3 or Cy5 dyes and then separated on the same 2D gel, which eliminates gel-to-gel variations [
181]. The development of sophisticated DIGE analysis software programs and improved protein identification approaches has greatly increased the quality of quantitative assessments of multiple proteomes on the same 2D gel [
196,
197,
198].
An illustrative example of analysing changes in the skeletal muscle proteome by 2D-DIGE methodology is presented in
Figure 6. Shown are 2D gel images from an optimization study of human
gastrocnemius muscle. The figure clearly demonstrates the differing expression levels of distinct 2D protein spots in the gel electrophoretically separated Cy3-labelled controls (Sample A) versus physiologically challenged and Cy5-labelled specimens (Sample B). The protein clusters in the marked 2D-DIGE Zones 1 to 3 were densitometrically analysed and show unaltered proteoforms (Zone 1 and 2) versus drastically changed expression levels of distinct proteoforms (Zone 3).
3.3. Top-Down Proteomics of Troponins and Tropomyosins
As already described above for isoforms of myosins, the application of 2DGE top-down proteomics has also established regulatory proteins of the actin-containing thin filament as robust markers of fibre type shifting. Fast versus slow fibre specification in
gastrocnemius and
soleus muscle is clearly reflected by altered expression levels of the fast versus the slow isoform of tropomyosin-1-alpha [
114]. During chronic electro-stimulation induced fast-to-slow muscle transitions, an increase in slow troponin TnT and TnI and concomitant decrease in fast troponin TnI were established by fluorescence 2D-DIGE analysis [
142]. Troponins and tropomyosins are critical factors of the calcium-associated regulation of the excitation-contraction-relaxation cycle [
199,
200]. Tropomyosin interacts directly with actin filaments in an inhibitory role and is linked to troponin TnT, while inhibitory troponin TnI is regulated by the calcium-sensing TnC subunit [
201].
In mature skeletal muscles, tropomyosin (TM) exists as slow and fast isoform combinations and troponin (TN) can be categorised as fast TnCf, slow TnCs, fast TnT1f to TnT4f, slow TnT1s, slow TnT2s, fast TnIf and slow TnIs [
200]. These individual subunits relate in human muscle to slow TnT (P13805;
TNNT1 gene), fast TnT (P45378;
TNNT3 gene), slow TnI (P19237;
TNNI1 gene), fast TnI (P48788;
TNNI2 gene), slow TnC (P63316;
TNNC1 gene), fast TnC (P02585;
TNNC2 gene), TM-alpha-1 (P09493;
TPM1 gene), TM-alpha-3 (P06753;
TPM3 gene) and TM-alpha-4 (P67936;
TPM4 gene). The recent top-down proteomic analysis of tropomyosins in rat, pig and human muscle has established that tropomyosin isoforms Tpm1.1 and Tpm2.2 are the two major tropomyosin isoforms in swine and rat skeletal fibres [
202]. In contrast, isoforms Tpm1.1, Tpm2.2, and Tpm3.12 were identified in human muscles. Since the age-related loss of skeletal muscle mass and contractile strength [
203] plays an essential role in the frailty syndrome [
204,
205,
206], top-down proteomics is instrumental for studying skeletal muscle aging. Gel-based proteomic surveys of senescent human skeletal muscles and established animal models of sarcopenia strongly suggest that a gradual shift to more oxidative metabolism and a slower mode of fibre contraction occurs during aging [
176]. Besides myosin heavy and light chains, the regulatory elements tropomyosin and troponin also showed distinct fast-to-slow changes in proteoform expression levels [
164,
165,
166,
168,
207]. Decreases in fast TnT and tropomyosin TM-alpha agree with the idea that age-associated abnormalities in the peripheral nervous system trigger denervation, faulty patterns of re-innervation and excitation-contraction-uncoupling, which indirectly affects the skeletal muscle phenotype and causes a gradual transformation to a slower-contracting muscle [
176]. The phosphorylation status of the sarcomeric Z-disk component cypher, as well as PTMs in other sarcomeric proteins, was determined to be altered in aged muscle by quantitative top-down proteomics [
208].
3.4. Analysis of Post-Translational Modifications Using Two-Dimensional Gel Electrophoresis
There are many PTMs including phosphorylation, glycosylation, acetylation and ubiquitination that are involved in a large variety of processes such as signal transduction pathways, protein trafficking, enzymatic activity or cell motility to name but a few [
209]. Determining the precise role of PTMs in physiology and pathophysiology can decisively increase our biochemical understanding and help to take the correct approach when trying to modulate these modifications with respect to therapeutic actions. 2DGE is a highly effective top-down approach to resolve proteoforms, including post-translationally modified proteins. Thousands of proteins can be identified using optimized 2DGE when combined with advanced MS, and potentially many PTMs can be detected. Specific stains have been developed for phosphoproteins (Pro-Q Diamond) and glycoproteins (Pro-Q Emerald) to study specific PTMs using fluorescence 2DGE [
210,
211,
212,
213,
214]. By using a combination of background-adjusted Pro-Q Diamond staining/total protein ratio values, differential phosphorylation for specific proteins can be detected. Phosphorylated proteins of interest can subsequently be digested, phospho-peptides enriched using titanium dioxide (TiO
2) chromatography and specific phosphorylation sites identified by MS analysis. The advantages of separating protein by 2DGE and then identifying augmented phosphorylation signals means that researchers can focus on individual proteins and characterise their PTM status specifically, as opposed to high-throughput LC-MS/MS analysis of enriched peptides that represents a more global approach.
Reversible protein phosphorylation, predominantly involving serine, threonine or tyrosine residues, is one of the most important and intensely investigated PTMs [
215]. As an established model system of sarcopenia of old age, 30-month old rat
gastrocnemius muscle fibres were evaluated using phospho-specific Pro-Q Diamond staining of 2D gels [
216]. Increased phosphorylation levels were shown for various contractile proteins including slow myosin light chain MLC2, tropomyosin alpha and actin, providing evidence that age-related muscle wasting has a complex pathology that is associated with significant changes in the abundance of phosphorylated proteins [
177,
178,
208]. The importance of contractile protein phosphorylation was confirmed by a study on myosin binding protein MyBP-C. Verduyn and co-workers [
217] induced differences in the degree of phosphorylation of the myofilaments to explore the role of phosphorylation of the cMyBP-C isoform in isolated rat ventricular myocytes under different physiological conditions, namely phosphorylation of myosin binding protein in quiescent hearts perfused with lidocaine at a relatively low calcium concentration, higher levels of phosphorylation obtained by β-adrenergic stimulation, enhancement of calmodulin-dependent protein kinase CaMK II activity by an increase in the time averaged intracellular calcium concentration, and/or by blockade of phosphatase activity. Using 2DGE and ProQ Diamond staining procedures, the endogenous phosphorylation levels of contractile proteins was determined for troponin cTnT, myosin light chain MLC1 and myosin light chain MLC2. Phosphorylation levels of the contractile proteins varied considerably with the experimental conditions used, with an approximate 2-fold increase in MLC2 phosphorylation in perfused contractile tissues stimulated at 5 Hz. Addition of the beta-agonist isoprenaline to the stimulated hearts resulted in 3.7-fold increases in both cMyBP-C and troponin cTnI phosphorylation [
217].
Protein acetylation is involved in the regulation of protein stability and function during various cellular and physiological processes [
218,
219], including skeletal muscle atrophy [
220] and age-related fibre wasting [
221]. Acetylation of lysine residues on both histones and non-histone proteins is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases, controlling a range of different diverse biological functions including protein-protein interactions, protein-DNA interactions, enzymatic activity and subcellular localization. High levels of dihydrolipoyllysine-residue acetyltransferase were detected in the nucleus in aged rat
triceps muscle by fluorescence 2D-DIGE analysis and shown to be associated with increased protein acetylation [
221]. Acetylation of lysine residues removes the positive charge of the side chain and therefore directly impacts the electrostatic status of the modified protein, allowing for separation associated with isoelectric focusing. Protein p
I-shifts due to PTMs revealed that the theoretical p
I-value of the non-acetylated form of tropomyosin-beta is close to the p
I-value of its experimentally acetylated counterpart, while additional acetylation of tropomyosin (LEKTIDDLEETLASAK + acetyl (K); acetyl (N-term)) does not significantly affect the p
I-value [
222]. Interestingly, a variety of contractile proteins are changed in chronic Chagas disease, an often fatal outcome of
Trypanosoma cruzi infection, which is characterized by severe cardiomyopathy and chronic skeletal muscle myositis and vasculitis [
223,
224]. Protein changes were evaluated using 2DGE of specimens from end-stage chronic Chagas disease patients to gain insight into its pathophysiology [
225]. Several gel spots with differing p
I-values and comparable molecular mass were identified by peptide mass fingerprinting as proteoforms of key structural and contractile proteins, including several forms of actin (ACTA1, ACTA2, ACTC, ACTG2, ACTN2), desmin, myosin (MYL3 and MYL7) and vimentin. The variations in their position within 2D slab gels could be a result of PTMs, such as acetylation or other processes.
Glycosylation is one of the most complex PTMs and is involved in many biological mechanisms including cell differentiation, intracellular signalling and protein trafficking [
226]. Cieniewski-Bernard and co-workers [
227] demonstrated the utility of 2DGE for the analysis of O-linked N-acetylglucosaminylation (O-GlcNAc) in skeletal muscle proteins, focusing on rat
gastrocnemius muscle that is composed of both fast- and slow-contracting fibres. O-GlcNAc is an abundant and reversible type of glycosylation and especially found within the cytosolic and the nuclear compartments. The total level of O-GlcNAc proteins in rat after hind-limb unloading, a model of skeletal muscle atrophy, were also evaluated. The O-GlcNAc modification of myosin was found to be significant and verified using immunoblot analysis with an anti-O-GlcNAc antibody. The authors suggested that O-GlcNAc could be involved in the regulation of the polymerization of myosin in the thick filament assembly. The variation in the O-GlcNAc level measured after skeletal muscle unloading suggests a role for dynamic glycosylation patterns in muscle plasticity [
227].
Ubiquitination is a key type of reversible PTM in proteins and plays a significant role in the regulation of many biological processes, including protein degradation and signal transduction [
228]. Ubiquitin, a highly conserved 76-amino acid protein, can be added to substrate protein as a protein tag by the sequential actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). Using fractions of
gastrocnemius muscles isolated from Trim32 knockout mice (T32KO) compared to wild type, 36 proteins with altered abundance in T32KO muscle were identified using a combination of fluorescence 2D-DIGE and LC-MS/MS analysis [
229]. Mutations in the
TRIM32 gene, which encodes an E3 ubiquitin ligase containing the tripartite motif, have been implicated in the pathogenesis of limb girdle muscular dystrophies. Of the significant proteins identified using this model system, the fast muscle isoform of troponin TnT was shown to decrease in abundance in T32KO muscle, a result that supports selective type II (fast) fibre atrophy in T32KO mice.