Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 89
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To date, all identified FHC and NM mutations arewithin the myofilament proteins (both structural and contractile proteins; e.g.,a-actinin, nebulin, Tm, TnT, actin). They vary in severity (mild to lethal) with thetime of onset (neonatal, childhood, or adult) reflecting, in part, the isoform(s) affected (adult vs. fetal), site of mutation, and the nature of the amino acid change.Mutations in cardiac myofilament proteins cause cardiac hypertrophy and arethought to be involved in some cases of sudden death syndrome.
For example,some cases of FHC are attributed to different single amino acid mutations tocTnT (for review, see [38]) and/or cTnI [39–41] while five different known skeletalactin mutations can cause NM [42]. In skeletal muscle (for review, see [43]), rare(and perhaps under-represented) cases of NM occur as the result of mutations inskeletal isoforms that can cause facial, limb/locomotor, postural and respiratorymuscle weakness. Although many of these problems affect quality of life (e.g.,limb muscle weakness can limit activity and affect balance), they are generally notlife threatening. In contrast, respiratory muscle dysfunction can, by impairingventilation, predispose the individual to respiratory infections and, in severe cases,respiratory failure.
Remarkably, FHC and NM are both caused by a change in justone amino acid of almost any one of the myofilament proteins. That changingone amino acid is sufficient to cause profound changes in contractile performanceillustrates the importance of myofilament protein-protein interactions.19.2.2Myofilament Protein Isoform Alterations in DiseaseDisease states are often associated with changes in the expression of various myofilament protein isoforms. In COPD and HF patients, depending on the severityof the disease, changes in both limb and respiratory muscles occur. In limb mus-32132219 Myofilament Proteomicscles, there is a shift to type II (fast glycolytic) fibers while the diaphragm shifts towards type I (slow oxidative) fibers (see [44] for review).
However, a detailed analysis of alterations in expression of fiber type-specific proteins (e.g., co-expression ofmyosin isoforms in some cases of NM; [42]) or in myofilament PTM (e.g., phosphorylation, glycosylation) is still unavailable.In the failing myocardium, there is selective re-expression of the fetal isoformof cardiac TnT (cTnT) but not cTnI. Furthermore, the extent to which the fetal isoforms are re-expressed varies between species and types of HF. In addition, denovo isoform expression can occur. For example, during HF, the atrial isoform ofMLC-1 is abnormally expressed in the ventricles [45].
Remarkably, as little as 3%expression of the atrial isoform of MLC-1 in ventricle is sufficient to double in vitro force generation (when myosin light chain 2 (MLC-2) is completely dephosphorylated) (for review, see [46]).19.2.3Myofilament Protein Post-translational Modifications in DiseaseThere is an accumulating number of disease-induced PTMs of myofilament proteins that correlate with contractile dysfunction. For example, specific and progressive cTnI proteolysis occurs with ischemia/reperfusion injury in the isolated ratheart (see [47] for review).
Proteolysis of cTnI has been reported in the myocardium of patients undergoing bypass surgery [48] and in the serum from patientswith acute myocardial infarction (AMI) [49]. Interestingly, expression in mousemyocardium of the first proteolytic fragment observed with ischemia/reperfusioninjury of the isolated rat heart (cTnI1-192) is sufficient to recapitulate the contractile dysfunction observed during HF in animal models and humans [50]. Moreover, this dysfunction occurs with only 9–17% expression of cTnI1-192; greater levels may not have been achieved due to lethality.
In skeletal muscle, proteolysis ofsTnI and sTnT occurs only in diaphragm, not in accessory respiratory muscles orlimb muscles, in severely hypoxemic dogs [9]. In addition, proteolytic fragmentsof both fast and slow sTnI have also been reported in the serum of a patient withrhabdomyolysis [51]. Although the skeletal muscle pool (i.e., axial, limb, respiratory) from which the sTnI originated could not be identified, the proteolytic fragments observed in the serum likely originated from the diseased skeletal muscleproteome. Whether the observed protein alterations are the cause or the result ofthe disease remains to be investigated. Complete analysis of the myofilament proteome is critical for a full understanding of the contractile dysfunction associatedwith each disease state.19.3Proteomic Analysis of the Myofilament ProteinsProteomics is the study of the proteome (the protein complement of a cell) at agiven time.
Proteomics most often involves separating all the proteins (and their19.3 Proteomic Analysis of the Myofilament Proteinsalterations) for subsequent analysis (which “spots” are unique to disease andwhich unique to health), identification (e.g., myosin, Tm), and characterization(e.g., native-Tm and phosphorylated-Tm at serine 122). Two-dimensional gel electrophoresis (2-DE) is widely used for protein separation.
In 2-DE, proteins are separated in the first (horizontal) dimension by isoelectric focusing (IEF) which separates proteins based on their intrinsic charge, or pI. In the second (vertical) dimension, proteins are separated based on their relative molecular masses usingsodium dodecyl sulfate-polyacrylamide gel electrophoresis. After protein staining,the different proteins, as well as the various forms of the same protein (isoformsor PTMs), appear as discrete and, one hopes, unique spots, since they differ intheir pI and/or molecular mass. 2-DE is a powerful method for the separation ofproteins which, for striated muscle, enables us to resolve over 1,500 protein spotson a single gel (Fig.
19.1). However, alternative methods of protein separation, exploiting hydrophobicity or other intrinsic protein characteristics, are attractive asthey overcome limitations associated with 2-DE (e.g., very large molecular weightproteins, hydrophobic proteins, and proteins with extreme pIs). Mass spectrometry (peptide mass fingerprinting and amino acid sequencing), western blotting, Nterminal amino acid sequencing, or some combination, is used to determine theidentity of each protein spot and, when applicable, its modification.Proteomic analysis (separation and identification) of myofilament proteins withhigh molecular weights (e.g., myosin heavy chain), extreme pIs (e.g., TnI), andstrong protein-protein interactions (Tn complex) is especially challenging. In addition, it is necessary to detect and quantify all the myofilament protein alterations,even those present in modest quantities, which can occur in disease.
In the restof this chapter, we discuss the complications of myofilament proteins and theirproteomic analysis, and how technological advances are overcoming these limitations.19.3.1Creating a Myofilament ProteomeSubproteomic analysis simplifies the daunting task of attempting to simultaneously resolve thousands of cellular proteins. It is particularly important forstriated muscle because of the enormous difference in abundances between themyofilament proteins (high) and the vast majority of other cellular proteins (low)(Fig.
19.1). 2-DE gel separations of total muscle homogenates will reveal only avery limited part of the cytosolic and membrane proteome; the shear quantity ofthe few myofilament proteins makes it impossible to observe properly the rest ofthe cellular proteome. Fig. 19.1 b shows that using low protein loads (whole cell/tissue) allows one to resolve many of the myofilament proteins but the rest of theproteome is virtually undetectable. In other words, there is a range of abundanceof the different cellular proteins, with a pronounced disparity between the contractile proteins (high) and the other cellular proteins (low). Theoretically, higher protein loads would reveal the rest of the proteome but the quantity of protein required to visualize it would result in overloading of the myofilament proteins32332419 Myofilament ProteomicsFig. 19.1 2-DE silver-stained gels of cardiactissue with high (a) and low (b) protein loads(pH 3–10, 12% SDS page).
Note: higher protein loads permitted more proteins to be visu-alized although analysis was limited becausemany spots overlapped and/or were poorlyfocused. Expanded views of boxed sections ofgels shown on right.which will distort the focusing (Fig. 19.1 a) and cause overlap of the spots associated with many of the cellular proteins, thus preventing or limiting their detection and analysis.
Therefore, a major goal is to separate the abundant myofilament proteins from the rest of the proteome, while “preserving” the proteome;this should allow more efficient observation and analysis of the less abundant cellular proteins.Fractionation methods are often used to produce a discrete soluble protein fraction of biological interest; the myofilament proteins constitute, in some regards, aperfect subproteome to study. As previously discussed, they are the final effectorsof contraction and have a complex array of protein-protein interactions such thateven a (seemingly) modest change can drastically affect efficiency and/or forceproduction.
Fractionation or extraction exploits specific protein characteristicssuch as their inherent chemical properties (biospecificity, hydrophobicity, charge)or differential cellular compartmentalization. Currently, for myofilament proteo-19.3 Proteomic Analysis of the Myofilament ProteinsFig. 19.2 2-DE silver-stained gels of whole tissue (a) and cytoplasmic-enriched (b) andmyofilament-enriched (c) extracts from cardiac tissue using IN sequence. Note: high pro-tein loads of cytoplasmic proteins were possible without the high abundance myofilamentproteins altering focusing or obscuring thelower abundance proteins.Fig.
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