Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 88
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The role(s) of each of the various protein alterations associated with disease, either transcriptional (expression levels and/orisoform changes) or post-translational modification(s) (PTM), may contribute to orcompensate for contractile dysfunction. Knowing if a modification is compensatory or detrimental (pathological) is crucial for the development of therapeuticstrategies which may either prevent or delay the onset of disease.19.1Regulation of Muscle ContractionMuscle contraction is the result of the complex interactions of many myofilamentproteins. It is, ultimately, the interplay between the thick and thin filaments, in aCa-dependant manner, which produces force at the expense of ATP hydrolysis (for19.1 Regulation of Muscle Contractionreviews, see [10–13]). Even though many agents can affect contraction (e.g., catecholamines, angiotensin II), the interaction between the thick and thin filamentsis tightly regulated. The thick filament is composed primarily of myosin, including the heavy chain and two associated light chains, which together form themechanoeynzyme.
The thin filament is comprised of filamentous actin (a polymerized filament composed of monomeric actin), tropomyosin (Tm, a dimer of the aisoform or a heterodimer composed of the a and b isoforms), and the troponincomplex (Tn). Tn consists of troponin I (TnI), termed the inhibitory protein due toits ability to block actin-myosin interaction, troponin T (TnT), named for its extensive binding to Tm, and troponin C (TnC), which binds calcium and triggers contraction (for reviews, see [14–19]). In addition, such structural proteins as titin,desmin and a-actinin contribute to the spatial orientation of the thick and thin filaments and, possibly, the transmission of force. Changes in quantity, isoform expression, or the status of PTM of one or more of the myofilament proteins candramatically influence protein-protein interactions, thus affecting force productionand/or contractile economy.The myofilament proteins are also called the contractile proteins; they make upthe myofibrils.
They produce force as a result of the formation of actin-myosin crossbridges between the thick and thin filaments. In striated (cardiac and skeletal) muscle, contraction occurs in response to increasing intracellular concentrations of Ca2+.Tn works in concert with Tm to regulate Ca-dependent muscle contraction which isinitiated by the binding of Ca2+ to TnC. TnC is an EF-hand Ca2+ binding protein withtwo domains (N- and C-terminals).
When intracellular Ca2+ increases, Ca2+ binds tothe regulatory site(s) within the N-terminus of TnC (cardiac TnC has one regulatoryCa2+ binding site; skeletal TnC has two). This alters the conformation of TnC (andhence the whole Tn complex), which induces the inhibitory region of TnI (the twelveamino acid sequence comprising residues 136–147 in human cTnI and 104–115 inhuman sTnI) to “switch” from its specific binding site on actin-Tm to a site on TnC.There are extensive interactions between the various troponin subunits, some withextremely high affinities, as well as between Tn and the other members of the thinfilament, contributing to the complexity of this system.The Ca-dependent change in the Tn-Tm complex results in its movement acrossthe actin filament, increasing the probability of its interaction with myosin. Tm isa long coiled-coil dimer extending along the actin filament covering every sevenactin monomers.
There is one Tn complex per Tm (dimer) and their control of actin-myosin interactions involves the intricate interplay of both steric and allostericmechanisms. The exact structural changes involved in signaling, and changes inthe interaction between the myofilament proteins involved in regulation and muscle contraction, are complicated and still being elucidated. Clearly, the striatedcontractile system constitutes a finely-tuned array of proteins that regulates forceproduction in response to changes in intracellular Ca2+ concentration.The differences in functional and mechanical properties of cardiac and skeletalmuscle are due, in part, to the different myofilament protein isoforms expressed.The isoforms have different amino acid sequences which affect their interactionswith other proteins in the myofilament. For instance, cardiac muscle contains only31932019 Myofilament Proteomicsaa-Tm while skeletal muscle contains varying ratios of aa-Tm and ab-Tm dependingon the fiber type.
Lehman and colleagues [20] observed that the different Tm isoforms adopt different positions on the actin filament, indicating that the interactionbetween the various Tm isoforms and actin is distinctive despite the extensive aminoacid sequence homology between these two isoforms. TnT and TnI have three isoforms (slow skeletal, fast skeletal, and cardiac) which are expressed in their corresponding fiber types, the single exception being the expression of the slow and cardiac isoforms of TnI in the embryonic heart until just after birth when the cardiacisoform predominates [21, 22]. The situation associated with TnT is more complex; it has many RNA splice sites with the potential to generate a remarkable diversity of isoforms.
For example, there are at least six mammalian and up to thirteenavian exons that can be alternatively spliced for fast skeletal TnT [23–26]. Furthermore, these TnT isoforms change during development and disease (for reviews,see [27–30]). TnC has two isoforms arising from two separate genes [31]. The cardiacand slow skeletal isoforms share one gene while fast skeletal has the other.
Thus,various combinations of isoforms of these different Tn subunits will influence themolecular regulation of contraction in the different striated muscle types.Several PTMs are specific to cardiac myofilament proteins. Cardiac muscle ismodulated by catecholamines through the activation of protein kinase A (PKA)(for review, see [32]). PKA phosphorylates several proteins, including cTnI at aminoacid residues serine 22 and serine 23. These two amino acid residues are located inthe unique 30 amino acid N-terminal extension of cTnI and are, therefore, not present in either the fast or slow skeletal isoforms.
There is an interplay between PKAmediated phosphorylation of cTnI and other functional regions of the molecule, asdemonstrated by the changes in the extent of cTnI phosphorylation in the mutationcTnIR145G (a cardiomyopathy mutant) as compared to wild type cTnI [33]. Also,we recently identified two novel phosphorylation sites of cardiac myosin lightchain 1 (MLC-1, a protein widely considered to be unphosphorylatable) while undertaking proteomic analysis of the myofilament proteins [34]. Not only was abasal level of MLC-1 phosphorylation observed (sham), the extent of phosphorylation was modulated during pharmacological preconditioning of rabbit cardiomyocytes.
We have also observed novel PTM of skeletal myofilament proteins,including the in vivo phosphorylation of fast skeletal TnI (unpublished data).This illustrates the potential of proteomic analysis to uncover novel aspects ineven a well-studied system like the myofilament proteins. One can only speculate on the number of changes that will be uncovered in various disease states.19.2Disease States and the Myofilament ProteinsA disease state (encompassing all types of injury), whether acute or chronic, ischaracterized by stress-induced (e.g., hypoxia, infection, toxins, drugs, hypertension, ischemia) dysfunction of the whole muscle system. This functional deficitcan be global (e.g., type II skeletal muscle fibers in different locomotor muscles)19.2 Disease States and the Myofilament Proteinsor restricted (e.g., all muscle fibers but in a single muscle).
Common diseasestates include HF, fatigue, myocardial infarction, myocarditis, COPD, sepsis, andinherited myopathies (e.g., skeletal muscle nemaline myopathy (NM) and cardiacmuscle hypertrophic myopathy). For each disease state, there is a specific underlying molecular cause related to a proteomic change responsible for the dysfunction. As stated earlier, many studies indicate that myofilament protein alterations(e.g., mutations, isoform switching, re-expression of fetal isoforms, and PTMs) areassociated with various disease states and most of these alterations are associatedwith contractile dysfunction (for reviews, see [35–37]). The next section deals withalterations specific to cardiac and skeletal muscle.19.2.1Myofilament Protein Mutations Cause DiseaseInherited muscle disease represents a spectrum of pathological conditions arisingfrom many different etiologies with unique as well as overlapping characteristics.Familial hypertrophic cardiomyopathy (FHC) and NM are inherited disorders resulting from mutations in genes encoding specific cardiac and skeletal myofilament proteins, respectively.
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