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Academic literature on the topic 'Sarcomeric protein stoichiometry'
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Journal articles on the topic "Sarcomeric protein stoichiometry"
1
Flashman, Emily, Hugh Watkins, and Charles Redwood. "Localization of the binding site of the C-terminal domain of cardiac myosin-binding protein-C on the myosin rod." Biochemical Journal 401, no. 1 (2006): 97–102. http://dx.doi.org/10.1042/bj20060500.
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cMyBP-C [cardiac (MyBP-C) myosin-binding protein-C)] is a sarcomeric protein involved both in thick filament structure and in the regulation of contractility. It is composed of eight IgI-like and three fibronectin-3-like domains (termed C0–C10). Mutations in the gene encoding cMyBP-C are a principal cause of HCM (hypertrophic cardiomyopathy). cMyBP-C binds to the LMM (light meromyosin) portion of the myosin rod via its C-terminal domain, C10. We investigated this interaction in detail to determine whether HCM mutations in β myosin heavy chain located within the LMM portion alter the binding of cMyBP-C, and to define the precise region of LMM that binds C10 to aid in developing models of the arrangement of MyBP-C on the thick filament. In co-sedimentation experiments recombinant C10 bound full-length LMM with a Kd of 3.52 μM and at a stoichiometry of 1.14 C10 per LMM. C10 was also shown to bind with similar affinity to LMM containing either the HCM mutations A1379T or S1776G, suggesting that these HCM mutations do not perturb C10 binding. Using a range of N-terminally truncated LMM fragments, the cMyBP-C-binding site on LMM was shown to lie between residues 1554 and 1581. Since it had been reported previously that acidic residues on myosin mediate the C10 interaction, three clusters of acidic amino acids (Glu1554/Glu1555, Glu1571/Glu1573 and Glu1578/Asp1580/Glu1581/Glu1582) were mutated in full-length LMM and the proteins tested for C10 binding. No effect of these mutations on C10 binding was however detected. We interpret our results with respect to the localization of the proposed trimeric collar on the thick filament.
2
Sussman, Mark A. "Analysis of Myofibrillar Organization and Degeneration by Fluorescence Confocal Microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 18–19. http://dx.doi.org/10.1017/s0424820100162557.
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Myofibrillar degeneration is an important pathological process in progressive cardiomyopathy leading to heart failure. Loss of myofibrils in vivo has been observed in both adaptive cardiac responses (i.e. hypertrophy) as well as in chemotherapeutic use of antitumor drugs with cardiotoxic side effects (i.e. doxorubicin). The molecular mechanism(s) of myofibrillar degeneration are poorly understood in comparison with the sequence of events involved in myofibrillar assembly and organization. Maintenance of myofibril integrity is dependent upon a variety of factors, including contractile protein stoichiometry and protein kinase activity.The repeating sarcomeric architecture of myofibrils is well suited to structural analysis, since disruption of normal organization is easily visualized by fluorescence microscopy. Many antibodies are available for use in observation of myofibril structure (see Table at right). Confocal microscopy provides advantages in studies of myofilament organization by allowing for direct and accurate measurement of distances to within 0.2 βm and the ability to perform vertical sectioning through individual cells. This vertical (Z-axis) sectioning can be used to select the focal plane within the cell for observation, resulting in higher resolution images by reducing fluorescent signals from above and below the plane. Image analysis software enables the user to create projections of optically sectioned cardiomyocytes which can be rotated to reveal interior structural relationships previously unobserved with single sections or conventional epifluorescence microscopy. Examples of image analysis will highlight useful features such as distance measurement, periodicity, pixel intensity, colocalization of dual labels, and three dimensional reconstruction.
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Warmke, J., M. Yamakawa, J. Molloy, S. Falkenthal, and D. Maughan. "Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila." Journal of Cell Biology 119, no. 6 (1992): 1523–39. http://dx.doi.org/10.1083/jcb.119.6.1523.
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We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.
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Agarwal, Radhika, Joao A. Paulo, Christopher N. Toepfer, et al. "Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation." Circulation Research 129, no. 7 (2021): 751–66. http://dx.doi.org/10.1161/circresaha.120.317076.
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Rationale: Dominant heterozygous variants in filamin C ( FLNC ) cause diverse cardiomyopathies, although the underlying molecular mechanisms remain poorly understood. Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (wild-type), ablated expression ( FLNC −/− ), or haploinsufficiency ( FLNC +/− ) that causes dilated cardiomyopathy. We also studied a heterozygous in-frame deletion ( FLNC +/Δ7aa ) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy. FLNC −/− hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. Although sarcomere formation and function were unaffected in FLNC +/ − and FLNC +/Δ7aa hiPSC-CMs, these heterozygous variants caused increases in lysosome content, enhancement of autophagic flux, and accumulation of FLNC-binding partners and Z-disc proteins. Conclusions: FLNC expression is required for sarcomere organization and physiological function. Variants that produce misfolded FLNC proteins cause the accumulation of FLNC and FLNC-binding partners which leads to increased lysosome expression and activation of autophagic pathways. Surprisingly, similar pathways were activated in FLNC haploinsufficient hiPSC-CMs, likely initiated by the loss of stoichiometric FLNC protein interactions and impaired turnover of proteins at the Z-disc. These results indicate that both FLNC haploinsufficient variants and variants that produce misfolded FLNC protein cause disease by similar proteotoxic mechanisms and indicate the therapeutic potential for augmenting protein degradative pathways to treat a wide range of FLNC -related cardiomyopathies.
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MARCO-FERRERES, Raquel, Juan J. ARREDONDO, Benito FRAILE, and Margarita CERVERA. "Overexpression of troponin T in Drosophila muscles causes a decrease in the levels of thin-filament proteins." Biochemical Journal 386, no. 1 (2005): 145–52. http://dx.doi.org/10.1042/bj20041240.
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Formation of the contractile apparatus in muscle cells requires co-ordinated activation of several genes and the proper assembly of their products. To investigate the role of TnT (troponin T) in the mechanisms that control and co-ordinate thin-filament formation, we generated transgenic Drosophila lines that overexpress TnT in their indirect flight muscles. All flies that overexpress TnT were unable to fly, and the loss of thin filaments themselves was coupled with ultrastructural perturbations of the sarcomere. In contrast, thick filaments remained largely unaffected. Biochemical analysis of these lines revealed that the increase in TnT levels could be detected only during the early stages of adult muscle formation and was followed by a profound decrease in the amount of this protein as well as that of other thin-filament proteins such as tropomyosin, troponin I and actin. The decrease in thin-filament proteins is not only due to degradation but also due to a decrease in their synthesis, since accumulation of their mRNA transcripts was also severely diminished. This decrease in expression levels of the distinct thin-filament components led us to postulate that any change in the amount of TnT transcripts might trigger the down-regulation of other co-regulated thin-filament components. Taken together, these results suggest the existence of a mechanism that tightly co-ordinates the expression of thin-filament genes and controls the correct stoichiometry of these proteins. We propose that the high levels of unassembled protein might act as a sensor in this process.
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Marín, María-Cruz, José-Rodrigo Rodríguez, and Alberto Ferrús. "Transcription of Drosophila Troponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements." Molecular Biology of the Cell 15, no. 3 (2004): 1185–96. http://dx.doi.org/10.1091/mbc.e03-09-0663.
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The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.
7
Li, Amy, Shane R. Nelson, Sheema Rahmanseresht, et al. "Skeletal MyBP-C isoforms tune the molecular contractility of divergent skeletal muscle systems." Proceedings of the National Academy of Sciences 116, no. 43 (2019): 21882–92. http://dx.doi.org/10.1073/pnas.1910549116.
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Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein, localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle type-specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague–Dawley rats? By mass spectrometry, 4 MyBP-C isoforms (1 fast-type MyBP-C and 3 N-terminally spliced slow-type MyBP-C) were expressed in EDL, but only the 3 slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that, only in the C-zone, MyBP-C Ca2+ sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-C’s N terminus as N-terminal proteolysis attenuated MyBP-C’s functional capacities. To determine each MyBP-C isoform’s contribution to thin filament Ca2+ sensitization and slowing in the C-zone, we used a combination of in vitro motility assays using expressed recombinant N-terminal fragments and in silico mechanistic modeling. Our results suggest that each skeletal MyBP-C isoform’s N terminus is functionally distinct and has modulatory capacities that depend on the muscle type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.
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Dumka, D., J. Talent, I. Akopova, G. Guzman, D. Szczesna-Cordary, and J. Borejdo. "E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics." American Journal of Physiology-Heart and Circulatory Physiology 291, no. 5 (2006): H2098—H2106. http://dx.doi.org/10.1152/ajpheart.00396.2006.
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Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca2+-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (τ1) of myosin heads from thin filaments, 2) rebinding time (τ2) of the cross bridges to actin, and 3) dissociation time (τ3) of ADP from the active site of myosin. τ1 was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. τ2 was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. τ3 was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. τ1 was statistically greater in Tg-m than in controls. τ2 was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. τ3 was the same in Tg-m and controls. To determine whether the difference in τ1 was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in τ1 was probably caused by myofibrillar disarray. The decrease in τ2 of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.
9
Helms, Adam S., Vi Tang, Jonathan Hernandez, et al. "Abstract 17079: MYBPC3 Truncation Mutations Cause Contractile Dysregulation in iPSC-Derived Cardiomyocytes." Circulation 138, Suppl_1 (2018). http://dx.doi.org/10.1161/circ.138.suppl_1.17079.
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The mechanism of MYBPC3 (encoding cardiac myosin binding protein C, MyBP-C) truncation mutations, the most common genetic cause of hypertrophic cardiomyopathy, has been incompletely resolved. We hypothesized that truncating MYBPC3 mutations cause myofibrillar protein assembly defects and/or contractile dysfunction. Methods and Results: Control iPSCs were CRISPR/Cas9-edited to create cell lines with homozygous (homCT) and heterozygous (hetCT) C-terminal MYBPC3 truncation mutations and heterozygous MYBPC3 promoter knock-out (hetPROM). HetCT was further edited to add an N-terminal flag tag (FhetCT). Purified cardiomyocytes were assayed at day ~25. RNAseq showed a 50% reduction in MYBPC3 mRNA in hetCT (p<0.01). Full ablation of MyBP-C was demonstrated in homCT but MyBP-C content was not reduced in hetCT or hetPROM by quantitative mass spectrometry. Immunofluorescence and co-IP of FhetCT showed no truncated MyBP-C. Sarcomere assembly, quantified by aligned myofilament number after replating onto 7:1 rectangular micropatterns, did not differ between lines; homCT (23±3), hetCT (23±3), hetPROM (22±3 myofilaments), control (22±3), p=NS. Quantitative mass spectrometry demonstrated that the stoichiometry of other major thick and thin filament proteins was not altered (all p=NS). Maximum force was reduced in homCT (866±366 uN, p<0.001) and hetCT (1213±545 uN, p=0.03) vs controls (1509±441 uN), and not significantly different for hetPROM (1238±360 uN, p=0.07), when measured in single micropatterned iPSC-CMs on 8.7 kPa hydrogels by traction force microscopy. Time to peak contraction was shorter in homCT (0.27 s, p=0.02) vs control (0.42 sec), but not in hetCT (0.46 s) and hetPROM (0.49 s). Contractile deceleration time was reduced only in homCT (0.12 s vs 0.25 s, p=0.002). Conclusions: Heterozygous MYBPC3 truncation mutations result in haploinsufficent mRNA with steady state compensation of MYBP3 protein and no MYBPC3 truncated peptide. Even complete ablation of MyBP-C loss does not alter the overall stoichiometry of other sarcomeric thick and thin filament proteins. MyBP-C ablation and heterozygous truncating mutations impair maximal contractile force, strongly implicating contractile dysregulation as the primary mechanism of MYBPC3-HCM.
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Zhang, Zhentao, Wenhui Zhang, and Young-Jae Nam. "Stoichiometric optimization of Gata4, Hand2, Mef2c, and Tbx5 expression for contractile cardiomyocyte reprogramming." Scientific Reports 9, no. 1 (2019). http://dx.doi.org/10.1038/s41598-019-51536-8.
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Abstract Reprogramming of fibroblasts to induced cardiomyocyte-like cells (iCMs) offers potential strategies for new cardiomyocyte generation. However, a major challenge of this approach remains its low efficiency for contractile iCMs. Here, we showed that controlled stoichiometric expression of Gata4 (G), Hand2 (H), Mef2c (M), and Tbx5 (T) significantly enhanced contractile cardiomyocyte reprogramming over previously defined stoichiometric expression of GMT or uncontrolled expression of GHMT. We generated quad-cistronic vectors expressing distinct relative protein levels of GHMT within the context of a previously defined splicing order of M-G-T with high Mef2c level. Transduction of the quad-cistronic vector with a splicing order of M-G-T-H (referred to as M-G-T-H) inducing relatively low Hand2 and high Mef2c protein levels not only increased sarcomeric protein induction, but also markedly promoted the development of contractile structures and functions in fibroblasts. The expressed Gata4 and Tbx5 protein levels by M-G-T-H transduction were relatively higher than those by transductions of other quad-cistronic vectors, but lower than those by previously defined M-G-T tri-cistronic vector transduction. Taken together, our results demonstrate the stoichiometric requirement of GHMT expression for structural and functional progresses of cardiomyocyte reprogramming and provide a new basic tool-set for future studies.