Abstract MP102: Skeletal Muscle Alpha Actin Acetylation Enhances Myosin Binding and Increases Calcium Sensitivity in vitro

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Samantha S Romanick ◽  
Allison Matsumura ◽  
Travis Stewart ◽  
Kianna Boc ◽  
Jonathan Baker ◽  
...  
Keyword(s):  
Calcium Sensitivity ◽  
Actin Binding ◽  
Cardiac Myosin ◽  
Sliding Velocity ◽  
Lysine Residues ◽  
Skeletal Myosin ◽  
Myosin Binding ◽  
Alpha Actin ◽  
Positively Charged

Actin and myosin are key proteins for muscle contraction/relaxation, while tropomyosin regulates actin-myosin interactions in the presence or absence of calcium. Mutations or dysregulation of any of these proteins results in cardiomyopathy. We previously showed that skeletal muscle alpha actin (ACTA1) was significantly deacetylated on lysine residues, K52, K317, and K328 in remodeled left ventricular tissue of obese mice. Computational modeling suggests that the positively charged lysine residues K328 and K317 of ACTA1 can interact electrostatically with the negatively charged glutamic acid residue E181 of tropomyosin and E286 of myosin. As acetylation is predicted to neutralize the positively charged lysine, ACTA1 acetylation would be postulated to decrease actin-myosin or actin-tropomyosin electrostatic interactions. To test this hypothesis, we used an in vitro actin motility assay to determine myosin sliding velocity, calcium sensitivity, and attachment/detachment kinetics of acetylated/deacetylated ACTA1. In addition, an actin binding protein spin-down assay was used to determine actin-myosin binding affinity using skeletal and cardiac myosin. In these assays, ACTA1 was chemically acetylated with acetic anhydride. In vitro actin motility analysis showed a significant decrease in sliding velocity with acetylated ACTA1 and skeletal myosin (1.709±0.210 μm/s) compared with deacetylated ACTA1 (4.427±0.275 μm/s). A similar significant decrease was also noted with cardiac myosin. Further analysis showed a significant increase in calcium sensitivity with ACTA1 acetylation (3.197x10-7 Kd compared to deacetylated ACTA1 1.191x10-6 Kd) and a loss of tropomyosin regulation with increasing ACTA1 acetylation. Lastly, ACTA1 acetylation enhanced actin binding affinity to cardiac and skeletal myosin. Investigation of attachment/detachment kinetics are currently underway. These data suggest that ACTA1 acetylation disrupts tropomyosin’s ability to inhibit myosin binding in the absence of calcium and further regulates actin-myosin interactions. Lastly, these data highlight acetylation as an additional post-translational modification outside of phosphorylation in the regulation of muscle contraction.

scholarly journals Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction

2018 ◽  
Vol 475 (24) ◽  
pp. 3933-3948 ◽  
Author(s):  
Sahar I. Da'as ◽  
Khalid Fakhro ◽  
Angelos Thanassoulas ◽  
Navaneethakrishnan Krishnamoorthy ◽  
Alaaeldin Saleh ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Binding Protein ◽  
Clinical Severity ◽  
Actin Binding ◽  
Cardiac Myosin ◽  
C2 Domains ◽  
Cardiac Phenotype ◽  
Myosin Binding Protein ◽  
Myosin Binding ◽  
The Impact

The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0–C2 protein fragments revealed that c-MYBPC3 variants alter the C0–C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0–C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations.

Sliding velocity of isolated rabbit cardiac myosin correlates with isozyme distribution

1992 ◽  
Vol 263 (2) ◽  
pp. H464-H472 ◽  
Author(s):  
H. Yamashita ◽  
S. Sugiura ◽  
T. Serizawa ◽  
T. Sugimoto ◽  
M. Iizuka ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Motility Assay ◽  
Cardiac Myosin ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Actin Cables

To investigate the relationship between the mechanical and biochemical properties of cardiac myosin, the sliding velocity of isolated cardiac myosin obtained from both euthyroid and hyperthyroid rabbits on actin cables was measured with an in vitro motility assay system. Ten rabbits (T) were treated with L-thyroxine to induce hyperthyroidism, and eight nontreated animals (N) were used as controls. Myosin was purified from the left ventricles of anesthetized animals. Myosin isozyme content was analyzed by the pyrophosphate gel electrophoresis method, and myosin adenosinetriphosphatase (ATPase) activity was determined on the same sample. Long well-organized actin cables of green algae, Nitellopsis, were used in the in vitro motility assay. Small latex beads were coated with purified cardiac myosin and introduced onto the Nitellopsis actin cables. Active unidirectional movement of the beads on the actin cables was observed under a photomicroscope, and the velocity was measured. The velocity was dependent on ATP concentrations, and the optimal pH for bead movement was approximately 7.0-7.5. The mean velocity was higher in T than in N (0.66 +/- 0.12 vs. 0.32 +/- 0.09 micron/s, P less than 0.01). Both Ca(2+)-activated ATPase activity and the percentage of alpha-myosin heavy chain were also higher in T than in N (0.691 +/- 0.072 vs. 0.335 +/- 0.072 microM Pi.mg-1.min-1, P less than 0.01, and 79 +/- 12 vs. 26 +/- 7%, P less than 0.01, respectively). The velocity of myosin closely correlated with both Ca(+2)-activated myosin ATPase activity (r = 0.87, P less than 0.01) and the percentage of alpha-myosin heavy chain (r = 0.87, P less than 0.01).

scholarly journals N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro

2016 ◽  
Vol 99 ◽  
pp. 47-56 ◽  
Author(s):  
Christoph Lipps ◽  
Jenine H. Nguyen ◽  
Lukas Pyttel ◽  
Thomas L. Lynch ◽  
Christoph Liebetrau ◽  
...  
Keyword(s):  
Protein C ◽  
Inflammatory Responses ◽  
Binding Protein ◽  
Cardiac Myosin ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Terminal Fragment ◽  
Myosin Binding

scholarly journals Single cardiac ventricular myosins are autonomous motors

Open Biology ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 170240 ◽  
Author(s):  
Yihua Wang ◽  
Chen-Ching Yuan ◽  
Katarzyna Kazmierczak ◽  
Danuta Szczesna-Cordary ◽  
Thomas P. Burghardt
Keyword(s):  
Actin Binding ◽  
Control Force ◽  
Cardiac Myosin ◽  
Step Size ◽  
Size Selection ◽  
Essential Light Chain ◽  
N Terminus ◽  
Increasing Load

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.

scholarly journals Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity.

1995 ◽  
Vol 128 (5) ◽  
pp. 837-848 ◽  
Author(s):  
C B Shuster ◽  
I M Herman
Keyword(s):  
Calcium Sensitivity ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Free Calcium ◽  
Cell Extracts ◽  
Beta Actin ◽  
Wide Range ◽  
Sepharose 4B

Whereas it has been demonstrated that muscle and nonmuscle isoactins are segregated into distinct cytoplasmic domains, the mechanism regulating subcellular sorting is unknown (Herman, 1993a). To reveal whether isoform-specific actin-binding proteins function to coordinate these events, cell extracts derived from motile (Em) versus stationary (Es) cytoplasm were selectively and sequentially fractionated over filamentous isoactin affinity columns prior to elution with a KCl step gradient. A polypeptide of interest, which binds specifically to beta-actin filament columns, but not to muscle actin columns has been conclusively identified as the ERM family member, ezrin. We studied ezrin-beta interactions in vitro by passing extracts (Em) over isoactin affinity matrices in the presence of Ca(2+)-containing versus Ca(2+)-free buffers, with or without cytochalasin D. Ezrin binds and can be released from beta-actin Sepharose-4B in the presence of Mg2+/EGTA and 100 mM NaCl (at 4 degrees C and room temperature), but not when affinity fractionation of Em is carried out in the presence of 0.2 mM CaCl2 or 2 microM cytochalasin D. N-acetyl-(leucyl)2-norleucinal and E64, two specific inhibitors of the calcium-activated protease, calpain I, protect ezrin binding to beta actin in the presence of calcium. Moreover, biochemical analysis of endothelial lysates reveals that a calpain I cleavage product of ezrin emerges when cell locomotion is stimulated in response to monolayer injury. Immunofluorescence analysis of leading lamellae reveals that anti-ezrin and anti-beta-actin IgGs can be simultaneously co-localized, extending the results of isoactin affinity fractionation of Em-derived extracts and suggesting that ezrin and beta-actin interact in vivo. To test the hypothesis that ezrin binds directly to beta-actin, we performed three sets of studies under a wide range of physiological conditions (pH 7.0-8.5) using purified pericyte ezrin and either alpha- or beta-actin. These included co-sedimentation, isoactin affinity fractionation, and co-immunoprecipitation. Results of these experiments reveal that purified ezrin does not directly bind to beta-actin filaments, either in solution or while isoactins are covalently cross-linked to Sepharose-4B. This is in contrast to our finding that ezrin and beta-actin could be co-immunoprecipitated or co-sedimented from Em-derived cell lysates. To explore whether calcium transients occur in cellular domains enriched in ezrin and beta-actin, we mapped cellular free calcium in endothelial monolayers crawling in response to injury.(ABSTRACT TRUNCATED AT 400 WORDS)

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