Do cardiac actin mutations lead to altered actomyosin interactions?

2015 ◽  
Vol 93 (4) ◽  
pp. 330-334 ◽  
Author(s):  
Marissa Dahari ◽  
John F. Dawson
Keyword(s):  
Molecular Mechanisms ◽  
Duty Ratio ◽  
Actin Gene ◽  
Step Size ◽  
Mutant Proteins ◽  
In Vitro Motility ◽  
The Core ◽  
Cardiac Actin ◽  
Myosin S1

It is currently hypothesized that increased heart muscle contractility leads to hypertrophic cardiomyopathy (HCM), and reduced contractility leads to dilated cardiomyopathy (DCM). To determine if changes in the core interaction between actin and myosin occur due to mutations in the cardiac actin gene (ACTC), we measured the interactions between myosin and 8 ACTC mutant proteins found in patients with HCM or DCM. R312H showed a decreased actin-activated myosin S1 ATPase rate (13.1 ± 0.63 μmol/L/min) compared to WT (15.3 ± 1.6 μmol/L/min), whereas the rate with E99K was significantly higher (20.1 ± 1.5 μmol/L/min). In vitro motility assays with varying ATP concentrations showed that the KM for E99K remains unchanged with a significantly decreased Vmax (1.90 ± 0.37 μm/sec) compared to WT (3.33 ± 0.46 μm/sec). Based on a 5 nm myosin step size, we calculated a duty ratio of approximately 0.04 for WT and the majority of mutant actins; however, the duty ratio for E99K was twice as high. Based on our analysis of 8 ACTC mutants, we infer that mutations in ACTC lead to disease through various molecular mechanisms. While changes in actomyosin interactions with the E99K mutation might cause increased ATP usage and tension leading to HCM, measurable changes in the basic interaction between actin and myosin do not appear to be involved in the mechanisms of disease development for the other ACTC mutants tested.

scholarly journals Cardiac actin changes in the actomyosin interface have different effects on myosin duty ratio

2018 ◽  
Vol 96 (1) ◽  
pp. 26-31 ◽  
Author(s):  
Haidun Liu ◽  
Mary Henein ◽  
Maria Anillo ◽  
John F. Dawson
Keyword(s):  
Cardiovascular Disease ◽  
Hypertrophic Cardiomyopathy ◽  
Left Ventricle ◽  
Duty Ratio ◽  
Actomyosin Atpase ◽  
In Vitro Motility ◽  
Cardiac Actin ◽  
Myosin Binding ◽  
Cardiac Sarcomeres

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disease (CD) that commonly causes an increased size of cardiomyocytes in the left ventricle. The proteins myosin and actin interact in the myocardium to produce contraction through the actomyosin ATPase cycle. The duty ratio (r) of myosin is the proportion of the actomyosin ATPase cycle that myosin is bound to actin and does work. A common hypothesis is that HCM mutations increase contraction in cardiac sarcomeres; however, the available data are not clear on this connection. Based on previous work with human α-cardiac actin (ACTC), we hypothesize that HCM-linked ACTC variants with alterations near the myosin binding site have an increased r, producing more force. Myosin duty ratios using human ACTC variant proteins were calculated with myosin ATPase activity and in-vitro motility data. We found no consistent changes in the duty ratio of the ACTC variants, suggesting that other factors are involved in the development of HCM when ACTC variants are present.

scholarly journals Facilitated Cross-Bridge Interactions with Thin Filaments by Familial Hypertrophic Cardiomyopathy Mutations inα-Tropomyosin

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Fang Wang ◽  
Nicolas M. Brunet ◽  
Justin R. Grubich ◽  
Ewa A. Bienkiewicz ◽  
Thomas M. Asbury ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Molecular Mechanisms ◽  
Helical Structure ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Maximum Speed ◽  
Sliding Speed ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Cardiac Sarcomeres

Familial hypertrophic cardiomyopathy (FHC) is a disease of cardiac sarcomeres. To identify molecular mechanisms underlying FHC pathology, functional and structural differences in three FHC-related mutations in recombinantα-Tm (V95A, D175N, and E180G) were characterized using both conventional and modified in vitro motility assays and circular dichroism spectroscopy. Mutant Tm's exhibited reducedα-helical structure and increased unordered structure. When thin filaments were fully occupied by regulatory proteins, little or no motion was detected at pCa 9, and maximum speed (pCa 5) was similar for all tropomyosins. Ca2+-responsiveness of filament sliding speed was increased either by increasedpCa50(V95A), reduced cooperativityn(D175N), or both (E180G). When temperature was increased, thin filaments with E180G exhibited dysregulation at temperatures ~10°C lower, and much closer to body temperature, than WT. When HMM density was reduced, thin filaments with D175N required fewer motors to initiate sliding or achieve maximum sliding speed.

scholarly journals Cardiac and Skeletal Actin Substrates Uniquely Tune Cardiac Myosin Strain-Dependent Mechanics

2018 ◽  
Author(s):  
Yihua Wang ◽  
Katalin Ajtai ◽  
Thomas P. Burghardt
Keyword(s):  
Resistive Load ◽  
Step Size ◽  
Actin Isoforms ◽  
Cardiac Actin ◽  
N Terminus ◽  
Human Adults ◽  
Force Velocity ◽  
Strain Dependent

ABSTRACTNative cardiac ventricular myosin (βmys) translates actin under load by transducing ATP free energy into mechanical work on actin during muscle contraction. Unitary βmys translation of actin is the myosin step-size. In vitro and in vivo βmys regulates contractile force and velocity by remixing 3 different step-sizes with stepping frequencies autonomously adapted to workload. Cardiac and skeletal actin isoforms have a specific 1:4 stoichiometry in normal adult human ventriculum. Human adults with inheritable hypertrophic cardiomyopathy (HCM) up-regulate skeletal actin in ventriculum suggesting that increasing skeletal/cardiac actin stoichiometry also adapts myosin force-velocity to respond to the muscle’s inability to meet demand.Nanometer scale displacement of quantum dot (Qdot) labeled actin under resistive load when impelled by βmys measures single myosin force-velocity in vitro in the Qdot assay. Unitary displacement classification constraints introduced here better separates myosin based signal from background upgrading step-size spatial resolution to the sub-nanometer range. Single βmys force-velocity for skeletal vs cardiac actin substrates was compared using the Qdot assay.Two competing myosin strain-sensitive mechanisms regulate step-size choices dividing mechanical characteristics into low- and high-force regimes. The actin isoforms alter myosin strain-sensitive regulation such that onset of the high-force regime, where a short step-size is a large or major contributor, is offset to higher loads by a unique cardiac ELC N-terminus/cardiac-actin contact at Glu6/Ser358. It modifies βmys force-velocity by stabilizing the ELC N-terminus/cardiac-actin association. Uneven onset of the high-force regime for skeletal vs cardiac actin dynamically changes force-velocity characteristics as skeletal/cardiac actin fractional content increases in diseased muscle.

scholarly journals Velocities of unloaded muscle filaments are not limited by drag forces imposed by myosin cross-bridges

2015 ◽  
Vol 112 (36) ◽  
pp. 11235-11240 ◽  
Author(s):  
Richard K. Brizendine ◽  
Diego B. Alcala ◽  
Michael S. Carter ◽  
Brian D. Haldeman ◽  
Kevin C. Facemyer ◽  
...  
Keyword(s):  
Duty Ratio ◽  
Step Size ◽  
Drag Forces ◽  
Contraction Speed ◽  
Myosin Filaments ◽  
Adp Release ◽  
Muscle Types ◽  
Cross Bridges ◽  
Muscle Myosin

It is not known which kinetic step in the acto-myosin ATPase cycle limits contraction speed in unloaded muscles (V0). Huxley’s 1957 model [Huxley AF (1957) Prog Biophys Biophys Chem 7:255–318] predicts that V0 is limited by the rate that myosin detaches from actin. However, this does not explain why, as observed by Bárány [Bárány M (1967) J Gen Physiol 50(6, Suppl):197–218], V0 is linearly correlated with the maximal actin-activated ATPase rate (vmax), which is limited by the rate that myosin attaches strongly to actin. We have observed smooth muscle myosin filaments of different length and head number (N) moving over surface-attached F-actin in vitro. Fitting filament velocities (V) vs. N to a detachment-limited model using the myosin step size d = 8 nm gave an ADP release rate 8.5-fold faster and ton (myosin’s attached time) and r (duty ratio) ∼10-fold lower than previously reported. In contrast, these data were accurately fit to an attachment-limited model, V = N·v·d, over the range of N found in all muscle types. At nonphysiologically high N, V = L/ton rather than d/ton, where L is related to the length of myosin’s subfragment 2. The attachment-limited model also fit well to the [ATP] dependence of V for myosin-rod cofilaments at three fixed N. Previously published V0 vs. vmax values for 24 different muscles were accurately fit to the attachment-limited model using widely accepted values for r and N, giving d = 11.1 nm. Therefore, in contrast with Huxley’s model, we conclude that V0 is limited by the actin–myosin attachment rate.

scholarly journals Identify Molecular Mechanisms of Jiangzhi Decoction on Nonalcoholic Fatty Liver Disease by Network Pharmacology Analysis and Experimental Validation

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Lei Wang ◽  
Yin Zhi ◽  
Ying Ye ◽  
Miao Zhang ◽  
Xing Ma ◽  
...  
Keyword(s):  
Liver Disease ◽  
Fatty Liver Disease ◽  
Molecular Mechanisms ◽  
Enrichment Analysis ◽  
Functional Enrichment Analysis ◽  
Functional Enrichment ◽  
Active Ingredients ◽  
Nonalcoholic Fatty Liver ◽  
The Core

Background. Jiangzhi Decoction (JZD), a traditional herb mixture, has shown significant clinical efficacy against nonalcoholic fatty liver disease (NAFLD). However, its multicomponent and multitarget characteristics bring difficulty in deciphering its pharmacological mechanisms. Our study is aimed at identifying the core molecular mechanisms of JZD against NAFLD. Methods. The active ingredients were searched from Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and Traditional Chinese Medicine Integrated Database (TCMID). The targets of those ingredients were identified using ChemMapper database based on 3D structure similarity. NAFLD-related genes were searched from DisGeNET database and Gene Expression Omnibus (GEO) database. Then, we performed protein-protein interaction (PPI) analysis, functional enrichment analysis, and constructed pathway networks of “herbs-active ingredients-candidate targets” and identified the core molecular mechanisms and key active ingredients in the network. Also, molecular docking was carried out to predict the ligands of candidate targets using SwissDock. Finally, the human hepatic L02 cell line was used to establish the NAFLD model in vitro. The effect and key molecules were validated by Oil Red O staining, biochemical assays, and quantitative real-time PCR (qRT-PCR). Results. We found 147 active ingredients in JZD, 1285 targets of active ingredients, 401 NAFLD-related genes, and 59 overlapped candidate targets of JZD against NAFLD. 22 core targets were obtained by PPI analysis. Finally, nuclear receptor transcription and lipid metabolism regulation were found as the core molecular mechanisms of JZD against NAFLD by functional enrichment analysis. The candidate targets PPARα and LXRα were both docked with hyperin as the most favorable interaction, and HNF4α was docked with linolenic acid ethyl ester. According to in vitro experiments, it was found that JZD had an inhibitory effect on lipid accumulation and regulatory effects on cholesterol and triglycerides. Compared with OA group, the mRNA expression levels of PPARα and HNF4α were significantly upregulated in JZD group ( P < 0.05 ), and LXRα was significantly downregulated ( P < 0.001 ). Conclusion. JZD might alleviate hepatocyte steatosis by regulating some key molecules related to nuclear receptor transcription and lipid metabolism, such as PPARα, LXRα, and HNF4α. Our study will provide the scientific evidences of the clinical efficacy of JZD against NAFLD.

Molecular Mechanisms of Mutations in Factor XIII A-subunit Deficiency: In vitro Expression in COS-cells Demonstrates Intracellular Degradation of the Mutant Proteins

1997 ◽  
Vol 77 (06) ◽  
pp. 1068-1072 ◽  
Author(s):  
Aarno Palotie ◽  
Hanna Mikkola ◽  
Laszlo Muszbek ◽  
Gizela Haramura ◽  
Eija Hämäläinen ◽  
...  
Keyword(s):  
Factor Xiii ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Subunit Gene ◽  
Three Dimensional Model ◽  
Mutant Proteins ◽  
Cos Cells ◽  
Intracellular Degradation ◽  
A Subunit

SummaryFactor XIII deficiency is an autosomal recessive bleeding disorder that is largely caused by various mutations in FXIII A-subunit gene. Characteristically, the patients lack both A-subunit activity and antigen in the circulation. Here we have analysed the consequences of four mis-sense mutations (Met242→Thr, Arg252→Ile, Arg326→Gln, Leu498 to Pro) and one stop mutation (Arg661→Stop) in the FXIII A-subunit gene by expression in COS-cells. After transient transfection each mutant cDNA expressed mRNA at an equal level to the wild type FXIII. However, the mutant polypeptides accumulated in the cells in significantly reduced quantities and demonstrated only very low enzymatic activity. Analysis of immunoprecipitated metabolically labelled polypeptides demonstrated remarkable instability and intracellular degradation of all mutant FXIII proteins. These results verify the deleterious nature of the individual amino acid changes and confirm that protein instability and susceptibility to proteolysis are consequences of the mutations, as predicted from the three-dimensional model of crystallised FXIII A-subunit.

scholarly journals Apigenin Inhibits the Growth of Hepatocellular Carcinoma Cells by Affecting the Expression of microRNA Transcriptome

2021 ◽  
Vol 11 ◽  
Author(s):  
Shou-Mei Wang ◽  
Pei-Wei Yang ◽  
Xiao-Jun Feng ◽  
Yi-Wei Zhu ◽  
Feng-Jun Qiu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Dependent Manner ◽  
Regulatory Pathways ◽  
The Core ◽  
Huh7 Cells

BackgroundApigenin, as a natural flavonoid, has low intrinsic toxicity and has potential pharmacological effects against hepatocellular carcinoma (HCC). However, the molecular mechanisms involving microRNAs (miRNAs) and their target genes regulated by apigenin in the treatment of HCC have not been addressed.ObjectiveIn this study, the molecular mechanisms of apigenin involved in the prevention and treatment of HCC were explored in vivo and in vitro using miRNA transcriptomic sequencing to determine the basis for the clinical applications of apigenin in the treatment of HCC.MethodsThe effects of apigenin on the proliferation, cell cycle progression, apoptosis, and invasion of human hepatoma cell line Huh7 and Hep3B were studied in vitro, and the effects on the tumorigenicity of Huh7 cells were assessed in vivo. Then, a differential expression analysis of miRNAs regulated by apigenin in Huh7 cells was performed using next-generation RNA sequencing and further validated by qRT-PCR. The potential genes targeted by the differentially expressed miRNAs were identified using a curated miRTarBase miRNA database and their molecular functions were predicted using Gene Ontology and KEGG signaling pathway analysis.ResultsCompared with the control treatment group, apigenin significantly inhibited Huh7 cell proliferation, cell cycle, colony formation, and cell invasion in a concentration-dependent manner. Moreover, apigenin reduced tumor growth, promoted tumor cell necrosis, reduced the expression of Ki67, and increased the expression of Bax and Bcl-2 in the xenograft tumors of Huh7 cells. Bioinformatics analysis of the miRNA transcriptome showed that hsa-miR-24, hsa-miR-6769b-3p, hsa-miR-6836-3p, hsa-miR-199a-3p, hsa-miR-663a, hsa-miR-4739, hsa-miR-6892-3p, hsa-miR-7107-5p, hsa-miR-1273g-3p, hsa-miR-1343, and hsa-miR-6089 were the most significantly up-regulated miRNAs, and their key gene targets were MAPK1, PIK3CD, HRAS, CCND1, CDKN1A, E2F2, etc. The core regulatory pathways of the up-regulated miRNAs were associated with the hepatocellular carcinoma pathway. The down-regulated miRNAs were hsa-miR-181a-5p and hsa-miR-148a-3p, and the key target genes were MAPK1, HRAS, STAT3, FOS, BCL2, SMAD2, PPP3CA, IFNG, MET, and VAV2, with the core regulatory pathways identified as proteoglycans in cancer pathway.ConclusionApigenin can inhibit the growth of HCC cells, which may be mediated by up-regulation or down-regulation of miRNA molecules and their related target genes.

Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein

2009 ◽  
Vol 39 (3) ◽  
pp. 195-201 ◽  
Author(s):  
Hee Keun Lee ◽  
Mee Hyun Song ◽  
Myengmo Kang ◽  
Jung Tae Lee ◽  
Kyoung-Ah Kong ◽  
...  
Keyword(s):  
Dna Binding ◽  
Inner Ear ◽  
Molecular Mechanisms ◽  
Tertiary Structure ◽  
In Vitro Assays ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dna Binding Domains ◽  
Binding Domains

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

scholarly journals Uncured PDMS Inhibits Myosin In Vitro Motility in a Microfluidic Flow Cell

2018 ◽  
Author(s):  
Yihua Wang ◽  
Thomas P. Burghardt
Keyword(s):  
Low Cost ◽  
Flow Cell ◽  
Small Sample ◽  
Cardiac Contraction ◽  
Chemical Extraction ◽  
Cell Aging ◽  
Step Size ◽  
In Vitro Motility ◽  
Microfluidic Flow

ABSTRACTThe myosin motor powers cardiac contraction and is frequently implicated in hereditary heart disease by its mutation. Principal motor function characteristics include myosin unitary step size, duty cycle, and force-velocity relationship for translating actin under load. These characteristics are sometimes measured in vitro with a motility assay detecting fluorescent labeled actin filament gliding velocity over a planar array of surface immobilized myosin. Assay miniaturization in a polydimethylsiloxane/glass (PDMS/glass) hybrid microfluidic flow channel is an essential component to a small sample volume assay applicable to costly protein samples however the PDMS substrate dramatically inhibits myosin motility. Myosin in vitro motility in a PDMS/glass hybrid microfluidic flow cell was tested under a variety of conditions to identify and mitigate the effect of PDMS on myosin. Substantial contamination by the monomeric species in polymerized PDMS flow cells is shown to be the cause of myosin motility inhibition. Normal myosin motility recovers by either extended cell aging (∼20 days) to allow more complete polymerization or by direct chemical extraction of the free monomers from the polymer substrate. PDMS flow cell aging is the low cost alternative compatible with the other PDMS and glass modifications needed for in vitro myosin motility assaying.

scholarly journals Effect of low pH on single skeletal muscle myosin mechanics and kinetics

2008 ◽  
Vol 295 (1) ◽  
pp. C173-C179 ◽  
Author(s):  
E. P. Debold ◽  
S. E. Beck ◽  
D. M. Warshaw
Keyword(s):  
Skeletal Muscle ◽  
Single Molecule ◽  
Low Ph ◽  
Muscular Fatigue ◽  
Step Size ◽  
Skeletal Muscle Myosin ◽  
In Vitro Motility ◽  
Adp Release ◽  
Muscle Myosin

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity ( Vactin) in the IVM by 36%. Single molecule experiments, at 1 μM ATP, decreased the average unitary step size of myosin ( d) from 10 ± 2 nm (pH 7.4) to 2 ± 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 μM to 1 mM at pH 6.4 restored d (9 ± 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. Vactin, however, was not restored by raising the [ATP] (1–10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding ( ton). Measurement of ton as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs ton by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., Vactin ∼ d/ ton), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in Vactin, suggesting a molecular explanation for this component of muscular fatigue.

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