scholarly journals Microscopic heat pulses activate cardiac thin filaments

2019 ◽  
Vol 151 (6) ◽  
pp. 860-869 ◽  
Author(s):  
Shuya Ishii ◽  
Kotaro Oyama ◽  
Tomomi Arai ◽  
Hideki Itoh ◽  
Seine A. Shintani ◽  
...  
Keyword(s):  
Laser Irradiation ◽  
Thin Filament ◽  
Structural Changes ◽  
Rapid Heating ◽  
Temperature Sensitive ◽  
In Vitro Motility Assay ◽  
Thin Filaments ◽  
Rhodamine Phalloidin ◽  
Ir Laser

During the excitation–contraction coupling of the heart, sarcomeres are activated via thin filament structural changes (i.e., from the “off” state to the “on” state) in response to a release of Ca2+ from the sarcoplasmic reticulum. This process involves chemical reactions that are highly dependent on ambient temperature; for example, catalytic activity of the actomyosin ATPase rises with increasing temperature. Here, we investigate the effects of rapid heating by focused infrared (IR) laser irradiation on the sliding of thin filaments reconstituted with human α-tropomyosin and bovine ventricular troponin in an in vitro motility assay. We perform high-precision analyses measuring temperature by the fluorescence intensity of rhodamine-phalloidin–labeled F-actin coupled with a fluorescent thermosensor sheet containing the temperature-sensitive dye Europium (III) thenoyltrifluoroacetonate trihydrate. This approach enables a shift in temperature from 25°C to ∼46°C within 0.2 s. We find that in the absence of Ca2+ and presence of ATP, IR laser irradiation elicits sliding movements of reconstituted thin filaments with a sliding velocity that increases as a function of temperature. The heating-induced acceleration of thin filament sliding likewise occurs in the presence of Ca2+ and ATP; however, the temperature dependence is more than twofold less pronounced. These findings could indicate that in the mammalian heart, the on–off equilibrium of the cardiac thin filament state is partially shifted toward the on state in diastole at physiological body temperature, enabling rapid and efficient myocardial dynamics in systole.

Abstract 1464: Cardiac Myosin Binding Protein-C (cMyBP-C) Directly Modulates Actomyosin Binding and Kinetics Independent of LMM and S2 Binding.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Walid Saber ◽  
Kelly J Begin ◽  
David M Warshaw ◽  
Peter VanBuren
Keyword(s):  
Actin Filament ◽  
Thin Filament ◽  
Motility Assay ◽  
Actin Binding ◽  
Maximal Velocity ◽  
In Vitro Motility Assay ◽  
Thin Filaments ◽  
Myosin Binding ◽  
Cross Bridges

BACKGROUND: While mutations in cMyBP-C constitute a common cause of FHC, its role in sarcomere contraction remains unclear. cMyBP-C binds to actin, titin and the S2 and LMM proteolytic domains of myosin. Through its numerous binding interactions cMyBP-C may act as a tether, restricting myosin and/or actomyosin function. We directly tested this hypothesis in the in vitro motility assay using either whole myosin or the myosin subfragments, HMM and S1 (which lack LMM and LMM-S2, respectively). METHODS AND RESULTS: The motility assay is an in vitro model of muscle contraction in which thin filaments are propelled across a myosin coated surface. The addition of cMyBP-C to the motility assay resulted in a concentration dependent reduction in actin filament velocity when using either whole myosin, HMM or S1, demonstrating that cMyBP-C inhibits thin filament velocity independent of LMM or S2 binding. Using whole myosin and thin filaments reconstituted with troponin/tropomyosin, the addition of cMyBP-C resulted in a 29% reduction in maximal velocity (P=0.002) with no effect on maximal force. At sub-maximal calcium, the pCa50 for velocity was increased (6.64 ± 0.06 vs. control, 6.44 ± 0.03, P=0.003) whereas the pCa50 for force was decreased (6.25 ± 0.09 vs. control, 6.55 ± 0.02, P=0.008). Thin filament activation by myosin strong-binding demonstrated an increased amount of myosin required to half maximally activate the thin filament in the presence of cMyBP-C, indicating that myosin binding to the thin filament is reduced with cMyBP-C. These findings were supported by co-sedimentation experiments which demonstrate that cMyBP-C competes with S1 for actin binding in the presence of ATP, with no effect on S1/actin binding in the absence of ATP. Finally, while the number of cross-bridges interacting with the thin filament is rate limiting for velocity at shorter filament lengths, this was not observed at longer filament lengths indicating that cMyBP- C directly modulates the kinetics of actomyosin. CONCLUSIONS: The effects of cMyBP-C on velocity and force demonstrate that cMyBP-C does not simply act as a tether but likely affects both the kinetics and the recruitment of myosin cross-bridges through its direct interaction with the myosin head and/or the actin filament.

scholarly journals Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics

2019 ◽  
Vol 30 (2) ◽  
pp. 268-281 ◽  
Author(s):  
Thu Ly ◽  
Christopher T. Pappas ◽  
Dylan Johnson ◽  
William Schlecht ◽  
Mert Colpan ◽  
...  
Keyword(s):  
Thin Filament ◽  
Structural Changes ◽  
Striated Muscle ◽  
In Vitro Assays ◽  
Missense Mutations ◽  
Thin Filaments ◽  
Thin Filament Regulation ◽  
Pointed End ◽  
Kinetics Of

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin’s thin-filament pointed-end assembly but does not affect tropomodulin’s assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin’s affinity for F-actin and affects leiomodin’s function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin’s function.

scholarly journals Cardioinotropic Effects in Subchronic Intoxication of Rats with Lead and/or Cadmium Oxide Nanoparticles

2021 ◽  
Vol 22 (7) ◽  
pp. 3466
Author(s):  
Svetlana V. Klinova ◽  
Boris A. Katsnelson ◽  
Ilzira A. Minigalieva ◽  
Oksana P. Gerzen ◽  
Alexander A. Balakin ◽  
...  
Keyword(s):  
Cadmium Oxide ◽  
Male Rats ◽  
Sliding Velocity ◽  
Muscle Type ◽  
In Vitro Motility Assay ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Toxic Impact ◽  
Cdo Nanoparticles

Subchronic intoxication was induced in outbred male rats by repeated intraperitoneal injections with lead oxide (PbO) and/or cadmium oxide (CdO) nanoparticles (NPs) 3 times a week during 6 weeks for the purpose of examining its effects on the contractile characteristics of isolated right ventricle trabeculae and papillary muscles in isometric and afterload contractions. Isolated and combined intoxication with these NPs was observed to reduce the mechanical work produced by both types of myocardial preparation. Using the in vitro motility assay, we showed that the sliding velocity of regulated thin filaments drops under both isolated and combined intoxication with CdO–NP and PbO–NP. These results correlate with a shift in the expression of myosin heavy chain (MHC) isoforms towards slowly cycling β–MHC. The type of CdO–NP + PbO–NP combined cardiotoxicity depends on the effect of the toxic impact, the extent of this effect, the ratio of toxicant doses, and the degree of stretching of cardiomyocytes and muscle type studied. Some indices of combined Pb–NP and CdO–NP cardiotoxicity and general toxicity (genotoxicity included) became fully or partly normalized if intoxication developed against background administration of a bioprotective complex.

Compositional and structural changes of human dentin following caries removal by Er, Cr: YSGG laser irradiation in primary teeth

2002 ◽  
Vol 26 (4) ◽  
pp. 377-382 ◽  
Author(s):  
Mozammal Hossain ◽  
Yukio Nakamura ◽  
Yoshishige Yamada ◽  
Yoshiko Murakami ◽  
Koukichi Matsumoto
Keyword(s):  
Laser Irradiation ◽  
Structural Changes ◽  
Primary Teeth ◽  
Thermal Damage ◽  
In Vitro Study ◽  
Surface Characteristic ◽  
Human Dentin ◽  
Caries Removal ◽  
Induced Changes

In this in vitro study, the compositional and structural changes of human dentin, and knoop harness of cavity floor following the removal of dental caries by Er,Cr:YSGG laser irradiation in primary teeth was compared with that of the conventional bur cavity. The results confirmed that laser irradiation revealed minimal thermal damage to the surrounding tissues, minimal thermal induced changes of dental hard tissue compositions, and favorable surface characteristic.

scholarly journals Calcium regulation of thin filament movement in an in vitro motility assay

1996 ◽  
Vol 70 (4) ◽  
pp. 1881-1892 ◽  
Author(s):  
E. Homsher ◽  
B. Kim ◽  
A. Bobkova ◽  
L.S. Tobacman
Keyword(s):  
Thin Filament ◽  
Calcium Regulation ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

scholarly journals A Troponin T Variant Linked with Pediatric Dilated Cardiomyopathy Reduces the Coupling of Thin Filament Activation to Myosin and Calcium Binding

2021 ◽  
pp. mbc.E21-02-0082
Author(s):  
Samantha K. Barrick ◽  
Lina Greenberg ◽  
Michael J. Greenberg
Keyword(s):  
Dilated Cardiomyopathy ◽  
Calcium Binding ◽  
Thin Filament ◽  
Troponin T ◽  
Calcium Sensitivity ◽  
Cellular Level ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Fluorescence Measurements

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well-understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an in vitro motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.

Formation of a distinct connexin43 phosphoisoform in mitotic cells is dependent upon p34cdc2 kinase

1998 ◽  
Vol 111 (6) ◽  
pp. 833-841 ◽  
Author(s):  
P.D. Lampe ◽  
W.E. Kurata ◽  
B.J. Warn-Cramer ◽  
A.F. Lau
Keyword(s):  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cyclin B ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Cdc2 Kinase ◽  
Junction Protein ◽  
P34 Cdc2 ◽  
Mitotic Cells

The gap junction protein connexin43 is a phosphoprotein that typically migrates as three bands (nonphosphorylated, P1 and P2) during polyacrylamide gel electrophoresis. The electrophoretic mobility of connexin43 from mitotic cells was distinctly reduced to a form (P3) that migrated slower than P2 from Rat1 cells prepared by shakeoff of nocodazole-treated and untreated cultures. Mitotic FT210 cells, which contain a temperature-sensitive mutation in the p34(cdc2) kinase, showed abundant levels of the P3 connexin43 when maintained at the permissive temperature where p34(cdc2) is active. In contrast, nocodozole-treated FT210 cells grown at the nonpermissive temperature did not contain P3 connexin43. These results indicated that generation of the P3 connexin43 was dependent upon active p34(cdc2)/cyclin B kinase. Although the p34(cdc2)kinase phosphorylated connexin43 in vitro on peptides containing serine 255, the major phosphotryptic peptides in P3 connexin43 from mitotic cells appeared to be the consequence of another protein kinase(s), which may be activated by the p34(cdc2)/cyclin B kinase. The P3 connexin43 exhibited a marked redistribution from cell-cell plasma membrane interfaces to multiple, distinctly stained cytoplasmic structures. These events may be part of the dramatic structural changes observed in mitotic cells undergoing cell rounding and cytokinesis. Results of initial studies using inhibitors of protein degradative and synthetic pathways suggested the likelihood that protein degradation and synthesis participate in the disappearance of the P3 connexin43 and restoration of the pattern of connexin43 isoforms observed in nonmitotic cells.

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