Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model
Copyright © 2017. Published by Elsevier Inc..
The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k-4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics-possibly caused by changes in the α-myosin heavy chain phosphorylation profile-as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2017 |
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| Erschienen: |
2017 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:112 |
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| Enthalten in: |
Biophysical journal - 112(2017), 8 vom: 25. Apr., Seite 1726-1736 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Kawai, Masataka [VerfasserIn] |
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| Links: |
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| Themen: |
8L70Q75FXE |
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| Anmerkungen: |
Date Completed 07.08.2017 Date Revised 13.11.2018 published: Print Citation Status MEDLINE |
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| doi: |
10.1016/j.bpj.2017.02.045 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM271319232 |
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| 100 | 1 | |a Kawai, Masataka |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model |
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| 520 | |a Copyright © 2017. Published by Elsevier Inc. | ||
| 520 | |a The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k-4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics-possibly caused by changes in the α-myosin heavy chain phosphorylation profile-as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM | ||
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| 700 | 1 | |a Johnston, Jamie R |e verfasserin |4 aut | |
| 700 | 1 | |a Karam, Tarek |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Li |e verfasserin |4 aut | |
| 700 | 1 | |a Singh, Rakesh K |e verfasserin |4 aut | |
| 700 | 1 | |a Pinto, Jose R |e verfasserin |4 aut | |
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