Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

Masataka Kawai; Jamie R Johnston; Tarek Karam; Li Wang; Rakesh K Singh; Jose R Pinto

doi:10.1016/j.bpj.2017.02.045

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Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

Journal article

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Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

Masataka Kawai, Jamie R Johnston, Tarek Karam, Li Wang, Rakesh K Singh and Jose R Pinto

Biophysical journal, Vol.112(8), pp.1726-1736

04/25/2017

DOI: 10.1016/j.bpj.2017.02.045

PMCID: PMC5406373

PMID: 28445763

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Abstract

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 Ca -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 Ca -sensitivity and a decrease (0.8×) in cooperativity (n ) 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 (K ) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k ) and its equilibrium constant (K ) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k ) decreased (0.5×), reversal step (k ) increased (2×), and the phosphate-release step (1/K ) 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.

Phosphorylation

Adenosine Triphosphate - metabolism

Animals

Calcium - metabolism

Cardiomyopathy, Hypertrophic - metabolism

Cations, Divalent - metabolism

Chromatography, Liquid

Disease Models, Animal

Gene Knock-In Techniques

Immunoblotting

Kinetics

Mice, Transgenic

Myofibrils - metabolism

Myosin Heavy Chains - metabolism

Myosin Subfragments - metabolism

Papillary Muscles - metabolism

Tandem Mass Spectrometry

Troponin C - genetics

Troponin C - metabolism

Details

Title: Subtitle: Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model
Creators: Masataka Kawai - University of Iowa
Jamie R Johnston - Florida State University
Tarek Karam - University of Iowa
Li Wang - University of Iowa
Rakesh K Singh - Florida State University
Jose R Pinto - Florida State University
Resource Type: Journal article
Publication Details: Biophysical journal, Vol.112(8), pp.1726-1736
DOI: 10.1016/j.bpj.2017.02.045
PMID: 28445763
PMCID: PMC5406373
NLM abbreviation: Biophys J
ISSN: 0006-3495
eISSN: 1542-0086
Grant note: R00 HL103840 / NHLBI NIH HHS R01 HL128683 / NHLBI NIH HHS K99 HL103840 / NHLBI NIH HHS
Language: English
Date published: 04/25/2017
Academic Unit: Anatomy and Cell Biology; Internal Medicine
Record Identifier: 9984284451802771

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