Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart

Priya Muthu, Prasad Mettikolla, Nils Calander, Rafal Luchowski, Ignacy Gryczynski, Zygmunt Gryczynski, Danuta Szczesna-Cordary, J. Borejdo

Research output: Contribution to journalArticle

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Abstract

One of the sarcomeric mutations associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC) is the D166V point mutation in the ventricular myosin regulatory light chain (RLC) encoded by the MYL2 gene. In this report we show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricles of transgenic (Tg)-D166V and Tg-WT mice. We have derived the myosin cross-bridge kinetic rates by tracking the orientation of a fluorescently labeled single actin molecule. Orientation (measured by polarized fluorescence) oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rate of cross-bridge attachment during isometric contraction decreased from 3 s-1 in myofibrils from Tg-WT to 1.4 s-1 in myofibrils from Tg-D166V. The rate of detachment decreased from 1.3 s-1 (Tg-WT) to 1.2 s-1 (Tg-D166V). We also showed that the level of RLC phosphorylation was largely decreased in Tg-D166V myofibrils compared to Tg-WT. Our findings suggest that alterations in the myosin cross-bridge kinetics brought about by the D166V mutation in RLC might be responsible for the compromised function of the mutated hearts and lead to their inability to efficiently pump blood.

Original languageEnglish
Pages (from-to)989-998
Number of pages10
JournalJournal of Molecular and Cellular Cardiology
Volume48
Issue number5
DOIs
StatePublished - 1 May 2010

Fingerprint

Familial Hypertrophic Cardiomyopathy
Myofibrils
Myosins
Actins
Ventricular Myosins
Light
Myosin Light Chains
Mutation
Isometric Contraction
Point Mutation
Transgenic Mice
Heart Ventricles
Fluorescence
Phosphorylation
Phenotype
Genes

Keywords

  • Familial hypertrophic cardiomyopathy
  • Fluorescence correlation spectroscopy
  • Fluorescence lifetime
  • Regulatory light chain of myosin
  • Sarcomere
  • Single molecule detection

Cite this

Muthu, Priya ; Mettikolla, Prasad ; Calander, Nils ; Luchowski, Rafal ; Gryczynski, Ignacy ; Gryczynski, Zygmunt ; Szczesna-Cordary, Danuta ; Borejdo, J. / Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart. In: Journal of Molecular and Cellular Cardiology. 2010 ; Vol. 48, No. 5. pp. 989-998.
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Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart. / Muthu, Priya; Mettikolla, Prasad; Calander, Nils; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Szczesna-Cordary, Danuta; Borejdo, J.

In: Journal of Molecular and Cellular Cardiology, Vol. 48, No. 5, 01.05.2010, p. 989-998.

Research output: Contribution to journalArticle

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T1 - Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart

AU - Muthu, Priya

AU - Mettikolla, Prasad

AU - Calander, Nils

AU - Luchowski, Rafal

AU - Gryczynski, Ignacy

AU - Gryczynski, Zygmunt

AU - Szczesna-Cordary, Danuta

AU - Borejdo, J.

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N2 - One of the sarcomeric mutations associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC) is the D166V point mutation in the ventricular myosin regulatory light chain (RLC) encoded by the MYL2 gene. In this report we show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricles of transgenic (Tg)-D166V and Tg-WT mice. We have derived the myosin cross-bridge kinetic rates by tracking the orientation of a fluorescently labeled single actin molecule. Orientation (measured by polarized fluorescence) oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rate of cross-bridge attachment during isometric contraction decreased from 3 s-1 in myofibrils from Tg-WT to 1.4 s-1 in myofibrils from Tg-D166V. The rate of detachment decreased from 1.3 s-1 (Tg-WT) to 1.2 s-1 (Tg-D166V). We also showed that the level of RLC phosphorylation was largely decreased in Tg-D166V myofibrils compared to Tg-WT. Our findings suggest that alterations in the myosin cross-bridge kinetics brought about by the D166V mutation in RLC might be responsible for the compromised function of the mutated hearts and lead to their inability to efficiently pump blood.

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