Functional consequences of hypertrophic and dilated cardiomyopathy-causing mutations in α-tropomyosin

Audrey N. Chang, Keita Harada, Michael J. Ackerman, James D. Potter

Research output: Contribution to journalArticle

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Abstract

To study the functional consequences of various cardiomyopathic mutations in human cardiac α-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.

Original languageEnglish
Pages (from-to)34343-34349
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number40
DOIs
StatePublished - Oct 7 2005
Externally publishedYes

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Tropomyosin
Hypertrophic Cardiomyopathy
Dilated Cardiomyopathy
Adenosine Triphosphatases
Mutation
Troponin
Calcium-Transporting ATPases
Myofibrils
Regain
Mutant Proteins
Recombinant Proteins
Electrophoresis
Gels
Calcium
Phenotype

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional consequences of hypertrophic and dilated cardiomyopathy-causing mutations in α-tropomyosin. / Chang, Audrey N.; Harada, Keita; Ackerman, Michael J.; Potter, James D.

In: Journal of Biological Chemistry, Vol. 280, No. 40, 07.10.2005, p. 34343-34349.

Research output: Contribution to journalArticle

Chang, Audrey N. ; Harada, Keita ; Ackerman, Michael J. ; Potter, James D. / Functional consequences of hypertrophic and dilated cardiomyopathy-causing mutations in α-tropomyosin. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 40. pp. 34343-34349.
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