Familial dilated cardiomyopathy mutations uncouple troponin i phosphorylation from changes in myofibrillar Ca2+ sensitivity

Massimiliano Memo, Man Ching Leung, Douglas G. Ward, Cristobal Dos Remedios, Sachio Morimoto, Lianfeng Zhang, Gianina Ravenscroft, Elyshia McNamara, Kristen J. Nowak, Steven B. Marston, Andrew E. Messer

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Abstract

AimsThe pure form of familial dilated cardiomyopathy (DCM) is mainly caused by mutations in genes encoding sarcomeric proteins. Previous measurements using recombinant proteins suggested that DCM mutations in thin filament proteins decreased myofibrillar Ca2+ sensitivity, but exceptions were reported. We re-investigated the molecular mechanism of familial DCM using native proteins.Methods and resultsWe used the quantitative in vitro motility assay and native troponin and tropomyosin to study DCM mutations in troponin I, troponin T, and -tropomyosin. Four mutations reduced myofilament Ca2+ sensitivity, but one mutation (TPM1 E54K) did not alter Ca2+ sensitivity and another (TPM1 D230N) increased Ca2+ sensitivity. In thin filaments from normal human and mouse heart, protein kinase A (PKA) phosphorylation of troponin I caused a two- to three-fold decrease in myofibrillar Ca2+ sensitivity. However, Ca2+ sensitivity did not change with the level of troponin I phosphorylation in any of the DCM-mutant containing thin filaments (E40K, E54K, and D230N in -tropomyosin; R141W and ΔK210 in cardiac troponin T; K36Q in cardiac troponin I; G159D in cardiac troponin C, and E361G in cardiac -actin). This 'uncoupling' was observed with native mutant protein from human and mouse heart and with recombinant mutant protein expressed in baculovirus/Sf9 systems. Uncoupling was independent of the fraction of mutated protein present above 0.55.ConclusionWe conclude that DCM-causing mutations in thin filament proteins abolish the relationship between myofilament Ca2+ sensitivity and troponin I phosphorylation by PKA. We propose that this blunts the response to β-adrenergic stimulation and could be the cause of DCM in the long term.

Original languageEnglish
Pages (from-to)65-73
Number of pages9
JournalCardiovascular research
Volume99
Issue number1
DOIs
Publication statusPublished - Jul 1 2013

Fingerprint

Troponin
Troponin I
Dilated Cardiomyopathy
Phosphorylation
Tropomyosin
Mutation
Troponin T
Myofibrils
Mutant Proteins
Cyclic AMP-Dependent Protein Kinases
Proteins
Recombinant Proteins
Troponin C
Baculoviridae
Adrenergic Agents
Familial dilated cardiomyopathy
Actins
Genes

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

Memo, M., Leung, M. C., Ward, D. G., Dos Remedios, C., Morimoto, S., Zhang, L., ... Messer, A. E. (2013). Familial dilated cardiomyopathy mutations uncouple troponin i phosphorylation from changes in myofibrillar Ca2+ sensitivity. Cardiovascular research, 99(1), 65-73. https://doi.org/10.1093/cvr/cvt071

Familial dilated cardiomyopathy mutations uncouple troponin i phosphorylation from changes in myofibrillar Ca2+ sensitivity. / Memo, Massimiliano; Leung, Man Ching; Ward, Douglas G.; Dos Remedios, Cristobal; Morimoto, Sachio; Zhang, Lianfeng; Ravenscroft, Gianina; McNamara, Elyshia; Nowak, Kristen J.; Marston, Steven B.; Messer, Andrew E.

In: Cardiovascular research, Vol. 99, No. 1, 01.07.2013, p. 65-73.

Research output: Contribution to journalArticle

Memo, M, Leung, MC, Ward, DG, Dos Remedios, C, Morimoto, S, Zhang, L, Ravenscroft, G, McNamara, E, Nowak, KJ, Marston, SB & Messer, AE 2013, 'Familial dilated cardiomyopathy mutations uncouple troponin i phosphorylation from changes in myofibrillar Ca2+ sensitivity', Cardiovascular research, vol. 99, no. 1, pp. 65-73. https://doi.org/10.1093/cvr/cvt071
Memo, Massimiliano ; Leung, Man Ching ; Ward, Douglas G. ; Dos Remedios, Cristobal ; Morimoto, Sachio ; Zhang, Lianfeng ; Ravenscroft, Gianina ; McNamara, Elyshia ; Nowak, Kristen J. ; Marston, Steven B. ; Messer, Andrew E. / Familial dilated cardiomyopathy mutations uncouple troponin i phosphorylation from changes in myofibrillar Ca2+ sensitivity. In: Cardiovascular research. 2013 ; Vol. 99, No. 1. pp. 65-73.
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abstract = "AimsThe pure form of familial dilated cardiomyopathy (DCM) is mainly caused by mutations in genes encoding sarcomeric proteins. Previous measurements using recombinant proteins suggested that DCM mutations in thin filament proteins decreased myofibrillar Ca2+ sensitivity, but exceptions were reported. We re-investigated the molecular mechanism of familial DCM using native proteins.Methods and resultsWe used the quantitative in vitro motility assay and native troponin and tropomyosin to study DCM mutations in troponin I, troponin T, and -tropomyosin. Four mutations reduced myofilament Ca2+ sensitivity, but one mutation (TPM1 E54K) did not alter Ca2+ sensitivity and another (TPM1 D230N) increased Ca2+ sensitivity. In thin filaments from normal human and mouse heart, protein kinase A (PKA) phosphorylation of troponin I caused a two- to three-fold decrease in myofibrillar Ca2+ sensitivity. However, Ca2+ sensitivity did not change with the level of troponin I phosphorylation in any of the DCM-mutant containing thin filaments (E40K, E54K, and D230N in -tropomyosin; R141W and ΔK210 in cardiac troponin T; K36Q in cardiac troponin I; G159D in cardiac troponin C, and E361G in cardiac -actin). This 'uncoupling' was observed with native mutant protein from human and mouse heart and with recombinant mutant protein expressed in baculovirus/Sf9 systems. Uncoupling was independent of the fraction of mutated protein present above 0.55.ConclusionWe conclude that DCM-causing mutations in thin filament proteins abolish the relationship between myofilament Ca2+ sensitivity and troponin I phosphorylation by PKA. We propose that this blunts the response to β-adrenergic stimulation and could be the cause of DCM in the long term.",
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AU - Dos Remedios, Cristobal

AU - Morimoto, Sachio

AU - Zhang, Lianfeng

AU - Ravenscroft, Gianina

AU - McNamara, Elyshia

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AU - Marston, Steven B.

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