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
Cardiovascular Research 2013 July 1, 99 (1): 65-73

AIMS: The 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 Ca(2+) sensitivity, but exceptions were reported. We re-investigated the molecular mechanism of familial DCM using native proteins.

METHODS AND RESULTS: We used the quantitative in vitro motility assay and native troponin and tropomyosin to study DCM mutations in troponin I, troponin T, and α-tropomyosin. Four mutations reduced myofilament Ca(2+) sensitivity, but one mutation (TPM1 E54K) did not alter Ca(2+) sensitivity and another (TPM1 D230N) increased Ca(2+) 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 Ca(2+) sensitivity. However, Ca(2+) 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 α-tropomyosin; R141W and ΔK210 in cardiac troponin T; K36Q in cardiac troponin I; G159D in cardiac troponin C, and E361G in cardiac α-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.

CONCLUSION: We conclude that DCM-causing mutations in thin filament proteins abolish the relationship between myofilament Ca(2+) 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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