Functional characterization of TNNC1 rare variants identified in dilated cardiomyopathy

Abstract

TNNC1, which encodes cardiac troponin C (cTnC), remains elusive as a dilated cardiomyopathy (DCM) gene. Here, we report the clinical, genetic, and functional characterization of four TNNC1 rare variants (Y5H, M103I, D145E, and I148V), all previously reported by us in association with DCM (Hershberger, R. E., Norton, N., Morales, A., Li, D., Siegfried, J. D., and Gonzalez-Quintana, J. (2010) Circ. Cardiovasc. Genet. 3, 155-161); in the previous study, two variants (Y5H and D145E) were identified in subjects who also carried MYH7 and MYBPC3 rare variants, respectively. Functional studies using the recombinant human mutant cTnC proteins reconstituted into porcine papillary skinned fibers showed decreased Ca(2+) sensitivity of force development (Y5H and M103I). Furthermore, the cTnC mutants diminished (Y5H and I148V) or abolished (M103I) the effects of PKA phosphorylation on Ca(2+) sensitivity. Only M103I decreased the troponin activation properties of the actomyosin ATPase when Ca(2+) was present. CD spectroscopic studies of apo (absence of divalent cations)-, Mg(2+)-, and Ca(2+)/Mg(2+)-bound states indicated that all of the cTnC mutants (except I148V in the Ca(2+)/Mg(2+) condition) decreased the α-helical content. These results suggest that each mutation alters the function/ability of the myofilament to bind Ca(2+) as a result of modifications in cTnC structure. One variant (D145E) that was previously reported in association with hypertrophic cardiomyopathy and that produced results in vivo in this study consistent with prior hypertrophic cardiomyopathy functional studies was found associated with the MYBPC3 P910T rare variant, likely contributing to the observed DCM phenotype. We conclude that these rare variants alter the regulation of contraction in some way, and the combined clinical, molecular, genetic, and functional data reinforce the importance of TNNC1 rare variants in the pathogenesis of DCM.

FIGURE 1.

TNNC1 (cTnC) gene structure and amino acid conservation resulting from the identified rare variants. The TNNC1 gene shows a remarkable degree of conservation between mammalian, avian, amphibian, and fish species. Altered amino acids are indicated for each missense mutation in a box above the first line, and the human wild-type sequence is shown in the first line, followed by chicken, mouse, rat, frog, and zebrafish sequences. All variants were conserved throughout these various species. Information concerning the helices and sites can be found under UniProt accession number P63316.

FIGURE 2.

Pedigrees of cTnC-associated DCM. Pedigrees have been labeled by letters, which correspond to their respective mutation as shown in Fig. 1 and are given in Tables 1 and 2. Squares represent males, and circles represent females. Arrowheads denote the probands. Diagonal line mark deceased individuals. Solid symbols indicate DCM with or without heart failure; shaded symbols represent any cardiovascular abnormality; and open symbols represent unaffected individuals. The presence or absence of the family's TNNC1 rare variant is indicated by + or −, respectively. Obligate carriers are noted in parentheses (+). The asterisk in Pedigree A denotes an MYH7 R1045C rare variant and that in Pedigree C denotes an MYBPC3 P910T rare variant thought to be possibly disease-causing as reported previously (19).

FIGURE 3.

pCa-force relationship measured in porcine skinned papillary fiber containing DCM TnC mutants. Native porcine TnC was extracted, and the skinned fibers were reconstituted with WT TnC or the DCM mutant. The Ca²⁺ sensitivity of force development was measured at pH 7.0 before (A) and after (B) PKA treatment. The fiber data are summarized in Table 3.

FIGURE 4.

Activation and inhibition of the actomyosin-Tm ATPase activity as a function of Tn. A, activation of the ATPase activity is shown in the presence of Ca²⁺. B, inhibition of the ATPase activity is shown in the absence of Ca²⁺. Data are reported as means ± S.E. (n = 6–8).

FIGURE 5.

Far-UV CD spectra. Experiments were performed in apo-, Mg²⁺-, and Ca²⁺/Mg²⁺-bound states. Spectra were recorded in a Jasco-720 spectropolarimeter at room temperature (21 °C). For each independent measurement, 10 scans were averaged. Data are summarized in Table 4. deg, degrees.