Functional Consequences of a Novel Cardiac Troponin T Mutation Linked to Infantile Restrictive Cardiomyopathy

Abstract

A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (N100 and E101) was identified in the cardiac cDNA of a pediatric transplant recipient. The patient previously presented with restrictive and hypertrophic cardiomyopathy. Family work-up was negative, and she was found to harbor a de novo mutation. Electron microscopy revealed the presence of myofibrillar disarray and fibrosis. To further define this cTnT mutation as a cause of the disease, functional studies were performed. Functional effects of the single and double cTnT mutants (ΔN100, ΔE101 and ΔN100/ΔE101) were analyzed in porcine skinned papillary muscle. Fibers were displaced with exogenous cTnT mutants or WT, Ca2+ unregulated force was measured and then reconstituted with binary cTnI.cTnC complex. The ΔN100 and ΔE101 mutations showed opposing changes in the Ca2+ sensitivity of force development compared to WT. The ΔN100 mutation increased this by 0.29 pCa units and the ΔE101 mutation, in contrast, decreased it by 0.28 pCa units. Interestingly, the ΔN100/ΔE101 mutation shifted the Ca2+ sensitivity to the left (+ 0.19 pCa units). This finding is compatible with the preserved systolic function in this patient. ΔE101 was the only mutation that decreased the maximal force recovery compared to WT. In contrast, ΔN100 and ΔN100/ΔE101 did not show significant changes in this parameter. Both ΔN100 and ΔN100/ΔE101 exhibited decreased cooperativity of force development, suggesting altered intra-filament protein-protein interactions. These data show that residue N100 has a predominant effect over E101 and its absence is much more deleterious for cTnT function. In addition, the strength of the functional data validates this novel cTnT deletion mutant as the cause of this cardiomyopathy. Supported by NIH HL-42325(JDP), AHA 0825368E (JRP), AHA 09POST2300030 (MSP) and CIHR GMHD 79045 (GA).