Fetal Cardiac Troponin Isoforms Rescue the Increased Ca 2+ Sensitivity Produced by a Novel Double Deletion in Cardiac Troponin T Linked to Restrictive Cardiomyopathy

Abstract

A novel double deletion in cardiac troponin T (cTnT) of two highly conserved amino acids (N100 and E101) was found in a restrictive cardiomyopathic (RCM) pediatric patient. Clinical evaluation revealed the presence of left atrial enlargement and marked left ventricle diastolic dysfunction. The explanted heart examined by electron microscopy revealed myofibrillar disarray and mild fibrosis. Pedigree analysis established that this mutation arose de novo. Functional studies were carried out in the fetal (cTnT1 + slow skeletal troponin I (ssTnI)) and adult (cTnT3 and cardiac TnI (cTnI)) troponin (Tn) backgrounds. The single and double cTnT mutants (cTnT3-ΔN100, cTnT3-ΔE101, cTnT3-ΔN100/ΔE101, cTnT1-ΔN110, cTnT1-ΔE111 and cTnT1-ΔN110/ΔE111) were generated and their functional effects analyzed in skinned cardiac muscle. In the adult Tn enviroment, the single cTnT3-ΔN100 and cTnT3-ΔE101 mutations in the presence of cTnI caused opposing changes on the Ca2+ sensitivity of force development compared to WT with respective values of +0.29 and −0.28 pCa units; yet the cTnT3-ΔN100/ΔE101 mutation shifted the Ca2+ sensitivity to the left (+ 0.19 pCa units). In addition, cTnT3-ΔN100/ΔE101 decreased cooperativity of force development suggesting altered intra-filament protein-protein interactions. In the fetal Tn eviroment, the single (cTnT1-ΔN110) and double (cTnT1-ΔN110/ΔE111) deletion in the presence of ssTnI did not show any change in the Ca2+ sensitivity compared to its control. In an attempt to recreate the patient's heterozygous genotype we performed a reconstituted ATPase activity assay. Thin filaments containing 50:50% of cTnT3-ΔN100/ΔE101:cTnT3-WT also showed increased Ca2+ sensitivity of the myofilament compared to the WT. This report reveals the protective role of the Tn fetal isoforms since they rescue the increased Ca2+ sensitivity produced by a cTnT-RCM mutation and may account for this mutation not being lethal during gestation.