Abstract 921: Cardiomyopathies Secondary to Cardiac Troponin T Mutations Result from Abnormal Protein Function Rather than Haploinsufficiency
BACKGROUND: Hypertrophic (HCM) and dilated (DCM) cardiomyopathies result from sarcomere protein mutations, e.g. cardiac troponin T (cTnT, TNNT2), but mechanisms are uncertain. Using mouse models, we tested the hypotheses (1) TNNT2 mutations cause cardiomyopathies by altering cTnT function, not reducing quantity; (2) myocyte Ca2+ desensitization occurs in DCM; (3) early embryonic cardiogenesis does not require contractility.
METHODS: By homologous recombination, we ablated 1 Tnnt2 allele to produce heterozygous Tnnt2+/− mice (129/SvEv strain). Crossbreeding Tnnt2+/− mice produced homozygous null Tnnt2−/− embryos. We generated transgenic mice with cDNA encoding mutant (TGMut) or wildtype (TGWT) Tnnt2 driven by the αMHC promoter. TGMut had a human DCM mutation, K210 deletion. Crossbreeding produced mice with heterozygous ablations of Tnnt2 and mutant (Tnnt2+/−/TGMut) or WT (Tnnt2+/−/TGWT) transgenes.
RESULTS: Tnnt2+/− mice had a 20% deficit in transcript (0.82±0.06[SD] vs. 1.00±0.12 arbitrary units; p=0.025), but no deficit in protein (1.8±1.3 vs. 1.0±0.4 arbitrary units; p=0.27) vs. WT (n=4/group). Tnnt2+/− mice had normal hearts (histology, mass, fractional shortening [FS], absence of arrhythmias). However, Tnnt2+/−/TGMut mice (n=20) had severe DCM vs. Tnnt2+/−/TGWT (n=4). Echocardiography showed LV end diastolic dilation (4.66±0.49 vs. 3.29±0.56 mm; p=10-3) and depressed FS (19±6 vs. 45±7%; p=10-6). Biomechanical studies on Tnnt2+/−/TGMut papillary muscles (n=4) showed Ca2+ desensitization vs. WT (n=8) (pCa50=5.48±0.04 vs. 5.83±0.06 at sarcomere length (SL)= 1.9 μm; 5.55±0.06 vs. 5.92±0.05 at SL=2.3 μm; p=10-5), but no difference in maximum force generation. Tnnt2−/− embryos (n=20) had normally looped hearts at embryo day 9.5, but thin ventricular walls, large pericardial effusions, noncontractile hearts, and severely disorganized sarcomeres.
CONCLUSIONS: Absence of 1 Tnnt2 allele leads to only a mild deficit in transcript and no deficit in protein, leading to a normal phenotype. DCM results from abnormal function of a mutant protein and is associated with myocyte Ca2+ desensitization in vivo. cTnT is essential for sarcomere formation, but normal looping of the embryonic heart occurs in the absence of contractile activity.