Journal
PLOS ONE
Volume 3, Issue 7, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0002642
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Funding
- American Heart Association [Postdoctoral Fellowships]
- Hillgrove Foundation [Postdoctoral Fellowships]
- Howard Hughes Medical Institute
- McGinnis Chair Endowment Funds
- Cardiovascular Institute of the University of Pittsburgh
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Background: Hypertrophic (HCM) and dilated (DCM) cardiomyopathies result from sarcomeric protein mutations, including cardiac troponin T (cTnT, TNNT2). We determined whether TNNT2 mutations cause cardiomyopathies by altering cTnT function or quantity; whether the severity of DCM is related to the ratio of mutant to wildtype cTnT; whether Ca(2+) desensitization occurs in DCM; and whether absence of cTnT impairs early embryonic cardiogenesis. Methods and Findings: We ablated Tnnt2 to produce heterozygous Tnnt2(+/-) mice, and crossbreeding produced homozygous null Tnnt2(-/-) embryos. We also generated transgenic mice overexpressing wildtype (TG(WT)) or DCM mutant (TG(K210 Delta)) Tnnt2. Crossbreeding produced mice lacking one allele of Tnnt2, but carrying wildtype ( Tnnt2(+/-)/TG(WT)) or mutant (Tnnt2(+/-)/ TG(K210 Delta)) transgenes. Tnnt2(+/-) mice relative to wildtype had significantly reduced transcript (0.82 +/- 0.06[SD] vs. 1.00 +/- 0.12 arbitrary units; p = 0.025), but not protein (1.01 +/- 0.20 vs. 1.00 +/- 0.13 arbitrary units; p = 0.44). Tnnt2(+/-) mice had normal hearts ( histology, mass, left ventricular end diastolic diameter [LVEDD], fractional shortening [FS]). Moreover, whereas Tnnt2(+/-)/TGK(210 Delta) mice had severe DCM, TGK(210 Delta) mice had only mild DCM (FS 18 +/- 4 vs. 29 +/- 7%; p<0.01). The difference in severity of DCM may be attributable to a greater ratio of mutant to wildtype Tnnt2 transcript in Tnnt2(+/-)/TGK(210 Delta) relative to TGK(210 Delta) mice (2.42 +/- 0.08, p = 0.03). Tnnt2(+/-)/TGK(210 Delta) muscle showed Ca(2+) desensitization (pCa(50) = 5.34 +/- 0.08 vs. 5.58 +/- 0.03 at sarcomere length 1.9 mu m, p<0.01), but no difference in maximum force generation. Day 9.5 Tnnt2(-/-) embryos had normally looped hearts, but thin ventricular walls, large pericardial effusions, noncontractile hearts, and severely disorganized sarcomeres. Conclusions: Absence of one Tnnt2 allele leads to a mild deficit in transcript but not protein, leading to a normal cardiac phenotype. DCM results from abnormal function of a mutant protein, which is associated with myocyte Ca(2+) desensitization. The severity of DCM depends on the ratio of mutant to wildtype Tnnt2 transcript. cTnT is essential for sarcomere formation, but normal embryonic heart looping occurs without contractile activity.
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