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Altered regulatory function of two familial hypertrophic cardiomyopathy troponin T mutants
P Mukherjea1, L Tong, J G Seidman
1Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Insights
Familial hypertrophic cardiomyopathy mutations create truncated cardiac troponin T. These troponin T mutants impair muscle contraction by reducing Ca2+ sensitivity and troponin I binding, suggesting a molecular basis for the disease.
Area of Science:
- Cardiology
- Molecular Biology
- Biochemistry
Background:
- Familial hypertrophic cardiomyopathy (HCM) is a genetic heart disease linked to mutations in cardiac troponin T.
- Cardiac troponin T is crucial for regulating muscle contraction by binding to tropomyosin.
- Specific mutations can lead to truncated troponin T variants, affecting cardiac function.
Purpose of the Study:
- To investigate the functional consequences of two C-terminal deletion mutants of human cardiac troponin T.
- To determine how these mutations impact the regulation of actomyosin ATPase activity and Ca2+ sensitivity.
- To explore the potential molecular basis of HCM caused by these troponin T variants.
Main Methods:
- Expression of wild-type and mutant cardiac troponin T cDNAs in Escherichia coli.
- Functional analysis of troponin T complexes with cardiac troponin C and troponin I.
- Measurement of actomyosin MgATPase activity under varying Ca2+ concentrations.
Main Results:
- Both C-terminal deletion mutants formed complexes with troponin C and troponin I, regulating actomyosin MgATPase similarly to wild-type.
- Mutant troponin Ts exhibited severely reduced activation of actomyosin in the presence of Ca2+.
- Mutant troponin Ts showed reduced affinity for troponin I, with one mutant being at least 6-fold weaker.
Conclusions:
- C-terminal deletions in cardiac troponin T impair the thin filament's "on" state switching mechanism.
- Reduced affinity for troponin I is a likely molecular basis for the contractile dysfunction observed in HCM patients with these mutations.
- These findings provide insights into the pathobiology of familial hypertrophic cardiomyopathy.
Abstract:
Mutations in the gene encoding human cardiac troponin T can cause familial hypertrophic cardiomyopathy, a disease that is characterized by ventricular hypertrophy and sudden, premature death. Troponin T is the tropomyosin-binding subunit of troponin required for thin filament regulation of contraction. One mutation, a change in the intron 15 splice donor site, results in two truncated forms of troponin T [Thierfelder et al. (1994) Cell 77, 701-712]. In one form, the mRNA skips exon 16 that encodes the C-terminal 14 amino acids; in the other, seven novel residues replace the exon 15- and 16-encoded C-terminal 28 amino acids. The two troponin T cDNAs were expressed in Escherichia coli for functional analysis. Both C-terminal deletion mutants formed a complex with cardiac troponin C and troponin I that exhibited the same concentration dependence as wild-type for regulation of the actomyosin MgATPase. However, both mutants showed severely reduced activation of the regulated actomyosin in the presence of Ca2+, though the inhibition in the absence of Ca2+ and the Ca(2+)-dependence of activation were not altered. The C-terminal deletions reduce the effectiveness of Ca(2+)-troponin to switch the thin filament from the "off" to the "on" state. Both mutant troponin Ts have reduced affinity for troponin I; the shorter mutant is at least 6-fold weaker than wild-type. The low level of activation of the ATPase would be consistent with reduced contractile performance, and the results suggest reduced troponin I affinity may be the molecular basis for the disease.