Deficiency of slow skeletal muscle troponin T causes atrophy of type I slow fibres and decreases tolerance to fatigue.

The total loss of slow skeletal muscle troponin T (ssTnT encoded by TNNT1 gene) due to a nonsense mutation in codon Glu(180) causes a lethal form of recessively inherited nemaline myopathy (Amish nemaline myopathy, ANM). To investigate the pathogenesis and muscle pathophysiology of ANM, we studied the phenotypes of partial and total loss of ssTnT in Tnnt1 gene targeted mice. An insertion of neomycin resistance cassette in intron 10 of Tnnt1 gene caused an approximately 60% decrease in ssTnT protein expression whereas cre-loxP-mediated deletion of exons 11-13 resulted in total loss of ssTnT, as seen in ANM muscles. In diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that ssTnT deficiency resulted in significantly decreased levels of other slow fibre-specific myofilament proteins whereas fast fibre-specific myofilament proteins were increased correspondingly. Immunohistochemical studies revealed that ssTnT deficiency produced significantly smaller type I slow fibres and compensatory growth of type II fast fibres. Along with the slow fibre atrophy and the changes in myofilament protein isoform contents, ssTnT deficiency significantly reduced the tolerance to fatigue in soleus muscle. ssTnT-deficient soleus muscle also contains significant numbers of small-sized central nuclei type I fibres, indicating active regeneration. The data provide strong support for the essential role of ssTnT in skeletal muscle function and the causal effect of its loss in the pathology of ANM. This observation further supports the hypothesis that the function of slow fibres can be restored in ANM patients if a therapeutic supplement of ssTnT is achieved.

Suppression of cardiac troponin T induces reduction of contractility and structural disorganization in chicken cardiomyocytes.

We herein examine the effect of cardiac troponin T (CTnT) suppression in cultured chicken cardiomyocytes derived from embryonic cardiac ventricular muscle. TnT is an important protein participating in regulation of striated muscle contraction, but it is not clear whether TnT contributes to the formation of sarcomere structure in myofibrils. Double-stranded RNA homologous to the nucleotide sequence of CTnT (CTnT-siRNA) was introduced into cultured muscle cells two days after plating. Transfection efficiency was above 80%. Immunoblot analyses suggested that the expression of CTnT progressively falls for the three consecutive days after transfection, but partly reappears on the fourth day. Maximum suppression occurs three days after transfection, with almost invisible CTnT protein on immunoblots in all the examined conditions: 0.5-2 nmol CTnT-siRNA towards 1-3 x 10(6) cells. The suppression was specific to CTnT, and the other myofibrillar proteins such as myosin, connectin/titin, tropomyosin, alpha-actinin, and troponin I were all present in transfected cells. The following functional and morphological changes were detected in CTnT-suppressed cells. The population of beating cells decreased significantly after transfection, when compared to control cells. A part of CTnT-suppressed cells showed two non-overlapping types of morphological changes: 1) myofibrils presenting unusually long Z-Z intervals; 2) myofibrils with irregular small striations in cells not connected at their adhesion interfaces of a jagged-appearance. Thus, our results reveal that CTnT is important for stable beating in cultured ventricular muscle cells, and also to some extent, for maintaining myofibrillar structure and cell-to-cell adhesion.

Hypertrophy, fibrosis, and sudden cardiac death in response to pathological stimuli in mice with mutations in cardiac troponin T.

BACKGROUND: Transgenic mouse models expressing a missense mutation (R92Q) or a splice donor site mutation (trunc) in the cardiac troponin T (cTnT) model familial hypertrophic cardiomyopathy (FHC) in humans. Although males from these strains share the unusual property of having significantly smaller ventricles and cardiac myocytes, they differ with regard to systolic function, fibrosis, and gene expression. Little is known about how these phenotypes affect the responses to additional pathological stimuli. METHODS AND RESULTS: We tested the ability of hearts of both sexes of wild-type and mutant mice to respond to defined pathological, pharmacological, hypertrophic stimuli in vivo. Hearts of mutant cTnT models of both sexes were able to undergo hypertrophy in response to at least one stimulus, but the extent differed between the 2 mutants and was sex specific. Interestingly, the trunc-mutant mouse heart was resistant to the development of fibrosis in response to pharmacological stimuli. Stimulation with 2 adrenergic agonists led to sudden cardiac death of all male but not female mutant animals, which suggests altered adrenergic responsiveness in these 2 models of FHC. CONCLUSIONS: Hypertrophic signaling is differentially affected by distinct mutations in cTnT and is sex modified. Hearts can respond with either an augmented hypertrophic and fibrotic response or a diminished hypertrophy and resistance to fibrosis. Sudden cardiac death is related to adrenergic stress and is independent of the development of fibrosis but occurred only in male mice. These results suggest that patients with certain TnT mutations may respond to certain pathological situations with a worsened phenotype.

Cardiac troponin T is essential in sarcomere assembly and cardiac contractility.

Mutations of the gene (TNNT2) encoding the thin-filament contractile protein cardiac troponin T are responsible for 15% of all cases of familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes. Mutant proteins are thought to act through a dominant-negative mode that impairs function of heart muscle. TNNT2 mutations can also lead to dilated cardiomyopathy, a leading cause of heart failure. Despite the importance of cardiac troponin T in human disease, its loss-of-function phenotype has not been described. We show that the zebrafish silent heart (sih) mutation affects the gene tnnt2. We characterize two mutated alleles of sih that severely reduce tnnt2 expression: one affects mRNA splicing, and the other affects gene transcription. Tnnt2, together with alpha-tropomyosin (Tpma) and cardiac troponins C and I (Tnni3), forms a calcium-sensitive regulatory complex within sarcomeres. Unexpectedly, in addition to loss of Tnnt2 expression in sih mutant hearts, we observed a significant reduction in Tpma and Tnni3, and consequently, severe sarcomere defects. This interdependence of thin-filament protein expression led us to postulate that some mutations in tnnt2 may trigger misregulation of thin-filament protein expression, resulting in sarcomere loss and myocyte disarray, the life-threatening hallmarks of TNNT2 mutations in mice and humans.

Manipulating the contractile apparatus: genetically defined animal models of cardiovascular disease.

Within the last 10 years via gene targeting and transgenesis, numerous models of cardiovascular disease have been established and used to determine if a protein's presence or absence causes cardiovascular disease. By affecting the heart's protein complement in a defined manner, the function of the different mutated proteins or protein isoforms present in the contractile apparatus can be determined and pathogenic mechanism(s) explored. We can now remodel the cardiac protein profile and effect replacement of even the most abundant contractile proteins. Precise genetic manipulation allows exploration of the structure-function relationships which underlie cardiac function, and the consequences of defined mutations at the molecular, biochemical, cytological and physiologic levels can be determined.