In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron.

Differentiation of mesenchymal stem cells (MSC) into specialised cells in vitro before transplantation may improve the engraftment efficiency of the transplanted cells as well as the safety and efficacy of treatment. To understand the differentiation process and the functional identities of cells in an animal model, we examined the in vitro differentiation capacity of porcine MSC (3–6 passage) into cardiomyocyte-like and neuron-like cells. The MSC isolated from the bone marrow of postnatal miniature piglets [T-type, PWG Micro-pig (R), PWG Genetics, Korea] exhibited a typical fibroblast-like morphology and expressed the specific markers, such as CD29, CD44, and CD90. After 21 days of culture in induction media, MSC revealed the appropriate phenotype of osteocytes (von Kossa and Alizarin red), adipocytes (Oil red O), and chondrocytes (Alcian blue). Ther MSC were further induced into cardiomyogenic and neurogenic differentiation following the protocols described earlier (Tomita et al. 2002 J. Thorac. Cardiovasc. Surg. 123, 1132–1140) and (Woodbury et al. 2002 J. Neurosci. Res. 96, 908–917), respectively, with minor modifications. Expression of lineage-specific markers was evaluated by immunocytochemistry, and RT-PCR and quantitative PCR (RT-qPCR). For cardiomyogenic differentiation, MSC were stimulated with 10 μM 5-azacytidine for 24 h, 3 days, or 7 days, and the cells were maintained in culture for 21 days. Upon induction, MSC exhibited elongated and stick-like morphology with extended cytoplasmic processes, and toward the end of culture, cells formed aggregates and myotube-like structures. Immunostaining was positive for the markers of cardiomyocyte-like cells, such as α-smooth muscle actin, cardiac troponin T, desmin, and α-cardiac actin. The RT-PCR and RT-qPCR analysis showed the expression and a time dependent up-regulation of cardiac troponin T, desmin, α-cardiac actin, and β-myosin heavy chain genes. Following induction with neuronal-specific media for 3 days, above 80% of MSC acquired the morphology of neuron-like cells with bi- or multipolar cell processes forming a network-like structure. Induced cells with neuronal phenotype were positively stained for nestin, neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and neurofilament-M (NF-M). The expression of neural transcripts, such as nestin, GFAP, and NF-M, was further confirmed by RT-PCR and RT-qPCR. In conclusion, our results showed the potential of porcine MSC to differentiate in vitro into cardiomyocyte-like and neuron-like cells, thus offering a useful model for studying their functional and molecular properties before transplantation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.

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Cardiac troponin I but not cardiac troponin T adheres to polysulfone dialyser membranes in an in vitro haemodialysis model: explanation for lower serum cTnI concentrations following dialysis

Open Heart ◽

10.1136/openhrt-2014-000108 ◽

2014 ◽

Vol 1 (1) ◽

pp. e000108 ◽

Cited By ~ 14

Author(s):

David C Gaze ◽

Paul O Collinson

Keyword(s):

Cardiac Troponin ◽

Troponin I ◽

Troponin T ◽

Cardiac Troponin I ◽

Cardiac Troponin T ◽

Lower Serum ◽

Model Explanation

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Abstract 62: Cardiac Troponin T Isoform Switching in Early Childhood Tropomyosin-linked Dilated Cardiomyopathy

Circulation Research ◽

10.1161/res.117.suppl_1.62 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Melissa Lynn ◽

Lauren Tal-Grinspan ◽

J.-P. Jin ◽

Jil Tardiff

Keyword(s):

Mouse Model ◽

Cardiac Troponin ◽

Troponin T ◽

Systolic Function ◽

Phenotypic Variability ◽

Bimodal Distribution ◽

General Mechanism ◽

Cardiac Troponin T ◽

Human Heart Failure

An oft-noted component of sarcomeric DCM is the observation that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. In a recent study of two unrelated multigenerational families with the tropomyosin (Tm) mutation Asp230Asn (D230N), a striking “bimodal” distribution of severity was observed. In these families, many children (<1 year) with the mutation presented with a severe form of DCM that led to sudden, often fatal CHF while adults developed a mild to moderate DCM in mid-life. Of note, children who survived the initial presentation often recovered significant systolic function into young adulthood. A potential hypothesis to explain this improvement despite the continued presence of the mutant Tm, is that the phenotype is modified by other thin filament isoforms. Thus we propose that the age-dependent remodeling seen in children with D230N Tm is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). Our initial biophysical studies (Regulated-IVM) revealed a decreased Ca2+ sensitivity in filaments containing D230N Tm that is more severe in the presence of fetal TnT (cTnT1), suggesting a modulatory role for cTnT1. Cardiac performance, assessed via 2D echo, in our novel D230N Tm x cTnT1 double transgenic (DTg) mouse model found a significantly reduced % FS for DTg (17%) mice as compared to D230N Tm (21%) littermates. This reduction in %FS was seen at 4 months but not 2 suggesting a progressive cardiomyopathy. Current efforts aim to model the early phase of this “bimodal” phenotype and assess the potential for disease reversibility using a cardiac specific inducible cTnT1 transgenic mouse model. Furthermore, we propose that modulation by cTnT1 could represent a more general mechanism for the progressive remodeling seen in human heart failure. Preliminary in vitro studies with human tissue found that RNA levels of cTnT1 are significantly higher in failing hearts as compared to non-failing. Thus these data suggest an isoform dependent mechanism for the “bimodal” phenotype in patients carrying D230N Tm that could translate to other sarcomeric cardiomyopathies.

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A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4898 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4898-4907 ◽

Cited By ~ 92

Author(s):

J Ramchatesingh ◽

A M Zahler ◽

K M Neugebauer ◽

M B Roth ◽

T A Cooper

Keyword(s):

Cardiac Troponin ◽

Troponin T ◽

Sr Proteins ◽

Alternative Exon ◽

Cross Linking ◽

Cardiac Troponin T ◽

Sr Protein ◽

Exon Inclusion ◽

Enhancer Rna

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion.

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Development and In Vitro Characterization of a New Immunoassay of Cardiac Troponin T

Clinical Chemistry ◽

10.1093/clinchem/38.3.386 ◽

1992 ◽

Vol 38 (3) ◽

pp. 386-393 ◽

Cited By ~ 258

Author(s):

H A Katus ◽

S Looser ◽

K Hallermayer ◽

A Remppis ◽

T Scheffold ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Cardiac Troponin ◽

Solid Phase ◽

Troponin T ◽

Immunoblot Analysis ◽

T Antigen ◽

Cardiac Troponin T ◽

In Vitro Characterization ◽

Hybridoma Technology

Abstract We describe a new one-step enzyme immunoassay of troponin T that uses two specific monoclonal antibodies and streptavidin-coated tubes as the solid phase. The monoclonal antibodies were obtained by conventional hybridoma technology, with human troponin T as antigen. The identity of the cardiac troponin T antigen was confirmed by analysis of the amino acid composition and by partial sequence analysis. The specificity of the monoclonal antibodies for cardiac troponin T was proved by immunoblot analysis and displacement curves. The capture antibody is labeled with biotin. The second antibody is conjugated to horseradish peroxidase (EC 1.11.1.7). The assay [Enzymun-TestR system (Boehringer Mannheim GmbH)] is performed in only 90 min at room temperature; the measuring range for troponin T is 0.1 to 15 micrograms/L. The assay shows excellent between-run precision (CV = 3.3-4.9%).

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