A Cardiac-Specific Troponin I Promoter. Distinctive Patterns of Regulation in Cultured Fetal Cardiomyocytes, Adult Heart and Transgenic Mice

We have produced seven lines of transgenic mice carrying the quail gene encoding the fast skeletal muscle-specific isoform of troponin I (TnIf). The quail DNA included the entire TnIf gene, 530 base pairs of 5'-flanking DNA, and 1.5 kilobase pairs of 3'-flanking DNA. In all seven transgenic lines, normally initiated and processed quail TnIf mRNA was expressed in skeletal muscle, where it accumulated to levels comparable to that in quail muscle. Moreover, in the three lines tested, quail TnIf mRNA levels were manyfold higher in a fast skeletal muscle (gastrocnemius) than in a slow skeletal muscle (soleus). We conclude that the cellular mechanisms directing muscle fiber type-specific TnIf gene expression are mediated by cis-regulatory elements present on the introduced quail DNA fragment and that they control TnIf expression by affecting the accumulation of TnIf mRNA. These elements have been functionally conserved since the evolutionary divergence of birds and mammals, despite the major physiological and morphological differences existing between avian (tonic) and mammalian (twitch) slow muscles. In lines of transgenic mice carrying multiple tandemly repeated copies of the transgene, an aberrant quail TnIf transcript (differing from normal TnIf mRNA upstream of exon 2) also accumulated in certain tissues, particularly lung, brain, spleen, and heart tissues. However, this aberrant transcript was not detected in a transgenic line which carries only a single copy of the quail gene.

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Morphometry and muscle gene expression in hypertrophied hearts from polyomavirus large T antigen transgenic mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.269.1.h86 ◽

1995 ◽

Vol 269 (1) ◽

pp. H86-H95 ◽

Cited By ~ 1

Author(s):

E. Holder ◽

B. Mitmaker ◽

L. Alpert ◽

L. Chalifour

Keyword(s):

Transgenic Mice ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Light Chain ◽

Myosin Light Chain ◽

Troponin I ◽

T Antigen ◽

Large T Antigen ◽

Cross Sectional

Transgenic mice expressing polyomavirus large T antigen (PVLT) in cardiomyocytes develop a cardiac hypertrophy in adulthood. Morphometric analysis identified cardiomyocytes enlarged up to ninefold in cross-sectional area in the adult transgenic hearts compared with normal age-matched nontransgenic hearts. Most enlarged cardiomyocytes were found in the subendocardium, whereas normal-sized cardiomyocytes were localized to the midmyocardium. Transgenic hearts did not express detectable skeletal muscle actin mRNA or protein, or skeletal troponin I isoform mRNA. Some, but not all, transgenic hearts expressed an increase in the beta-myosin heavy chain mRNA. All five transgenic mice tested had increased expression of atrial natriuretic factor (ANF) mRNA. Whereas normal hearts expressed three myosin light chain proteins of 19, 16, and 15 kDa, we found that the 19-kDa myosin light chain was not observed in the transgenic hearts. We conclude that adult, PVLT-expressing, transgenic mice developed enlarged cardiomyocytes with an increase in beta-myosin heavy chain and ANF mRNA expression, but a widespread skeletal isoform usage was not present in these transgenic mice. The adult transgenic hearts thus display histological and molecular changes similar to those found in hypertrophy induced by a pressure overload in vivo.

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The Human Troponin I Slow Promoter Directs Slow Fiber-Specific Expression in Transgenic Mice

DNA and Cell Biology ◽

10.1089/dna.1995.14.599 ◽

1995 ◽

Vol 14 (7) ◽

pp. 599-607 ◽

Cited By ~ 16

Author(s):

LINDA K. LEVITT ◽

JOHN V. O'MAHONEY ◽

KAREN J. BRENNAN ◽

JOSEPHINE E. JOYA ◽

LEI ZHU ◽

...

Keyword(s):

Transgenic Mice ◽

Troponin I ◽

Specific Expression ◽

Slow Fiber

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Systematic evaluation of a novel model for cardiac ischemic preconditioning in mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00472.2006 ◽

2006 ◽

Vol 291 (5) ◽

pp. H2533-H2540 ◽

Cited By ~ 94

Author(s):

Tobias Eckle ◽

Almut Grenz ◽

David Köhler ◽

Andreas Redel ◽

Melanie Falk ◽

...

Keyword(s):

Coronary Artery ◽

Transgenic Mice ◽

Infarct Size ◽

Ischemic Preconditioning ◽

Troponin I ◽

Coronary Artery Occlusion ◽

Systematic Evaluation ◽

Weight System ◽

Area At Risk ◽

Triphenyltetrazolium Chloride

Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of transgenic mice seems critical. Due to technical difficulty associated with performing cardiac IP in mice, we developed an in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion. This technique has the major advantage of eliminating the necessity of intermittently occluding the coronary artery with a knotted suture. To systematically evaluate this model, we first demonstrated correlation of ischemia times (10–60 min) with infarct sizes [3.5 ± 1.3 to 42 ± 5.2% area at risk (AAR), Evan’s blue/triphenyltetrazolium chloride staining]. IP (4 × 5 min) and cold ischemia (27°C) reduced infarct size by 69 ± 6.7% and 84 ± 4.2%, respectively ( n = 6, P < 0.01). In contrast, lower numbers of IP cycles did not alter infarct size. However, infarct sizes were distinctively different in mice from different genetic backgrounds. In addition to infarct staining, we tested cardiac troponin I (cTnI) as marker of myocardial infarction in this model. In fact, plasma levels of cTnI were significantly lower in IP-treated mice and closely correlated with infarct sizes ( R2 = 0.8). To demonstrate transcriptional consequences of cardiac IP, we isolated total RNA from the AAR and showed repression of the equilibrative nucleoside transporters 1–4 by IP in this model. Taken together, this study demonstrates highly reproducible infarct sizes and cardiac protection by IP, thus minimizing the variability associated with knot-based coronary occlusion models. Further studies on cardiac IP using transgenic mice may consider this technique.

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Functional Consequences of the Human Cardiac Troponin I Hypertrophic Cardiomyopathy Mutation R145G in Transgenic Mice

Journal of Biological Chemistry ◽

10.1074/jbc.m801661200 ◽

2008 ◽

Vol 283 (29) ◽

pp. 20484-20494 ◽

Cited By ~ 43

Author(s):

Yuhui Wen ◽

Jose Renato Pinto ◽

Aldrin V. Gomes ◽

Yuanyuan Xu ◽

Yingcai Wang ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Transgenic Mice ◽

Cardiac Troponin ◽

Troponin I ◽

Cardiac Troponin I ◽

Functional Consequences

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cis-acting sequences of the rat troponin I slow gene confer tissue- and development-specific transcription in cultured muscle cells as well as fiber type specificity in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.7019 ◽

1993 ◽

Vol 13 (11) ◽

pp. 7019-7028 ◽

Cited By ~ 44

Author(s):

S Banerjee-Basu ◽

A Buonanno

Keyword(s):

Skeletal Muscle ◽

Transgenic Mice ◽

Transcription Initiation ◽

Troponin I ◽

Regulatory Elements ◽

Initiation Site ◽

C2c12 Cells ◽

Fiber Types ◽

Tibialis Muscle ◽

Flanking Sequences

Transcription of the genes coding for troponin I slow (TnIslow) and other contractile proteins is activated during skeletal muscle differentiation, and their expression is later restricted to specific fiber types during maturation. We have isolated and characterized the rat TnIslow gene in order to begin elucidating its regulation during myogenesis. Transcriptional regulatory regions were delineated by using constructs, containing TnIslow gene sequences driving the expression of the chloramphenicol acetyltransferase (CAT) reporter gene, that were transiently transfected into undifferentiated and differentiated C2C12 cells. TnIslow 5'-flanking sequences directed transcription specifically in differentiated cells. However, transcription rates were approximately 10-fold higher in myotubes transfected with constructs containing the 5'-flanking sequences plus the intragenic region residing upstream of the translation initiation site (introns 1 and 2), indicative of interactions between elements residing upstream and in the introns of the gene. Deletion analysis of the 5' region of the TnIslow gene showed that the 200 bp upstream of the transcription initiation site is sufficient to confer differentiation-specific transcription in C2C12 myocytes. MyoD consensus binding sites were found both in the upstream 200-bp region and in a region residing in the second intron that is highly homologous to the quail TnIfast enhancer. Transactivation experiments using transfected NIH 3T3 fibroblasts with TnI-CAT constructs containing intragenic and/or upstream sequences and with the myogenic factors MyoD, myogenin, and MRF4 showed different potentials of these factors to induce transcription. Transgenic mice harboring the rat TnI-CAT fusion gene expressed the reporter specifically in the skeletal muscle. Furthermore, CAT levels were approximately 50-fold higher in the soleus than in the extensor digitorum longus, gastrocnemius, or tibialis muscle, indicating that the regulatory elements that restrict TnI transcription to slow-twitch myofibers reside in the sequences we have analyzed.

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Abstract 268: Myofilament Tyrosine Phosphorylation by Src Kinase is upregulated in ErbB2 Transgenic Mice

Circulation Research ◽

10.1161/res.117.suppl_1.268 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Mingguo Xu ◽

Genaro A Ramirez-Correa ◽

Zongming Fu ◽

Polina S Shah ◽

Frances Belmonte ◽

...

Keyword(s):

Transgenic Mice ◽

Cardiac Hypertrophy ◽

Western Blot ◽

Cell Signaling ◽

Light Chain ◽

Blot Analysis ◽

Western Blot Analysis ◽

Troponin I ◽

Src Kinase ◽

Post Translational Modification

Objectives: Tyrosine (Tyr) phosphorylation of the myofilament is an emerging, and potentially important, post-translational modification in cardiomyopathy. ErbB2, a Tyr receptor kinase, was overexpressed in transgenic mice (ErbB2-Tg) resulting in significant cardiac hypertrophy. We hypothesize that the development of cardiac hypertrophy in ErbB2-Tg is associated with increased myofilament Tyr phosphorylation and may implicate myofilament Tyr phosphorylation in cardiac hypertrophy. Methods: Proteins were isolated from ErbB2-Tg and Ntg heart homogenates ( n =4 per group). Reduction/alkylation was followed by trypsinization. Resulting peptides were desalted in C 18 columns and lyophilized. Phosphorylated Tyr (p-Tyr) enrichment was performed on 20 mg of peptides using a p-Tyr Mouse mAb kit (Cell Signaling). Immuno-precipitated and desalted peptides were analyzed by LC-MS/MS (Orbitrap Elite, Thermo). Raw data were searched with Mascot 2.3. Label-free quantification with MS1 extracted ion chromatograms was performed using Skyline. Western blot analysis for total and phosphorylated Src kinase was performed per manufacturer’s protocol (Cell Signaling). Results: We found a total of 286 p-Tyr modified peptides in ErbB2-Tg compared to 226 in control NTg mice. Over 70 p-Tyr sites on myofilament protein were up-regulated in ErbB2-Tg, including troponin I, myosin heavy chain, titin, α-tropomyosin, myosin-binding protein-C3, myosin regulatory light chain-2 and myosin light chain-1. We used PhosphoMotif Finder to search the potential responsible kinase. Most of the p-Tyr sites were consistent with Src kinase motifs. Furthermore, Western blot analysis showed that total, and phospho-Src (Y416) expression was increased in ErbB2-Tg mice. Conclusion: We concluded that these novel p-Tyr sites on myofilament proteins are increased in ErbB2-Tg mice and correlate with up-regulated Src kinase activity. Thus increased tyrosine myofilament phosphorylation may be involved in the development of cardiac hypertrophy. Since ErbB2 is a therapeutic target of trastuzumab therapy this may also have translational implications to ameliorate off target effects of cancer treatment.

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Changes in force and calcium sensitivity in the developing avian heart

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-251 ◽

1991 ◽

Vol 69 (11) ◽

pp. 1692-1697 ◽

Cited By ~ 29

Author(s):

R. E. Godt ◽

R. T. H. Fogaça ◽

T. M. Nosek

Keyword(s):

Young Adult ◽

Heart Development ◽

Troponin I ◽

Calcium Sensitivity ◽

Extracellular Calcium ◽

Contractile Apparatus ◽

Chicken Heart ◽

Twitch Force ◽

Adult Heart ◽

Isoform Switching

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45–3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I. Moreover, the decrease in maximal calcium-activated force with pH was also greater in trabeculae from young adult heart. Thus the contractile apparatus of embryonic chicken heart is less sensitive to the depressant effects of acidosis than that of the adult heart. These developmental changes in calcium sensitivity are analogous to those reported by others in mammalian heart.Key words: muscle contraction, heart development, acidosis, chicken, contractile proteins.

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IL-15 Overexpression Promotes Endurance, Oxidative Energy Metabolism, and Muscle PPARδ, SIRT1, PGC-1α, and PGC-1β Expression in Male Mice

Endocrinology ◽

10.1210/en.2012-1773 ◽

2013 ◽

Vol 154 (1) ◽

pp. 232-245 ◽

Cited By ~ 50

Author(s):

LeBris S. Quinn ◽

Barbara G. Anderson ◽

Jennifer D. Conner ◽

Tami Wolden-Hanson

Keyword(s):

Skeletal Muscle ◽

Insulin Sensitivity ◽

Energy Metabolism ◽

Transgenic Mice ◽

Oxidative Metabolism ◽

Troponin I ◽

Expression Patterns ◽

Sirtuin 1 ◽

Ppar Γ ◽

Oxidative Energy Metabolism

Endurance exercise initiates a pattern of gene expression that promotes fat oxidation, which in turn improves endurance, body composition, and insulin sensitivity. The signals from exercise that initiate these pathways have not been completely characterized. IL-15 is a cytokine that is up-regulated in skeletal muscle after exercise and correlates with leanness and insulin sensitivity. To determine whether IL-15 can induce any of the metabolic adaptations associated with exercise, substrate metabolism, endurance, and molecular expression patterns were examined in male transgenic mice with constitutively elevated muscle and circulating IL-15 levels. IL-15 transgenic mice ran twice as long as littermate control mice in a run-to-exhaustion trial and preferentially used fat for energy metabolism. Fast muscles in IL-15 transgenic mice exhibited high expression of intracellular mediators of oxidative metabolism that are induced by exercise, including sirtuin 1, peroxisome proliferator-activated receptor (PPAR)-δ, PPAR-γ coactivator-1α, and PPAR-γ coactivator-1β. Muscle tissue in IL-15 transgenic mice exhibited myosin heavy chain and troponin I mRNA isoform expression patterns indicative of a more oxidative phenotype than controls. These findings support a role for IL-15 in induction of exercise endurance, oxidative metabolism, and skeletal muscle molecular adaptations induced by physical training.

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