© 1996 American Heart Association, Inc.
Articles |
Transgenic Remodeling of the Contractile Apparatus in the Mammalian Heart
From the Children's Hospital Research Foundation, Department of Pediatrics, Division of Molecular Cardiovascular Biology, Cincinnati, Ohio.
Correspondence to Dr Jeffrey Robbins, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail teachdna@aol.com.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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Abstract The structure-function relationships of the sarcomeric proteins in the mammalian cardiac compartment remain ill-defined because of the lack of a suitable model in which they can be readily manipulated or exchanged in vivo. To establish the validity of the transgenic paradigm for remodeling the mammalian heart, the murine
-cardiac myosin heavy chain gene promoter was used to
express a ventricular myosin light chain-2 transgene
(MLC2v) in both the atria and ventricles of the adult
animal. Expression resulted in high levels of the transgene's
transcript in both compartments. In the ventricle, the transgene was
expressed against the background expression of the normal isoform. In
the atrium, the transgene's expression would be ectopic, in that
normally, MLC2v expression is restricted to the ventricle.
Ectopic expression of the transgene in the atria resulted in a complete
replacement of the atrial myosin light chain-2 protein isoform,
although the endogenous isoform's steady state transcript
levels were unchanged. In contrast, ventricular expression
of the transgene had no effect at the protein level, despite an
eightfold increase in MLC2v transcript levels. The data show that
sarcomeric protein stoichiometry is maintained rigorously via
posttranscriptional regulation and that protein replacement can be
achieved through a single transgenic manipulation.
Key Words: transgenic • myosin light chain • gene • muscle
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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The ability to modify the genetic apparatus in a defined manner underlies many of the approaches in molecular cardiovascular biology and biochemistry. Transgenesis has the potential to delineate the molecular, cellular, and functional consequences of protein replacement in the heart by creating animals in which modified genes or the cognate cDNAs are placed into the genome and the encoded proteins can be subsequently expressed in an organ-specific manner. The approach necessarily relies on the action of a strong cardiac-specific promoter that is able to drive high levels of cardiac-restricted expression. We have recently focused our attention on defining such promoters and have delineated the transcriptional elements in both
-MyHC and ß-MyHC that are responsible for
high levels of cardiac-specific gene
expression.1 2 3 In
the mouse, the animal that is most widely used for transgenic
approaches, -MyHC is constitutively expressed before and
after birth in the atria. ß-MyHC is expressed in the
ventricle during gestation but is downregulated at birth as
transcription is initiated from the -MyHC
locus.4 5
Thus, the rigorous characterization of the -MyHC
promoter1 2 6 provides a potentially
useful reagent for
the efficient overexpression of a target transgene in both cardiac
compartments in the adult.
The -MyHC promoter has already been used successfully to drive high
levels of expression of a ß-adrenergic receptor7 and
ß-adrenergic receptor kinase.8 We wished to
determine whether or not it was possible to achieve levels of transgene
expression such that some of the most abundant components of the
cardiomyocyte, the contractile proteins, could be
effectively remodeled. Although the experiment appears straightforward,
a number of possible outcomes could complicate the overall approach and
subsequent interpretation of the data. For example, it is well
documented in Drosophila that by merely altering contractile
protein gene dosage, one significantly perturbs the overall
stoichiometry of protein production with subsequent dramatic
effects on function.9 10 Thus, if transgenesis were
used
in a structure-function or replacement study in the cardiac
compartment, it is possible that an observed phenotype
might be misleading. By merely overexpressing a normal form of
the endogenous protein, the stoichiometry of the
contractile apparatus could be perturbed significantly,
with the resultant phenotype brought about by this change
rather than by difference(s) in protein function due to a mutated
polypeptide.
To begin to explore the usefulness of the transgenic paradigm in
remodeling the cardiac compartment's contractile
apparatus, we overexpressed MLC2v11 in both
cardiac compartments. The rationale was that expression of the
transgene would be ectopic in the atrium but occur against the normal
expression of the ventricular isoform in the ventricle.
Such an experiment would allow examination of gene "cross talk"
and perturbation of contractile protein stoichiometry to be considered
against the backdrop of effective increases in gene dosage specifically
in the cardiac compartment. Using the -MyHC promoter linked to a
full-length MLC2v cDNA, high levels of transgene expression were
achieved in both cardiac compartments. The data show that the
contractile apparatus can be effectively remodeled using
this methodology and that sarcomeric protein stoichiometry can be
maintained despite changes in the steady state transcript levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Construction of Transgenic Mice and Analyses of Transgene Expression
LacZ-expressing transgenic mice were generated using a construct that contained the
-MyHC promoter12 driving
the expression of a lacZ fusion gene.13 Embryos were
removed at 9.5 and 10.5 dpc (noon on the day of the vaginal plug is
taken as 0.5 day). Embryos or hearts were fixed and processed for
ß-galactosidase staining as described
previously.13 14 Older hearts were clipped at their
apex
to allow penetration of the substrate. For the transgene encoding
MLC2v, a full-length murine cDNA isolated from a BALB/c adult mouse
cardiac cDNA library (Clonetech), was sequenced, linked to the -MyHC
promoter, and used to generate transgenic mice. A "tagged" MLC2v
cDNA was also made (used to generate line 97; see Fig 2
) by
including,
at the 3' end of the construct, a polyA tail and the exon sequences
that make up the ß-MyHC 3'-UTR. This results in a transcript that is
200 to 250 bp longer than the endogenous MLC2v mRNA. The
constructs were digested free of vector sequence with Not I
and prepared for microinjection as described previously.15
The founder mice were identified using the polymerase chain
reaction16 and confirmed by genomic Southern blots using
DNA obtained from tail clips. Stable transgenic lines were generated by
breeding the founder mice with nontransgenic littermates.
Subsequent offspring were screened by the polymerase chain reaction.
For the RNA analyses, Northern and dot blots were carried out
as described previously.6 17 End-labeled
oligonucleotides specific to MLC2v
(5'-CACAGCCCTGGGATGGAGAGTGGGCTGTGGGTCACCTGAGGCTGTGGTTCAG-3'),
GAPDH
(5'-GGAACATGTAGACCATGTAGTTGAGGTCAATGAAG-3'),
MLC2a (5'-GAGGTGACCTCAGCCTGTCTACTCCTCTTTCTCATCCCCG-3'), and
-MyHC17 were used, and hybridization signals were
quantified on a PhosphorImager (Molecular Dynamics).
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Sarcomeric Protein Analyses
The atrial flaps and ventricular
apex were excised
from adult transgenic and nontransgenic littermates. Myofilament
protein was extracted,18 19 and all washes were
collected
to obtain the entire complement of cardiac proteins. Myofilament
protein (20 µg) and the corresponding wash fractions were
electrophoresed on a 15% polyacrylamide gel in the presence of
0.1% SDS and stained with Coomassie brilliant blue R250.
MLC Immunofluorescence
Hearts were excised from adult
(>8-week-old) transgenic and
nontransgenic littermates, drop-fixed in 4%
paraformaldehyde, and embedded in either paraffin for
immunoperoxidase analyses or O.C.T. compound (Miles) for
immunofluorescence. Sections (5 or 10 µm) were
incubated with rabbit polyclonal antisera against either MLC2a or
MLC2v.20 Immunoperoxidase staining was accomplished by use
of an avidin-biotin ABC kit (Vector Laboratories, Inc) to increase
the sensitivity of the primary antibodies.
Immunofluorescence was performed without
amplification using a goat anti-rabbit (IgG) secondary antibody
conjugated to L-rhodamine (Boehringer-Mannheim).
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Compartment-Specific Expression of the
-MyHC
Promoter–Driven
Transgene During Development To ensure that the
-MyHC promoter
fragment does, in fact, drive
transgene expression in a compartment-restricted fashion during
early development and that ectopic expression is absent, the 5.5-kb
fragment that lies between ß- and -MyHC and contains
the active -MyHC promoter1 6 was linked to
lacZ. Transgene expression was determined by staining for
ß-galactosidase activity. The data show that expression of
lacZ recapitulates exactly the pattern of -MyHC gene
expression in the heart (Fig 1). As is the case for
endogenous -MyHC expression,4 5 reporter
gene activity is limited to the atrial compartments during gestation
and is rapidly activated in the postnatal ventricles.
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Expression of a Transgene Encoding an Ectopic and
Endogenous Contractile Protein Isoform
To assess the potential of the
-MyHC promoter to effect a
remodeling of the heart's contractile apparatus, we tested
the effects of transgenic overexpression under conditions in which the
transgene's product is endogenously produced and in
which transgene expression might lead to ectopic expression of the
protein product. The ventricular and atrial isoforms of
MLC2 are encoded by two members of a multigene family of
Ca2+ binding proteins whose cardiac expression is chamber
specific throughout development.11 21 Each MLC2 is a
small
phosphoprotein that is intimately associated at stoichiometric levels
with the globular head region of the MyHC22 23 and
may
modulate striated muscle contractility by increasing
the sensitivity of the sarcomere to
Ca2+.24 25 26
We isolated a full-length MLC2v cDNA (see "Materials and
Methods") and sequenced it completely to confirm the open reading
frame.27
We anticipated that the -MyHC promoter would
drive ectopic
expression of MLC2v in the atrium while expression in the ventricle
would occur against the endogenous gene's activity.
Hemizygous transgenic mice overexpressing MLC2v message in the atria
and ventricles were generated by using the -MyHC gene promoter to
drive transcription of the full-length mouse MLC2v cDNA (Fig
2A
). The integrity of the transgenic transcript was
confirmed by Northern analysis (Fig 2B), and the data show that
the amount of full-length MLC2v mRNA is significantly increased in
both the atria and ventricles of the transgenic animals.
Analysis of a transgenic construct (line 97), which carries an
MLC2v transgene "tagged" at its 3'-UTR with an additional
sequence to distinguish it from its endogenous counterpart,
shows that MLC2v overexpression does not significantly alter
endogenous MLC2v mRNA levels in the transgenic ventricles
(Fig 2B). To quantify possible changes in other cardiac
transcript
levels that might arise as a result of MLC2v overexpression, RNA blots
were prepared from atrial and ventricular tissue and
hybridized with transcript-specific probes (Fig 2C). Comparison
of
nontransgenic and transgenic tissues show that MLC2v mRNA in the
transgenic atria and ventricles was eightfold and sevenfold higher,
respectively, than in nontransgenic ventricles. Despite the high levels
of MLC2v mRNA in the atria, MLC2a transcript levels were identical in
the nontransgenic and transgenic hearts, suggesting that accumulation
of the ectopic transcript does not affect endogenous gene
expression. The data also show that the -MyHC–driven transgene did
not titrate out transcription factors essential for the
endogenous gene promoter's activity, since -MyHC
transcript levels were identical in the transgenic and nontransgenic
hearts.
Ectopic Replacement of a Sarcomeric Protein
Sarcomeric and
total protein pools in the transgenic hearts were
analyzed by electrophoresis to examine the effects of transgene
expression on component protein accumulation. Contractile protein
isoform levels are controlled mainly at the transcriptional
level28 or, in some cases, by alternative splicing of the
primary transcript.29 Thus, under normal circumstances the
amount of protein correlates with the level of its cognate mRNA.
Transgenic overexpression clearly perturbs this relationship, as
evidenced by a complete MLC2 isoform switch (MLC2a
MLC2v) in the
atria (Fig 3A) despite the persistence of normal MLC2a
transcript levels. Even more surprising is the fact that no increase in
MLC2v protein levels occurs in the transgenic ventricles (Fig
3A)
despite a sevenfold excess in the transcript level (Fig 2B and
2C). Nor
do there appear to be pools of nonsarcomeric-associated MLC2
proteins in the transgenic hearts (Fig 3B, lanes b, d, f, and
h).
Moreover, the myofilament preparations and washes show that there are
no overt changes in the other sarcomeric proteins due to MLC2v
overexpression in either cardiac compartment.
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To ensure that these
changes (or the lack thereof) in the MLC2
isoforms, identified by SDS-PAGE, were uniform throughout the atria and
ventricles, immunohistochemical studies were carried out with
antibodies directed against either MLC2a or MLC2v (Fig 4). The
normal patterns of isoform expression seen in
the nontransgenic hearts (Fig 4A and 4E) are
clearly modified in the
transgenic animals. The ectopic replacement of MLC2a with MLC2v in the
transgenic atria is confirmed by a total loss of staining with the
anti-MLC2a antibody (Fig 4B and 4C) and by the
high levels of
immunoreactivity with anti-MLC2v in this compartment (Fig 4F
and 4G).
At higher magnification (Fig 4G), it is apparent that the
transgenic
atria are homogeneously transformed, and the isoform switch
results in a level of MLC2v expression comparable to that observed in
the transgenic ventricle. Immunofluorescent
analysis confirmed that anti-MLC2v staining of the transgenic
atrial cardiomyocytes (Fig 4H) decorated the sarcomeres,
producing striations identical to anti-MLC2a staining in the control
atria (Fig 4D).
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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These data indicate that it is possible to remodel the protein complement of the contractile apparatus with directed transgenic overexpression. A total of six lines carrying the MLC2v transgene were generated. Of these, three generated high levels of the transgenic transcript and were selected for further study and subsequently shown to produce high levels of the cognate protein that resulted in either a partial or complete MLC2a
MLC2v switch in the
atria.27 Similar results have been obtained in parallel
experiments in which the -MyHC promoter was used to overexpress the
isoform present in the fast fibers of skeletal muscle (MLC2f) in
both cardiac compartments. Line-to-line variation was again
observed, but in 30% of the lines, an essentially complete switch
(MLC2aMLC2f) was observed in the atria. In all cases, the overall
stoichiometry of the MLC2 isoform was maintained (J. Gulick and J.
Robbins, unpublished data, 1996). To date, expression of the transgenes, although they vary from line to line, have remained stable within a line. Our working hypothesis is that the line-to-line variation is due to the differences in copy number and/or position effects, and this can be formally tested as multiple lines are accumulated and analyzed for a variety of transgenes. The variation in expression can be used to one's experimental advantage, as the different degrees of replacement observed between multiple lines generated from the identical transgene can be used to carry out a dose-response curve for the phenotypic consequences of the altered protein complement.
Structure-function relationships can thus be explored directly, both within a protein and between the different contractile isoforms directly using a one-step transgenic procedure. Indeed, we have noted that ectopic replacement of MLC2a with MLC2v leads to significant changes in heart function as assessed by a working heart preparation (authors' unpublished data, 1995). Transgenic overexpression can offer an alternative to the more laborious two-step gene targeting/replacement approach,30 31 and its efficacy for exploring the functional aspects of the contractile apparatus is increased by the rigorous posttranscriptional controls.
The data are significant in light of the stoichiometric concerns outlined above when a transgenic approach to remodeling the heart is being considered. Data obtained from experiments carried out in Drosophila, in which the gene dosage of the myosin heavy chain was methodically varied, showed that as few as four functional copies of the gene resulted in elevated accumulation of the protein and a flightless phenotype. Further increases in copy number were lethal.32 Particularly intriguing in light of those data is the observation that the transgenic mice used in the present study are characterized by low (2 to 10 copies per diploid genome) transgene copy number.27 Other viable lines we have generated carrying transgenes encoding contractile proteins also have copy numbers in this range (authors' unpublished data, 1996). This contrasts with the wide variability seen in copy number when reporter transgenes, such as cat, were used with the MyHC promoters.1 6 33 We have not yet generated a sufficient number of lines expressing a transgene encoding a sarcomeric protein to allow us to establish a statistically significant link between copy number and expression levels or lethality. Nevertheless, in our experience, the narrow range of transgene copy number is striking.
The mechanism by which normal MLC2v protein levels in the ventricle are
maintained in the presence of significantly higher steady state
transcript levels in this cardiac compartment is presently unknown.
The transcripts present are polyadenylated and intact
(Fig 2B) and presumably are accessible to the translational
apparatus. Our working hypothesis is that increased protein
turnover may account for the maintenance of the stoichiometry
in the presence of increased de novo protein synthesis. Alternatively,
it is possible that the translational efficiency of these transcripts
is somehow modified. It will be necessary to measure the rate of MLC2v
protein synthesis in the transgenic animals and calculate the flux
through the protein pool in the intact hearts to rigorously demonstrate
the validity of this idea. If MLC2 turnover is not increased, the
translational efficiency of the transgenic transcripts will need to be
assessed by comparing the fraction of the transcripts in polysomes
derived from the transgenic and nontransgenic hearts. The relative
abilities of RNA derived from these fractions could also be tested in
in vitro translation assays.
If high copy numbers of the transgene do prove to be lethal, it may well be that this control point (or others, as yet undetermined) can be eventually overwhelmed with a resultant change in the contractile protein stoichiometry, leading to cardiac insufficiency during the latter half of gestation. The transgenic system will certainly be a useful tool for exploring in detail these questions of gene dosage and the resultant contractile protein stoichiometries in the whole-animal context.
These considerations, should they prove germane, do not, however,
negate the general usefulness of transgenesis for remodeling the
contractile apparatus in the heart. We have demonstrated
that the -MyHC promoter can drive very high levels of contractile
protein expression and can be used to effectively replace an atrial
isoform with the corresponding ventricular protein.
Furthermore, despite high levels of "wild-type" transgenic
transcript (ie, MLC2v), an increase in the cognate protein level, with
the attendant possibilities for modifying function via changes in
contractile protein levels, does not occur. This should allow a
straightforward assessment of the functional correlates with isoform
replacements/mutations that can be made, free of the confounding
variable of changes in the relative levels of the sarcomere's
components.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-22619, HL-41496, HL-52318, and HL-46826 (to Dr Robbins); by the Marion Merrell-Dow Foundation (to Dr Palermo); and by National Institutes of Health training grant HL-07752 (to Dr Fewell). We thank K. Chien and S. Kubalak for the MLC2a and MLC2v antibodies.
Received November 6, 1995; accepted December 26, 1995.
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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M. G. Trivieri, G. Y. Oudit, R. Sah, B.-G. Kerfant, H. Sun, A. O. Gramolini, Y. Pan, A. D. Wickenden, W. Croteau, G. Morreale de Escobar, et al. Cardiac-specific elevations in thyroid hormone enhance contractility and prevent pressure overload-induced cardiac dysfunction PNAS, April 11, 2006; 103(15): 6043 - 6048. [Abstract] [Full Text] [PDF]
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J. Xu, N. L. Gong, I. Bodi, B. J. Aronow, P. H. Backx, and J. D. Molkentin Myocyte Enhancer Factors 2A and 2C Induce Dilated Cardiomyopathy in Transgenic Mice J. Biol. Chem., April 7, 2006; 281(14): 9152 - 9162. [Abstract] [Full Text] [PDF]
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 T. Oka, M. Maillet, A. J. Watt, R. J. Schwartz, B. J. Aronow, S. A. Duncan, and J. D. Molkentin Cardiac-Specific Deletion of Gata4 Reveals Its Requirement for Hypertrophy, Compensation, and Myocyte Viability Circ. Res., March 31, 2006; 98(6): 837 - 845. [Abstract] [Full Text] [PDF]
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 N. S. Dhalla, M. R. Dent, P. S. Tappia, R. Sethi, J. Barta, and R. K. Goyal Subcellular Remodeling as a Viable Target for the Treatment of Congestive Heart Failure Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2006; 11(1): 31 - 45. [Abstract] [PDF]
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J. Xu, T. R. Kimball, J. N. Lorenz, D. A. Brown, A. R. Bauskin, R. Klevitsky, T. E. Hewett, S. N. Breit, and J. D. Molkentin GDF15/MIC-1 Functions As a Protective and Antihypertrophic Factor Released From the Myocardium in Association With SMAD Protein Activation Circ. Res., February 17, 2006; 98(3): 342 - 350. [Abstract] [Full Text] [PDF]
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 H. Kiriazis, X.-J. Du, X. Feng, E. Hotchkin, T. Marshall, S. Finch, X.-M. Gao, G. Lambert, J. K. Choate, and D. M. Kaye Preserved left ventricular structure and function in mice with cardiac sympathetic hyperinnervation Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1359 - H1365. [Abstract] [Full Text] [PDF]
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S. Sakthivel, N. L. Finley, P. R. Rosevear, J. N. Lorenz, J. Gulick, S. Kim, P. VanBuren, L. A. Martin, and J. Robbins In Vivo and in Vitro Analysis of Cardiac Troponin I Phosphorylation J. Biol. Chem., January 7, 2005; 280(1): 703 - 714. [Abstract] [Full Text] [PDF]
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M. Krenz, A. Sanbe, F. Bouyer-Dalloz, J. Gulick, R. Klevitsky, T. E. Hewett, H. E. Osinska, J. N. Lorenz, C. Brosseau, A. Federico, et al. Analysis of Myosin Heavy Chain Functionality in the Heart J. Biol. Chem., May 2, 2003; 278(19): 17466 - 17474. [Abstract] [Full Text] [PDF]
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 P. E.M.H. Habets, A. F.M. Moorman, and V. M. Christoffels Regulatory modules in the developing heart Cardiovasc Res, May 1, 2003; 58(2): 246 - 263. [Abstract] [Full Text] [PDF]
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A. Sanbe, J. Gulick, M. C. Hanks, Q. Liang, H. Osinska, and J. Robbins Reengineering Inducible Cardiac-Specific Transgenesis With an Attenuated Myosin Heavy Chain Promoter Circ. Res., April 4, 2003; 92(6): 609 - 616. [Abstract] [Full Text] [PDF]
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S. M. Raidel, C. Haase, N. R. Jansen, R. B. Russ, R. L. Sutliff, L. W. Velsor, B. J. Day, B. D. Hoit, A. M. Samarel, and W. Lewis Targeted myocardial transgenic expression of HIV Tat causes cardiomyopathy and mitochondrial damage Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1672 - H1678. [Abstract] [Full Text] [PDF]
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 T. Shioi, J. R. McMullen, P. M. Kang, P. S. Douglas, T. Obata, T. F. Franke, L. C. Cantley, and S. Izumo Akt/Protein Kinase B Promotes Organ Growth in Transgenic Mice Mol. Cell. Biol., April 15, 2002; 22(8): 2799 - 2809. [Abstract] [Full Text] [PDF]
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 M. C. Ferreira-Cornwell, Y. Luo, N. Narula, J. M. Lenox, M. Lieberman, and G. L. Radice Remodeling the intercalated disc leads to cardiomyopathy in mice misexpressing cadherins in the heart J. Cell Sci., April 15, 2002; 115(8): 1623 - 1634. [Abstract] [Full Text] [PDF]
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O. F Bueno, E. van Rooij, J. D Molkentin, P. A Doevendans, and L. J De Windt Calcineurin and hypertrophic heart disease: novel insights and remaining questions Cardiovasc Res, March 1, 2002; 53(4): 806 - 821. [Abstract] [Full Text] [PDF]
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T. Osugi, Y. Oshima, Y. Fujio, M. Funamoto, A. Yamashita, S. Negoro, K. Kunisada, M. Izumi, Y. Nakaoka, H. Hirota, et al. Cardiac-specific Activation of Signal Transducer and Activator of Transcription 3 Promotes Vascular Formation in the Heart J. Biol. Chem., February 15, 2002; 277(8): 6676 - 6681. [Abstract] [Full Text] [PDF]
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J. M. Nerbonne, C. G. Nichols, T. L. Schwarz, and D. Escande Genetic Manipulation of Cardiac K+ Channel Function in Mice: What Have We Learned, and Where Do We Go From Here? Circ. Res., November 23, 2001; 89(11): 944 - 956. [Abstract] [Full Text] [PDF]
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Q. Yang, T. E. Hewett, R. Klevitsky, A. Sanbe, X. Wang, and J. Robbins PKA-dependent phosphorylation of cardiac myosin binding protein C in transgenic mice Cardiovasc Res, July 1, 2001; 51(1): 80 - 88. [Abstract] [Full Text] [PDF]
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I. Kishimoto, K. Rossi, and D. L. Garbers A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy PNAS, February 8, 2001; (2001) 51625598. [Abstract] [Full Text]
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J. James, Y. Zhang, H. Osinska, A. Sanbe, R. Klevitsky, T. E. Hewett, and J. Robbins Transgenic Modeling of a Cardiac Troponin I Mutation Linked to Familial Hypertrophic Cardiomyopathy Circ. Res., October 27, 2000; 87(9): 805 - 811. [Abstract] [Full Text] [PDF]
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A. Sanbe, J. Gulick, E. Hayes, D. Warshaw, H. Osinska, C.-B. Chan, R. Klevitsky, and J. Robbins Myosin light chain replacement in the heart Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1355 - H1364. [Abstract] [Full Text] [PDF]
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A. Sanbe, D. Nelson, J. Gulick, E. Setser, H. Osinska, X. Wang, T. E. Hewett, R. Klevitsky, E. Hayes, D. M. Warshaw, et al. In Vivo Analysis of an Essential Myosin Light Chain Mutation Linked to Familial Hypertrophic Cardiomyopathy Circ. Res., August 18, 2000; 87(4): 296 - 302. [Abstract] [Full Text] [PDF]
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D. J. Sheridan, D. J. Autelitano, B. Wang, E. Percy, E. A. Woodcock, and X.-J. Du {beta}2-Adrenergic receptor overexpression driven by {alpha}-MHC promoter is downregulated in hypertrophied and failing myocardium Cardiovasc Res, July 1, 2000; 47(1): 133 - 141. [Abstract] [Full Text] [PDF]
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J. James, A. Sanbe, K. Yager, L. Martin, R. Klevitsky, and J. Robbins Genetic Manipulation of the Rabbit Heart via Transgenesis Circulation, April 11, 2000; 101(14): 1715 - 1721. [Abstract] [Full Text] [PDF]
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M. Funamoto, Y. Fujio, K. Kunisada, S. Negoro, E. Tone, T. Osugi, H. Hirota, M. Izumi, K. Yoshizaki, K. Walsh, et al. Signal Transducer and Activator of Transcription 3 Is Required for Glycoprotein 130-mediated Induction of Vascular Endothelial Growth Factor in Cardiac Myocytes J. Biol. Chem., March 31, 2000; 275(14): 10561 - 10566. [Abstract] [Full Text] [PDF]
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H. Li, J. Wang, H. Wilhelmsson, A. Hansson, P. Thoren, J. Duffy, P. Rustin, and N.-G. Larsson Genetic modification of survival in tissue-specific knockout mice with mitochondrial cardiomyopathy PNAS, March 28, 2000; 97(7): 3467 - 3472. [Abstract] [Full Text] [PDF]
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P. Paradis, N. Dali-Youcef, F. W. Paradis, G. Thibault, and M. Nemer Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling PNAS, January 18, 2000; 97(2): 931 - 936. [Abstract] [Full Text] [PDF]
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 M. C. Hart and J. A. Cooper Vertebrate Isoforms of Actin Capping Protein {beta} Have Distinct Functions in Vivo J. Cell Biol., December 13, 1999; 147(6): 1287 - 1298. [Abstract] [Full Text] [PDF]
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Q. Yang, A. Sanbe, H. Osinska, T. E. Hewett, R. Klevitsky, and J. Robbins In Vivo Modeling of Myosin Binding Protein C Familial Hypertrophic Cardiomyopathy Circ. Res., October 29, 1999; 85(9): 841 - 847. [Abstract] [Full Text] [PDF]
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H. Xu, D. M. Barry, H. Li, S. Brunet, W. Guo, and J. M. Nerbonne Attenuation of the Slow Component of Delayed Rectification, Action Potential Prolongation, and Triggered Activity in Mice Expressing a Dominant-Negative Kv2 {alpha} Subunit Circ. Res., October 1, 1999; 85(7): 623 - 633. [Abstract] [Full Text] [PDF]
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 H. Xu, H. Li, and J. M Nerbonne Elimination of the transient outward current and action potential prolongation in mouse atrial myocytes expressing a dominant negative Kv4 {alpha} subunit J. Physiol., August 15, 1999; 519(1): 11 - 21. [Abstract] [Full Text] [PDF]
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M. V. Westfall, F. P. Albayya, and J. M. Metzger Functional Analysis of Troponin I Regulatory Domains in the Intact Myofilament of Adult Single Cardiac Myocytes J. Biol. Chem., August 6, 1999; 274(32): 22508 - 22516. [Abstract] [Full Text] [PDF]
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A. Sanbe, J. G. Fewell, J. Gulick, H. Osinska, J. Lorenz, D. G. Hall, L. A. Murray, T. R. Kimball, S. A. Witt, and J. Robbins Abnormal Cardiac Structure and Function in Mice Expressing Nonphosphorylatable Cardiac Regulatory Myosin Light Chain 2 J. Biol. Chem., July 23, 1999; 274(30): 21085 - 21094. [Abstract] [Full Text] [PDF]
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S. Engelhardt, L. Hein, F. Wiesmann, and M. J. Lohse Progressive hypertrophy and heart failure in beta 1-adrenergic receptor transgenic mice PNAS, June 8, 1999; 96(12): 7059 - 7064. [Abstract] [Full Text] [PDF]
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 H. Xu, W. Guo, and J. M. Nerbonne Four Kinetically Distinct Depolarization-activated K+ Currents in Adult Mouse Ventricular Myocytes J. Gen. Physiol., May 1, 1999; 113(5): 661 - 678. [Abstract] [Full Text] [PDF]
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S. Minamisawa, Y. Gu, J. Ross Jr., K. R. Chien, and J. Chen A Post-transcriptional Compensatory Pathway in Heterozygous Ventricular Myosin Light Chain 2-Deficient Mice Results in Lack of Gene Dosage Effect during Normal Cardiac Growth or Hypertrophy J. Biol. Chem., April 9, 1999; 274(15): 10066 - 10070. [Abstract] [Full Text] [PDF]
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S. H. Buck, P. J. Konyn, J. Palermo, J. Robbins, and R. L. Moss Altered kinetics of contraction of mouse atrial myocytes expressing ventricular myosin regulatory light chain Am J Physiol Heart Circ Physiol, April 1, 1999; 276(4): H1167 - H1171. [Abstract] [Full Text] [PDF]
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D. M. Barry, H. Xu, R. B. Schuessler, and J. M. Nerbonne Functional Knockout of the Transient Outward Current, Long-QT Syndrome, and Cardiac Remodeling in Mice Expressing a Dominant-Negative Kv4 {alpha} Subunit Circ. Res., September 7, 1998; 83(5): 560 - 567. [Abstract] [Full Text] [PDF]
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C. M. Pawloski-Dahm, G. Song, D. L. Kirkpatrick, J. Palermo, J. Gulick, G. W. Dorn II, J. Robbins, and R. A. Walsh Effects of Total Replacement of Atrial Myosin Light Chain-2 With the Ventricular Isoform in Atrial Myocytes of Transgenic Mice Circulation, April 21, 1998; 97(15): 1508 - 1513. [Abstract] [Full Text] [PDF]
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D. Franco, W. H Lamers, and A. F.M Moorman Patterns of expression in the developing myocardium: towards a morphologically integrated transcriptional model Cardiovasc Res, April 1, 1998; 38(1): 25 - 53. [Full Text] [PDF]
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J. F. James, T. E. Hewett, and J. Robbins Cardiac Physiology in Transgenic Mice Circ. Res., March 9, 1998; 82(4): 407 - 415. [Abstract] [Full Text] [PDF]
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J. Robbins {alpha}-Tropomyosin Knockouts : A Blow Against Transcriptional Chauvinism Circ. Res., January 23, 1998; 82(1): 134 - 136. [Full Text] [PDF]
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J. Chen, S. W. Kubalak, S. Minamisawa, R. L. Price, K. D. Becker, R. Hickey, J. Ross Jr., and K. R. Chien Selective Requirement of Myosin Light Chain 2v in Embryonic Heart Function J. Biol. Chem., January 9, 1998; 273(2): 1252 - 1256. [Abstract] [Full Text] [PDF]
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J. James and J. Robbins Molecular remodeling of cardiac contractile function Am J Physiol Heart Circ Physiol, November 1, 1997; 273(5): H2105 - H2118. [Abstract] [Full Text] [PDF]
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J. Gulick, T. E. Hewett, R. Klevitsky, S. H. Buck, R. L. Moss, and J. Robbins Transgenic Remodeling of the Regulatory Myosin Light Chains in the Mammalian Heart Circ. Res., May 19, 1997; 80(5): 655 - 664. [Abstract] [Full Text]
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R. Kelly and M. Buckingham Manipulating Myosin Light Chain 2 Isoforms In Vivo : A Transgenic Approach to Understanding Contractile Protein Diversity Circ. Res., May 19, 1997; 80(5): 751 - 753. [Full Text]
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A. Sanbe, J. Gulick, J. Fewell, and J. Robbins Examining the in Vivo Role of the Amino Terminus of the Essential Myosin Light Chain J. Biol. Chem., August 24, 2001; 276(35): 32682 - 32686. [Abstract] [Full Text] [PDF]
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Q. Liang, L. J. De Windt, S. A. Witt, T. R. Kimball, B. E. Markham, and J. D. Molkentin The Transcription Factors GATA4 and GATA6 Regulate Cardiomyocyte Hypertrophy in Vitro and in Vivo J. Biol. Chem., August 3, 2001; 276(32): 30245 - 30253. [Abstract] [Full Text] [PDF]
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I. Kishimoto, K. Rossi, and D. L. Garbers A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy PNAS, February 27, 2001; 98(5): 2703 - 2706. [Abstract] [Full Text] [PDF]
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D. S. Sohal, M. Nghiem, M. A. Crackower, S. A. Witt, T. R. Kimball, K. M. Tymitz, J. M. Penninger, and J. D. Molkentin Temporally Regulated and Tissue-Specific Gene Manipulations in the Adult and Embryonic Heart Using a Tamoxifen-Inducible Cre Protein Circ. Res., July 6, 2001; 89(1): 20 - 25. [Abstract] [Full Text] [PDF]
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