Transgenic animal models for cardiac hypertrophy and methods...

Details Transgenic animal models for cardiac hypertrophy and methods... Transgenic animal models for cardiac hypertrophy and methods...

1998-10-15

2001-03-13

Chambers, Jasemine (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Method of using a transgenic nonhuman animal in an in vivo...

C435S004000, C435S006120, C435S007100, C435S008000, C435S325000, C435S354000, C435S366000, C800S018000

Reexamination Certificate

active

06201165

ABSTRACT:

TECHNICAL FIELD
The present invention relates to transgenic animal models for cardiac hypertrophy. It further relates to the use of such transgenic animals to detect substances with therapeutic activity toward cardiac hypertrophy. It further relates to assays to detect substances for use in treating cardiac hypertrophy. It further relates to methods for treating cardiac hypertrophy.
BACKGROUND ART
Cardiac hypertrophy
Cardiac hypertrophy is an adaptive response of the heart to virtually all forms of cardiac disease, including those arising from hypertension, mechanical load, myocardial infarction, cardiac arrhythmias, endocrine disorders, and genetic mutations in cardiac contractile protein genes. While the hypertrophic response is initially a compensatory mechanism that augments cardiac output, sustained hypertrophy can lead to dilated cardiomyopathy, heart failure, and sudden death. In the United States, approximately half a million individuals are diagnosed with heart failure each year, with a mortality rate approaching 50%.
Despite the diverse stimuli that lead to cardiac hypertrophy, there is a prototypical final molecular response of cardiomyocytes to hypertrophic signals that involves an increase in cell size and protein synthesis, enhanced sarcomeric organization, up-regulation of fetal cardiac genes, and induction of immediate-early genes, such as c-fos and c-myc. See, Chien et al. (1993)
Ann. Rev. Physiol.
55:77-95; and Sadoshima and Izumo (1997)
Ann. Rev. Physiol.
59:551-571. The causes and effects of cardiac hypertrophy have been extensively documented, but the underlying molecular mechanisms that couple hypertrophic signals initiated at the cell membrane to the reprogramming of cardiomyocyte gene expression remain poorly understood. Elucidation of these mechanisms is a central issue in cardiovascular biology and will be critical for designing new strategies for prevention or treatment of cardiac hypertrophy and heart failure.
Numerous studies have implicated intracellular Ca
2+
as a signal for cardiac hypertrophy. In response to myocyte stretch or increased loads on working heart preparations, intracellular Ca
2+
concentrations increase (Marban et al. (1987)
Proc. Natl. Acad. Sci. USA
84:6005-6009; Bustamante et al. (1991)
J. Cardiovasc. Pharmacol.
17:S110-S113; and Hongo et al. (1995)
Am J. Physiol.
269:C690-C697, consistent with a role of Ca
2+
in coordinating physiologic responses with enhanced cardiac output. A variety of humoral factors, including angiotensin II (AngII), phenylephrine (PE), and endothelin-1 (ET-1), which induce the hypertrophic response in cardiomyocytes, also share the ability to elevate intracellular Ca
2+
concentrations. Karliner et al. (1990)
Experientia
46:81-84; Sadoshima and Izumo (1993)
Circ. Res.
73:424-438; Sadoshima et al. (1993)
Cell
75:977-984; and Leite et al. (1994)
Am. J. Physiol.
267:H2193-H2203.
Hypertrophic stimuli result in reprogramming of gene expression in the adult myocardium, such that genes encoding fetal protein isoforms like &bgr;-myosin heavy chain (MHC) and &agr;-skeletal actin are up-regulated, whereas the corresponding adult isoforms, &agr;-MHC and &agr;-cardiac actin, are down-regulated. The natriuretic peptides, atrial natriuretic factor (ANF), and b-type natriuretic peptide (BNP), which decrease blood pressure by vasodilation and natriuresis, are also rapidly up-regulated in the heart in response to hypertrophic signals. Komuro and Yazaki (1993)
Ann. Rev. Physiol.
55:55-75. The mechanisms involved in coordinately regulating these cardiac genes during hypertrophy are unknown, although binding sites for several transcription factors, including serum response factor (SRF), TEF-1, AP-1, and Sp1, are important for activation of fetal cardiac genes in response to hypertrophy. Sadoshima and Izumo (1993); Sadoshima et al. (1993); Kariya et al. (1994)
J. Biol. Chem.
269:3775-3782; Karns et al. (1995)
J. Biol. Chem.
270:410-417; and Kovacic-Milivojevic et al. (1996)
Endocrinol.
137:1108-1117. Most recently, the cardiac-restricted zinc finger transcription factor GATA4 has also been shown to be required for transcriptional activation of the genes for Ang II type 1a receptor and &bgr;-MHC during hypertrophy. Herzig et al. (1997)
Proc. Natl. Acad. Sci. USA
94:7543-7548; Hasegawa et al. (1997)
Circulation
96:3943-3953; and Molkentin and Olson (1997)
Circulation
96:3833-3835.
A number of intracellular signaling pathways have been implicated in transduction of hypertrophic stimuli. For example, occupancy of the cell surface receptors for AngII, PE, and ET-1 leads to activation of phospholipase C, resulting in the production of diacylglycerol and inositol triphosphate, which in turn results in mobilization of intracellular Ca
2+
and activation of protein kinase C (PKC). Sadoshima and Izumo (1993); Yamazaki et al. (1996)
J. Biol. Chem.
271:3221-3228; and Zou et al. (1996)
J. Biol. Chem.
271:33592-33597. There is also evidence that the Ras and mitogen-activated protein (MAP) kinase pathways are transducers of hypertrophic signals. Thorburn et al. (1993)
J. Biol. Chem.
268:2244-2249; and Force et al. (1996)
Circ. Res.
78:947-953. The extent to which these signaling pathways are coordinated during cardiac hypertrophy is unknown. However, all of these pathways are associated with an increase in intracellular Ca
2+
, consistent with a central regulatory role of Ca
2+
in coordinating the activities of multiple hypertrophic signaling pathways.
In B and T cells, the Ca
2+
, calmodulin-dependent phosphatase calcineurin has been shown to link intracellular signaling pathways that result in elevation of intracellular Ca
2+
with activation of the immune response. Calcineurin regulates immune response genes through dephosphorylation of a family of transcription factors known as NF-ATs (nuclear factors of activated T cells). Rao et al. (1997)
Ann. Rev. Immunol.
15:707-747. Once dephosphorylated by calcineurin, NF-AT transcription factors translocate to the nucleus and directly activate immune response genes. Flanagan et al. (1991)
Nature
352:803-807; Loh et al. (1996)
Mol. Cell. Biol.
16:3945-3954; and Loh et al. (1996)
J. Biol. Chem.
271:10884-10891. The immunosuppressant drugs cyclosporin A (CsA) and FK506 suppress the immune response by inhibiting calcineurin's ability to activate NF-AT transcription factors. Shaw et al. (1995)
Proc. Natl. Acad. Sci. USA
92:11205-11209; Loh et al. (1996)
J. Biol. Chem.
271:10884-10891.
There are no spontaneous mouse mutations with sufficient similarities to cardiac hypertrophy to be useful as experimental models. A transgenic rodent line has been produced that overexpresses calmodulin under the control of the human atrial natriuretic factor gene. Gruver et al. (1993)
Endocrinology
133:376-388. In these mice, developmental overexpression of CaM in mouse cardiomyocytes produced a markedly exaggerated cardiac growth response, characterized by the presence of cardiomyocyte hypertrophy in regions demonstrated to overexpress CaM and by cardiomyocyte hyperplasia, apparent at early developmental stages. However, the expression of calmodulin in these animals is constitutive and is thus not reflective of physiological conditions in humans. Transgenic mice have been created in which a reporter gene is regulated by a tetracycline-controlled transactivator (tTA), which in turn is under the transcriptional control of 2.9 kb of 5′ flanking sequence from the rat &agr;-myosin heavy chain gene. Yu et al. (1996)
Circ. Res.
79:691-697.
Current medical management of cardiac hypertrophy includes the use of three types of drugs: calcium channel blocking agents, &bgr;-adrenergic blocking agents, and disopyramide. Kikura and Levy (1995) Int. Anesthesiol. Clin. 33:21-37. Therapeutic agents for heart failure include angiotensin II converting enzyme (ACE) inhibitors and diuretics. Other pharmaceutical agents which have been disclosed for treatment of cardiac hypertrophy include angiotensin II receptor antagonists

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