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5-integrin results in perinatal lethality
Mayo Clinic Scottsdale, Scottsdale, Arizona 85259
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Communication between the
extracellular matrix and the intracellular signal transduction and
cytoskeletal system is mediated by integrin receptors.
5
1-Integrin and its cognate ligand
fibronectin are essential in development of mesodermal structures,
myocyte differentiation, and normal cardiac development. To begin to
explore the potential roles of
51-integrin specifically in
cardiomyocytes, we used a transgenic expression strategy. We
overexpressed two forms of the human 5-integrin in
cardiomyocytes: the full-length wild-type
5-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated
5-1-integrin. Overexpression of the wild-type
5-integrin has no detectable adverse effects in the
mouse, whereas expression of 5-1-integrin caused
electrocardiographic abnormalities, fibrotic changes in the ventricle,
and perinatal lethality. Thus physiological regulation of integrin
function appears essential for maintenance of normal cardiomyocyte
structure and function. This strengthens the role of inside-out
signaling in regulation of integrins in vivo and suggests that
integrins and associated signaling molecules are important in
cardiomyocyte function.
inside-out signaling; electrocardiogram abnormalities; cardiomyopathy; fibrosis
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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INTEGRINS are
divalent, cation-dependent transmembrane -heterodimeric
glycoprotein receptors for matrix components and other cell surface
molecules. Integrins couple the intracellular cytoskeleton with the
extracellular matrix, regulating cell growth, migration, and
differentiation. Their pivotal role in development is underscored by
the failure of embryos lacking 1-integrin to develop
beyond blastocyst implantation (7). Although
1-integrin-null embryonic stem cells differentiate into
cardiomyocytes in vitro, they have disorganized sarcomeres, and
pacemaker cell development is impaired (8). Several
specific ligands and 1-integrins are implicated in
cardiac development, including fibronectin (4, 9, 10, 24,
26) and 51-integrin, a fibronectin
receptor (36). The
41-integrin and an
41-ligand, vascular cell adhesion
molecule-1, are essential in epicardial migration and coronary vessel
development (19, 35).
Integrins are often expressed in spatial domains more extensively than
the biological events they modulate (e.g., in the case of
51-integrin, cell migration and
fibronectin matrix assembly), consistent with regulatory mechanisms in
addition to expression. Indeed, the avidity of integrins for ligand is
modulated from within the cell via "inside-out" signaling
(23), mediated via highly conserved, charged,
membrane-proximal sequences in integrin cytoplasmic domains (-GFFKR
and -LLv-iHDR). Deleting these sequences locks integrins into a
high-affinity state. Physiologically, this regulation presumably serves
to regulate cell adhesion, for example, maintaining the platelet
integrin receptor for fibrinogen, IIb3, in a low-affinity state until platelet activation. The role of inside-out signaling in regulation of
51-integrin function in vivo has not been
determined. We asked whether inappropriate expression of a
gain-of-function mutation in 5-integrin altered
cardiomyocyte structure in mice. We transgenically expressed two forms
of the human 5-integrin subunit in mice: the full-length
physiologically regulated 5-integrin and an activated
subunit lacking the carboxy-terminal cytoplasmic domain [denoted
5-1 (3)]. Deletion of the
membrane-proximal negative regulatory GFFKR sequence in the
5-integrin subunit creates a gain-of-function mutation
(17), as shown by increased activity in fibronectin matrix
assembly (34). The results were striking and unequivocal.
All animals expressing the 5-integrin survived for 6 mo
in good health. In contrast, mice expressing the
5-1-integrin exhibited electrocardiographic (ECG)
abnormalities, cardiomyopathy, fibrosis, and sudden death before 1 mo
of age.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Mice were housed in compliance with the Guide to the Care and Use of Laboratory Animals [DHHS Publ. No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892] in an American Association for Accreditation of Laboratory Animal Care-approved facility. FVB/N mice (Taconic Farms, Germantown, NY) were used in all transgenic experiments and as controls. Standard transgenic techniques were used (11). All mouse procedures were approved by the Mayo Foundation Animal Care and Use Committee.
Transgenic constructs.
cDNA encoding the full-length human 5-integrin subunit
(2) or a cDNA with a truncation of the cytoplasmic domain,
5-1 (29), was subcloned 3' to the 5.5-kb
BamH I-Sal I 5' regulatory sequence of the human
cardiac -myosin heavy chain (MHC) promoter (13). We
refer to the cytoplasmic domain truncation 5-1-integrin as "activated."
PCR analysis.
Mice were genotyped by PCR (21) with the use of the
following primers: 5'-GGGGAGTTAAGACCTGGCAG-3' (forward primer for
5-integrin transgene), 5'-GCGTCCCACTGTGGATCATC-3'
(forward primer for 5-1-integrin), and
5'-GAGTGGACCCAACGCATGTG-3' (common reverse primer annealing with the
human growth hormone polyadenylation signal present in both
transgenes). The 5-integrin-expressing mice were
identified by the presence of a 666-bp amplicon, and the
5-1-integrin-expressing mice were identified by a 676-bp amplicon.
Antibodies.
Mouse anti-human 5-integrin monoclonal antibody 6F4 was
provided by Dr. Ralph Isberg (Howard Hughes Medical Institute, Tufts University) (32), and anti-human 5-integrin
antibody I55220 was purchased from Transduction Laboratories
(Lexington, KY). The cytoplasmic domain-specific antibody AB47
(25) was used to compare the levels of mouse vs. human
5-integrin, inasmuch as they have identical
carboxy-terminal sequences (2, 16). A
1D-integrin-specific antibody (AB186) was obtained from
Dr. Robert Ross (University of California, Los Angeles, CA).
Electrocardiography. Three-lead ECG with standard lead II configuration was performed under 1.75% isoflurane anesthesia.
Histology. Formalin-fixed, paraffin-embedded tissue was sectioned and stained with hematoxylin and eosin or with Masson's trichrome. Cryostat sections (7 µm) were postfixed for 5 min in acetone.
Immunostaining. Sections were deparaffinized and stained with the mouse Histo-Kit (Zymed, South San Francisco, CA). Bright-field microscopy was performed using a Nikon FXA. Images were collected using Kodak film or a Diagnostics Instruments Spot camera.
Immunoblotting. Hearts were homogenized in 1 ml of 50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM orthovanadate, 100 µM leupeptin, 5 kallikrein-inactivating units/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 10 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride (TNEN buffer) and incubated for 10 min on ice. Lysates were clarified by centrifugation (16,000 g, 15 min, 4°C). Protein was determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL). Equal amounts of protein (10 µg) were electrophoresed on a 4-12% gradient bis-Tris SDS-PAGE gel (NOVEX, Carlsbad, CA) and transferred onto Hybond-C (Amersham, UK). Immunoblots were blocked in 5% milk-PBS and incubated with indicated antibodies. Densitometry was performed using pdi Quantity One (pdi, Huntington Station, NY).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiac expression of an activated 5-1-integrin
subunit results in perinatal lethality.
Twenty-two of eighty-two (27%) potential founders integrated the
full-length human 5-integrin construct and survived to
weaning. In contrast, the first zygote injection of the
5-1-integrin transgene construct yielded 74 pups
surviving to weaning but no transgenic animals, suggesting that
expression of 5-1-integrin in the heart might be lethal.
A second injection of the 5-1-integrin construct was
performed, and pups were screened for integration of the
5-1-integrin transgene at birth. Thirteen (18%) of 72 animals integrated the 5-1-integrin construct (Table
1). Of these, five died perinatally, three died between 3 and 5 wk of age, and five survived to sexual maturity (mortality P < 0.0001 compared with
5-integrin transgenic animals). Potential founders were
mated to FVB/N mice, and F1 animals were screened. Eighteen of
twenty-two founders expressing 5-integrin and four of
five founders expressing 5-1-integrin exhibited germline
transmission (Table 1). However, the surviving 5-1-integrin-expressing lines had low-level expression
(less than endogenous 5-integrin) of
5-1-integrin protein (data not shown).
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Expression of the 5- and 5-1-integrin
transgenes at similar levels results in different phenotypes.
Transgene copy number ranged from 3 to 40 for the
5-1-integrin transgene and from 6 to 24 for the
wild-type 5-integrin transgene (Table
2). Comparison of two higher-expressing
lines (JAM40 for 5-1-integrin and JAM443 for
5-integrin) revealed similar levels of transgene mRNA
(Fig. 1A) and protein (Fig.
1C). The ratio of 5- to
5-1-integrin transgene protein was 1:1.7 by quantitative densitometry. Thus differences in expression of the two forms of
5-integrin transgenes are not a likely cause for the
lethal effect of 5-1-integrin. Relative to the level of
endogenous 5-integrin, both transgenes were
overexpressed (Fig. 1B). Comparison of total 1 (1A + 1D)-integrin
expression (data not shown) or specific 1D-integrin
expression from a nontransgenic animal (Fig. 1D, lane 1)
with that of 5-1 (lane 2)- and
5 (lane 3)-integrin transgenic animals
revealed no change in 1-integrin expression to
compensate for the increased 5-integrin expression.
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5-integrin was expressed exclusively in the
cardiovascular system. Staining of a neonate with the human-specific 5-integrin monoclonal antibody 6F4 (32)
revealed human 5-integrin expression in the pulmonary
and internal jugular veins, atria, and ventricles (Fig.
2A). The 5- and
5-1-integrin subunits localized in the plasma membrane,
Z-disks, intercalated junctions, and T-tubule system, similar to
endogenous integrins within cardiomyocytes (18). Figure
2B shows expression of the human 5-integrin
in an F1 animal from the JAM443 line, and Fig. 2C shows
expression of 5-1-integrin in a transgenic F1 animal
from the JAM40 line. In both lines, the transgene was expressed in all
cardiomyocytes. The staining intensity of the transgene protein
correlated well with copy number (Table 2). JAM40 transgenic offspring
expressed 5-1-integrin intensely in all cardiomyocytes.
In contrast, the JAM40 founder and JAM37 offspring expressed
5-1-integrin intensely, but only in approximately
one-half of cardiomyocytes (Fig. 2D). Given the normal life
span (~2.5 yr) and the inevitable early death of transgenic JAM40
offspring, this was of particular interest. These results demonstrate
that high-level expression of 5-1-integrin restricted to
a subset of cardiomyocytes is not associated with any apparent cardiac
phenotype, while expression in all cardiomyocytes is lethal.
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ECG analysis and histopathology.
All independently derived potential founders expressing the
5-1-integrin protein at high levels in all
cardiomyocytes died at an early age. However, we were able to perform a
more detailed evaluation of the JAM40 line, inasmuch as the male
founder was healthy and fertile. JAM40 F1 transgenic animals inevitably
died between 18 and 22 days of age, without prior overt signs of ill health. At the time of death, the hearts of JAM40 animals exhibited massive atrial enlargement (Fig.
3, C and
D). All cardiomyocytes exhibited strong staining for
5-1-integrin expression (Fig. 2C). Serial
sections revealed no gross anatomic defects or valvular abnormalities
in JAM40 transgenic mice (data not shown). As JAM40 offspring
approached the age of weaning, there was striking disorganization of
atrial and ventricular myocytes, expansion of the interstitium, and
fibrosis (Fig. 3, H-J). As noted above, two
independently derived animals expressing 5-1-integrin,
JAM35 and JAM50, exhibited histopathology indistinguishable from that
of JAM40 F1 animals (data not shown). In contrast, no histological
changes were observed in hearts from animals expressing wild-type
5-integrin (data not shown). The JAM40 F1 line also
exhibited an approximately twofold increase in steady-state mRNA
encoding atrial natriuretic factor, a fetal gene reexpressed during
hypertrophy (Fig. 3K).
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5-integrin were indistinguishable from ECGs
obtained from nontransgenic animals before 3 mo of age.
Although ECG abnormalities were evident before the onset of
histological changes, we sought to determine whether abrogating the
pathological changes would prevent or diminish the severity of ECG
abnormalities. The MHC promoter is transcriptionally regulated by
thyroid hormone (31). Three litters of JAM40 offspring
were treated with propylthiouracil (PTU, 1 mg/ml in drinking water) to
induce hypothyroidism (22) and evaluated for ECG
abnormalities, histopathological changes, and survival. Ten JAM40
animals treated with PTU were followed by ECG analysis to monitor the
development of conduction abnormalities. Characteristic ECG
abnormalities were noted in two animals by 10 days of age and in all
animals by 20 days of age. At 28 days, five animals were killed for
histopathology. Interestingly, no histological abnormalities were
observed in PTU-treated mice, despite the presence of significant ECG
abnormalities (data not shown). The remaining five PTU-treated
transgenic animals survived for 20, 26, 36, 42, and 45 days (the last
died during anesthesia), unprecedented survival for JAM40 transgenic
animals. Thus PTU administration prolonged survival and prevented
cardiac fibrosis but did not abrogate the ECG abnormalities or prevent premature death. Thus the ECG abnormalities appeared to develop independently of histological abnormalities in the myocardium.
The tissue renin-angiotensin system is prominently implicated in the
fibroproliferative response accompanying pathological cardiac
hypertrophy (28). Overexpressing the angiotensin
AT1 receptor under the control of the MHC
promoter results in a phenotype quite similar to the JAM40 line
(14). We treated three litters of JAM40 F1 mice with
[2S]-1-[3-mercapto-2-methylpropionyl]-L-proline (Captopril), an inhibitor of angiotensin-converting enzyme. Captopril neither suppressed the development of ECG abnormalities nor prolonged survival, inasmuch as transgenic animals died at 17 days of age. However, Masson's trichrome staining of sections revealed no evidence of mature connective tissue deposition (data not shown). We conclude that, as suggested by the results of PTU administration, cardiac fibrosis is not required for the ECG abnormalities or for a fatal outcome in this model. However, the renin-angiotensin system may be
involved in expansion of the fibroblast component of the heart and
connective tissue deposition associated with expression of the
activated 5-1-integrin in cardiac myocytes.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
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We show that transgenic overexpression of a human
5-integrin subunit lacking the cytoplasmic domain in
cardiomyocytes results in perinatal lethality, while overexpression of
the full-length 5-integrin does not. Potential
5-1-integrin-expressing founder animals exhibited a
consistent phenotype, including ECG abnormalities, atrial enlargement,
and fibrosis. The absence of a phenotype in animals expressing the
wild-type 5-integrin subunit demonstrates that this
phenotype is not due to a transgenic artifact. Moreover, the
5-1-integrin phenotype cannot result from competition
for endogenous 1-integrin subunits with endogenous mouse
-integrin subunits. Thus the human 5-1-integrin acts
as a gain-of-function rather than a classic dominant negative mutant
(15), consistent with the enhanced biological activity of
5-11-integrin compared with the wild-type
51-integrin (33). If
integrins function as mechanotransducers in cardiomyocytes (27,
28, 30), a physiological input signal could be erroneously
interpreted as pathological by cardiomyocytes, as schematized in Fig.
4.
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Alternatively, the 5-11-integrin complex
presents an unopposed 1-integrin cytoplasmic domain
within cardiomyocytes. Heterologous fusion proteins consisting of
nonintegrin extracellular and transmembrane domains (e.g., -subunit
of the interleukin-2 receptor) and 1 cytoplasmic domains
inhibit endogenous integrin function while activating integrin-mediated
signaling pathways (1, 20). Indeed, we have found that
expression of chimeric 1A- or
1D-integrins in cardiomyocytes also results in severe
perinatal lethality (unpublished observations). Thus it will be
necessary to create a third class of -subunit transgenic animals
with a deleted cytoplasmic domain and a mutation that prevents ligand
binding to distinguish between these two alternatives. Conditional
expression strategies are required to definitively address the effect
of gain- and loss-of-function mutations in integrins on cardiomyocyte
function during development and in the adult heart.
The mechanism of death in 5-1-integrin-expressing
animals appears to be related to abnormalities in the conduction system of the heart. We base this on the appearance of distinctive ECG abnormalities before the onset of cardiomyocyte disorganization and
fibrosis and the severity of the electrical abnormalities. The
suppression of histological abnormalities with PTU or captopril did not
abrogate the ECG abnormalities. This strongly suggests that the ECG
abnormalities are not secondary to fibrosis. Rather, it may reflect the
myogenic origin of the proximal conducting system (5, 12).
Altering integrin function could impair the ability of myocytes to
migrate or differentiate into the conducting cells. Regulation of
5-integrin is known to play a role in myogenesis in
other systems. In fact, an activating antibody mimicking the constitutive activation of 5-integrin, achieved here by
mutation, inhibited myogenesis (6). The absence of any
pathological findings in the JAM40 founder and in the JAM37 line
demonstrates a remarkable protective effect resulting from the presence
of normal cardiomyocytes adjacent to those expressing
5-1-integrin. The absence of effects on the conduction
system could be explained by preferential incorporation of
cardiomyocyte precursors lacking in transgene expression into the
conducting system. This still leaves unexplained the protection against
the fibroproliferative changes. Clearly, additional studies using
conditional expression strategies are necessary to sort out the
mechanisms and consequences of ectopic expression and activation of
integrins in cardiomyocytes.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Anita Jennings and the Histology Core Facility, Marv Ruona (Medical Graphics), and Suresh Savarirayan and Stephanie Munger (MCS Transgenic Core Facility).
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FOOTNOTES |
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This work was supported by the Mayo Foundation for Medical Education and Research and American Heart Association, Arizona Affiliate, Grants AZGS-43-96, SWA-GS-13-98, and AZFW-13-97.
Address for reprint requests and other correspondence: J. A. McDonald, Samuel C. Johnson Medical Research Bldg., Mayo Clinic Scottsdale, 13400 East Shea Blvd., Scottsdale, AZ 85259 (E-mail: mcdonald.john{at}mayo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 March 2000; accepted in final form 3 August 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Rayburn H,
and
Hynes RO.
Embryonic mesodermal defects in 5 integrin-deficient mice.
Development
119:
1093-1105,
1993[Abstract].
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