U.S. patent application number 11/429204 was filed with the patent office on 2007-06-07 for cardiac muscle function and manipulation.
This patent application is currently assigned to Zensun (Shanghai) Science and Technology Ltd.. Invention is credited to Mingdong Zhou.
Application Number | 20070129296 11/429204 |
Document ID | / |
Family ID | 3812054 |
Filed Date | 2007-06-07 |
United States Patent
Application |
20070129296 |
Kind Code |
A1 |
Zhou; Mingdong |
June 7, 2007 |
Cardiac muscle function and manipulation
Abstract
A method of causing cardiomyocyte growth and/or differentiation,
the method comprising exposing a cardiomyocyte to neuregulin (NRG)
thereby activating the MAP kinase pathway in the cardiomyocyte and
causing growth and/or differentiation of the cardiomyocyte. Use of
neuregulin, neuregulin polypeptide, neuregulin derivatives, or
compounds which mimic the activities of neuregulins in the
treatment or management of heart disease and heart failure in a
mammal.
Inventors: |
Zhou; Mingdong; (La Jolla,
CA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Zensun (Shanghai) Science and
Technology Ltd.
|
Family ID: |
3812054 |
Appl. No.: |
11/429204 |
Filed: |
May 4, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09980672 |
Mar 8, 2002 |
|
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PCT/AU99/01137 |
Dec 21, 1999 |
|
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11429204 |
May 4, 2006 |
|
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Current U.S.
Class: |
514/9.7 ;
514/16.2; 514/16.4; 514/9.6 |
Current CPC
Class: |
G01N 33/74 20130101;
G01N 2333/4756 20130101; G01N 33/6887 20130101; A61K 38/1883
20130101; G01N 2800/32 20130101; G01N 2800/325 20130101; A61P 9/00
20180101; C12N 5/0657 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/18 20060101
A61K038/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 1998 |
AU |
PP 7850 |
Claims
1.-21. (canceled)
22. A method for treating heart failure in a mammal in need
thereof, comprising administering an effective amount of a
neuregulin or a neuregulin analog to the mammal, wherein the
neuregulin or neuregulin analog is a neuregulin isoform or a
polypeptide comprising a neuregulin EGF domain.
23.-24. (canceled)
25. The method of claim 22, wherein the neuregulin or neuregulin
analog comprises the EGF-like domain of human neuregulin .beta.2
isoform.
26. The method of claim 25, wherein the neuregulin or neuregulin
analog comprises the amino acid sequence of SEQ ID NO:2.
27. The method of claim 25, wherein the neuregulin is encoded by
the nucleic acid sequence set forth in SEQ ID NO: 1.
28. The method of claim 22, wherein the mammal is a human.
29. The method of claim 22, wherein the heart failure is a disease
state selected from the group consisting of congestive heart
failure, myocardial infarction, tachyarrhythmia, familial
hypertrophic cardiomyopathy, ischaemic heart disease, idiopathic
dilated cardiomyopathy and myocarditis.
30. The method of claim 22, wherein the heart failure is ischaemic,
congenital, rheumatic or idiopathic.
31. The method of claim 22, wherein the heart failure results from
disassociation of cardiac muscle cell-cell adhesion and/or the
disarray of sarcomeric structures in the human.
32. The method of claim 22, wherein the heart failure results from
an agent that causes cardiac hypertrophy or congestive heart
failure.
33. The method of claim 32, wherein the agent that causes cardiac
hypertrophy or congestive heart failure is fludrocortisone acetate
or herceptin.
34. The method of claim 32, wherein the neuregulin or neuregulin
analog is administered prior to, during or after exposure to said
agent.
35. The method claim 22, wherein the neuregulin or neuregulin
analog is administered prior to or after the diagnosis of heart
failure in said mammal.
36. The method of claim 22, wherein the neuregulin or neuregulin
analog is administered after the mammal has suffered ischemia or
heart attack.
37. The method of claim 22, wherein administration of the
neuregulin or neuregulin analog maintains left ventricular
hypertrophy.
38. The method of claim 22, wherein said method prevents
progression of myocardial thinning.
39. The method of claim 22, wherein administration of the
neuregulin or neuregulin analog inhibits cardiomyocyte
apoptosis.
40. The method of claim 22, wherein administration of the
neuregulin or neuregulin analog decreases DNA synthesis in cardiac
muscle cells.
41. The method of claim 22, wherein the neuregulin or neuregulin
analog is administered in an amount of at least 10-8 M.
42. The method of claim 22, wherein the neuregulin or neuregulin
analog is administered with a pharmaceutically acceptable carrier,
excipient or stabilizer.
43. The method of claim 22, wherein the neuregulin or neuregulin
analog is administered by gene therapy methods.
44. The method of claim 22 further comprising administering an
effective amount of an agent that acts to suppress a hypertrophy
induction pathway different from the pathway suppressed by the
neuregulin or neuregulin analog, an angiotensin-converting enzyme
(ACE) inhibitor, or an agent for treating hypertension.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/980,672, now pending, which is the U.S. national stage
of International Application No. PCT/AU99/01137, filed Dec. 21,
1999, which claims priority to Australian Patent Application No. PP
7850, filed Dec. 21, 1998, the contents of which are incorporated
by reference herein in their entirety.
TECHNICAL FIELD
[0002] This invention relates to polypeptides which affect
myocardial cell differentiation and organisation of cardiac muscle
contractile units, assay for identifying such polypeptides, and
methods for improving cardiac function by the administration of
such polypeptides to patients with heart disease.
BACKGROUND OF THE INVENTION
[0003] Heart failure affects 1.5% of populations, approximately
three million Americans, developing at a rate of approximately
400,000 new cases per year in USA. Current therapy for heart
failure is primarily directed to using angiotensin-converting
enzyme (ACE) inhibitors and diuretics. ACE inhibitors appear to
slow progress to end-stage heart failure; however, they are unable
to relieve symptoms in more than 60% of heart failure patients and
reduce mortality of heart failure only by approximately 15-20%.
Heart transplantation is limited by the availability of donor
hearts. With the exception of digoxin, the chronic administration
of positive isotropic agents has not resulted in a useful drug
without adverse side effects, including increased arrhythmias, or
sudden death. These deficiencies in current therapy suggest the
need for additional therapeutic approaches.
[0004] Growth of cardiac muscle cells switches from proliferation
to hypertrophy during heart development. The former process is
characterised by an increase in cardiac muscle cell number, and the
latter by an increase in cell size without DNA synthesis or cell
division. This switch is associated with terminal differentiation
of cardiac muscle cells and occurs gradually during heart
development, starting during the late embryonic stages and ending a
few weeks after birth. During this period, gene expression,
particularly that involving the cell cycle and signalling, is
reprogrammed. For example, expression of a number of receptor
protein tyrosine kinases and other cell cycle components decreases.
Cell phenotype is also changed as cell-cell adhesions and
contractile proteins are more organised in terminal differentiated
myocardial cells.
[0005] Adult heart hypertrophy is an important adaptive
physiological response to increased demands for cardiac work or
after a variety of pathological stimuli that lead to cardiac
injury. Normal hypertrophic cells have a large size with increased
and well organised contractile units, as well as strong cell-cell
adhesions. Although pathologically hypertrophic cells also have
large size and accumulation of proteins, they often display
disorganisation of contractile proteins (disarray of sarcomeric
structures) and poor cell-cell adhesions (disarray of myofibers).
Thus, in pathological hypertrophy, the increase in size and
accumulation of contractile proteins are associated with
disorganised assembly of sarcomeric structures and a lack of robust
cell-cell interactions (Braunwald (1994) in Pathphysiology of Heart
Failure, (Braunwald, ed.); Saunsers, Philadelphia; Vol. 14, pp
393-402).
[0006] The disarray of myofibers and sarcomeres are important
features of cardiomyopathy. The former is a disorder of cell-cell
association, and the latter is disorganisation of heart muscle
contractile proteins. They are influenced by specific cell signals.
Thus, a number of signals, like growth factors and hormones, alter
cell adhesion and sarcomeric structure. Without these stimuli,
cardiomyocytes display disarray of the cytoskeleton and sarcomeric
structures, as well as disassociation of cell-cell interactions. As
cardiac muscle cell differentiation is tightly associated with
cardiac cell remodelling, adhesion and contractile protein
organisations, factors that stimulate myocardial cell
differentiation may be critical for enhancing the assembly of adult
cardiac muscle cell sarcomeric structures.
[0007] Studies in an in vitro model system of cardiac muscle cell
have led to the identification of a number of mechanical, hormonal,
growth factor, and pathological stimuli which can activate several
independent phenotype features of cardiac hypertrophy (Chien et al.
(1991) FASEB J. 5:3037-3046; Zhou et al., (1995) PNAS. USA,
92:7391-7395). Currently, there are at least three signal
transduction pathways, involving both ras-, rho- and G.sub.q
protein-dependent downstream effectors implicated in the activation
of features of the hypertrophic response in these in vitro model
systems. While a great deal of progress has been made in uncovering
the signalling pathways which activate the ventricular muscle cell
hypertrophic response, relatively little is known about the
mechanisms which specifically stimulate terminal differentiation of
cardiac muscle cells and the terminal differentiation-associated
assembly of contractile proteins. Compounds that could influence
these processes may be form a major new class of therapeutics for
the treatment of a variety of cardiac diseases.
[0008] Neuregulins, a family of EGF-like growth factors, activate
ErbB receptor tyrosine kinases that belong to the EGF receptor
superfamily, and are involved in an array of biological responses:
stimulation of breast cancer cell differentiation and secretion of
milk proteins; induction of neural crest cell differentiation to
Schwann cells; stimulation of skeletal muscle cell synthesis of
acetylcholine receptors; and, promotion of myocardial cell survival
and DNA synthesis. In vivo studies of neuregulin gene-targeted
homozygous mouse embryos with severe defects in ventricular
trabeculae formation and dorsal root ganglia development indicate
that neuregulin is essential for heart and neural development.
However, information on how neuregulin controls cell
differentiation and its downstream signalling pathways is
limited.
[0009] Within the heart, neuregulin and ErbB receptors are
respectively expressed in the endocardial lining and cardiac muscle
layer in early stages of development. Since these two layers are
widely separated, the neuregulin ligand must transverse the space
between the two cell layers to activate their cognate ErbB
receptors. Activation of these receptors in myocardial cells is
necessary for promoting muscle cell growth or migration toward the
endocardium, which results in the formation of finger-like
structures (ventricular trabeculae) between these two layers. It is
not clear previously if neuregulin stimulates myocardial cell
differentiation.
[0010] The present inventor has now found that neuregulin and/or
its cellular action may be suitable for use in detection, diagnosis
and treatment of heart disease. Moreover, the inventor believes
that potential beneficial effects of neuregulin and/or its cellular
action may be specific for heart muscle cells and not necessarily
applicable to skeletal or smooth muscle cells since 1) heart,
skeletal and smooth muscle are both embryological and functionally
distinct; 2) factors involved in skeletal muscle growth and
differentiation, such as MyoD, play little or no role in cardiac
muscle growth and differentiation; 3) inactivation of the genes for
ErbB2 or 4 receptors or neuregulin produces major defects in
cardiac but not skeletal or smooth muscle development; 4) as shown
here, the growth factor, insulin like growth factor-I (IGF-I)
causes embryonic myocyte proliferation but unlike neuregulin does
not stimulate differentiation of these cells. By contrast, IGF-I
but not neuregulin, has been shown to induce muscle
hypertrophy.
SUMMARY OF THE INVENTION
[0011] The present invention is based in part on the discovery that
neuregulin enhances cardiac muscle cell differentiation and
organisation of sarcomeric and cytoskeleton structures, as well as
cell-cell adhesion. Neuregulin, neuregulin polypeptide, neuregulin
derivatives, or compounds which mimic the activities of
neuregulins, fall within the scope of the methods of the present
invention and are abbreviated hereinafter as NRG.
[0012] In a first aspect, the present invention consists in a
method of causing cardiomyocyte growth and/or differentiation, the
method comprising exposing the cardiomyocyte to NRG thereby
activating the MAP kinase pathway in the cardiomyocyte and causing
growth and/or differentiation of the cardiomyocyte.
[0013] In a second aspect, the present invention consists in a
method of inducing remodelling of muscle cell sarcomeric and
cytoskeleton structures, or cell-cell adhesions, the method
comprising treating the cells with neuregulin thereby activating
the MAP kinase pathway in the cells and causing remodelling of the
cell structures or the cell-cell adhesions.
[0014] It will be appreciated that neuregulin may be provided
directly to the cell or provided indirectly by causing neuregulin
to be produced in cells by inducing expression of the gene(s)
involved in neuregulin production. The production may be in the
same cell to which the method is directed in autocrine manner or by
some other cell in a paracrine manner.
[0015] In a third aspect, the present invention consists in a
method of identifying polypeptides or compounds which stimulate
cardiac muscle cell differentiation, the method comprising
contacting the cardiac muscle with a test polypeptide or compound
in the presence of an inducer of cardiac muscle cell proliferation
in the form of neuregulin, and measuring the development of cardiac
muscle cell differentiation.
[0016] The differentiation of cardiac muscle cells is preferably
measured in cells exposed to neuregulin or other test polypeptides,
or to a mixture of neuregulin with a test polypeptide.
Differentiation of cardiac muscle cell can be measured in a variety
of ways, including by calculation of increases or decreases in DNA
synthesis, analysis of the time-course of phosphorylation of MAP
kinases in cardiac muscle cells, evaluation of the expression of
cell cycle inhibitor, p21.sup.CIP1, phenotypic organisation of
contractile units, accumulation of contractile units, phenotypic
alteration of cytoskeleton actin fibers, and the phenotype of
cell-cell adhesions.
[0017] In one preferred embodiment of the method of identifying
polypeptides or compounds which stimulate cardiac muscle cell
differentiation, cells are incubated with different concentrations
of various peptides or compounds and the effect of the test peptide
or compound in different concentrations on cardiac muscle cell
differentiation measured.
[0018] In another preferred embodiment of identifying polypeptides
or compounds which induce cardiac muscle cell differentiation that
dominates over that of the putative inducer of cardiac muscle
proliferation, insulin-like growth factor-1 (IGF-1), cells are
incubated with IGF-1, with and without the test polypeptide or
compound, and the ability of the test polypeptide or compound to
inhibit IGF-1-mediated cardiac muscle cell DNA synthesis, assembly
of sarcomeric structures and cell-cell adhesions are measured.
[0019] In a further embodiment, the cells are incubated with
phenylephrine (PE) with and without the test polypeptide or
compound, and the ability of the test polypeptide or compound to
augment PE-mediated cardiac muscle cell differentiation is
determined. A test polypeptide which stimulates cardiac muscle cell
differentiation may stimulate the assembly of sarcomeres and thus
enhance heart function in a variety of ways, including by
activating neuregulin-specific receptors, e.g., ErbB2, ErbB3 and
ErbB4.
[0020] In a fourth aspect, the present invention consists in a
method of identifying polypeptides or compounds which inhibit
neuregulin stimulation of ventricular muscle cell differentiation,
the method comprising contacting the ventricular muscle cell with
the test polypeptide or compound in the presence neuregulin and
measuring any inhibition of neuregulin stimulation of the
ventricular muscle cell.
[0021] A compound may inhibit neuregulin stimulation of ventricular
muscle cell differentiation by blocking, suppressing, reversing, or
antagonising the action of neuregulin. In one embodiment, the
measurement is by detecting DNA synthesis of ventricular muscle
cells.
[0022] In a fifth aspect, the present invention consists in a
therapeutic method of treating or preventing disassociation of
cardiac muscle cell-cell adhesion and/or the disarray of sarcomeric
structures in a mammal, the method comprising administering to the
mammal a therapeutically effective amount of a neuregulin or its
derivatives.
[0023] In one preferred embodiment, the therapeutic method is
directed to treating heart failure resulting from disassociation of
cardiac muscle cell-cell adhesion and/or the disarray of sarcomeric
structures in the mammal.
[0024] In a sixth aspect, the present invention consists in a
method of preventing or lowering the incidence of heart disease in
a mammal, the method comprising preventing or lowering the
interference or effects of polypeptides or compounds on the action
of neuregulin and its receptors, ErbBs, that produces heart
failure.
[0025] In another embodiment, a therapeutic agent which mimics the
effects of neuregulin is used to treat or prevent PE, or
IGF-1-mediated cardiac muscle cell dysfunction.
[0026] In an seventh aspect, the present invention consists in a
method of determining predisposition to heart disease or heart
failure in a subject, the method comprising testing cardiac or
related cells of the subject for the ability to express and/or
produce normal or adequate levels of neuregulin or its cognate ErbB
receptors. The inability to express and/or produce normal or
adequate levels of neuregulin being indicative of predisposition to
heart disease or heart failure.
[0027] In a eighth aspect, the present invention consists in the
use of neuregulin, neuregulin polypeptide, neuregulin derivatives,
or compounds which mimic the activities of neuregulins in the
treatment or management of heart disease and heart failure.
[0028] In a ninth aspect, the present invention consists in the use
of neuregulin, neuregulin polypeptide, neuregulin derivatives, or
compounds which mimic the activities of neuregulins in the
manufacture of a medicament for the treatment or management of
heart disease and heart failure.
[0029] By using primary cultured myocardial cells as a model
system, the present inventor evaluated neuregulin signalling in
cardiac myocyte differentiation, maturation and assembly or
maintenance of sarcomeric and cytoskeleton structures. To assay the
neuregulin effect on cell signalling, embryonic cardiac muscle
cells were incubated with recombinantly purified human neuregulin
ligand (rhNRG.beta.2). Neuregulin at 10.sup.-8M resulted in
sustained activation of MAP kinases for at least 21 hours, whereas
only transient activation was observed with a lower concentration
(10.sup.-10M) of rhNRG.beta.2. Expression of the Cdk inhibitor,
p21.sup.CIP1, was enhanced by the 10.sup.-8M, but not the
10.sup.-10M concentration of the ligand. The higher ligand
concentration, concomitant with this increase in p21.sup.CIP1
expression, resulted in a decrease in DNA synthesis, that was
associated with terminal differentiation, whereas an increase in
DNA synthesis and continued proliferation was observed with the
lower dose. Furthermore, when neuregulin was mixed with IGF-1,
rhNRG.beta.2 at either concentrations (10.sup.-8M, or 10.sup.-10M)
did not show a negative effect on the DNA synthesis and
significantly blocked IGF-I-stimulated cardiomyocyte proliferation.
To further evaluate the NRG-stimulated myocardial cell
differentiation, sarcomeric and cytoskeleton structures of cultured
neonatal rat cardiac muscle cells were examined by Phalloidin
staining and immunofluorescent staining with anti-.alpha.-actinin
antibody. rhNRG.beta.2 dramatically improved sarcomeric and
cytoskeleton structures, as well as cell-cell adhesions. Such an
effect was not found from the cells stimulated with either insulin
IGF-1 or PE. When rhNRG.beta.2 was mixed with either IGF-1 or PE,
rhNRG.beta.2 improved the cell structures. The 10.sup.-8M
concentration of rhNRG.beta.2 showed maximal effect on improvements
of sarcomeres and cell-cell adhesions. In addition, neuregulin
overrode the negative regulation of MHC-.alpha. expression mediated
by PE stimulation. These findings indicate that NRG function
through two distinct pathways: one activated at lower ligand
concentrations results in cardiomyocyte growth, whereas the other,
activated with higher concentrations, is mediated by sustained
activation of the MAP kinase pathway and results in terminal
differentiation and maturation.
[0030] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element, integer or step, or group of elements, integers or
steps, but not the exclusion of any other element, integer or step,
or group of elements, integers or steps.
[0031] In order that the present invention may be more clearly
understood, preferred forms will be described with reference to the
following examples and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1. Growth factor-stimulated DNA synthesis. DNA
synthesis ([.sup.3H] thymidine incorporation) by cultured embryonic
mouse cardiomyocytes in response to 20 hr of treatment with the
indicated concentrations of vehicle (purified Flag-peptide) (open
square), recombinant human NRG.beta.2 (rhNRG.beta.2) (closed
triangle) or insulin-like growth factor-I (IGF-1) (open circle).
Data shown are the mean.+-.S.E of five determinations with each
treatment and at each concentration. All rhNRG.beta.2 responses are
significantly greater than control (P<0.001) except at
10.sup.-7M, and the rhNRG.beta.2 responses to concentration .sup.3
10.sup.-9M are significant than the respective IGF-I responses
(P<0.01).
[0033] FIG. 2. NRG-mediated ErbB receptor phosphorylation. (a)
Serum-starved cardiomyocytes were stimulated with vehicle (0) or
rhNRG.beta.32 at a concentration of either 10.sup.-10M or
10.sup.-8M for the times indicated. Phosphorylation of ErbB
receptors was then determined as described in Methods using an
anti-phosphotyrosine antibody (RC20H). Fold increases in immunoblot
intensities are shown, which were normalised for protein load based
on the intensities of simultaneously determined immunoblots of
ErbB2 shown below the phosphotyrosine species. (b) Phosphorylation
of immunoprecipitated ErbB2 (top panel) or ErbB4 (bottom panel)
resulting from the stimulation of embryonic cardiomyocytes for 5
min with 10.sup.-10M or 10.sup.-8M rhNRG.beta.2. Studies were
performed as detailed in Methods and the immunoprecipitated
products evaluated by immuno-blotting with anti-phosphotyrosine,
anti-ErbB2 or anti-ErbB4 antibodies. Fold changes in immunoblot
intensities are shown, which were normalised for protein loading
based on the intensities of simultaneously determined immunoblots
of ErbB2 or ErbB4 shown below the phosphotyrosine species.
[0034] FIG. 3. NRG or IGF-I stimulated MAP kinase activation. (a)
MAP kinase phosphorylation resulting from embryonic cardiomyocyte
stimulation with rhNRG.beta.2 (10.sup.-10M or 10.sup.-8M) for the
times shown. After treatment with rhNR.beta.2, cell extracts were
prepared and evaluated for MAP kinase phosphorylation using an
anti-phospho MAP kinase antibody, as described in Methods. Fold
changes in immunoblot intensities are shown below the
phosphotyrosine species. To control for protein loading, cell
extracts were simultaneously evaluated for ErbB2 expression by
immunoblot analysis using anti-ErbB2 antibody. (b) MAP kinases
catalytic activity was determined as described in Methods using
extracts prepared from cardiomyocytes treated with rhNRG.alpha.2
(10.sup.-10M or 10.sup.-8M) for the times indicated, and shown as
fold increase over the basal level activity of cells which were not
stimulated with rhNRG.alpha.2. Values of the fold shown as the
means.+-.SE of five determinations with each treatment and at each
concentration. (c) MAP kinase phosphorylation resulting from IGF-I
(10.sup.-9M) stimulation of embryonic cardiomyocytes for the times
indicated, was determined as in (a).
[0035] FIG. 4. Effects of NRG on IGF-I stimulated DNA synthesis and
MAP kinase phosphorylation. (a) DNA synthesis ([.sup.3H) thymidine
incorporation) was examined in cultured cells stimulated with a
maximal concentration of IGF-I (10.sup.-9M) in the absence or
presence of rhNRG.alpha.2 at concentrations of either 10.sup.-10M
or 10.sup.-8M for 20 hrs. Bars show the mean values of data from
five samples .+-.1S.E (error bars). Similar results were obtained
from three independent experiments. Significant difference ( * * *
, P<0.001) from the control are indicated. (b) Time course of
MAPK phosphorylation in embryonic cardiac muscle cells in response
to a mixture of 10.sup.-9M IGF-I and 10.sup.-8M rhNRG.beta.2 was
determined by immunoblotting using a specific anti-phospho-MAPK
antibody. ErbB2 expression was evaluated simultaneously to control
for protein loading.
[0036] FIG. 5. NRG-mediated induction of p21.sup.CIP1 expression.
(a) p21.sup.CIP1 expression in cultured cardiac muscle cells
stimulated either with 10.sup.-10M or 10.sup.-8M rhNRG.beta.2 in
the absence or presence of serum (5% of FBS) for 24 hrs; or (b)
with 10.sup.-10M or 10.sup.-8M IGF-I. After the various treatments,
p21.sup.CIP1 expression was evaluated by immunoblot analysis using
an anti-p21.sup.CIP1 antibody. ErbB2 expression was evaluated
simultaneously to control for protein loading. Fold changes in
p21.sup.CIP1 expression, normalised for protein loading, are shown.
(c) Effect of the MEK inhibitor (PD98059) (50 .mu.M) on
rhNRG.beta.2 (either at 10.sup.-10M or 10.sup.-8M)-mediated
stimulation of p21.sup.CIP1 expression in cultured embryonic
cardiac muscle cells in the absence of serum. p21.sup.CIP1 was
detected by immunoblot analysis using an anti-p21.sup.CIP1
antibody. Fold changes in p21.sup.CIP1 expression, normalised for
protein loading, are shown. (d) Effects of PD98059 on the
inhibition of NRG- or IGF-I-activated MAP kinase activities were
monitored by a measurement of p42/44 MAP kinase phosphorylation
after cells were stimulated with NRG or IGF-I for 5 min. p42/44
kinase phosphorylation was evaluated by immunoblot analysis using
anti-phospho-p42/44 or anti-p42/44 MAP kinase antibodies. The same
amount of whole cell extract (20 .mu.g protein) was loaded, and
normalised for p42/44 MAP kinase expression, using an anti-p42/44
MAP kinase antibody.
[0037] FIG. 6. Effects of NRG on cardiac sarcomere assembly and
myosin heavy chains expression. (a) E12.5 mouse cardiac muscle
cells were cultured in serum-free medium (control) or stimulated
with 10.sup.-10 or 10.sup.-8M rhNRG.beta.2 (NRG) for 48 hrs. Cells
were then stained with phalloidin (left panels) or evaluated for
anti-.alpha.-actinin immunoflorescency (right panels). (b)
Sarcomeric myosin heavy chain .alpha.-actin expression
rhNRG.beta.2-stimulated embryonic mouse cardiac muscle cells were
evaluated by immunoblot analysis using an anti-sarcomeric myosin
heavy chain antibody (MF20) or an anti-a-actinin antibody. The same
amount of whole cell extracts (20 fig proteins) was loaded into
each lane for SDS-PAGE fractionation.
MODES FOR CARRYING OUT THE INVENTION
[0038] Utilising an in vitro system of cardiac muscle cell
differentiation, a role for neuregulin in stimulating the
activation of the differentiation response in comparison with two
well-defined hormonal and growth factor stimuli,
.alpha..sub.1-adrenergic agonists and IGF-1 has been demonstrated.
The present inventor has demonstrated that neuregulin
differentiation pathways exist within cardiac muscle cells, and
that neuregulin polypeptides can activate these pathways. Since
cardiac muscle cell differentiation includes the processes of
organisation of sarcomeric structures and cell-cell adhesions, the
invention, thus, provides a useful method for the treatment and
prevention of cardiac muscle cell with disorganisation of the
sarcomeric structures and cell-cell adhesions, and the enhancement
of heart function in cardiomyopathy, and for identifying
polypeptides or compounds which activate cardiac muscle
differentiation pathways.
[0039] Before the methods of the invention are described, it is to
be understood that this invention is not limited to the particular
methods described. The terminology used herein is for the purpose
of describing particular embodiments only.
[0040] As used in this specification, the singular forms "a", "an",
and "the" include plural references unless the context clearly
dictates otherwise. Thus, for example, references to "neuregulin"
or "a neuregulin peptide" includes mixtures of such neuregulins,
neuregulin isoforms, and/or neuregulin-like polypeptides. Reference
to "the formulation" or "the method" includes one or more
formulations, methods, and/or steps of the type described herein
and/or which will become apparent to those persons skilled in the
art upon reading this disclosure.
[0041] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference for the purpose of disclosing and describing material for
which the reference was cited in connection with.
Definitions
[0042] "Neuregulin or neuregulin analogs" are molecules that can
activate ErbB2/ErbB4 or ErbB2/ErbB3 heterodimer protein tyrosine
kinases, such as all neuregulin isoforms, neuregulin EGF domain
alone, neuregulin mutants, and any kind of neuregulin-like gene
products that also activate the above receptors. The "neuregulin"
used in this invention is the following polypeptide which is a
fragment of human neuregulin .beta.2 isoform containing the
EGF-like domain, the receptor binding domain.
[0043] The cDNA sequence: TABLE-US-00001 (SEQ ID NO:1)
AGCCATCTTGTAAATGTGCGGAGAAGGAGAAAACTTTCTGTGTGAATGGA
GGGGAGTGCTTCATGGTGAAAGACCTTTCAAACCCCTCGAGATACTTGTG
AGGAGCTGTACCAG
[0044] The amino acid sequence encoded by the above DNA sequence:
SHLVKCAEKEK TFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELNYQ
(SEQ ID NO:2).
[0045] "Cardiac muscle cell differentiation" is a condition
characterised by the decrease in DNA synthesis by more than 10%,
inhibition of other factor-stimulated DNA synthesis more than 10%,
well organised sarcomeric structures and cell-cell adhesions,
sustained activation of MAP kinases, and enhanced expression of
p21.sup.CIP1.
[0046] "Organised, or enhanced organisation of sarcomeres or
sarcomeric structures" is a condition characterised by the straight
array of contractile proteins revealed by immunofluorescent
staining of .alpha.-actinin in cardiac muscle cells. The straight
array of a-actinin proteins in cells can be distinguished by
microscopy and its connected photography as exampled in Figures of
this specification.
[0047] "Disorganised or disarray of sarcomeres or sarcomeric
structures" is the opposite meaning of the above definitions.
[0048] "Organised, or enhanced organisation of sarcomeres or
sarcomeric structures" is a condition characterised by the straight
actin fibers revealed by phalloidin staining of cardiac muscle
cells. The straight actin fibers in cells can be distinguished by
microscopy and its connected photography as exampled in Figures of
this specification.
[0049] "Disorganised or disarray of cytoskeleton structures" is the
opposite meaning of the above definitions.
[0050] "Sustained activation of MAP kinases" is that phosphorylated
state of MAP kinases, p42/44, is maintained for at least 21 hr in
cells.
[0051] "Enhanced expression of p21.sup.CIP1" is that expression of
p21.sup.CIP1 is increased at least 50% that is maintained for at
least 24 hr in cells.
[0052] "The treatment of heart diseases" includes all suitable
kinds of methods, such as vein injection of the neuregulin
polypeptide, and gene therapy methods, in which heart or other
cells were forced to contain a gene encoding neuregulin or
derivatives for the treatment of heart diseases. For example,
Adenovirus or Adeno-Associated-Virus can be used as a carrier to
deliver neuregulin gene into infected heart or other cells. The
infected cell can then express and secret neuregulin polypeptide to
activate ErbBs on cardiac muscle cells.
[0053] "Ventricular muscle cell hypertrophy" is a condition
characterised by an increase in the size of individual ventricular
muscle cells, the increase in cell size being sufficient to result
in a clinical diagnosis of the patient or sufficient as to allow
the cells to be determined as larger (e.g., 2-fold or more larger
than non-hypertrophic cells). It may be accompanied by accumulation
of contractile proteins within the individual cardiac cells and
activation of embryonic gene expression.
[0054] In vitro and in vivo methods for determining the presence of
ventricular muscle cell hypertrophy are known. In vitro assays for
ventricular muscle cell hypertrophy include those methods described
herein, e.g., increased cell size and increased expression of
atrial natriuretic factor (AND). Changes in cell size are used in a
scoring system to determine the extent of hypertrophy. These
changes can be viewed with an inverted phase microscope, and the
degree of hypertrophy scored with an arbitrary scale of 7 to 0,
with 7 being fully hypertrophied cells, and 3 being non-stimulated
cells. The 3 and 7 states may be seen in Simpson et al. (1982)
Circulation Res. 51: 787-801, FIG. 2, A and B, respectively. The
correlation between hypertrophy score and cell surface area
(.mu.m.sup.2) has been determined to be linear (correlation
coefficient=0.99). In phenylephrine-induced hypertrophy,
non-exposed (normal) cells have a hypertrophy score of 3 and a
surface area/cell of 581 .mu.m.sup.2 and fully hypertrophied cells
have a hypertrophy score of 7 and a surface area/cell of 1811
.mu.m.sup.2, or approximately 200% of normal. Cells with a
hypertrophy score of 4 have a surface area/cell of 771 .mu.m , or
approximately 30% greater size than non-exposed cells; cells with a
hypertrophy score of 5 have a surface area/cell of 1109
.mu.m.sup.2, or approximately 90% greater size than non-exposed
cells; and cells with a hypertrophy score of 6 have a surface
area/cell of 1366 .mu.m , or approximately 135% greater size than
non-exposed cells. The presence of ventricular muscle cell
hypertrophy preferably includes cells exhibiting an increased size
of about 15% (hypertrophy score 3.5) or more. Inducers of
hypertrophy vary in their ability to induce a maximal hypertrophic
response as scored by the above-described assay. For example, the
maximal increase in cell size induced by endothelin is
approximately a hypertrophy score of 5.
[0055] "Suppression" of ventricular muscle cell hypertrophy means a
reduction in one of the parameters indicating hypertrophy relative
to the hypertrophuc condition, or a prevention of an increase in
one of the parameters indicating hypertrophy relative to the normal
condition. For example, suppression of ventricular muscle cell
hypertrophy can be measured as a reduction in cell size relative to
the hypertrophic condition. Suppression of ventricular muscle cell
hypertrophy means a decrease of cell size of 10% or greater
relative to that observed in the hypertrophic condition. More
preferably, suppression of hypertrophy means a decrease in cell
size of 30% or greater; most preferably, suppression of hypertrophy
means a decrease of cell size of 50% or more. Relative to the
hypertrophy score assay when phenylephrine is used as the inducing
agent, these decreases would correlate with hypertrophy scores of
about 6.5 or less, 5.0-5.5, and 4.0-5.0, respectively. When a
different agent is used as the inducing agent, suppression is
measured relative to the maximum cell size (or hypertrophic score)
measured in the presence of that inducer.
[0056] Prevention of ventricular muscle cell hypertrophy is
determined by preventing an increase in cell size relative to
normal cells, in the presence of a concentration of inducer
sufficient to fully induce hypertrophy. For example, prevention of
hypertrophy means a cell size increase less than 200% greater than
non-induced cells in the presence of maximally-stimulating
concentration of inducer. More preferably, prevention of
hypertrophy means a cell size increase less than 135% greater than
non-induced cells; and most preferably, prevention of hypertrophy
means a cell size increase less than 90% greater than non-induced
cells. Relative to the hypertrophy score assay when phenylephrine
is used as the inducing agent, prevention of hypertrophy in the
presence of a maximally-stimulating concentration of phenylephrine
means a hypertrophic score of about 6.0-6.5, 5.0-5.5, and 4.0-4.5,
respectively.
[0057] In vivo determination of hypertrophy include measurement of
cardiovascular parameters such as blood pressure, heart rate,
systemic vascular resistance, contractility, force of heart beat,
concentric or dilated hypertrophy, left ventricular systolic
pressure, left ventricular mean pressure, left ventricular
end-diastolic pressure, cardiac output, stroke index, histological
parameters, and ventricular size and wall thickness. Animal models
available for determination of development and suppression of
ventricular muscle cell hypertrophy in vivo include the
pressure-overload mouse model, RV murine dysfunctional model,
transgenic mouse model, and post-myocardial infarction rat model.
Medical methods for assessing the presence, development, and
suppression of ventricular muscle cell hypertrophy in human patents
are known, and include, for example, measurements of diastolic and
systolic parameters, estimates of ventricular mass, and pulmonary
vein flows.
[0058] The terms "treatment", "treating" and the like are used
herein to generally mean obtaining a desired pharmacological and/or
physiological effect. The effect may be prophylactic in terms of
completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete cure
for a disease and/or adverse effect attributable to the disease.
"Treatment" as used herein covers any treatment of a disease in a
mammal, particularly a human, and includes:
[0059] preventing the disease from occurring in a subject which may
be predisposed to the disease but has not yet been diagnosed as
having it;
[0060] inhibiting the disease, i.e., arresting its development;
or
[0061] relieving the disease, i.e., causing regression of the
disease.
[0062] The invention is directed to treating patients with or at
risk for development of heart disease and related conditions, e.g.,
heart failure. More specifically, "treatment" is intended to mean
providing a therapeutically detectable and beneficial effect on a
patient suffering from heart disease.
[0063] By the term "heart failure" is meant an abnormality of
cardiac function where the heart does not pump blood at the rate
needed for the requirements of metabolising tissues. Heart failure
includes a wide range of disease states such as congestive heart
failure, myocardial infarction, tachyarrythmia, familial
hypertrophic cardiomyopathy, ischaemic heart disease, idiopathic
dilated cardiomyopathy, and myocarditis. The heart failure can be
caused by any number of factors, including ischaemic, congenital,
rheumatic, or idiopathic forms. Chronic cardiac hypertrophy is a
significantly diseased state which is a precursor to congestive
heart failure and cardiac arrest.
[0064] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) hypertrophy. Those in need of
treatment include those already with the disorder as well as those
prone to have the disorder or those in which the disorder is to be
prevented. The hypertrophy may be from any cause which is
responsive to retinoic acid, including congenital, viral,
idiopathic, cardiotrophic, or myotrophic causes, or as a result of
ischaemia or ischaemic insults such as myocardial infarction.
Typically, the treatment is performed to stop or slow the
progression of hypertrophy, especially after heart damage, such as
from ischaemia, has occurred. Preferably, for treatment of
myocardial infarctions, the agent(s) is given immediately after the
myocardial infarction, to prevent or lessen hypertrophy.
[0065] The terms "synergistic", "synergistic effect" and like are
used herein to describe improved treatment effects obtained by
combining one or more therapeutic agents with one or more retinoic
acid compounds. Although a synergistic effect in some fields is
meant an effect which is more than additive (e.g., 1+1=3), in the
field of medical therapy an additive (1+1=2) or less than additive
(1+1=1.6) effect may be synergistic. For example, if each of two
drugs were to inhibit the development of ventricular muscle cell
hypertrophy by 50% if given individually, it would not be expected
that the two drugs would be combined to completely stop the
development of ventricular muscle cell hypertrophy. In many
instances, due to unacceptable side effects, the two drugs cannot
be administered together. In other instances, the drugs counteract
each other and slow the development of ventricular muscle cell
hypertrophy by less than 50% when administered together. Thus, a
synergistic effect is said to be obtained if the two drugs slow the
development of ventricular muscle cell hypertrophy by more than 50%
while not causing an unacceptable increase in adverse side
effects.
Materials and Methods
Reagents and Antibodies
[0066] The following antibodies and reagents were used: IGF
(Boehringer); collagenase (Worthington); pancreatin (Gibco BRL);
MEK1 (MAPKK) inhibitor (PD98059) (New England);
[methyl-.sup.3H]thymidine (Amersham); monoclonal anti-erbB2
antibody (Novocastra); monoclonal IgG.sub.2b p21.sup.CIP1 (F-5)
(Santa Cruz), monoclonal anti-phospho-tyrosine horse radish
peroxidase (HRPO)-conjugated antibody, RC20 (Transduction
Laboratories); monoclonal anti-.alpha.-sacromeric actin antibody
(clone 5c5), HRPO-conjugated anti-rabbit Ig, and anti-mouse Ig
(Sigma); PhosphoPlus.RTM. p44/42 MAP kinase (Thr202/Tyr204)
antibody kit (New England); anti-FLAG.RTM. M1 affinity gel and
anti-FLAG M2 monoclonal antibody (Eastman Kodak) mAb MF20 to
sarcomeric myosin heavy chain (kindly provided by R. P. Harvey,
Victor Chang Cardiac Research Institute); anti-sarcomeric
.alpha.-actin antibody (Sigma).
Recombinant Human NRG12 Expression and Purification
[0067] A cDNA encoding the EGF-like domain of human NRG.beta.2
isoform (rhNRG.beta.2, residues 177-237, was inserted into the
pFLAG1 expression vector (IBI) (a gift from Dr. Rodney J. Fiddes,
Co-operative Research Centre for Biopharmaceutical Research,
Australia). rhNRG.beta.2 with a FLAG-peptide attached at its
N-terminus, was expressed in the periplasmic space of E. coli
DH5.alpha., and purified by affinity chromatography using anti-FLAG
M1 monoclonal antibody according to the manufacturer's
instructions. The purity of rhNRG.beta.2 was more than 90% as
evidenced by SDS-PAGE separation and Coomassie Blue staining of
purified protein samples. The concentration of purified proteins
was determined using a Bio-Rad protein assay kit. Activity of the
purified proteins was assayed by stimulation of MCF-7 breast cancer
cell ErbB receptors with various ligand doses. This revealed
increased ErbB receptor phosphorylation with increasing ligand
concentration (10.sup.-12M to 10.sup.-8M).
Primary Cultures of Mouse Cardiac Myocytes
[0068] Mouse embryos (El 1.5-12.5) were used to prepare primary
cardiac myocytes. Heart tissue was isolated aseptically from
embryos. Myocardial cells were isolated by collagenase digestion
and separated from non cardiomyocytes by preattachments on culture
dishes that was performed three times. Cells were then cultured as
described previously. Using this method, it was routinely possible
to obtain primary cultures with >90% myocytes.
ErbB and MAP Kinase Phosphorylation, and MAP Kinase Activity
[0069] Embryonic myocardial cells were cultured in serum-free
medium for at least 24 hrs and then stimulated with rhNRG.beta.2 or
IGF-I for various times. Stimulation was terminated by washing
cells rapidly with cold PBS. To block the MAP kinase activation,
the MEK inhibitor, PD98059, was added to the medium 30 mins prior
to the administration of rhNRG.beta.2 or IGF-I. Cells were then
harvested as previously described for Western blot analysis with
HRPO-conjugated monoclonal antibody RC20H (1:2,000) for detection
of phosphorylated ErbB receptors, or a phospho-specific p42/p44 MAP
kinase antibody (dilution ratio 1:1,000) for detection of
phosphorylated MAP kinases. The same amount of cell extract protein
was loaded into each lane and separated by SDS-PAGE. Immunobloting
with an anti-ErbB receptor or anti-p42/44 MAP kinase antibodies was
also used to normalise for protein loading. MAP-kinase (p42/p44)
activity was measured using a p42/44 MAP kinase enzyme assay kit
(RPN84; Amersham, Bucks., U.K.) according to the manufacturer's
instructions.
Detection of p21.sup.CIP1 Protein
[0070] Embryonic myocardial cells cultured in serum-free or 5% FBS
medium were stimulated with various concentrations of rhNRG.beta.2
with or without the MEK inhibitor, PD98059, for 24 or 48 hrs,
harvested as described above and subjected to immunoblot analysis
using an anti-p21.sup.CIP1 antibody (1:100). The same amount of
protein was loaded into each well of a SDS polyacrylamide gel.
After immunobloting, the membrane was stripped and probed further
with an antibody to the ErbB2 receptor for normalisation of protein
loading.
Thymidine Incorporation
[0071] Embryonic myocardial cells were cultured with rhNRG.beta.2
or IGF-I containing serum-free DMEM for 20 hrs. [methyl-.sup.3H]
Thymidine (0.5 .mu.Ci/well) was added and cells were then cultured
for a further 12 hrs. After rinsing twice with cold PBS, once with
ice-cold 10% trichloroacetic acid, and then five times with
ice-cold PBS, the cells were dissolved in 100 .mu.l of 1% SDS, and
counted in a liquid scintillation counter.
Immunofluorescent and Phalloidin Staining
[0072] Myocardial cells were plated in 2-well Novex plates (Nunc),
and cultured with or without rhNRG.beta.2 in serum-free DMEM medium
for 24-48 hour. After rinsing the cells with PBS, they were fixed
with 4% paraformaldehyde and 0.1% Triton X-100 at room temperature
for 30 minutes. The fixed cells were then blocked with 5% skim milk
in PBS for 1 hr, followed by incubation with an anti-a-actinin
monoclonal antibody (Sigma), for 45 minutes at room temperature.
After washing, anti-mouse IgG conjugated with FITC (Sigma) was
added and the cells were incubated for another half hour. For
phalloidin staining, cells were fixed with 4% formadehyde for 1 hr,
washed, and stained with phalloidin buffer (100 .mu.l PBS, 10 .mu.l
rhodarmine phalloidin (6.6 .mu.M in MeOH)) for 1 hr. After PBS
washing, cells were mounted with 1% p-phenylenediamine (1 mg/ml,
Sigma) in glycerol, and then covered and sealed. Cells were
examined using a UV fluorescent microscope and photographed with a
40.times. power objective.
[0073] All of the above assays were repeated at least three times
for each experiments. Data for DNA synthesis and MAP kinase
activity are presented as the mean.+-.S.E of five replicate
samples. Statistical significance was determined by ANOVA using the
SAS statistical package with P<0.05 being considered
significant. Immunoblots were quantitated by densitometry analysis
with the intensity of the evaluated protein bands being shown below
the blots as the fold changes over control (see Figures).
Results
NRG Regulates Embryonic Myocardial Cell DNA Synthesis
[0074] DNA synthesis in primary embryonic mouse cardiomyocytes
(E11.5-12.5) was evaluated to investigate their growth response to
NRG following stimulation with rhNRG.beta.2. As shown in FIG. 1,
rhNRG.beta.2 at a concentration of 10.sup.-10 produced an
approximately 2-fold increase in the DNA synthesis. However, DNA
synthesis decreased with ligand concentrations >10.sup.-10M. In
contrast to the response to rhNRG.beta.2, myocardial cells showed
only a proliferative response to recombinant human insulin-like
growth factor I (IGF-I), in concentrations ranging from 10.sup.-11M
to 10.sup.-7M. Inhibition of DNA synthesis by the higher
concentrations of NRG was not due to E. coli proteins contaminating
the bacterially-expressed rhNRG.beta.2, since proteins purified
from bacteria transformed with FLAG-vector alone did not inhibit
DNA synthesis. Moreover, to avoid possible effects of E. coli
proteins, both commercially obtained IGF-I and purified
rhNRG.beta.2 were disolved or diluted with anti-FLAG-protein
preparations (10.sup.-8M of FLAG peptide). These reagents showed
identical activities to those prepared with PBS in stimulating
myocardial cell DNA synthesis.
NRG Activates Embryonic Myocardial Cell ErbB Receptors
[0075] Of the four members of the ErbB receptor family (ErbB 1-4),
ErbB2 and ErbB4 are most abundantly expressed in cardiac myocytes.
Phosphorylation of ErbB2 and ErbB4 receptors was evaluated by
Western blot analysis of cell lysates, following stimulation with
either 10.sup.-8M or 10.sup.-10M rhNRG.beta.2. As shown in FIG. 2a,
a higher level of phosphorylated 180-185 kDa proteins corresponding
to ErbB2/ErbB4 receptors, was evident with the higher concentration
of NRG. The levels of phosphorylation gradually decreased with
time. The concentration dependence of p180-185 protein
phosphorylation corresponded to that for the decrease in DNA
synthesis with rhNRG.beta.2 treatment (FIG. 1). ErbB2 and ErbB4
receptors were also immunoprecipitated using anti-ErbB2 or ErbB4
antibodies, and examined by Western blotting with
anti-phospho-tyrosine antibodies. As shown in FIG. 2b,
phosphorylation of both receptors was dependent on rhNRB32
concentrations. Although ErbB2 and ErbB4 phosphorylation levels
differed slightly between experiments, the relative phosphorylation
difference between high and low concentrations of rhNRG.beta.2
persisted.
NRG Concentration-dependent Activation of MAP Kinases
[0076] Activation of the ErbB receptor family initiates a cascade
of molecular interactions, ultimately resulting in the stimulation
of MAP kinases. The duration of MAP kinase activation is critical
for cell-fate decisions. Therefore, the present inventor
investigated the time course of MAP kinase phosphorylation after
either 10.sup.-8M or 10.sup.-10M rhNRG.beta.32 treatment, using a
specific-phospho-MAP kinase antibody, which recognises
phosphorylated p42/p44 MAP kinases. As shown in FIG. 3a,
phosphorylation of p42/p44 MAP kinases was sustained for at least
21 hours with the higher dose of rhNRG.beta.2. MAP kinase
activation was transient at the lower ligand concentration, and
fell to the basal level in less than three hours. As shown in FIG.
3b, MAP kinase catalytic activity paralleled these changes in
phosphorylation. Thus, MAP kinase activity was sustained for at
least 21 hours in cells stimulated with 10.sup.-8M rhNRG.beta.2,
but was only transient in cells treated with 10.sup.-10M
rhNRG.beta.2. In contrast to these NRG responses, MAP kinase
phosphorylation was transient both with low (10.sup.-9M) (FIG. 3c)
and with high concentrations (10.sup.-8M or 10.sup.-7M) of
IGF-I.
Effect of NRG on IGF-I-stimulated Myocardial Cell Proliferation
[0077] Since myocardial cells are exposed to multiple peptide
hormones and growth factors in vivo, the present inventor
investigated if the growth inhibitory effects of a high
concentration of NRG could oppose the proliferative response of
other growth factors. This was achieved by evaluating the effects
of both rhNRG.beta.2 and IGF-I on cardiac myocyte DNA synthesis. As
shown in FIG. 4a, a 10.sup.-10M concentration of NRG had little
effect on IGF-I (10.sup.-9M)-stimulated DNA synthesis. However, the
10.sup.-8M concentration significantly blocked the IGF-I response.
This indicated that a specific intracellular pathway was activated
by the higher concentration of NRG. Interestingly, no additive
effect was observed when both IGF-I and the lower concentration of
NRG were applied to cells, indicating that the 10.sup.-9M
concentration of IGF-I may already be maximal. That the pathway(s)
activated by the higher concentration of NRG may be dominant over
that activated by IGF-I was further supported by the observation
that the combination of IGF-I (10.sup.-9M) and rhNRG.beta.2
(10.sup.-8M) resulted in sustained MAP kinase phosphorylation
(compare FIG. 4b and FIG. 3c)
NRG and p21.sup.CIP1 Expression
[0078] Since sustained activation of MAP kinase is directly related
to the expression of p21.sup.CIP1 in other types of cells,.sup.31
and accumulation of p21.sup.CIP1 leads to cell cycle arrest at the
G1 phase,.sup.32,33 it was asked if the sustained activation of MAP
kinases leads to a higher level of p21.sup.CIP1 expression in
embryonic cardiac muscle cells. As shown in FIG. 5a, an increase in
p21.sup.CIP1 expression was observed only with the higher
concentration of rhNRG.beta.2. This effect on p21.sup.CIP1
expression was independent of the cell culture conditions used,
since similar effects were observed with both serum-free and
serum-containing culture medium. Enhanced p21.sup.CIP1 expression
with 10.sup.-8M rhNRG.beta.2 was sustained for at least 24 hours (a
48 hour incubation of cells with rhNRG.beta.2 results in an
identical expression of p21.sup.CIP1), and thus, may be critical
for the inhibition of DNA synthesis in cardiac muscle cells treated
with the high concentration of NRG. As shown in FIG. 5b, IGF-I did
not stimulate p21.sup.CIP1 expression. To evaluate if the
p21.sup.CIP1 response involves MAP kinase activation,
cardiomyocytes were treated with the specific MAP kinase kinase
(MEK1) inhibitor (PD98059). Both in the presence or absence of
serum, PD98059 blocked the increase in p21.sup.CIP1 expression
induced by 10.sup.-8M rhNRG.beta.2 (FIG. 5c), as well as the
increase in p42/44 MAP kinase phosphorylation (FIG. 5d).
NRG Sarcomeric Structure and MHC Expression
[0079] To examine if NRG also affects embryonic myocardial cell
structure and function, the effects of NRG on cardiomyocyte
cytoskeletal and sarcomeric structures were evaluated. As shown in
FIG. 6a, rhNRG.beta.2 (10.sup.-8M) stimulated both sarcomeric actin
reorganisation (phalloidin staining) and cardiac contractile unit
assembly (staining of .alpha.-actinin in Z-bands). In contrast,
effects of 10.sup.-10M rhNRG.beta.32 were much less evident (FIG.
6a). A role for NRG in the regulation of myocardial cell function
was also evident by the observation that rhNRG.beta.2 enhanced
expression of sarcomeric myosin heavy chains, while sarcomeric
actin expression remained unchanged (FIG. 6b). Moreover, the
effects of rhNRG.beta.2 on cardiomyocytes were also sensitive to
MEKI inhibition by PD98059.
Discussion
[0080] Evidence provided indicates that ligand (NRG) concentration
is an important factor in determining either the transient or
sustained activation states of MAP kinases. The latter results in
increased expression level of the Cdk inhibitor, p21.sup.CIP1, and
is associated with decreased DNA synthesis in embryonic myocardial
cells. This finding provides clear support that the ligand gradient
may decide cell fate in cell differentiation and embryo
development, and further furnishes molecular insights on how
intracellular signalling pathways distinguish the signal strength
based on ligand concentrations.
[0081] The importance of ligand concentration in cell
differentiation has been suspected for some time based on the
following observations:
[0082] i) embryo developmental patterning is associated with a
ligand gradient;
[0083] ii) ligand concentration is critical for cell
differentiation in vitro, and
[0084] iii) overexpression of receptors in cells changes their fate
in response to ligand stimulation.
[0085] Taking these observations into consideration, NRG
concentration-dependent MAP kinase activation in embryonic
myocardial cells establishes a model for further delineating the
mechanisms of erbB receptor-coupled cell signalling in reaction to
changes in ligand concentration.
[0086] The notion that NRG is a myocardial cell differentiation
factor is supported by the finding that NRG induces expression of
p21.sup.CIP1 in embryonic myocardial cells. As p21.sup.CIP1 is well
documented to be an inhibitor of Cdk, which promotes entry from the
G1 to the S phase of the cell cycle, increased expression of this
protein in myocardial cells could be critical for the initiation of
terminal differentiation. This is also supported by previous
findings that p21.sup.CIP1 expression increases in vivo with the
onset of myocardial cell terminal differentiation (Parker et al.
(1995) Science 267:1024-1027), as well as with skeletal muscle cell
differentiation (Dias et al. (1994) Semin. Diagn Pathol. 11:3-14).
In the latter process, increased p21.sup.CIP1 expression eventually
results in an exit from the cell cycle and differentiation. Since
increase in p21.sup.CIP1 expression occurs prior to that of other
cell cycle regulators, it is used as an early marker for skeletal
muscle differentiation. As demonstrated here, expression of
p21.sup.CIP1 is concomitant with the decrease in DNA synthesis in
NRG-stimulated myocardial cells, suggesting the physiological role
of NRG-stimulated p21.sup.CIP1 CILP expression in these cells.
Furthermore, the inhibition of both MAP kinases and p21.sup.CIP1 by
the ERK kinases inhibitor assessed that NRG-stimulated p21.sup.CIP1
expression is a direct result of activation of MAP kinases.
[0087] The sustained activation of MAP kinases is required for
induction of p21.sup.CIP1 constitutive expression in cultured
myocardial cells, whereas transient MAP kinase activation results
in temporal expression of p21.sup.CIP1. The latter is presumably
insufficient to regulate the Cdk activity, since p21.sup.CIP1 will
be quickly degraded and constitutive expression is essential for
blocking the cyclin/Cdk complex. In PC12 cells, sustained
activation of the MAP kinase pathway is confined to a response to
specific signals from NGF receptors. The sustained activation of
MAP kinases causes PC12 cell differentiation becoming neuronal
cells. This pathway in cardiac myocytes, however, is able to
differentially respond to NRG concentration-based signal
strength.
[0088] Further evidence support that NRG is a differentiation
factor is that NRG stimulate assembly of sarcomeric and
cytoskeleton structures, which occur as myocardial progenitor cells
differentiate to cardiac muscle cells. Previous observation also
indicated that more differentiated cells have more organised
sarcomeres (Rumynatsev, P. P. (1977) in International Review
Cytology 51, pp 187-273). In a comparison of cells stimulated with
either PE or IGF-1, NRG-stimulated cells have the best organised
sarcomeres. More importantly, when NRG is mixed with PE or IGF-1,
NRG greatly improved sarcomeres, indicating that NRG is dominant in
stimulation of sarcomere assembly in presence of other cell
signals. NRG overrides the PE-mediated negative regulation of
MHC-.alpha. expression, indicating that NRG is involved in the
maintenance of adult type of contractile proteins. As previous
studies indicated that NRG, ErbB2 and ErbB4 are expressed in adult
heart, NRG should play a role in the maintenance of myocardial cell
differentiation state.
[0089] Two very important features of heart failure associated with
cardiomyopathy in patients are disarrays of myofibers and
sarcomeres. The former is the loose of the cell-cell adhesion and
the latter is the loose of the sarcomere organisation. These
pathological conditions widely exist from congestive heart failure
to dilated cardiomyopathy and severely affect heart function.
Currently no treatment is target on the assembly of cell-cell
adhesion and sarcomere structures. NRG clearly plays a role in the
process of the assembly and maintenance of cell-cell adhesion and
sarcomeric structures. That NRG stimulates myocardial cell
differentiation and the assembly of sarcomeric structures indicates
that cardiac muscle cell differentiation is associated with its
cell structure remodelling. Such a conclusion is consistent with
general observation from heart muscle cell differentiation during
heart development: differentiated muscle cells always contain well
organised sarcomeres.
[0090] In summary, that NRG is a differentiation factor for
myocardial cells is supported by following evidence:
[0091] i) NRG stimulates sustained activation of MAP kinases;
[0092] ii) NRG enhances p21.sup.CIP1 expression;
[0093] ii) NRG inhibits IGF-1-stimulated DNA synthesis;
[0094] iv) NRG stimulates the myocardial cell assembly of
sarcomeric and cytoskeleton structures; and
[0095] v) NRG stimulates expression of the adult-type MHC gene.
Therapeutic use
[0096] The present invention provides methods for treating or
preventing heart failure or cardiac muscle cell hypertrophy in a
mammal by providing an effective amount of a neuregulin.
Preferably, the mammal is a human patient suffering from or at risk
of developing heart failure.
[0097] The present invention is useful in preventing heart failure
and cardiomyopathy in patients being treated with a drug which
cause cardiac hypertrophy or congestive heart failure, e.g.,
fludrocortisone acetate or herceptin. In the method of the
invention, a neuregulin polypeptide can be given prior to,
simultaneously with, or subsequent to a drug which causes cardiac
diseases.
[0098] In the therapeutic method of the invention, a neuregulin
polypeptide is administered to a human patient chronically or
acutely, for example by injection into the patient's vein.
Optionally, neuregulin is administered chronically in combination
with an effective amount of a compound that acts to suppress a
different hypertrophy induction pathway than a neuregulin.
Additional optional components include a cardiotrophic inhibitor
such as a Ct-1 antagonist, an ACE inhibitor, such as captopril,
and/or human growth hormone and/or IGF-I in the case of congestive
heart failure, or with another anti-hypertrophic, myocardiotrophic
factor, anti-arrhythmic, or inotropic factor in the case of other
types of heart failure or cardiac disorder.
[0099] The present invention can be combined with current
therapeutic approaches for treatment of heart failure, e.g., with
ACE inhibitor treatment. ACE inhibitors are angiotensin-converting
enzyme inhibiting drugs which prevent the conversion of angiotensin
I to angiotensin II. The ACE inhibitors may be beneficial in
congestive heart failure by reducing systemic vascular resistance
and relieving circulatory congestion. ACE inhibitors include drugs
designated by the trademarks Accupril.RTM. (quinapril), Altace.RTM.
(ramipril), Capoten.RTM. (captopril), Lotensin.RTM. (benazepril),
Monopril.RTM. (fosinopril), Prinivil.RTM. (lisinopril),
Vasotec.RTM. (enalapril), and Zestril.RTM. (lisinopril).
[0100] The present invention can be combined with the
administration of drug therapies for the treatment of heart
diseases such as hypertension. For example, a neuregulin
polypeptide can be administered with endothelin receptor
antagonists, for example, and antibody to the endothelin receptor,
and peptide or other such small molecule antagonists;
.beta.-adrenoreceptor antagonists such as carvedilol;
.alpha..sub.1-adrenoreceptor antagonists; anti-oxidants; compounds
having multiple activities (e.g.,
.beta.-blocker/.alpha.-blocker/anti-oxidant); carvedilol-like
compounds or combinations of compounds providing multiple functions
found in carvedilol; growth hormone, etc.
[0101] Neuregulin agonists alone or in combination with other
hypertrophy suppressor pathway agonists or with molecules that
antagonise known hypertrophy induction pathways, are useful as
drugs for in vivo treatment of mammals experiencing heart failure,
so as to prevent or lessen heart failure effects.
[0102] Therapeutic formulations of agonist(s) for treating heart
disorders are prepared for storage by mixing the agonist(s) having
the desired degree of purity with optional physiologically
acceptable carriers, excipients, or stabilisers (Remington's
Pharmaceutical Sciences, 16.sup.th edition, Oslo, A., Ed., 1980),
in the form of lyophilised cake or aqueous solutions. Acceptable
carriers, excipients, or stabilisers are non-toxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid; low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counter ions such as sodium; and/or
non-ionic surfactants such as Tween, Pluronics, or polyethylene
glycol (PEG). The antagonist(s) are also suitably linked to one of
a variety of nonproteinaceous polymers, e.g., polyethylene glycol,
polypropylene glycol, or polyalkylenes, in the manner set forth in
U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337. The amount of carrier used in a formulation
may range from about 1 to 99%, preferably from about 80 to 99%,
optimally between 90 and 99% by weight.
[0103] The agonist(s) to be used for in vivo administration should
be sterile. This is readily accomplished by methods known in the
art, for example, by filtration through sterile filtration
membranes, prior to or following lyophilisation and reconstitution.
The agonist(s) ordinarily will be stored in lyophilised form or in
solution.
[0104] Therapeutic agonist compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle. The agonist(s) administration is in a chronic
fashion only, for example, one of the following routes: injection
or infusion by intravenous, intraperitoneal, intracerebral,
intramuscular, intraocular, intraarterial, or intralesional routes,
orally or using sustained-release systems as noted below.
Agonist(s) are administered continuously by infusion or by periodic
bolus injection if the clearance rate is sufficiently slow, or by
administration into the blood stream or lymph. The preferred
administration mode is targeted to the heart, so as to direct the
molecule to the source and minimise side-effects of the
agonists.
[0105] Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
protein, which matrices are in form of shaped articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate) as described by Langer et al.
(1981) J. Biomed. Mater. Res. 15: 167-277 and Langer (1982) Chem.
Tech. 12:98-105, or poly(vinyl alcohol)), polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al. (1983) Biopolymers 22: 547-556),
non-degradable ethylene-vinyl acetate (Langer et al. (1981) supra)
degradable lactic acid-glycolic acid copolymers such as the Lupron
Depot.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0106] The agonist(s) also may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerisation (for example, hydroxymethyl cellulose or
gelatin-microcapsules and poly-[methylmethacylate] microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, supra.
[0107] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release molecules for shorter time periods. When
encapsulated molecules remain in the body for a long time, they may
denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for stabilisation depending on the mechanism involved,
e.g., using appropriate additives, and developing specific polymer
matrix compositions.
[0108] Sustained-release agonist(s) compositions also include
liposomally entrapped agonists(s). Liposomes containing agonists(s)
are prepared by methods known per se: DE 3,218,121; Epstein et al.
(1985) Proc. Natl. Acad. Sci. USA 82: 3688-3692; Hwang et al.
(1980) Proc. Natl. Acad. Sci. USA 77: 4030-4034: EP 52,322; EP
36676; EP 88,046; EP 143,949; EP 142,641; Japanese patent
application 83-118008;U.S. Pat. Nos. 4,485,045 and 4,544,545; and
EP 102, 324. Ordinarily the liposomes are of the small (about
200-800 .ANG.) unilamellar type in which the lipid content is
greater than about 30 mol % cholesterol, the selected proportion
being adjusted for the optimal agonist therapy. A specific example
of suitable sustained-release formulation is in EP 647,449.
[0109] An effective amount of NRG to be employed therapeutically
will depend, for example, upon the therapeutic objectives, the
route of administration, and the condition of the patient.
Accordingly, it will usually be necessary for the clinician to
titre the dosage and modify the route of administration as required
to obtain the optimal therapeutic effect.
[0110] NRG optionally is combined with or administered in concert
with other agents for treating congestive heart failure, including
ACE inhibitors, CT-1 inhibitors, human growth hormone, and/or
IGF-1. The effective amounts of such agents, if employed, will be
at the clinician's discretion. Dosage administration and adjustment
are determined by methods known to those skilled in the art to
achieve the best management of congestive heart failure and ideally
takes into account use of diuretics or digitalis, and conditions
such as hypotension and renal impairment. The dose will
additionally depend on such factors as the type of drug used and
the specific patient being treated. Typically the amount employed
will be the same dose as that used if the drug were to be
administered without agonist; however, lower doses may be employed
depending on such factors as the presence of side-effects, the
condition being treated, the type of patient, and the type of
agonists and drug, provided the total amount of agents provides an
effective dose for the condition being treated.
[0111] Thus, for example, in the case of ACE inhibitors, a test
dose of enalapril is 5 mg, which is then increased up to 10-20 mg
per day, once a day, as the patient tolerates it. As another
example, captopril is initially administered orally to human
patients in a test dose of 6.25 mg and the dose is then escalated,
as the patient tolerates it to 25 mg twice per day (BID) or three
times per day (TID) and may be titrated to 50 mg BID or TID.
Tolerance level is estimated by determining whether decrease in
blood pressure is accompanied by signs of hypotension. If
indicated, the dose may be increased up to 100 mg BID or TID.
Captopril is
Sequence CWU 1
1
2 1 114 DNA Homo sapiens 1 agccatcttg taaatgtgcg gagaaggaga
aaactttctg tgtgaatgga ggggagtgct 60 tcatggtgaa agacctttca
aacccctcga gatacttgtg aggagctgta ccag 114 2 61 PRT Homo sapiens 2
Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn 1 5
10 15 Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg
Tyr 20 25 30 Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys
Gln Asn Tyr 35 40 45 Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu
Tyr Gln 50 55 60
* * * * *