U.S. patent application number 14/893920 was filed with the patent office on 2016-04-21 for therapies for cardiomyopathy.
The applicant listed for this patent is UNIVERSITY COLLEGE DUBLIN, NATIONAL UNIVERSITY OF IRELAND. Invention is credited to John Baugh, Mark Ledwidge, Ken McDonald, Chris Watson.
Application Number | 20160106771 14/893920 |
Document ID | / |
Family ID | 48784738 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160106771 |
Kind Code |
A1 |
Watson; Chris ; et
al. |
April 21, 2016 |
THERAPIES FOR CARDIOMYOPATHY
Abstract
The present invention relates to therapies and therapeutic
agents for use in the treatment of cardiomyopathies. In particular,
the invention is concerned with, but not limited to therapies and
therapeutic agents for use in the treatment of hypertrophic
cardiomyopathy. Such therapeutic agents comprise hypomethylating
agents.
Inventors: |
Watson; Chris; (Dublin,
IE) ; Baugh; John; (Dublin, IE) ; Ledwidge;
Mark; (Cork, IE) ; McDonald; Ken; (Dublin,
IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY COLLEGE DUBLIN, NATIONAL UNIVERSITY OF IRELAND |
Belfield, Dublin |
|
IE |
|
|
Family ID: |
48784738 |
Appl. No.: |
14/893920 |
Filed: |
May 26, 2014 |
PCT Filed: |
May 26, 2014 |
PCT NO: |
PCT/EP2014/060853 |
371 Date: |
November 24, 2015 |
Current U.S.
Class: |
514/43 ;
435/6.12; 435/6.13; 435/7.92; 536/28.3 |
Current CPC
Class: |
C12Q 2600/136 20130101;
A61K 31/713 20130101; A61K 31/7064 20130101; A61P 9/04 20180101;
G01N 2500/10 20130101; A61K 31/706 20130101; A61P 9/10 20180101;
G01N 2800/325 20130101; C12Q 2600/158 20130101; C12Q 2600/154
20130101; G01N 2500/04 20130101; A61P 9/00 20180101; G01N 2500/02
20130101; C12Q 1/6883 20130101; G01N 33/5023 20130101 |
International
Class: |
A61K 31/7064 20060101
A61K031/7064; G01N 33/50 20060101 G01N033/50; C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2013 |
GB |
1309444.6 |
Claims
1. A hypomethylating agent for use in the prevention or treatment
of cardiomyopathy.
2. The hypomethylating agent of claim 1 wherein the agent comprises
5-Azacytidine or 5-Aza-2'-Deoxycytidine.
3. The hypomethylating agent of claim 1 for use in the prevention
or treatment of ischemic or non-ischemic cardiomyopathy.
4. The hypomethylating agent of claim 1 for use in the prevention
or treatment of a cardiomyopathy selected from the group consisting
of hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive
cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy and
endomyocardial fibrosis.
5. The hypomethylating agent of claim 1 for use in the prevention
or treatment of hypertrophic cardiomyopathy.
6. The hypomethylating agent of claim 1 for use in the prevention
or treatment of cardiomyopathy, which is independent of
hypertension.
7. The hypomethylating agent of claim 5 wherein the agent has the
effect of inhibiting the expression or action of DNA
methyltransferase1, DNA methyltransferase3A, or DNA
methyltransferase3B.
8. A method of identifying a therapeutic agent capable of
preventing or treating cardiomyopathy comprising testing the
ability of possible therapeutic agents to modify expression and/or
action of one or more of the following genes involved in
hypertrophic cardiomyopathy: MYH7, MYBPC3, TNNT2, TNNI3, TPM 1,
MYL2, MYL3, ACTC1, CSRP3, TTN, ACTN2, MYH6, TCAP, TNNC1, ACTA1.
9. The method of claim 8 comprising testing the ability of possible
therapeutic agents to increase the expression and/or action of one
or both of the genes cardiac myosin binding protein-C(MYBPC3) and
ACTA1.
10. An agent capable of reducing the expression of, silencing or
degrading HIF protein or HIF mRNA for use in the prevention or
treatment of cardimyopathy or myocardial fibrosis.
11. The agent of claim 10 comprising a monoclonal antibody, a
peptide or a small molecule capable of interacting with HIF protein
and preventing its binding to hypoxia responsive elements within
the promoter regions of DNA methyltransferase genes.
12. The agent of claim 10 comprising a monoclonal antibody, a
peptide, an oligonucleotide or a small molecule capable of binding
to hypoxia responsive elements within the promoter regions of DNA
methyltransferase genes, thereby preventing binding of HIF to said
elements.
13. The agent of claim 10 comprising the siRNA of SEQID 31.
14. A method of identifying a therapeutic agent capable of
preventing or treating cardiomyopathy or fibrosis comprising:
testing the ability of putative therapeutic agents to reduce the
expression of, degrade or silence HIF protein or HIF mRNA; and/or
testing the ability of putative therapeutic agents to prevent HIF
protein from binding to hypoxia responsive elements within the
promoter regions of DNA methyltransferase genes.
15. A method of preventing or treating cardiomyopathy or myocardial
fibrosis comprising the use of a hypomethylating agent.
16. A method of preventing or treating cardiac hypertrophy or
myocardial fibrosis comprising administering to a subject in need
thereof an agent capable of reducing the expression of, silencing
or degrading HIF protein or HIF mRNA, or an agent capable of
preventing HIF protein from binding to hypoxia responsive elements
within the promoter regions of DNA methyltransferase genes.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to therapies and therapeutic
agents for use in the treatment of cardiomyopathies. In particular,
the invention is concerned with, but not limited to therapies and
therapeutic agents for use in the treatment of hypertrophic
cardiomyopathy.
BACKGROUND TO THE INVENTION
[0002] The strict definition of cardiomyopathy is a myocardial
disorder in which heart muscle is structurally and functionally
abnormal. The vast majority of cardiomyopathy is extrinsic
(external causes) and of these, the ischemic form is the most
common. The term "ischemic cardiomyopathy" typically refers to
remodeled and hypertrophied myocardium as a result of inadequate
oxygen delivery to the myocardium, with coronary artery disease
being the most common cause. Ischemic cardiomyopathy may therefore
be regarded as a first subtype of cardiomyopathy.
[0003] The term "cardiomyopathy" is also sometimes used to
specifically describe non-ischemic forms of cardiomyopathy, often
with intrinsic/idiopathic causes. These non-ischemic
cardiomyopathies may therefore be regarded as a second subtype of
cardiomyopathy. Even though these diseases may be classified as
non-ischemic, a relative myocardial hypoxia may nevertheless occur
with such diseases. The classification of these forms is often on a
functional/structural basis. Many of the forms of non-ischemic
cardiomyopathy are associated with abnormalities of the genes
encoding sarcomeric proteins, the commonest types of which are
hypertrophic cardiomyopathy and dilated cardiomyopathy, and
combinations of myocardial hypertrophy and/or fibrosis occur in
these diseases. Other types of non-ischemic cardiomyopathy include
restrictive cardiomyopathy, arrhythmogenic right ventricular
cardiomyopathy and endomyocardial fibrosis. A brief summary of
these diseases is as follows.
[0004] Hypertrophic cardiomyopathy (HCM) is often inherited and is
discussed in more detail below. Patients present with dyspnoea due
to diastolic dysfunction, chest pain, impaired diastole of
hypertrophied myocardium, arrhythmias and presyncope/syncope due to
inadequate cardiac output. HCM is characterised by disorganised
cardiac myocytes and unexplained left ventricular hypertrophy,
often due to mutations in the genes encoding sarcomeric proteins,
such as cardiac beta-myosin heavy chain gene, myosin binding
protein C3, troponin and alpha-tropomyosin. HCM patients undergo
risk stratification using regular exercise testing and Holter
monitoring, family screening and drug therapy, often with avoidance
of physical exertion. Indicators of a high risk for poor outcome
such as sudden cardia death include diagnosis at age <30 years,
unexplained syncope, family history of early sudden cardiac death,
cardiac arrest, spontaneous ventriculary arrhythmias, LV thickness
>3 cm and an abnormal BP response on exercise testing.
Beta-blockers, and non-dihydropiridine calcium channel blockers
such as verapamil are used in management to decrease the heart
rate, though the use of calcium channel blockers in patients with
Hypertrophic Obstructive Cardiomyopathy ("HOCM", a severe subtype
of HCM), heart failure (HF) and low blood pressures should be done
cautiously. Agents for arrhythmia can be considered for additional
symptom relief. Diuretics may be considered for patients with
evidence of fluid overload--but cautiously, especially in patients
with HOCM. As yet there have been no large-scale randomised trials
comparing alcohol septal ablation with surgical myectomy for
symptom relief in drug-refractory patients with HCM and the
American College of Cardiology and the European Society of
Cardiology (ACC/ESC) guidelines recommend surgical myectomy as the
primary treatment option, and alcohol ablation in patients with
high surgical risks.
[0005] Dilated cardiomyopathy (DCM) is characterised by an LV
ejection fraction <45 percent with increased (dilated) left
ventricular (LV) dimension. DCM presents with dyspnoea, orthopnoea,
ankle oedema and weight gain. A viral prodrome may be present. DCM
is hereditary in one third of cases (usually autosomal dominant);
however, it can be caused by acute viral (usually
entero-/adenoviruses) myocarditis leading to chronic inflammation,
ventricular remodelling and dysfunction. Five-year survival for DCM
patients is about 30 percent and features such as mitral
regurgitation or diastolic dysfunction are markers of poor
outcome.
[0006] Many factors predictive of sudden death in ischaemic LV
dysfunction are not predictive in non-ischaemic DCM. A
meta-analysis of five trials (1,854 patients with non-ischaemic
cardiomyopathy) suggested that ICD therapy reduces all-cause
mortality compared with medical therapy (relative risk
reduction--31 percent, absolute risk reduction 2 percent per
year).
[0007] Other rarer causes of cardiomyopathy are arrhythmogenic,
restrictive or unclassified. Restrictive cardiomyopathy (RCM) is
sub-classified by some researchers into primary (Loeffier's
endocarditis, endomyocardial fibrosis) and secondary (e.g.
infiltrative causes: amyloidosis, sarcoidosis; storage disorders:
haemochromatosis, glycogen storage disorder, Fabry's disease;
post-radiation). Loeffler's endocarditis is caused by acute
eosinophilic myocarditis with mural thrombosis and fibrotic
thickening at the apex of one or both ventricles. Endomyocardial
fibrosis is the chronic form. Prognosis depends on aetiology, but
is generally poor.
[0008] Arrhythmogenic right ventricular cardiomyopathy (ARVC) is
caused by fibro-fatty replacement of right ventricular myocytes due
to apoptosis, inflammation or a genetic cause (familial in up to
half usually with autosomal dominant inheritance). Though ARVC is
uncommon (1 in 5,000), it is reported to have regional clustering
in places such as northern Italy and Greece.
[0009] Unclassified cardiomyopathy includes left ventricular
non-compaction (LVNC) and Takotsubo cardiomyopathy. LVNC is caused
by embryogenic arrest of normal myocardial maturation resulting in
non-compacted myocardial fibres with deep recesses communicating
with the LV cavity. Takotsubo cardiomyopathy predominantly affects
women and arises from catecholamine surges causing coronary
vasospasm and severe apical, mid-LV dysfunction.
[0010] For completeness, secondary cardiomyopathies that may
overlap with the forms of cardiomyopathy described above include
metabolic storage disease causes (e.g. amyloidosis,
haemochromatosis), inflammatory causes (e.g. Chagas disease),
endocrine causes (e.g. diabetic cardiomyopathy, hyperthyroidism,
acromegaly), toxicity causes (e.g. anthracycline chemotherapy,
alcohol), neuromuscular (e.g. muscular dystrophy) and nutritional
diseases (obesity related). These can be either ischemic or
non-ischemic, inherited or acquired (secondary) in various
combinations.
[0011] As will be readily appreciated, the above classification
ontology is but one way in which cardiomyopathies may be ordered.
This is particularly the case in view of the fact that many
cardiomyopathies are multifactorial in nature, which makes it
difficult to place every cardiomyopathy in a discrete class.
Specifically recognised cardiomyopathies that may overlap and/or be
synonymous with the forms of cardiomyopathy described above
include: Arrhythmogenic right ventricular dysplasia (Arrhythmogenic
right ventricular cardiomyopathy); Atrial stand still (Atrial
cardiomyopathy with heart block); Cirrhotic cardiomyopathy;
Congenital cataract--hypertrophic cardiomyopathy--mitochondrial
myopathy; Dilated cardiomyopathy; Dilated
cardiomyopathy--hypergonadotropic hypogonadism; Dilated
cardiomyopathy with ataxia; Early-onset myopathy with fatal
cardiomyopathy; Encephalopathy--hypertrophic cardiomyopathy--renal
tubular disease; Familial dilated cardiomyopathy; Familial dilated
cardiomyopathy with conduction defect due to LMNA mutation;
Familial hypertrophic cardiomyopathy; Familial hypertrophic
cardiomyopathy (Familial hypertrophic obstructive cardiomyopathy)
Familial isolated arrhythmogenic right ventricular dysplasia
(Familial isolated arrhythmogenic right ventricular
cardiomyopathy); Familial isolated arrhythmogenic right ventricular
dysplasia (Familial isolated arrhythmogenic ventricular
cardiomyopathy); Familial isolated arrhythmogenic ventricular
dysplasia, biventricular form (Familial isolated arrhythmogenic
ventricular cardiomyopathy, biventricular form); Familial isolated
arrhythmogenic ventricular dysplasia, left dominant form (Familial
isolated arrhythmogenic ventricular cardiomyopathy, left dominant
form); Familial isolated arrhythmogenic ventricular dysplasia,
right dominant form (Familial isolated arrhythmogenic ventricular
cardiomyopathy, classic form); Familial isolated arrhythmogenic
ventricular dysplasia, right dominant form (Familial isolated
arrhythmogenic ventricular cardiomyopathy, right dominant form);
Familial isolated dilated cardiomyopathy; Familial isolated dilated
cardiomyopathy (Familial or idiopathic dilated cardiomyopathy);
Familial isolated hypertrophic cardiomyopathy; Familial isolated
hypertrophic cardiomyopathy (Familial isolated hypertrophic
obstructive cardiomyopathy); Familial isolated hypertrophic
cardiomyopathy (Familial or idiopathic hypertrophic obstructive
cardiomyopathy); Familial isolated hypertrophic cardiomyopathy
(Hypertrophic obstructive cardiomyopathy); Familial isolated
hypertrophic cardiomyopathy (Primitive hypertrophic obstructive
cardiomyopathy); Familial isolated restrictive cardiomyopathy;
Familial isolated restrictive cardiomyopathy (Familial or
idiopathic restrictive cardiomyopathy); Familial restrictive
cardiomyopathy; Familial restrictive cardiomyopathy type 1;
Familial restrictive cardiomyopathy type 2; Familial restrictive
cardiomyopathy type 3; Fatal infantile hypertrophic cardiomyopathy
due to mitochondrial complex I deficiency; Fatal infantile
hypertrophic cardiomyopathy due to mitochondrial complex I
deficiency (Fatal infantile hypertrophic cardiomyopathy due to
NADH-CoQ reductase deficiency); Fatal infantile hypertrophic
cardiomyopathy due to mitochondrial complex I deficiency (Fatal
infantile hypertrophic cardiomyopathy due to NADH-coenzyme Q
reductase deficiency); Fatty acid oxidation and ketogenesis
disorder with dilated cardiomyopathy; Fatty acid oxidation and
ketogenesis disorder with hypertrophic cardiomyopathy; Glycogen
storage disease with hypertrophic cardiomyopathy; Glycogen storage
disease with hypertrophic cardiomyopathy (GSD with hypertrophic
cardiomyopathy); Glycogen storage disease with hypertrophic
cardiomyopathy (Glycogenesis with hypertrophic cardiomyopathy);
Heart-hand syndrome, Slovenian type (Cardiac conduction
disease--dilated cardiomyopathy--brachydactyly); Histiocytoid
cardiomyopathy; Histiocytoid cardiomyopathy (Infantile
cardiomyopathy with histiocytoid change); Histiocytoid
cardiomyopathy (Infantile xanthomatous cardiomyopathy);
Histiocytoid cardiomyopathy (Oncocytic cardiomyopathy);
Hypertrophic cardiomyopathy; Hypertrophic cardiomyopathy
(Obstructive hypertrophic cardiomyopathy); Hypertrophic
cardiomyopathy and renal tubular disease due to mitochondrial DNA
mutation; Hypertrophic cardiomyopathy and renal tubular disease due
to mitochondrial DNA mutation (Hypertrophic cardiomyopathy and
renal tubular disease due to mtDNA mutation); Hypertrophic
cardiomyopathy due to intensive athletic training; Leigh syndrome
with cardiomyopathy; Leigh syndrome with cardiomyopathy
(Cardiomyopathy with hypotonia due to cytochrome C oxidase
deficiency); Leigh syndrome with cardiomyopathy (Cardiomyopathy
with myopathy due to COX deficiency); Lipoatrophy with diabetes,
leukomelanodermic papules, liver steatosis, and hypertrophic
cardiomyopathy; Lysosomal disease with hypertrophic cardiomyopathy;
Lysosomal disease with restrictive cardiomyopathy;
Maternally-inherited cardiomyopathy and hearing loss;
Maternally-inherited cardiomyopathy and hearing loss
(Maternally-inherited cardiomyopathy and deafness);
Maternally-inherited cardiomyopathy and hearing loss
(tRNA-LYS-related cardiomyopathy--hearing loss);
Maternally-inherited mitochondrial hypertrophic cardiomyopathy;
Microcephaly--cardiomyopathy; Mitochondrial disease with dilated
cardiomyopathy; Mitochondrial disease with hypertrophic
cardiomyopathy; Mitochondrial hypertrophic cardiomyopathy with
lactic acidosis due to MTO1 deficiency; Naxos disease (Keratosis
palmoplantaris with arrythmogenic cardiomyopathy); Naxos disease
(Palmoplantar hyperkeratosis with arrythmogenic cardiomyopathy);
Naxos disease (Palmoplantar keratoderma with arrythmogenic
cardiomyopathy); Neuromuscular disease with dilated cardiomyopathy;
Non-familial dilated cardiomyopathy; Non-familial hypertrophic
cardiomyopathy; Non-familial rare disease with dilated
cardiomyopathy; Non-familial restrictive cardiomyopathy; Peripartum
cardiomyopathy; Peripartum cardiomyopathy (Postpartum
cardiomyopathy); Progressive sensorineural hearing
loss--hypertrophic cardiomyopathy; Progressive sensorineural
hearing loss--hypertrophic cardiomyopathy (Progressive neurosensory
deafness--hypertrophic cardiomyopathy); Progressive sensorineural
hearing loss--hypertrophic cardiomyopathy (Progressive neurosensory
hearing loss--hypertrophic cardiomyopathy); Progressive
sensorineural hearing loss--hypertrophic cardiomyopathy
(Progressive sensorineural deafness--hypertrophic cardiomyopathy);
Restrictive cardiomyopathy; Sensorineural deafness with dilated
cardiomyopathy; Sensorineural deafness with dilated cardiomyopathy
(Neurosensory deafness with dilated cardiomyopathy); Sensorineural
deafness with dilated cardiomyopathy (Neurosensory hearing loss
with dilated cardiomyopathy); Sensorineural deafness with dilated
cardiomyopathy (Sensorineural hearing loss with dilated
cardiomyopathy); Severe dilated cardiomyopathy due to lamin A/C
mutation; Severe dilated cardiomyopathy due to lamin A/C mutation
(Severe dilated cardiomyopathy with or without myopathy); Syndrome
associated with dilated cardiomyopathy; Syndrome associated with
hypertrophic cardiomyopathy; Tako-Tsubo cardiomyopathy; Tako-Tsubo
cardiomyopathy (Ampulla cardiomyopathy); Tako-Tsubo cardiomyopathy
(Ballooning cardiomyopathy); Tako-Tsubo cardiomyopathy (Stress
cardiomyopathy); Tako-Tsubo cardiomyopathy (Takotsubo
cardiomyopathy); Transthyretin-related familial amyloid
cardiomyopathy; Transthyretin-related familial amyloid
cardiomyopathy (ATTR cardiomyopathy); Transthyretin-related
familial amyloid cardiomyopathy (TTR-related amyloid
cardiomyopathy); Tubular renal disease--cardiomyopathy;
Unclassified cardiomyopathy; Woolly hair-palmoplantar
keratoderma-dilated cardiomyopathy syndrome; Woolly
hair-palmoplantar keratoderma-dilated cardiomyopathy syndrome
(Woolly hair-palmoplantar hyperkeratosis-dilated cardiomyopathy
syndrome); Woolly hair-palmoplantar keratoderma-dilated
cardiomyopathy syndrome (Wooly hair--palmoplantar
keratoderma--dilated cardiomyopathy); and Woolly hair-palmoplantar
keratoderma-dilated cardiomyopathy syndrome (Wooly
hair-palmoplantar hyperkeratosis-dilated cardiomyopathy
syndrome).
[0012] Turning now to HCM in more detail, this disease has a
prevalence of 1 in 500, which makes it more prevalent overall than
an orphan disease. However it is very heterogenous not least in
view of the fact that it can be described as idiopathic, familial
or acquired.
[0013] It is widely accepted that most forms of HCM are caused by
mutations in one of the genes currently known to encode different
components of the sarcomere. It is usually characterized by left
ventricular hypertrophy (LVH) in the absence of predisposing
cardiac conditions (e.g., aortic stenosis) or cardiovascular
conditions (e.g., long-standing hypertension). The clinical
manifestations of HCM range from asymptomatic to progressive HF to
sudden cardiac death and vary from individual to individual even
within the same family. Common symptoms include shortness of breath
(particularly with exertion), chest pain, palpitations,
orthostasis, presyncope, and syncope. Most often the LVH of HCM
becomes apparent during adolescence or young adulthood, although it
may also develop late in life, in infancy, or in childhood.
[0014] The diagnosis of HCM is most often established when
two-dimensional echocardiography detects LVH in a nondilated
ventricle; it can also be established by pathognomonic
histopathologic findings in cardiac tissue. Familial HCM (FHCM)
without multisystem involvement is diagnosed by family history and
molecular genetic testing of any of the 14 genes currently known to
encode different components of the sarcomere for which testing is
clinically available. FHCM caused by mutation in at least one of
the genes currently known to encode different components of the
sarcomere is inherited in an autosomal dominant manner.
[0015] HCM has been extensively reviewed by Cirino and Ho (2011,
http://www.ncbi.nlm.nih.gov/books/NBK1768/) and the following has
been adapted from their work. As described above, medical
management of diastolic dysfunction using beta blockers and
non-dihydropyridine calcium channel blockers is used along with
medical or surgical management of ventricular outflow obstruction
if it exists; important also is achievement and maintenance of
sinus rhythm in those with atrial fibrillation; implantable
cardioverter-defibrillators (ICDs) are used in survivors of cardiac
arrest and those at high risk of cardiac arrest; if HF develops,
medical treatment for HF and consideration for cardiac
transplantation may be necessary. Prevention of secondary
complications may involve anticoagulation in those with persistent
or paroxysmal atrial fibrillation to reduce the risk of
thromboembolism and antibiotic prophylaxis when necessary. During
the pregnancy of a woman with HCM, care by an experienced
cardiologist and specialist obstetrician is required.
[0016] Monitoring and ongoing management also involves reassessment
of risk for sudden cardiac death (SCD) approximately once a year or
more frequently based on clinical findings. Patients are advised to
avoid competitive endurance training, burst activities (e.g.,
sprinting), intense isometric exercise (e.g., heavy weight
lifting), dehydration, hypovolemia (i.e., use diuretics with
caution), and medications that decrease afterload (e.g.,
ACE-inhibitors, angiotensin receptor blockers, and other direct
vasodilators).
[0017] To date, HCM is known to be caused by mutation in one of the
14 genes (see Table 1) encoding different components of the
sarcomere. More than 900 individual mutations have been
identified.
TABLE-US-00001 TABLE 1 genes implicated in FHCM (Cirino and Ho
(2011, http://www.ncbi.nlm.nih.gov/books/NBK1768/) Other disorder %
of HCM phenotypes Caused by caused by Locus Gene Mutations in
mutation in Name Symbol Protein Name This Gene same gene CMH1 MYH7
Myosin heavy 40% Dilated chain, cardiac Cardiomyopathy muscle beta
(DCM), Laing isoform distal myopathy CMH4 MYBPC3 Myosin-binding 40%
DCM protein C, cardiac-type CMH2 TNNT2 Troponin T, 5% DCM cardiac
muscle CMH7 TNNI3 Troponin I, 5% DCM, cardiac muscle restrictive
cardiomyopathy CMH3 TPM1 Tropomyosin 1 2% DCM alpha chain CMH10
MYL2 Myosin Unknown regulatory light chain 2, ventricular/ cardiac
muscle isoform CMH8 MYL3 Myosin light 1% polypeptide 3 ACTC1 Actin,
alpha Unknown DCM cardiac muscle 1 CSRP3 Cysteine and Unknown
glycine-rich protein 3, muscle LIM protein CMH9 TTN Titin DCM, Udd
distal myopathy ACTN2 Alpha-actinin-2 Unknown DCM Myosin heavy DCM
chain, cardiac muscle alpha isoform TCAP Telothonin limb-girdle
muscular dystrophy, DCM Other genes potentially implicated in HCM
TNNC1 Troponin C, Unknown DCM slow skeletal and cardiac muscles
[0018] Cirino and Ho describe the sarcomere as the basic
contractile unit of the cardiac myocyte. Cardiac contraction occurs
when calcium binds the troponin complex (subunits I, C, and T) and
.alpha.-tropomyosin and releases the inhibition of myosin-actin
interactions by troponin I. ATPase activity and binding of actin by
the globular myosin head result in conformational changes that bend
the neck (also termed the lever arm) and result in the sliding of
thick filaments in relation to thin filaments (solid arrows) to
generate the power stroke.
[0019] As previously stated, HCM is the commonest inherited cardiac
disease with an estimated prevalence of 1 in 500. Reports suggest
that it is caused by autosomal dominant mutations in the cardiac
sarcomeric proteins (FHCM) in 60% of cases. Within FHCM, the most
frequent genes to be implicated are myosin heavy chain cardiac
muscle beta isoform (MHY7) and cardiac myosin binding
protein-C(MYBPC3). Several studies suggest that MYBPC3 mutations
are associated with up to 40% of FHCM cases (see Table 1).
Accordingly, the prevalence of MYBPC3 related FHCM is arguably
>1 in 2000.
[0020] According to recent studies examining FHCM associated with
MYBPC3, there is a very varied clinical presentation. The disease
tends to occur later in life and has a more benign prognosis in
comparison with FHCM associated with other sarcomeric protein
mutations such as MYH7. This has been used to suggest periodic
screening of families with MYBPC3 abnormalities. By determining
clinical disease expression, penetrance, and outcomes in a large
cohort of patients and relatives with mutations in MYBPC3, these
studies demonstrated marked heterogeneity with incomplete,
age-related, and gender specific penetrance (Cardiac Myosin Binding
Protein-C Mutations in Families With Hypertrophic Cardiomyopathy:
Disease Expression in Relation to Age, Gender, and Long Term
Outcome Stephen P. Page, Stavros Kounas, et al. Circ Cardiovasc
Genet 2012; 5; 156-166). These studies concluded that disease
expression (clinical) is heterogeneous and unrelated to mutation
type or specific mutation, that disease penetrance is incomplete on
average over a carrier's lifetime and that disease penetrance is
higher in males than females. Accordingly, because neither mutation
type (eg, nonsense, missense, etc) nor specific mutation appeared
to predict a particular clinical phenotype in these studies, and
because marked phenotypic diversity was seen in families sharing
identical mutations, it may be suggested that epigenetic mechanisms
(frequently occurring in genes with high mutability) may play a
role in the acquired phenotype of FHCM associated with MYBPC3.
[0021] There are more than 1,000 causative mutations identified in
20 sarcomere and myofilament related genes associated with HCM
and--as stated above--more than 900 associated with 14 genes in the
sarcomere. The most frequently occurring gene mutations are
associated with MYBPC3. The high density of mutations found in
genes associated with HCM may suggest that mechanisms promoting
increased mutability play a role in disease prevalence.
[0022] DNA methylation is an epigenetic modification that involves
the addition of methyl groups to cytosine residues already
incorporated into DNA sequences, forming 5-methylcytosine (5MeC).
This can either physically prevent transcription factor binding or
reduces access through local chromatin condensation, with both
processes resulting in gene repression. DNA methylation is
regulated by a family of DNA methyltransferase (DNMT) enzymes, one
of which, DNMT1, preferentially methylates hemi-methylated DNA and
is required to maintain the methylation pattern of the genome in
daughter cells during cell division. DNMT3A and DNMT3B are both de
novo methylating enzymes, and are responsible for establishing the
initial methylation patterns of the genome during development, and
their expression re-emerges in disease states. Dysregulation of
this process has been extensively studied in cancer.
[0023] Differential DNA methylation of CpG sites in two isoforms of
MYBPC has been evaluated in studies that also evaluated if the
methylation level is gene specific and possibly involved with gene
mutability (Meurs and Kuan (Differential methylation of CpG sites
in two isoforms of myosin binding protein C, an important
hypertrophic cardiomyopathy gene. Environ Mol Mutagen. 2011 March;
52(2):161-4). These studies also evaluated the methylation of the
CpGs within the exonic regions of the cardiac (MYBPC3) and skeletal
muscle (MYBPC2) isoforms of the myosin binding protein C gene. In
the case of MYBPC2, there are no known mutations that lead to the
development of FHCM. It was demonstrated that although the mean
number of CpGs was similar in the two proteins, the mean
methylation level of CpGs was significantly higher in MYBPC3 than
MYBPC2 (P<0.0001) suggesting that there may be epigenetic
involvement resulting in increased genetic mutability.
[0024] This hypothesis has been supported by other studies. For
example, in one study a significant increase in the number of
methylated CpG islands was identified in murine physiologic cardiac
hypertrophy (3762.+-.500 vs. 2499.+-.299, P=0.02) associated with
741 promoters differentially methylated. Of these, 634 were
hypermethylated and 107 were hypomethylated. Promoter DNA
methylation data was integrated with the gene expression profiles,
and it was discovered that 142 genes showed both altered DNA
methylation patterns and expression levels. Gene ontology analysis
of these genes reveals an overrepresentation of gene categories
involved in actin filament-based process, cytoskeleton organization
and programmed cell death (all P<0.001). Another study noted
that there was methylation in all cytosine residues within CpG
dinucleotides found in exons 8, 9, and intron 8 of cardiac troponin
T (cTNT) gene in subjects with FHCM. In work that recognizes that
epigenetic mechanisms such as microRNA and histone modification are
crucially responsible for dysregulated gene expression in HF, a
further study evaluated DNA methylation in this setting. They
studied ischaemic and idiopathic end-stage cardiomyopathic left
ventricular (LV) explants from patients who had undergone cardiac
transplantation were profiled compared to normal control. Using a
preliminary analysis with methylated-DNA immunoprecipitation-chip
(MeDIP-chip), differential methylation loci were then validated by
bisulfite-(BS) PCR and high throughput sequencing. It was found
that a large population of CpG islands (CGIs) and gene promoters
are more significantly hypomethylated in end-stage cardiomyopathic
hearts. This study also identified 3 angiogenesis-related genetic
loci that were differentially methylated (ARHGAP24, PECAMand
AMOTL2). The hypomethylation of the angiomotin-like 2 gene (AMOTL2)
and hypermethylation of the 5' promoter region in the
platelet/endothelial cell adhesion molecule gene (PECAM1) led to
decreased expression of both, whereas hypermethylation within the
gene body of Rho GTPase-activating protein 24 (ARHGAP24) favored
its expression. Quantitative RT-PCR, found that the expression of
these genes differed significantly between CM hearts and normal
control (p<0.01). There was a correlation between methylation
and differential expression of the corresponding gene. It has been
noted that this provides the first evidence for a difference in
methylation status between human cardiomyopathic hearts and
controls.
[0025] A subsequent study demonstrated global epigenomic profiles
in human cardiomyopathy. DNA methylation and H3K36me3 maps were
generated from human hearts, their profiles characterized and it
was found that DNA methylation differs between end stage
cardiomyopathy and control hearts in CpG islands and gene bodies
regions. A significant decrease in global gene promoter methylation
also correlated with genes that were upregulated in cardiomyopathy
but not with genes that were downregulated, suggesting that
demethylation leads to increased expression of the corresponding
gene and downregulation of genes in cardiomyopathy occurs
independently of promoter methylation.
[0026] Of possible relevance to this study is another study in
which TNF-.alpha. treated HL1 murine atrial cardiomyocytes which
exhibited decreased Atp2a2 expression, increased CpG methylation of
the gene's promoter, and elevated levels of the DNA
(cytosine-5-)-methyltransferase 1 (Dnmt1)--findings combined that
suggest that methylation plays an important role in the negative
transcriptional regulation of Atp2a2.
[0027] It is known that MYBPC3 is hypermethylated, has mutations
associated with HCM and when its expression is decreased
hypertrophy is worsened. However, for another gene implicated in
HCM, ACTA1, increased expression is associated with HCM (mutations
of ACTA1 are not known to be associated with HCM, but variations in
its level of expression are).
[0028] Myocardial fibrosis can be a characteristic feature of HCM
and is believed to contribute to the higher risk of sudden cardiac
death, arrhythmias and cardiac dysfunction. The fibrosis pattern is
associated with increased interstitial and focal fibrosis patterns
and can be visualized using cardiac magnetic resonance imaging
(MRI) with gadolinium enhancement.
[0029] However, fibrosis and hypertrophy are separate but linked
processes in the HCM syndrome. For example, it has been shown that
one third of HCM patients present with only hypertrophy and do not
have evidence of fibrosis on cardiac MRI with late gadolinium
enhancement. Nonetheless, the same study also showed that HCM with
fibrosis is associated with >3 fold increase in cardiovascular
death, unplanned cardiovascular admission, sustained ventricular
tachycardia or ventricular fibrillation, or appropriate implantable
cardioverter-defibrillator discharge. Furthermore, within the
patient cohort with fibrosis, a greater extent of fibrosis was
associated with worse outcome.
[0030] The cause of myocardial fibrosis in HCM is unclear. Some
reports attribute it to premature myocyte death and expansion of
the interstitial matrix as a result of the stress created by
sarcomere mutations resulting in fibrosis accruing in HCM hearts.
Other work has associated the mutations with other pathologic
changes such as ischemia--for example compromised coronary flow due
to hypertrophy, microvascular dysfunction, increased oxidative
stress, and increased metabolic demands imposed by abnormal
biophysical properties of mutant sarcomeres resulting in an
imbalance of oxygen demand and supply. This can create an ischemic
milieu further contributing to the cycle of premature myocyte death
and the emergence of focal fibrosis in HCM.
[0031] Recent data in mouse models of HCM has shown that sarcomere
gene mutations activate proliferative and pro-fibrotic signals in
non-myocyte cells from HCM mouse hearts resulting in increased
levels of pro-fibrotic proteins including TGF.beta. resulting in
fibrosis.
[0032] However, some animal work has also shown that a pro-fibrotic
state is already present in animal models when which later develop
cardiac hypertrophy before cardiac histologic findings are
abnormal. This work is now supported in human patients by recent
work that has confirmed the frequent co-existence of fibrosis and
hypertrophy and early signals of myocardial fibrosis before
clinical hypertrophy is evident. They conclude that elevated levels
of serum PICP indicated increased myocardial collagen synthesis in
sarcomere-mutation carriers without overt disease and that this
profibrotic state preceded the development of left ventricular
hypertrophy or fibrosis visible on MRI. Furthermore, while cardiac
MRI studies showed late gadolinium enhancement, indicating
myocardial fibrosis, in 71% of subjects with overt hypertrophic
cardiomyopathy, conversely there was none in the mutation carriers
without left ventricular hypertrophy.
[0033] Finally, this recent work shows that the pattern and natural
history of fibrosis and cardiac dysfunction associated with the two
most frequent mutations associated with HCM (MYBPC3 and MYH7) are
different. In this work comparing patients with MYBPC3 mutations
and MYH7 mutations, but no evidence of HCM, markers of myocardial
fibrosis, (PICP levels) and diastolic function (diastolic
velocities) were worse in the MYH7 mutation patients. Furthermore,
in those patients with the MYBPC3 mutations with HCM, the age of
onset of hypertrophy was later, the LVMI was lower, and diastolic
function was better than in those patients with MYH7 mutations.
These data suggest that MYH7 mutations trigger earlier/more
extensive fibrosis and myocardial remodeling than MYBPC3
mutations.
[0034] In conclusion: [1] while myocardial fibrosis and hypertrophy
often co-exist in patients with HCM, they are distinct
pathophysiological features of the disease and in one third of
consecutive patients in a large HCM cohort, hypertrophy is evident
without fibrosis; [2] this underlines the co-existence but distinct
nature of hypertrophy and fibrosis and recent work in HCM mice
demonstrates that myocyte expression of sarcomere protein mutations
alters gene transcription in non-myocyte cells, inducing
proliferation and expression of profibrotic molecules that produce
pathologic remodeling and fibrosis in HCM that is dependent on
TGF.beta.; [3] the fibrotic response to sarcomere
mutations/abnormalities appears to occur early in the natural
history of HCM and this often precedes the development of clinical
hypertrophy; [4] while MYBPC3 mutations are associated with HCM,
including hypertrophy and fibrosis, there is marked heterogeneity
in phenotypic response to mutations of this gene even within
families and this could be explained by epigenetic factors such as
hypermethylation; [5] based on certain studies, it is known that
MYBPC3 is associated with hypermethylation and this is consistent
with a high frequency of gene mutations associated with HCM; [6]
the hypertrophic myocardium in HCM is in a state of relative
hypoxia and this can have important implications for the
pathophysiology and natural history of the disease.
[0035] With respect to epigenetic therapies, successfully developed
epigenetic drugs exist, but they are mostly applied to cancer and
haematological diseases. These include (but are not limited to):
hypomethylating agents, such as 5-azacytidine (referred to as 5-aza
or azacitidine), 5-aza-2'deoxycytidine (referred to as 2'deoxy,
5azaDC, or dectiabine) zebularine; histone deacetylase inhibitors
such as suberoylanilide hydroxamic acid; and histone methylation
inhibitors such as 3-Deazaneplanocin.
[0036] A hypomethylating agent is one that inhibits DNA
methylation, and is often referred to as a demethylating agent or a
DNA methylation inhibitor. While the terms are used interchangeably
in the art, it is at least theoretically possible that
hypomethylation/demethylation may also take place by way of
mechanisms other than by DNA methylation inhibition. DNA
methylation inhibitors (for example, but not limited to, 5-aza,
2'deoxy and zebularine) work by preventing the establishment of new
methylation patterns onto newly synthesised DNA during the cell
cycle. With every cell division, the methylation pattern of the
parent strand is copied to the newly synthesising daughter strand
during S-phase of the dividing cell. The enzymes that regulate this
process are the DNA methyltransferases (DNMTs). The DNMTs maintain
the integrity of the methylation pattern of the genome within each
cell. DNA methylation inhibitors prevent or inhibit this process,
so that as cells divide they gradually lose their methylation
pattern because they are not copied to the newly formed DNA. The
DNA methylation inhibitors interact with the DNMT enzymes to
achieve this effect. Therefore with continual rounds of cell
division, the genes gradually become hypomethylated. Additional
hypomethylating mechanisms can occur by which DNA demethylation
occurs outside the cell cycle.
[0037] Other hypomethylating agents of note include clofarabine and
hydralazine. Clofarabine is a cytotoxic purine nucleoside analogue
that primarily works by inducing apoptosis. However, the compound
is under investigation in clinical trials for its potential
hypomethylating effect, and for use in combination with other
epigenetic drugs. Clofarabine thus potentially exhibits a dual mode
of action, and may be the first of other drugs that may be found to
exert a secondary epigenetic mode of action in addition to their
primary effect. In clinical trials, this drug has elicited
responses in patients with pre-treated relapsed and/or refractory
(rr-)ALL, rr-AML, and high-risk myelodysplasia syndrome (MDS).
[0038] Hydralazine was originally developed as an oral
antihypertensive drug. It is currently being used to treat
pregnancy-associated hypertension and is also used in combination
with nitrates in the management of HF. Hydralazine has shown the
ability to reactivate tumour suppressor gene expression that has
been silenced by hypermethylation, both in vitro and in vivo.
Hydralazine's extensive previous use as a hypertensive provided the
safety and tolerability data that lead to testing in Phase I trial
with cancer patients, which confirmed its demethylating ability. It
is currently being evaluated in combination therapies with HDAC
inhibitors, chemotherapy, or radiation in Phase III trials for
haematological and solid tumours.
[0039] Bacanamwo et al. Circulation (2007) vol 116, p124 describes
demethylation effects of 5aza on genes associated with hypertension
in an Angiotensin II hypertensive disease model, in which an
antihypertensive effect of 5aza by putative demethylation effects
on genes associated with hypertension such as 11.beta.-HSD2 which
was methylated in the arteries and kidneys is reported. The authors
conclude the data "suggest that DNA methylation plays a critical
role in the coordinate regulation of genes involved in the
pathogenesis of hypertension and vascular remodeling." However,
hypertension is known to increase hypertrophy and fibrosis and
antihypertensive therapies are well known to reduce hypertrophy and
fibrosis. The effects described in the present invention are not
dependent on hypertension. All genes can be methylated and there is
no way to predict that because of the described antihypertensive
effects on 11.beta.-HSD2, there could be an antihypertrophic effect
of the 5aza independent of hypertension, nor a beneficial effect
on, for example, the expression of MYBPC3. Taylor et al New England
Journal of Medicine (2004) vol 351 pp 2049-2057, describes the
antihypertrophic effects of isosorbide dinitrate and hydralazine in
combination. Hydralazine is an antihypertensive agent and
antihypertensive agents can reduce hypertrophy and fibrosis.
Isosorbide dinitrate has independent pharmacological effects which
contribute to and promote BP reduction and protect against
myocardial ischemia. There is no evidence that the observed effect
on cardiac hypertrophy is related to hydralazine alone and also to
epigenetic effects. US A 2011/0319466 describes the use of a HDAC
inhibitor (phenylbutyrate) in combination with an ACE inhibitor for
the treatment of heart failure, cardiac hypertrophy and cardiac
dysfunction. Esha et al. FASEB Journal (2002) vol 16, pA491
describes a DNMT inhibitor (5azaDC) to manage ischemic injury in
dogs and an improvement in post-ischemic myocardial function and
"scar formation." There is no disclosure of an impact on
hypertrophy. Furthermore, hypertrophy can occur in the absence of
ischemia and in the case of familial cardiac hypertrophies, the
pathophysiological driver is not ischemia.
[0040] US A 2012/0014962 describes the antifibrotic effects of DNMT
inhibitors on models of liver fibrosis and antifibrotic effects of
the drug on cardiac fibrosis and endomyocardial fibrosis. The
antifibrotic effects of DNMT inhibitors 5aza and 5azaDC are known,
but this is quite distinct to the antihypertrophic effects
described in the present application. Kao et al Laboratory
Investigation (2011) vol 91, pp 1291-1297 describes the DNMT
inhibiting effects of hydralazine on SERCa2. There is no evidence
of an antihypertrophic effect independent of SERCAa2 and so it is
not possible to ascribe the reduction in cardiac hypertrophy to DNA
demethylation, nor could one anticipate the effects of 5aza on
MYBPC3. UA A 2009/0105168 describes the use of a DNMT inhibitor for
ischemic injury and treatment of hypertrophy and post ischemic
injury including cardiac surgery, stroke and myocardial infarction.
As mentioned above hypertrophy can occur in the absence of
ischemia. In the case of familial cardiac hypertrophies, the
pathophysiological driver is often not ischemia.
[0041] Fibrosis is often implicated in hypertrophic cardiomyopathy,
but there is a substantial number of HCM instances where there is
no evidence of fibrosis. Thus, what can be used to treat fibrosis
cannot necessarily be used to treat all instances of HCM.
[0042] There remains a need for a better understanding of the
mechanisms of action of cardiomyopathies, and the possible
therapies that may be developed to treat cardiomyopathies in light
of such mechanisms of action. There also remains a need for a
better understanding of the mechanisms of hypertrophy and/or
fibrosis and how possible therapies may be developed in light of
such mechanisms of action.
SUMMARY OF THE INVENTION
[0043] In one aspect, the present invention provides a
hypomethylating agent for use in the prevention or treatment of
cardiomyopathy. In one aspect, the hypomethylating agent may
comprise 5-Azacytidine or 5-Aza-2'-Deoxycytidine. In an aspect, the
hypomethylating agent may be for use in the prevention or treatment
of non-ischemic cardiomyopathy. In an aspect the hypomethylating
agent may be for use in the prevention or treatment of a
cardiomyopathy selected from the group consisting of hypertrophic
cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy,
arrhythmogenic right ventricular cardiomyopathy and endomyocardial
fibrosis. In a further aspect the invention provides a
hypomethylating agent for use in the prevention or treatment of
cardiomyopathy which is independent of hypertension.
[0044] In an aspect, the hypomethylating agent may have the effect
of inhibiting the expression of DNA methyltransferase3B.
[0045] In another aspect, the present invention provides a method
of identifying a therapeutic agent capable of preventing or
treating cardiomyopathy comprising testing the ability of putative
therapeutic agents to hypomethylate one or more of the 14 genes
encoding components of the sarcomere. In one aspect, the method may
comprise testing the ability of putative therapeutic agents to
hypomethylate the gene cardiac myosin binding protein-C.
[0046] In another aspect, the invention provides an agent capable
of reducing the expression of, silencing or degrading HIF protein
or HIF mRNA for use in the prevention or treatment of cardimyopathy
or myocardial fibrosis. The agent may comprise a monoclonal
antibody, a peptide or a small molecule capable of interacting with
HIF protein and preventing its binding to hypoxia responsive
elements within the promoter regions of DNA methyltransferase
genes. The agent may also comprise a monoclonal antibody, a
peptide, an oligonucleotide or a small molecule capable of binding
to hypoxia responsive elements within the promoter regions of DNA
methyltransferase genes, thereby preventing binding of HIF to said
elements. The agent may comprise the siRNA of SEQID 31.
[0047] One aspect of the invention comprises prodrugs or
pharmaceutically acceptable salts of any of the agents comprised in
the invention, or compositions comprising any of said agents and a
pharmaceutically acceptable excipient.
[0048] In another aspect, the invention comprises a method of
identifying a therapeutic agent capable of preventing or treating
cardiomyopathy or fibrosis comprising: testing the ability of
putative therapeutic agents to reduce the expression of, degrade or
silence HIF protein or HIF mRNA; and/or testing the ability of
putative therapeutic agents to prevent HIF protein from binding to
hypoxia responsive elements within the promoter regions of DNA
methyltransferase genes.
[0049] A further aspect of the invention comprises a method of
preventing or treating cardiomyopathy or myocardial fibrosis
comprising the use of a hypomethylating agent as described
above.
[0050] In another aspect, the invention comprises the use of a
hypomethylating agent as described above in a method of treating or
preventing cardiomyopathy or myocardial fibrosis.
[0051] A further aspect of the invention comprises a method of
preventing or treating cardiac hypertrophy or myocardial fibrosis
comprising the use of an agent capable of reducing the expression
of, silencing or degrading HIF protein or HIF mRNA, or comprising
the use of an agent capable of preventing HIF protein from binding
to hypoxia responsive elements within the promoter regions of DNA
methyltransferase genes. An additional aspect of the invention
comprises the use of an agent in such a method. Such agents may
include but are not exclusive to, monoclonal antibodies, peptides
(whether synthetic or from the animal kingdom), or small molecule
inhibitors, that can interact with HIF protein preventing its
binding to hypoxia responsive elements (HRE) within DNA
methyltransferase (DNMT) promoter regions. Further such agents may
include but are not exclusive to agents that can bind to HRE sites
on the DNMT promoter regions preventing binding of HIF. Such agents
may comprise monoclonal antibodies, peptides (whether synthetic or
from the animal kingdom), small molecule inhibitors, or
oligonucleotides.
[0052] An additional aspect of the invention comprises an agent in
accordance with any aspect of the invention for use in the
preparation of a medicament for the prevention or treatment of
cardiomyopathy, myocardial fibrosis, or other forms of fibrosis. As
aspect of the invention further comprises the method of preparing
such a medicament, and the use of such an agent in the preparation
of such a medicament.
[0053] It has not previously been demonstrated that 5-aza and
2'deoxy can modulate cardiac hypertrophy in accepted
cardiomyopathic models, something the inventors have now done.
Although fibrosis and hypertrophy may co-exist, these are separate
disease entities often responding to common stimuli explaining
co-existence. Furthermore, it is known that fibrosis occurs as a
consequence of abnormal sarcomeric protein expression and that this
process heralds a poorer prognosis.
[0054] Therefore, the present invention proposes in one aspect that
cardiomyopathy associated with abnormal MYBPC3 gene expression
arising from gene mutations and hypermethylation results in
hypertrophy, fibrosis and a hypoxic environment. It demonstrates
for the first time that demethylation using agents such as 5-aza
and 2'deoxy increases MYBPC3 expression, and reduces hypertrophy
and fibrosis by at least one of the following four mechanisms, or a
combination thereof: [0055] (i) direct inhibition of
hypermethylation of genes involved in hypertrophy and/or
myofibrosis; [0056] (ii) amelioration of relative hypoxia response
in the hypertrophic myocardium resulting in reduced HIF induced
activation of DNA methyltransferase enzymes which can promote
further hypermethylation and activation of hypertrophy and/or
fibrosis; [0057] (iii) direct or indirect up-regulation of MYBPC3
gene expression, resulting in reduced activation of a pro-fibrotic
response in non-myocyte cells; [0058] (iv) alteration in
phosphorylation status of MYBPC3
[0059] While hypertrophy and fibrosis are separate disease
processes, abnormal expression of sarcomeric genes in the
myocardium results in hypertrophy, activates fibrosis through
mechanisms described above and results in a more aggressive disease
phenotype and poorer prognosis. A therapeutic intervention that
could inhibit both hypertrophy and/or fibrosis by one or a
combination of the mechanisms described above would be of important
clinical value and could present opportunities to manage and
perhaps even prevent morbidity and mortality due to cardiomyopathy.
Furthermore, the implication of altered MYBPC3 expression and/or
other proteins involved in cardiomyopathy extends beyond myocardial
hypertrophy and fibrosis. Therapeutic interventions that could
inhibit diseases with abnormal methylation patterns could have
important use in a number of chronic diseases of other organs such
as the eye, gastrointestinal tract, liver, kidney and lung and
diseases which are also associated with inflammatory autoimmune
conditions. Emerging evidence exemplifies this potential
particularly in idiopathic pulmonary fibrosis models, suggesting
that DNA hypermethylation changes are reversible with
hypomethylating agents such as 5-aza and 2'deoxy resulting disease
suppression in-vitro and in-vivo.
[0060] The present invention envisages 5-aza, 2'deoxy and other
hypomethylating agents for use in the prevention and treatment of
cardiomyopathies described and listed in the background section of
this application above. The present invention also envisages such
hypomethylating agents for use in the prevention and treatment of
myocardial fibrosis and other forms of fibrosis. The present
invention further envisages therapeutic agents capable of impacting
on the interaction mechanism between HIF protein and the DNMT3b
gene promoter, affecting the levels of HIF protein and/or affecting
the levels of DNMT3b protein for use in the treatment of
cardiomyopathies, myocardial fibrosis and other forms of fibrosis.
Further still, the invention envisages a method of treating
cardiomyopathy, myocardial fibrosis or another fibrotic condition
using any agent comprised in this invention. The invention further
envisages the screening for possible therapeutic agents based on
the agents' capacity to impact on the HIF-DNMT3b/HRE pathway, or on
its capacity of affect the methylation levels or expression
patterns of genes implicated in HCM.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] Embodiments of the invention will be described, by way of
example only, with reference to the accompanying drawings in
which:
[0062] FIG. 1 depicts results illustrating that tissue hypoxia is
associated with an enhanced fibrotic expression profile.
[0063] FIG. 2 depicts the results of in vitro hypoxia studies
utilising a human primary cardiac fibroblast cell line (HCF)
[0064] FIG. 3 depicts the results of DNA methylation studies
carried out to ascertain whether the pro-fibrotic effects of
hypoxia was associated with epigenetic changes within the HCF
cells
[0065] FIG. 4 depicts the results of an investigation into the
mechanism by which hypoxia regulates DNMT3B expression
[0066] FIG. 5 depicts the results of treatment of HCF cells the
HIF-1.alpha. stabilisation compound DMOG in normoxia
[0067] FIG. 6 depicts results illustrating that siRNA mediated
reduction in DNMT3B (siDNMT3B) resulted in reduced expression
levels of the pro-fibrotic proteins ASMA and Collagen 1.
[0068] FIG. 7 depicts results illustrating that 5aza treatment
reverses the pro-fibrotic impact of hypoxia.
[0069] FIGS. 8-11 depict the effect of 5AZA treatment on
echocardiographic parameters. Ejection fraction (EF) was
significantly improved in SHR-5AZA compared with SHR-V. A
significant reduction in cardiac hypertrophy was observed. 5AZA
treatment reduced left ventricular mass index (LVMi) and the
interventricular septum diameter. Diastolic dysfunction was
observed in SHR-V, as measured by E prime (E') and this was
significantly improved with 5AZA treatment. Results represent mean
and standard error of the mean. *p<0.05, **p<0.01,
***p<0.001.
[0070] FIG. 12 depicts the effect of 5AZA treatment on collagen
content within the heart. Hydroxyproline analysis was carried out
to assess total collagen within the myocardium of SHR-V and
SHR-5AZA animals. A significant reduction in collagen deposition
was detected in SHR-5AZA animals. Results represent mean and
standard error of the mean. *p<0.05.
[0071] FIGS. 13-15 depict the effect of 5AZA treatment on
perivascular collagen. Digital analysis of the slides revealed a
significant decrease in perivascular collagen in SHR-5AZA compared
to SHR-V. Representative images of picrosirius red stained tissue
from one animal is shown. Collagen staining appears pink/red in
colour. A mark-up image is generated following digital
quantification of positive pixels, highlighting negative pixels as
blue and positive pixels as either yellow, orange or red. Results
represent mean and standard error of the mean. *p<0.05.
[0072] FIG. 16 depicts the effect of 5AZA treatment on myocyte area
in vivo. Myocyte area digital analysis of cardiac tissue sections
stained with haematoxylin and A. A eosin revealed a significant
decrease in cell area in SHR animals treated with 5AZA compared to
control animals SHR-V. Results represent mean and standard error of
the mean. ***p<0.001
[0073] FIGS. 17 and 18 depict the effect of 5AZA treatment on
cardiac expression levels of MYBP-C and ACTA1. Gene expression
levels of MYBP-C and ACTA1 were assessed using quantitative
real-time PCR in the cardiac tissue of SHR-V and SHR-AZA. Analysis
reveals that treatment of SHR animals with 5 AZA resulted in a
significant 4-fold increase in MYBP-C and ACTA1. Results represent
mean and standard error of the mean. *p<0.05, ***p<0.001.
[0074] FIGS. 19-22 depict the effect of 5AZA and 5AZA_DC treatment
on cardiac myoblast expression levels of MYBP-C and ACTA1 in vitro.
Gene expression levels of MYBP-C and ACTA1 were assessed using
quantitative real-time PCR following 4 day treatment with DNA
demethylating agents 5AZA and 5AZA_DC. Analysis reveals that
treatment with these compounds resulted in a significant increase
in both MYBP-C and ACTA1 when using either of the two DNA
methylation inhibitors. Results represent mean and standard error
of the mean. *p<0.05, **p<0.01, ***p<0.001.
[0075] FIG. 23 is an illustration of the wild type promoter of
DNMT1 showing forward and reverse primer locations and a putative
hypoxia response element where HIF protein binds.
[0076] FIG. 24 is an illustration of the promoter of DNMT1 showing
forward and reverse primer locations and a putative hypoxia
response element (HRE) where HIF protein binds, bearing
site-directed mutations in the HRE.
[0077] FIG. 25 is an illustration of the wild type promoter of
DNMT3b showing forward and reverse primer locations and a putative
hypoxia response element where HIF protein binds.
[0078] FIG. 26 is an illustration of the promoter of DNMT3b showing
forward and reverse primer locations and a putative hypoxia
response element (HRE) where HIF protein binds, bearing
site-directed mutations in the HRE.
DETAILED DESCRIPTION OF THE DRAWINGS
Example 1
Hypoxia Alters the DNA Methylation Profile of Cardiac Fibroblasts
via HIF-1a Regulation of DNMT3b
[0079] Methods
[0080] Human Cardiac Tissue Collection and Handling
[0081] Human tissue samples were collected from the hearts of 26
stable patients undergoing elective cardiac-bypass surgery.
Specifically, right atrial appendages were obtained adjacent to the
venous cannulation site in either coronary artery bypass grafting
patients (n=18) or valve repair/replacement patients (n=8). All
subjects gave written informed consent to participate in the study.
The study protocol conformed to the principles of the Helsinki
Declaration and received local ethical committee approval. Tissue
samples were collected at onset of surgery and immediately divided
into two parts and either stored in Allprotect Tissue Stabilisation
Reagent (Qiagen) for subsequent RNA extraction, or formalin-fixed
for histological staining.
[0082] Cardiac Tissue Analysis
[0083] For analysis of gene expression within the myocardial
samples, the tissue was first individually disrupted and
homogenized using an Ultra Turrax T25 Dispersing Instrument (IKA)
before the RNA was extracted using the AllPrep DNA/RNA extraction
kit (Qiagen), according to the manufacturer's instructions. First
strand cDNA synthesis was carried out using SuperScript II RT
(Invitrogen). Quantitative real-time PCR (QPCR) primers were
designed so that one of each primer pair was exon/exon boundary
spanning to ensure only mature mRNA was amplified. The sequences of
the gene-specific primers used are as follows; ASMA,
5'-CGTTACTACTGCTGAGCGTGA-3' (forward) (SEQID 13),
5'-AACGTTCATTTCCGATGGTG-3' (reverse) (SEQID 14); collagen 1
.alpha.1 (COL1A1), 5'-GAACGCGTGTCATCCCTTGT-3' (forward) (SEQID 15),
5'-GAACGAGGTAGTCTTTCAGCAACA-3' (reverse) (SEQID 16); collagen 3
.alpha.1 (COL3A1), 5'-AACACGCAAGGCTGTGAGACT-3' (forward) (SEQID
17), 5'-GAACGAGGTAGTCTTTCAGCAACA-3' (reverse) (SEQID 18); carbonic
anhydrase IX (CAIX), 5'-AGGTCCCAGGACTGGACATA-3' (forward) (SEQID
19), 5'-GAGGGTGTGGAGCTGCTTAG-3' (reverse) (SEQID 20); DNMT1,
5'-TATCCGAGGAGGGCTACCTG-3' (forward) (SEQID 21),
5'-CACTTCCCGGTTGTAAGCAT-3' (reverse) (SEQID 22); DNMT3A,
5'-AGCCCAAGGTCAAGGAGATT-3' (forward) (SEQID 23),
5'-GTTCTTGCAGTTTTGGCACA-3' (reverse) (SEQID 24); DNMT3B,
5'-TCAGGATGGGAAGGAGTTTG-3' (forward) (SEQID 25),
5'-CTGCAGAGACCTCGGAGAAC-3' (reverse) (SEQID 26). QPCR reactions
were normalized by amplifying the same cDNA with
beta-2-microglobulin (B2M) primers, 5'-AGGCTATCCAGCGTACTCCA-3'
(forward) (SEQID 27), 5'-CCAGTCCTTGCTGAAAGACA-3' (reverse) (SEQID
28).
[0084] QPCR was performed using Platinum SYBR Green qPCR
SuperMix-UDG (Invitrogen). Amplification and detection were carried
out using Mx3000P System (Stratagene). The PCR cycling program
consisted of 40 three-step cycles of 15 seconds/95.degree. C., 30
seconds/TA and 30 seconds/72.degree. C. Each sample was amplified
in duplicate. In order to confirm signal specificity, a melting
program was carried out after the PCR cycles were completed.
[0085] For analysis of interstitial collagen within the myocardial
sample, formalin-fixed tissue was paraffin-embedded and stained
using a Masson's trichrome (MTC) Stain Kit (Dako) optimised for use
on an Artisan staining system according to the manufacturer's
Instructions.
[0086] Manual Scoring of MTC Stained Cardiac Biopsies
[0087] Microscope slides containing 8 .mu.M thick sections of
myocardial tissue stained with MTC were examined by a pathologist
(OSE) and manually assessed for the degree of fibrosis in a blinded
fashion. MTC for collagen type I detection (fibrosis) was graded
from 1 to 5, where 1 is the least and 5 is the most severe
interstitial fibrosis. Only interstitial fibrosis was considered,
where invasive fibrosis with muscle replacement or compression
would count Subendocardial or epicardial fibrosis were
excluded.
[0088] Automated Image Analysis of MTC Stained Cardiac Biopsies
[0089] The Aperio ScanScope XT Slide Scanner (Aperio Technologies)
system was used to capture whole slide digital images with a
20.times. objective. Automated image analysis was performed using
Imagescope (Aperio). A positive pixel count algorithm was used to
automatically quantify the area occupied by stain colours within
each scanned slide image. Calibration of individual staining
patterns was performed by specifying the requisite colour (range of
hues and saturation) and limits for the desired Intensity range.
Required input parameters for each stain were based on the HSI
(Hue, Saturation and intensity) colour model. To detect the blue
colour of collagen with MTC stain, a hue value of 0.66 was
specified. The equivalent value for detection of red stained
myocytes was 0.0. The default hue width value of 0.5 was used to
allow inclusion of a moderate range of colour shades. A collagen
volume fraction was calculated based on the percent of blue
collagen staining quantified within a tissue section.
[0090] Primary Cell Culture
[0091] Primary human cardiac fibroblast cells from the adult
ventricle (HCF) were purchased from ScienCell Research
Laboratories. Primary cells were derived from a single female donor
aged 20. Until required for experiments, cells were cultured and
maintained in Dulbecco's modified eagles medium (DMEM) (Gibco),
supplemented with 10% Fetal Bovine Serum (Gibco) and
penicillin-streptomycin antibiotics (Gibco) in a 5% CO2 humidified
incubator kept at 37.degree. C.
[0092] Cell Culture Treatments
[0093] Where indicated, HCF cells were exposed to a 1% oxygen
environment for up to 8 days using a hypoxic chamber (Coy
Laboratories). The effects of 10 ng/ml recombinant TGF.beta.1
treatment (R&D Systems) under these conditions were
investigated. When required, cells were treated for 24 hours with 1
mM of the prolyl hydroxylase inhibitor DMOG (Sigma) to simulate
hypoxia through the induction of HIF under normoxic conditions.
[0094] Quantitative Flow Cytometry
[0095] The impact of hypoxia on HCF cells global methylation
profile was investigated using an antibody specific to methylated
DNA and quantified using flow cytometry. Briefly, HCF cells exposed
to either normoxia (21% oxygen) or hypoxia (1% oxygen) were fixed
in Camoy's solution prior to 60 minutes acid hydrolysis in 1 M HCl
at 37.degree. C. Following this DNA denaturation step, cells were
immunostained using anti-5'methylcytidine (5MeC) monoclonal
antibody (Eurogentec). IgG1 negative controls were used at the same
concentration as the primary antibody. Secondary immunostaining was
conducted using an FITC conjugated rabbit anti-mouse secondary
antibody (Dako). Analysis was performed on a CYAN flow cytometer
and results assessed using SUMMIT software (Dako).
[0096] Quantitative Real-Time PCR Analysis of Primary HCF Cells
[0097] Gene expression changes were measured in HCF cells using
QPCR as described in human cardiac tissue analysis methods section.
RNA isolation from cells was achieved using NucleoSpin RNA II Kit
(Macherey-Nagel). QPCR data was analysed using the delta delta CT
method.
[0098] Western Blotting
[0099] Whole cell protein lysates were generated using RIPA Lysis
Buffer (Millipore), containing a protease inhibitor cocktail
(Roche). Nuclear protein extracts were obtained using the Ne-PeR
Nuclear and Cytoplasmic extraction Reagents, according to
manufacturer's instructions (Pierce Biotechnology). Protein
concentrations were determined using the BCA Protein Assay Kit
(Pierce). 10-20 .mu.g of protein lysates were denatured, reduced
and resolved on SDS-polyacrylamide gels by SDS-PAGE before transfer
onto 0.45 .mu.m pore size Immobilon-P polyvinylidene fluoride
(PVDF) membranes (Millipore).
[0100] Membranes were incubated with blocking buffer (TBS, 0.25%
Tween-20, 0.1% serum from species that secondary antibody was
raised in, and 5% fat free skimmed milk) for 1 hour at room
temperature. Membranes were subsequently probed overnight with
either anti-ASMA (Sigma), anti-collagen 1, anti-DNMT3B (Imgenex),
or anti-hypoxia inducible factor 1.alpha. (HIF-1.alpha.) (Novus
Biologicals). Detection of the specific binding of the primary
antibody was achieved using HRP-conjugated secondary antibodies,
followed by signal detection with Immobilon Western
chemiluminescent HRP substrate (Millipore) according to the
manufacturer's instructions. Anti-alpha tubulin (Sigma) was used to
verify equal loading.
[0101] Generating a Functional and Mutated DNMT3B Luciferase
Construct
[0102] A 250 bp fragment of the DNMT3B promoter containing a
putative hypoxia response element (HRE) was cloned and inserted
into a pGL3 luciferase construct (Promega), generating pDNMT3B-Luc.
In addition, a mutated version of this construct was created by
site-directed mutagenesis using QuikChange II Site-Directed
Mutagenesis Kit (Stratagene) to mutate the putative HRE within the
DNMT3B promoter. Specifically, 2 cytosine bases within the putative
HRE were replaced with adenine bases, generating
pDNMT3B-.DELTA.HRE-Luc. DNA sequencing was used to confirm the
presence of the mutation. Either pDNMT3B-Luc,
pDNMT3B-.DELTA.HRE-Luc, or empty vector was subsequently
transiently transfected into Hela cells using FUGENE HD (Promega).
24 hours post transfection, cells were either exposed to 1% oxygen
for 24 hours, or left in normaxia (21% oxygen). The degree of
DNMT3B promoter activity was quantified using the
luciferin/luciferase bioluminescence assay following cell lysis
with Passive Lysis Buffer (Promega) and incubating with Luciferase
Assay Reagent (Promega), with luciferase activity measured on a
GloMax 20/20 Luminometer (Promega).
[0103] siRNA Targeted DNMT3B Knock Down
[0104] HCF cells were transfected with either 20 nM siDNMT3B of the
sequence 5'-AGAUGACGGAUGCCUAGAGUU-3' (SEQID 31), or control siRNA
using DharmaFect4 transfection reagent (Dharmacon). Transfected
cells were incubated for 12 hours prior to hypoxic exposure of 1%
oxygen for 4 days, or left in normoxia (21% oxygen). To ensure
DNMT3B levels remained low over the time course, siRNA was
replenished on day 2. The impact or 10 ng/ml TGF.beta. under these
experimental conditions was also investigated.
[0105] DNA Demethylation with 5-Aza-2'-Deoxycytidine
[0106] For demethylation analysis, cells were treated with 1-5
.mu.M 5-aza-2'-deoxycytidine (5-azadc; Sigma) for up to eight days.
The effect of global DNA methylation inhibition on pro-fibrotic
protein expression was assessed using Western blotting.
[0107] Results
[0108] Myocardial Tissue Hypoxia is Associated with an Enhanced
Fibrotic Gene Expression Profile.
[0109] Following ethical approval and informed patient consent,
human right atrial tissue samples were collected from the hearts of
26 patients undergoing elective cardiac-bypass surgery. RNA was
extracted from this tissue and was used to look at the relationship
between hypoxia and collagen production using quantitative
real-time PCR. The degree of carbonic anhydrase IX (CAIX)
expression was used as a validated surrogate marker for cardiac
tissue hypoxia (Holotnakova 2008). A significant positive
correlation between collagen 1 gene expression with CAIX was
detected (r=0.50, p<0.01), as highlighted in FIG. 1B. Relative
CAIX gene expression levels also correlated positively with ASMA
(r=0.42, p<0.05), FIG. 1A. Collagen 3 gene expression was not
statistically associated with changes in CAIX expression, although
a positive trend was observed (r=0.35, p=0.07).
[0110] Tissue Hypoxia is Associated with Increased Collagen
Deposition
[0111] The degree of collagen deposition within right atrial tissue
samples was assessed using Masson's trichrome (MTC) staining.
Examples of myocardial tissue stained with MTC can be observed in
FIG. 1D, with positive blue staining for collagen being evident,
and myocytes staining red. This figure highlights examples of both
interstitial and perivascular fibrosis. Two methods were used to
analyse the collagen content within the MTC stained slides, namely,
digital quantification of positive pixels (FIG. 1E, an example were
the positive pixel algorithm has been applied to a MTC stained
slide generating a mark up image detailing the positive blue pixels
representing collagen) and blinded manual scoring by a Pathologist
(OSE) (FIG. 1F, examples of manual scoring images grading collagen
deposition between 0-5).
[0112] The relationship between collagen deposition and hypoxia was
assessed by dividing the MTC slides into two groups based on median
CAIX gene expression levels. Results indicate that increased tissue
hypoxia (higher CAIX expression) was associated with a significant
increase in collagen (p<0.05). This was observed in both the
automated digitally analysed positive pixel quantification of
collagen (FIG. 1G) and the blinded manual scoring of MTC staining
(FIG. 1H).
[0113] These results indicate that the degree of hypoxia within
myocardial tissue is associated with increased expression of the
pro-fibrotic genes ASMA and collagen 1, as well as increased
deposition of fibrillar collagen protein. As the likely source of
the increased collagen within the myocardium is the cardiac
fibroblast we undertook in vitro hypoxia studies utilising a human
primary cardiac fibroblast cell line (HCF). HCF cells were cultured
in either 21% oxygen or 1% oxygen for up to 8 days. Culturing cells
in 1% oxygen stabilised nuclear HIF-1.alpha., indicating that the
cells were experiencing a hypoxic environment, FIG. 2A. Under these
conditions, an increase in cell proliferation was observed, another
pathological feature of fibrosis, FIG. 2B. Using quantitative
real-time PCR, a significant increase in both ASMA and collagen 1
gene expression was detected at 4 and 8 days post hypoxia, compared
with cells grown under normoxia, FIGS. 2C and 2D. Interestingly,
the pro-fibrotic effects of TGF.beta.1 treatment was enhanced in
hypoxia. TGF.beta.1 treatment of HCF significantly increased ASMA
and collagen 1 gene expression, and this was further enhanced if
the cells were exposed to 8 days hypoxia, FIGS. 2E and 2F,
respectively.
[0114] To determine whether the pro-fibrotic effects of hypoxia was
associated with epigenetic changes within the HCF cells, DNA
methylation studies were carried out. Global DNA methylation was
analysed in cells exposed to either 21% oxygen, 1% oxygen for 4
days, or 1% oxygen for 8 days. Application of an antibody directed
to methylated DNA (anti-5MeC) and quantification using flow
cytometry revealed significant DNA hypermethylation in hypoxic
cells (p<0.001), FIG. 3A. Confirmation of specific nuclear
staining was achieved using immunofluorescent microscopy, as
highlighted in FIG. 3B, with specific nuclear anti-5MeC positivity
over a background of the blue nuclear counterstain DAPI. To
investigate possible mechanisms of hypoxia induced DNA
hypermethylation, the enzymes that catalyze this process were
quantified, namely the DNA methyltransferases (DNMT). The gene
expression levels of DNMT1, the enzyme primarily responsible for
maintaining the methylation status of daughter cells during cell
cycle, and the de novo methylating enzyme DNMT3B, were both
significantly up-regulated as early as 24 hours following exposure
to a 1% hypoxic environment (p<0.001), FIGS. 3C and 3E. Levels
of the de novo methylating enzyme DNMT3A was unchanged, FIG. 3D.
Western blot analysis of hypoxic nuclear extracts showed that
DNMT3B protein was significantly up-regulated in hypoxia,
suggesting a possible role in regulating the global changes in DNA
methylation in hypoxia. Interestingly, when examining the human
cardiac tissue samples, DNMT3B gene expression levels significantly
correlated with CAIX levels (r=0.50, p<0.001), FIG. 3F.
[0115] The mechanism by which hypoxia regulates DNMT3B expression
was explored due to the potential importance of this de novo
methylating enzyme in directing epigenetic changes and
susceptibility to hypoxia induced fibrosis. Following analysis of
the DNMT3B promoter, a consensus sequence for a HIF-1.alpha.
binding site, referred to a hypoxia response element (HRE) was
identified. Accordingly, this hypoxia mediated transcription factor
posed as a likely candidate for regulating DNMT3B expression. To
investigate this further, a luciferase expression construct
containing the human DNMT3B promoter was generated (pDNMT3B-Luc).
An additional construct was generated were the HIF-1.alpha. binding
site was mutated (pDNMT3B-.DELTA.HRE-Luc). Hela cells were selected
as a suitable vehicle to investigate hypoxia regulation of DNMT3B
as they possess a functional HIF-1.alpha. responsive pathway when
exposed to 1% hypoxia, as indicated by nuclear protein
stabilisation, FIG. 4A. Hela cells were utilised and transfected
with either a luciferase construct with a functional or HRE mutated
DNMT3B promoter sequence. Cells were cultured in normoxia or 1%
hypoxia for 24 hours, prior to analysis of luciferase activity.
Results indicate that the DNMT3B promoter activity is increased
3-fold in hypoxia (p<0.05), FIG. 4B. Hypoxia mediated DNMT3B
promoter activity was significantly attenuated when the
HIF-1.alpha. binding site was mutated (p<0.001), FIG. 4C.
[0116] As supportive evidence for a role for HIF-1.alpha. in
regulating DNMT3B expression in hypoxia, HCF cells were treated
with the HIF-1.alpha. stabilisation compound DMOG in normoxia. As
highlighted in FIG. 5A, DMOG treatment stabilises nuclear
HIF-1.alpha. protein expression, and also results in increased
expression of DNMT3B.
[0117] Collectively, these data indicate that hypoxia induced
expression of DNMT3B in cardiac fibroblast cells is at least in
part regulated by the hypoxia inducible transcription factor
HIF-1.alpha..
[0118] Based on these results, the potential anti-fibrotic impact
of blocking either hypoxia induced HIF-1.alpha. expression or
DNMT3B expression on the pro-fibrotic effects of hypoxia on HCF was
investigated. These experiments were carried out using siRNA to
prevent/reduce DNMT3B expression. Human cardiac fibroblast cells
were transfected with either siDNMT3B or control siRNA using.
Transfected cells were analysed four days later using Western
blotting to quantify cellular levels of collagen 1 and alpha smooth
muscle actin (ASMA). siRNA targeted DNMT3B knock down resulted in a
significant reduction in expression of pro-fibrotic proteins
including ASMA and collagen 1. Successful siRNA knockdown of DNMT3B
was confirmed by Western blotting (FIG. 6). Alpha tubulin was
utilised as a loading control.
[0119] Treating hypoxic fibroblasts with the DNMT inhibitor
5-aza2dC inhibited the pro-fibrotic effects of TGF.beta., one of
the most potent pro-fibrotic agonists. 5-aza2dC significantly
reduced expression of ASMA, collagen 1 and collagen 3.
DISCUSSION
[0120] The detrimental impact of ischemia on the structure and
function of the myocardium is well acknowledged. Initial cardiac
responses to regional hypoxia, usually after myocardial infarction
or following prolonged coronary artery disease are organ
protective. However, chronic insults or maladaption to the
pathophysiological process can lead to reactive cardiac fibrosis
and over time may potentially lead to heart failure 1, 3. The
importance of this led us to investigate the relationship between
hypoxic myocardial tissue and fibrosis, and to determine whether
hypoxia can regulate the fibrotic phenotype in cardiac fibroblast
cells. We also sought to examine whether any hypoxia induced
pro-fibrotic responses were related to changes in the epigenetic
profile of cardiac fibroblast cells.
[0121] Following ethical approval, we acquired atrial myocardial
tissue samples from 26 patients undergoing elective cardiac bypass
surgery for either coronary artery bypass grafting or mitral valve
repair/replacement. With this tissue we were able to look at the
relationship between the degree of tissue hypoxia and fibrosis. The
extent of myocardial tissue hypoxia was assessed by quantifying the
gene expression levels of carbonic anhydrase IX (CAIX). CAIX is
involved in cell adhesion and pH regulation with its expression
being controlled by hypoxia inducible factor 1 (HIF-1). The
importance of CAIX within the hypoxic myocardium has been shown 8.
Although HIF-1 is the main mediator of hypoxic responses, its
fluctuating stability and rapid degradation would have proved
difficult to study in tissue samples collected at time of surgery,
therefore CAIX was adopted as a surrogate marker of hypoxia 9, 10.
Analysis of the myocardial tissue highlighted a significant
positive correlation between gene expression levels of CAIX with
both collagen 1 and the myofibroblast differentiation marker
alpha-smooth muscle actin (ASMA). Collagen 3 gene expression did
not significantly correlate, however a close relationship was
observed. To further investigate this, the cardiac tissue samples
were stained with Masson's trichrome blue (MTC) in order to
quantify fibrillar collagen 1 protein deposition. Staining for
collagen, blue in appearance, was quantified using two approaches,
namely automated digital analysis which involved applying a
positive pixel algorithm to quantify blue pixels within digitalised
stained sections, and a manual blinded scoring method carried out
by an experienced clinical pathologist (OSE). Using the median CAIX
expression level, the tissue cohort was divided into low and high
CAIX expressers, signifying low and high tissue hypoxia. Using this
division, the degree of collagen deposition was compared. Using
automated quantification, a significantly higher level of collagen
deposition was detected in the more hypoxic tissue (high CAIX) and
these findings were confirmed by the blinded manual scoring. In
light with previously published work these findings support other
studies exploring the impact of hypoxia and fibrosis.
[0122] As the main source of enhanced ASMA and collagen with the
degree of hypoxia in cardiac tissue is the fibroblast, we carried
out in vitro studies utilising a primary human cardiac fibroblast
cell line (HCF) and subjecting it to hypoxia. In support with the
myocardial tissue findings, we exposed HCF cells to 1% oxygen for
up to 8 days. Importantly, hypoxia stabilised HIF-1.alpha. protein
under these conditions demonstrating the cells were experiencing a
hypoxic environment. Interestingly, we also observed an increase in
cell proliferation in hypoxia, a process that could contribute to
the overall fibrotic burden in vivo. Other than an increase in
fibroblast numbers within fibrotic diseases (increased
proliferation or reduced apoptosis), an enhanced profibrotic
phenotype is an important contributor to the disease with
myofibroblast differentiation and increased extracellular matrix
deposition. Here we report an increase in collagen 1 and ASMA
expression in fibroblasts exposed to chronic hypoxia (1%, 8 days).
Importantly, these cells are also more responsive to exogenous
TGF.beta.1 treatment, exhibiting greater levels of collagen 1 and
ASMA induction. Although these results are limited to studying
collagen, ASMA, and proliferation as indicators of chronic hypoxia
generating a pro-fibrotic phenotype, it is likely that these
cellular responses involve multiple alterations in gene expression
profiles. As such, we sought to determine whether these findings
occurred on a background of epigenetic changes, and thus providing
a potential platform to support fibrogenesis.
[0123] The importance of epigenetics in cardiovascular disease is
becoming ever apparent, with some studies exploring the impact of
histone modifications on gene expression in diseases. However,
studies into the impact of epigenetic modifications in cardiac
fibroblasts are limited, particularly investigations into DNA
methylation changes and subsequent pro-fibrotic phenotypes. Given
the importance of this epigenetic phenomenon, and previously
published literature highlighting that hypoxia can impact the
epigenetic machinery, including DNA methylation in cancer this
study set out to examine whether hypoxia can alter the DNA
methylation profile of cardiac fibroblast cells. As highlighted in
FIG. 3A, it was found that chronic hypoxia significantly increased
global DNA methylation within the genome of cardiac fibroblast
cells. Subsequent analysis of the DNA methyltransferase enzymes
(DNMT) revealed that both DNMT1 and DNMT3B gene and protein
expression was increased, highlighting a potential mechanism by
which hypoxia can hypermethylate the DNA of cardiac fibroblast
cells. Interestingly, DNMT1 and DNMT3B gene expression levels
within the cardiac tissue significantly correlated with CAIX
levels, highlighting that the in vitro relationship between hypoxia
and DNMTs are also apparent in vivo.
[0124] During embryogenesis, expression levels of the de novo
methylation enzyme DNMT3B are high when the methylation pattern of
the genome is being established. Whereas in normal healthy adult
tissue DNMT3B expression is usually low, however, increased
expression has been implicated in the pathogenesis of numerous
diseases, including cancer. Therefore, its re-emergence in disease
states is likely causing pathological DNA methylation and aberrant
gene silencing. It was therefore examined whether there was a
direct role for DNMT3B in hypoxia mediated cardiac fibrosis. Upon
examination of the DNMT3B promoter, a putative hypoxia response
element (HRE) was discovered, providing a mechanism by which
hypoxia can drive its expression via the transcription factor
HIF-1.alpha.. Three approaches were undertaken to confirm this
theory; (I) a study generating DNMT3B promoter driven luciferase
constructs with either a functional (pDNMT3B-Luc) or mutated
(pDNMT3B-.DELTA.HRE-Luc) HRE site, (II) a study looking at the
impact of siHIF-1.alpha., and (III) a study using the compound DMOG
to stabilise HIF-1.alpha. in normoxia. As shown in FIG. 4, Hela
cells transfected with pDNMT3B-Luc exhibit a significant increase
in luciferase expression compared with cells maintained in
normoxia. Importantly, when the HRE site is mutated, there is a
significant blunting in luciferase activity. Of note, DNMT3B
up-regulation is not completely abolished, suggesting the
possibility that additional hypoxia mediated transcription factors
may contribute to DNMT3B regulation. A role for HIF-1.alpha. in
regulating DNMT3B is further supported by siRNA and DMOG
experiments, as shown in FIG. 5.
[0125] Collectively, all three approaches confirm that hypoxia
mediated up-regulation of DNMT3B is regulated by the hypoxia
inducible transcription factor HIF-1.alpha.. The importance of this
stems well beyond the scope of cardiac fibrosis and into the realms
of other diseases, including cancer biology, and other fibrotic
entities.
[0126] Having shown that hypoxia is associated with a pro-fibrotic
phenotype, which occurs against a background of epigenetic changes,
including HIF-1.alpha. mediated DNMT3B up-regulation, it was sought
to determine whether global demethylation or a DNMT3B targeted
approach could be potentially anti-fibrotic. Firstly, the impact of
reversing DNA methylation on cellular responses to the pro-fibrotic
cytokine TGF.beta. was investigated. Using the DNA demethylation
drug 5-aza-2-deoxycytidine, it was shown that responses to
TGF.beta. were significantly reduced. Specifically, reduced
expression of ASMA, collagen 1 and collagen 3 was observed. The
impact of directly targeting DNMT3B was also investigated using
siRNA. Results show that by reducing or 5 suppressing DNMT3B
expression protein levels of ASMA and collagen 1 are significantly
reduced. Collectively these novel data highlight the potential use
of epigenetic mediated therapies, specifically those that target
DNA methylation, to reduce, prevent or reverse cardiac
fibrosis.
[0127] Characteristics of the patients from whom cardiac tissue was
obtained are summarized in Table 2. Relative tissue hypoxia was
determined based on above and below the median gene expression
levels of the hypoxic marker CA9.
TABLE-US-00002 TABLE 2 Characteristics of the patients from whom
cardiac tissue was obtained. Hypoxic Normoxic Total tissue tissue
Demographics Population (High CAIX) (Low CAIX) N 26 13 13 Age, yr
72 .+-. 10 72 .+-. 9 72 .+-. 11 Gender, male 18 (69%) 9 (69%) 9
(69%) SBP, mmHg 134 .+-. 6 134 .+-. 6 132 .+-. 7 DBP, mmHg 78 .+-.
7 76 .+-. 6 80 .+-. 7 BMI, kg/m.sup.2 27 .+-. 3 27 .+-. 3 27 .+-. 3
Atrial Fibrillation 5 (19%) 2 (15%) 3 (23%) Diabetes Mellitus 5
(19%) 3 (23%) 2 (15%) Smoking History 10 (38%) 7 (54%) 3 (23%)
Hypercholesterolemia 9 (35%) 6 (46%) 3 (23%) Coronary Artery 20
(77%) 10 (77%) 10 (77%) Disease Valvular Heart 12 (46%) 5 (38%) 7
(54%) Disease Hypertension 9 (35%) 4 (31%) 5 (38%) RAAS Inhibitor
12 (41%) 6 (46%) 6 (46%) Beta-Blocker 16 (55%) 9 (69%) 7 (54%)
Statin 16 (55%) 11 (85%) * 5 (38%) Creatinine, .mu.mol/l 90 .+-. 13
90 .+-. 4 90 .+-. 17 BNP, pg/ml 104 (17:127) 84 (15:116) 135
(26:171) LVEF, % 60 .+-. 7 60 .+-. 7 60 .+-. 8 LVIDd, mm 53.0 .+-.
5.0 53.0 .+-. 4.8 55.0 .+-. 5.2 IVS, mm 9.7 .+-. 1.5 9.6 .+-. 1.3
9.9 .+-. 1.7 PW, mm 10.7 .+-. 1.6 10.5 .+-. 2.0 11.0 + 1.1 E/e' 8.7
.+-. 2.8 8.7 .+-. 3.0 8.6 .+-. 2.6 LAVI, mls/m.sup.2 28.4 .+-. 4.4
28.1 .+-. 5.2 28.6 .+-. 3.4 Values are mean .+-. SD, median
(25th:75th percentiles) or n (%). SBP/DBP, systolic and diastolic
blood pressure; BMI, body mass index; RAAS Inhibitor, renin
angiotensin system inhibitor; BNP, b-type natriuretic peptide;
LVEF, left ventricular ejection fraction; LVIDd, left ventricular
end diastolic dimension; IVS, intraventricular septum; PW,
posterior wall; E/e', ratio of mitral early diastolic flow velocity
over tissue Doppler lateral mitral annular lengthening velocity;
LAVI, left atrial volume index.
Example 2
Epigenetic Therapy for the Treatment of Cardiac Fibrosis and
Hypertrophy
[0128] Methods
[0129] Primary Cell Culture and Treatments
[0130] Primary human cardiac fibroblast cells from the adult
ventricle (HCF) were purchased from ScienCell Research
Laboratories. Cells were cultured and maintained in Dulbecco's
modified eagles medium (DMEM) (Gibco), supplemented with 10% Fetal
Bovine Serum (Gibco) and penicillin-streptomycin antibiotics
(Gibco) in a 5% CO2 humidified incubator kept at 37.degree. C. When
required, HCF cells were treated for up to 8 days with either 10
ng/ml human recombinant transforming growth factor beta 1
(TGF.beta.1) (R&D Systems), 1 .mu.M 5-azacytidine(5-aza)
(Sigma), or with both compounds simultaneously.
[0131] Quantitative Real-Time PCR
[0132] RNA isolation from HCF cells was achieved using NucleoSpin
RNA II Kit (Macherey-Nagel). First strand cDNA synthesis was
carried out using SuperScript II RT (Invitrogen). Quantitative
real-time PCR (QPCR) primers were designed so that one of each
primer pair was exon/exon boundary spanning to ensure only mature
mRNA was amplified. The sequences of the gene-specific primers used
are as follows; ASMA, 5'-CGTTACTACTGCTGAGCGTGA-3' (forward),
5'-AACGTTCATTTCCGATGGTG-3' (reverse); collagen 1 .alpha.1 (COL1A1),
5'-GAACGCGTGTCATCCCTTGT-3' (forward),
5'-GAACGAGGTAGTCTTTCAGCAACA-3' (reverse); collagen 3 .alpha.1
(COL3A1), 5'-AACACGCAAGGCTGTGAGACT-3' (forward),
5'-GAACGAGGTAGTCTTTCAGCAACA-3' (reverse). QPCR reactions were
normalized by amplifying the same cDNA with beta-2-microglobulin
(B2M) primers, 5'-AGGCTATCCAGCGTACTCCA-3' (forward),
5'-CCAGTCCTTGCTGAAAGACA-3' (reverse).
[0133] QPCR was performed using Platinum SYBR Green qPCR
SuperMix-UDG (Invitrogen). Amplification and detection were carried
out using Mx3000P System (Stratagene). The PCR cycling program
consisted of 40 three-step cycles of 15 seconds/95.degree. C., 30
seconds/TA and 30 seconds/72.degree. C. Each sample was amplified
in duplicate. In order to confirm signal specificity, a melting
program was carried out after the PCR cycles were completed.
Relative fold changes in gene expression was calculated using the
delta delta CT method.
[0134] Western Blotting
[0135] Whole cell protein lysates were generated using RIPA Lysis
Buffer (Millipore), containing a protease inhibitor cocktail
(Roche). Protein concentrations were determined using the BCA
Protein Assay Kit (Pierce). Protein lysates were denatured, reduced
and resolved on SDS-polyacrylamide gels by SDS-PAGE before transfer
onto 0.45 .mu.m pore size Immobilon-P polyvinylidene fluoride
(PVDF) membranes (Millipore).
[0136] Membranes were incubated with blocking buffer (TBS, 0.25%
Tween-20, 0.1% serum from species that secondary antibody was
raised in, and 5% fat free skimmed milk) for 1 hour at room
temperature. Membranes were subsequently probed overnight with
anti-ASMA (Sigma). Detection of the specific binding of the primary
antibody was achieved using HRP-conjugated secondary antibodies,
followed by signal detection with Immobilon Western
chemiluminescent HRP substrate (Millipore) according to the
manufacturer's instructions. Anti-alpha tubulin (Sigma) was used to
verify equal loading.
[0137] 5-Azacytidine Treatment of a Rat Model of Hypertensive Heart
Disease
[0138] Approval from the local Animal Research Ethics Committee was
sought and obtained to investigate the anti-fibrotic impact of
5-aza on cardiac function and fibrosis. Male spontaneously
hypertensive rats (SHR) and their normotensive counterpart Wistar
Kyote rats (WKY) were purchased from Charles River and utilised for
the study. From 10 weeks of age, the animals received alternate day
intra-peritoneal injection of either PBS (vehicle) or 5-aza (10
mg/kg) for 12 weeks. The 5-aza was purchased from Sigma, diluted in
sterile PBS and filtered through a 0.22 .mu.m filter. Aliquots were
stored at -20.degree. C. and were used within 5 days of
reconstitution. The study design consisted of three groups of 10
animals; group 1 included 10 SHR rats who received 5-aza
(SHR-5-aza); group 2 included 10 SHR rats who received PBS vehicle
(SHR-V); group 3 included 10 WKY rats who received PBS vehicle
(WKY-V). All rats were housed in the animal facility under
identical conditions, with a 12 hour light-dark cycle.
[0139] Systolic Blood Pressure Measurements
[0140] Systolic blood pressure was measured using the non-invasive
tail-cuff method (Letica Scientific Instruments LE 5001). Blood
pressure values were recorded while the animals were under inhaled
anaesthesia (2% isoflurane). The mean of three consecutive
measurements was obtained for each animal at study mid-point (6
weeks) and end of study (12 weeks).
[0141] Doppler Echocardiography
[0142] Cardiac structure and function was assessed at baseline and
at the end of the study (12 weeks) using Echocardiography. During
the procedure, the animals were under inhaled anaesthesia
(isoflurane 2%) and body temperature was maintained using a heat
mat. Echocardiography assessment was performed using a Vevo 770
High-Resolution In Vivo Micro-Imaging System (Visualsonics) with a
10 mHz transducer. M-mode and 2-dimensional (2D) images were
obtained in the parasternal long- and short-axis views. The
interventricular septal thickness, posterior wall thickness, and LV
diameter were measured in systole and diastole at the tips of the
papillary muscle. Measurements were taken over three consecutive
cardiac cycles and averaged. Left ventricular mass (LVM) was
calculated according to Devereux's formula and indexed to tibial
length (LVMi). Blinded analysis was performed by two separate
independent observers.
[0143] Serum Collection and BNP Analysis
[0144] Blood was collected at baseline via the tail vein method and
at the end of the study (12 weeks) during terminal bleed (abdominal
aorta). Serum isolation was achieved using Microvette serum tubes
(Sarstedt) with centrifugation at 10,000 rpm for 5 minutes at room
temperature. Serum levels of the cardiac stress hormone B-type
natriuretic peptide (BNP) was quantified using an ultra-sensitive
immunoassay with electrochemiluminescence detection as instructed
by the manufacturer (Meso Scale Discovery). The assay sensitivity
was 1.5 pg/mL.
[0145] Myocardial Rat Tissue Collection and Preparation
[0146] On completion of the in vivo rat study, animals were
sacrificed (terminal bleed while under inhaled 4% isoflurane
anaesthesia), and the heart was removed en-bloc to study the impact
of 5-aza on collagen deposition within the myocardium. Two
methodological approaches were used to quantify collagen
deposition, namely, Immunostaining of cardiac tissue sections using
picrosirus red, and the hydroxyproline assay using tissue
lysates.
[0147] For picrosirius red staining, the left ventricular
mid-sections (papillary level) of the heart were dissected
immediately following sacrifice, rinsed in PBS, and fixed with 10%
formalin (Sigma). Formalin fixed tissue was embedded in paraffin
and 5 .mu.m thick tissue sections were created for collagen
analysis using picrosirius red.
[0148] For hydroxyproline quantification, the left ventricular base
of the heart was dissected immediately following sacrifice, rinsed
in PBS, and snap frozen in liquid nitrogen until required for
analysis. Frozen hearts were thawed on ice and individually
disrupted and homogenized using an Ultra Turrax T25 Dispersing
Instrument (IKA). Total protein within the tissue lystates were
quantified using the BCA method and 10 .mu.g of homogenate was used
to determine hydroxyproline content. Cardiac tissue was homogenised
in PBS at a ratio of 100 mg tissue to 1 ml PBS.
[0149] Picrosirius Red Staining and Automated Digital
Quantification
[0150] Tissue sections were deparaffinised and rehydrated prior to
incubating with 0.2% phosphomolibid acid (PMA) for 2 minutes. After
rinsing in distilled water, the slides were stained with picrosirus
red (Direct Red 80 dissolved in picric acid, Sigma) for 90 minutes.
Finally, the slides were placed in 0.4% hydrochloric acid (HCl) for
2 minutes, 70% ethanol for 45 seconds, dehydrated and coverslipped
for analysis.
[0151] The degree of collagen deposition was quantified by
automated digital Image analysis. The Aperio ScanScope XT Slide
Scanner (Aperio Technologies) system was used to capture whole
slide digital images with a 20.times. objective. Automated image
analysis was performed using Imagescope (Aperio). A positive pixel
count algorithm was used to automatically quantify the area
occupied by the dark pink stain colours representing collagen
within each scanned slide image. Calibration of individual staining
patterns was performed by specifying the requisite colour (range of
hues and saturation) and limits for the desired intensity range.
Required input parameters for each stain were based on the HSI
(Hue, Saturation and intensity) colour model. To detect the dark
pink colour of collagen with picrosirus red, a hue value of 0.8 was
specified. The hue width value of 0.5 was used to allow inclusion
of a moderate range of colour shades. A collagen volume fraction
was calculated based on the percent of dark pink collagen staining
quantified within a tissue section.
[0152] Hydroxyproline Assay
[0153] In brief, 500 .mu.l of homogenised cardiac tissue sample
(ratio of 100 mg tissue homogenised in 1 ml PBS) was incubated at
37.degree. C. in a vacuum oven overnight in 1 ml 6N HCl. Five
microlitres of citrate/acetate buffer (7.24% sodium acetate, 5%
citric add, 3.4% sodium hydroxide, 1.2% glacial acetic add, pH 6.0)
and 100 .mu.l chloramine T-solution (282 mg chloramine T, 2 ml
n-propanol, 2 ml H2O, 16 ml citrate/acetate buffer) were added to 5
.mu.l of the digested cardiac tissue sample, and incubated for 20
minutes at room temperature. Following incubation, 100 .mu.l of
Ehrlich's solution (2.5 g 4-[dimethylamino]benzaldehyde [4-DMAB],
9.3 ml N-propanol, 3.9 ml 70% perchloric acid) was added to each
sample and incubated for 20 minutes at 65.degree. C. Samples were
subsequently cooled for 10 minutes and read at 550 nm using a
SpectraMax M2 plate reader (Molecular Devices) with SoftMax Pro
software (Molecular Devices, version 4.7.1). In parallel, a
hydroxyproline standard curve was created to generate quantifiable
data. Hydroxyproline (Sigma) concentrations from 0-200 .mu.g/ml
were used and were handled in a similar fashion to the digested
homogenised cardiac tissue samples.
[0154] Results
[0155] Echocardiography Data
[0156] Effect of 5-Aza on Left Ventricular Hypertrophy and
Diastolic Dysfunction
[0157] As seen from FIGS. 8-11, Echocardiographic analysis revealed
a significant reduction in interventricular septum diameter in
diastole and left ventricular mass index (LVMI) in the SHR-5AZA
group compared to the SHR-V group. The WKY-V control group had
significantly less LVH than both SHR groups. Ejection fraction was
significantly lower in the SHR-V group compared to the WKY-V group.
Treatment with 5AZA significantly increased ejection fraction in
SHR animals. E prime (E'), a robust and established
echocardiographic measure of diastolic dysfunction, was
significantly reduced in SHR-V compared to normotensive WKY-V
control. Importantly, this echocardiographic marker of diastolic
dysfunction was significantly approved with treatment of 5AZA,
equivalent to that of the normotensive WKY-V control.
[0158] Cardiac Tissue Analysis:
[0159] Effect of 5AZA on Myocardial Interstitial Disease
[0160] Total collagen content within the myocardium was estimated
using the hydroxyproline assay. As highlighted in FIG. 12, a
significant reduction in total collagen was observed in SHR rats
treated with 5AZA compared to SHR vehicle controls.
[0161] As an indication of collagen distribution and localisation,
tissue sections were stained using picrosirius red. The degree of
collagen deposition (area occupied by the dark pink stain colours)
was quantified by automated digital image analysis through the
application of a modified positive pixel count algorithm.
Application of the algorithm generated a mark-up image highlighting
positive pixels as yellow, orange or red, and negative pixels as
blue. A collagen volume fraction was calculated based on the
percent of dark pink collagen staining quantified within a tissue
section. Results highlight that perivascular collagen was
significantly reduced in SHR-5AZA animals compared to SHR-V animals
(FIGS. 13-15).
[0162] Effect of 5AZA on Cardiac Myocytes In Vivo
[0163] Within the cardiac tissue, myocyte area was quantified to
assess the ability of 5AZA to reduce myocyte hypertrophy. In this
regard, FIG. 16 highlights that the average myocyte area within the
SHR-5AZA animals are significantly smaller than that of their
counterpart controls SHR-V.
[0164] A potential mechanism by which 5AZA improves cardiac
function, including hypertrophy and diastology, is through
up-regulation of cardiac myosin binding protein C (MYBP-C). It has
previously been shown that decreased expression of MYBP-C in the
heart results in abnormal contractile function at the myofilament
level, thereby contributing to the development of hypertrophic
cardiomyopathy in humans. This reduction may be linked with gene
mutations as well as through other potential mechanisms. One
mechanism yet to be explored is epigenetic changes, including DNA
methylation. Re-expressing or increasing the expression levels of
MYBP-C through gene transfer in vivo has recently been shown to
improve both systolic and diastolic contractile function as well as
reduce left ventricular wall thickness (Merkulov et al 2012 Circ
HF; PMID:22855556). Our novel data has shown that administration of
the demethylating agent 5AZA to an in vivo murine model of
hypertensive heart disease resulted in a significant increase in
MYBP-C expression within the myocardium (FIG. 17). This highlights
a novel mechanism by which MYBP-C expression can be enhanced, and
thus has the potential to improve cardiac dysfunction. Treatment
with DNA methylation inhibitors, such as 5-azacytidine and
5-aza-2-deoxycytidine could also impact the phosphorylation status
of MYBP-C.
[0165] In addition to an increase in MYBP-C gene expression, 5AZA
treatment of SHR animals surprisingly resulted in a significant
increase in Actin, Alpha 1, Skeletal Muscle (ACTA1), FIG. 18.
Up-regulation of MYBP-C and ACTA1 expression were associated with a
reduction in hypertrophy and diastolic dysfunction, as indicated by
LVMI, E prime, and inter ventricular septum diameter changes.
[0166] Cardiac Myoblast Data:
[0167] Confirmation of the impact of DNA methylation inhibitors can
up-regulate the expression levels of MYBP-C and ACTA1 was assessed
in vitro using cardiac myoblast cells. Cells were grown in culture
for 4 days either in the presence or absence of 5-azacytidine
(5AZA) or 5-aza-2-deoxycytidine (5AZA_DC). Gene expression was
quantified using real-time PCR. Treatment with 5AZA_DC resulted in
a significant increase in both MYBP-C and ACTA1, as indicated in
FIGS. 19-22.
[0168] The present invention illustrates the possible utility of
hypomethylating agents such as 5-aza and 2'deoxy in the prevention
or treatment of cardiomyopathies. In particular, such agents have
shown potential for use in treating HCM and myocardial fibrosis.
This is particularly surprising, because 5-aza and 2'deoxy have
been shown to increase expression of ACTA1, elevated levels of
which are implicated in exacerbated hypertrophy. In spite of this,
the current data show that these agents nevertheless are capable of
reversing the effects of hypertrophy and also the pro-fibrotic
impact of hypoxia.
[0169] The term "oligonucleotide" is meant to include both standard
and modified oligoribonucleotides, oligodeoxyribonucleotides and
analogs or combinations thereof. Examples are standard and modified
DNA, RNA, and combinations thereof.
[0170] The term "or" is intended to be used as listing a number of
non-mutually exclusive alternatives. As such, the term "or" should
be interpreted to mean "and/or". For example, a statement that an
aspect of the invention comprises a method for treating cardiac
hypertrophy or myocardial fibrosis, is to be understood as meaning
a method for treating any one taken from the set of: cardiac
hypertrophy alone; myocardial fibrosis alone; and both cardiac
hypertrophy and myocardial fibrosis together.
[0171] The words "comprises/comprising" and the words
"having/including" when used herein with reference to the present
invention are used to specify the presence of stated features,
integers, steps or components but do not preclude the presence or
addition of one or more other features, integers, steps, components
or groups thereof.
[0172] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
sub-combination.
* * * * *
References