U.S. patent application number 13/947800 was filed with the patent office on 2014-02-27 for cardioprotective effects of ghrh agonists.
This patent application is currently assigned to United States of America, Represented by The Department of Veterans Affairs. The applicant listed for this patent is United States of America, Represented by The Department of Veterans Affairs, University of Miami. Invention is credited to Norman L. Block, Joshua M. Hare, Rosemeire Miyuki Kanashiro-Takeuchi, Andrew V. SCHALLY.
Application Number | 20140057847 13/947800 |
Document ID | / |
Family ID | 48916594 |
Filed Date | 2014-02-27 |
United States Patent
Application |
20140057847 |
Kind Code |
A1 |
SCHALLY; Andrew V. ; et
al. |
February 27, 2014 |
CARDIOPROTECTIVE EFFECTS OF GHRH AGONISTS
Abstract
Disclosed herein are methods demonstrating that growth-hormone
releasing hormone (GHRH) directly activates cellular reparative
mechanisms within the injured heart, in a GH/IGF-I independent
fashion. Following experimental myocardial infarction (MI), rats
were randomly assigned to receive, during a 4 week period, either
placebo (n=14), rat recombinant GH (rrGH, n=8) or JI-38 (n=8; 50
.mu.g/Kg/day), a potent GHRH-agonist. JI-38 did not elevate serum
levels of GH or IGF-I, but markedly attenuated the degree of
cardiac functional decline and remodeling after injury. In
contrast, GH administration markedly elevated body weight, heart
weight, circulating GH and IGF-I, but did not offset the decline in
cardiac structure and function. Whereas, both JI-38 and GH
augmented levels of cardiac precursor cell proliferation, only
JI-38 increased anti-apoptotic gene expression. Collectively, these
findings demonstrate that within the heart, GHRH-agonists can
activate cardiac repair following MI.
Inventors: |
SCHALLY; Andrew V.; (Miami
Beach, FL) ; Block; Norman L.; (Hollywood, FL)
; Hare; Joshua M.; (Miami Beach, FL) ;
Kanashiro-Takeuchi; Rosemeire Miyuki; (Miami, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
United States of America, Represented by The Department of Veterans
Affairs
University of Miami |
Washington
Miami |
DC
FL |
US
US |
|
|
Assignee: |
United States of America,
Represented by The Department of Veterans Affairs
Washington
DC
University of Miami
Miami
FL
|
Family ID: |
48916594 |
Appl. No.: |
13/947800 |
Filed: |
July 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12914023 |
Oct 28, 2010 |
8507433 |
|
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13947800 |
|
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|
61289949 |
Dec 23, 2009 |
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Current U.S.
Class: |
514/11.2 |
Current CPC
Class: |
C07K 14/60 20130101;
A61P 9/10 20180101; A61K 38/25 20130101 |
Class at
Publication: |
514/11.2 |
International
Class: |
A61K 38/25 20060101
A61K038/25 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This work was supported by NIH grant R01-AG025017,
RO1-HL084275, RO1-HL65455, RO1-HL094848 and by National Heart,
Lung, and Blood Institute Grants U54-HL081028. The studies in the
laboratory of AVS were supported in part by The Medical Research
Service of the Veterans Affairs Department and South Florida
Veterans Affairs Foundation for Research and Education and
University of Miami, Miller School of Medicine, Departments of
Pathology and Medicine, Division of Hematology/Oncology.
Claims
1. A method of treating a subject having myocardial ischemia
comprising administering to the subject a therapeutic amount of a
peptide selected from synthetic growth hormone releasing hormone
(GHRH) and GHRH-A.
2. The method of claim 1, wherein the method comprises
administering GHRH-A.
3. The method of claim 1, wherein the peptide administration is at
a dosage of 50 .mu.g/kg.
4. The method of claim 1, wherein the peptide is administered
subcutaneously.
5. A method of treating a subject having myocardial fibrosis
comprising administering to the subject a therapeutic amount of a
peptide selected from synthetic growth hormone releasing hormone
(GHRH) and GHRH-A.
6. The method of claim 5, wherein the method comprises
administering GHRH-A.
7. The method of claim 5, wherein the peptide administration is at
a dosage of 50 .mu.g/kg.
8. The method of claim 5, wherein the peptide is administered
subcutaneously.
9. A method of treating a subject having congestive heart failure
comprising administering to the subject a therapeutic amount of a
peptide selected from synthetic growth hormone releasing hormone
(GHRH) and GHRH-A.
10. The method of claim 9, wherein the method comprises
administering GHRH-A.
11. The method of claim 9, wherein the peptide administration is at
a dosage of 50 .mu.g/kg.
12. The method of claim 9, wherein the peptide is administered
subcutaneously.
13. A method of regenerating damaged cardiac myocytes in a subject
having a cardiac disease comprising administering to the subject a
therapeutic amount of a peptide selected from synthetic growth
hormone releasing hormone (GHRH) and GHRH-A.
14. The method of claim 13, wherein the method comprises
administering GHRH-A.
15. The method of claim 13, wherein the peptide administration is
at a dosage of 50 .mu.g/kg.
16. The method of claim 13, wherein the peptide is administered
subcutaneously.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 12/914,023 filed on Oct. 28, 2010, which
claims priority to U.S. Provisional Application No. 61/289,949
filed Dec. 23, 2009, the contents of each of the foregoing
applications are incorporated by reference herein.
FIELD OF THE INVENTION
[0003] Cardioprotective Effects of GHRH Agonists
Sequence Listing
[0004] The instant application contains a Sequence Listing which
was submitted in ASCII format via EFS-Web in the parent
application, U.S. application Ser. No. 12/914,023 filed Oct. 28,
2010, and is hereby incorporated by reference in its entirety. Said
ASCII copy, created on Nov. 23, 2010, is named SHAL3038.txt and is
1,706 bytes in size.
Discussion of the Prior Art
[0005] Congestive heart failure remains a leading cause of
morbidity and mortality in developed countries. Despite major
therapeutic advances, current therapies fail to fully reverse heart
failure and/or left ventricular (LV) dysfunction. One major
therapeutic avenue is that of cytokine and/or hormonal signaling
pathways, and in this regard, various experimental and clinical
studies have suggested an important role for the GH/IGF-I axis in
the regulation of cardiac growth regeneration and function (41,
42). Moreover, several clinical studies have tested the impact of
GH replacement on the failing human heart, with controversial
results (3,4).
[0006] In addition to GH itself and IGF-I, GH releasing peptides
such as ghrelin and synthetic GH secretagogues are also suggested
to have cardiac effects (45-48) and GHRH mRNA is detected in
peripheral tissues, including the heart (40, 10) consistent with
widespread biological signaling potential beyond the
hypothalamic-pituitary axis. Recently, Granata et al. (10) reported
that rat GHRH (1-44) promoted survival of cardiomyocytes in vitro
and protected rat hearts from ischemia-reperfusion injury. The
detection of the GHRH receptor on the cardiomyocyte sarcolemmal
membrane supports the view that GHRH may elicit direct signal
transduction within the heart, independent of the GH/IGF-I axis,
per se (10). Ghrelin and other GH secretagogues may have
pharmacological potential (10), but also have pleiotropic actions
with a high possibility of unexpected side effects and potentially
serious disadvantages. Thus, the administration of GHRH offers a
potentially highly physiological approach based on direct action
without known side effects or the necessity to activate the
GH/IGF-I axis (11, 12). Furthermore, synthetic GHRH agonists, such
as JI-38 (GHRH-A) are more potent and longer acting agents than
native GHRH (13). Here we tested the hypothesis that GHRH-A has a
favorable cardiac effect, attenuating the progressive decrease of
cardiac function associated with post-Myocardial infarction left
ventricular (MI LV) remodeling. In addition, we investigated the
conjecture that GHRH directly activates signaling within the heart
(10) and exerts effects on cellular reparative pathways.
SUMMARY
[0007] There is provided method of treating cardiac disease by
activating the growth hormone releasing hormone pathway,
particularly where the cardiac disease is selected from the group
consisting myocardial infarction, myocardial ischemia, myocardial
fibrosis, cardiac weakness, cardiac failure and cardiac
inefficiency, especially where heart disease is congestive heart
failure. The method comprises administering to the patient in need
of same a growth hormone releasing hormone receptor activating
agent such as a peptide in particular a synthetic peptide.
[0008] The main finding of the present study is that GHRH-A has a
cardioprotective role in vivo following acute MI. Animals receiving
GHRH-A had improved cardiac structure and function and reduced
infarct size. In addition, cardiac fibrosis, which is one of the
main biological determinants of poor prognosis in heart failure,
and strongly associated with severe arrhythmias, diastolic
dysfunction and sudden death (14), was markedly reduced in the
GHRH-A group but not in the rrGH group. The cardiac effects of
GHRH-agonist appear to be direct, not involving the GH/IGF-I axis,
since the circulating levels of these hormones were not increased
by GHRH-A treatment.
[0009] The current findings can be viewed in the context of
previous evaluations of the GH/IGF-I axis that have yielded
variable results. The inconsistent and contradictory effects of GH
or IGF-I administration on experimental post-MI models have been
shown to be dependent on the timing of the treatment, the stage of
the disease at treatment initiation, different dosing regimens (8)
and might be related to the heterogeneous origin of treatment (15).
In most of the studies early treatment with recombinant human GH
have shown improvement in cardiac function and reduction on the LV
remodeling (45, 16, 17) while other studies did not show beneficial
effects (15, 18). Similarly, treatments with rat recombinant GH did
not show beneficial effect in rats with large MI (19). Conversely,
in rats all studies starting late after MI showed improvement on
cardiac function (20-22). Importantly, all treatments with
recombinant human GH in rats had a clear limitation due perhaps to
the production of anti-GH antibodies after 2 weeks of treatment
(23). Therefore, the long-term effects (either beneficial or
deleterious) remain unknown in these models (48).
[0010] Our findings demonstrate that rrGH markedly increases body
weight (BW), heart weight (HW) and circulating levels of GH and
IGF-I, but does not improve cardiac function or prevent remodeling;
on the contrary, rats treated with rrGH exhibited larger chambers
and worse ejection fraction (EF). These results are in agreement
with a study which showed that GH caused adverse effects on the
process of LV remodeling (18).
[0011] An alternative approach for increasing systemic levels of GH
is the administration of GH secretogues (GHS) such as Ghrelin (24)
or a synthetic GHS peptides such as hexarelin (20, 25). Nagaya et
al. (24) showed that ghrelin improved LV function and attenuated
cardiac remodeling in a chronic heart failure model; however, these
results were attributed to both GH/IGF-I dependent and
GH-independent vasodilatory effects of ghrelin. Similarly, Tivesten
et al (46) showed that hexarelin increased stroke volume and
reduced total peripheral resistance. In contrast, Shen et al (19)
reported increased survival rate, but no hemodynamic beneficial
effect of GH-releasing peptide in dogs subjected to transient
coronary occlusion, suggesting that these effects were mediated by
GHS receptors rather than through the GH/IGF-I axis (that is by a
GH independent pathway). To date, only one study in vitro has shown
cardioprotective and a direct effect of GHRH (10). In that study,
GHRH cardioprotection was demonstrated in isolated rat hearts
subjected to ischemia-reperfusion injury, while in our work,
cardiac function was assessed by echocardiography and in vivo
closed-chest LV catheterization in rats subjected to a permanent
occlusion. The mechanism underlying the differences between GHRH
and GH effects is unclear. Post-receptor signaling cascades can be
one reason for differences in activity between GHRH and GH. GHRH
actions involve the stimulation of its receptor (GHRHR), a G
protein-coupled receptor that activates at least two transduction
pathways, the adenyl cyclase (AC)/cAMP/protein kinase A (PKA) via
the Gs subunit (26), and the Ras/MAPK pathway through the subunits
(27).
[0012] The activation of the ERK1/2 signaling pathway has been
connected with several cellular activities such as proliferation,
differentiation, and survival, and ghrelin has previously been
shown to activate both ERK1/2 and the serine threonine kinase Akt
(28). GHRH induces activation of cAMP and a significant activation
of the Akt and ERK1/2 survival pathways as has been demonstrated by
Western blotting after GHRH administration. The PI3k/Akt pathway is
a well-known signaling pathway for cell protection, and recently,
Granata et al (10) reported that ERK1/2 and PI3k/Akt are involved
in survival effects induced by GHRH and found that GHRH increased
ERK1/2 and Akt phosphorylation, cAMP, and phosphorylation on serine
133 of CREB. Recently, Lorenz et al (29) proposed that specific
phosphorylation events on ERK 1/2 integrate differing upstream
signals to induce hypertrophy. Hexarelin has also previously been
shown to promote neuroprotection through activation of the PI3/Akt
pathway (30). Moreover, the PI3k/Akt pathway controls cell size,
including cardiomyocyte size (31) and is associated with
cardiomyocyte hypertrophy and apoptosis (30,32).
[0013] Traditionally, the adult heart has been considered a
post-mitotic organ where the cardiac myocytes were terminally
differentiated without ability to divide. However, several
investigators (33-35) have suggested that at least a subpopulation
of myocytes re-enter the cell cycle and divide, and also that a
pool of cardiac stem cells may reside in the myocardium. In the
present study, the expression of Ki67 positive cells was
significantly higher at the remote zone but only in the rrGH group
and this was accompanied by an increase in capillary density in the
same group. Previous study has documented that GH is able to
stimulate mature cardiac myocytes to re-enter the cell cycle,
divide and thereby increase their number in rat myocardium (36). A
reduction in apoptosis would also lead to an increased number of
cardiac myocytes but in our study, surprisingly, the reduction in
apoptosis in both treated groups was lower and not statistically
significant when assessed by TUNEL assay; however, at the molecular
level, changes in the expression of Bax and Bc12 supported an
anti-apoptotic effect of GHRH-A.
[0014] We also examined the abundance of cardiac precursor cells
which showed increased c-kit positive cells expression (clusters)
in the infarct zone in both treated groups; recruitment of c-kit
positive stem cells is associated with improvement in cardiac
performance (37). Bru'el et al (36) also reported that the number
of c-kit positive cells in a GH treated group was 31% higher than
that of the control group, but it was not statistically
significant. Given the observation of similar increases in c-kit
cells with GH and GHRH, yet greater reverse remodeling with GHRH,
it is attractive to speculate that GHRH may stimulate cardiopoiesis
to a greater extent. An alternate explanation is that the c-kit
cells may traffic and/or proliferate to a greater or earlier
extent. Finally, the findings of an anti-apoptotic milieu might
suggest improved survival of differentiation cardiac precursor
cells (CPCs). Future work is required to evaluate the direct
effects of GHRH on CPC). Besides CPCs possess the IGF-I/IGF-I
receptor system (38) which potentiates their survival and growth
(39). Further studies are needed to ascertain whether GHRH-A or
rrGH stimulated existing cardiac stem cells to differentiate into
mature cardiac myocytes.
[0015] In summary, the present findings document that GHRH
activation in the heart leads to reverse remodeling and recovery of
functional performance to a greater degree than that due to GH, and
that this occurs without stimulation of body weight or heart
weight. These findings support ongoing basic and translational
research into GHRH signal transduction mechanisms within the
heart.
BRIEF DESCRIPTION OF FIGURES
[0016] FIG. 1. Changes over time in LV diastolic (A: *p<0.05 vs.
placebo, f p<0.01 vs. GHRH-A) and systolic (B: *p<0.01 vs.
GHRH-A) dimensions, EF (C: *p<0.05 vs. placebo) and FS (D:
*p<0.05 vs. placebo and GHRH-A).
[0017] FIG. 2. Representative pressure-volume loops from Sham, MI
Placebo, rrGH and GHRH-A groups.
[0018] FIG. 3. Bar graphs showing infarct size (A) and percentage
of fibrosis (B). Representative Masson's trichrome-stained
histological sections (C) for infarct size measurement. The infarct
size (MI %) was significantly attenuated in GHRH-A group (*p=0.011
vs. placebo and rrGH). Similarly, the percentage of fibrosis was
reduced in GHRH-A group (*p=0.0002 vs. placebo and rrGH).
[0019] FIG. 4. A: Cryosections (A) of pituitary (top panel), heart
(mid panel) and skeletal muscle (bottom panel) incubated with GHRHR
(green). Scale bar: 50 jam. GHRHR specificity is demonstrated by
intense immunohistochemical reactivity in pituitary (positive
control) and heart; negative results are observed in skeletal
muscle (negative control). B: Western blotting detected a 47 kDa
protein corresponding to GHRHR. Molecular weight markers are
indicated on the left side of the panel (NC: negative control). C:
GHRHR protein abundance measured by Western blotting analysis and
expressed in arbitrary units. D: Representative confocal micrograph
image showing the presence of GHRHR (green) on cardiomyocyte
sarcolemmal membrane. Scale bar: 10 m.
[0020] FIG. 5. Bar graphs showing the expression of cells
positively stained for Ki.sub.67 (A) in the remote zone (*p<0.05
vs. placebo and GHRH-A). Panel B depicts a representative confocal
micrograph image of Ki.sub.67 positive cells (green, white arrows),
tropomyosin (red) and DAPI (blue). Scale bar: 20 m. Panel C: Bar
graphs showing the expression of TUNEL positive cells per unit area
(mm.sup.3).
[0021] FIG. 6. Representative images of c-kit.sup.+ cells in the
infarct zone observed under confocal microscopy. Panels depict
representative triple staining for mast cell tryptase (red), c-kit
(green) and nuclei (blue) obtained from placebo (top right), rrGH
(bottom left) and GHRH-A (bottom right). Arrows correspond to
examples of mast cell Scale bar: 20 m. Bar graph showing the high
expression of c-kit.sup.+ cells per unit area (mm.sup.3) in rrGH
and GHRH-A treated rats. (*p<0.05 vs. placebo).
[0022] FIG. 7. Changes over time in body weight (BW) after MI (A:
*p<0.05 vs. baseline; .dagger.p<0.01 vs. BSN and placebo at
week 4). Effects of 4-week treatment with rrGH or GHRH-A on heart
weight (HW) and the ratio HW/BW (B and C, respectively;
*p<0.0001 vs. placebo and GHRH-A). All values represent
means.+-.SEM.
[0023] FIG. 8. Serum concentration (ng/ml) of GH (A) and IGF-I (B)
measured after 4-week treatment with placebo, rrGH or GHRH-A. All
values represent means.+-.SEM. (*p<0.01 vs. placebo and
GHRH-A).
[0024] FIG. 9. Bar graphs showing capillary density (A) and myocyte
width (B) measurements (*p<0.001 vs. placebo and GHRH-A).
[0025] FIG. 10. Representative confocal micrograph of the
expression of GHRHR on cardiomyocytes from rats treated with
placebo, rrGH or GHRH-A. Scale bar: 50 .left brkt-top.m. Bar graphs
show the intensity of fluorescence (intensity/pixel). *p<0.01
vs. placebo and .dagger.p<0.0001 vs. GHRH-A.
[0026] FIG. 11. RT-PCR analysis of GHRH-R (a) and .beta.-actin (b)
in representative samples of rat heart tissues. A. Lanes 1, 2, 3
represent placebo samples and lanes 4, 5 and 6 represent rrGH (A)
treated samples. B. Lanes 1, 2, 3 represent placebo samples and
lanes 4, 5 and 6 represent JI-38 (B) treated samples. DNA molecular
weight marker is presented in lane M.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Material and Methods
[0027] Animal Model
[0028] MI induced by coronary artery ligation was performed in
female 6-month-old Fisher-344 rats as described previously (40).
Animals were randomly assigned to receive placebo, GHRH-agonist
(GHRH-A [JI-38], 50 .mu.g/kg) or rat recombinant GH (rrGH, 0.5
mg/kg) starting 2 hours post-surgery. All treatment was given
subcutaneously twice daily for 4 weeks. The Institutional Animal
Care and Use committee of University of Miami approved all
protocols and experimental procedures.
[0029] Drugs
[0030] Rat recombinant GH (rrGH) was supplied by Dr. A. F. Parlow
from National Hormone and Pituitary Program (NHPP) (UCLA-Harbor,
Torrance, Calif.) and GHRH-A (JI-38) ([Dat.sup.1 Gln.sup.8,
Orn.sup.12,21, Abu.sup.15, Nle.sup.27, Asp.sup.28,
Agm.sup.29]hGH-RH(1-29)NH.sub.2, the non-coded amino acids are
abbreviated as follows: Dat: desaminotyrosine, Orn: ornithine, Abu:
aminobutyric acid, Nle: norleucine, Agm: agmatine) was made in the
laboratory of one of us (AVS) (12, 13).
Results
[0031] As depicted in FIG. 7A, baseline body weight (BW) was
similar in all groups. In the placebo group, MI significantly
reduced BW from 225.+-.4 to 208.+-.3 g (p<0.05), an effect that
was fully prevented by administration of GHRH-A (from 231.+-.5 to
225.+-.3 g). Conversely, rrGH increased BW from 217.+-.4 to
256.+-.3 g (p<0.01). Heart weight (HW) was increased in concert
by rrGH (850.+-.38 mg) in comparison to placebo (674.+-.14 mg) or
GHRH-A (695.+-.26 mg) (p<0.0001 for both, FIG. 7B). Accordingly,
the HW/BW ratios (FIG. 7C) were similar in all groups.
[0032] Growth Hormone and Insulin-Like Growth Factor-I Levels
[0033] To test the impact of rrGH and GHRH-A on the GH-IGF-I axis
we measured circulating levels of these hormones (FIG. 8A-B).
Whereas treatment with GHRH-A did not increase serum levels of
either GH or IGF-I relative to placebo, treatment with rrGH led to
marked increases in GH (679.+-.196 vs. 64.+-.23 ng/ml, P<0.01)
and IGF-I (1052.+-.91 vs. 553.+-.46 ng/ml, p<0.01) compared to
placebo.
[0034] Echocardiographic Measurements
[0035] Next, we measured the impact of GHRH-A and rrGH on cardiac
structure and function following MI. Baseline echocardiography
documented similar parameters of LV dimension and function in all
groups (FIG. 1A-D, Table S1). As expected, MI led to a
time-dependent increase in LV chamber dimensions and a reduction in
ejection fraction (EF) and fraction shortening (FS). Treatment with
GHRH-A, but not with rrGH, attenuated the MI-induced increase in LV
end-systolic dimension (LVESD). In addition, the reduction in EF
due to MI was ameliorated by GHRH-A (47.+-.4% vs. 38.+-.3%,
p<0.05) but not by rrGH (44.+-.2%, p=NS), both compared to
placebo. Similarly, a reduction in FS from 57.+-.1 to 18.5.+-.0.9%
(p<0.05) due to MI was improved in the GHRH-A (28.7.+-.3.3%,
p<0.05) but not in the rrGH group (20.3.+-.1.3%, p=NS) both
compared to placebo.
[0036] Hemodynamic Measurements
[0037] To directly assess the impact of these interventions on
cardiac contractile performance and to separate the effects of
GHRH-A on cardiac contractility and cardiovascular loading
conditions, we performed in vivo hemodynamic analysis (Table 1,
FIG. 2). Treatment with GHRH-A but not rrGH caused an increase in
both stroke volume (SV) and cardiac output (CO) relative to
placebo. This increase in cardiac performance was attributed, at
least partially, to a reduction in ventricular afterload, measured
as arterial elastance (Ea). Interestingly, Ea was actually
increased with rrGH. LV end-systolic (LVESP) and end-diastolic
(LVEDP) pressures were similar in all groups. Consistent with the
echocardiographic data, EF was higher in GHRH-A than in placebo or
rrGH group. Similarly, stroke work (SW) was increased in GHRH-A
group vs. placebo or rrGH. With regard to myocardial contractility,
the peak rate of pressure rise (dP/dt.sub.max) was increased in the
GHRH-A group in comparison to placebo and rrGH groups, while there
were no significant difference in the peak rate of pressure decline
(dP/dt.sub.min) and the relaxation time constant (Tau); however,
treatment with GHRH-A trended to increase preload-recruitable
stroke work (PRSW) and the relationship between dP/dt.sub.max and
end-diastolic volume (EDV) (dP/dt.sub.max EDV). Conversely, the
ratio between arterial elastance (Ea) and end-systolic elastance
(Ees) trended to be lower in the GHRH-A group.
[0038] Histopathology
[0039] MI size (FIG. 3A) in rrGH and placebo groups was similar
(45.+-.2 vs. 41.+-.1%, respectively) while GHRH-A rats had reduced
MI size (36.+-.3%, p<0.05 vs. placebo and rrGH). The reduced
infarct burden was also reflected in the percentage of ventricular
fibrosis (FIG. 3B), which was strikingly reduced with GHRH-A
(20.+-.1%) in comparison to placebo (29.+-.1%) and rrGH
(27.+-.1%)(p<0.01 for both), whereas, capillary density (FIG.
9A) was higher in rrGH (0.02.+-.0.002/mm.sup.2) than in placebo or
GHRH-A groups (0.01.+-.0.001 and 0.006.+-.0.001/mm.sup.2,
respectively) (p<0.001 for both). The width of myocytes was not
different among groups (FIG. 9B).
[0040] GHRH Receptor
[0041] The presence or absence of GHRH-R was detected in frozen
sections of pituitary, heart and, skeletal muscle under fluorescent
and confocal microscopy (FIG. 4A) and the intensity of the
fluorescence of the GHRHR was measured in paraffin tissues of
treated and non-treated rats (FIG. 10). The expression of GHRH-R
was confirmed by Western blotting (FIG. 4B-C) and the GHRH-R was
also detected within cardiomyocytes (FIG. 4D). In addition, using
real-time polymerase chain reaction (rt-qPCR) we demonstrated the
presence of mRNA for GHRH receptor in rat heart (Tables S2-S3, FIG.
11A-B) and the radioligand binding studies revealed that the
ischemic rat heart samples showed specific high affinity binding
sites for GHRH antagonist, JV-1-42 ligand, characterized by a
K.sub.d of 0.86 nM and a B.sub.max of 51.28 fmol/mg protein.
[0042] Impact on Cellular Division and Proliferation
[0043] Immunostaining for Ki.sub.67 positive myocytes and
non-myocytes revealed no differences between the border and infarct
zones, however, in the remote zone, the expression of Ki.sub.67
positive cells was higher in the rrGH relative to placebo and
GHRH-A groups (p<0.01 for both) (FIG. 5A-B). Next we measured
the proliferation of endogenous c-kit.sup.+ cardiac precursor
cells. Importantly, the expression of c-kit.sup.+ cells (mast cells
excluded) per mm.sup.3 was higher (p=0.02) in both treated groups
than in placebo (FIG. 6).
[0044] TUNEL staining (FIG. 5C) did not show differences between
groups. On the other hand, rt-qPCR, revealed that the expression of
an anti-apoptotic gene (Bc12) was upregulated in GHRH-A (p=0.07),
while the pro-apoptotic gene (Bax) trended to be downregulated in
the same group (p=0.207). Accordingly, the ratio between Bax and
Bc12 expression was significantly reduced in the GHRH-A group in
comparison to placebo or rrGH treated rats (p=0.03).
TABLE-US-00001 TABLE 1 Hemodynamic parameters and indices of
systolic and diastolic function derived from pressure-volume
relationships. Placebo rrGH GHRH-A (8) (6) (8) Heart rate 256 .+-.
6.6 247 .+-. 6.8 270 .+-. 17.sup. (bpm) Integrated performance EF
(%) 29.8 .+-. 1.4 26.2 .+-. 1.7 .sup. 36.9 .+-. 2.6 * .dagger. SW
(mmHg .times. .mu.l) 9424 .+-. 1158 6920 .+-. 790 12000 .+-. 866 *
.dagger. SV (.mu.l) 131 .+-. 20 98 .+-. 13 161 .+-. 12 .dagger-dbl.
CO (ml/min) 30.5 .+-. 5.0 22.5 .+-. 3.4 .sup. 40.1 .+-. 3.1 *
.dagger. Ea/Ees 4.1 .+-. 0.7 4.4 .+-. 0.6 3.2 .+-. 0.3 Afterload
LVESP (mmHg) .sup. 85 .+-. 1.8 .sup. 91 .+-. 2.3 83 .+-. 1.1 Ea
(mmHg/.mu.l) 0.8 .+-. 0.1 1.1 .+-. 0.2 0.5 .+-. 0.004 * .dagger.
Preload LVEDP (mmHg) 9.8 .+-. 0.6 .sup. 10 .+-. 1.8 8 .+-. 0.5
LVEDV (.mu.l) 413 .+-. 56 351 .+-. 38 421 .+-. 26.sup.
Contractility dP/dt.sub.max 6198 .+-. 194 6243 .+-. 313 6986 .+-.
163 .dagger-dbl. (mmHg/s) dP/dt.sub.max.sub.--EDV 22.3 .+-. 8.1
17.5 .+-. 4.3 42.5 .+-. 12.9 (mmHg/s/.mu.l) Ees (mmHg/.mu.l) 0.26
.+-. 0.09 0.33 .+-. 0.12 0.19 .+-. 0.02 PRSW(mmHg) .sup. 45 .+-.
3.6 .sup. 48 .+-. 5.0 53 .+-. 2.1 Lusitropy dP/dtmin 3986 .+-. 177
4028 .+-. 334 3989 .+-. 106.sup. (mmHg/s) TAU(G)(ms) 16.8 .+-. 0.9
19.9 .+-. 0.4 16.9 .+-. 0.5.sup.
[0045] Ejection fraction (EF), stroke work (SW), stroke volume
(SV), cardiac output (CO), ratio between arterial elastance and
end-systolic elastance (Ea/Ees), left ventricular end-systolic
pressure (LVESP), arterial elastance (Ea), left ventricular
end-diastolic pressure (LVEDP), left ventricular end-diastolic
volume (LVEDV), peak rate of the pressure rise (dP/dtmax),
relationship between dP/dtmax and end-diastolic volume
(dP/dtmax_EDV), end-systolic elastance (Ees), preload recruitable
stroke work (PRSW), peak rate of pressure decline (dP/dtmin),
relaxation time constant calculated by Glantz method (TAU).
Supporting Information Material and Methods
[0046] Echocardiographic Measurements
[0047] Echocardiographic measurements were obtained at baseline, 2
days, 1, 2 and 4 weeks. Echocardiographic assessments were
performed in anesthetized rats (2% isoflurane inhalation) using a
Vevo-770 echocardiogram (Visual Sonics Inc., Toronto, Ontario,
Canada) equipped with a 17.5-MHz transducer. Cardiac dimensions: LV
end diastolic (LVEDD), end systolic (LVESD) diameters and
fractional shortening (FS) were recorded from M-mode images using
averaged measurements from 3 to 5 consecutive cardiac cycles
according to the American Society of Echocardiography (1). Ejection
fraction (EF) was calculated from bi-dimensional long-axis
parasternal views taken through the infarcted area. All images were
analyzed using Vevo 770 3.0.0 software (Visual Sonics Inc.,
Toronto, Ontario, Canada).
[0048] Hemodynamic Measurements
[0049] Rats were anesthetized by intramuscular injection of a
mixture of ketamine (100 mg/kg), xylazine (20 mg/kg) and
acepromazine (10 mg/kg). A 2-F micromanometer tipped catheter
(SPR-838, Millar Instruments, Houston, Tex.) was inserted into the
right carotid artery and advanced retrograde into the left
ventricle. Measurements were calibrated by injecting a hypertonic
saline (15%) bolus to determine extra-ventricular conductance;
relative volume units were converted to actual volume using the
cuvette calibration method (2). All analyses were performed using
PVAN 3.0 software (Millar Instruments, Houston, Tex.). Left
ventricular pressure-volume relations were assessed by transiently
compressing the inferior vena cava.
[0050] Tissue Collection
[0051] At the end of the study, rat hearts were harvested for
further analysis. Hearts were weighted and the basal portion, free
of fibrotic tissue, was flash-frozen in liquid nitrogen for total
RNA isolation and protein analysis. Remaining tissue was fixed with
10% formalin for histology.
[0052] Histology
[0053] Slides were prepared with H&E and Masson's trichrome
stain to assess cardiac structure and the presence and extent of
fibrosis and myocardial scar, respectively. The size of MI was
determined using NIH Image version 1.30v for Windows to quantify
the percentage area of fibrosis. An image-processing software
(Imaging Processing Toolkit 5.0, Reindeer Graphics, Asheville,
N.C.) and Adobe Photoshop CS2 (San Jose, Calif.) were used to
assess the slides as previously described with minor modifications
(3). The percentage of fibrosis was calculated by using the
following formula:
%fibrosis=fibrotic area/(fibrotic area+healthy area).
[0054] H&E stained sections of hearts from midventricular level
were used to measure the myocyte width. At least 35-50
cardiomyocytes were counted and averaged at the level of the nuclei
in non-infarcted remote myocardium.
[0055] Total RNA Isolation
[0056] Total RNA from heart tissue was extracted using Trizol
(Invitrogen, Carlsbad, Calif.). The quality of RNA isolated was
tested using NanoDrop1000 (Thermo Fisher Scientific Inc.,
Wilmington, Del.). OD 260/280 ratio was in the range of 1.8 to 2.1
for all samples.
[0057] Myocyte Isolation
[0058] The isolation of myocytes was performed as previously
described (4). Briefly; the rats were anesthetized with
pentobarbital (100 mg/Kg, Sigma, St. Louis, Mo.) with heparin (4000
U/Kg, APP Pharmaceuticals, Schaumburg, Ill.). For the isolation of
myocytes, the hearts were cannulated and perfused through the aorta
with Ca.sup.2+ free bicarbonate buffer containing 120 mM NaCl, 5.4
KCl, 1.2 mM MgSO.sub.4, 1.2 mM NaH.sub.2PO.sub.4, 20 mM
NaHCO.sub.3, 10 mM 2,3-butanedione monoxime, 5 mM taurine and, 5.6
mM glucose, gassed with 95% O2-5% CO2. This was followed by
enzymatic digestion with collagenase type-2 (1 mg/ml, Worthington
Biochemical Co., Lakewood, N.J.) and protease type-XIV (0.1 mg/ml,
Sigma, St. Louis, Mo.).
[0059] Cardiomyocytes were obtained from digested hearts followed
by mechanical disruption, by filtration, centrifugation, and
resuspension in a Tyrode solution containing 0.125 mM CaCl.sub.2,
144 mM NaCl, 1 mM MgCl.sub.2, 10 mM HEPES, 5.6 mM glucose, 1.2 mM
NaHPO.sub.4, 5 mM KCl, pH7.4.
[0060] GH and IGF-I Measurements
[0061] At the end of the study, blood was drawn 1-2 hours after the
last rrGH or GHRH-A injection and the serum was stored at
-80.degree. C. until the measurements were done. All the samples
were assayed together and each sample was assayed in duplicate. Rat
serum GH was measured using a rat GH Enzyme-Linked Immunosorbent
Assay (ELISA) Kit (DSL-10-72100, DSL Webster, Tex.), following the
manufacturer's recommendations. This test is an enzymatically
amplified "one-step" sandwich-type enzyme immunoassay, where
standards, controls and unknown samples are incubated in
microtitration wells precoated with the anti-rat GH antibody. The
standard curve of the assay was established with samples provided
by the manufacturer. Rat serum IGF-I was measured using a rat IGF-I
Radioimmunoassay Kit (DSL-2900, DSL Webster, Tex.), after
extraction with acid ethanol, following the manufacturer's
recommendations. The IGF-I assay included quality controls provided
by the manufacturer. The standard curve of the assay was
established with samples provided by the manufacturer.
[0062] GHRH Receptors
[0063] The expression of GHRHR was measured by immunofluorescence
and real time PCR. The detection of GHRHR protein was carried out
by Western blotting using the method of Schulz et al (45). The
binding affinity of GHRHR was demonstrated by radioligand binding
assay (see details in each section, respectively).
[0064] Immunostaining
[0065] Cardiomyocytes were stained as described. Briefly, after
isolation, 150 .mu.l of cardiomyocytes in suspension were allowed
to sediment and then fixed for 10 minutes (2% paraformaldehyde).
Cells were stained with rabbit polyclonal antibody against human
GHRH-R at 4.degree. C. for 24 hours followed by the secondary
antibody at 37.degree. C. for 1 hour (see table with a list of
antibodies in Supplemental Information). Frozen sections were used
for positive controls (pituitary) and negative controls (skeletal
muscle).
[0066] Paraffin sections were deparaffinized and rehydrated by
immersion in xylene and a graded series of ethanols. Antigen
retrieval was performed by a heat-induced method with citrate
buffer (Dako, Carpinteria, Calif.). After blocking with 10% normal
donkey serum, sections were incubated with a primary antibody
(table S3), at 37.degree. C. for 1 hour, followed by application of
secondary antibody. Omission of the primary antibodies on parallel
sections was used as negative control. Nuclei were counterstained
with DAPI (Invitrogen, Carlsbad, Calif.). The total numbers of
positively-stained cells were quantified per slide to calculate the
number of cells per unit volume (mm.sup.3) on each sample.
Morphometric analysis was performed by using Adobe Photoshop CS3
(San Jose, Calif.)
[0067] To quantify apoptosis of cardiac cells, terminal
deoxynucleotidyl transferase-mediated dUTP nick end-labeling
(TUNEL) staining was performed on paraffin embedded tissue sections
according to the manufacturer's protocol using a commercially
available kit (In Situ Cell Death Detection Kit, POD, Roche
Diagnostics GmbH, Germany). Slides were analyzed by fluorescent
microscopy under 40.times. magnifications. Apoptotic nuclei were
identified by green fluorescence staining and expressed as a
percentage per millimeter cubic (mm.sup.3) from tissue sections per
animal. All images were obtained with both fluorescent (Olympus
IX81, Olympus America Inc., Center Valley, Pa.) and a LSM710 Zeiss
confocal laser scanning module (Carl Zeiss MicroImaging GmbH,
Germany).
[0068] Quantification of Immunohistochemistry Staining for
GHRHR
[0069] All images were obtained using a 40.times. objective and the
settings were kept the same for the entire study. Ten high power
fields of confocal images were taken from each sample (n=3 for each
group) (FIG. S4). The quantification of the fluorescence intensity
was performed following deconvolution, using Huygen Essential
software, version 3.4 (Scientific Volume Imaging, Hilversum, The
Netherlands). An optical density plot of the selected area was
generated using the histogram tool in the Image Pro plus version
6.3 (Media Cybernetics, MD) and the mean staining intensity
(intensity/pixel) was recorded.
[0070] Real Time PCR
[0071] The expression of GHRHR was measured using real-time
quantitative PCR as described previously by Havt et al (46).
[0072] We evaluated the mRNA expression of rat GHRH receptor
(GHRH-R) and .beta.-actin. The probes designed to evaluate the
expression of GHRH-R and .beta.-actin are 5'-/Cy5/ACC TCC GAC TTT
CTC AGT TCC TGT ATG CCC/BHQ.sub.--2/-3' (SEQ ID NO:1) and
5'-/6-FAM/ATC CTG CGT CTG GAC CTG GCT GGC/BHQ.sub.--1/-3' (SEQ ID
NO: 2), respectively. Gene specific primer sequences were the
following: GHRH-R (sense) 5'-TCTGCTTTCTCTAGGTCCCTGT-3' (SEQ ID NO:
3) and 5'-TGGTTTCCCTGGGCCTTGG-3' (SEQ ID NO: 4) (antisense) with a
product size of 110 bp, .beta.-actin: (sense)
5'-GGGTTACGCGCTCCCTCAT-3' (SEQ ID NO: 5) and
5'-GTCACGCACGATTTCCCTCTC-3' (SEQ ID NO: 6) (antisense) with a
product size of 133 bp.
[0073] All real-time PCR reactions were performed in the iCycler
iQ.TM. Real-Time PCR Detection System (Bio-Rad, Hercules, Calif.).
Thermal cycling conditions comprised an initial denaturation step
at 95.degree. C. for 3 min followed by 45 cycles at 95.degree. C.
for 30 sec and an annealing temperature at 60.degree. C. for GHRH-R
and .beta.-actin for 1 min. As final steps, we included two cycles:
one at 95.degree. C. and the other at 60.degree. C., both for 1
min. All samples were run in triplicate and each well of PCR
reactions contained 25 .mu.L as final volume including 2 .mu.L of
cDNA, 200 nM of gene specific primers and 400 nM of probes. iQ.TM.
Supermix (Bio-Rad) was used in the PCR. The efficiencies of all
primers (Invitrogen Life Technologies, Carlsbad, Calif.) and probes
(Integrated DNA Technologies, Coralville, Iowa) were tested prior
to the experiments and they were all efficient in the range of
95-105%. Normal rat pituitary was used as positive control and rat
.beta.-actin as housekeeping gene. Negative samples were run in
each reaction consisting of no-RNA in reverse transcriptase
reaction and no-cDNA in PCR reaction. Two microliters of each
amplification reaction was electrophoretically separated on 1.5%
agarose gel, stained with SYBR.RTM. Green I (Lonza, Rockland, Me.),
and visualized under UV light.
[0074] The mathematical method described by Pfaffl (47) was used to
evaluate the relative expression ratio for GHRH-R compared with
.beta.-actin, with the efficiencies for each set. of real-time PCR
reactions and the threshold cycle (C.sub.T).
[0075] The monitoring of pro-apoptotic and anti-apoptotic genes was
also assessed by real time PCR. First-strand cDNA was synthesized
from 1 .mu.g total RNA using the High-Capacity cDNA
Reverse-Transcription Kit (Applied Biosystems, Inc., Foster City,
Calif., USA), and ribosomal 18S RNA served as the housekeeping
gene. We used TaqMan probes labeled with 6-carboxyfluorescein (FAM)
for real-time RT-PCR reactions, according to manufacturer's
protocol (Applied Biosystems, Inc., Foster City, Calif., USA). Data
were analyzed by the threshold cycle (Ct) relative quantification
method.
[0076] Western Blotting
[0077] The detection of GHRHR protein Immunoblot analysis was
performed as described. Equal amount of proteins (80 .mu.g) from
rat pituitary, brain, heart and liver for negative control were
resolved in 12% SDS-PAGE and incubated overnight with rabbit
polyclonal anti-human GHRHR antibody (Abcam, 1/1000) at 4.degree.
C.
[0078] Radioligand Binding Studies
[0079] Radioiodinated derivatives of GHRH antagonist JV-1-42 were
prepared by the chloramine-T method as described by Halmos et al
(48, 49). Preparation of membrane fractions from ischemic rat heart
samples was performed as reported by Halmos et al (48). Binding
characteristics of GHRH binding sites were determined by in vitro
ligand competition assays based on the binding of radiolabeled
JV-1-42 to heart membrane fractions. Binding affinity (K.sub.d) and
capacity (B.sub.max) were calculated by the Prism 4.0.1 (GraphPad
Software, Inc., La Jolla, Calif.).
[0080] Statistical Analysis
[0081] All values are shown as mean.+-.SEM. Echocardiographic
parameters during a 4-week follow-up were compared within and
between groups using one-way ANOVA for repeated measurements and
two-way ANOVA followed by post-hoc tests, respectively. For a given
parameter, p<0.05 was considered significant. All tests were
carried out using Sigma Stat 3.5 (Jandel, San Rafael, Calif.).
TABLE-US-00002 TABLE 2 Echocardiographic measurements at baseline
(BSL) and at 4 weeks (W4) post-MI. PLACEBO rrGH GHRH-A (10) (8) (8)
LVEDD (mm) BSL 6.1 .+-. 0.1 5.9 .+-. 0.1 6.3 .+-. 0.2 W4 7.9 .+-.
0.1 8.6 .+-. 0.1 * 7.7 .+-. 0.2 LVESD (mm) BSL 2.7 .+-. 0.1 2.3
.+-. 0.1 2.9 .+-. 0.1 W4 6.4 .+-. 0.1 6.8 .+-. 0.2 .dagger. .sup.
5.7 .+-. 0.3 .dagger-dbl. FS (%) BSL 55.5 .+-. 1.2 58.2 .+-. 0.7
53.1 .+-. 1.0 W4 18.5 .+-. 0.9 20.3 .+-. 1.3 28.7 .+-. 3.3 .sctn.
EF (%) BSL 84.5 .+-. 1.4 89.2 .+-. 0.4 85.7 .+-. 1.6 W4 38.3 .+-.
3.4 44.3 .+-. 2.3 47.2 .+-. 4.0 Left ventricle end-diastolic
diameter (LVEDD), left ventricle end-systolic diameter (LVESD),
fraction shortening (FS) and ejection fraction (EF).
TABLE-US-00003 TABLE 3 Real-Time PCR values of the relative
expression of mRNA for GHRH-R and .beta.-actin in rat heart tissue
samples after treatment with rrGH. Placebo rrGH Ct values Ct
values, mean_SEM, mean_SEM Ratio GHRH-R 28.74 .+-. 0.12 27.81 .+-.
0.05 2.10* .beta.-actin 17.87 .+-. 0.11 17.84 .+-. 0.12 -- The
ratio represents the gene expression level in the treatment group
as compared to the placebo group (*p < 0.05 versus placebo).
TABLE-US-00004 TABLE 4 Real-time PCR values of the relative
expression of mRNA for GHRH-R and .beta.-actin in rat heart tissue
samples after treatment with GHRH-A (JI-38). Placebo JI-38 Ct
values, Ct values, mean_SEM mean_SEM Ratio GHRH-R 32.38 .+-. 0.42
32.96 .+-. 0.18 .91 .beta.-actin 20.72 .+-. 0.61 22.33 .+-. 0.03 --
The ratio represents the gene expression level in the treatment
group as compared to the placebo group.
TABLE-US-00005 TABLE 5 Antibody list Protein Antibody Labeling
Fluorochromes GHRHR rabbit polyclonal indirect F c-kit goat
polyclonal indirect F Mast cell tryptase mouse polyclonal indirect
T Ki67 rabbit polyclonal indirect F Tropomyosin mouse polyclonal
indirect T Nuclear DNA DAPI N/A TUNEL Tdt/dUTP direct F Direct
labeling: primary antibody conjugated with the fluorochrome
Indirect labeling: species-specific secondary antibody with the
fluorochrome. F: fluorescein isothiocyanate, T: tetramethyl
rhodamine isothiocyanate, Cy5: cyanine 5
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Sequence CWU 1
1
6130DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 1acctccgact ttctcagttc ctgtatgccc
30224DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 2atcctgcgtc tggacctggc tggc 24322DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3tctgctttct ctaggtccct gt 22419DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 4tggtttccct gggccttgg
19519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5gggttacgcg ctccctcat 19621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6gtcacgcacg atttccctct c 21
* * * * *