Patent Application: US-91402310-A

Abstract:
whether the growth hormone / insulin - like growth factor 1 axis exerts cardioprotective effects remains controversial ; and the underlying mechanism for such actions are unclear . here we tested the hypothesis that growth - hormone releasing hormone directly activates cellular reparative mechanisms within the injured heart , in a gh / igf - i independent fashion . following experimental myocardial infarction , rats were randomly assigned to receive , during a 4 week period , either placebo , rat recombinant gh or ji - 38 , 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 . the receptor for ghrh was detectable on myocytes supporting direct activation of cardiac signal transduction . collectively , these findings demonstrate that within the heart ghrh - agonists can activate cardiac repair following mi , suggesting the existence of a potential signaling pathway based on ghrh in the heart . the phenotypic profile of the response to a potent ghrh agonist has therapeutic implications .

Description:
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 μg / 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 . 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 1 , gln 8 , orn 12 , 21 , abu 15 , nle 27 , asp 28 , agm 29 ] hgh - rh ( 1 - 29 ) nh 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 ). as depicted in fig . s1a , 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 & lt ; 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 & lt ; 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 & lt ; 0 . 0001 for both , fig . s1b ). accordingly , the hw / bw ratios ( fig . s1c ) were similar in all groups . to test the impact of rrgh and ghrh - a on the gh - igf - i axis we measured circulating levels of these hormones ( fig . s2a - 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 & lt ; 0 . 01 ) and igf - i ( 1052 ± 91 vs . 553 ± 46 ng / ml , p & lt ; 0 . 01 ) compared to placebo . 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 ( fig1 a - 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 & lt ; 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 & lt ; 0 . 05 ) due to mi was improved in the ghrh - a ( 28 . 7 ± 3 . 3 %, p & lt ; 0 . 05 ) but not in the rrgh group ( 20 . 3 ± 1 . 3 %, p = ns ) both compared to placebo . 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 , fig2 ). 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 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 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 max and end - diastolic volume ( edv ) ( dp / dt max — edv ). conversely , the ratio between arterial elastance ( ea ) and end - systolic elastance ( ees ) trended to be lower in the ghrh - a group . mi size ( fig3 a ) in rrgh and placebo groups was similar ( 45 ± 2 vs . 41 ± 1 %, respectively ) while ghrh - a rats had reduced mi size ( 36 ± 3 %, p & lt ; 0 . 05 vs . placebo and rrgh ). the reduced infarct burden was also reflected in the percentage of ventricular fibrosis ( fig3 b ), which was strikingly reduced with ghrh - a ( 20 ± 1 %) in comparison to placebo ( 29 ± 1 %) and rrgh ( 27 ± 1 %)( p & lt ; 0 . 01 for both ), whereas , capillary density ( fig . s3a ) was higher in rrgh ( 0 . 02 ± 0 . 002 / mm 2 ) than in placebo or ghrh - a groups ( 0 . 01 ± 0 . 001 and 0 . 006 ± 0 . 001 / mm 2 , respectively ) ( p & lt ; 0 . 001 for both ). the width of myocytes was not different among groups ( fig . s3b ). the presence or absence of ghrh - r was detected in frozen sections of pituitary , heart and , skeletal muscle under fluorescent and confocal microscopy ( fig4 a ) and the intensity of the fluorescence of the ghrhr was measured in paraffin tissues of treated and non - treated rats ( fig . s4 ). the expression of ghrh - r was confirmed by western blotting ( fig4 b - c ) and the ghrh - r was also detected within cardiomyocytes ( fig4 d ). 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 . s5a - 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 d of 0 . 86 nm and a b max of 51 . 28 fmol / mg protein . immunostaining for ki 67 positive myocytes and non - myocytes revealed no differences between the border and infarct zones , however , in the remote zone , the expression of ki 67 positive cells was higher in the rrgh relative to placebo and ghrh - a groups ( p & lt ; 0 . 01 for both ) ( fig5 a - b ). next we measured the proliferation of endogenous c - kit + cardiac precursor cells . importantly , the expression of c - kit + cells ( mast cells excluded ) per mm 3 was higher ( p = 0 . 02 ) in both treated groups than in placebo ( fig6 ). tunel staining ( fig5 c ) did not show differences between groups . on the other hand , rt - qpcr , revealed that the expression of an anti - apoptotic gene ( bcl2 ) 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 bcl2 expression was significantly reduced in the ghrh - a group in comparison to placebo or rrgh treated rats ( p = 0 . 03 ). 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 / dt max ), relationship between dp / dt max and end - diastolic volume ( dp / dt max — edv ), end - systolic elastance ( ees ), preload recruitable stroke work ( prsw ), peak rate of pressure decline ( dp / dt min ), relaxation time constant calculated by glantz method ( tau ). 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 ). 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 . 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 . slides were prepared with h & amp ; e and masson &# 39 ; 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 : h & amp ; 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 . 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 . 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 2 + free bicarbonate buffer containing 120 mm nacl , 5 . 4 kcl , 1 . 2 mm mgso 4 , 1 . 2 mm nah 2 po 4 , 20 mm nahco 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 .). cardiomyocytes were obtained from digested hearts followed by mechanical disruption , by filtration , centrifugation , and resuspension in a tyrode solution containing 0 . 125 mm cacl 2 , 144 mm nacl , 1 mm mgcl 2 , 10 mm hepes , 5 . 6 mm glucose , 1 . 2 mm nahpo 4 , 5 mm kcl , ph7 . 4 . 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 ° 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 &# 39 ; 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 &# 39 ; 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 . 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 ( 5 ). the binding affinity of ghrhr was demonstrated by radioligand binding assay ( see details in each section , respectively ). cardiomyocytes were stained as described . briefly , after isolation , 150 μl 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 ° c . for 24 hours followed by the secondary antibody at 37 ° 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 ). 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 ° 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 3 ) on each sample . morphometric analysis was performed by using adobe photoshop cs3 ( san jose , calif .) 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 &# 39 ; 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 × magnifications . apoptotic nuclei were identified by green fluorescence staining and expressed as a percentage per millimeter cubic ( mm 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 microlmaging gmbh , germany ). all images were obtained using a 40 × 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 ) ( figure 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 . the expression of ghrhr was measured using real - time quantitative pcr as described previously by havt et al ( 6 ). we evaluated the mrna expression of rat ghrh receptor ( ghrh - r ) and β - actin . the probes designed to evaluate the expression of ghrh - r and β - actin are 5 ′-/ cy5 / acc tcc gac ttt ctc agt tcc tgt atg ccc / bhq — 2 /- 3 ′ and 5 ′-/ 6 - fam / atc ctg cgt ctg gac ctg gct ggc / bhq — 1 /- 3 ′, respectively . gene specific primer sequences were the following : ghrh - r ( sense ) 5 ′- tctgctttctctaggtccctgt - 3 ′ and 5 ′- tggtttccctgggccttgg - 3 ′ ( antisense ) with a product size of 110 bp , β - actin : ( sense ) 5 ′- gggttacgcgctccctcat - 3 ′ and 5 ′- gtcacgcacgatttccctctc - 3 ′ ( antisense ) with a product size of 133 bp . all real - time pcr reactions were performed in the icycler iq ™ real - time pcr detection system ( bio - rad , hercules , calif .). thermal cycling conditions comprised an initial denaturation step at 95 ° c . for 3 min followed by 45 cycles at 95 ° c . for 30 sec and an annealing temperature at 60 ° c . for ghrh - r and β - actin for 1 min . as final steps , we included two cycles : one at 95 ° c . and the other at 60 ° c ., both for 1 min . all samples were run in triplicate and each well of pcr reactions contained 25 μl as final volume including 2 μl of cdna , 200 nm of gene specific primers and 400 nm of probes . iq ™ 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 β - 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 ® green i ( lonza , rockland , me . ), and visualized under uv light . the mathematical method described by pfaffl ( 7 ) was used to evaluate the relative expression ratio for ghrh - r compared with β - actin , with the efficiencies for each set of real - time pcr reactions and the threshold cycle ( c t ). the monitoring of pro - apoptotic and anti - apoptotic genes was also assessed by real time pcr . first - strand cdna was synthesized from 1 μg 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 &# 39 ; s protocol ( applied biosystems , inc ., foster city , calif ., usa ). data were analyzed by the threshold cycle ( ct ) relative quantification method . the detection of ghrhr protein immunoblot analysis was performed as described . equal amount of proteins ( 80 μg ) 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 ° c . radioiodinated derivatives of ghrh antagonist jv - 1 - 42 were prepared by the chloramine - t method as described by halmos et al ( 8 , 9 ). preparation of membrane fractions from ischemic rat heart samples was performed as reported by halmos et al ( 8 ). 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 d ) and capacity ( b max ) were calculated by the prism 4 . 0 . 1 ( graphpad software , inc ., la jolla , calif .). 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 & lt ; 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