lysosomal integral membrane protein - 2 is a novel component of intercalated discs and prevents cardiomyopathy from 10 - week - old ren - 2 and sprague - dawley ( sd ) rats ( mollegard , lille skensveld , denmark ), a biopsy of the lv was taken as described previously ( van haaften et al ., 2006 ). rats were followed by serial echocardiography at 10 , 12 , 15 , 16 , 18 , 19 and 21 weeks of age and sacrificed at 15 - 18 weeks upon clinical signs of heart failure ( heart failure - prone / hf - prone rats ) or at 21 weeks when clinical signs of failure had not appeared ( compensated / comp rats ). total rna was isolated from lv biopsies and amplified as previously described ( schroen et al ., 2004 ; heymans et al ., 2005 ), hybridized to affymetrix rat 230 2 . 0 genechips and analyzed with microarray analysis suite software 5 . 0 . lv protein extracts ( 50 μg ) were immunoblotted with polyclonal rabbit anti - limp - 2 ( novus biologicals , littleton , colo ., 1 : 500 ) and polyclonal rabbit anti - gapdh ( abcam , leusden , netherlands ; 1 : 10 , 000 ). ten - to twelve - week - old male limp - 2 ko and wt c57 / b16 mice weighing 20 - 25 grams were used . to study blood pressure effects of angii , arterial pressures were monitored during intravenous infusions at doses of 0 . 5 , 1 . 5 , 5 , 15 , and 50 ng per minute . to study development of lv hypertrophy , angii ( 1 . 5 μg / g / day ) was infused subcutaneously by osmotic minipump 2004 ( alzet osmotic pumps , cupertino , calif .) for 28 days . echocardiography was performed at day 0 , day 14 and day 28 . at day 28 , mice were hemodynamically monitored ( dp / dt ) using millar ® under basal and dobutamin - stimulated conditions ; afterward , lvs were removed . rna was isolated with rneasy mini kit ( qiagen , valencia , calif .) and sybr green quantitative pcr analysis was performed on a biorad icycler to determine bnp , anf and alpha - skeletal actin ( aska ) expression ( table 1 ). lv sections were stained with hematoxylin - eosin ( he ) and picro serious red ( sr ) as described before ( junqueira et al ., 1979 ), or were immunohistochemically stained with monoclonal mouse anti - pan - cadherin ( sigma , saint louis , usa ; 1 : 500 ) and monoclonal mouse anti - human desmin ( dako cytomation , denmark , 1 : 50 ). ultrastructural analysis was performed by transmission electron microscopy as described previously ( schroen et al ., 2004 ). rna was isolated from transmural biopsies obtained from 20 aortic stenosis patients and seven non - hypertrophic control patients as described before ( heymans et al ., 2005 ), and sybr green quantitative pcr analysis was performed to determine limp - 2 expression ( table 1 ). double immunofluorescent stainings with rabbit anti - limp - 2 ( 1 : 250 , cy2 ) and mouse anti - pan - cadherin ( 1 : 500 , cy3 ) were done on sections of one control subject and two patients that died of overt heart failure , as defined by an ejection fraction of less than 35 %. nuclear counterparts were stained with topro - 3 ( invitrogen , breda , the netherlands ). sections were imaged with a laser scanning confocal system ( leica , rijswijk , the netherlands ), digitized at a final magnification of × 126 and analyzed with leica confocal software . the ethics committees of the academic hospital maastricht and of university hospital leuven approved the study , and all patients gave informed consent . a rat - limp - 2 shrna expressing lentiviral vector was generated by annealing complementary shlimp - 2 oligonucleotides ( table 1 ) and ligating them into hpai xhoi digested pll3 . 7 puro vector dna ( modified from a kind donation by luk van parijs , massachusetts institute of technology , cambridge , usa ). lentiviral production was performed by co - transfection of 3 μg shlimp - 2 / pll3 . 7 puro or empty pll3 . 7puro and packaging vectors into 293ft cells by lipofectamine 2000 ( invitrogen ) and virus - containing supernatant was harvested after 48 hours . rat ventricular cardiac myocytes ( rcms ) were isolated by enzymatic disassociation of 1 - to 2 - day - old neonatal rats as described previously ( de windt et al ., 1997 ). for lentiviral infection , rcms were plated on gelatinized six - well plates with 5 * 10 5 cells per well , cultured overnight in dmem / m199 ( 4 : 1 ) media supplemented with 10 % horse serum , 5 % newborn calf serum , glucose , gentamycin and arac , and next day infected with shlimp - 2 or empty lentivirus , facilitated by polybrene ( sigma ). after puromycin selection ( 3 μg / ml ), infection efficiencies were above 80 %. after ten culture days , cellular protein was isolated for immunoprecipitation ( ip ) with anti - limp - 2 ( 1 : 100 ), monoclonal mouse anti - pan - cadherin ( sigma , 1 : 100 ) or igg . ip lysates were immunoblotted with monoclonal anti - pan - cadherin ( 1 : 5000 ), polyclonal anti - phospho - beta - catenin ( ser33 / 37 / thr41 ; cell signaling technology , danvers , mass ., usa , 1 : 1000 ) and monoclonal anti - beta - catenin ( bd transduction laboratories , franklin lakes , usa , 1 : 1000 ). for stretch experiments , rcms were cultured on a collagen type i - coated silastic membrane ( specialty manufacturing , inc ., usa ) and subjected to static , equibiaxial stretch during a six - hour period . rna was isolated with rneasy mini kit ( qiagen ) for limp - 2 sybr green quantitative rt - pcr ( table 1 ). all study protocols involving animal experiments were approved by the animal care and use committee of the universiteit maastricht , and were performed according to the official rules formulated in the dutch law on care and use of experimental animals , highly similar to those of the nih . data are presented as average ± sem . the data for each study group were compared using mann whitney or student &# 39 ; s t - test where appropriate . p & lt ; 0 . 05 was considered to be statistically significant . in table 2 , a list is presented of genes differentially expressed in failure - prone as compared to compensated ren - 2 rats . the differential expression of these genes precedes the development of heart failure in ren - 2 rats , because it is derived from cardiac biopsies taken at 10 weeks of age , when all rats still have compensatory hypertrophy . in table 3 , elaborate echocardiographic data are presented of limp - 2 wt and ko mice at baseline , and after 14 and 28 days of angii treatment . cardiac biopsies in ten homozygous ren - 2 rats were obtained at a stage of compensated lv hypertrophy at 10 weeks of age . four rats rapidly progressed toward heart failure within five weeks after the biopsy was taken , while the remaining six rats remained compensated for 11 weeks after biopsy ( fig1 a ). after linear t7 based amplification and subsequent affymetrix 230 2 . 0 gene expression analysis in these biopsies ( geo number gse4286 ), 143 differentially expressed genes that were up - or down - regulated only in the hypertrophied hearts that progressed towards heart failure were identified ( table 2 ). limp - 2 , a lysosomal membrane protein , was one of the up - regulated mrnas in heart failure - prone rats ( fig1 b ), and of particular interest given its ability to interact with thrombospondins ( tsp ) 1 ( crombie et al ., 1998 ) and 2 ( data not shown ), the latter has been shown earlier to be crucial in the transition from hypertrophy towards heart failure . fig1 c shows that limp - 2 protein also has a role in end - stage heart failure in ren - 2 rats . since loss - of - function mutations in lysosomal proteins have been linked to heart failure ( eskelinen et al ., 2003 ; nishino et al ., 2000 ; stypmann et al ., 2002 ), the role of limp - 2 in a mouse model of angiotensin ii - ( angii -) induced hypertension was further investigated . angii was given subcutaneously for four weeks to limp - 2 knockout and control mice . angii treatment resulted in a 30 % increase in lv mass index in wild - type mice , but the hypertrophic response was attenuated in the angii - treated limp - 2 knockout mice ( 14 % increase in lv mass index ; p & lt ; 0 . 01 ) ( fig2 a ). this was confirmed by measurement of individual cardiac myocyte area . lv myocyte area was significantly smaller in angii - treated knockout mice than in angii - treated wt controls ( myocyte area in arbitrary units : 264 ± 42 in angii - treated knockout mice , versus 308 ± 14 in angii - treated wild - types ; p & lt ; 0 . 01 ). in addition , while angii induced comparable increases in perivascular fibrosis in limp - 2 knockout and wild - type mice ( data not shown ), angii induced a massive interstitial fibrotic response in the lv of limp - 2 knockout mice as opposed to wild - type control littermates ( interstitial fibrosis : 15 . 0 ± 6 . 0 % in angii - treated knockout mice versus 1 . 8 ± 0 . 1 % in angii - treated controls ; p & lt ; 0 . 002 ) ( fig3 ). immunohistochemical staining for desmin showed myocyte disarray in angii - treated limp - 2 null mice ( fig4 ). it was confirmed that angii induced a similar blood pressure response in both wild - type and knockout mice ( fig2 b ). despite decreased lv hypertrophy , limp - 2 null mice demonstrated a normal response of the classical markers for hypertrophy brain natriuretic peptide ( bnp ) and atrial natriuretic factor ( anf ) ( fig2 c ), suggesting that the hypertrophic gene expression program was normally initiated upon angii treatment . in contrast , the structural cellular hypertrophy marker alpha - skeletal actin was induced to a significantly lesser extent in angii - treated limp - 2 knockouts as compared to angii - treated wild - types ( fig2 c ), reflecting the reduced hypertrophic response of cardiac myocytes ( stilli et al ., 2006 ). serial echocardiography revealed that angii induced significant cardiac dilatation in angii - treated limp - 2 null mice , whereas angii - treated wild - types showed concentric lv hypertrophy without dilatation ( fig2 d and table 3 ). in addition , angii induced loss of contractile reserve in limp - 2 null mice as demonstrated by a reduced contractile response to dobutamine infusion (+ dp / dt 79 . 8 / second ± 5 . 1 in angii - treated knockout mice versus 100 . 0 / second ± 4 . 0 in angii - treated controls ; p & lt ; 0 . 005 ) ( fig2 e ). taken together , in limp - 2 null mice hypertension did not induce the normal hypertrophic response but rather dilated cardiomyopathy with reactive interstitial fibrosis and loss of cardiac function . the finding that angii - treated limp - 2 null mice failed to mount a hypertrophic response , yet normally induced expression of bnp and anf suggested that limp - 2 is a crucial part of the normal response to mechanical loading . indeed , it was also shown that limp - 2 expression increased significantly after cardiac myocyte stretch in vitro ( p = 0 . 02 ) and also increased in exercise - induced physiological hypertrophy ( p = 0 . 04 ) ( fig5 a and 5b ). to ascertain that limp - 2 is also involved in the human adaptation to cardiac pressure loading , the expression of limp - 2 was analyzed by quantitative rt - pcr in cardiac biopsies of twenty aortic stenosis patients with overt cardiac hypertrophy and seven controls . this experiment showed a significant limp - 2 up - regulation in the hypertrophic hearts of aortic stenosis patients as compared to controls by mann - whitney test ( 1 . 23 - fold ; p = 2 . 3e − 4 ). next , the expression pattern of limp - 2 in pressure - overloaded murine myocardium was analyzed by immunohistochemistry . the protein is expressed , as expected , in intracellular vacuole - shaped compartments of cardiac myocytes and endothelial cells , but was also found to be atypically distributed on the plasma membrane of cardiac myocytes ( fig6 a ). immuno - electron microscopy confirmed this finding ( fig6 b ). strikingly , electron - microscopy of angii - treated limp - 2 knockout and control left ventricular sections revealed abnormal morphology of the id in limp - 2 null mice , suggesting that limp - 2 may be involved in normal id biology . at cell - cell contacts , the membrane at the angii - treated ko - id showed a higher degree of convolution with a higher concentration of adherens junction proteins ( fig6 c ), indicative of disturbed id architecture ( perriard et al ., 2003 ). since alterations in the id have been shown to cause dilated cardiomyopathy ( perriard et al ., 2003 ), it was surmised that limp - 2 may be crucial for proper functioning of the id . immunoprecipitation of neonatal rat cardiac myocytes protein showed that limp - 2 physically interacts with n - cadherin , a vital constituent of adherens junctions ( fig7 a ). this finding was translated to the human situation as confocal microscopy of control as well as failing human myocardium confirmed the interaction between cadherin and limp - 2 and showed that this interaction takes place at the site of the id , where cadherin and limp - 2 co - localize ( fig7 b ). this suggested that limp - 2 may be important for proper id function by mediating the role of cadherin . indeed , histochemical analysis of cadherin in hearts of limp - 2 null mice showed aberrant cadherin distribution ( fig7 c ), but normal distribution in angii - treated wild - types . angii - treated wild - type mice show cadherin expression at the contact sites between two longitudinal cardiac myocytes , while this expression is less organized and more diffuse in cardiac myocytes of angii - treated limp - 2 knockout mice . these data establish that limp - 2 is crucial for the proper structural organization of the intercalated disc . to identify which regulatory mechanism depends on limp - 2 , lentivirally introduced short - hairpin rna against limp - 2 ( shlimp - 2 ) was used to obtain a separate model of limp - 2 inactivation in neonatal rat cardiac myocytes . after ten days of culture , limp - 2 protein expression was diminished by 92 % in shlimp - 2 - treated cardiac myocytes as compared to control - treated cardiac myocytes ( fig8 a ). it has been reported that the functional integrity of the intercalated disc depends on the proper interaction between p ( ser37 )- β - catenin and cadherin . therefore , it was investigated whether the absence of limp - 2 affected the binding of p - β - catenin to cadherin . immunoprecipitation of cadherin in lysates of cardiac myocytes showed that knock - down of limp - 2 indeed diminished the interaction between p - β - catenin and cadherin ( fig8 b ). immunoprecipitation was specific for cadherin ( fig8 c ). it was demonstrated in this study that the lysosomal protein limp - 2 is an important and novel component of the cardiac myocyte intercalated disc , in particular , adherens junctions . according to the present invention , it has been shown that limp - 2 binds to n - cadherin , and that limp - 2 null mice develop dilated cardiomyopathy upon angii - induced hypertension , accompanied by disturbed localization of n - cadherin in the heart . confirming this in vitro , it was shown that knock - down of limp - 2 in cultured myocytes disturbs interactions between n - cadherin and β - catenin . this suggests that limp - 2 , which was initially known as a lysosomal protein , is an important part of the intercalated disc . limp - 2 stands out among id proteins . complete loss of other major constituents of the id ( cadherin , β - catenin , plakoglobin ) results in lethal developmental cardiac derangements , suggesting that these components of the id are essential for normal cardiac development . in contrast , according to the invention , it was found that limp - 2 null mice have normal cardiac development , but that its loss only affects postnatal cardiac remodeling . this suggests that limp - 2 represents a different type of id protein , whose role is essential mainly under increased loading conditions . this specific role for limp - 2 is underlined by the finding that expression of limp - 2 further rises in hypertrophied rat hearts that are on the brink to progress to failure , which suggests that limp - 2 expression particularly increases in cardiac myocytes that seem unable to normalize loading conditions . taken together , it was suggested that limp - 2 is a novel mediator of id function , and represents a hitherto unidentified class of mediators that are essential for the id and the myocyte to respond to increased loading conditions . limp - 2 increased particularly in those hypertrophied hearts that would later progress to failure , in comparison to the hypertrophied hearts that remained compensated . this indicates that cardiac limp - 2 expression may be an early molecular sign of excessive loading . that limp - 2 constitutes a defensive mechanism against excessive loading is suggested by the finding that when limp - 2 null mice were subjected to pressure loading by chronic angiotensin ii infusion , they developed cardiac dilatation and fibrosis , yet very little cardiac myocyte hypertrophy . natriuretic peptides were normally induced , which suggests that the cardiac myocytes of limp - 2 null mice do sense increased loading conditions , yet fail to mount an adequate hypertrophic response , as evidenced by the attenuated expression of alpha - skeletal actin . this suggests that limp - 2 is essential for a normal response to cardiac loading , and that limp - 2 expression is strongly increased when loading conditions exceed compensatory mechanisms . these findings were translated to the human situation , which showed that limp - 2 is also robustly increased in patients with clinically severe pressure loading . taken together , limp - 2 is a novel constituent of the id and seems to represent a novel type of id protein , essential for the response to loading rather than for normal cardiac function . intercalated disc abnormalities were documented in pressure - loaded limp - 2 null mice , characteristic of a disturbed cardiac intercalated disc , which normally acts to organize adjoining myocytes . remodeling of the intercalated disc has been shown previously during the transition from compensated lv hypertrophy towards heart failure , while structural perturbations of the intercalated disc have been linked to dilated cardiomyopathy in humans , hamsters and pigs . it was shown that limp - 2 binds n - cadherin , suggesting a role for limp - 2 via this id constituent . indeed , pressure - overloaded limp - 2 ko mice show abnormal intercalated discs on electron microscopy and their n - cadherin distribution is disturbed , suggesting a defect in the adherens junctions . the strength of adherens junctions is determined by the binding affinity between n - cadherin and β - catenin ( gumbiner et al ., 2000 ), which is regulated by phosphorylation of the latter . it was shown in vitro by knock down of limp - 2 in cultured myocytes , that loss of limp - 2 disturbs this n - cadherin / β - catenin complex . given the linkage of adherens junctions to myofibrils , a loss of limp - 2 is expected to lead to less efficient force transduction across the plasma membrane ( ferreira et al ., 2002 ). it has been suggested that limp - 2 is essential for the proper binding of n - cadherin to β - catenin , and that this role is particularly important under loading conditions . however , the precise way by which limp - 2 assures binding of n - cadherin to β - catenin remains to be elucidated . limp - 2 contains two transmembrane domains , a cytoplasmic loop and two luminal glycosylated domains . it is known that lysosomal membrane proteins can shuttle between lysosomal and plasma membranes , where limp - 2 can bind to tsp1 and tsp2 ( data not shown ). the latter is intriguing , as it has been documented earlier that tsp2 is also essential for the response to cardiac pressure loading and increases in failure - prone forms of lv hypertrophy ( schroen et al ., 2004 ). this suggests that both limp - 2 and tsp2 may be part of a complex that is needed for the cardiac myocyte to mount an adaptive response to loading . according to the present invention , a novel role for limp2 has been uncovered as an important mediator of the id when the myocardium faces increased loading conditions . apart from this novel biological insight , the finding that expression of limp - 2 rises in hypertrophied rat hearts that are on the brink to progress to failure , makes it tempting to speculate that increased limp - 2 expression by cardiac myocytes demonstrates their inability to normalize loading conditions . as such , increased limp - 2 expression may signify imminent failure . since it has been shown that limp - 2 expression is also robustly increased in patients with clinically severe pressure loading , and is located at the plasma membrane , limp - 2 may be an attractive target for molecular imaging to identify already in a very early stage , the myocardium that is about to succumb to the pressure . tgf - beta promotes cardiac hypertrophy by suppressing krüppel - like factor 15 , a novel inhibitor of cardiac hypertrophy eighteen male homozygous ren - 2 rats and five age - matched sprague - dawley ( sd ) ( mollegard breeding center , lille skensveld , denmark ) were studied . three ren - 2 rats were sacrificed at 10 to 12 weeks of age upon clinical signs of heart failure and excluded from the study . from the remaining healthy 15 ren - 2 rats and five sd controls , a biopsy of the left ventricle was taken at 10 weeks of age , as described previously . rats were followed by serial echocardiography at 10 , 12 , 15 , 16 , 18 , 19 and 21 weeks of age as described above . nine ren - 2 rats were sacrificed at 15 to 18 weeks of age upon clinical signs of heart failure and designated “ heart failure - prone ” rats . the remaining six ren - 2 rats were monitored and sacrificed at 21 weeks when clinical signs of failure had not appeared , and they were designated “ compensated ” rats . total rna was isolated and amplified as previously described from lv biopsies taken at 10 weeks of age of four sd controls , of six rats that remained compensated and of four heart failure - prone rats . amplified crna was then hybridized to affymetrix rat 230 2 . 0 genechips . gene transcript levels of sd controls , compensated and hf rats were determined with microarray analysis suite software version 5 . 0 ( mas5 . 0 ). lentiviral vectors were generated by annealing complementary shklf - 15 oligonucleotides ( sense 5 ′- gatgtacaccaagagcagc - 3 ′ ( seq id no : 1 ) and antisense 5 ′- gctgctcttggtgtacat - 3 ′ ( seq id no : 2 )) and cloning them into digested pll3 . 7 puro vector dna ( kindly donated by luk van parijs , department of biology , massachusetts institute of technology , cambridge , usa ) using e . coli dh5α - competent cells . constructs were purified using qiagen plasmid midi kit . 293ft cells were cultured in dmem with 10 % fcs , 2 mm l - glut , 10 mm non - essential amino acids , 1 mm sodium pyruvate and pen / strep antibiotics . lentiviral production was performed by co - transfection of 3 μg shklf15 or shtgfb1 / pll3 . 7 puro or empty pll3 . 7 puro and packaging vectors into 293ft cells by lipofectamine 2000 ( invitrogen life technology , breda , the netherlands ) and virus containing supernatant was harvested , filtrated and snap - frozen after 48 hours . neonatal rat ventricular myocytes ( nrvm ) were isolated by enzymatic disassociation of one - to three - day - old neonatal rat hearts as previously described ( schroen et al ., 2004 ). nrvms were cultured in dmem / m199 ( 4 : 1 ) media supplemented with 10 % horse serum ( hs ), 5 % newborn calf serum ( nbcs ), glucose , gentamycin and 2 % antibiotic / antimycotic on a gelatinized six - well plate with 5 * 10 5 cells per well . for shklf15 infection , nrvm were cultured overnight and the next day infected with shklf15 and an empty lentiviral control vector , facilitated by polybrene ( sigma ). after 48 hours , cells were washed free of vector and placed under puromycin selection for another 48 hours . then , cells were kept under quiescent conditions overnight in dmem / m199 ( 4 : 1 ), glucose , gentamycin and 10 % antibiotic / antimycotic . the next day , medium was replaced by medium containing dmem / m199 , glucose , gentamycin , 5 % antibiotic / antimycotic , insulin , l - carnitin and bsa . after one hour , tgf - b ( 10 ng / ml medium ) was added for 1 hour and whereafter rna was isolated using the rneasy mini protocol ( qiagen ) for sybr green quantitative pcr with klf15 or bnp primers ( f 5 ′- gct gct ttg ggc aga aga tag a - 3 ′ ( seq id no : 3 ) or r 5 ′- gcc agg agg tct tcc taa aac a - 3 ′ ( seq id no : 4 ). knock - down efficiency of shklf15 is about 80 % compared to levels in nrvm infected with an empty lentiviral vector . nrvm were isolated as described above . cells were cultured in dmem / m199 ( 4 : 1 ) media supplemented with 10 % horse serum ( hs ), 5 % newborn calf serum ( nbcs ), glucose , gentamycin and 2 % antibiotic / antimycotic on a gelatinized six - well plate with 5 * 10 5 cells per well . for shklf15 infection , nrvm were cultured overnight and the next day infected with shklf15 and an empty lentiviral control vector , facilitated by polybrene ( sigma ). after 48 hours , cells were washed free of vector and placed under puromycin selection for another 48 hours . a mef2 reporter plasmid ( pgl2 - 3 × mef2 - luciferase ) containing three mef2 binding sites cloned upstream of the tata - box and luciferase in the cells via transient transfection . cells were washed and per well , 1 . 6 μg of the mef2 construct was added together with opti - mem i media ( invitrogen ) and lipofectamine 2000 , and antibiotics free media . the next morning , cells were washed and placed under normal culture media for another two days . cells were kept overnight under low serum conditions ( see above ) and the next morning angii ( x grams / nil ) was added for 4 hours . the luciferase assay was performed using the luciferase assay protocol ( promega ). tgfβri f / f mice ( c57b1 / 6 background ) generated by flanking exon 3 of tgfβri with lox - p site ( sohal et al ., 2001 ) were crossed with mice ( c57b1 / 6 fvb background ) containing cre - recombinase under the control of α - mhc promoter ( mercremer cre / wt ( larsson et al .) to generate heterozygous double transgenic mice that contained tgfβri fl / wtcre genes . these mice were then back - crossed with tgfβri f / f mice resulting in a colony with tgfβri f / cre and tgfβri f / wcre in a mixed background of c57b1 / 6 fvb , and c57bl / 6 . dna was isolated from the mouse tail using genomic dna purification kit ( promega ) according to the manufacturer &# 39 ; s instruction . we used pcr to assess the genotype of tβri flox mice , using the three primers , 5 - atg agt tat tag aag ttg ttt ( seq id no : 5 ), 3 ′- acc ctc tca ctc ttc ctg agt ( seq id no : 6 ), and 3 ′- gga act ggg aaa gga gat aac ( seq id no : 7 ) as previously described ( sohal et al ., 2001 ). to induce α - mhc - coupled cre recombinase in cardiomyocytes , adult tgfβri f / fcre and tgfβri f / wtcre double transgenic mice were treated with tamoxifen ( sigma ) at a dose of 20 mg / kg per day for seven days by subcutaneous insertion of mini - osmotic pumps ( alzet , model 2001 ). a group of wild - type mice was treated with tamoxifen to check whether tamoxifen itself had any effects on cardiac morphology and function . tamoxifen was dissolved in 10 % ethanol and 90 % polyethyleneglycol - 400 followed by a brief sonification . mice were allowed to recover for two weeks prior to treatment with ang ii or vehicle . mice of either sex weighing 24 - 32 g were anesthetized with 2 . 5 % isofluorane . under sterile conditions , a midscapular incision was made , a pocket was created in the subcutaneous tissue by a blunt dissection and a mini - osmotic pump ( alzet model 2004 ; alza corp ., palo alto , calif ., usa ) filled with saline or ang ii ( 0 . 5 mg / kg / day ) was inserted . the contents of the mini - osmotic pump were delivered into the local subcutaneous space at a rate of 0 . 25 μl / hour for four weeks . in each group , seven to nine mice were recruited for experiments and at least five mice from each group completed the experiments . all the dropouts were due to death from anesthesia except three animals in which lv catheterization did not succeed . transthoracic echocardiography was performed preoperatively and after four weeks of angii infusion in wild - type tgfβri −/− and tgfβrl −/+ mice under 2 . 5 % isofluorane anesthesia . standard views were obtained in 2d - echocardiography , end - diastolic and end - systolic internal diameters were measured and ejection fraction and fractional shortening were calculated . mice were anesthetized with intraperitoneal injection of urethan . a millar ( 1 . 4 f ) catheter ( millar instruments inc ., houston , tex ., usa ) was placed in the right common carotid artery and advanced into the left ventricle for the measurement of left intraventricular pressure . body temperature was maintained at 37 ° c . using a thermally controlled surgical table and monitored with a rectal probe . the mice were then allowed to stabilize for 30 minutes prior to hemodynamic measurements . following hemodynamic measurements , hearts were rapidly excised , washed in 0 . 9 % sodium chloride solution , atria were removed and the ventricles were cut into pieces . for rna and protein isolation , samples were snap frozen in liquid nitrogen and stored in − 80 ° c . for histological analysis , left ventricles were fixed in paraformaldehyde ( 1 %) and embedded in paraffin . for the visualization of total collagen , picrosirius staining was performed as described previously . p38 was localized by immunostaining using anti - p38 antibody according to the manufacturer &# 39 ; s instruction ( cell signaling technology , leusden , the netherlands ). frozen ventricles were crushed and homogenized in radioimmunoassay buffer according to the standard protocol ( santacruz biotechnology , leiden , the netherlands ). western blotting was performed using specific antibodies against tβri ( 1 : 1000 ), total and phospho ( p )- smad2 , total and p - p38 ( 1 : 1000 , cell signaling technology , leusden , the netherlands ), p - smad3 ( 1 : 5000 , a kind gift from professor e . leof and dr . m . wilkes , mayo clinic cancer research , rochester , minn ., usa ), collagen i ( 1 : 3000 ) and iii ( 1 : 500 ) antibodies ( abeam , leusden , the netherlands ). cardiac tissue sections were deparaffinized and rehydrated and antigen retrieval tissue was incubated overnight with the primary antibody ( rabbit anti tgfβ receptor 1 ( santa cruz sc - 398 ) and subsequently the secondary antibody ( goat anti rabbit - biotine ( dakocytomation e0432 ), whereafter they were treated with streptavidin - horseradish peroxidase ( renaissance tsa ™ biotin system , perkin elmer precisely , tyramide signal amplification kit ). all study protocols described above involving animal experiments were approved by the animal care and use committee of the maastricht university , and were performed according to the official rules formulated in the dutch law on care and use of experimental animals , highly similar to those of the nih . data are shown as mean ± sem . unpaired t - test was performed to compare the difference between the means of ang ii / tgfβ / shklf15 and vehicle - treated animals and cells . p - values of ≦ 0 . 05 were considered statistically significant . it has previously been shown that the outbred homozygous hypertensive tgr ( mren2 ) 27 rat ( ren - 2 ) enables study of the transition from hypertrophy towards heart failure ( schroen et al ., 2004 ). myocardial biopsies obtained at the age of ten weeks were used to investigate whether altered gene expression can predict which rat later will later progress to heart failure . expression profiling of these biopsies revealed that suppression of the gene coding for krüppel - like factor 15 ( klf15 ) characterized the hypertrophied hearts that would quickly progress to failure . this was confirmed by real - time pcr , which showed that klf15 was down - regulated in compensated lvh , but that it was significantly further suppressed in the hypertrophied hearts that quickly progressed to failure ( fig9 a ). in situ hybridization showed that expression of klf15 was particularly down - regulated in cardiac myocytes ( fig9 b ). these findings extend earlier observations that klf15 is constitutively expressed in the heart , but down - regulated in hypertrophy . that more intense suppression of klf15 preceded the transition toward heart failure has led to the suggestion that klf15 has important protective properties . to explore the functional role of klf15 , a short hairpin rna ( shrna ) against klf15 was stably introduced . spontaneous expression of bnp , a molecular hallmark of the hypertrophy gene program , was induced more than ten - fold upon shrna - mediated suppression of klf15 in cultured cardiac myocytes ( fig9 c ). this suggests that the constitutive presence of klf15 is important to prevent the expression of the hypertrophy gene program . in a parallel study , it has been shown that klf15 null mice develop hypertrophy and cardiac function loss upon pressure loading , underlining that constitutively expressed klf15 is essential to protect against maladaptive forms of lvh . to explore the mechanism by which klf15 can repress the hypertrophy gene program , its role in activation of mef2 was studied . mef2 is a target for hypertrophic signaling conveyed by the calcineurin and the mapk pathway and is recognized as one of the crucial transcriptional activators of the hypertrophy gene program . a mef2 reporter construct was used to address whether altered levels of klf15 affect mef2 activity in cardiac myocytes . this reporter only weakly responds to stimulation by mef2 ( creemers , olson unpublished data ). indeed , only minor increases in mef2 activity were observed in response to angiotensin ii . however , knockdown of klf15 significantly increased mef2 activity ( fig9 d ), suggesting that klf15 acts as a repressor of mef2 . it was next sought to explore which mechanism suppresses klf15 in cardiac myocytes . therefore , known mediators of cardiac hypertrophy were screened for their ability to inhibit klf15 expression in cardiac myocytes . in cultured cardiac myocytes , tgfβ very robustly suppressed klf15 , so that expression of klf15 was almost completely abolished after addition of tgfβ . knockdown of the tgfβ type i receptor by inhibitory rna prevented the suppression of klf15 by tgfβ ( fig1 a ), demonstrating that classical tgfβ signaling involving its type i receptor is essential for this effect . therefore , to address the regulation of klf15 by the tgfβ type i receptor , in vivo mice carrying a floxed tgfβ receptor type i gene , combined with the mercremer allele , were generated , which allows activation of cre specifically in cardiac myocytes by administration of tamoxifen ( larsson et al ., sohal et al ., 2001 ). this allowed deletion of the tgfβ type i receptor specifically in cardiac myocytes in adult mice , avoiding the developmental effects of an embryonic loss of the tgfβ i receptor . hypertension was induced by chronic angiotensin ii infusion as described above in these mice to provoke hypertrophy and down - regulation of klf15 . western blotting of whole heart homogenate revealed a significant down - regulation of the tgfβ type i receptor ( fig1 b ). immunohistochemistry confirmed the myocyte - specific down - regulation of the tgfβ type i receptor , and showed the expression of this receptor in other cell types explaining the residual signal of the tgfβ type i receptor found in the whole heart homogenate ( fig1 c ). angiotensin ii induced lvh in wild - type mice , but the development of lvh was prevented in the mercremer - tgfβ type i mice . while in wt mice angiotensin ii decreased fractional shortening , fractional shortening remained preserved in the mercremer - tgfβ type i mice . this indicates that loss of the tgfβ type i receptor from cardiac myocytes can prevent hypertension - induced hypertrophy and function loss . as expected , the expression of klf15 was suppressed in the hypertrophied hearts from wt mice , but this suppression was absent in the hearts of mercremer - tgfβ type i mice ( fig1 f ). this shows that the tgfβ type i receptor on cardiac myocytes is important for the development of hypertensive hypertrophy , and at the same time for the suppression of klf15 . taken together , klf15 is the first krüppel - like factor to have a role in cardiac myocytes as a suppressor of cardiac hypertrophy . klf15 inhibits mef2 and parallel work shows it inhibits other prohypertrophic transcription factors like gat4 as well . consequently , it is conceivable that loss of klf15 very robustly induces hypertrophic gene expression and is related to an adverse outcome . suppression of klf15 may , therefore , be a novel and crucial step in the development of failure prone forms of hypertrophy . it has been shown that tgfβ very robustly can suppress klf15 . inhibitors of tgfβ , which are currently being developed in different fields , thus may have unexpected therapeutic potential as to prevent cardiac hypertrophy from progressing toward heart failure . the heart hypertrophies in response to loading and injury , which often progresses towards overt heart failure . according to the present invention , a novel mechanism in this process is unveiled , where the cytokine tgfβ suppresses a novel inhibitor of hypertrophy , krüppel - like factor 15 ( klf - 15 ). loss of the tgfβ type i receptor in vivo and in vitro prevents the suppression of klf - 15 and the development of cardiac hypertrophy and failure . the finding that tgfβ can hinder this novel mechanism that suppresses cardiac hypertrophy , opens exciting possibilities for inhibition of tgfβ signaling to prevent adverse forms of cardiac hypertrophy . according to the invention , it has been shown that the zinc - finger transcription factor , krüppel - like factor 15 ( klf - 15 ), is a potent transcriptional repressor of lv hypertrophy . gene - targeting studies showed that klf15 null mice develop normally , but in response to pressure overload , develop an exaggerated form of cardiac hypertrophy , characterized by increased heart weight , increased expression of hypertrophic genes , left ventricular cavity dilatation with increased myocyte size and reduced left ventricular systolic function . all together , these studies demonstrate a role for klf15 in lv hypertrophy , in vivo . interestingly , klf15 is down - regulated in several forms of pathological but not physiological hypertrophy , indicating that klf15 is a regulator of pathological hypertrophy , but not of physiological hypertrophy . the fact that klf15 counteracts hypertrophy and the additional observation that klf15 is significantly down - regulated in pathological hypertrophy and heart failure led to the exciting possibility that interventions aimed at preventing the decrease of klf15 levels could prevent or even reverse pathological growth . to test the intriguing possibility that preventing the loss of klf15 during pathological hypertrophy may limit pathological growth of the heart , klf15 was over - expressed specifically in the mouse heart using recombinant adeno - associated virus ( raav )- mediated gene delivery under the control of the cardiac troponin i promoter ( vandedriessche et al ., 2007 ). in particular , raav9 vectors have been shown to achieve a robust increase of transgene expression in cardiac tissue for several weeks following intravenous administration . mice were intravenously injected with 1 × 10 10 vg aav9 - klf15 or aav9 - gfp , after which hypertrophy was induced by angiotensin ii ( angii ) treatment ( four weeks , through osmotic mini - pumps ). as shown in fig1 ( upper panel ), klf15 was over - expressed in the heart . strikingly , mice allocated to aav9 - klf15 gene transfer developed significantly less hypertrophy upon angii stimulation , compared to angii - treated mice that received aav9 - gfp . ( see fig1 , lower panel .) together , these data show that forced expression of klf - 15 in cardiac myocytes suffices to reduce cardiac hypertrophy . loss of klf15 is a vital step in the development of hypertrophy and the transition toward heart failure . the observation that cardiac over - expression of klf15 inhibits the development of pathological hypertrophy opens exciting possibilities for strategies that prevent the down - regulation of klf15 in vivo to prevent hypertrophy and subsequent heart failure . a housekeeping genes : cyclophilin a ( ppia ), glyceraldehyde 3 - phosphate dehydrogenase ( gapdh ), phosphoglycerate kinase 1 ( pgk - 1 ) c fold - change , fold - change in gene expression of failure - prone ren - 2 rats as compared to compensated ren - 2 rats . for example , negative sign means down - regulated in failure - prone ren - 2 rats . d gene name , name of gene associated with the probe set id
the present invention relates to a method for identifying a subject at risk of developing heart failure , comprising : determining the level of one or more biological markers in a biological sample of the subject ; comparing the level of the biological marker to a standard level of the same biological marker ; and determining whether the level of the marker is indicative of a risk for developing heart failure , wherein the biological marker is krüppel - like factor 15 and / or lysosomal integral membrane protein - 2 and / or fragments and / or variants thereof , and / or wherein the biological marker is a gene coding for klf 15 and / or limp - 2 , and / or fragments and / or variants thereof . the invention further relates to use of the klf15 and / or limp - 2 protein , and / or the gene coding for klf15 and / or limp2 , and / or fragments , and / or variants of the genes and / or proteins , for the preparation of a medicament for a prophylactic and / or a therapeutic medicament for prevention and / or treatment of heart failure .