Patent Application: US-73721309-A

Abstract:
the invention relates to the fields of molecular biology and medicine , more specifically to treatment and prevention of heart disease . the invention provides alternative methods for counteracting , diminishing , treating , delaying and / or preventing heart disease .

Description:
as experimental animal models we made use of two - month - old wild - type b6cba and mhc - cna transgenic mice , which express an activated mutant of calcineurin under control of the 5 . 5 kb murine cardiac a - myosin heavy chain ( myh6 ) promoter . 1 , 2 all protocols were performed according to institutional guidelines and were approved by local animal care and use committees . transverse aortic banding ( tac ) or sham surgery was performed in two - month - old wild - type b6cba by subjecting the aorta to a defined , 27 gauge constriction between the first and second truncus of the aortic arch as described previously in detail . 3 doppler echocardiography was used to calculate the pressure gradient between the proximal and distal sites of the transverse aortic constriction using the doppler - estimated bernoulli &# 39 ; s equation , 4 and only mice with a pressure gradient & gt ; 20 mm hg were included . we isolated total rna from different mouse tissues or from cultured mammalian cells . wild - type and mhc - cna transgenic mice were sacrificed by cervical dislocation under isofluorane anesthesia . whole hearts and small samples of brain , thymus , kidney , intestine , colon and testis were removed , cleaned in pbs , placed in a labeled tube containing 1 ml of trizol reagent ( invitrogen ) and immediately put into liquid nitrogen . tissues were homogenized several times at maximum speed , each time for about one minute ( to prevent overheating ), until complete disruption . cells cultured in six - well plates to 100 % of confluency were washed twice with pbs before adding 1 ml of trizol per well and collecting the cell lysates in rnase - free tubes . after shaking the homogenates for ten minutes at 4 ° c . ( to permit the complete dissociation of nucleoprotein complexes ), 0 . 3 ml of chloroform per 1 ml of trizol were added to each sample . centrifugation at 12 , 000 g for 15 minutes at 4 ° c . results in the separation of rna ( upper aqueous phase ) from dna and proteins ( organic lower and intermediate phase ). aqueous phases ( 60 % of the sample volume ) were collected in new rnase - free tubes and rna was precipitated with 0 . 5 ml of isopropanol by incubation at − 20 ° c . for at least one hour and centrifugation at 12 , 000 g for 30 minutes at 4 ° c . the pellets , containing the rna , were washed twice with 1 ml of 70 % ethanol at 12 , 000 g for five minutes at 4 ° c . after decantation of the ethanol and total removal by evaporation , samples were dissolved in 20 - 30 μl of rnase - free water . rna quantity from the individual tissues was measured with a nanodrop ® nd - 1000 uv - vis spectrophotometer ( wilmington ), and rna quality was monitored using an agilent 2100 bioanalyzer . the expression analysis of 483 mirna was performed by a mirna - profiling service ( exiqon , denmark ) using mircury lna arrays . in short , two μg of total rna pooled from three samples ( three mhc - cna transgenic hearts ) and reference pool ( three nontransgenic hearts ) were labeled with hy3 ™ and hy5 ™ fluorescent label , respectively , using the mircury ™ lna array labeling kit . the hy3 ™- labeled samples and a hy5 ™- labeled reference pool rna sample were mixed pair - wise and hybridized to the mircury ™ lna array version 8 . 1 , which contains capture probes targeting all mirnas for all species registered in the mirbase version 8 . 1 at the sanger institute . the hybridization was performed according to the mircury ™ lna array manual using a tecan hs4800 hybridization station ( tecan , austria ). after hybridization , the microarray slides were scanned and stored in an ozone free environment ( ozone level below 2 . 0 ppb ) in order to prevent potential bleaching of the fluorescent dyes . the lna array slides were scanned using the agilent g2505b microarray scanner system ( agilent technologies , inc ., usa ) and image analysis was carried out using the imagene 7 . 0 software ( biodiscovery , inc ., usa ). the raw signal for each probe was obtained by subtracting the maximum of the local background and negative control signals from the foreground signal . the data was pre - processed to remove poor - quality spots and normalization was used to remove any systematic bias . quantified signals were normalized using the global lowess ( locally weighted scatterplot smoothing ) regression algorithm ( exiqon ). three micrograms of total rna from heart or other different tissues were fractionated on a denaturing 12 % polyacrylamide gel containing 8 m urea , transferred to nytran n membrane ( schleicher & amp ; schuell , germany ) by capillary method and fixed by uv cross - linking according to the manufacturer &# 39 ; s instructions . membranes were hybridized with specific 5 ′- digoxigenin ( dig )- labeled lna detection probes ( exiqon ) for mir - 199b or u6 ( loading control ). detection was performed with an antibody against dig ( roche ). an adenovirus expressing an activated mutant of calcineurin ( adcna ) was described earlier . 5 adlacz was described previously . 6 antisense oligonucleotides targeting mir - 199b were obtained from exiqon ( mircury lna knockdown oligo mmu - mir - 199b , lna - mir - 199b ) and mir - 199b precursor molecules were obtained from ambion ( pre - mir ™ mmu - mir - 199b mirna precursor , pre - mir - 199b ). neonatal rat ventricular myocytes were obtained by enzymatic dissociation of one - to two - day - old rat neonatal ventricles as described previously in detail . 7 ventricles were stored in hepes buffered dmem ( ph 7 . 4 ) prior to multiple rounds of enzymatic digestion in dmem nutrient mixture f - 12 ham base ( sigma ) supplemented with 0 . 7 mg / ml collagenase type 2 ( invitrogen ) and 1 mg / ml pancreatin ( sigma ). cells were collected by centrifugation at 61 × g for ten minutes , resuspended in neonatal calf serum ( invitrogen ) and stored in an incubator at 37 ° c . all cell suspensions were pooled , centrifuged at 61 × g for ten minutes and resuspended in dmem ( invitrogen ) supplemented with 10 % horse serum ( invitrogen ) and 5 % fetal calf serum ( invitrogen ). subsequently , the cells were differentially plated for three hours in uncoated cell culture dishes to remove contaminating non - myocytes . the cardiomyocytes ( containing less than 5 % non - myocytes ) were then plated on fibronectin ( sigma )- coated six - well culture dishes . approximately 24 hours after plating the media was replaced by dmem : m199 ( 4 : 1 ) medium ( serum free medium ). for transfection , neonatal rat cardiomyocytes were plated in dmem supplemented with nutridoma ( roche ) in six - well fibronectin - coated plates with density of 2 * 10 5 cells per well . the next day , cells were transiently transfected with 30 nm of lna - mir - 199b , pre - mir - 199b or respective scrambled controls , with oligofectamine reagent ( invitrogen ) according to the manufacturer &# 39 ; s recommendations . cells were washed the next day and left untreated , stimulated with 10 μm phenylephrine ( pe ), or infected with adlacz or adcna for 24 hours before cell fixation or rna isolation . to visualize cardiomyocyte size and sarcomeric organization , cultured cardiomyocytes were fixed for ten minutes in 4 % paraformaldehyde and permeabilized with 0 . 2 % triton x - 100 in pbs for five minutes . primary and secondary antibodies were diluted using 1 % bsa in tbs and incubations were carried out at room temperature for one hour . cells were washed three times with pbs for five minutes , mounted with coverslips in vectashield mounting medium for fluorescence ( vector laboratories ), and analyzed by confocal microscopy using a zeiss lsm 510 meta microscope . antibodies used included mouse monoclonal anti α - actinin ( sigma , 1 : 500 ); rabbit polyclonal anti anf ( peninsula laboratories ) cy5 goat anti - rabbit and cy3 goat anti - mouse ( jackson immuno research , 1 : 100 and 1 : 500 , respectively ); and topro - 3 ( 1 : 100 , invitrogen ). cell surface areas were determined using spot - imaging software ( diagnostic instruments ) on 80 - 100 cardiomyocytes in 10 to 20 fields in three independent experiments . to find the target genes of a specific microrna we made use of several web servers based on predictive bioinformatics algorithms ( pictar , miranda , mirbase ). these are intuitive interfaces that incorporate processing algorithms and powerful mirna targets search tools to search the mirna targets against the most conserved 3 ′ utr sequences from ucsc genome browser . by comparing the target gene lists resulting from each algorithm we shortened the initial lists of hundreds of potential target genes to a list of 32 genes , common to all algorithms used . we designed primers targeted against transcripts of 20 of the predicted genes and l7 . the primers were specific for mouse sequences ( www . ensembl . org ) and selected using beacon designer software ( invitrogen ) based on the following requirements : i ) primer melting temperature of ˜ 60 ° c ., ii ) gc - content of ˜ 55 %, iii ) preferably no g at 5 ′ end , iv ) avoid runs of more than three identical nucleotides , and v ) amplicon length of ˜ 100 nucleotides . specificity was checked with the basic local alignment search tool ( blast ) and the specific melting point of the amplicons was analyzed using biorad dissociation curve software ( icycler , biorad ). all primer sets were tested for pcr efficiency and alternative primers were designed in case they fell outside the 5 % efficiency range ( 3 . 14 ≦ slope ≦ 3 . 47 ). three μg of rna from indicated hearts was reverse - transcribed using superscript ii reverse transcriptase ( invitrogen ). pcr amplification was performed ( in duplicate ) as a singleplex reaction with 400 nm forward and reverse primers on 40 ng cdna , in a total reaction volume of 25 μl . the pcr was cycled between 95 ° c ./ 30 seconds and 60 ° c ./ 30 seconds for 40 cycles , following an initial denaturation step at 95 ° c . for three minutes . real time pcr results were verified by electrophoresis of the reverse transcribed material in 1 . 2 % agarose gels and visualized under uv illumination after ethidium bromide staining . transcript quantities were compared to the amount of endogenous control ( l7 ). we developed a cell model to validate several of the predicted target genes . double stable , mir - 199b - inducible cells were generated using the t - rex system ( invitrogen ) with modifications . briefly , cells were transfected using fugene 6 reagent ( roche ) with 8 μg pcagβtrs - hygro , a vector expressing the tet - repressor ( tr ) under control of a ( 3 - actin promoter ( generously provided by hans clevers , the hubrecht institute ) and stable clones were selected with 250 μg / μl hygromycin . selected colonies were transiently transfected with 0 . 2 μg pcdna4 / to - luciferase ( invitrogen ), using fugene 6 reagent ( roche ), to test their responsiveness to doxycyclin ( dox ) using the dual luciferase assay system ( promega ). two different tet - repressor clones ( tr1 and tr4 ), showing high luciferase activity and low background , were subsequently transfected with 8 . 5 μg pcdna4 / to - mir - 199b and cultured in the presence of hygromycin and 750 μg / μl of zeocin to generate double stable cell lines . zeocin / hygromycin resistant clones were transiently transfected with a reporter construct encoding firefly luciferase under control of the proximal promoter region of the rat anf gene ( base pairs − 3003 to + 1 relative to the beginning of exon 1 ) to test their dox - inducible mir - 199b transcriptional activation profile . we selected two clones ( tr1 - 2 and tr4 - 7 ), which systematically showed significant induction of mir - 199b expression levels in the presence of doxycyclin in the culture media . in this way , we established a cellular system with inducible activation of mir - 199b and concomitant translational repression of the endogenous mir - 199b target genes ( see fig4 ). proteins were extracted from clones tr1 - 2 and tr4 - 7 , left untreated or treated with dox , using cell lysis buffer ( 20 mm tris ph 8 . 0 , 150 mm nacl , 1 mm edta , 1 mm egta , 1 % triton x - 100 ) supplemented with a protease inhibitor cocktail ( complete mini , roche ). sds page electrophoresis and blotting was performed as described in detail . 8 antibodies used included rabbit polyclonal against dyrk1a and mouse - monoclonal antibody to gapdh ( both from santa cruz ), followed by corresponding horseradish peroxidase ( hrp )- conjugated secondary antibodies ( dako ) and ecl detection . 3 ′ utr regulatory sequences have been shown to be important for mrna stability , translation , and transport . we designed primers specific for mouse sequences ( http :// ensembl . org ) targeting the specific binding site of mir - 199b on the 3 ′ utr of d yrkl a ( nucleotides 1536 - 1365 , http :// cbio . mskcc . org / cgi - bin / mirnaviewer /). after pcr amplification of this specific sequence , a pcr product with the expected size ( 286 bp ) was visualized and isolated from a 1 . 2 agarose gel . after purification , the 3 ′ utr fragment was cloned into a pmir - report ™ mirna expression reporter vector ( ambion ). this vector contains firefly luciferase under the control of the cmv mammalian promoter , with a mirna target cloning region downstream of the luciferase translation sequence . this vector is optimized for cloning of mirna targets and evaluation of mirna regulation and , therefore , can be used as a screening tool to identify mirna targets . after plasmid isolation and sequencing , the plasmid was used to transfect the double stable tr - mirl99b clones . cells were cultured in 96 - well plates , transfected with the pmir - reporter - 3 ′ utr dyrk1a plasmid or the empty vector and incubated for 24 hours at 37 ° c . after one wash with pbs , cells were left untreated or were treated with dox for 48 hours before measuring luciferase activity . the results are presented as mean values ± standard error of the mean ( sem ). statistical analyses were performed using prism 5 software ( graphpad software inc .) and consisted of anova followed by turkey &# 39 ; s post - test when group differences were detected at the 5 % significance level , or student &# 39 ; s t - test when comparing two experimental groups . we profiled the expression levels of cardiac mirnas in calcineurin transgenic mice . rna was isolated from hearts of two - month - old wild - type and mhc - cna transgenic mice and we performed mirna profiling on these samples . the hy3 - labeled samples and a hy5 - labeled reference pool rna sample were mixed pair - wise and hybridized to mircury lna arrays , which contain capture probes targeting all mirnas registered in the mirbase version 8 . 1 at the sanger institute ( 345 mirnas ) and probes targeting licensed human sequences not yet annotated in mirbase ( 138 mirplus , exiqon ). we detected micrornas that are co - regulated with the development of calcineurin - induced heart failure using commercially available oligonucleotide microrna microarrays ( fig1 , panel a ), and we have analyzed the genomic localization of one specific microrna : mmu - mir - 199b and the human orthologue hsa - mir - 199b ( fig1 , panel b ). human mir - 199b is an intragenic microrna encoded in the dynamin 1 ( dnm1 ) gene on the opposite strand in between exon 14 and 15 ( fig1 , panel b ). northern blot analysis of cardiac tissue isolated from mhc - cna transgenic and transverse aortic constriction ( tac )- operated pressure overloaded mice , as two well established models of pathological cardiac hypertrophy , confirmed that mir - 199b - 5p is indeed strongly up - regulated in the diseased heart ( fig2 , panels a and c ). next , we analyzed its expression pattern in different murine tissues including heart , brain , thymus , kidney , intestine , colon and testis by northern blotting ( fig2 , panel b ). although not cardiac specific , mir - 199b - 5p emerged as being highly abundant in the cardiac tissue . further , mir - 199b - 5p is also more abundantly expressed in biopsies of human cardiac tissues of heart failure patients , compared to control , human healthy heart tissue ( fig2 , panel d ). finally , mir - 199b is an immediate target gene of the calcineurin / nfat pathways , since hearts from mice harboring a null allele of nfatc2 showed less mir - 199b expression , both under baseline conditions as well as following chronic activation of calcineurin signaling ( fig2 , panel e ). to address the role of mir - 199b in cardiomyocyte remodeling we transfected primary neonatal rat cardiomyocytes with mir - 199b precursor molecules to overexpress mir - 199b and compared them with cardiomyocyte cultures that have been infected with an adenovirus expressing lacz , and adenovirus expressing an activated form of calcineurin ( adcna ), or that have been exposed to 10 mm phenylephrine ( pe ; fig3 , panel a ). to monitor the change in cell size or sarcomere organization induced by the different treatments , cardiomyocytes were stained for sarcomeric a - actinin ( fig3 , panel b ). as expected , adcna or pe treatment resulted in robust hypertrophy response as shown by a significant increase in cell size and in perinuclear presence of anf . surprisingly , a similar increase in cell size and in anf expression was observed in cardiomyocytes overexpressing mir - 199b ( fig3 , panel b ). to begin to assess the requirement of mir - 199b downstream of ( calcineurin - mediated ) cardiomyocyte hypertrophy , we used antisense oligonucleotides targeting endogenous mir - 199b ( lna - mir - 199b ), and transfected these oligonucleotides into primary cardiomyocyte cultures . as a control , cardiomyocytes were also transfected with a non - specific control oligonucleotide ( fig3 , panels c , d , and e ). next , we infected the cardiomyocyte cultures with an adenovirus expressing an activated form of calcineurin ( adcna ), or exposed to 10 mm phenylephrine ( pe ). to monitor the change in cell size or sarcomere organization , cardiomyocytes were stained for sarcomeric a - actinin . adcna or pe treatment resulted in a strong hypertrophic response when cells were treated with the control oligonucleotide . in contrast , pretreatment with the lna - mir - 199b completely abrogated the classical hypertrophy phenotype in response to adcna - infection or pe treatment ( fig3 , panel d ). quantification of the data indicated a two - fold increase in cell surface area in adcna - infected or pe - treated cells pretreated with the control oligonucleotide . these prohypertrophic effects of adcna and pe were abrogated by blocking the binding of mir - 199b to its target mrna ( fig3 , panel e ). mir - 199b is predicted to target different genes downstream of the calcineurin - nfat signaling pathway . despite the large number of identified mirnas in several disease situations , only a handful of mirnas have been functionally characterized . complicated expression patterns and large numbers of predicted targets genes preclude a straightforward analysis of their precise biological function . to understand the role of mir - 199b in calcineurin - induced cardiac failure we undertook an expression analysis of predicted mmu - mir - 199b mrna targets listed in several public datasets developed based on several studies . 8 - 17 by rt - pcr we found that not all the predicted target mrnas for mir - 199b were differentially expressed in mhc - cna transgenic hearts , compared to the wild - type hearts ( fig4 , panel a ). however , genes like mylb6 , m11s1 , grpc5a , and , in particular , dyrk1a were strongly down - regulated in mhc - cna transgenic hearts . however , none of these genes , except for dyrk1a , have been described to be linked to calcineurin / nfat signaling pathway . mir - 199b overexpression results in down - regulation of the dual - specificity tyrosine - phosphorylation regulated kinase , dyrk1a . from the genes that were down - regulated in mhc - cna transgenic hearts at the transcript level , only dyrk1a has been shown to be directly connected to this pathway . recently , two independent groups obtained evidence linking dysregulation of nfat signaling in down &# 39 ; s syndrome . 18 - 2 ° the nfat family of transcription factors , which are critical to development , reside in the cytoplasm in a hyperphosphorylated form ; they are dephosphorylated by calcineurin in response to calcium influx and translocate to the nucleus to activate target genes . mice lacking various nfatc genes showed abnormalities comparable to those of people with down &# 39 ; s syndrome . examination of the region of human chromosome 21 believed to contain genes responsible for the down syndrome phenotype revealed two potential regulators of nfat signaling : dscr1 ( which encodes a calcineurin inhibitor ) and dyrk1a ( dual - specificity tyrosine - phosphorylation regulated kinase ), which encodes a nuclear serine / threonine kinase . dyrk1a and dscr1 synergistically inhibited nfat - dependent transcription in cultured neurons . moreover , dyrk1a was shown to phosphorylate nfat and prime it for further phosphorylation by glycogen synthase kinase 3 ( gsk3 ) and , therefore , to promote its export from the nucleus . transgenic mice that overexpressed dyrk1a and dscrl showed cardiovascular abnormalities most likely related to the cytoplasmic localization of endocardial nfat . based on these findings , we hypothesized that calcineurin / nfat - dependent activation of mir - 199b results in direct down - regulation of dyrk1a expression . being true , this would result in decreased phosphorylation of nuclear nfat , decreased translocation of phosphorylated nfat to cytoplasm and subsequent induction of the cardiac remodeling and hypertrophic response . to test this hypothesis we generated a cellular system with inducible activation of mir - 199b ( fig4 , panel b ) and , in theory , concomitant translational repression of the endogenous mir - 199b target genes . indeed , treatment of these cells with dox showed an increase in mir - 199b expression by northern blotting analysis , in contrast with the untreated cells that expressed very low levels of the mir ( fig4 , panel c , nb ). correlated with an increase in mir - 199b expression we observed a concomitant decrease in protein levels for dyrk1a , showing that dyrk1a is indeed a direct target of mir - 199b ( fig4 , panel c , wb ). to further analyze whether dyrk1a is a direct target gene of mir - 199b we looked more carefully at the 3 ′ utr sequence of dyrk1a , more specifically to the mir - 199b seed region . fig5 , panel a , shows that this region is highly conserved between human ( seq id nos : 10 and 11 ) and mouse ( seq id nos : 8 and 9 ), suggesting that this is indeed a target sequence of mir - 199b ( seq id nos : 6 and 7 ). to confirm this , we made use of a mirna expression reporter vector ( pmir - reporter , ambion ). this vector contains firefly luciferase under the control of the cmv mammalian promoter . the 3 ′ utr of the luciferase gene contains a multiple cloning site for insertion of predicted mirna binding targets or other nucleotide sequences . by cloning the sequence of the 3 ′ utr of dyrk1a , to which mir199b is predicted to bind , into the pmir - report vector , the luciferase reporter will be subjected to regulation that will mimic regulation of the mirna target ( in this case , dyrk1a ; fig5 , panel a ). if overexpression of mir - 199b would result in a decrease in luciferase activity , this would show that the 3 ′ utr sequence of dyrk1a would be a direct target of this mir . indeed , this is what we observed ( fig5 , panel b ). in addition , we created a vector where we introduced two point mutations in the mir - 199b seed region within the 3 ′ utr sequence of dyrk1a as a control ( seq id nos : 8 and 12 ) ( fig5 , panel a ). the p - mir - reporter - 3 ′ utr dyrk1a was sensitive to mir - 199b expression by expression of mir - 199b upon dox addition to inducible mir - 199b expressing clones ( fig5 , panel b ) and in a dose - dependent manner by transient co - transfection of a vector expressing mir - 199b ( fig5 , panel c ), while no sensitivity was observed for a co - expression of and unrelated microrna , mir - 216a ( fig5 , panel c ). in addition , mir - 199b - inducible clones treated or not with dox for 48 hours were pre - transfected with the p - mir - reporter - 3 ′ utr dyrk1a . luciferase activity was strongly inhibited in the cells overexpressing mir - 199b , compared to the cells left untreated or transfected with the empty vector , while a mutated p - mir - reporter - 3 ′ utr dyrk1a showed no sensitivity to mir - 199b expression ( fig5 , panel d ). combined , these data demonstrate the presence of a functional , and evolutionary conserved mir - 199b seed region in the 3 ′ utr of dyrk1a . finally , we made use of an antagomir approach designed to block endogenous mir - 199b expression in vivo ( fig6 , panel a ). to this end , we performed an experiment in which a chemically modified antisense oligonucleotide specific for mir - 199b ( antagomir - 199b ) was delivered by ip injection on three consecutive days to wild - type and calcineurin transgenic mice at the age of 14 days after birth ( p 14 ; fig6 , panel b ). mice tolerated antagomir - 199b well without any obvious signs of illness or discomfort . four days after the last injection we analyzed the gross morphology of the hearts , where we found calcineurin transgenic mice treated with antagomir - 199b to have near normalized heart size ( fig6 , panels c and d ) compared to vehicle - treated littermates . northern blotting of cardiac tissue revealed a near completion of mir - 199b expression in both wild - type and calcineurin transgenic mice , indicating the effectiveness of the antagomir - 199b design ( fig6 , panel e ). interestingly , dyrk1a expression levels were down - regulated to about 50 % in vehicle - treated calcineurin transgenic mice compared to vehicle - treated wild - type mice ( fig6 , panel f ). in contrast , antagomir - 199b - treated animals demonstrated restored dyrk1a protein levels . this was accompanied by normalized nfat activity levels , as measured by the relative expression levels of rcan1 . 4 transcript abundance ( fig6 , panel g ). conversely , we generated transgenic mouse lines overexpressing mir - 199b in the post - natal myocardium using the alpha - myosin heavy chain promoter ( fig7 , panel a ). we were able to generate three transgenic lines , each with differing overexpression of mir - 199b as assessed by northern blotting ( fig7 , panel a ). at the age of three weeks , mir - 199b overexpressors did not show an obvious cardiac phenotype . when we crossbred mir - 199b transgenic mice with calcineurin transgenic mice , however , we observed a more exaggerated cardiac phenotype than mice only harboring the calcineurin transgene ( fig7 , panel b ). the cardiac phenotype was reflected at the level of relative mir - 199b expression level ( fig7 , panel c ), as well as by relative heart weights . finally , we also observed lowered dyrk1a protein expression levels in biopsies of patients with ischemic heart failure , which correlated inversely with their expression of mir - 199b ( fig7 , panel d ). all together , our data show for the first time that mir - 199b plays an important role in calcineurin - induced cardiac hypertrophy . more importantly , we have identified the mechanism whereby mir - 199b enhances calcineurin / nfat - induced cardiomyocyte hypertrophy and , therefore , pathological cardiac hypertrophy , by active down - regulation of its direct target gene , dyrk1a . 1 . palermo j ., j . gulick , m . colbert , j . fewell , and j . robbins . transgenic remodeling of the contractile apparatus in the mammalian heart . circ . res . 1996 ; 78 : 504 - 509 . 2 . molkentin j . d ., j . r . lu , c . l . antos , b . markham , j . richardson , j . robbins , s . r . grant , and e . n . olson . a calcineurin - 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