apoptosis , a distinct form of cell death , is at the heart of both mechanical and molecular mechanisms of cardiomyocyte loss during ischemia .
after mi , there is heightened apoptosis in the peri - infarct and noninfarcted myocardium , and this coincides with increased left ventricular diameter and decreased cardiac function . to this effect
, therapies that inhibit apoptosis in mi have been shown to improve cardiac function [ 2 , 3 ] .
micrornas ( mir ) are small noncoding ribonucleic acids ( rnas ) measuring 1825 nucleotides that are involved in posttranscription regulation of gene expression [ 4 , 5 ] .
mir-208a is a cardiac specific microrna coded for in an intron of myosin heavy chain 6 ( myh6 ) gene and regulates cardiac conduction , stress response , and gene expression [ 69 ] .
several researches have shown that its inhibition or deletion is associated with downregulation of cardiac fibrosis and hypertrophy in response to various stress stimuli [ 68 ] .
it has also been shown to be dysregulated in several cardiac diseases including myocardial infarction and dilated cardiomyopathy , in which it is associated with adverse outcomes [ 10 , 11 ] .
however , no study as yet has been done to investigate its role in cardiomyocyte apoptosis despite these cardiac pathologies being associated with increased myocyte apoptosis .
we therefore investigated the effect of mir-208a on cardiomyocyte apoptosis and apoptosis related genes and if its silencing has any therapeutic benefit in myocardial infarction .
we report that mir-208a upregulation alters apoptosis genes ' expression and promotes cardiomyocyte apoptosis , while its silencing attenuates apoptosis and improves cardiac function after mi . to the best of our knowledge ,
our study is the first to explore the antiapoptotic effects of mir-208a silencing and the therapeutic benefits of mir-208a antagomir in myocardial infarction .
cardiomyocytes were transfected with 0.2  nmols / ml of mir-208a agomir for gain of function or 0.4  nmols / ml of mir-208a antagomir for loss of function , by directly adding the agomir or antagomir formulation to the cell culture medium according to manufacturer 's instructions ( ribobio co. ltd . ,
silencing , cardiomyocyte cells were transfected with bax sirna ( 5-aacagaucaugaagacagggg-3 ) using ribofectamine reagent according to manufacturer 's instructions ( ribobio co. ltd . ,
after 48 hours , the different treatment groups underwent simulated ischemia for 12 hours by placing the cardiomyocytes in a hypoxia chamber with 5% co2 and 95% n2 at 37c in glucose - free dmem medium .
myocytes were harvested , fixed , and assayed for apoptosis according to previously described protocol .
microarray profiling was done on custom - made array chips by a service provider ( ribobio co. , guangzhou , china ) .
rna was isolated from cultured control or mir-208a agomir transfected cardiomyocytes , 72 hours after transfection .
differential gene expression between controls and agomir transfected cardiomyocytes was analyzed using permutation analysis of differential expression .
gene clustering was performed with cluster 3.0 and heat map images generated in java tree view .
gene ontology analysis was done using david online tool [ 14 , 15 ] , and apoptosis pathway analysis was done using ingenuity pathway analysis ( http://www.ingenuity.com/ ) .
all experiments were conducted according to the guide for the care and use of laboratory animals and approved by the animal care and utilization committee of union hospital , tongji medical college , huazhong university of science and technology , wuhan , china .
the investigation conformed to the guide for the care and use of laboratory animals , published by the us national institute of health ( 2011 ) .
adult male c57bl/6 mice , 8-week - old , were purchased from wuhan university animal research center .
mice were anesthetized by an intraperitoneal injection of pentobarbital sodium ( 50  mg / kg ) , orally intubated , and connected to a rodent ventilator , and ecg monitoring was done using rm6240 data acquisition system sampling at 1  khz .
a left thoracotomy was performed by a horizontal incision at the third intercostal space and myocardial infarction induced by ligation of the left anterior descending coronary artery ( lad ) according to previously described protocol   and then randomly assigned to antagomir group ( n = 6 ) or control group ( n = 7 ) . sham group mice ( n = 7 ) had the same procedure done but without ligation of the lad . starting within 2 hours after mi , each antagomir group mouse received a total of 300  nmols of mir-208a antagomir given over 3 consecutive days ( days 0 , 1 , and 2 ) using 0.2  mls of normal saline as vehicle , by low pressure tail vein injection , while control and sham group mice each received 0.2  mls of vehicle only by the same method over the same period of time .
echocardiography was performed at 7 and 28 days , and thereafter animals were euthanized and hearts harvested for further analysis . at 28 days , animals were euthanized and hearts harvested , cut transversely into blocks , and fixed in 4% paraformaldehyde .
these were then embedded in oct compound ( bhd , uk ) and then transversely cut into 5  m thick cryosections for histological analysis .
two cryosections from each of the blocks were stained with masson 's trichrome for assessment of fibrosis and infarct size .
the infarct size was measured as the sum of the epicardial and endocardial scar length , divided by the sum of lv epicardial and endocardial circumferences . for assessment of fibrosis
, we randomly selected 4 fields per slide in the peri - infarct zone ( piz ) or 5 fields per slide in the noninfarct zone ( niz ) and calculated collagen volume fraction ( cvf ) as the ratio of blue stained ( collagen ) area to the total tissue area .
the collagen - rich border zone of vessels and the scar were excluded from the analysis .
cardiomyocyte hypertrophy was assessed by capturing cardiomyocyte images over 5 different fields per slide ( 40x objective ) . using image - pro plus software version 6 , we obtained mean cardiomyocyte cross - sectional areas , which were compared between groups . at 28 days after mi or after 12 hours of simulated cardiomyocyte ischemia , tissues or cells , respectively ,
were fixed with 4% paraformaldehyde , and apoptosis assay was done by tunel method according to previously described protocol .
total rna was extracted from cells and tissue samples using trizol reagent ( invitrogen , usa ) according to the manufacturer 's protocol .
cdna was synthesized from 2  g of total rna with takara 's reverse transcription kit ( takara , china ) , according to manufacturer 's instructions in a 25  l volume including 2  g total rna , and reverse transcription primer for mrna genes , or random primers for u6 rrna and mir-208a specific primers ( bulge - loop mirna qpcr primers from ribobio , china ) .
real - time pcr was carried out with the reagents of a sybr green i mix ( takara , china ) in a 20  l reaction volume ( 10  l sybr green i mix , 200  mm forward and reverse primer , and 2  l cdna template ) on an mj opticon monitor chromo4 instrument ( bio - rad , usa ) using the following protocol : 95c for 20  s ; 40 cycles of 95c for 10  s , 60c for 20  s , and 70c for 1  s. normalization for mir-208a was done using u6 while bax expression was normalized using gapdh .
the following primer sets were used for bax and gapdh :  bax :  forward : tgtttgctgatggcaacttc  reverse : gatggttctgatcagctcgg  gapdh :  forward : 5-gctggcgctgagtacgtcgtggagt-3  reverse : 5-cacagtcttctgggtggcagtgatgg-3data analyses were performed using the 2 method .  forward : tgtttgctgatggcaacttc  reverse : gatggttctgatcagctcgg forward : tgtttgctgatggcaacttc reverse : gatggttctgatcagctcgg  forward : 5-gctggcgctgagtacgtcgtggagt-3  reverse : 5-cacagtcttctgggtggcagtgatgg-3 forward : 5-gctggcgctgagtacgtcgtggagt-3 reverse : 5-cacagtcttctgggtggcagtgatgg-3 to assess if therapeutic silencing of mir-208a improves cardiac function in mi , 2-dimensional transthoracic echocardiography studies were performed on 4 - 5 mice from each group , lightly anesthetized with pentobarbital sodium ( 25  mg / kg ) , on days 7 and 28 , using a ge voluson ultrasound system equipped with an 11.5  mhz high frequency probe .
cardiac imaging was done in a parasternal short - axis view at the level of the papillary muscles to record m - mode and determine fractional shortening ( fs ) and ejection fraction ( ef ) which are measures of cardiac function .
groups were compared using independent student 's t - test or one - way anova with the tukey or games - howell post hoc tests as appropriate .
statistical significance was defined as a p value < 0.05 and values are presented as mean  sem .
expression of mir-208a in vivo and in vitro was analyzed using quantitative rt - pcr .
we found that 0.4  nmol / ml of mir-208a antagomir significantly downregulated mir-208a expression in cultured cardiomyocytes ( figure 1(a ) ) .
similarly , mir-208a agomir upregulated mir-208a expression in neonatal cardiomyocytes 48 hours after transfection ( figure 1(b ) ) . to silence mir-208a ,
300  nmols of mir-208a antagomir per mouse was administered after mi , and this significantly downregulated mir-208a expression at 28 days after mi ( figure 1(c ) ) .
apoptosis , a distinct form of cell death , is a contributor to cardiac dysfunction , remodeling , and heart failure following mi [ 17 , 18 ] .
simulated ischemia of neonatal cardiomyocytes following transfection with mir-208a agomir or antagomir showed that mir-208a upregulation significantly increases apoptosis while its silencing blunts apoptosis during ischemia ( figures 2(a ) and 2(b ) ) . to gain insight into the effect of mir-208a on apoptosis related genes , we employed custom - built microarrays ( ribobio co. , guangzhou , china ) .
hierarchical clustering and heat map visualization of the differentially expressed genes are shown in figure 3(a ) .
cluster analysis of the differentially expressed genes revealed that eight apoptosis related genes were upregulated , while twelve apoptosis related genes were downregulated ( table 1 ) .
a closer inspection of the dysregulated genes showed that mir-208a tended to downregulate genes favoring the extrinsic apoptosis pathway , while upregulating those that favor the intrinsic pathway of apoptosis .
analysis of differentially expressed apoptosis genes highlighted bax , casp8 , and hras1 as involved in the canonical apoptosis pathways ( figure 3(b ) ) .
if bax was essential for mir-208a induced apoptosis , we used sirna to silence bax gene .
bax sirna attenuated the proapoptotic effect of mir-208a agomir ( figure 2(c ) ) , suggesting that mir-208a functions to promote apoptosis at least in part by upregulating bax .
apoptosis has been observed to occur in the peri - infarct and noninfarcted myocardium and is a contributor to cardiac dysfunction , remodeling , and heart failure following mi [ 17 , 18 ] . to investigate the antiapoptotic effects of mir-208a silencing in an mi setting , we treated mice with 300  nmols of mir-208a antagomir .
antagomir attenuated peri - infarct apoptosis at 28 days , a finding that is novel and may have therapeutic significance ( figures 4(a ) and 4(b ) ) . in the noninfarct area ,
however , the difference in apoptosis between antagomir and control group hearts only showed a trend towards reduction but did not reach statistical significance ( figure 4(c ) ) .
qpcr showed that mir-208a silencing in mi also decreased bax levels at 28 days ( figure 4(d ) ) .
a comparison of infarct area sizes between control and antagomir groups showed a trend towards reduction in infarct size by antagomir , but this did not reach statistical significance ( figure 4(e ) ) . at one month after mi ,
antagomir treatment significantly decreased cardiomyocyte hypertrophy ( figures 5(a ) and 5(b ) ) , a known pathological response associated with heart failure and sudden death [ 19 , 20 ] .
moreover , mir-208a antagomir also decreased percentage fibrosis at day 28 ( figures 5(c ) , 5(d ) , and 5(e ) ) .
increased fibrosis is known to reduce cardiac contractility and to contribute to reduction of cardiac function after mi , and thus its reduction by antagomir is potentially beneficial .
the above results were consistent with previous reports which showed that mir-208a silencing or knockout attenuates cardiac fibrosis and hypertrophy in response to cardiac stress [ 6 , 7 ] .
aiming to see any beneficial effects of long term silencing of mir-208a on cardiac function after mi , mice received a total of 300  nmols of mir-208a antagomir , and echo was done at 7 and 28 days .
antagomir treatment significantly attenuated cardiac dysfunction after mi ( table 2 and figures 5(f ) and 5(g ) ) .
the current study investigated the role of mir-208a in apoptosis during cardiomyocyte ischemia and the therapeutic potential of mir-208a antagomir in myocardial infarction . here
we reveal for the first time that mir-208a promotes myocyte apoptosis during ischemia and further show that therapeutic administration of mir-208a antagomir attenuates cardiomyocyte apoptosis , hypertrophy , and fibrosis , coupled with improvement in cardiac function after mi .
following myocardial infarction , nonischemic myocyte apoptosis ensues resulting in further myocardial loss and ventricular dysfunction [ 2 , 19 , 20 ] . because apoptosis plays a critical role in both mechanical and molecular mechanisms of cardiac dysfunction and remodeling after mi
our finding that mir-208a antagomir mitigates ischemia induced apoptosis opens a window for its possible therapeutic application in mi . despite noting no difference in infarct size between the groups , the observed decrease in apoptosis levels could still be a contributor to improved contractile performance , as other studies have shown .
interestingly , some researchers have shown that cardiac mir-208a in human mi is persistently upregulated , with the highest levels observed in patients who died within 24 hours .
thus , targeted silencing of mir-208a in human mi may be a potentially viable and beneficial therapeutic option .
although mir-208a has been shown to be proproliferative under certain conditions , we demonstrated that it is proapoptotic in the ischemic setting [ 22 , 23 ] .
it is not uncommon for micrornas or other genes to have opposite effects under different conditions .
for example , mir-494 has been shown to have both antiapoptosis and proapoptosis effects under different circumstances .
similarly , p21 ( waf 1 ) , a target of mir-208a , has also been reported to be both anti- and proapoptosis depending on prevailing conditions . to investigate the genetic basis of mir-208a induced apoptosis , we employed custom - built microarrays , which provided an insight into the mir-208a regulated genes associated with apoptosis
. there was a noted trend towards downregulation of genes involved in the extrinsic apoptosis pathway and upregulation of those in the intrinsic apoptosis pathway . among those upregulated was bax , an essential member in the intrinsic pathway , which was also highlighted by pathway analysis of the differentially expressed apoptosis genes ( figure 3(a ) ) . in mi
, bax was also shown to be downregulated by mir-208a silencing and may thus be playing a role in mir-208a mediated myocyte apoptosis during ischemia .
bax functions by controlling access of upstream apoptotic signals to the mitochondria and together with bak is essential for intrinsic pathway mediated apoptosis [ 17 , 26 ] . given that micrornas can upregulate gene expression by binding to the promoter regions and target sites in the mrna or indirectly by downregulating repressors [ 2729 ] , we scanned the bax promoter regions and mrna for any mir-208a binding sites .
results revealed no predicted mir-208a binding sites both in the gene promoter regions and in mrna , suggesting that mir-208a may be acting indirectly to alter bax expression .
our findings only open a window and more studies will be needed to further decipher the mechanisms involved in mir-208a mediated apoptosis . upon histological examination ,
we observed significantly reduced myocyte hypertrophy in the noninfarcted area of the lv following antagomir treatment . given that heightened cardiomyocyte hypertrophy in the noninfarcted region
is associated with remodeling and cardiac dysfunction , the noted decrease in myocyte hypertrophy may be a contributor to the observed improvement in cardiac function . moreover , antagomir therapy attenuated fibrosis , a known contributor to post - mi cardiac dysfunction .
studies have shown that mir-208a functions via upregulation of endoglin to increase myocardial fibrosis , and its silencing downregulates endoglin and reduces type 1 collagen synthesis [ 31 , 32 ] .
the observed decrease in fibrosis provides yet another possible mechanism by which mir-208a antagomir may contribute to improving cardiac function after mi . using 2d echocardiography
, we demonstrated improved cardiac function at 28 days after mi in animals therapeutically treated with mir-208a antagomir .
the observed improvement in cardiac function is likely due to a combination of factors including decrease in apoptosis , hypertrophy , and fibrosis . to the best of our knowledge
, our study is the first to demonstrate that antagomir based mir-208a silencing can attenuate cardiomyocyte apoptosis during ischemia and improve cardiac function after mi . we believe that this will provide a basis for development of therapies targeting mir-208a in mi .
 
 aims . mir-208a is associated with adverse outcomes in several cardiac pathologies known to have increased apoptosis , including myocardial infarction ( mi ) . 
 we investigated if mir-208a has proapoptotic effects on ischemic cardiomyocytes and if its silencing has therapeutic benefits in mi . 
 methods and results . 
 the effect of mir-208a on apoptosis during ischemia was studied in cultured neonatal mice myocytes transfected with agomir or antagomir . 
 differential gene expression was assessed using microarrays . 
 mi was induced in male c57bl/6 mice randomly assigned to antagomir ( n = 6 ) or control group ( n = 7 ) , while sham group ( n = 7 ) had sham operation done . 
 antagomir group received mir208a antagomir , while control and sham group mice received vehicle only . at 7 and 28 days 
 , echocardiography was done and thereafter hearts were harvested for analysis of apoptosis by tunel method , fibrosis using masson 's trichrome , and hypertrophy using hematoxylin and eosin . 
 mir-208a altered apoptosis genes expression and increased apoptosis in ischemic cardiomyocytes . 
 therapeutic inhibition of mir-208a decreased cardiac fibrosis , hypertrophy , and apoptosis and significantly improved cardiac function 28 days after mi . 
 conclusion . 
 mir-208a alters apoptosis genes expression and promotes apoptosis in ischemic cardiomyocytes , and its silencing attenuates apoptosis , fibrosis , and hypertrophy after mi , with significant improvement in cardiac function . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion