BACKGROUND
from late gestation, the majority of human cardiomyocytes cease proliferating due to either an absence of karyokinesis and/or cytokinesis .
consequently, and in contrast to the neonatal mammalian heart, the adult heart has limited capacity to regenerate cardiomyocytes that may be lost due to damage or aging .
this is dissimilar to zebrafish, whose cardiomyocytes retain the ability to proliferate throughout life and are therefore able to regenerate the heart after significant damage  <cit> .
the majority of regenerated zebrafish and neonatal mouse cardiomyocytes originate from existing cardiomyocytes rather than cardiac stem cells  and are less differentiated and have less contractile organisation compared to those from un-injured myocardium  <cit> .
recent evidence suggests that microrna  regulate cardiomyocyte proliferation and can induce reversion of adult cardiomyocytes to a pro-proliferative state  <cit> .
mirna are small  non-coding rnas that are often evolutionarily conserved between species.
mirna originate from longer gene transcripts that form hairpin loops, which are cleaved by the rnaase enzymes, drosher and dicer, to generate mature mirna.
once bound to the argonaut protein, mirna guide the rna-induced silencing complex , sometimes referred to as mirnp , to mrna targets and subsequently regulate the expression of genes by suppressing translation and/or initiating the degradation of the mrna.
there are in excess of a thousand mirna encoded by the human genome and each mirna can inhibit multiple genes, thereby potentially allowing the regulation of functional or developmental pathways .
a comparison of mirna expression in regenerated versus un-injured zebrafish myocardium identified mir- <dig> as being specifically down-regulated during the period of cardiomyocyte proliferation and regeneration  <cit> .
there are three genomic loci producing mir- <dig> with only mir-133a- <dig> and mir-133a- <dig> being expressed in the heart  <cit> .
mir- <dig> is one of the most abundant cardiac mirna  <cit>  and is essential for normal cardiogenesis in mice, through regulation of serum response factor   dependent transcription  <cit> .
interestingly, srf together with cyclin d <dig>   was associated with increased cardiomyocyte proliferation in mir-133a-1/mir-133a- <dig> double-knockout mice  <cit> .
mir-133a also regulates connexin- <dig>    <cit> , phosphoglycerate mutase    <cit>  and connective tissue growth factor    in regenerative and proliferative cardiomyocytes.
mir-133a has also been implicated as an inhibitor of vascular smooth muscle  <cit>  and ovarian cancer  <cit>  cell proliferation, through the inhibition of insulin-like growth factor receptor  <dig>  , which is a potent stimulator of cardiomyocyte proliferation  <cit> .
murine cardiomyocytes transition from a proliferative to a quiescent state from days  <dig> to  <dig> after birth  <cit> .
comparisons of mirna expression across this period identified mir- <dig>  a member of the mir- <dig> family, as the most up-regulated mirna and therefore possibly implicated in the inhibition of cardiomyocyte proliferation  <cit> .
furthermore, across the period of diminished proliferation in mice, the mrna expression was reduced for both conserved and non-conserved target genes of the mir- <dig> family, such as checkpoint kinase  <dig>  , cyclin dependent kinase- <dig>  , survivin/baculoviral inhibitor of apoptosis repeat-containing  <dig>   and sperm-associated antigen  <dig>  .
furthermore, inhibiting the mir- <dig> family in vivo increased the number of mitotic cardiomyocytes in mouse  <cit> .
both mir-133a and the mir- <dig> family are associated with inhibition of cardiomyocyte proliferation and myocardial regeneration, however, there are also mirna that promote proliferation.
mir-199a and mir- <dig> are involved in promoting proliferation in rodents  <cit> .
in addition, treating adult rat cardiomyocytes with mimics for mir-199a and mir- <dig> promotes cell cycle re-entry and promotion of cardiac regeneration in vivo  <cit> .
using short interfering rna that were specific for each gene target of mir-199a and mir- <dig>  the knockdown of  <dig> genes increased the percentage of cardiomyocytes undergoing dna synthesis by approximately 2-fold.
of the genes that were associated with the up-regulation of dna synthesis, three genes  , homeodomain-only protein   and homer protein homolog  <dig>  ) were targets of both mir-199a and mir- <dig> and are therefore believed to be important regulators of cell cycle activity in cardiomyocytes.
mouse and rat cardiomyocytes become quiescent in postnatal life and this process is complete by day  <dig>  <cit>  and  <dig>  <cit> , respectively.
the timing of quiescence in rodents corresponds to a period just after birth when there is significant cardiac remodelling triggered by changes in the circulation due to air breathing and the switch in cardiomyocyte metabolism from predominantly glycolysis in utero to fatty acid oxidation in postnatal life  <cit> .
hence, changes in mirna and target gene expression during this period in rodents are difficult to interpret due to metabolic changes occurring with developmental processes.
quiescent human cardiomyocytes have been identified from as early as  <dig>  of gestation  <cit>  and the process is generally near complete by birth, however, recent studies demonstrate that a very low level cardiomyocyte proliferation may extend to 20 years of age  <cit> .
the in utero transition to quiescence in humans is similarly timed to sheep, a species where cardiomyocytes become quiescent due to binucleation from  <dig>  of gestation  <cit>  and the percentage of cardiomyocytes in the cell cycle decreases from 7 % at 110 days gestation to 1 % close to birth  <cit> .
the current investigation aimed to gain greater insight into the regulation of human cardiomyocyte proliferation by determining the expression of specific mirna and their target genes, from the aforementioned zebrafish and murine studies, in sheep myocardium across late gestation and early postnatal life.
through the use of a mirna microarray, we further aimed to determine the expression of all mirna across this developmental window, thereby isolating the mirna that may regulate cardiomyocyte quiescence from those associated with the major physiological and biochemical changes that occur just after birth.
RESULTS
body and heart weight measurements
body weight and heart weight data were collected at each time point and, as expected, increased with age .
heart and body growth patterns were in unison until 5 days of age, such that heart weight relative to body weight was maintained, however, at 21 days of age and furthermore at 173 days of age, heart weight relative to body weight decreased .table  <dig> fetal body and heart weight measurements
f fetal age, p postnatal age in days; values are mean ± sem.
values with a different letter signify that age points are significantly different from each other.
p < <dig>  was considered significant
expression of mirna involved in regulation of cardiac proliferation and their target genes
two complementary experimental strategies were used to profile ovine mirna and their target genes over the cardiac developmental period from 91 days of gestation to 173 days of age.
first, qrt-pcr was used to measure the expression of specific ovine mirna and target genes associated with rodent and zebrafish cardiomyocyte quiescence.
second, for comprehensive analysis a mirna microarray was used to profile over  <dig> probes representing mirna from several mammalian species, including sheep and a bioinformatics approach was undertaken to predict enriched functions associated with clusters of developmentally regulated mirna.
developmental profiling of rodent and zebrafish mirna by qrt-pcr
the ovine cardiac expression of three sets of mirna, which are highly conserved across species, and their target genes that were previously associated with regulation of cardiomyocyte proliferation in rodent and zebrafish  <cit>  were measured by qrt-pcr at each developmental age to investigate their roles in prenatal cardiomyocyte quiescence in a larger mammal, sheep, characterised by quiescence of cardiomyocytes in late gestation.
mir-133a
the expression of mir-133a increased with age and reached its maximum expression by 5 days of age .
counterintuitively, the expression of the mir-133a gene targets that promote cardiomyocyte proliferation in rodents, ccnd <dig>  srf, pgam <dig> and gja <dig>  had their highest expression after birth , when the majority of sheep cardiomyocytes were binucleated and quiescent.
furthermore, the expression of mir-133a gene target ctfg did not change with age .
these results suggest that these genes are not directly regulated by mir-133a in sheep or are subject to more complex and overriding developmental regulators.
the result also indicates that these genes are unlikely to be directly involved in ovine cardiomyocyte quiescence in sheep.
the increased expression of mir-133a, however, coincided with decreased expression of igf1r, a known target for this mirna  <cit>  .fig.
<dig> expression of mir-133a and its target mrnas during heart development.
mir-133a increased with age  and the expression of its target genes, pgam <dig>  gja <dig>  srf and ccnd <dig> increased between  <dig> and 21 days of age  and igf1r decreases with age .
data are expressed relative to 91 days gestation.
f fetal, p postnatal, d age in days, ns not significant.
values with a different letter signify that age points are significantly different from each other.
p < <dig>  was considered significant
mir- <dig> family
the mir- <dig> family contains five mirna, mir-15a, mir-15b, mir- <dig>  mir- <dig> and mir- <dig>  ovine cardiac expression of mir-15a expression was lowest at 140 days gestation and highest at 21 days of age , the expression of mir-15b was lowest at 173 days of age and highest at 5 days of age  and mir- <dig> expression was lowest at 140 days gestation and highest at 91 days gestation,  <dig> and 21 days of age .
the expression of both mir- <dig> and mir- <dig> did not change with age .
despite the variable expression patterns of members of the mir- <dig> family, the expression of their target mrnas  decreased with increasing age, reaching a minimum and then plateauing at 140 days gestation .
given that these genes are involved in cell cycle progression and mitosis , these results suggest that the decreased expression of these genes may be involved in ovine cardiomyocyte quiescence, however, the mir- <dig> family may not be the principal developmental regulator of these genes or ovine cardiomyocyte quiescence.fig.
<dig> expression of mir- <dig> family members and their target mrna during heart development.
the expression of mir- <dig> family members is variable with age , however, the expression of their target genes chek <dig>  cdc2a, birc <dig> and spag <dig> decreased with age .
data are expressed relative to 91 days gestation; f fetal, p postnatal, d age in days, ns not significant.
values with a different letter signify that age points are significantly different from each other.
p < <dig>  was considered significant
mir-199a and mir-590
the expression of mir-199a peaked around birth, but then decreased by 21 days of age to the level seen at 91 days gestation .
the expression of mir- <dig>  however, did not change with age .
the expression of hopx, a target of both mir-199a and mir- <dig>  <cit> , did not change with age , however, the expression of clic <dig> and homer <dig> increased with age and reached their maximum expression by 5 days and 173 days of age, respectively .
these data suggest that mir-199a and mir- <dig> mirna and their target genes may not be directly involved in ovine cardiomyocyte quiescence.fig.
<dig> expression of pro-proliferative mir, mir-199a and mir- <dig>  and their target mrna during heart development.
mir-199a increases with age, but is then reduced to the level seen at 91 days gestation by 21 days of age .
the expression of clic <dig> and homer <dig> is increased in postnatal life .
data are expressed relative to 91 days gestation; f, fetal; p, postnatal; d, age in days.
values with a different letter signify that age points are significantly different from each other.
p < <dig>  was considered significant
mirna microarray analysis
the targeted profiling of mirna identified in rodent and zebrafish investigations showed limited correspondence with the developmental program of ovine cardiomyocyte quiescence occurring during late gestation in sheep.
consequently, mirna microarrays were used to investigate age related changes in cardiac mirna in a subset of the animals   <dig>  f <dig>  postnatal   <dig> and p173).
this approach complements the targeted mirna approach and has the potential to identify additional mirna and possibly broader functional themes associated with cardiac development in sheep.
a custom designed mirna microarray that incorporated multiple replicates of  <dig>  unique probes for sheep and other mammalian mirna was used.
technical limitations of the approach are listed in the methods.
a pca analysis of the microarray data shows separation of the four developmental groups and clustering of the biological replicates within these groups .
the first component in the analysis explained 97 % of the variation.
a total of  <dig>  differentially expressed probes  were identified, which included  <dig> anonymous probes and some redundant probes.
the full analysis is presented in additional file  <dig>  the analysis clearly indicates that multiple mirna are involved in cardiac development and age related functions, and suggests substantial complexity in the regulation of target gene expression orchestrating these developmental and physiological changes.fig.
<dig> principal components analysis of the mirna microarray data.
the data represented in a pca plot show separation of the four developmental age groups and clustering of the biological replicates within these groups.
red, fetal 91 days gestation; green, fetal 140 days gestation; blue, postnatal 5 days; yellow, postnatal 173 days
k-means clustering of the differentially expressed mirna separated the developmental data into six discrete clusters .
this analysis posits that mirna within a cluster have functions pertaining to the specific developmental transition represented by the cluster profile.
the largest clusters were  <dig> and  <dig> containing  <dig> and  <dig> probes, respectively .
the former cluster represented a progressive decline in overall signal with all developmental time points while the latter cluster represented a progressive increase of signal with development.
clusters  <dig>   <dig> and  <dig>  showed more complex developmental profiles that may reflect key developmental transitions in the myocardium and the indirect impacts of the major physiological and biochemical changes occurring after birth.
in particular, these clusters are characterised by major transitions between the late gestation fetus at 140 days gestation and the lamb at 5 days after birth.
cluster  <dig> showed a profile characterised as a small continuous decline until 5 days after birth followed by a more substantial decline to 173 days after birth.fig.
<dig> k-means clustering of the differentially expressed mirna.
mirna were grouped into co-expression clusters using k-means clustering.
a summary of non-redundant mirna present in each k-means cluster can be found in table  <dig>  f fetal, p postnatal, d age in days
aall species identifiers were removed.
fourteen anonymous probes beginning with the designation ‘pc’ were excluded, but can be found with species identifiers in additional file  <dig> b mirna included in the targeted qrt-pcr investigatation highlighted in bold.
in general, the patterns of the mirna measured by qrt-pcr were consistent with the cluster profiles.
mir-133a was present in cluster  <dig> and displayed the same pattern of expression in both qrt-pcr and microarray analysis.
mir- <dig> family members mir-15b, mir- <dig>  mir- <dig> and mir- <dig> were identified in clusters  <dig>   <dig>   <dig> and  <dig>  respectively.
mir-199a was identified in cluster  <dig> and the expression pattern of mir- <dig> did not allow its inclusion in any cluster.
mirna are often genomically positioned in groups on chromosomes and we therefore investigated whether any of the mirna clusters were associated with specific chromosomal regions.
by mapping the mirna to ovine chromosomes , we determined that cluster  <dig> was strongly overrepresented by mirna  associated with a large and complex group of mirna in a 250 kb region located toward the telomeric end of oar <dig> .
the broader  <dig>  mb region encompassing the protein encoding genes begain to dio <dig> is imprinted and the mirna within this region are maternally expressed .
the biological and molecular roles of these mirna are unclear.fig.
<dig> schematic representation of an imprinted genomic segment on the telomeric arm of ovine chromosome  <dig>  the genomic region highlights multiple mirna many of which were identified as differentially expressed in cluster  <dig>  blue, paternally expressed protein encoding genes.
pink, maternally expressed noncoding rna.
the central region contains a number of maternally expressed mirna including a large cluster designated mirg.
meg <dig> contains a large cluster of snorna while meg <dig> is a long noncoding rna.
rtl1as transcribes a noncoding rna containing five mirna, which are known to cause risc mediated cleavage of the sense protein encoding gene rtl <dig>  all maternally expressed genes are transcribed in the same direction
to identify putative mrna targets for the mirna, a non-redundant set of mirna corresponding to each k-means cluster  was used for mrna target prediction using mirwalk.
the strategy undertaken was highly conservative as mirwalk was chosen to include a consensus of at least seven of ten different mirna target prediction methods  <cit> .
the numbers of unique target mrnas for the six mirna clusters were  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  respectively.
enriched gene ontology  terms and kegg pathways were then identified using david  <cit>  .
the full kegg pathway analysis and the enriched go categories biological process, cellular component and molecular function for each cluster are listed in additional file  <dig> fig.
<dig> representations of enriched kegg pathways for predicted target genes associated with each k-means cluster.
david was used to identify enriched kegg pathways  <cit> .
an fdr adjusted p-value < <dig>  was considered significant.
the top ranked five pathways are shown.
clusters  <dig> and  <dig> did not produce any significantly enriched kegg pathways.
each cluster is identified by a number.
fdr corrected p-values are shown on a –log <dig> scale .
full data for enriched kegg pathways and the three go categories biological process, cellular component and molecular function are listed in additional file 2
cluster  <dig> was strongly associated with an extracellular matrix theme in the enriched kegg pathway and go cellular component  analyses.
there was also a theme related to regulation of transcription revealed by the go biological process  and molecular function  categories.
kegg pathway analysis for cluster  <dig> showed enrichment for proliferative activity and insulin signalling while regulation of transcription was a common theme in the go bp and mf analyses.
go bp showed enrichment for aspects of macromolecule metabolism and biosynthesis.
cluster  <dig> showed enrichment for protein transport  and synapse , the latter possibly indicative of innervation changes.
cluster  <dig> was associated with regulation of transcription , regulation of biosynthetic processes  and neuron projection .
enrichment for the tgfβ pathway was linked with cluster  <dig> as well as regulation of phosphate metabolic processes , golgi  and regulation of transcription .
cluster  <dig> showed mixed themes including mapk signalling and extracellular matrix , regulation of transcription  and golgi .
importantly, clusters  <dig>   <dig> and  <dig> contained enriched pathways that have been associated with “cancer”, which likely reflect aspects of cell proliferation, while clusters  <dig>   <dig> and 4– <dig> contained enrichment for terms associated with transcription factor regulation, which also may reflect transcriptional regulation of cardiomyocyte proliferative activity.
discussion
this study is the first to analyse the expression of mirna in a mammalian model  where, as in humans, cardiomyocyte quiescence occurs in utero.
the current study highlights species specific differences and some similarities in the expression patterns of specific mirna previously implicated in the regulation of cardiomyocyte proliferation and regeneration in rodents and zebrafish.
the interpretation of mirna and target gene developmental profiles in different species may be complex.
first, the sheep and rodent models of cardiac development are inherently different in terms of the timing of cardiomyocyte quiescence and moreover the latter models are potentially confounded by superimposition of developmental changes with large scale postnatal metabolic changes and cardiomyocyte hypertrophy.
second, some mirna target genes may vary in the sequence of their potential mirna recognition sites leading to changes in the binding affinity and stoichiometry of mirna with target genes.
this could generate altered molecular processes regulating similar developmental processes in a species specific manner.
third, changes in mirna abundance may impact other related mirna through direct competition for target mrna.
the use of a mirna microarray in combination with targeted analyses in the sheep model has revealed enrichment for clusters of mirna belonging to functional categories possibly associated with cardiomyocyte proliferation/quiescence such as ‘proliferative activity’ and ‘cancer’  and provided broad insight into the program of cardiac development in a large mammal where cardiomyocyte quiescence occurs during late gestation, similar to human and unlike rodents and zebrafish.
identification of mirna with expression profiles consistent with involvement in inhibition of cardiomyocyte proliferation in sheep
cardiomyocyte quiescence is observed in sheep from  <dig>  of gestation  <cit>  and only ~1 % of cardiomyocytes are in the cell cycle near term  <cit> .
mirna that are key inhibitors of cardiomyocyte proliferation would likely fall predominantly into mirna cluster  <dig> or possibly  <dig> and  <dig>  since their expression patterns increase with age.
consistent with this view, mirna implicated in rodent cardiomyocyte quiescence, mir-133a, mir- <dig> and mir- <dig>  were identified in clusters  <dig>   <dig> and  <dig>  respectively, from the mirna microarray analysis.
in particular, mir-133a, the highly abundant cardiac mirna implicated in regulation of cardiomyocyte proliferation in zebrafish  <cit>  and mice  <cit> , was confirmed by qrt-pcr and microarray analysis as increasing with developmental age in sheep.
the role of mir-133a as a suppressor of cardiomyocyte proliferation is supported by both mir-133a double-knockout  mouse studies showing that mir-133a is essential for regulating cardiomyocyte proliferation and normal heart development  <cit> , and zebrafish studies, where mir-133a was significantly reduced in regenerative/proliferative cardiac tissue  <cit> .
the increased abundance of mir-133a coincided with the decreased expression of one of its gene targets, igf1r.
the igf1r signalling pathway is an important regulator of cardiac development  <cit>  and stimulates cardiomyocyte proliferation  <cit> .
the current investigation is the first to highlight a potential link between mir-133a and igf1r in the regulation of cardiomyocyte proliferation in a large mammal.
however, the expression of other mir-133a target genes  that are pro-proliferative , either increased in postnatal life  or did not change with age  in the sheep.
these observations suggest that these genes are not target genes for mir-133a in sheep or that there are additional contributors to cardiomyocyte quiescence that counter the effects of these pro-proliferative genes.
additional limitations associated with cross species comparative analyses of mirna function are listed in the methods section.
although the mir- <dig> family has been implicated in regulation of cardiomyocyte proliferation in rodents  <cit> , there was remarkable variation in the expression profiles for the family members, being represented in clusters  <dig>   <dig>   <dig> and  <dig>  since these family members have highly similar target mrna, it is difficult to interpret the relevance of individual family members in regulating cardiomyocyte proliferation.
one possibility is that the different expression patterns represent fine tuning of the ratio of this mirna family to target mrna at different development ages.
the expression profiles of mir- <dig> family target mrna  were consistent with decreased cardiomyocyte proliferation perhaps representing their specific roles in cell cycle regulation.
the current findings in sheep however suggest that the mir- <dig> family may not be pivotal or direct regulators of the specific decrease in cardiomyocyte proliferation that occurs in late gestation and in early postnatal life in sheep.
while the exact role of each mirna in cluster  <dig>   <dig> and  <dig> is yet to be defined, it is noteworthy that mir- <dig>  which has been implicated as an inhibitor of cell proliferation by down-regulating igf1r in bladder cancer cells  <cit>  and other cancers , was present in cluster  <dig>  further analysis into the functional role of each mirna identified in these clusters is required in order to determine which mirna have a role in developmental cardiomyocyte quiescence.
identification of mirna that may promote cardiomyocyte proliferation in sheep
in sheep, mirna that have been previously identified as key promoters of proliferation in rodents are predicted to have decreased expression with age and therefore reside in clusters  <dig>   <dig> or  <dig>  mir-199a, which has been shown to stimulate proliferation in cultured rat cardiomyocytes  <cit>  was identified in cluster  <dig>  however, further analysis using qrt-pcr demonstrated that mir-199a peaked around birth, then decreased into postnatal life to the level of that seen at 91 days gestation.
furthermore, the expression pattern of pro-proliferative mir- <dig>  <cit>  did not allow its inclusion in any cluster and qrt-pcr demonstrated a similar expression at all ages.
considering the varied expression of their target genes, the outcomes of the present study suggest that mir-199a and mir- <dig> are not major regulators of developmental cardiomyocyte quiescence in sheep.
the roles of every mirna in clusters  <dig>   <dig> and  <dig> have not been identified, however, some mirna in these clusters have been involved in aspects of the regulation of cell proliferation in other species.
recent studies have implicated the mir-17- <dig> group as an important regulator of cardiomyocyte proliferation and capable of stimulating previously quiescent, adult mice cardiomyocytes  <cit> .
there are six members of the mir-17- <dig> group, which includes mir- <dig>  mir-18a, mir-19a, mir-20a, mir-19b- <dig> and mir-92a- <dig>  in the present study, mir- <dig> and mir- <dig> were in cluster  <dig> , which suggests a potential role of these mirna in the inhibition of cardiomyocyte proliferation in late gestation, however, mir-20a and mir-19b were in cluster  <dig>  and the expression of mir-18a and 19a did not change with age.
thus, the specific roles of mirna in the mir-17- <dig> group in regulating cardiomyocyte quiescence are unclear.
the developmental regulation of an imprinted mirna cluster during heart development
the present study has allowed us to isolate the period when most cardiomyocytes lose the ability to proliferate from other cellular processes, such as hypertrophy, that are upregulated after birth.
cluster  <dig> was particularly enriched for mirna derived from an imprinted locus located on the telomeric end of oar <dig> in sheep .
the syntenic region is conserved in placental mammals and it contains a large cluster of maternally expressed mirna that may have complex roles in the cis- and trans-regulation of expression of genes within the broader imprinted ~ <dig>  mb region or direct involvement in the maintenance of the imprinting status of the locus  <cit> .
a mutation  in this broader imprinted region in sheep strongly deregulates expression of multiple genes within the core of this locus, including the maternally expressed mirna, and causes postnatal skeletal muscle hypertrophy in select muscles in a rostro-caudal gradient across the animal but only when the mutation is inherited in the context of the paternal heterozygote  <cit> .
the mutation apparently does not impact cardiac function, although specific tests have not been undertaken to date.
whether the mirna within this genomic locus are targeted at mrna or involved in imprinting maintenance is not clear, but it is interesting to note that the mirna from this cluster putatively target the kegg tgfβ pathway.
multiple members of this pathway have strong roles in regulating muscle development  <cit>  and cardiac development and disease  <cit> .
maternally expressed genes typically limit aspects of offspring tissue growth and are often down-regulated after birth  <cit> .
we therefore postulate that the down-regulation of these mirna after birth may help coordinate the activation of negative regulators of growth thereby limiting cardiomyocyte size and possibly heart size relative to body size.
functional analysis of predicted targets of developmentally regulated mirna clusters
the functional enrichment analyses for the predicted target genes for each mirna cluster from the mirna microarray study often showed enrichment for “cancer” related themes, which is likely indirectly reflecting proliferative potential, and also enrichment for the related terms transcription factor regulation and proliferative activity.
thus, there is a consistent functional theme relating to regulation of proliferation in the predicted target genes of the differentially expressed mirna.
one of the difficulties in the biological interpretation of mirna data relates to the abundance of a mirna, its mrna targets and the effects of competing mirna, which together interplay in a complex manner to regulate gene expression.
therefore, understanding the enriched biological themes in the current investigation will require functional modulation of multiple mirna in an appropriate cell culture model of cardiomyocyte cell proliferation and differentiation, coupled with biochemical validation of putative mirna target genes.
CONCLUSIONS
this study has identified mirna and their target genes associated with cardiac development across late gestation and into postnatal life in sheep, thereby isolating the transition of cardiomyocytes to quiescence that occurs in late gestation from the major physiological and biochemical changes that occur after birth.
the investigation has identified a number of clusters of differentially expressed mirna, some of which are consistent in timing with the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep.
the present study is also consistent with a coordinated and complex role of mirna in the regulation of cardiomyocyte quiescence.
the study identified substantial differences between sheep and rodent cardiac development that may reflect species differences underpinning the very different timing relative to birth of cardiomyocyte quiescence in these species.
importantly, through the use of mirna microarrays, the current study highlights the potential involvement of many mirna in the coordination of this pivotal developmental transition.

 from late gestation, the majority of human cardiomyocytes cease proliferating due to either an absence of karyokinesis and/or cytokinesis. consequently, and in contrast to the neonatal mammalian heart, the adult heart has limited capacity to regenerate cardiomyocytes that may be lost due to damage or aging. this is dissimilar to zebrafish, whose cardiomyocytes retain the ability to proliferate throughout life and are therefore able to regenerate the heart after significant damage. the majority of regenerated zebrafish and neonatal mouse cardiomyocytes originate from existing cardiomyocytes rather than cardiac stem cells and are less differentiated and have less contractile organisation compared to those from un-injured myocardium. recent evidence suggests that microrna regulate cardiomyocyte proliferation and can induce reversion of adult cardiomyocytes to a pro-proliferative state. mirna are small non-coding rnas that are often evolutionarily conserved between species. mirna originate from longer gene transcripts that form hairpin loops, which are cleaved by the rnaase enzymes, drosher and dicer, to generate mature mirna. once bound to the argonaut protein, mirna guide the rna-induced silencing complex, sometimes referred to as mirnp, to mrna targets and subsequently regulate the expression of genes by suppressing translation and/or initiating the degradation of the mrna. there are in excess of a thousand mirna encoded by the human genome and each mirna can inhibit multiple genes, thereby potentially allowing the regulation of functional or developmental pathways. a comparison of mirna expression in regenerated versus un-injured zebrafish myocardium identified mir- as being specifically down-regulated during the period of cardiomyocyte proliferation and regeneration. there are three genomic loci producing mir- with only mir-133a- and mir-133a- being expressed in the heart. mir- is one of the most abundant cardiac mirna and is essential for normal cardiogenesis in mice, through regulation of serum response factor dependent transcription. interestingly, srf together with cyclin d was associated with increased cardiomyocyte proliferation in mir-133a-1/mir-133a- double-knockout mice. mir-133a also regulates connexin-, phosphoglycerate mutase and connective tissue growth factor in regenerative and proliferative cardiomyocytes. mir-133a has also been implicated as an inhibitor of vascular smooth muscle and ovarian cancer cell proliferation, through the inhibition of insulin-like growth factor receptor, which is a potent stimulator of cardiomyocyte proliferation. murine cardiomyocytes transition from a proliferative to a quiescent state from days to after birth. comparisons of mirna expression across this period identified mir- a member of the mir- family, as the most up-regulated mirna and therefore possibly implicated in the inhibition of cardiomyocyte proliferation. furthermore, across the period of diminished proliferation in mice, the mrna expression was reduced for both conserved and non-conserved target genes of the mir- family, such as checkpoint kinase, cyclin dependent kinase-, survivin/baculoviral inhibitor of apoptosis repeat-containing and sperm-associated antigen. furthermore, inhibiting the mir- family in vivo increased the number of mitotic cardiomyocytes in mouse. both mir-133a and the mir- family are associated with inhibition of cardiomyocyte proliferation and myocardial regeneration, however, there are also mirna that promote proliferation. mir-199a and mir- are involved in promoting proliferation in rodents. in addition, treating adult rat cardiomyocytes with mimics for mir-199a and mir- promotes cell cycle re-entry and promotion of cardiac regeneration in vivo. using short interfering rna that were specific for each gene target of mir-199a and mir- the knockdown of genes increased the percentage of cardiomyocytes undergoing dna synthesis by approximately 2-fold. of the genes that were associated with the up-regulation of dna synthesis, three genes, homeodomain-only protein and homer protein homolog ) were targets of both mir-199a and mir- and are therefore believed to be important regulators of cell cycle activity in cardiomyocytes. mouse and rat cardiomyocytes become quiescent in postnatal life and this process is complete by day and, respectively. the timing of quiescence in rodents corresponds to a period just after birth when there is significant cardiac remodelling triggered by changes in the circulation due to air breathing and the switch in cardiomyocyte metabolism from predominantly glycolysis in utero to fatty acid oxidation in postnatal life. hence, changes in mirna and target gene expression during this period in rodents are difficult to interpret due to metabolic changes occurring with developmental processes. quiescent human cardiomyocytes have been identified from as early as of gestation and the process is generally near complete by birth, however, recent studies demonstrate that a very low level cardiomyocyte proliferation may extend to 20 years of age. the in utero transition to quiescence in humans is similarly timed to sheep, a species where cardiomyocytes become quiescent due to binucleation from of gestation and the percentage of cardiomyocytes in the cell cycle decreases from 7 % at 110 days gestation to 1 % close to birth. the current investigation aimed to gain greater insight into the regulation of human cardiomyocyte proliferation by determining the expression of specific mirna and their target genes, from the aforementioned zebrafish and murine studies, in sheep myocardium across late gestation and early postnatal life. through the use of a mirna microarray, we further aimed to determine the expression of all mirna across this developmental window, thereby isolating the mirna that may regulate cardiomyocyte quiescence from those associated with the major physiological and biochemical changes that occur just after birth.  body and heart weight measurements body weight and heart weight data were collected at each time point and, as expected, increased with age. heart and body growth patterns were in unison until 5 days of age, such that heart weight relative to body weight was maintained, however, at 21 days of age and furthermore at 173 days of age, heart weight relative to body weight decreased.table fetal body and heart weight measurements f fetal age, p postnatal age in days; values are mean ± sem. values with a different letter signify that age points are significantly different from each other. p < was considered significant expression of mirna involved in regulation of cardiac proliferation and their target genes two complementary experimental strategies were used to profile ovine mirna and their target genes over the cardiac developmental period from 91 days of gestation to 173 days of age. first, qrt-pcr was used to measure the expression of specific ovine mirna and target genes associated with rodent and zebrafish cardiomyocyte quiescence. second, for comprehensive analysis a mirna microarray was used to profile over probes representing mirna from several mammalian species, including sheep and a bioinformatics approach was undertaken to predict enriched functions associated with clusters of developmentally regulated mirna. developmental profiling of rodent and zebrafish mirna by qrt-pcr the ovine cardiac expression of three sets of mirna, which are highly conserved across species, and their target genes that were previously associated with regulation of cardiomyocyte proliferation in rodent and zebrafish were measured by qrt-pcr at each developmental age to investigate their roles in prenatal cardiomyocyte quiescence in a larger mammal, sheep, characterised by quiescence of cardiomyocytes in late gestation. mir-133a the expression of mir-133a increased with age and reached its maximum expression by 5 days of age. counterintuitively, the expression of the mir-133a gene targets that promote cardiomyocyte proliferation in rodents, ccnd srf, pgam and gja had their highest expression after birth, when the majority of sheep cardiomyocytes were binucleated and quiescent. furthermore, the expression of mir-133a gene target ctfg did not change with age. these results suggest that these genes are not directly regulated by mir-133a in sheep or are subject to more complex and overriding developmental regulators. the result also indicates that these genes are unlikely to be directly involved in ovine cardiomyocyte quiescence in sheep. the increased expression of mir-133a, however, coincided with decreased expression of igf1r, a known target for this mirna.fig. expression of mir-133a and its target mrnas during heart development. mir-133a increased with age and the expression of its target genes, pgam gja srf and ccnd increased between and 21 days of age and igf1r decreases with age. data are expressed relative to 91 days gestation. f fetal, p postnatal, d age in days, ns not significant. values with a different letter signify that age points are significantly different from each other. p < was considered significant mir- family the mir- family contains five mirna, mir-15a, mir-15b, mir- mir- and mir- ovine cardiac expression of mir-15a expression was lowest at 140 days gestation and highest at 21 days of age, the expression of mir-15b was lowest at 173 days of age and highest at 5 days of age and mir- expression was lowest at 140 days gestation and highest at 91 days gestation, and 21 days of age. the expression of both mir- and mir- did not change with age. despite the variable expression patterns of members of the mir- family, the expression of their target mrnas decreased with increasing age, reaching a minimum and then plateauing at 140 days gestation. given that these genes are involved in cell cycle progression and mitosis, these results suggest that the decreased expression of these genes may be involved in ovine cardiomyocyte quiescence, however, the mir- family may not be the principal developmental regulator of these genes or ovine cardiomyocyte quiescence.fig. expression of mir- family members and their target mrna during heart development. the expression of mir- family members is variable with age, however, the expression of their target genes chek cdc2a, birc and spag decreased with age. data are expressed relative to 91 days gestation; f fetal, p postnatal, d age in days, ns not significant. values with a different letter signify that age points are significantly different from each other. p < was considered significant mir-199a and mir-590 the expression of mir-199a peaked around birth, but then decreased by 21 days of age to the level seen at 91 days gestation. the expression of mir- however, did not change with age. the expression of hopx, a target of both mir-199a and mir-, did not change with age, however, the expression of clic and homer increased with age and reached their maximum expression by 5 days and 173 days of age, respectively. these data suggest that mir-199a and mir- mirna and their target genes may not be directly involved in ovine cardiomyocyte quiescence.fig. expression of pro-proliferative mir, mir-199a and mir- and their target mrna during heart development. mir-199a increases with age, but is then reduced to the level seen at 91 days gestation by 21 days of age. the expression of clic and homer is increased in postnatal life. data are expressed relative to 91 days gestation; f, fetal; p, postnatal; d, age in days. values with a different letter signify that age points are significantly different from each other. p < was considered significant mirna microarray analysis the targeted profiling of mirna identified in rodent and zebrafish investigations showed limited correspondence with the developmental program of ovine cardiomyocyte quiescence occurring during late gestation in sheep. consequently, mirna microarrays were used to investigate age related changes in cardiac mirna in a subset of the animals f postnatal and p173). this approach complements the targeted mirna approach and has the potential to identify additional mirna and possibly broader functional themes associated with cardiac development in sheep. a custom designed mirna microarray that incorporated multiple replicates of unique probes for sheep and other mammalian mirna was used. technical limitations of the approach are listed in the methods. a pca analysis of the microarray data shows separation of the four developmental groups and clustering of the biological replicates within these groups. the first component in the analysis explained 97 % of the variation. a total of differentially expressed probes were identified, which included anonymous probes and some redundant probes. the full analysis is presented in additional file the analysis clearly indicates that multiple mirna are involved in cardiac development and age related functions, and suggests substantial complexity in the regulation of target gene expression orchestrating these developmental and physiological changes.fig. principal components analysis of the mirna microarray data. the data represented in a pca plot show separation of the four developmental age groups and clustering of the biological replicates within these groups. red, fetal 91 days gestation; green, fetal 140 days gestation; blue, postnatal 5 days; yellow, postnatal 173 days k-means clustering of the differentially expressed mirna separated the developmental data into six discrete clusters. this analysis posits that mirna within a cluster have functions pertaining to the specific developmental transition represented by the cluster profile. the largest clusters were and containing and probes, respectively. the former cluster represented a progressive decline in overall signal with all developmental time points while the latter cluster represented a progressive increase of signal with development. clusters and showed more complex developmental profiles that may reflect key developmental transitions in the myocardium and the indirect impacts of the major physiological and biochemical changes occurring after birth. in particular, these clusters are characterised by major transitions between the late gestation fetus at 140 days gestation and the lamb at 5 days after birth. cluster showed a profile characterised as a small continuous decline until 5 days after birth followed by a more substantial decline to 173 days after birth.fig. k-means clustering of the differentially expressed mirna. mirna were grouped into co-expression clusters using k-means clustering. a summary of non-redundant mirna present in each k-means cluster can be found in table  f fetal, p postnatal, d age in days aall species identifiers were removed. fourteen anonymous probes beginning with the designation ‘pc’ were excluded, but can be found with species identifiers in additional file b mirna included in the targeted qrt-pcr investigatation highlighted in bold. in general, the patterns of the mirna measured by qrt-pcr were consistent with the cluster profiles. mir-133a was present in cluster and displayed the same pattern of expression in both qrt-pcr and microarray analysis. mir- family members mir-15b, mir- mir- and mir- were identified in clusters and respectively. mir-199a was identified in cluster and the expression pattern of mir- did not allow its inclusion in any cluster. mirna are often genomically positioned in groups on chromosomes and we therefore investigated whether any of the mirna clusters were associated with specific chromosomal regions. by mapping the mirna to ovine chromosomes, we determined that cluster was strongly overrepresented by mirna associated with a large and complex group of mirna in a 250 kb region located toward the telomeric end of oar. the broader  mb region encompassing the protein encoding genes begain to dio is imprinted and the mirna within this region are maternally expressed. the biological and molecular roles of these mirna are unclear.fig. schematic representation of an imprinted genomic segment on the telomeric arm of ovine chromosome the genomic region highlights multiple mirna many of which were identified as differentially expressed in cluster blue, paternally expressed protein encoding genes. pink, maternally expressed noncoding rna. the central region contains a number of maternally expressed mirna including a large cluster designated mirg. meg contains a large cluster of snorna while meg is a long noncoding rna. rtl1as transcribes a noncoding rna containing five mirna, which are known to cause risc mediated cleavage of the sense protein encoding gene rtl all maternally expressed genes are transcribed in the same direction to identify putative mrna targets for the mirna, a non-redundant set of mirna corresponding to each k-means cluster was used for mrna target prediction using mirwalk. the strategy undertaken was highly conservative as mirwalk was chosen to include a consensus of at least seven of ten different mirna target prediction methods. the numbers of unique target mrnas for the six mirna clusters were and respectively. enriched gene ontology terms and kegg pathways were then identified using david. the full kegg pathway analysis and the enriched go categories biological process, cellular component and molecular function for each cluster are listed in additional file fig. representations of enriched kegg pathways for predicted target genes associated with each k-means cluster. david was used to identify enriched kegg pathways. an fdr adjusted p-value < was considered significant. the top ranked five pathways are shown. clusters and did not produce any significantly enriched kegg pathways. each cluster is identified by a number. fdr corrected p-values are shown on a –log scale. full data for enriched kegg pathways and the three go categories biological process, cellular component and molecular function are listed in additional file 2 cluster was strongly associated with an extracellular matrix theme in the enriched kegg pathway and go cellular component analyses. there was also a theme related to regulation of transcription revealed by the go biological process and molecular function categories. kegg pathway analysis for cluster showed enrichment for proliferative activity and insulin signalling while regulation of transcription was a common theme in the go bp and mf analyses. go bp showed enrichment for aspects of macromolecule metabolism and biosynthesis. cluster showed enrichment for protein transport and synapse, the latter possibly indicative of innervation changes. cluster was associated with regulation of transcription, regulation of biosynthetic processes and neuron projection. enrichment for the tgfβ pathway was linked with cluster as well as regulation of phosphate metabolic processes, golgi and regulation of transcription. cluster showed mixed themes including mapk signalling and extracellular matrix, regulation of transcription and golgi. importantly, clusters and contained enriched pathways that have been associated with “cancer”, which likely reflect aspects of cell proliferation, while clusters and 4– contained enrichment for terms associated with transcription factor regulation, which also may reflect transcriptional regulation of cardiomyocyte proliferative activity. discussion this study is the first to analyse the expression of mirna in a mammalian model where, as in humans, cardiomyocyte quiescence occurs in utero. the current study highlights species specific differences and some similarities in the expression patterns of specific mirna previously implicated in the regulation of cardiomyocyte proliferation and regeneration in rodents and zebrafish. the interpretation of mirna and target gene developmental profiles in different species may be complex. first, the sheep and rodent models of cardiac development are inherently different in terms of the timing of cardiomyocyte quiescence and moreover the latter models are potentially confounded by superimposition of developmental changes with large scale postnatal metabolic changes and cardiomyocyte hypertrophy. second, some mirna target genes may vary in the sequence of their potential mirna recognition sites leading to changes in the binding affinity and stoichiometry of mirna with target genes. this could generate altered molecular processes regulating similar developmental processes in a species specific manner. third, changes in mirna abundance may impact other related mirna through direct competition for target mrna. the use of a mirna microarray in combination with targeted analyses in the sheep model has revealed enrichment for clusters of mirna belonging to functional categories possibly associated with cardiomyocyte proliferation/quiescence such as ‘proliferative activity’ and ‘cancer’ and provided broad insight into the program of cardiac development in a large mammal where cardiomyocyte quiescence occurs during late gestation, similar to human and unlike rodents and zebrafish. identification of mirna with expression profiles consistent with involvement in inhibition of cardiomyocyte proliferation in sheep cardiomyocyte quiescence is observed in sheep from of gestation and only ~1 % of cardiomyocytes are in the cell cycle near term. mirna that are key inhibitors of cardiomyocyte proliferation would likely fall predominantly into mirna cluster or possibly and since their expression patterns increase with age. consistent with this view, mirna implicated in rodent cardiomyocyte quiescence, mir-133a, mir- and mir- were identified in clusters and respectively, from the mirna microarray analysis. in particular, mir-133a, the highly abundant cardiac mirna implicated in regulation of cardiomyocyte proliferation in zebrafish and mice, was confirmed by qrt-pcr and microarray analysis as increasing with developmental age in sheep. the role of mir-133a as a suppressor of cardiomyocyte proliferation is supported by both mir-133a double-knockout mouse studies showing that mir-133a is essential for regulating cardiomyocyte proliferation and normal heart development, and zebrafish studies, where mir-133a was significantly reduced in regenerative/proliferative cardiac tissue. the increased abundance of mir-133a coincided with the decreased expression of one of its gene targets, igf1r. the igf1r signalling pathway is an important regulator of cardiac development and stimulates cardiomyocyte proliferation. the current investigation is the first to highlight a potential link between mir-133a and igf1r in the regulation of cardiomyocyte proliferation in a large mammal. however, the expression of other mir-133a target genes that are pro-proliferative, either increased in postnatal life or did not change with age in the sheep. these observations suggest that these genes are not target genes for mir-133a in sheep or that there are additional contributors to cardiomyocyte quiescence that counter the effects of these pro-proliferative genes. additional limitations associated with cross species comparative analyses of mirna function are listed in the methods section. although the mir- family has been implicated in regulation of cardiomyocyte proliferation in rodents, there was remarkable variation in the expression profiles for the family members, being represented in clusters and since these family members have highly similar target mrna, it is difficult to interpret the relevance of individual family members in regulating cardiomyocyte proliferation. one possibility is that the different expression patterns represent fine tuning of the ratio of this mirna family to target mrna at different development ages. the expression profiles of mir- family target mrna were consistent with decreased cardiomyocyte proliferation perhaps representing their specific roles in cell cycle regulation. the current findings in sheep however suggest that the mir- family may not be pivotal or direct regulators of the specific decrease in cardiomyocyte proliferation that occurs in late gestation and in early postnatal life in sheep. while the exact role of each mirna in cluster and is yet to be defined, it is noteworthy that mir- which has been implicated as an inhibitor of cell proliferation by down-regulating igf1r in bladder cancer cells and other cancers, was present in cluster further analysis into the functional role of each mirna identified in these clusters is required in order to determine which mirna have a role in developmental cardiomyocyte quiescence. identification of mirna that may promote cardiomyocyte proliferation in sheep in sheep, mirna that have been previously identified as key promoters of proliferation in rodents are predicted to have decreased expression with age and therefore reside in clusters or mir-199a, which has been shown to stimulate proliferation in cultured rat cardiomyocytes was identified in cluster however, further analysis using qrt-pcr demonstrated that mir-199a peaked around birth, then decreased into postnatal life to the level of that seen at 91 days gestation. furthermore, the expression pattern of pro-proliferative mir- did not allow its inclusion in any cluster and qrt-pcr demonstrated a similar expression at all ages. considering the varied expression of their target genes, the outcomes of the present study suggest that mir-199a and mir- are not major regulators of developmental cardiomyocyte quiescence in sheep. the roles of every mirna in clusters and have not been identified, however, some mirna in these clusters have been involved in aspects of the regulation of cell proliferation in other species. recent studies have implicated the mir-17- group as an important regulator of cardiomyocyte proliferation and capable of stimulating previously quiescent, adult mice cardiomyocytes. there are six members of the mir-17- group, which includes mir- mir-18a, mir-19a, mir-20a, mir-19b- and mir-92a- in the present study, mir- and mir- were in cluster, which suggests a potential role of these mirna in the inhibition of cardiomyocyte proliferation in late gestation, however, mir-20a and mir-19b were in cluster and the expression of mir-18a and 19a did not change with age. thus, the specific roles of mirna in the mir-17- group in regulating cardiomyocyte quiescence are unclear. the developmental regulation of an imprinted mirna cluster during heart development the present study has allowed us to isolate the period when most cardiomyocytes lose the ability to proliferate from other cellular processes, such as hypertrophy, that are upregulated after birth. cluster was particularly enriched for mirna derived from an imprinted locus located on the telomeric end of oar in sheep. the syntenic region is conserved in placental mammals and it contains a large cluster of maternally expressed mirna that may have complex roles in the cis- and trans-regulation of expression of genes within the broader imprinted ~  mb region or direct involvement in the maintenance of the imprinting status of the locus. a mutation in this broader imprinted region in sheep strongly deregulates expression of multiple genes within the core of this locus, including the maternally expressed mirna, and causes postnatal skeletal muscle hypertrophy in select muscles in a rostro-caudal gradient across the animal but only when the mutation is inherited in the context of the paternal heterozygote. the mutation apparently does not impact cardiac function, although specific tests have not been undertaken to date. whether the mirna within this genomic locus are targeted at mrna or involved in imprinting maintenance is not clear, but it is interesting to note that the mirna from this cluster putatively target the kegg tgfβ pathway. multiple members of this pathway have strong roles in regulating muscle development and cardiac development and disease. maternally expressed genes typically limit aspects of offspring tissue growth and are often down-regulated after birth. we therefore postulate that the down-regulation of these mirna after birth may help coordinate the activation of negative regulators of growth thereby limiting cardiomyocyte size and possibly heart size relative to body size. functional analysis of predicted targets of developmentally regulated mirna clusters the functional enrichment analyses for the predicted target genes for each mirna cluster from the mirna microarray study often showed enrichment for “cancer” related themes, which is likely indirectly reflecting proliferative potential, and also enrichment for the related terms transcription factor regulation and proliferative activity. thus, there is a consistent functional theme relating to regulation of proliferation in the predicted target genes of the differentially expressed mirna. one of the difficulties in the biological interpretation of mirna data relates to the abundance of a mirna, its mrna targets and the effects of competing mirna, which together interplay in a complex manner to regulate gene expression. therefore, understanding the enriched biological themes in the current investigation will require functional modulation of multiple mirna in an appropriate cell culture model of cardiomyocyte cell proliferation and differentiation, coupled with biochemical validation of putative mirna target genes.  this study has identified mirna and their target genes associated with cardiac development across late gestation and into postnatal life in sheep, thereby isolating the transition of cardiomyocytes to quiescence that occurs in late gestation from the major physiological and biochemical changes that occur after birth. the investigation has identified a number of clusters of differentially expressed mirna, some of which are consistent in timing with the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep. the present study is also consistent with a coordinated and complex role of mirna in the regulation of cardiomyocyte quiescence. the study identified substantial differences between sheep and rodent cardiac development that may reflect species differences underpinning the very different timing relative to birth of cardiomyocyte quiescence in these species. importantly, through the use of mirna microarrays, the current study highlights the potential involvement of many mirna in the coordination of this pivotal developmental transition. 
the cardiac expression of mir-133a increased and its target gene igf1r decreased with increasing age, reaching their respective maximum and minimum abundance when the majority of ovine cardiomyocytes were quiescent. the expression patterns of ‘pro-proliferative’ mir-199a and mir- were also inconsistent with their involvement in cardiomyocyte quiescence. the expression patterns of the mir- family, mir-199a and mir- were inconsistent with direct involvement in the regulation cardiomyocyte proliferation in sheep, despite studies in rodents demonstrating that their manipulation can influence the degree of cardiomyocyte proliferation. these latter profiles are inconsistent with the direct involvement of this family of mirna in cardiomyocyte quiescence in late gestation sheep. mirna microarray analysis suggests a coordinated and potentially more complex role of multiple mirna in the regulation of cardiomyocyte quiescence and highlights significant differences between species that may reflect their substantial differences in the timing of this developmental process. the results of this study show that the expression of mir-133a and one of its target genes is consistent with it being involved in the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep. the functions of predicted target genes for the mirna in four of the six clusters were enriched for aspects of cell division and regulation of cell proliferation suggesting a potential role of these mirna in regulating cardiomyocyte proliferation. the expression of the mir- family members was variable with age, however, four of their target genes decreased with age. recent studies in zebrafish and rodents implicate microrna in the regulation of genes responsible for cardiac cell cycle progression and regeneration, in particular, mir-133a, the mir- family, mir-199a and mir- however, the significance of these mirna and mirna in general in the regulation of cardiomyocyte proliferation in large mammals, including humans, where the timing of heart development relative to birth is very different than in rodents, is unclear. consequently, mirna microarray analysis was undertaken, which identified six discrete clusters of mirna with characteristic developmental profiles. to determine the involvement of mirna in the down-regulation of cardiomyocyte proliferation occurring before birth in large mammals, we investigated mirna and target gene expression in sheep hearts before and after birth. the experimental approach included targeted transcriptional profiling of mirna and target mrna previously identified in rodent studies as well as genome-wide mirna profiling using microarrays.
 there is a limited capacity to repair damage in the mammalian heart after birth, which is primarily due to the inability of cardiomyocytes to proliferate after birth. this is in contrast to zebrafish and salamander, in which cardiomyocytes retain the ability to proliferate throughout life and can regenerate their heart after significant damage. recent studies in zebrafish and rodents implicate microrna in the regulation of genes responsible for cardiac cell cycle progression and regeneration, in particular, mir-133a, the mir- family, mir-199a and mir- however, the significance of these mirna and mirna in general in the regulation of cardiomyocyte proliferation in large mammals, including humans, where the timing of heart development relative to birth is very different than in rodents, is unclear. to determine the involvement of mirna in the down-regulation of cardiomyocyte proliferation occurring before birth in large mammals, we investigated mirna and target gene expression in sheep hearts before and after birth. the experimental approach included targeted transcriptional profiling of mirna and target mrna previously identified in rodent studies as well as genome-wide mirna profiling using microarrays.  the cardiac expression of mir-133a increased and its target gene igf1r decreased with increasing age, reaching their respective maximum and minimum abundance when the majority of ovine cardiomyocytes were quiescent. the expression of the mir- family members was variable with age, however, four of their target genes decreased with age. these latter profiles are inconsistent with the direct involvement of this family of mirna in cardiomyocyte quiescence in late gestation sheep. the expression patterns of ‘pro-proliferative’ mir-199a and mir- were also inconsistent with their involvement in cardiomyocyte quiescence. consequently, mirna microarray analysis was undertaken, which identified six discrete clusters of mirna with characteristic developmental profiles. the functions of predicted target genes for the mirna in four of the six clusters were enriched for aspects of cell division and regulation of cell proliferation suggesting a potential role of these mirna in regulating cardiomyocyte proliferation.  the results of this study show that the expression of mir-133a and one of its target genes is consistent with it being involved in the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep. the expression patterns of the mir- family, mir-199a and mir- were inconsistent with direct involvement in the regulation cardiomyocyte proliferation in sheep, despite studies in rodents demonstrating that their manipulation can influence the degree of cardiomyocyte proliferation. mirna microarray analysis suggests a coordinated and potentially more complex role of multiple mirna in the regulation of cardiomyocyte quiescence and highlights significant differences between species that may reflect their substantial differences in the timing of this developmental process. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords microarraymir-133mir- familymir-590mir-199acardiomyocyteproliferationissue-copyright-statement© the author 2015 