- morphophysiological differences in the fully formed heart of these species, such as chamber septation, heart rate frequency, blood pressure, and cardiac output volume. - Conclusions: Both the conserved and group-specific miRNAs can be considered modulators orchestrating the core and peripheral genes of heart GRNs of vertebrates, which can be related to the morphophysiological differences and similarities existing in the heart of distinct vertebrate groups. - We propose a hypothesis to explain evolutionary differences in the putative functional roles of miRNAs in the heart GRNs analyzed. - The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. - insights into the molecular mechanisms that could be helping modulate the diversity of morphophysiology in the heart organ of vertebrate species.. - This organ is the first to form and function in the developing embryo [1]. - “reptiles”, due to the differences in the heart morphology and the control of body temperature characteristic. - However, the molecular mechanisms controlling the refinement in the expression of TBX5 and other important genes have yet to be fully uncovered. - In fact, diverse inter- actions and regulatory mechanisms acting in the heart GRNs responsible for heart species-specific singularities remain unclear. - Particularly, little is known about the role played by non-coding RNAs in shaping the heart distinc- tive morphology among species, both at the onset of heart formation and later in the adult heart.. - However, only a small fraction of miRNAs expressed in the heart of vertebrates have been deeply examined, implying that functional roles of miRNAs and bona-fide miRNA-target interactions in heart GRNs are still largely unknown.. - All these data indicate that the whole set of miR- NAs are important modulators across numerous GRNs governing the design of distinct phenotypes, including the GRNs responsible for the observed heart shape in the vertebrate species.. - In order to understand the roles played by miRNAs in the evolution of heart GRNs of vertebrates, we used publicly available data from 17 vertebrate species and expanded the set of species analyzed by sequencing miR- NAs from the heart of Nile tilapia, Xenopus laevis, and one lizard species. - Tracing the birth age of miRNAs expressed in the heart of vertebrates revealed that conserved miRNA families have representatives that can be traced back to 400- 690 Million Years Ago (MYA). - In this sense, our analysis sug- gests that conserved miRNAs, which present high expres- sion and target several genes, may be acting on several processes in the heart GRN, whereas the group-specific miRNAs, which present lower expression and target few genes, may be fine-tuning specific processes.. - Our pipeline to identify miRNA-target interactions were designed to integrate both predictions of TargetScan and miRanda, followed by filtering genes not expressed in the heart. - Results from all pre- dicted and validated interactions identified along with the centrality analysis were organized in the Supplementary Tables S1–S12 in Additional file 4.. - In the fish heart network (Fig. - These miRNAs may be acquiring a central role in the network by targeting several genes and helping to fine-tune various biological processes. - How- ever, most miRNAs may be acting as peripheral genes in the network, which suggests that they play roles in specific biological processes in the heart of fishes. - Table S2 in Additional file 4), suggesting these miRNAs are important modulators in the heart of fish species.. - In the amphibian network (Fig. - Interestingly, these miRNAs were predicted to target the kernel genes of heart GRN, which suggests that their functions may be related to core functions in the heart of amphib- ians. - miRNAs and HAND1 were only predicted in amphib- ians (Table S1 in Additional file 4), indicating that the modulation of expression of HAND1 by miR-129 and miR-221 is occurring specifically in the amphibian heart GRN. - Moreover, the miR-338, miR-191, and let-7 were predicted to target CX40, indicating that those miRNAs may be playing roles in the heart contraction rate. - Fur- thermore, we detected pairs of previously validated inter- actions in the heart such as between miR-1 and HAND2 and miR-128 and ISL1 ([38]. - Table S2 in Additional file 4), which shows that both miRNAs may be important regulators in the amphibian heart GRN.. - In the reptile network (Fig. - The numbers at the right bottom indicate the number of miRNA families in the groups indicated at the left bottom. - In the bird heart network (Fig. - 3 Expression level and the number of predicted targets of miRNAs with conserved and group-specific expression in the heart of vertebrates.. - Moesin), which indicates that those miRNAs may be acting together to modulate the cellular proliferation process in the heart of birds. - In the mammal heart network (Fig. - More- over, among the mammal-specific miRNAs, the miR- 154 presents a high degree and closeness score, which indicates that this miRNA may be playing a central role in the heart GRN of mammals by targeting sev- eral genes, suggesting roles for miR-154 in several path- ways of the mammal heart. - miR-26 and miR-142 present binding sites in the 3’UTR of SMAD1, suggesting an integrative effort among both miRNAs to possibly modulate the expression of SMAD1 to control the cardiomyocyte proliferation, differentia- tion, and tissue homeostasis processes. - Comparative analysis of miR-target interactions in the heart GRN of vertebrates. - We were able to detect conserved miR-target interactions among heart networks of vertebrate groups in the com- parative analysis (Additional file 5). - This reveals that the miR-target interactions in the fish network present. - This suggests that miR-target interactions in the heart of amphibians are more similar to reptiles than to other vertebrate groups, which may reflect a shared morphophysiological trait among these groups.. - Comparative analysis of the heart miRNome of vertebrates Our large-scale comparative analysis of miRNAs com- position and expression breadth in the vertebrate heart. - In fact, miRNAs are specialized in function by dis- playing an organ/cell-specific expression patterns [40], which indicates that the heterogeneity of miRNAs may be related to the distinct traits observed in the heart of vertebrates, including variable regenerative capacity. - By contrast, similarities in the set of miRNAs may correlate with core heart functions across species, such as blood pumping, electric patterning [2], and other pathways related to the basal metabolism of the cardiac cells.. - We noticed that most of the conserved miRNA families expressed in the heart of the 20 species investigated can be traced back to 400-690 Million Years Ago (MYA). - These observations are consistent with the previous hypoth- esis that ancient miRNAs show higher expression and regulate a wider range of targets than younger miRNAs suggesting that the functional roles of ancient miRNAs are well established in the regulation of heart GRNs. - MicroRNAs and the heart GRN. - The heart networks obtained for each group indicate that most of the miRNAs may be acting as peripheral genes in the network, suggesting that miRNAs tend to act in spe- cific biological processes. - Otherwise, few miRNAs may be added to their group-specific heart GRNs, due to the high number of interactions with genes expressed in the heart, including the kernel genes. - In this topic, we discuss the miRNAs that may be playing important functional roles in the heart GRN of vertebrates and should be functionally analyzed in future experiments.. - The miR-1 is one of the most expressed miRNAs in the heart of all vertebrate species analyzed, which suggests that miR-1 has key roles in the maintenance and home- ostasis processes of the heart of vertebrates. - In this sense, the present data indicate that we can include miR- 1 in the heart GRN by targeting HAND2 in a conserved manner for all vertebrate groups and targeting Gata4 in a mammal-specific manner. - In this sense, the interactions between miR-1 and FZD7 may help to generate the dis- tinct phenotypes observed in the heart of vertebrates.. - Moreover, knockout of miR-1 in mice results in postnatal lethality due to abnormalities in the heart [52, 53], which suggests an important functional role for miR-1 during cardiogenesis and homeostasis of heart biology.. - For the miR-133, we identified a validated interaction between miR-133 and SRF in mice indicating a role for miR-133 in the heart of mammals and putatively other vertebrates. - We detected that members of the let-7 family are highly expressed in the heart and let-7 can present a poten- tial function in the heart GRN of mammals, once we detected a validated interaction with BMP4 in humans and predicted a conserved interaction with HAND1 in all mammal species. - Interestingly, a study analyzing miRNAs expressed during mouse heart development detected that let-7 targets HAND1 and may play key roles in the heart development network of mammals [60]. - In this sense, let-7 may be included in the heart GRN of mammals due to their interactions with the kernel genes.. - The miR-8 presented a putative functionality as a cen- tral gene in the heart network of vertebrates (i.e., high degree and closeness score in all groups). - In this sense, miR-8 may be an important mod- ulator of the myocyte proliferation and differentiation in the heart GRN of vertebrates.. - The miR-26 is a conserved miRNA that may be added to the kernel due to the validated interaction with SMAD1 detected in humans and the conserved interaction pre- dicted in 10 species from the 14 analyzed in the present study. - In fact, miR-26 may play pivotal roles in the heart (reviewed by [69. - In this sense, miR-26 should be the focus of further experiments to elucidate its functional roles in the heart GRN of vertebrates.. - Looking at the group-specific miRNAs in the fish heart GRN, we noticed that the miR-724 may be regulating the gene ENSP SGCG), which suggests roles on the heart contraction rate, whereas the miR-722 is puta- tively targeting the gene ENSP MSN), which indicates roles in the myocyte proliferation process.. - The reptile-specific miR-5399 was predicted to target SMAD1, which indicates a role, specific to reptiles, for this miRNA in the processes of cardiomyocyte proliferation, differentiation, and tissue homeostasis in an adult context [34].. - Alongside the bird-specific miRNAs, we were able to detect that the miR-1552 and miR-12223 may be acting together on the gene ENSP MSN) and that miR-1552 is possibly targeting SRF, which indicates that both miRNAs may be acting on controlling the cellular proliferation process in the cardiac cells of birds.. - The mammal-specific miR-154 was predicted to target several genes expressed in the heart of mammals, includ- ing a few kernel genes. - Such interactions indicate that miR-154 may be acting on controlling the myocyte pro- liferation processes and is playing central roles in the mammal heart GRN. - In fact, several studies showed that miR-154 controls the myocyte proliferation, fibrosis, and cardiac remodeling processes in the heart of mice [71–73].. - Overall, we were able to show that several miRNAs may be included in the heart GRN of vertebrates and noticed that most of the interactions detected are sup- ported by previous experimental data. - One limitation of the pipeline applied in the present study is related to the fact that we only used interac- tions available from Human data in the STRING database due to the higher number of interactions available for this species when compared to other species. - However, we believe that this issue may not heavily dis- rupt the results obtained and discussed in the present study once we focused our analysis on miRNA-target interactions.. - MicroRNAs in the evolution of heart GRN. - In this sense, it is possible that miRNAs act as CRMs to shape the evolution of GRNs governing the morphophysiological differences observed in the heart of vertebrates [7].. - Then after the inclusion of miRNAs in the heart GRN, the expression level of miRNAs and its targets could shape the appearance and maintenance of novel phenotypic traits in the heart of vertebrates along evolution.. - Here, we present a comprehensive annotation and com- parative analysis of miRNAs expressed in the heart of 20 species representative of all major vertebrate groups.. - We showed that conserved miRNAs may act distinc- tively in the heart GRNs of vertebrates, whereas some of the group-specific miRNAs may be playing singular roles in the heart of groups they are being expressed. - Our data indicate that some miRNAs may be playing central roles in the heart GRN, by acting in several processes, whereas most of the miRNAs are participating as peripheral genes, by acting in specific pathways.. - These developmental studies will be helpful to improve knowl- edge about heart GRN evolution and will lead to insight into the addition of miRNAs in the heart evolution pro- cess of vertebrates.. - The raw reads were used in the subse- quent data analysis. - We applied a common pipeline workflow to all datasets analyzed in the present study, which is described below and summarized in the Additional file 7.. - In the case where the species had no previ- ous miRNA annotation available, we referred the annota- tion to the closely related species. - We excluded those loci detected by miRD- eep2 pipeline that did not present any unique reads in the ShortStack analysis. - After the target prediction step, we filtered the list of putative targets by keeping only genes expressed in the heart organ of each species by using heart transcriptome data available at the SRA and ENA database (see Table S4 in Additional file 6 for more details). - The transcrip- tome analysis of heart datasets was performed using the Kallisto algorithm [95] and only genes with TPM higher or equal to 1 were considered expressed in the heart and kept as putative targets. - laevis, zebra finch, and rabbit), we only used 14 vertebrate species in the miRNA target prediction analysis. - After the target filtering step, we detected the human orthologous genes from each species using the BioMart tool from Ensembl and designed the networks based on the interactions available in the STRING database (v11.0;. - In the groups containing more than one species in the GRN analysis, we applied a conservation filter to keep only conserved interactions.. - The centrality measures are calculated based on the interactions of nodes in the network and indicate when a node is central or periph- eral. - Then, we manually check the differences and similarities in the interactions in the GRN of vertebrate groups. - Moreover, to comple- ment the network analysis and confirm the interactions observed, we checked for strong validated interactions among miRNAs and their putative targets in the miR- TarBase (release 7.0. - The heart miRNA-seq data of Nile tilapia, African clawed frog and lizard are available in the NCBI BioProject database under the accession number PRJNA560566.. - The heart miRNA-seq data from all other vertebrate species are available in the NCBI SRA database (accession numbers SRR1554476, SRR866605, SRR2473346, SRR1231994, SRR553599, SRR553594, SRR553589, SRR6662685, SRR4048260, SRR553584, SRR553579, and SRR553574) and EMBL-EBI ENA database (accession numbers SRR1736653, SRR1047498, SRR2062562, and SRR3587077).. - The heart RNA-seq data from vertebrate species analyzed are available in the NCBI SRA database (accession numbers SRR2013387, SRR391681, SRR2054794, SRR1524252, SRR579563, SRR2515151, SRR579558, SRR306714, SRR306730, SRR306749, SRR2226636, SRR388744, SRR087419, SRR306768, SRR306782, SRR306849).. - Gene regulatory networks in the evolution and development of the heart. - A meta-analysis of in vivo vertebrate cardiac performance: implications for cardiovascular support in the evolution of endothermy. - Dose-dependent Smad1, Smad5 and Smad8 signaling in the early mouse embryo. - Prdm1a and miR-499 act sequentially to restrict Sox6 activity to the fast-twitch muscle lineage in the zebrafish embryo. - microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart. - Multifaceted roles of miR-1 s in repressing the fetal gene program in the heart. - Are microRNAs responsible for cardiac hypertrophy in fish and mammals? What we can learn in the activation process in a zebrafish ex vivo model. - microRNA-133a regulates cardiomyocyte proliferation and suppresses smooth muscle gene expression in the heart
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