A dynamic spatiotemporal extracellular matrix facilitates epicardial-mediated vertebrate heart regeneration

Unlike humans, certain adult vertebrates such as newts and zebrafish possess extraordinary abilities to functionally regenerate lost appendages and injured organs, including cardiac muscle. Here, we present new evidence that a remodeled extracellular matrix (ECM) directs cell activities essential for cardiac muscle regeneration. Comprehensive mining of DNA microarrays and Gene Ontology term enrichment analyses for regenerating newt and zebrafish hearts revealed that distinct ECM components and ECM-modifying proteases are among the most significantly enriched genes in response to local injury. In contrast, data analyses for mammalian cardiac injury models indicated that inflammation and metabolic processes are the most significantly activated gene groups. In the regenerating newt heart, we show dynamic spatial and temporal changes in tenascin-C, hyaluronic acid, and fibronectin ECM distribution as early as 3 days postamputation. Linked to distinct matrix remodeling, we demonstrate a myocardium-wide proliferative response and radial migration of progenitor cells. In particular, we report dramatic upregulation of a regeneration-specific matrix in the epicardium that precedes the accumulation and migration of progenitor cells. For the first time, we show that the regenerative ECM component tenascin-C significantly increases newt cardiomyocyte cell cycle reentry in vitro. Thus, the engineering of nature-tested extracellular matrices may provide new strategic opportunities for the enhancement of regenerative responses in mammals.     

Regulation of zebrafish heart regeneration by miR-133

Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. Using pharmacological inhibition and EGFP sensor interaction studies, we found that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation.     

Cardiomyocyte overexpression of miR-27b induces cardiac hypertrophy and dysfunction in mice

Recent studies have begun to reveal critical roles of microRNAs (miRNAs) in the pathogenesis of cardiac hypertrophy and dysfunction. In this study, we tested whether a transforming growth factor-β (TGF-β)-regulated miRNA played a pivotal role in the development of cardiac hypertrophy and heart failure (HF). We observed that miR-27b was upregulated in hearts of cardiomyocyte-specific Smad4 knockout mice, which developed cardiac hypertrophy. In vitro experiments showed that the miR-27b expression could be inhibited by TGF-β1 and that its overexpression promoted hypertrophic cell growth, while the miR-27b suppression led to inhibition of the hypertrophic cell growth caused by phenylephrine (PE) treatment. Furthermore, the analysis of transgenic mice with cardiomyocyte-specific overexpression of miR-27b revealed that miR-27b overexpression was sufficient to induce cardiac hypertrophy and dysfunction. We validated the peroxisome proliferator-activated receptor-γ (PPAR-γ) as a direct target of miR-27b in cardiomyocyte. Consistently, the miR-27b transgenic mice displayed significantly lower levels of PPAR-γ than the control mice. Furthermore, in vivo silencing of miR-27b using a specific antagomir in a pressure-overload-induced mouse model of HF increased cardiac PPAR-γ expression, attenuated cardiac hypertrophy and dysfunction. The results of our study demonstrate that TGF-β1-regulated miR-27b is involved in the regulation of cardiac hypertrophy, and validate miR-27b as an efficient therapeutic target for cardiac diseases.     

Deficiency of Cardiomyocyte-specific MicroRNA-378 Contributes to the Development of Cardiac Fibrosis Involving a Transforming Growth Factor β (TGFβ1)-dependent Paracrine Mechanism

Understanding the regulation of cardiac fibrosis is critical for controlling adverse cardiac remodeling during heart failure. Previously we identified miR-378 as a cardiomyocyte-abundant miRNA down-regulated in several experimental models of cardiac hypertrophy and in patients with heart failure. To understand the consequence of miR-378 down-regulation during cardiac remodeling, our current study employed a locked nucleic acid-modified antimiR to target miR-378 in vivo. Results showed development of cardiomyocyte hypertrophy and fibrosis in mouse hearts. Mechanistically, miR-378 depletion was found to induce TGFβ1 expression in mouse hearts and in cultured cardiomyocytes. Among various secreted cytokines in the conditioned-media of miR-378-depleted cardiomyocytes, only TGFβ1 levels were found to be increased. The increase was prevented by miR-378 expression. Treatment of cardiac fibroblasts with the conditioned media of miR-378-depleted myocytes activated pSMAD2/3 and induced fibrotic gene expression. This effect was counteracted by including a TGFβ1-neutralizing antibody in the conditioned-medium. In cardiomyocytes, adenoviruses expressing dominant negative N-Ras or c-Jun prevented antimiR-mediated induction of TGFβ1 mRNA, documenting the importance of Ras and AP-1 signaling in this response. Our study demonstrates that reduction of miR-378 during pathological conditions contributes to cardiac remodeling by promoting paracrine release of profibrotic cytokine, TGFβ1 from cardiomyocytes. Our data imply that the presence in cardiomyocyte of miR-378 plays a critical role in the protection of neighboring fibroblasts from activation by pro-fibrotic stimuli.     

Ischemic Postconditioning-Mediated miRNA-21 Protects against Cardiac ischemia/reperfusion Injury via PTEN/Akt Pathway

Background

Ischemic postconditioning (IPost) protects the reperfused heart from infarction which has drawn much attention recently. However, studies to date have rarely investigated the role of microRNAs (miRNAs) in IPost. The aims of this study were to investigate whether miR-21 is involved in the protective effect of IPost against myocardial ischemia-reperfusion (I/R) injury and disclose the potential molecular mechanisms involved.

Methods and Results

We found that miR-21 was remarkably up-regulated in mouse hearts after IPost. To determine the protective role of IPost-induced miR-21 up-regulation, the mice were divided into the following four groups: I/R group; I/R+IPost group (I/R mice treated with IPost); Antagomir-21+IPost+I/R group (I/R mice treated with anagomir-21 and IPost); Scramble+IPost+I/R group (I/R mice treated with scramble and IPost). The results showed IPost could reduce I/R injury-induced infarct size of the left ventricle, improve cardiac function, and prevent myocardial apoptosis, while knockdown of miR-21 with antagomir-21 could reverse these protective effects of IPost against mouse I/R injury. Furthermore, we confirmed that miR-21 plays a protective role in myocardial apoptosis through PTEN/Akt signaling pathway, which was abrogated by the PI3K inhibitor LY294002. The protective effect of miR-21 on myocardial apoptosis was further revealed in mouse hearts after IPost treatment in vivo.

Conclusions

Our data clearly demonstrate that miR-21 is involved in IPost-mediated cardiac protection against I/R injury and dysfunction through the PTEN/Akt signaling pathway in vivo. Identifying the beneficial roles of IPost-regulated miRNAs in cardiac protection, which may be a rational target selection for ischemic cardioprotection.     

Cell-specific detection of microRNA expression during cardiomyogenesis by combined in situ hybridization and immunohistochemistry

MicroRNAs (miRNAs) regulate gene expression by mediating translational repression or mRNA degradation of their targets, and several miRNAs control developmental decisions through embryogenesis. In the developing heart, miRNA targets comprise key players mediating cardiac lineage determination. However, although several miRNAs have been identified as differentially regulated during cardiac development and disease, their distinct cell-specific localization remains largely undetermined, likely owing to a lack of adequate methods. We therefore report the development of a markedly improved approach combining fluorescence-based miRNA-in situ hybridization (miRNA-ISH) with immunohistochemistry (IHC). We have applied this protocol to differentiating embryoid bodies (EBs) as well as embryonic and adult mouse hearts, to detect miRNAs that were upregulated during EB cardiomyogenesis, as determined by array-based miRNA expression profiling. In this manner, we found specific co-localization of miR-1 to myosin positive cells (cardiomyocytes) of EBs, developing and mature hearts. In contrast, miR-125b and -199a did not localize to cardiomyocytes, as previously suggested for miR-199a, but were rather expressed in connective tissue cells of the heart. More specifically, by co-staining with α-smooth muscle actin (α-SMA) and collagen-I, we found that miR-125b and -199a localize to perivascular α-SMA⁻ stromal cells. Our approach thus proved valid for determining cell-specific localization of miRNAs, and the findings we present highlight the importance of determining exact cell-specific localization of miRNAs by sequential miRNA-ISH and IHC in studies aiming at understanding the role of miRNAs and their targets. This approach will hopefully aid in identifying relevant miRNA targets of both the heart and other organs.     

Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians.     

MiRNAs with Apoptosis Regulating Potential Are Differentially Expressed in Chronic Exercise-Induced Physiologically Hypertrophied Hearts

Physiological cardiac hypertrophy is an adaptive mechanism, induced during chronic exercise. As it is reversible and not associated with cardiomyocyte death, it is considered as a natural tactic to prevent cardiac dysfunction and failure. Though, different studies revealed the importance of microRNAs (miRNAs) in pathological hypertrophy, their role during physiological hypertrophy is largely unexplored. Hence, this study is aimed at revealing the global expression profile of miRNAs during physiological cardiac hypertrophy. Chronic swimming protocol continuously for eight weeks resulted in induction of physiological hypertrophy in rats and histopathology revealed the absence of tissue damage, apoptosis or fibrosis. Subsequently, the total RNA was isolated and small RNA sequencing was executed. Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during physiological hypertrophy. The expression profile of the significantly differentially expressed miRNAs was validated by qPCR. In silico prediction of target genes by miRanda, miRdB and TargetScan and subsequent qPCR analysis unraveled that miRNAs including miR-99b, miR-100, miR-19b, miR-10, miR-208a, miR-133, miR-191a, miR-22, miR-30e and miR-181a are targeting the genes that primarily regulate cell proliferation and cell death. Gene ontology and pathway mapping showed that the differentially expressed miRNAs and their target genes were mapped to apoptosis and cell death pathways principally via PI3K/Akt/mTOR and MAPK signaling. In summary, our data indicates that regulation of these miRNAs with apoptosis regulating potential can be one of the major key factors in determining pathological or physiological hypertrophy by controlling fibrosis, apoptosis and cell death mechanisms.     

Spiked-in Pulsed in Vivo Labeling Identifies a New Member of the CCN Family in Regenerating Newt Hearts

The newt Notophthalmus viridescens , which belongs to the family of salamanders (Urodela), owns remarkable regenerative capacities allowing efficient scar-free repair of various organs including the heart. Salamanders can regrow large parts of the myocardium unlike mammals, which cannot replace lost cardiomyocytes efficiently. Unfortunately, very little is known about the molecules and the regulatory circuits facilitating efficient heart regeneration in newts or salamanders. To identify proteins that are involved in heart regeneration, we have developed a pulsed SILAC-based mass spectrometry method based on the detection of paired peptide peaks after (13)C(6)-lysine incorporation into proteins in vivo. Proteins were identified by matching mass spectrometry derived peptide sequences to a recently established normalized newt EST library. Our approach enabled us to identify more than 2200 nonredundant proteins in the regenerating newt heart. Because of the pulsed in vivo labeling approach, accurate quantification was achieved for 1353 proteins, of which 72 were up- and 31 down-regulated with a (|log 2 ratio| > 1) during heart regeneration. One deregulated member was identified as a new member of the CCN protein family, showing a wound specific activation. We reason that the detection of such deregulated newt-specific proteins in regenerating hearts supports the idea of a local evolution of tissue regeneration in salamanders. Our results significantly improve understanding of dynamic changes in the complex protein network that underlies heart regeneration and provides a basis for further mechanistic studies.     

miR-1 Exacerbates Cardiac Ischemia-Reperfusion Injury in Mouse Models

Recent studies have revealed the critical role of microRNAs (miRNAs) in regulating cardiac injury. Among them, the cardiac enriched microRNA-1(miR-1) has been extensively investigated and proven to be detrimental to cardiac myocytes. However, solid in vivo evidence for the role of miR-1 in cardiac injury is still missing and the potential therapeutic advantages of systemic knockdown of miR-1 expression remained unexplored. In this study, miR-1 transgenic (miR-1 Tg) mice and locked nucleic acid modified oligonucleotide against miR-1 (LNA-antimiR-1) were used to explore the effects of miR-1 on cardiac ischemia/reperfusion injury (30 min ischemia followed by 24 h reperfusion). The cardiac miR-1 level was significantly increased in miR-1 Tg mice, and suppressed in LNA-antimiR-1 treated mice. When subjected to ischemia/reperfusion injury, miR-1 overexpression exacerbated cardiac injury, manifested by increased LDH, CK levels, caspase-3 expression, apoptosis and cardiac infarct area. On the contrary, LNA-antimiR-1 treatment significantly attenuated cardiac ischemia/reperfusion injury. The expression of PKCε and HSP60 was significantly repressed by miR-1 and enhanced by miR-1 knockdown, which may be a molecular mechanism for the role miR-1 in cardiac injury. Moreover, luciferase assay confirmed the direct regulation of miR-1 on protein kinase C epsilon (PKCε) and heat shock protein 60 (HSP60). In summary, this study demonstrated that miR-1 is a causal factor for cardiac injury and systemic LNA-antimiR-1 therapy is effective in ameliorating the problem.     

Relationship Between Downregulation of miRNAs and Increase of Oxidative Stress in the Development of Diabetic Cardiac Dysfunction: Junctin as a Target Protein of miR-1

Oxidative stress is involved in the etiology of diabetes-induced cardiac dysfunction while microRNAs (miRNAs) are known as regulators for genes involved in cardiac remodeling. However, a functional link between miRNAs and diabetes-induced cardiac dysfunction remains to be investigated. Here, we aimed to identify whether the expression levels of miRNAs are associated with oxidative stress/diabetic heart and if proteins responsible from contractile activity during diabetes might be directly modulated by miRNAs. Diabetic cardiomyopathy developed with streptozotocin, is characterized with marked changes in sarcomere and mitochondria, depressed left ventricular developed pressure, and a massive oxidative stress that is particularly evident in the heart. miRNA profiling was performed in freshly isolated left ventricular cells from diabetic rats. Using microarray analysis, we identified marked changes in the expression of 43 miRNAs (37 of them were downregulated while 6 miRNAs were upregulated) out of examined total of 351 miRNAs. Among them, 6 miRNAs were further validated by real-time PCR. The expression levels of miR-1, miR-499, miR-133a, and miR-133b were markedly depressed in the diabetic cardiomyocytes while miR-21 level increased and miR-16 level was unchanged. Notably, normalization of cardiac function and oxidant/antioxidant level after N-acetylcysteine (NAC)-treatment of diabetic rats resulted with a significant restoration in the expression levels of miR-499, miR-1, miR-133a, and miR-133b in the myocardium. Since changes in the level of muscle-specific miR-1 has been implicated in cardiac diseases and its specific molecular targets involved in its action, in part, associated with oxidative stress are limited, we first examined the protein levels of some SR-associated proteins such as junctin and triadin. Junctin but not triadin is markedly overexpressed in diabetic cardiomyocytes while its level was normalized in NAC-treated diabetics. Luciferase reporter assay showed that junctin is targetted by miR-1. Taken together, our data demonstrates that intervention with an antioxidant treatment for 4-week leads to significant cardioprotection against diabetes-induced injury, controlling oxidant/antioxidant level, which may directly control the levels of some miRNAs including miR-1 and its target protein junctin, which is involved in the development of diabetic cardiomyopathy.     

MiRNA-1/133a Clusters Regulate Adrenergic Control of Cardiac Repolarization

The electrical properties of the heart are primarily determined by the activity of ion channels and the activity of these molecules is permanently modulated and adjusted to the physiological needs by adrenergic signaling. miRNAs are known to control the expression of many proteins and to fulfill distinct functions in the mammalian heart, though the in vivo effects of miRNAs on the electrical activity of the heart are poorly characterized. The miRNAs miR-1 and miR-133a are the most abundant miRNAs of the heart and are expressed from two miR-1/133a genomic clusters. Genetic modulation of miR-1/133a cluster expression without concomitant severe disturbance of general cardiomyocyte physiology revealed that these miRNA clusters govern cardiac muscle repolarization. Reduction of miR-1/133a dosage induced a longQT phenotype in mice especially at low heart rates. Longer action potentials in cardiomyocytes are caused by modulation of the impact of β-adrenergic signaling on the activity of the depolarizing L-type calcium channel. Pharmacological intervention to attenuate β-adrenergic signaling or L-type calcium channel activity in vivo abrogated the longQT phenotype that is caused by modulation of miR-1/133a activity. Thus, we identify the miR-1/133a miRNA clusters to be important to prevent a longQT-phenotype in the mammalian heart.     

The role of microRNA-26b in human adipocyte differentiation and proliferation

Recent findings indicate that microRNAs (miRNAs) are involved in the regulatory network of adipogenesis and obesity. Thus far, only a few human miRNAs are known to function as adipogenic regulators, fanning interest in studies on the functional role of miRNAs during adipogenesis in humans. In a previous study, we used a microarray to assess miRNA expression during human preadipocyte differentiation. We found that expression of the miR-26b was increased in mature adipocytes. MiR-26b is an intronic miRNA located in the intron of CTDSP1 (carboxy terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1). Target prediction and Renilla luciferase analyses revealed the phosphatase and tensin homolog gene (PTEN) as a putative target gene. In this study, we found that miR-26b was gradually upregulated during adipocyte differentiation. To understand the roles of miR-26b in adipogenesis, we adopted a loss-of-function approach to silence miR-26b stably in human preadipocytes. We found that miR-26b inhibition effectively suppressed adipocyte differentiation, as evidenced by decreased lipid droplets and the ability of miR-26b to decrease mRNA levels of adipocyte-specific molecular markers and triglyceride accumulation. Furthermore, the cell growth assay revealed that miR-26b inhibition promoted proliferation. Nevertheless, it had no effect on apoptosis. Taken together, these data indicate that miR-26b may be involved in adipogenesis and could be targeted for therapeutic intervention in obesity.     

Recovery of the Xenopus laevis heart from ROS-induced stress utilizes conserved pathways of cardiac regeneration

Urodele amphibians and some fish are capable of regenerating up to a quarter of their heart tissue after cardiac injury. While many anuran amphibians like Xenopus laevis are not capable of such feats, they are able to repair lesser levels of cardiac damage, such as that caused by oxidative stress, to a far greater degree than mammals. Using an optogenetic stress induction model that utilizes the protein KillerRed, we have investigated the extent to which mechanisms of cardiac regeneration are conserved during the restoration of normal heart morphology post oxidative stress in X. laevis tadpoles. We focused particularly on the processes of cardiomyocyte proliferation and dedifferentiation, as well as the pathways that facilitate the regulation of these processes. The cardiac response to KillerRed-induced injury in X. laevis tadpole hearts consists of a phase dominated by indicators of cardiac stress, followed by a repair-like phase with characteristics similar to mechanisms of cardiac regeneration in urodeles and fish. In the latter phase, we found markers associated with partial dedifferentiation and cardiomyocyte proliferation in the injured tadpole heart, which, unlike in regenerating hearts, are not dependent on Notch or retinoic acid signaling. Ultimately, the X. laevis cardiac response to KillerRed-induced oxidative stress shares characteristics with both mammalian and urodele/fish repair mechanisms, but is nonetheless a unique form of recovery, occupying an intermediate place on the spectrum of cardiac regenerative ability. An understanding of how Xenopus repairs cardiac damage can help bridge the gap between mammals and urodeles and contribute to new methods of treating heart disease.     

Novel regulation and functional interaction of polycistronic miRNAs

The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11∼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11∼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response.