Reducing branched-chain amino acids improves cardiac stress response in mice by decreasing histone H3K23 propionylation.

Identification of branched-chain amino acid (BCAA) oxidation enzymes in the nucleus led us to predict that they are a source of the propionyl-CoA that is utilized for histone propionylation and, thereby, regulate gene expression. To investigate the effects of BCAAs on the development of cardiac hypertrophy and failure, we applied pressure overload on the heart in mice maintained on a diet with standard levels of BCAAs (BCAA control) versus a BCAA-free diet. The former was associated with an increase in histone H3K23-propionyl (H3K23Pr) at the promoters of upregulated genes (e.g., cell signaling and extracellular matrix genes) and a decrease at the promoters of downregulated genes (e.g., electron transfer complex [ETC I-V] and metabolic genes). Intriguingly, the BCAA-free diet tempered the increases in promoter H3K23Pr, thus reducing collagen gene expression and fibrosis during cardiac hypertrophy. Conversely, the BCAA-free diet inhibited the reductions in promoter H3K23Pr and abolished the downregulation of ETC I-V subunits, enhanced mitochondrial respiration, and curbed the progression of cardiac hypertrophy. Thus, lowering the intake of BCAAs reduced pressure overload-induced changes in histone propionylation-dependent gene expression in the heart, which retarded the development of cardiomyopathy.

Klf9 plays a critical role in GR -dependent metabolic adaptations in cardiomyocytes.

Glucocorticoids through activation of the Glucocorticoid receptor (GR) play an essential role in cellular homeostasis during physiological variations and in response to stress. Our genomic GR binding and transcriptome data from Dexamethasone (Dex) treated cardiomyocytes showed an early differential regulation of mostly transcription factors, followed by sequential change in genes involved in downstream functional pathways. We examined the role of Krüppel-like factor 9 (Klf9), an early direct target of GR in cardiomyocytes. Klf9-ChIPseq identified 2150 genes that showed an increase in Klf9 binding in response to Dex. Transcriptome analysis of Dex treated cardiomyocytes with or without knockdown of Klf9 revealed differential regulation of 1777 genes, of which a reversal in expression is seen in 1640 genes with knockdown of Klf9 compared to Dex. Conversely, only 137 (∼8%) genes show further dysregulation in expression with siKLf9, as seen with Dex treated cardiomyocytes. Functional annotation identified genes of metabolic pathways on the top of differentially expressed genes, including those involved in glycolysis and oxidative phosphorylation. Knockdown of Klf9 in cardiomyocytes inhibited Dex induced increase in glycolytic function and mitochondrial spare respiratory capacity, as measured by glycolysis and mito stress tests, respectively. Thus, we conclude that cyclic, diurnal GR activation, through Klf9 -dependent feedforward signaling plays a central role in maintaining cellular homeostasis through metabolic adaptations in cardiomyocytes.

G3bp1 - microRNA-1 axis regulates cardiomyocyte hypertrophy.

Adaptation of gene expression is one of the most fundamental response of cardiomyocytes to hypertrophic stimuli. G3bp1, an RNA binding protein with site-specific endoribonuclease activity regulates the processing of pre-miR-1 stem-loop, and thus levels of cardiomyocyte -enriched mature miR-1. Here, we examine the role of G3bp1 in regulating gene expression in quiescent cardiomyocytes and those undergoing growth-factor induced hypertrophy. Further, we determine if these changes are facilitated through G3bp1-mediated regulation of miR-1 in these cardiomyocytes. Using isolated cardiomyocytes with knockdown of endogenous G3bp1, we performed high throughput RNA sequencing to determine the change in cardiac transcriptome. Then, using gain and loss of function approach for both, G3bp1 and miR-1, alone or in combination we examine the G3bp1-miR-1 signaling in regulating gene expression and Endothelin (ET-1) -induced cardiomyocyte hypertrophy. We show that knockdown of endogenous G3bp1 results in inhibition of genes involved in calcium handling, cardiac muscle contraction, action potential and sarcomeric structure. In addition, there is inhibition of genes that contribute to hypertrophic and dilated cardiomyopathy development. Conversely, an increase is seen in genes that negatively regulate the Hippo signaling, like Rassf1 and Arrdc3, along with inflammatory genes of TGF-ß and TNF pathways. Knockdown of G3bp1 restricts ET-1 induced cardiomyocyte hypertrophy. Interestingly, concurrent silencing of G3bp1 and miR-1 rescues the change in gene expression and inhibition of hypertrophy seen with knockdown of G3bp1 alone. Similarly, expression of exogenous G3bp1 reverses the miR-1 induced inhibition of gene expression. Intriguingly, expression of Gfp tagged G3bp1 results in perinuclear accumulations of G3bp1-Gfp, resembling Stress Granules. Based on our results, we conclude that G3bp1 through its regulation of mature miR-1 levels plays a critical role in regulating the expression of essential cardiac-enriched genes and those involved in development of cardiomyocyte hypertrophy.

A protocol for transdifferentiation of human cardiac fibroblasts into endothelial cells via activation of innate immunity.

Endothelial cells (ECs) have emerged as key pathogenic players in cardiac disease due to their proximity with cardiomyocytes. Induced pluripotent stem cells (iPSCs) have been employed to generate ECs. However, it may be more clinically relevant to transdifferentiate fibroblasts into ECs directly without introducing pluripotent or virally driven transcription factors. Here, we present a protocol that describes the direct conversion of human cardiac fibroblasts into ECs by leveraging the innate immune system. Our protocol produces bona fide human ECs with 95%-98% purity by first passage. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020) and Sayed et al. (2015).

Acute NelfA knockdown restricts compensatory gene expression and precipitates ventricular dysfunction during cardiac hypertrophy.

Coordinated functional balance of negative and positive transcription complexes maintain and accommodate gene expression in hearts during quiescent and hypertrophic conditions, respectively. Negative elongation factor (Nelf) complex has been implicated in RNA polymerase II (pol II) pausing, a widespread regulatory transcriptional phenomenon observed across the cardiac genome. Here, we examine the role of NelfA aka, Wolf-Hirschhorn syndrome candidate 2 (Whsc2), a critical component of the negative elongation complex in hearts undergoing pressure-overload induced hypertrophy. Alignment of high-resolution genome-wide occupancy data of NelfA, Pol II, TFIIB and H3k9ac from control and hypertrophied hearts reveal that NelfA associates with active gene promoters. High NelfA occupancy is seen at promoters of essential and cardiac-enriched genes, expressed under both quiescent and hypertrophic conditions. Conversely, de novo NelfA recruitment is observed at inducible gene promoters with pressure overload, accompanied by significant increase in expression of these genes with hypertrophy. Interestingly, change in promoter NelfA levels correlates with the transcript output in hypertrophied hearts compared to Sham, suggesting NelfA might be playing a critical role in the regulation of gene transcription during cardiac hypertrophy. In vivo knockdown of NelfA (siNelfA) in hearts subjected to pressure-overload results in early ventricular dilatation and dysfunction, associated with decrease in expression of inducible and cardiac-enriched genes in siNelfA hypertrophied compared to control hypertrophied hearts. In accordance, in vitro knockdown of NelfA in cardiomyocytes showed no change in promoter pol II, however significant decrease in in-gene and downstream pol II occupancy was observed. These data suggest an inhibited pol II progression in transcribing and inducible genes, which reflects as a decrease in transcript abundance of these genes. These results indicate that promoter NelfA occupancy is essential for pol II -dependent transcription. Therefore, we conclude that NelfA is required for active transcription and gene expression during cardiac hypertrophy.

HIF1α Regulates Early Metabolic Changes due to Activation of Innate Immunity in Nuclear Reprogramming.

Innate immune signaling has recently been shown to play an important role in nuclear reprogramming, by altering the epigenetic landscape and thereby facilitating transcription. However, the mechanisms that link innate immune activation and metabolic regulation in pluripotent stem cells remain poorly defined, particularly with regard to key molecular components. In this study, we show that hypoxia-inducible factor 1α (HIF1α), a central regulator of adaptation to limiting oxygen tension, is an unexpected but crucial regulator of innate immune-mediated nuclear reprogramming. HIF1α is dramatically upregulated as a consequence of Toll-like receptor 3 (TLR3) signaling and is necessary for efficient induction of pluripotency and transdifferentiation. Bioenergetics studies reveal that HIF1α regulates the reconfiguration of innate immune-mediated reprogramming through its well-established role in throwing a glycolytic switch. We believe that results from these studies can help us better understand the influence of immune signaling in tissue regeneration and lead to new therapeutic strategies.

Oxoglutarate dehydrogenase and acetyl-CoA acyltransferase 2 selectively associate with H2A.Z-occupied promoters and are required for histone modifications.

Histone H2A.Z plays an essential role in regulating transcriptional rates and memory. Interestingly, H2A.Z-bound nucleosomes are located in both transcriptionally active and inactive promotors, with no clear understanding of the mechanisms via which it differentially regulates transcription. We hypothesized that its functions are mediated through recruitment of regulatory proteins to promoters. Using rapid chromatin immunoprecipitation-mass spectrometry, we uncovered the association of H2A.Z-bound chromatin with the metabolic enzymes, oxoglutarate dehydrogenase (OGDH) and acetyl-CoA acyltransferase 2 (ACAA2). Recombinant green florescence fusion proteins, combined with mutations of predicted nuclear localization signals, confirmed their nuclear localization and chromatin binding. Conclusively, chromatin immunoprecipitation-deep sequencing, confirmed the predominant association of OGDH and ACAA2 with H2A.Z-occupied transcription start sites and enhancers, the former of which we confirmed is conserved in both mouse and human tissue. Furthermore, H2A.Z-deficient human HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, accompanied with reduced posttranslational histone modifications, including acetylation and succinylation. Specifically, knockdown of OGDH diminished H4 succinylation. Thus, the data reveal that select metabolic enzymes are assembled at active, H2A.Z-occupied, promoters, for potential site-directed production of metabolic intermediates that are required for histone modifications.

Glucocorticoid Receptor-Binding and Transcriptome Signature in Cardiomyocytes.

Background An increase in serum cortisol has been identified as a risk factor for cardiac failure, which highlights the impact of glucocorticoid signaling in cardiomyocytes and its influence in the progression of failure. Dexamethasone, a synthetic glucocorticoid, is sufficient for induction of cardiomyocyte hypertrophy, but little is known of the glucocorticoid receptor (GR) genome-binding and -dependent transcriptional changes that mediate this phenotype. Methods and Results In this study using high-resolution sequencing, we identified genomic targets of GR and associated change in the transcriptome after 1 and 24 hours of dexamethasone treatment. We showed that GR associates with 6482 genes in the cardiac genome, with differential regulation of 738 genes. Interestingly, alignment of the chromatin immunoprecipitation and RNA sequencing data show that, after 1 hour, 69% of differentially regulated genes are associated with GR and identify as regulators of RNA pol II-dependent transcription. Conversely, after 24 hours only 45% of regulated genes are associated with GR and involved in dilated and hypertrophic cardiomyopathies as well as other growth-related pathways. In addition, our data also reveal that a majority of genes (76.42%) associated with GR show incremental changes in transcript abundance and are genes involved in basic cellular processes that might be regulated by the dynamics of promoter-paused RNA pol II, as seen in hearts undergoing hypertrophy. In vivo administration of dexamethasone resulted in similar changes in the cardiac transcriptome, as seen in isolated cardiomyocytes. Conclusions Our data reveal genome-wide GR binding sites in cardiomyocytes, identify novel targets and GR-dependent change in the transcriptome that induces and contributes to cardiomyocyte hypertrophy.

Recruitment of RNA Polymerase II to Metabolic Gene Promoters Is Inhibited in the Failing Heart Possibly Through PGC-1α (Peroxisome Proliferator-Activated Receptor-γ Coactivator-1α) Dysregulation.

BACKGROUND: Proper dynamics of RNA polymerase II, such as promoter recruitment and elongation, are essential for transcription. PGC-1α (peroxisome proliferator-activated receptor [PPAR]-γ coactivator-1α), also termed PPARGC1a, is a transcriptional coactivator that stimulates energy metabolism, and PGC-1α target genes are downregulated in the failing heart. However, whether the dysregulation of polymerase II dynamics occurs in PGC-1α target genes in heart failure has not been defined. METHODS AND RESULTS: Chromatin immunoprecipitation-sequencing revealed that reduced promoter occupancy was a major form of polymerase II dysregulation on PGC-1α target metabolic gene promoters in the pressure-overload-induced heart failure model. PGC-1α-cKO (cardiac-specific PGC-1α knockout) mice showed phenotypic similarity to the pressure-overload-induced heart failure model in wild-type mice, such as contractile dysfunction and downregulation of PGC-1α target genes, even under basal conditions. However, the protein levels of PGC-1α were neither changed in the pressure-overload model nor in human failing hearts. Chromatin immunoprecipitation assays revealed that the promoter occupancy of polymerase II and PGC-1α was consistently reduced both in the pressure-overload model and PGC-1α-cKO mice. In vitro DNA binding assays using an endogenous PGC-1α target gene promoter sequence confirmed that PGC-1α recruits polymerase II to the promoter. CONCLUSIONS: These results suggest that PGC-1α promotes the recruitment of polymerase II to the PGC-1α target gene promoters. Downregulation of PGC-1α target genes in the failing heart is attributed, in part, to a reduction of the PGC-1α occupancy and the polymerase II recruitment to the promoters, which might be a novel mechanism of metabolic perturbations in the failing heart.

Transcriptional regulation mediated by H2A.Z via ANP32e-dependent inhibition of protein phosphatase 2A.

The mechanisms that regulate H2A.Z and its requirement for transcription in differentiated mammalian cells remains ambiguous. In this study, we identified the interaction between the C-terminus of ANP32e and N-terminus of H2A.Z in a yeast two-hybrid screen. Knockdown of ANP32e resulted in proteasomal degradation and nuclear depletion of H2A.Z or of a chimeric green florescence protein fused to its N-terminus. This effect was reversed by inhibition of protein phosphatase 2A (PP2A) and, conversely, reproduced by overexpression of its catalytic subunit. Accordingly, knockdown of ANP32e inhibited phosphorylation of H2A.Z, whereas a mutation of serine-9 proved its requirement for both the protein's stability and nuclear localization, as did knockdown of the nuclear mitogen and stress-induced kinase 1. Moreover, ANP32e's knockdown also revealed its differential requirement for cell signaling and gene expression, whereas, genome-wide binding analysis confirmed its co-localization with H2A.Z at transcription start sites, as well as, gene bodies of inducible and tissue-specific genes. The data also suggest that H2A.Z restricts transcription, which is moderated by ANP32e at the promoter and gene bodies of expressed genes. Thus, ANP32e, through inhibition of PP2A, is required for nucleosomal inclusion of H2A.Z and the regulation of gene expression.

GTPase Activating Protein (Sh3 Domain) Binding Protein 1 Regulates the Processing of MicroRNA-1 during Cardiac Hypertrophy.

BACKGROUND: MicroRNAs (miR) are small, posttranscriptional regulators, expressed as part of a longer primary transcript, following which they undergo nuclear and cytoplasmic processing by Drosha and Dicer, respectively, to form the functional mature ~20mer that gets incorporated into the silencing complex. Others and we have shown that mature miR-1 levels decrease with pressure-induced cardiac hypertrophy, however, there is little or no change in the primary transcript encompassing miR-1 stem-loop, suggesting critical regulatory step in microRNA processing. The objective of this study was to investigate the underlying mechanisms regulating miR-1 expression in cardiomyocytes. RESULTS: Here we report that GTPase-activating protein (SH3 domain) binding protein 1 (G3bp1), an endoribonuclease regulates miR-1 processing in cardiomyocytes. G3bp1 is upregulated during cardiac hypertrophy and restricts miR-1 processing by binding to its consensus sequence in the pre-miR-1-2 stem-loop. In accordance, exogenous G3bp1 is sufficient to reduce miR-1 levels, along with derepression of miR-1 targets; General transcription factor IIB (Gtf2b), cyclin dependent factor 9 (Cdk9) and eukaryotic initiation factor 4E (Eif4e). While Cdk9 and Gtf2b are essential for transcription, Eif4e is required for translation. Thus, downregulation of miR-1 is necessary for increase in these molecules. Similar to miR-1 knockdown, G3bp1 overexpression is not sufficient for development of cardiac hypertrophy. Conversely, knockdown of G3bp1 in hypertrophying cardiomyocytes inhibited downregulation of miR-1 and upregulation of its targets along with restricted hypertrophy, suggesting that G3bp1 is necessary for development of cardiac hypertrophy. These results indicate that G3bp1-mediated inhibition of miR-1 processing with growth stimulation results in decrease in mature miR-1 and, thereby, an increase of its targets, which play fundamental roles in the development of hypertrophy. CONCLUSION: G3bp1 posttranscriptionally regulates miRNA-1 processing in the heart, and G3bp1 mediated downregulation of mature miRNA-1 levels is required for the derepression of its targets and increase in gene expression during cardiac hypertrophy.

miR-206 Mediates YAP-Induced Cardiac Hypertrophy and Survival.

RATIONALE: In Drosophila, the Hippo signaling pathway negatively regulates organ size by suppressing cell proliferation and survival through the inhibition of Yorkie, a transcriptional cofactor. Yes-associated protein (YAP), the mammalian homolog of Yorkie, promotes cardiomyocyte growth and survival in postnatal hearts. However, the underlying mechanism responsible for the beneficial effect of YAP in cardiomyocytes remains unclear. OBJECTIVES: We investigated whether miR-206, a microRNA known to promote hypertrophy in skeletal muscle, mediates the effect of YAP on promotion of survival and hypertrophy in cardiomyocytes. METHODS AND RESULTS: Microarray analysis indicated that YAP increased miR-206 expression in cardiomyocytes. Increased miR-206 expression induced cardiac hypertrophy and inhibited cell death in cultured cardiomyocytes, similar to that of YAP. Downregulation of endogenous miR-206 in cardiomyocytes attenuated YAP-induced cardiac hypertrophy and survival, suggesting that miR-206 plays a critical role in mediating YAP function. Cardiac-specific overexpression of miR-206 in mice induced hypertrophy and protected the heart from ischemia/reperfusion injury, whereas suppression of miR-206 exacerbated ischemia/reperfusion injury and prevented pressure overload-induced cardiac hypertrophy. miR-206 negatively regulates Forkhead box protein P1 expression in cardiomyocytes and overexpression of Forkhead box protein P1 attenuated miR-206-induced cardiac hypertrophy and survival, suggesting that Forkhead box protein P1 is a functional target of miR-206. CONCLUSIONS: YAP increases the abundance of miR-206, which in turn plays an essential role in mediating hypertrophy and survival by silencing Forkhead box protein P1 in cardiomyocytes.

Acute targeting of general transcription factor IIB restricts cardiac hypertrophy via selective inhibition of gene transcription.

BACKGROUND: We previously reported that specialized and housekeeping genes are differentially regulated via de novo recruitment and pause-release of RNA polymerase II, respectively, during cardiac hypertrophy. However, the significance of this finding remains to be examined. Therefore, the purpose of this study was to determine the mechanisms that differentially regulate these gene groups and exploit them for therapeutic targeting. METHODS AND RESULTS: Here, we show that general transcription factor IIB (TFIIB) and cyclin-dependent kinase 9 are upregulated during hypertrophy, both targeted by microRNA-1, and play preferential roles in regulating those 2 groups of genes. Chromatin immunoprecipitation-sequencing reveals that TFIIB is constitutively bound to all paused, housekeeping, promoters, whereas de novo recruitment of TFIIB and polymerase II is required for specialized genes that are induced during hypertrophy. We exploited this dichotomy to acutely inhibit induction of the latter set, which encompasses cardiomyopathy, immune reaction, and extracellular matrix genes, using locked nucleic acid-modified antisense TFIIB oligonucleotide treatment. This resulted in suppression of all specialized genes, while sparing the housekeeping ones, and, thus, attenuated pathological hypertrophy. CONCLUSIONS: The data for the first time reveal distinct general TFIIB dynamics that regulate specialized versus housekeeping genes during cardiac hypertrophy. Thus, by acutely targeting TFIIB, we were able to inhibit selectively the former set of genes and ameliorate pressure overload hypertrophy. We also demonstrate the feasibility of acutely and reversibly targeting cardiac mRNA for therapeutic purposes using locked nucleic acid-modified antisense oligonucleotides.

Transcriptional regulation patterns revealed by high resolution chromatin immunoprecipitation during cardiac hypertrophy.

Cardiac hypertrophy is characterized by a generalized increase in gene expression that is commensurate with the increase in myocyte size and mass, on which is superimposed more robust changes in the expression of specialized genes. Both transcriptional and posttranscriptional mechanisms play fundamental roles in these processes; however, genome-wide characterization of the transcriptional changes has not been investigated. Our goal was to identify the extent and modes, RNA polymerase II (pol II) pausing versus recruitment, of transcriptional regulation underlying cardiac hypertrophy. We used anti-pol II and anti-histone H3K9-acetyl (H3K9ac) chromatin immunoprecipitation-deep sequencing to determine the extent of pol II recruitment and pausing, and the underlying epigenetic modifications, respectively, during cardiac growth. The data uniquely reveal two mutually exclusive modes of transcriptional regulation. One involves an incremental increase (30-50%) in the elongational activity of preassembled, promoter-paused, pol II, and encompasses ∼25% of expressed genes that are essential/housekeeping genes (e.g. RNA synthesis and splicing). Another involves a more robust activation via de novo pol II recruitment, encompassing ∼5% of specialized genes (e.g. contractile and extracellular matrix). Moreover, the latter subset has relatively shorter 3'-UTRs with fewer predicted targeting miRNA, whereas most miRNA targets fall in the former category, underscoring the significance of posttranscriptional regulation by miRNA. The results, for the first time, demonstrate that promoter-paused pol II plays a role in incrementally increasing housekeeping genes, proportionate to the increase in heart size. Additionally, the data distinguish between the roles of posttranscriptional versus transcriptional regulation of specific genes.

GATA4 expression is primarily regulated via a miR-26b-dependent post-transcriptional mechanism during cardiac hypertrophy.

AIMS: GATA4 is a transcription factor that is up-regulated during cardiac hypertrophy and plays a fundamental role in myocyte growth and survival. In this study, we investigate the transcriptional vs. post-transcriptional mechanisms that are involved in regulating GATA4 in the heart during neonatal and pressure overload-induced hypertrophic growth. METHODS AND RESULTS: GATA4 protein is significantly higher during pressure overload-induced (2.9 ± 0.4-fold) and neonatal (6.8 ± 1-fold) hypertrophic growth vs. the normal adult mouse heart. Using RNA polymerase II immunoprecipitation combined with deep sequencing, we confirmed that active transcription of the Gata4 gene remained unchanged during hypertrophy, whereas it was two-fold higher in the neonatal vs. adult heart, commensurate with the mRNA levels. These results suggested a post-transcriptional mode of regulation of its expression, which prompted the identification of a conserved sequence in its 3'-untranslated region that was responsible for reduced translation via miR-26b. Overexpression of miR-26b reduced GATA4-dependent transcription, endothelin-induced hypertrophy, and sensitized the cells to apoptotic insults. Additionally, miR-26b targeted phospholipase C-ß1, which, in turn, inhibited miR-26b expression, creating a double-negative feedback loop. Accordingly, overexpression of miR-26b in the heart inhibited up-regulation of its targets and the development of hypertrophy. However, knockdown of miR-26b is not sufficient for inducing hypertrophy. CONCLUSION: Down-regulation of miR-26b in the heart is required for the up-regulation of GATA4 and the induction of pressure-induced cardiac hypertrophy. The results also underscore the functional relevance of miRNAs in regulating gene expression during cardiac hypertrophy.

MicroRNAs in development and disease.

MicroRNAs (miRNAs) are a class of posttranscriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. There are currently over 10,000 miRNAs that have been identified in a range of species including metazoa, mycetozoa, viridiplantae, and viruses, of which 940, to date, are found in humans. It is estimated that more than 60% of human protein-coding genes harbor miRNA target sites in their 3' untranslated region and, thus, are potentially regulated by these molecules in health and disease. This review will first briefly describe the discovery, structure, and mode of function of miRNAs in mammalian cells, before elaborating on their roles and significance during development and pathogenesis in the various mammalian organs, while attempting to reconcile their functions with our existing knowledge of their targets. Finally, we will summarize some of the advances made in utilizing miRNAs in therapeutics.

AKT-ing via microRNA.

MicroRNAs are involved in almost every aspect of a mammalian cell's functionality, from stem cell differentiation to aging and pathogenesis; however, their role in immediate cell signaling is less defined. This has been recently demonstrated by the rapid increase or decrease of miR-21's abundance within minutes of activation or inhibition of the AKT pathway, respectively, which mediates its regulation of Fas ligand (FasL) and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression, among other targets. Conversely, AKT induces rapid downregulation of miR-199a-5p to effect upregulation of hypoxia-inducible factor 1α Hif-1α and sirtuin 1 (Sirt1). This suggests that posttranscriptional mechanisms regulate miRNAs' processing and/or stability to induce the rapid fluctuation in their levels. In support, a growing number of studies are showing specific posttranscriptional regulation of miRNAs. The data potentially explain how AKT, and plausibly other signaling pathways, can specifically and promptly modulate a gene's translation while circumventing the need for transcription during transient signaling events. In this article we present our views regarding cell signaling via miRNAs.

MicroRNA-21 is a downstream effector of AKT that mediates its antiapoptotic effects via suppression of Fas ligand.

MicroRNA-21 (miR-21) is highly up-regulated during hypertrophic and cancerous cell growth. In contrast, we found that it declines in cardiac myocytes upon exposure to hypoxia. Thus, the objective was to explore its role during hypoxia. We show that miR-21 not only regulates phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but also targets Fas ligand (FasL). During prolonged hypoxia, down-regulation of miR-21 proved necessary and sufficient for enhancing expression of both proteins. We demonstrate here for the first time that miR-21 is positively regulated via an AKT-dependent pathway, which is depressed during prolonged hypoxia. Accordingly, hypoxia-induced down-regulation of miR-21 and up-regulation of FasL and PTEN were reversed by activated AKT and reproduced by a dominant negative mutant, wortmannin, or PTEN. Moreover, the antiapoptotic function of AKT partly required miR-21, which was sufficient for inhibition of caspase-8 activity and mitochondrial damage. In consensus, overexpression of miR-21 in a transgenic mouse heart resulted in suppression of ischemia-induced up-regulation of PTEN and FasL expression, an increase in phospho-AKT, a smaller infarct size, and ameliorated heart failure. Thus, we have identified a unique aspect of the function of AKT by which it inhibits apoptosis through miR-21-dependent suppression of FasL.

An antagonism between the AKT and beta-adrenergic signaling pathways mediated through their reciprocal effects on miR-199a-5p.

We have recently reported that downregulation of miR-199a-5p is necessary and sufficient for inducing upregulation of its targets, including hypoxia-inducible factor-1 alpha (Hif-1 alpha) and Sirt1, during hypoxia preconditioning (HPC). Conversely, others and we have reported that miR-199a-5p is upregulated during cardiac hypertrophy. Thus, the objective of this study was to delineate the signaling pathways that regulate the expression of miR-199a-5p and its targets, and their role in myocyte survival during hypoxia. Since HPC is mediated through activation of the AKT pathway, we questioned if AKT is sufficient for inducing downregulation of miR-199a-5p. Our present study shows that overexpression of a constitutively active AKT (caAKT) induced 70% reduction in miR-199a-5p and was associated with a robust increase in HiF-1 alpha (10+/-2 fold) and Sirt1 (4+/-0.8 fold) that was reversed by overexpression of miR-199a-5p. Similarly, insulin receptor-stimulated activation of the AKT pathway induced downregulation of miR-199a-5p and upregulation of its targets. In contrast, beta-adrenergic receptor (beta AR) activation in vitro and in vivo, induced 1.8-3.5-fold increase in miR-199a-5p. Accordingly, we predicted that beta AR would antagonize AKT-induced, miR-199a-5p-dependent, upregulation of Hif-1 alpha and Sirt1. Indeed, pre-treating the myocytes with isoproterenol before applying HPC, caAKT, or insulin resulted in 87+/-3%, 75+/-15%, and 100% reductions in Hif-1 alpha expression, respectively, and sensitized the cells to hypoxic injury. Thus, activation of beta-adrenergic signaling counteracts the survival effects of the AKT pathway via upregulating miR-199a-5p.

Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes.

MicroRNAs are posttranscriptional gene regulators that are differentially expressed during various diseases and have been implicated in the underlying pathogenesis. We report here that miR-199a is acutely downregulated in cardiac myocytes on a decline in oxygen tension. This reduction is required for the rapid upregulation of its target, hypoxia-inducible factor (Hif)-1alpha. Replenishing miR-199a during hypoxia inhibits Hif-1alpha expression and its stabilization of p53 and, thus, reduces apoptosis. On the other hand, knockdown of miR-199a during normoxia results in the upregulation of Hif-1alpha and Sirtuin (Sirt)1 and reproduces hypoxia preconditioning. Sirt1 is also a direct target of miR-199a and is responsible for downregulating prolyl hydroxylase 2, required for stabilization of Hif-1alpha. Thus, we conclude that miR-199a is a master regulator of a hypoxia-triggered pathway and can be exploited for preconditioning cells against hypoxic damage. In addition, the data demonstrate a functional link between 2 key molecules that regulate hypoxia preconditioning and longevity.