Ariana Foinquinos

Long noncoding RNA Chast promotes cardiac remodeling

Inhibition of the long noncoding RNA Chast prevents pressure overload–induced cardiac remodeling in mice. The missing lnc in cardiac hypertrophy RNA that does not code for a protein comprises a large portion of the human genome. These so-called noncoding RNAs are emerging as important players in disease pathogenesis, yet their functional roles are not always well known. Viereck et al. have discovered a new long noncoding RNA (lncRNA) that promotes cardiac remodeling and hypertrophy in mice, which could one day be targeted with therapeutics to treat human cardiovascular diseases. The identified lncRNA, which the authors named Chast (for “cardiac hypertrophy–associated transcript”), was discovered to be up-regulated in hypertrophic mouse hearts. When mouse and human heart cells expressed Chast, they tended to be larger than their normal counterparts. By silencing Chast with antisense oligonucleotides, mice either did not develop hypertrophy or were rescued from established disease. In a step toward translation, the authors discovered a human homolog, CHAST, that similarly caused cells in a dish to enlarge. Additional investigation in patients will confirm the relevance of this lncRNA in human disease and whether it is indeed a promising target for treating cardiac hypertrophy and heart failure. Recent studies highlighted long noncoding RNAs (lncRNAs) to play an important role in cardiac development. However, understanding of lncRNAs in cardiac diseases is still limited. Global lncRNA expression profiling indicated that several lncRNA transcripts are deregulated during pressure overload–induced cardiac hypertrophy in mice. Using stringent selection criteria, we identified Chast (cardiac hypertrophy–associated transcript) as a potential lncRNA candidate that influences cardiomyocyte hypertrophy. Cell fractionation experiments indicated that Chast is specifically up-regulated in cardiomyocytes in vivo in transverse aortic constriction (TAC)–operated mice. In accordance, CHAST homolog in humans was significantly up-regulated in hypertrophic heart tissue from aortic stenosis patients and in human embryonic stem cell–derived cardiomyocytes upon hypertrophic stimuli. Viral-based overexpression of Chast was sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. GapmeR-mediated silencing of Chast both prevented and attenuated TAC-induced pathological cardiac remodeling with no early signs on toxicological side effects. Mechanistically, Chast negatively regulated Pleckstrin homology domain–containing protein family M member 1 (opposite strand of Chast), impeding cardiomyocyte autophagy and driving hypertrophy. These results indicate that Chast can be a potential target to prevent cardiac remodeling and highlight a general role of lncRNAs in heart diseases.

Osteopontin is indispensible for AP1-mediated angiotensin II-related miR-21 transcription during cardiac fibrosis

Aims Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process. Methods and results OPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo. Conclusion In cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation.

Cardiac myocyte-secreted cAMP exerts paracrine action via adenosine receptor activation

Acute stimulation of cardiac β-adrenoceptors is crucial to increasing cardiac function under stress; however, sustained β-adrenergic stimulation has been implicated in pathological myocardial remodeling and heart failure. Here, we have demonstrated that export of cAMP from cardiac myocytes is an intrinsic cardioprotective mechanism in response to cardiac stress. We report that infusion of cAMP into mice averted myocardial hypertrophy and fibrosis in a disease model of cardiac pressure overload. The protective effect of exogenous cAMP required adenosine receptor signaling. This observation led to the identification of a potent paracrine mechanism that is dependent on secreted cAMP. Specifically, FRET-based imaging of cAMP formation in primary cells and in myocardial tissue from murine hearts revealed that cardiomyocytes depend on the transporter ABCC4 to export cAMP as an extracellular signal. Extracellular cAMP, through its metabolite adenosine, reduced cardiomyocyte cAMP formation and hypertrophy by activating A1 adenosine receptors while delivering an antifibrotic signal to cardiac fibroblasts by A2 adenosine receptor activation. Together, our data reveal a paracrine role for secreted cAMP in intercellular signaling in the myocardium, and we postulate that secreted cAMP may also constitute an important signal in other tissues.

Inhibition of the Cardiac Fibroblast-Enriched lncRNA Meg3 Prevents Cardiac Fibrosis and Diastolic Dysfunction

Rationale: Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. Recent studies described the role of long noncoding RNAs (lncRNAs) in cardiac pathologies. Nevertheless, detailed reports on lncRNAs regulating CF biology and describing their implication in cardiac remodeling are still missing. Objective: Here, we aimed at characterizing lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction. Methods and Results: Global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp-2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-&bgr; I. Chromatin immunoprecipitation showed that further induction of Mmp-2 expression by transforming growth factor-&bgr; I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance. Conclusions: Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling.

Quaking Inhibits Doxorubicin-Mediated Cardiotoxicity Through Regulation of Cardiac Circular RNA Expression

Rationale: RBPs (RNA-binding proteins) have been described to be expressed and regulated in various organs including the heart. Little is known about the role of RBPs in heart failure induced by the chemotherapy drug doxorubicin and their interaction with circular RNAs. Objective: We aimed to identify key RBPs involved in doxorubicin-mediated heart failure and to elucidate their function. Methods and Results: Global transcriptome profiling from murine myocardium exposed to doxorubicin identified 5 differentially expressed RBPs. Expression of the RBP QKI (Quaking) in response to doxorubicin was strongly downregulated in rodent cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes in vitro and in vivo in mice. Knockdown of Qki in primary cardiomyocytes increased apoptosis and atrophy after treatment with doxorubicin, whereas lentiviral mediated overexpression of Qki5 inhibited the doxorubicin-induced apoptosis in cardiomyocytes. In vivo, AAV9 (adeno-associated virus serotype 9)–mediated cardiac overexpression of Qki5 prevented cardiac apoptosis and cardiac atrophy induced by doxorubicin and improved cardiac function. Mechanistically, by lentiviral-based overexpression and CRISPR/Cas9-mediated silencing of Qki5, we identified regulated expression of specific circular RNAs derived from Ttn (Titin), Fhod3 (Formin homology 2 domain containing 3), and Strn3 (Striatin, calmodulin-binding protein 3). Moreover, inhibition of Ttn-derived circular RNA increased the susceptibility of cardiomyocytes to doxorubicin. Conclusions: We here show that overexpression of Qki5 strongly attenuates the toxic effect of doxorubicin via regulating a set of circular RNAs. Qki5 is, thus, an interesting target molecule to combat doxorubicin-induced cardiotoxicity.

Noncoding RNAs in Heart Failure

Heart failure is a major contributor to the healthcare burden and mortality worldwide. Current treatment strategies are able to slow down the transition of healthy heart into the failing one; nevertheless better understanding of the complex genetic regulation of maladaptive remodeling in the failing heart is essential for new drug discovery. Noncoding RNAs are key epigenetic regulators of cardiac gene expression and thus significantly influence cardiac homeostasis and functions.In this chapter we will discuss characteristics of noncoding RNAs, especially miRNAs, long noncoding RNAs, and circular RNAs, and review recent evidences proving their profound involvement during different stages of heart failure progression. Several open questions still prevent the extensive use of noncoding RNA-modulating therapies in clinics; yet they are becoming an attractive target to define novel regulatory mechanisms in the heart. In-depth study of their interaction with gene networks will refine our current view of heart failure and revolutionize the drug development in coming years.

MicroRNA-Based Therapy of GATA2-Deficient Vascular Disease

Background: The transcription factor GATA2 orchestrates the expression of many endothelial-specific genes, illustrating its crucial importance for endothelial cell function. The capacity of this transcription factor in orchestrating endothelial-important microRNAs (miRNAs/miR) is unknown. Methods: Endothelial GATA2 was functionally analyzed in human endothelial cells in vitro. Endogenous short interfering RNA–mediated knockdown and lentiviral-based overexpression were applied to decipher the capacity of GATA2 in regulating cell viability and capillary formation. Next, the GATA2-dependent miR transcriptome was identified by using a profiling approach on the basis of quantitative real-time polymerase chain reaction. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation, luciferase promoter assays, and bisulfite sequencing analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at the angiogenic cytokine level via proteome profiler and enzyme-linked immunosorbent assays. Downstream miR targets were identified via bioinformatic target prediction and luciferase reporter gene assays. In vitro findings were translated to a mouse model of carotid injury in an endothelial GATA2 knockout background. Nanoparticle-mediated delivery of proangiogenic miR-126 was tested in the reendothelialization model. Results: GATA2 gain- and loss-of-function experiments in human umbilical vein endothelial cells identified a key role of GATA2 as master regulator of multiple endothelial functions via miRNA-dependent mechanisms. Global miRNAnome-screening identified several GATA2-regulated miRNAs including miR-126 and miR-221. Specifically, proangiogenic miR-126 was regulated by GATA2 transcriptionally and targeted antiangiogenic SPRED1 and FOXO3a contributing to GATA2-mediated formation of normal vascular structures, whereas GATA2 deficiency led to vascular abnormalities. In contrast to GATA2 deficiency, supplementation with miR-126 normalized vascular function and expression profiles of cytokines contributing to proangiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of antiangiogenic miR-221 by GATA2-dependent demethylation of a putative CpG island in the miR-221 promoter. Mechanistically, a reverted GATA2 phenotype by endogenous suppression of miR-221 was mediated through direct proangiogenic miR-221 target genes ICAM1 and ETS1. In a mouse model of carotid injury, GATA2 was reduced, and systemic supplementation of miR-126–coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved reendothelialization of injured carotid arteries in vivo. Conclusions: GATA2-mediated regulation of miR-126 and miR-221 has an important impact on endothelial biology. Hence, modulation of GATA2 and its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.

MicroRNAs: Novel Therapeutic Targets for Diabetic Wound Healing

Diabetic foot ulcer (DFU) represents a major clinical challenge among diabetic complications and combines multiple physiological factors involved in the inhibition of wound healing. Healing of skin wounds is a complex and dynamic process in response to cutaneous injury, which involves a cascade of molecular events orchestrated with temporal and spatial gene regulation in different cell types. Abnormal patterns of tissue repair-related gene expression and resultant cellular malfunctions are key components of impaired healing in diabetic patients. Thus, to understand the pathophysiology of delayed healing in DFU, it is crucial to identify the functional regulators in individual cell types. Recent studies have demonstrated various genetic and epigenetic regulators of the cellular transcriptome and among them highly conserved, tiny noncoding RNAs, especially microRNAs, constitute an important class of master regulators to regulate diverse cellular functions essential for skin wound healing. Here, we will discuss the recent advancements on miRNA regulation of tissue repair processes and their potential as novel therapeutic targets to accelerate healing in diabetic patients.

MicroRNA-Mediated Regulation of Cardiovascular Differentiation and Therapeutic Implications

Some stem cells—for example, embryonic, mesenchymal, and some progenitor cells—have the capacity to differentiate into cardiovascular cells lines such as cardiomyocytes, smooth-muscle cells, and endothelial cells. Mesenchymal stem cells serve a cardio-protective function by secreting large amounts of paracrine factors that regulate wound healing. miRNAs are important in cardiac wound healing and regeneration mediated by stem/progenitor cells, in that they modulate or precondition cardiac stem cells and thus optimize stem cell–based therapies. Stem/progenitor cell–based therapies are promising approaches to overcoming the limited capacity of the adult heart to recover after injury, using miRNAs as key regulators in cardiac wound healing and regeneration.